Register or Login To Download This Patent As A PDF
United States Patent Application |
20110243937
|
Kind Code
|
A1
|
DONALD; Carlton D.
|
October 6, 2011
|
TARGETING PAX2 FOR THE TREATMENT OF BREAST CANCER
Abstract
The present application provides methods of prevention and/or treatment
of breast cancer in a subject by inhibiting expression of PAX2. In the
cancer treatment methods disclosed, the method of inhibiting expression
of PAX2 can be by administration of a nucleic acid encoding an siRNA for
PAX2. A method of treating cancer in a subject by administering DEFB1 is
also provided. Similarly, provided is a method of treating cancer in a
subject by increasing expression of DEFB1 in the subject.
Inventors: |
DONALD; Carlton D.; (Mount Pleasant, SC)
|
Assignee: |
PHIGENIX, INC.
Atlanta
GA
|
Serial No.:
|
116752 |
Series Code:
|
13
|
Filed:
|
May 26, 2011 |
Current U.S. Class: |
424/133.1; 424/172.1; 424/94.63; 514/19.2; 514/19.4; 514/303; 514/381; 514/43; 514/44A; 514/44R; 514/456; 514/523 |
Class at Publication: |
424/133.1; 514/19.4; 514/19.2; 514/44.R; 514/44.A; 514/381; 424/172.1; 424/94.63; 514/43; 514/303; 514/523; 514/456 |
International Class: |
A61K 39/395 20060101 A61K039/395; A61K 38/02 20060101 A61K038/02; A61P 35/00 20060101 A61P035/00; A61K 31/7088 20060101 A61K031/7088; A61K 31/713 20060101 A61K031/713; A61K 31/4178 20060101 A61K031/4178; A61K 38/48 20060101 A61K038/48; A61K 31/7056 20060101 A61K031/7056; A61K 31/437 20060101 A61K031/437; A61K 31/277 20060101 A61K031/277; A61K 31/352 20060101 A61K031/352 |
Claims
1-9. (canceled)
10. A method of treating breast cancer or mammary intraepithelial
neoplasia (MIN) in a subject, comprising enhancing expression of DEFB1 in
a breast cancer tissue or MIN tissue in the subject.
11. The method of claim 10, wherein the enhancing expression of DEFB1
comprises administering to the subject an efficient amount of DEFB1.
12. The method of claim 10, wherein the enhancing expression of DEFB1
comprises administering to the breast cancer tissue or MIN tissue in the
subject an efficient amount of an expression vector encoding DEFB1.
13. The method of claim 10, further comprising the step of: administering
to the subject an effective amount of an anti-hormonal agent.
14. The method of claim 13, wherein the anti-hormonal agent is Tamoxifen.
15. The method of claim 10, further comprising the step of: administering
to the subject an effective amount of an anti-ERBB-2 agent.
16. The method of claim 15, wherein the said anti-ERBB-2 agent is
Herceptin.
17. The method of claim 10, further comprising the step of: administering
to the subject an effective amount of an anti-Her-2 agent.
18. The method of claim 17, wherein the anti-Her-2 agent is Trastuzumab.
19. The method of claim 10, further comprising the step of: administering
to the subject an effective amount of an anti-AIB-1/SRC-3 agent.
20. (canceled)
21. A method for treating a breast condition in a subject, comprising:
(a) determining the PAX2-to-DEFB1 expression ratio in a diseased breast
tissue from said subject; (b) determining the ER/PR status of said
diseased breast tissue from said subject; and (c) based on the results of
(a) and (b), administering to a breast tissue of said subject, a first
composition that (1) inhibits PAX2 expression or PAX2 activity, (2)
expresses DEFB1 or (3) inhibits PAX2 expression or PAX2 activity and
expresses DEFB1.
22. The method of claim 21, wherein said breast condition is breast
cancer or MIN.
23-30. (canceled)
31. The method of claim 21, wherein the step (c) further comprises
administering a second composition comprising an anti-hormonal agent.
32. The method of claim 31, wherein the anti-hormonal agent is Tamoxifen.
33. The method of claim 21, wherein the step (c) further comprises
administering a second composition comprising an anti-ERBB-2 agent.
34. The method of claim 33, wherein the anti-ERBB-2 agent is Herceptin.
35. The method of claim 21, wherein the step (c) further comprises
administering a second composition comprising an anti-Her-2 agent.
36. The method of claim 35, wherein the anti-Her-2 agent is Trastuzumab
37. The method of claim 21, wherein the step (c) further comprises
administering a second a second composition comprising an
anti-AIB-1/SRC-3 agent.
38. The method of claim 10, wherein the enhancing expression of DEFB1
comprises administering to the breast cancer tissue or MIN tissue in the
subject an efficient amount of DEFB1.
Description
[0001] The present application is a continuation application of U.S.
patent application Ser. No. 12/708,294, filed on Feb. 18, 2010, which is
a continuation-in-part application of U.S. patent application Ser. No.
12/090,191, filed Sep. 15, 2008 as a national stage application of
PCT/US2006/040215 which claims priority to U.S. patent application Ser.
No. 60/726,921, filed Oct. 14, 2005. The entirety of all of the
aforementioned applications is incorporated herein by reference.
BACKGROUND
[0002] Breast cancer is the most common cause of cancer in women and the
second most common cause of cancer death in women in the U.S. While the
majority of new breast cancers are diagnosed as a result of an
abnormality seen on a mammogram, a lump or change in consistency of the
breast tissue can also be a warning sign of the disease. Heightened
awareness of breast cancer risk in the past decades has led to an
increase in the number of women undergoing mammography for screening,
leading to detection of cancers in earlier stages and a resultant
improvement in survival rates. Still, breast cancer is the most common
cause of death in women between the ages of 45 and 55.
[0003] It is known that many types of cancer are caused by genetic
aberrations, i.e., mutations. The accumulation of mutations and the loss
of cellular control functions cause progressive phenotypic changes from
normal histology to early pre-cancer such as intraepithelial neoplasia
(IEN) to increasingly severe IEN to superficial cancer and finally to
invasive disease. Although this process can be relatively aggressive in
some cases, it generally occurs relatively slowly over years and even
decades. Oncogene addiction is the physiologic dependence of cancer cells
on the continued activation or overexpression of single oncogenes for
maintaining the malignant phenotype. This dependence occurs in the milieu
of the other changes that mark neoplastic progression.
[0004] The long period of progression to invasive cancer provides an
opportunity for clinical intervention. Therefore, it is important to
identify biomarkers that are indicative of pre-cancerous conditions so
that treatment measures can be taken to prevent or delay the development
of invasive cancer.
SUMMARY
[0005] One aspect of the present invention relates to a method for
preventing or treating a breast condition in a subject. The method
comprises administering to a breast tissue of the subject, a composition
that inhibits PAX2 expression or PAX2 activity.
[0006] In one embodiment, the breast condition is breast cancer or mammary
intraepithelial neoplasia (MIN).
[0007] In another embodiment, the inhibiting expression of PAX2 comprises
administering to the breast cancer tissue or MIN tissue in the subject a
nucleic acid encoding an siRNA for PAX2.
[0008] In a related embodiment, the siRNA comprises a sequence selected
from the group consisting of SEQ ID NOS: 3-6 and 11-15.
[0009] In another embodiment, the composition comprises an oligonucleotide
that inhibits PAX2 binding to the DEFB1 promoter.
[0010] In a related embodiment, the oligonucleotide comprises SEQ ID NO:1
in forward or reverse orientation.
[0011] In a related embodiment, the oligonucleotide comprises the sequence
of X1GGAACX2, wherein X1 and X2 are 0 to 30 nucleotides complementary to
nucleotides contiguous to SEQ ID NO:1 in the DEFB1 coding sequence.
[0012] In a related embodiment, the oligonucleotide comprises a sequence
selected from the group consisting of SEQ ID NOS: 18-21, 25, 26, 28 and
29.
[0013] In another embodiment, the composition comprises a blocker of RAS
signaling pathway.
[0014] In another embodiment, the composition comprises an antagonist
selected from the group consisting of antagonists of angiotensin II,
antagonists of angiotensin II receptor, antagonists of
angiotensin-converting enzyme (ACE), antagonists of mitogen-activated
protein kinase (MEK), antagonists of (extracellular signal-regulated
kinase) ERK1,2, and antagonists of signal transducer and activator of
transcription 3 (STAT3).
[0015] Also disclosed is a method of treating breast cancer or MIN in a
subject, comprising enhancing expression of DEFB1 in a breast cancer
tissue or MIN tissue in the subject.
[0016] In one embodiment, the enhancing expression of DEFB1 comprises
administering to the breast cancer tissue or MIN tissue in the subject an
efficient amount of DEFB1.
[0017] In another embodiment, the enhancing expression of DEFB1 comprises
administering to the breast cancer tissue or MIN tissue in the subject an
efficient amount of an expression vector encoding DEFB1.
[0018] Also disclosed is a method for treating a breast condition in a
subject, comprising: (a) determining the PAX2-to-DEFB1 expression ratio
in a diseased breast tissue from said subject; (b) determining the ER/PR
status of said diseased breast tissue from said subject; and (c) based on
the result of (a) and (b), administering to a breast tissue of said
subject, a composition that (1) inhibits PAX2 expression or PAX2
activity, (2) expresses DEFB1 or (3) inhibits PAX2 expression or PAX2
activity and expresses DEFB1.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] The accompanying drawings illustrate one or more embodiments of the
invention and, together with the written description, serve to explain
the principles of the invention. Wherever possible, the same reference
numbers are used throughout the drawings to refer to the same or like
elements of an embodiment.
[0020] FIGS. 1A-1D show quantitative RT-PCR (QRT-PCR) analysis of
beta-defensin-1 (DEFB1) expression.
[0021] FIG. 2 shows microscopic analysis of DEFB1 induced changes in
membrane integrity and cell morphology. Membrane ruffling is indicated by
black arrows and apoptotic bodies are indicated white arrows.
[0022] FIG. 3 shows analysis of DEFB1 Cytotoxicity in Prostate Cancer
Cells. The prostate cell lines DU145, PC3 and LNCaP were treated with
PonA to induce DEFB1 expression for 1-3 days after which MTT assay was
performed to determine cell viability. Results represent mean.+-.s.d.,
n=9.
[0023] FIGS. 4A and 4B show induction of cell death in DU145 and PC3 cells
by DEFB1.
[0024] FIG. 5 shows pan-caspase analysis following DEFB1 induction.
[0025] FIG. 6 show silencing of paired box homeotic gene 2 (PAX2) protein
expression following PAX2 siRNA treatment.
[0026] FIG. 7 shows analysis of prostate cancer cells growth after
treatment with PAX2 siRNA.
[0027] FIG. 8 shows analysis of cell death following siRNA silencing of
PAX2. Results represent mean.+-.s.d., n=9.
[0028] FIG. 9 shows analysis of caspase activity.
[0029] FIG. 10 shows analysis of apoptotic factors following PAX2 siRNA
treatment.
[0030] FIG. 11 shows model of PAX2 binding to DNA recognition sequence.
[0031] FIG. 12 illustrates the DEFB1 reporter construct.
[0032] FIG. 13 shows inhibition of PAX2 results in DEFB1 Expression.
[0033] FIG. 14 shows that inhibition of PAX2 results in increased DEFB1
promoter activity.
[0034] FIG. 15 shows that DEFB1 expression causes loss of membrane
integrity.
[0035] FIG. 16 shows that PAX2 inhibition results in loss of membrane
integrity.
[0036] FIGS. 17A and 17B show ChIP analysis of PAX2 binding to DEFB1
promoter. In FIG. 17A, Lane 1 contains a 100 by molecular weight marker.
Lane 2 is a positive control representing 160 by region of the DEFB1
promoter amplified from DU145 before cross-linking and
immunoprecipitation. Lane 3 is a negative control representing PCR
performed without DNA. Lanes 4 and 5 are negative controls representing
PCR from immunoprecipitations performed with IgG from cross-linked DU145
and PC3, respectively. PCR amplification of 25 pg of DNA (lane 6 and 8)
and 50 pg of DNA (lane 7 and 9) immunoprecitipated with anti-PAX2
antibody after crosslinking show 160 by promoter fragment in DU145 and
PC3, respectively. In FIG. 17B, Lane 1 contains a 100 by molecular weight
marker. Lane 2 is a positive control representing 160 by region of the
DEFB1 promoter amplified from DU145 before cross-linking and
immunoprecipitation. Lane 3 is a negative control representing PCR
performed without DNA. Lane 4 and 5 are negative controls representing
PCR from immunoprecipitations performed with IgG from cross-linked DU145
and PC3, respectively. PCR amplification of 25 pg of DNA (lane 6 and 8)
and 50 pg of DNA (lane 7 and 9) immunoprecitipated with anti-PAX2
antibody after crosslinking show 160 by promoter fragment in DU145 and
PC3, respectively
[0037] FIG. 18 shows predicted structure of the PrdPD and PrdHD with DNA.
[0038] FIG. 19 shows comparison of consensus sequences of different paired
domains. At the top of the Figure is drawn a schematic representation of
protein.+-.DNA contacts described in the crystallographic analysis of the
Prd-paired-domain.+-.DNA complex. Empty boxes indicate a-helices, shaded
boxes indicates b-sheets and a thick line indicate a b-turn. Contacting
amino acids are shown by single-letter code. Only direct amino
acid.+-.base contacts are shown. Empty circles indicate major groove
contacts while red arrows indicate minor groove contacts. This scheme is
aligned to all known consensus sequences for paired-domain proteins (top
strands only are shown). Vertical lines between consensus sequences
indicate conserved base-pairs. Numbering of the positions is shown at the
bottom of the Figure.
[0039] FIG. 20 shows targeting PAX2 as a chemopreventive strategy.
[0040] FIG. 21 shows effect of angiotensin II (Ang II) on PAX2 expression
in DU145 Cells.
[0041] FIG. 22A shows effect of Losartan (Los) on PAX2 expression in
DU145.
[0042] FIG. 23 shows Los blocks AngII effect on PAX2 expression in DU145.
[0043] FIG. 24 shows AngII increases DU145 cell proliferation.
[0044] FIGS. 25A, 25B and 25C show effect of Los and MAP Kinase inhibitors
on PAX2 expression in DU145 cells. FIG. 25A shows treatment of DU145
cells with Losartan suppresses phosphor-ERK 1/2 and PAX2 expression; FIG.
25B shows MEK kinase inhibitors and AICAR suppresses PAX2 protein
expression; FIG. 25C shows MEK kinase inhibitors and Losartan suppresses
phospho-STAT3 protein expression.
[0045] FIGS. 26A and 26B show effect of Los and MEK kinase inhibitors on
PAX2 activation in DU145 cells
[0046] FIG. 27 shows AngII increases PAX2 and decreases DEFB1 expression
in hPrEC cells.
[0047] FIG. 28 shows schematic of AngII signaling and PAX2 prostate
cancer.
[0048] FIG. 29 shows schematic of blocking PAX2 expression as a therapy
for prostate cancer.
[0049] FIG. 30 shows comparison of DEFB1 and PAX2 expression with Gleason
Score.
[0050] FIGS. 31A and 31B show PAX2-DEFB1 ratio as a predictive factor for
prostate cancer development.
[0051] FIG. 32 shows the Donald Predictive Factor (DPF) is based on the
relative PAX2-DEFB1 expression ratio.
[0052] FIGS. 33A and 33B show analysis of hBD-1 expression in human
prostate tissue.
[0053] FIGS. 34A and 34B show analysis of hBD-1 expression in prostate
cell lines. FIG. 34A shows hBD-1 expression levels compared relative to
hPrEC cells in prostate cancer cell lines before and after hBD-1
induction. An asterisk represents statistically higher expression levels
compared to hPrEC. Double asterisks represent statistically significant
levels of expression compared to the cell line before hBD-1 induction
(Student's t-test, p <0.05). FIG. 34B shows ectopic hBD-1 expression
verified in the prostate cancer cell line DU145 by immunocytochemistry.
hPrEC cells were stained for hBD-1 as appositive control (A: DIC and B:
fluorescence). DU145 cells were transfected with hBD-1 and induced for 18
hours (C: DIC and D: fluorescence). Sizebar=20 .mu.M.
[0054] FIG. 35 shows analysis of hBD-1 cytotoxicity in prostate cancer
cells. Each bar represents the mean.+-.S.E.M. of three independent
experiments performed in triplicate.
[0055] FIGS. 36A and 36B show QRT-PCR analysis of hBD-1 and cMYC
expression in LCM human prostate tissue sections of normal, PIN and
tumor. Expression for each gene is presented as expression ratios
compared to .beta.-actin. FIG. 36A shows comparison of hBD-1 expression
levels in normal, PIN and tumor sections. FIG. 36B shows comparison of
cMYC expression level in normal, PIN and tumor sections.
[0056] FIG. 37 shows QRT-PCR analysis of hBD1 expression following PAX2
knockdown with siRNA. hBD-1 expression levels are presented as expression
ratios compared to .beta.-actin. An asterisk represents statistically
higher expression levels compared to the cell line before PAX2 siRNA
treatment (Student's t-test, p<0.05).
[0057] FIGS. 38A and 38B show silencing of PAX2 protein expression
following PAX2 siRNA treatment. FIG. 38A shows PAX2 expression examined
by Western blot analysis in HPrEC prostate primary cells (lane 1) and in
DU145 (lane 2), PC3 (lane 3) and LNCaP (lane 4) prostate cancer cells.
Blots were stripped and re-probed for -actin as an internal control to
ensure equal loading. FIG. 38B shows Western blot analysis of DU145, PC3
and LNCaP all confirmed knockdown of PAX2 expression following
transfection with PAX2 siRNA duplex. Again, blots were stripped and
re-probed for .beta.-actin as an internal control.
[0058] FIG. 39 shows analysis of prostate cancer cells growth after
treatment with PAX2 siRNA. Bar=20 .mu.m.
[0059] FIG. 40 shows analysis of cell death following siRNA silencing of
PAX2. Results represent mean.+-.SD, n=9.
[0060] FIG. 41 shows analysis of caspase activity. Bar=20 .mu.m.
[0061] FIGS. 42A-42C show analysis of apoptotic factors following PAX2
siRNA treatment. Results represent mean.+-.SD, n=9. Asterisks represents
statistical differences (p<0.05).
DETAILED DESCRIPTION
[0062] One aspect of the present invention provides a method of preventing
or treating breast cancer in a subject. The method includes administering
to the subject a composition comprising an inhibitor of PAX2 expression
or PAX2 activity, or an enhacer of DEFB-1 expression or DEFB-1 activity.
In one embodiment, the subject is diagnosed with mammary intraepithelial
neoplasia (MIN).
[0063] In some aspects, PAX2 is upregulated in breast tissue prior to MIN.
Thus, also provided is a method of treating or preventing MIN in a
subject. The method comprises administering to the subject a composition
comprising an inhibitor of PAX2 expression or PAX2 activity, or an
enhacer of DEFB-1 expression or DEFB-1 activity.
[0064] "Activities" of a protein include, for example, transcription,
translation, intracellular translocation, secretion, phosphorylation by
kinases, cleavage by proteases, hemophilic and heterophilic binding to
other proteins, ubiquitination. In some aspects, "PAX2 activity" refers
specifically to the binding of PAX2 to the DEFB-1 promoter.
Breast Cancer
[0065] The commonly used screening methods for breast cancer include self
and clinical breast exams, x-ray mammography, and breast Magnetic
Resonance Imaging (MRI). The most recent technology for breast cancer
screening is ultrasound computed tomography, which uses sound waves to
create a three-dimensional image and detect breast cancer without the use
of dangerous radiation used in x-ray mammography. Genetic testing may
also be used. Genetic testing for breast cancer typically involves
testing for mutations in the BRCA genes. It is not a generally
recommended technique except for those at elevated risk for breast
cancer.
[0066] The incidence of breast cancer, a leading cause of death in women,
has been gradually increasing in the United States over the last thirty
years. While the pathogenesis of breast cancer is unclear, transformation
of normal breast epithelium to a malignant phenotype may be the result of
genetic factors, especially in women under 30. The discovery and
characterization of BRCA1 and BRCA2 has recently expanded our knowledge
of genetic factors which can contribute to familial breast cancer.
Germ-line mutations within these two loci are associated with a 50 to 85%
lifetime risk of breast and/or ovarian cancer. However, it is likely that
other, non-genetic factors also have a significant effect on the etiology
of the disease. Regardless of its origin, breast cancer morbidity and
mortality increases significantly if it is not detected early in its
progression. Thus, considerable effort has focused on the early detection
of cellular transformation and tumor formation in breast tissue.
[0067] Currently, the principal manner of identifying breast cancer is
through detection of the presence of dense tumorous tissue. This may be
accomplished to varying degrees of effectiveness by direct examination of
the outside of the breast, or through mammography or other X-ray imaging
methods. The latter approach is not without considerable cost, however.
Every time a mammogram is taken, the patient incurs a small risk of
having a breast tumor induced by the ionizing properties of the radiation
used during the test. In addition, the process is expensive and the
subjective interpretations of a technician can lead to imprecision, e.g.,
one study showed major clinical disagreements for about one-third of a
set of mammograms that were interpreted individually by a surveyed group
of radiologists. Moreover, many women find that undergoing a mammogram is
a painful experience. Accordingly, the National Cancer Institute has not
recommended mammograms for women under fifty years of age, since this
group is not as likely to develop breast cancers as are older women. It
is compelling to note, however, that while only about 22% of breast
cancers occur in women under fifty, data suggests that breast cancer is
more aggressive in pre-menopausal women.
PAX2
[0068] PAX genes are a family of nine developmental control genes coding
for nuclear transcription factors. They play an important role in
embryogenesis and are expressed in a very ordered temporal and spatial
pattern. They all contain a "paired box" region of 384 base pairs
encoding a DNA binding domain which is highly conserved throughout
evolution (Stuart, E T, et al. 1994). The influence of Pax genes on
developmental processes has been demonstrated by the numerous natural
mouse and human syndromes that can be attributed directly to even a
heterozygous insufficiency in a Pax gene. A PAX2 sequence is given in
Dressler, et al. 1990. The amino acid sequences of the human PAX2 protein
and its variants, as well as the DNA sequences encoding the proteins, are
listed in SEQ ID NOS: 39-50 (SEQ ID NO:39, amino acid sequence encoded by
exon 1 of the human PAX2 gene; SEQ ID NO:40, human PAX2 gene promoter and
exon 1; SEQ ID NO:41, amino acid sequence of the human PAX2; SEQ ID
NO:42, human PAX2 gene; SEQ ID NO:43, amino acid sequence of the human
PAX2 gene variant b; SEQ ID NO:44, human PAX2 gene variant b; SEQ ID
NO:45, amino acid sequence of the human PAX2 gene variant c; SEQ ID
NO:46, human PAX2 gene variant c; SEQ ID NO:47, amino acid sequence of
the human PAX2 gene variant d; SEQ ID NO:48, human PAX2 gene variant d;
SEQ ID NO:49, amino acid sequence of the human PAX2 gene variant e; SEQ
ID NO:50 human PAX2 gene variant e). It has been reported that PAX2
suppresses DEFB-1 expression by binding to the DEFB-1 promoter (Bose S K
et al., Mol Immunol. 2009, 46:1140-8.) at a 5'-CCTTG-3' (SEQ ID NO:1)
recognition site immediately adjacent to the DEFB1 TATA box. In some
references, the binding site is also referred to as the 3'-GTTCC-5' (SEQ
ID NO:1) or 5'-CAAGG-3' (SEQ ID NO:2) recognition site, which is the
sequence on the opposite strand. The two sequences both refer to the PAX2
binding site on the DEFB1 promoter. Examples of cancers in which PAX2
expression has been detected are listed in Table 1
TABLE-US-00001
TABLE 1
PAX2-expressing cancers
Estimated Estimated Estimated Estimated
PAX2 Expressing New Deaths New Deaths
Cancers Cases in US in US Cases Global Global
Prostate 234,460 27,350 679,023 221,002
Breast 214,600 41,430 1,151,298 410,712
Ovarian 20,180 15,310 204,500 124,860
Renal 38,890 12,840 208,479 101,895
Brain 12,820 18,820 189,485 141,650
Cervical 9,710 3,700 493,243 273,505
Bladder 61,420 13,060 356,556 145,009
Leukemia 35,020 22,280 300,522 222,506
Kaposi Sarcoma Data Not Data Not Data Not Data Not
Available Available Available Available
TOTAL(approx.) 627,100 154,790 3,583,106 1,641,139
DEFB1
[0069] Beta-defensins are cationic peptides with broad-spectrum
antimicrobial activity that are products of epithelia and leukocytes.
These two exon, single gene products are expressed at epithelial surfaces
and secreted at sites including the skin, cornea, tongue, gingiva,
salivary glands, esophagus, intestine, kidney, urogenital tract, and the
respiratory epithelium. To date, five beta-defensin genes of epithelial
origin have been identified and characterized in humans: DEFB1 (Bensch et
al., 1995), DEFB2 (Harder et al., 1997), DEFB3 (Harder et al., 2001; Jia
et al., 2001), DEFB4, and HE2/EP2. The amino acid sequence of human DEFB1
and the human DEFB1 gene sequences are shown in SEQ ID NOS:63 and 64,
respectively.
[0070] The primary structure of each beta-defensin gene product is
characterized by small size, a six cysteine motif, high cationic charge
and exquisite diversity beyond these features. The most characteristic
feature of defensin proteins is their six-cysteine motif that forms a
network of three disulfide bonds. The three disulfide bonds in the
beta-defensin proteins are between C1-C5, C2-C4 and C3-C6. The most
common spacing between adjacent cysteine residues is 6, 4, 9, 6, 0. The
spacing between the cysteines in the beta-defensin proteins can vary by
one or two amino acids except for C5 and C6, located nearest the carboxy
terminus. In all known vertebrate beta-defensin genes, these two cysteine
residues are adjacent to each other.
[0071] A second feature of the beta-defensin proteins is their small size.
Each beta-defensin gene encodes a preproprotein that ranges in size from
59 to 80 amino acids with an average size of 65 amino acids. This gene
product is then cleaved by an unknown mechanism to create the mature
peptide that ranges in size from 36 to 47 amino acids with an average
size of 45 amino acids. The exceptions to these ranges are the EP2/HE2
gene products that contain the beta-defensin motif and are expressed in
the epididymis.
[0072] A third feature of beta-defensin proteins is the high concentration
of cationic residues. The number of positively charged residues
(arginine, lysine, histidine) in the mature peptide ranges from 6 to 14
with an average of 9.
[0073] The final feature of the beta-defensin gene products is their
diverse primary structure but apparent conservation of tertiary
structure. Beyond the six cysteines, no single amino acid at a given
position is conserved in all known members of this protein family.
However, there are positions that are conserved that appear to be
important for secondary and tertiary structures and function.
[0074] Despite the great diversity of the primary amino acid sequence of
the beta-defensin proteins, the limited data suggests that the tertiary
structure of this protein family is conserved. The structural core is a
triple-stranded, antiparallel beta-sheet, as exemplified for the proteins
encoded by BNBD-12 and DEFB2. The three beta-strands are connected by a
beta-turn, and an alpha-hairpin loop, and the second beta-strand also
contains a beta-bulge. When these structures are folded into their proper
tertiary structure, the apparently random sequences of cationic and
hydrophobic residues are concentrated into two faces of a globular
protein. One face is hydrophilic and contains many of the positively
charged side chains and the other is hydrophobic. In solution, the HBD-2
protein encoded by the DEFB2 gene exhibited an alpha-helical segment near
the N-terminus not previously ascribed to solution structures of
alpha-defensins or to the beta-defensin BNBD-12. The amino acids whose
side chains are directed toward the surface of the protein are less
conserved between beta defensin proteins while the amino acid residues in
the three beta-strands of the core beta-sheet are more highly conserved.
[0075] Beta-defensin peptides are produced as pre-pro-peptides and then
cleaved to release a C-terminal active peptide fragment; however the
pathways for the intracellular processing, storage and release of the
human beta-defensin peptides in airway epithelia are unknown.
Inhibitors of PAX2 Expression or PAX2 Activity
Functional Nucleic Acids
[0076] The inhibitor of the disclosed methods can be a functional nucleic
acid that inhibits PAX2 expression. Functional nucleic acids are nucleic
acid molecules that have a specific function, such as binding a target
molecule or catalyzing a specific reaction. Functional nucleic acid
molecules can be divided into the following categories, which are not
meant to be limiting. For example, functional nucleic acids include
antisense molecules, aptamers, ribozymes, triplex forming molecules,
RNAi, and external guide sequences. The functional nucleic acid molecules
can act as affectors, inhibitors, modulators, and stimulators of a
specific activity possessed by a target molecule, or the functional
nucleic acid molecules can possess a de novo activity independent of any
other molecules.
[0077] Functional nucleic acid molecules can interact with any
macromolecule, such as DNA, RNA, polypeptides, or carbohydrate chains.
Thus, functional nucleic acids can interact with the mRNA of PAX2 or the
genomic DNA of PAX2 or they can interact with the polypeptide PAX2. Often
functional nucleic acids are designed to interact with other nucleic
acids based on sequence homology between the target molecule and the
functional nucleic acid molecule. In other situations, the specific
recognition between the functional nucleic acid molecule and the target
molecule is not based on sequence homology between the functional nucleic
acid molecule and the target molecule, but rather is based on the
formation of tertiary structure that allows specific recognition to take
place.
[0078] Antisense molecules are designed to interact with a target nucleic
acid molecule through either canonical or non-canonical base pairing. The
interaction of the antisense molecule and the target molecule is designed
to promote the destruction of the target molecule through, for example,
RNAseH mediated RNA-DNA hybrid degradation. Alternatively the antisense
molecule is designed to interrupt a processing function that normally
would take place on the target molecule, such as transcription or
replication. Antisense molecules can be designed based on the sequence of
the target molecule. Numerous methods for optimization of antisense
efficiency by finding the most accessible regions of the target molecule
exist. Exemplary methods would be in vitro selection experiments and DNA
modification studies using DMS and DEPC. It is preferred that antisense
molecules bind the target molecule with a dissociation constant (Kd) less
than or equal to 10.sup.-6, 10.sup.-8, 10.sup.-10, or 10.sup.-12.
[0079] Aptamers are molecules that interact with a target molecule,
preferably in a specific way. Typically aptamers are small nucleic acids
ranging from 15-50 bases in length that fold into defined secondary and
tertiary structures, such as stem-loops or G-quartets. Aptamers can bind
small molecules, such as ATP and theophiline, as well as large molecules,
such as reverse transcriptase and thrombin. Aptamers can bind very
tightly with Kd's from the target molecule of less than 10.sup.-12 M. It
is preferred that the aptamers bind the target molecule with a Kd less
than 10.sup.-6, 10.sup.-8, 10.sup.-10, or 10.sup.-12. Aptamers can bind
the target molecule with a very high degree of specificity. For example,
aptamers have been isolated that have greater than a 10,000 fold
difference in binding affinities between the target molecule and another
molecule that differ at only a single position on the molecule. It is
preferred that the aptamer have a Kd with the target molecule at least
10, 100, 1000, 10,000, or 100,000 fold lower than the Kd with a
background binding molecule. It is preferred when doing the comparison
for a polypeptide for example, that the background molecule be a
different polypeptide.
[0080] Ribozymes are nucleic acid molecules that are capable of catalyzing
a chemical reaction, either intramolecularly or intermolecularly.
Ribozymes are thus catalytic nucleic acid. It is preferred that the
ribozymes catalyze intermolecular reactions. There are a number of
different types of ribozymes that catalyze nuclease or nucleic acid
polymerase type reactions which are based on ribozymes found in natural
systems, such as hammerhead ribozymes, hairpin ribozymes, and tetrahymena
ribozymes. There are also a number of ribozymes that are not found in
natural systems, but which have been engineered to catalyze specific
reactions de novo. Preferred ribozymes cleave RNA or DNA substrates, and
more preferably cleave RNA substrates. Ribozymes typically cleave nucleic
acid substrates through recognition and binding of the target substrate
with subsequent cleavage. This recognition is often based mostly on
canonical or non-canonical base pair interactions. This property makes
ribozymes particularly good candidates for target specific cleavage of
nucleic acids because recognition of the target substrate is based on the
target substrates sequence.
[0081] Triplex forming functional nucleic acid molecules are molecules
that can interact with either double-stranded or single-stranded nucleic
acid. When triplex molecules interact with a target region, a structure
called a triplex is formed, in which there are three strands of DNA
forming a complex dependant on both Watson-Crick and Hoogsteen
base-pairing. Triplex molecules are preferred because they can bind
target regions with high affinity and specificity. It is preferred that
the triplex forming molecules bind the target molecule with a Kd less
than 10.sup.-6, 10.sup.-8, 10.sup.-10 or 10.sup.-12.
[0082] External guide sequences (EGSs) are molecules that bind a target
nucleic acid molecule forming a complex, and this complex is recognized
by RNase P, which cleaves the target molecule. EGSs can be designed to
specifically target a RNA molecule of choice. RNAse P aids in processing
transfer RNA (tRNA) within a cell. Bacterial RNAse P can be recruited to
cleave virtually any RNA sequence by using an EGS that causes the target
RNA:EGS complex to mimic the natural tRNA substrate. Similarly,
eukaryotic EGS/RNAse P-directed cleavage of RNA can be utilized to cleave
desired targets within eukaryotic cells.
[0083] Gene expression can also be effectively silenced in a highly
specific manner through RNA interference (RNAi). This silencing was
originally observed with the addition of double stranded RNA (dsRNA).
Once dsRNA enters a cell, it is cleaved by an RNase III--like enzyme,
Dicer, into double stranded small interfering RNAs (siRNA) 21-23
nucleotides in length that contains 2 nucleotide overhangs on the 3'
ends. In an ATP dependent step, the siRNAs become integrated into a
multi-subunit protein complex, commonly known as the RNAi induced
silencing complex (RISC), which guides the siRNAs to the target RNA
sequence. At some point the siRNA duplex unwinds, and it appears that the
antisense strand remains bound to RISC and directs degradation of the
complementary mRNA sequence by a combination of endo and exonucleases.
However, the effect of iRNA or siRNA or their use is not limited to any
type of mechanism.
[0084] Short Interfering RNA (siRNA) is a double-stranded RNA that can
induce sequence-specific post-transcriptional gene silencing, thereby
decreasing or even inhibiting gene expression. In one example, an siRNA
triggers the specific degradation of homologous RNA molecules, such as
mRNAs, within the region of sequence identity between both the siRNA and
the target RNA. For example, WO 02/44321 discloses siRNAs capable of
sequence-specific degradation of target mRNAs when base-paired with 3'
overhanging ends, herein incorporated by reference for the method of
making these siRNAs. Sequence specific gene silencing can be achieved in
mammalian cells using synthetic, short double-stranded RNAs that mimic
the siRNAs produced by the enzyme dicer. siRNA can be chemically or in
vitro-synthesized or can be the result of short double-stranded
hairpin-like RNAs (shRNAs) that are processed into siRNAs inside the
cell. Synthetic siRNAs are generally designed using algorithms and a
conventional DNA/RNA synthesizer. Suppliers include Ambion (Austin,
Tex.), ChemGenes (Ashland, Mass.), Dharmacon (Lafayette, Colo.), Glen
Research (Sterling, Va.), MWB Biotech (Esbersberg, Germany), Proligo
(Boulder, Colo.), and Qiagen (Vento, The Netherlands). siRNA can also be
synthesized in vitro using kits such as Ambion's SILENCER.RTM. siRNA
Construction Kit. Disclosed herein are any siRNA designed as described
above based on the sequences for PAX2.
[0085] The production of siRNA from a vector is more commonly done through
the transcription of a short hairpin RNAs (shRNAs). Kits for the
production of vectors comprising shRNA are available, such as, for
example, Imgenex's GENESUPPRESSOR.TM. Construction Kits and Invitrogen's
BLOCK-IT.TM. inducible RNAi plasmid and lentivirus vectors. Disclosed
herein are any shRNA designed as described above based on the sequences
for the herein disclosed inflammatory mediators.
[0086] In certain embodiments, the functional nucleic acids include siRNAs
that inhibit expression of PAX 2 (anti-PAX2 siRNA). Examples of anti-PAX2
siRNAs include, but are not limited to, siRNAs having the sequences of
(5' to 3' direction):
TABLE-US-00002
AUAGACUCGACUUGACUUCUU, (SEQ ID NO: 3)
AUCUUCAUCACGUUUCCUCUU, (SEQ ID NO: 4)
GUAUUCAGCAAUCUUGUCCUU, (SEQ ID NO: 5)
GAUUUGAUGUGCUCUGAUGUU, (SEQ ID NO: 6)
ACCCGACTATGTTCGCCTGG, (SEQ ID NO: 11)
AAGCTCTGGATCGAGTCTTTG, (SEQ ID NO: 12)
ATGTGTCAGGCACACAGACG, (SEQ ID NO: 13)
GUCGAGUCUAUCUGCAUCCUU, (SEQ ID NO: 14)
GGAUGCAGAUAGACUCGACUU, (SEQ ID NO: 15)
and fragments of at least 10 nucleic acids and conservative variants
thereof; and combinations thereof.
[0087] In other embodiments, the functional nucleic acids include
antisense RNA to PAX2 and oligonuclotides that interfere with or inhibit
the binding of PAX2 to the DEFB1 promoter. The oligonucleotide can be
complementary to the sequence of PAX2 that binds to the DEFB1 promoter.
Alternatively, the oligonucleotide can interact with the PAX2 in a way
that inhibits binding to DEFB1. This interaction can be based on
three-dimensional structure rather than primary nucleotide sequence.
[0088] PAX proteins are a family of transcription factors conserved during
evolution and able to bind specific DNA sequences through a domains
called a "paired domain" and a "homeodomain". The paired domain (PD) is a
consensus sequence shared by certain PAX proteins (e.g., PAX2 and PAX6).
The PD directs DNA binding of amino acids located in the .alpha.3-helix
forming a DNA-Protein complex. For PAX2, the amino acids in the HD
recognize and interact specifically with a CCTTG (SEQ ID NO:1) DNA core
sequence. Oligonucleotides include this sequence or its complement are
expected to be inhibitors. A critical DNA region in the DEFB1 promoter
for PAX2 protein binding has the sequence of AAGTTCACCCTTGACTGTG (SEQ ID
NO: 16).
[0089] In one embodiment, the oligonucleotide has the sequence of
V-CCTTG-W (SEQ ID NO: 17), wherein V and W are nucleotide sequences of 1
to 35 nucleotides. In certain embodiments, V or W or both comprise
contiguous nucleotide sequences that normally flank the PAX2 binding site
of DEFB1 promoter. Alternatively, the nucleotide sequences of V and/or W
may be unrelated to the DEFB1 promoter, and selected randomly to avoid
interference with the PAX2 recognition sequence.
[0090] Other examples of oligonucleotides that inhibit PAX2 binding to the
DEFB1 promoter include, but are not limited to, oligonucleotide having
the sequences of (5' to 3' direction):
TABLE-US-00003
(SEQ ID NO: 18)
CTCCCTTCAGTTCCGTCGAC,
(SEQ ID NO: 19)
CTCCCTTCACCTTGGTCGAC,
(SEQ ID NO: 20)
ACTGTGGCACCTCCCTTCAGTTCCGTCGACGAGGTTGTGC,
and
(SEQ ID NO: 21)
ACTGTGGCACCTCCCTTCACCTTGGTCGACGAGGTTGTGC.
Other Inhibitors
[0091] Besides functional neucleotides, the inhibitors of PAX2 expression
or PAX2 activity can be any small molecule that interferes or inhibits
binding of PAX2 to the DEFB1 promoter. The inhibitors of PAX2 expression
or PAX2 activity can also be an antagonist of angiotensin II or an
antagonist of angiotensin-converting enzyme (ACE). For example, the
inhibitor can be enalapril or/and an antagonist of angiotensin II type 1
receptor (AT1R). The inhibitor can be valsartan, olmesartan, or/and
telmisartan. The inhibitor can be an antagonist of MEK, an antagonist of
ERK1,2 or/and an antagonist of STATS. In some aspects, the disclosed
inhibitor of PAX2 expression or activity is not an AT1R receptor
antagonist. The term "antagonist" refers to an agent that inhibits the
activity of the target.
[0092] The antagonists of MEK and/or ERK1,2 include U0126 and PD98059.
U0126 is a chemically synthesized organic compound that was initially
recognized as a cellular AP-1 antagonist, and found to be a very
selective and highly potent inhibitor of Mitogen-Activated Protein Kinase
(MAPK) cascade by inhibiting its immediate upstream activators, Mitogen
Activated Protein Kinase Kinase 1 and 2 (also known as MEK1 and MEK2,
IC50: 70 and 60 nM respectively). U0126 inhibits both active and inactive
MEK1,2, unlike PD98059 which only inhibits activation of inactive MEK.
Blockade of MEK activation would prevent downstream phosphorylation of a
number of factors including p62TCF (Elk-1), an upstream inducer of c-Fos
and c-Jun, components of the AP-1 complex. Inhibition of MEK/ERK pathway
by U0126 also prevents all effects of oncogenic H-Ras and K-Ras, inhibits
part of the effects triggered by growth factors and blocks the production
of inflammatory cytokines and matrix metalloproteinases.
[0093] PD98059 has been shown to act in vivo as a highly selective
inhibitor of MEK1 activation and the MAP kinase cascade. PD98059 binds to
the inactive forms of MEK1 and prevents activation by upstream activators
such as c-Raf. PD98059 inhibits activation of MEK1 and MEK2 with IC50
values of 4 .mu.M and 50 .mu.M, respectively.
[0094] In certain embodiments, the expression of PAX2 is inhibited by
administering to the breast cancer tissue or MIN tissue in the subject a
blocker of RAS signaling pathway.
[0095] In certain other embodiment, th inhibitor of PAX2 expression or
PAX2 activity is conjugated to an antibody, a receptor or a ligand to
target the tumor tissue.
Enhacer of DEFB-1 Expression or DEFB-1 Activity.
[0096] Enhancer of DEFB-1 expression or DEFB-1 activity can be vectors
that express DEFB-1 protein. Since PAX2 inhibits DEFB-1 expression,
inhibitors of PAX2 expression or PAX2 activity are also enhancers of
DEFB-1 expression.
Delivery Systems
[0097] There are a number of compositions and methods which can be used to
deliver nucleic acids to cells, either in vitro or in vivo. These methods
and compositions can largely be broken down into two classes: viral based
delivery systems and non-viral based delivery systems. For example, the
nucleic acids can be delivered through a number of direct delivery
systems such as, electroporation, lipofection, calcium phosphate
precipitation, plasmids, viral vectors, viral nucleic acids, phage
nucleic acids, phages, cosmids, or via transfer of genetic material in
cells or carriers such as cationic liposomes. Such methods are well known
in the art and readily adaptable for use with the compositions and
methods described herein. In certain cases, the methods will be modified
to specifically function with large DNA molecules. Further, these methods
can be used to target certain diseases and cell populations by using the
targeting characteristics of the carrier.
Nucleic Acid Based Delivery Systems
[0098] The inhibitors of PAX2 expression or PAX2 activity and enhancers of
DEFB1 expression or DEFB1 activity may be delivered to the target cells
using nucleic acid based delivery systems, such as plasmids and viral
vectors. As used herein, plasmid or viral vectors are agents that
transport the disclosed nucleic acids, such as PAX2 siRNA into the cell
without degradation and include a promoter yielding expression of the
gene in the cells into which it is delivered. In some embodiments the
vectors are derived from either a virus or a retrovirus. Viral vectors
are, for example, Adenovirus, Adeno-associated virus, Herpes virus,
Vaccinia virus, Polio virus, AIDS virus, neuronal trophic virus, Sindbis
and other RNA viruses, including these viruses with the HIV backbone.
Also preferred are any viral families which share the properties of these
viruses which make them suitable for use as vectors. Retroviruses include
Murine Maloney Leukemia virus, MMLV, and retroviruses that express the
desirable properties of MMLV as a vector. Retroviral vectors are able to
carry a larger genetic payload, i.e., a transgene or marker gene, than
other viral vectors, and for this reason are a commonly used vector.
However, they are not as useful in non-proliferating cells. Adenovirus
vectors are relatively stable and easy to work with, have high titers,
and can be delivered in aerosol formulation, and can transfect
non-dividing cells. Pox viral vectors are large and have several sites
for inserting genes, they are thermostable and can be stored at room
temperature. Viral vectors can have higher transaction (ability to
introduce genes) abilities than chemical or physical methods to introduce
genes into cells. Typically, viral vectors contain, nonstructural early
genes, structural late genes, an RNA polymerase III transcript, inverted
terminal repeats necessary for replication and encapsidation, and
promoters to control the transcription and replication of the viral
genome. When engineered as vectors, viruses typically have one or more of
the early genes removed and a gene or gene/promotor cassette is inserted
into the viral genome in place of the removed viral DNA. Constructs of
this type can carry up to about 8 kb of foreign genetic material. The
necessary functions of the removed early genes are typically supplied by
cell lines which have been engineered to express the gene products of the
early genes in trans.
[0099] The nucleic acids that are delivered to cells typically contain
expression controlling systems. For example, the inserted genes in viral
and retroviral systems usually contain promoters, and/or enhancers to
help control the expression of the desired gene product. A promoter is
generally a sequence or sequences of DNA that function when in a
relatively fixed location in regard to the transcription start site. A
promoter contains core elements required for basic interaction of RNA
polymerase and transcription factors, and may contain upstream elements
and response elements.
[0100] Preferred promoters controlling transcription from vectors in
mammalian host cells may be obtained from various sources, for example,
the genomes of viruses such as: polyoma, Simian Virus 40 (SV40),
adenovirus, retroviruses, hepatitis-B virus and most preferably
cytomegalovirus, or from heterologous mammalian promoters, e.g. beta
actin promoter.
[0101] Enhancer generally refers to a sequence of DNA that functions at no
fixed distance from the transcription start site and can be either 5' or
3' to the transcription unit. Furthermore, enhancers can be within an
intron as well as within the coding sequence. They are usually between 10
and 300 by in length, and they function in cis. Enhancers f unction to
increase transcription from nearby promoters. Enhancers also often
contain response elements that mediate the regulation of transcription.
Promoters can also contain response elements that mediate the regulation
of transcription. Enhancers often determine the regulation of expression
of a gene. While many enhancer sequences are now known from mammalian
genes (globin, elastase, albumin, -fetoprotein and insulin), typically
one will use an enhancer from a eukaryotic cell virus for general
expression. Preferred examples are the SV40 enhancer on the late side of
the replication origin (bp 100-270), the cytomegalovirus early promoter
enhancer, the polyoma enhancer on the late side of the replication
origin, and adenovirus enhancers.
[0102] The promotor and/or enhancer may be specifically activated either
by light or specific chemical events which trigger their function.
Systems can be regulated by reagents such as tetracycline and
dexamethasone. There are also ways to enhance viral vector gene
expression by exposure to irradiation, such as gamma irradiation, or
alkylating chemotherapy drugs.
[0103] In certain embodiments the promoter and/or enhancer region can act
as a constitutive promoter and/or enhancer to maximize expression of the
region of the transcription unit to be transcribed. In certain constructs
the promoter and/or enhancer region be active in all eukaryotic cell
types, even if it is only expressed in a particular type of cell at a
particular time. A preferred promoter of this type is the CMV promoter
(650 bases). Other preferred promoters are SV40 promoters,
cytomegalovirus (full length promoter), and retroviral vector LTR.
[0104] It has been shown that all specific regulatory elements can be
cloned and used to construct expression vectors that are selectively
expressed in specific cell types such as melanoma cells. The glial
fibrillary acetic protein (GFAP) promoter has been used to selectively
express genes in cells of glial origin.
[0105] Expression vectors used in eukaryotic host cells (yeast, fungi,
insect, plant, animal, human or nucleated cells) may also contain
sequences necessary for the termination of transcription which may affect
mRNA expression. These regions are transcribed as polyadenylated segments
in the untranslated portion of the mRNA encoding tissue factor protein.
The 3' untranslated regions also include transcription termination sites.
It is preferred that the transcription unit also contain a
polyadenylation region. One benefit of this region is that it increases
the likelihood that the transcribed unit will be processed and
transported like mRNA. The identification and use of polyadenylation
signals in expression constructs is well established. It is preferred
that homologous polyadenylation signals be used in the transgene
constructs. In certain transcription units, the polyadenylation region is
derived from the SV40 early polyadenylation signal and consists of about
400 bases. It is also preferred that the transcribed units contain other
standard sequences alone or in combination with the above sequences
improve expression from, or stability of, the construct.
[0106] The viral vectors may include nucleic acid sequence encoding a
marker product. This marker product is used to determine if the gene has
been delivered to the cell and once delivered is being expressed.
Preferred marker genes are the E. Coli lacZ gene, which encodes
.beta.-galactosidase, and green fluorescent protein.
[0107] In some embodiments the marker may be a selectable marker. Examples
of suitable selectable markers for mammalian cells are dihydrofolate
reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418,
hydromycin, and puromycin. When such selectable markers are successfully
transferred into a mammalian host cell, the transformed mammalian host
cell can survive if placed under selective pressure. There are two widely
used distinct categories of selective regimes. The first category is
based on a cell's metabolism and the use of a mutant cell line which
lacks the ability to grow independent of a supplemented media. Two
examples are: CHO DHFR-cells and mouse LTK-cells. These cells lack the
ability to grow without the addition of such nutrients as thymidine or
hypoxanthine. Because these cells lack certain genes necessary for a
complete nucleotide synthesis pathway, they cannot survive unless the
missing nucleotides are provided in a supplemented media. An alternative
to supplementing the media is to introduce an intact DHFR or TK gene into
cells lacking the respective genes, thus altering their growth
requirements. Individual cells which were not transformed with the DHFR
or TK gene will not be capable of survival in non-supplemented media.
[0108] The second category is dominant selection which refers to a
selection scheme used in any cell type and does not require the use of a
mutant cell line. These schemes typically use a drug to arrest growth of
a host cell. Those cells which have a novel gene would express a protein
conveying drug resistance and would survive the selection. Examples of
such dominant selection use the drugs neomycin, mycophenolic acid, or
hygromycin. The three examples employ bacterial genes under eukaryotic
control to convey resistance to the appropriate drug G418 or neomycin
(geneticin), xgpt (mycophenolic acid) or hygromycin, respectively. Others
include the neomycin analog G418 and puramycin.
Non-Nucleic Acid Based Systems
[0109] The inhibitors of PAX2 expression or PAX2 activity and enhancers of
DEFB1 expression or DEFB1 activity may also be delivered to the target
cells in a variety of ways. For example, the compositions can be
delivered through electroporation, or through lipofection, or through
calcium phosphate precipitation. The delivery mechanism chosen will
depend in part on the type of cell targeted and whether the delivery is
occurring for example in vivo or in vitro.
[0110] Thus, the compositions can comprise lipids such as liposomes, such
as cationic liposomes (e.g., DOTMA, DOPE, DC-cholesterol) or anionic
liposomes. Liposomes can further comprise proteins to facilitate
targeting a particular cell, if desired. Administration of a composition
comprising a compound and a cationic liposome can be administered to the
blood afferent to a target organ or inhaled into the respiratory tract to
target cells of the respiratory tract. Furthermore, the compound can be
administered as a component of a microcapsule that can be targeted to
specific cell types, such as macrophages, or where the diffusion of the
compound or delivery of the compound from the microcapsule is designed
for a specific rate or dosage.
[0111] In the methods described above which include the administration and
uptake of exogenous DNA into the cells of a subject (i.e., gene
transduction or transfection), delivery of the compositions to cells can
be via a variety of mechanisms. As one example, delivery can be via a
liposome, using commercially available liposome preparations such as
LIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, Md.), SUPERFECT
(Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (Promega Biotec, Inc.,
Madison, Wis.), as well as other liposomes developed according to
procedures standard in the art. In addition, the disclosed nucleic acid
or vector can be delivered in vivo by electroporation, the technology for
which is available from Genetronics, Inc. (San Diego, Calif.) as well as
by means of a SONOPORATION machine (ImaRx Pharmaceutical Corp., Tucson,
Ariz.).
[0112] The materials may be in solution, suspension (for example,
incorporated into microparticles, liposomes, or cells). These may be
targeted to a particular cell type via antibodies, receptors, or receptor
ligands. Vehicles such as "stealth" and other antibody conjugated
liposomes (including lipid mediated drug targeting to colonic carcinoma),
receptor mediated targeting of DNA through cell specific ligands,
lymphocyte directed tumor targeting, and highly specific therapeutic
retroviral targeting of murine glioma cells in vivo. In general,
receptors are involved in pathways of endocytosis, either constitutive or
ligand induced. These receptors cluster in clathrin-coated pits, enter
the cell via clathrin-coated vesicles, pass through an acidified endosome
in which the receptors are sorted, and then either recycle to the cell
surface, become stored intracellularly, or are degraded in lysosomes. The
internalization pathways serve a variety of functions, such as nutrient
uptake, removal of activated proteins, clearance of macromolecules,
opportunistic entry of viruses and toxins, dissociation and degradation
of ligand, and receptor-level regulation. Many receptors follow more than
one intracellular pathway, depending on the cell type, receptor
concentration, type of ligand, ligand valency, and ligand concentration.
[0113] Nucleic acids that are delivered to cells which are to be
integrated into the host cell genome, typically contain integration
sequences. These sequences are often viral related sequences,
particularly when viral based systems are used. These viral integration
systems can also be incorporated into nucleic acids which are to be
delivered using a non-nucleic acid based system of deliver, such as a
liposome, so that the nucleic acid contained in the delivery system can
be come integrated into the host genome.
[0114] Other general techniques for integration into the host genome
include, for example, systems designed to promote homologous
recombination with the host genome. These systems typically rely on
sequence flanking the nucleic acid to be expressed that has enough
homology with a target sequence within the host cell genome that
recombination between the vector nucleic acid and the target nucleic acid
takes place, causing the delivered nucleic acid to be integrated into the
host genome. These systems and the methods necessary to promote
homologous recombination are known to those of skill in the art.
[0115] The inhibitors of PAX2 expression or PAX2 activity and enhancers of
DEFB lexpression or DEFB1 activity can be delivered to the target cells
in a variety of ways. can be administered in a pharmaceutically
acceptable carrier and can be delivered to the subjects cells in vivo
and/or ex vivo by a variety of mechanisms well known in the art (e.g.,
uptake of naked DNA, liposome fusion, intramuscular injection of DNA via
a gene gun, endocytosis and the like).
[0116] If ex vivo methods are employed, cells or tissues can be removed
and maintained outside the body according to standard protocols well
known in the art. The compositions can be introduced into the cells via
any gene transfer mechanism, such as, for example, calcium phosphate
mediated gene delivery, electroporation, microinjection or
proteoliposomes. The transduced cells can then be infused (e.g., in a
pharmaceutically acceptable carrier) or homotopically transplanted back
into the subject per standard methods for the cell or tissue type.
Standard methods are known for transplantation or infusion of various
cells into a subject.
Composition and Kits
[0117] Another aspect of the present invention relates to compositions and
kits for treating or preventing cancer. The composition includes an
inhibitor of PAX2 expression or PAX2 activity, and/or an enhacer of
DEFB-1 expression or DEFB-1 activity, and a pharmaceutically acceptable
carrier.
[0118] By "pharmaceutically acceptable" is meant a material that is not
biologically or otherwise undesirable, i.e., the material may be
administered to a subject, along with the nucleic acid or vector, without
causing any undesirable biological effects or interacting in a
deleterious manner with any of the other components of the pharmaceutical
composition in which it is contained. The carrier would naturally be
selected to minimize any degradation of the active ingredient and to
minimize any adverse side effects in the subject, as would be well known
to one of skill in the art.
[0119] Suitable carriers and their formulations are described in
Remington: The Science and Practice of Pharmacy (19th ed.) ed. A. R.
Gennaro, Mack Publishing Company, Easton, Pa. 1995. Typically, an
appropriate amount of a pharmaceutically-acceptable salt is used in the
formulation to render the formulation isotonic. Examples of the
pharmaceutically-acceptable carrier include, but are not limited to,
saline, Ringer's solution and dextrose solution. The pH of the solution
is preferably from about 5 to about 8, and more preferably from about 7
to about 7.5. Further carriers include sustained release preparations
such as semipermeable matrices of solid hydrophobic polymers containing
the antibody, which matrices are in the form of shaped articles, e.g.,
films, liposomes or microparticles. It will be apparent to those persons
skilled in the art that certain carriers may be more preferable depending
upon, for instance, the route of administration and concentration of
composition being administered.
[0120] Pharmaceutical carriers are known to those skilled in the art.
These most typically would be standard carriers for administration of
drugs to humans, including solutions such as sterile water, saline, and
buffered solutions at physiological pH. The compositions can be
administered intramuscularly or subcutaneously. Other compounds will be
administered according to standard procedures used by those skilled in
the art.
[0121] Pharmaceutical compositions may include carriers, thickeners,
diluents, buffers, preservatives, surface active agents and the like in
addition to the molecule of choice. Pharmaceutical compositions may also
include one or more active ingredients such as antimicrobial agents,
anti-inflammatory agents, anesthetics, and the like.
[0122] Preparations for parenteral administration include sterile aqueous
or non-aqueous solutions, suspensions, and emulsions. Examples of
non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable
oils such as olive oil, and injectable organic esters such as ethyl
oleate. Aqueous carriers include water, alcoholic/aqueous solutions,
emulsions or suspensions, including saline and buffered media. Parenteral
vehicles include sodium chloride solution, Ringer's dextrose, dextrose
and sodium chloride, lactated Ringer's, or fixed oils. Intravenous
vehicles include fluid and nutrient replenishers, electrolyte
replenishers (such as those based on Ringer's dextrose), and the like.
Preservatives and other additives may also be present such as, for
example, antimicrobials, anti-oxidants, chelating agents, and inert gases
and the like.
[0123] Formulations for topical administration may include ointments,
lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
Conventional pharmaceutical carriers, aqueous, powder or oily bases,
thickeners and the like may be necessary or desirable.
[0124] Compositions for oral administration include powders or granules,
suspensions or solutions in water or non-aqueous media, capsules,
sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers,
dispersing aids or binders may be desirable.
[0125] Some of the compositions may potentially be administered as a
pharmaceutically acceptable acid- or base-addition salt, formed by
reaction with inorganic acids such as hydrochloric acid, hydrobromic
acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and
phosphoric acid, and organic acids such as formic acid, acetic acid,
propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid,
malonic acid, succinic acid, maleic acid, and fumaric acid, or by
reaction with an inorganic base such as sodium hydroxide, ammonium
hydroxide, potassium hydroxide, and organic bases such as mono-, di-,
trialkyl and aryl amines and substituted ethanolamines.
[0126] The materials may be in solution, suspension (for example,
incorporated into microparticles, liposomes, or cells). These may be
targeted to a particular cell type via antibodies, receptors, or receptor
ligands. Vehicles such as "stealth" and other antibody conjugated
liposomes (including lipid mediated drug targeting to colonic carcinoma),
receptor mediated targeting of DNA through cell specific ligands,
lymphocyte directed tumor targeting, and highly specific therapeutic
retroviral targeting of murine glioma cells in vivo. In general,
receptors are involved in pathways of endocytosis, either constitutive or
ligand induced. These receptors cluster in clathrin-coated pits, enter
the cell via clathrin-coated vesicles, pass through an acidified endosome
in which the receptors are sorted, and then either recycle to the cell
surface, become stored intracellularly, or are degraded in lysosomes. The
internalization pathways serve a variety of functions, such as nutrient
uptake, removal of activated proteins, clearance of macromolecules,
opportunistic entry of viruses and toxins, dissociation and degradation
of ligand, and receptor-level regulation. Many receptors follow more than
one intracellular pathway, depending on the cell type, receptor
concentration, type of ligand, ligand valency, and ligand concentration.
[0127] The materials described above as well as other materials can be
packaged together in any suitable combination as a kit useful for
performing, or aiding in the performance of, the disclosed method. It is
useful if the kit components in a given kit are designed and adapted for
use together in the disclosed method. For example disclosed are kits for
detecting, treating, or preventing prostate cancer, PIN, breast cancer,
and MIN. The kit comprising an inhibitor of PAX2 expression or PAX2
activity, and/or an enhancer of DEFB1 expression or DEFB1 activity. In
one embodiment, the kit contains a peptide or an antibody that
specifically bind PAX2 or DEFB1.
[0128] A composition disclosed herein may be administered in a number of
ways depending on whether local or systemic treatment is desired, and on
the area to be treated. For example, the compositions may be administered
orally, parenterally (e.g., intravenous, subcutaneous, intraperitoneal,
or intramuscular injection), by inhalation, extracorporeally, topically
(including transdermally, ophthalmically, vaginally, rectally,
intranasally) or the like.
[0129] As used herein, "topical intranasal administration" means delivery
of the compositions into the nose and nasal passages through one or both
of the nares and can comprise delivery by a spraying mechanism or droplet
mechanism, or through aerosolization of the nucleic acid or vector.
Administration of the compositions by inhalant can be through the nose or
mouth via delivery by a spraying or droplet mechanism. Delivery can also
be directly to any area of the respiratory system (e.g., lungs) via
intubation.
[0130] Parenteral administration of the composition, if used, is generally
characterized by injection. Injectables can be prepared in conventional
forms, either as liquid solutions or suspensions, solid forms suitable
for solution of suspension in liquid prior to injection, or as emulsions.
A more recently revised approach for parenteral administration involves
use of a slow release or sustained release system such that a constant
dosage is maintained.
[0131] The exact amount of the compositions required will vary from
subject to subject, depending on the species, age, weight and general
condition of the subject, the severity of the allergic disorder being
treated, the particular nucleic acid or vector used, its mode of
administration and the like. An appropriate amount can be determined by
one of ordinary skill in the art using only routine experimentation given
the teachings herein. Thus, effective dosages and schedules for
administering the compositions may be determined empirically, and making
such determinations is within the skill in the art. The dosage ranges for
the administration of the compositions are those large enough to produce
the desired effect in which the symptoms disorders are affected. The
dosage should not be so large as to cause adverse side effects, such as
unwanted cross-reactions, anaphylactic reactions, and the like.
Generally, the dosage will vary with the age, condition, sex and extent
of the disease in the patient, route of administration, or whether other
drugs are included in the regimen, and can be determined by one of skill
in the art. The dosage can be adjusted by the individual physician in the
event of any counter indications. Dosage can vary, and can be
administered in one or more dose administrations daily, for one or
several days. Guidance can be found in the literature for appropriate
dosages for given classes of pharmaceutical products.
[0132] For example, a typical daily dosage of the disclosed composition
used alone might range from about 1 .mu.g/kg to up to 100 mg/kg of body
weight or more per day, depending on the factors mentioned above. In
certain embodiments, the treatment method is tailored based on the
PAX2-to-DEFB1 expression ratio (P/D ratio) and estrogen-receptor
(ER)/progesterone-receptor (PR) status of the diseased tissue. Table 2
shows the treatment options based on the P/D ratio and ER/PR status.
There is a positive correlation between PAX2 status and ER status in
normal breast tissue, MIN and low grade breast carcinoma. PAX2 also
regulates ERBB2 expression and subsequently Her2/neu expression via the
oestogen receptor. Conversely, there is an inverse relationship between
PAX2 expression and high grade (or invasive) breast carcinoma. Therefore
monitoring PAX2 expression levels can be used to predict drug response or
resistance, as well as identify patients who may be candidates for DEFB1
or anti-PAX2 therapy. The term "anti-PAX2 therapy" refers to methods for
inhibiting PAX2 expression or PAX2 activity. The term "DEFB1 therapy"
refers to methods for increasing DEFB1 expression. The term "DEFB1
therapy" does not include methods for inhibiting PAX2 expression or PAX2
activity, although such methods also result in increase of DEFB1
expression.
[0133] As shown in Table 2, anti-PAX2 therapy and/or DFB1 therapy may be
used in conjunction with one or more other treatments for breast cancer,
such as anti-hormone treatment (e.g., Tamoxifen), anti-ERBB2 treatment
(e.g., Herceptin), anti-Her2 treatment (e.g., Trastuzumab), and
anti-AIB-1/SRC-3 treatment.
TABLE-US-00004
TABLE 2
Using PAX2-to-DEFB1 Ratio to Treat Breast Conditions
Change in
PAX2/DEFB1 ER/PR DEFB1 Anti-PAX2
Tissue Type Ratio* Status Therapy Therapy Adjuvant Therapy
MIN ER.sup.+/PR.sup.+ No Yes No
Low Grade ER.sup.+/PR.sup.+ Yes Yes Anti-ERBB2 (eg. Herceptin)
Cancer Anti-Her2 (eg. Trastuzumab)
Anti-AIB-1/SRC-3
Low Grade ER.sup.+/PR.sup.- Yes Yes Anti-ERBB2 (eg. Herceptin)
Cancer Anti-Her2 (eg. Trastuzumab)
Anti-AIB-1/SRC-3
High Grade ER.sup.+/PR.sup.+ Yes No Anti-hormone (eg. Tamoxifen)
Cancer Anti-ERBB2 (eg. Herceptin)
Anti-Her2 (eg. Trastuzumab)
Anti-AIB-1/SRC-3
High Grade ER.sup.+/PR.sup.- Yes No Anti-hormone (eg. Tamoxifen)
Cancer Anti-ERBB2 (eg. Herceptin)
Anti-Her2 (eg. Trastuzumab)
Anti-AIB-1/SRC-3
High Grade ER.sup.-/PR.sup.+ Yes No Anti-ERBB2 (eg. Herceptin)
Cancer Anti-Her2 (eg. Trastuzumab)
High Grade ER.sup.-/PR.sup.- Yes No Anti-ERBB2 (eg. Herceptin)
Cancer Anti-Her2 (eg. Trastuzumab)
*Compared to the PAX2/DEFB1 ratio in normal breast epithelium
PAX2-to-DEFB1 Expression Ratio
[0134] As used hereinafter, the term "PAX2-to-DEFB1 expression ratio"
refers to the ratio between the amount of functional PAX2 protein or its
variant and the amount of functional DEFB1 protein or its variant in a
given cell or tissue. Levels of PAX2 and DEFB1 expression in a cell or
tissue can be measured any method known in the art. In certain
embodiments, the levels of PAX2 and DEFB1 expression in breast tissue are
determined by determining the levels of PAX2 and DEFB1 in a cell or cells
obtained directly from the breast tissue.
[0135] The "PAX2-to-DEFB1 expression ratio" can be determined directly at
protein level or indirectly at the RNA level. The protein levels may be
measured with protein arrays, immunoassays and enzyme assays. The RNA
levels may be measured, for example, with DNA arrays, RT-PCR and Northern
Blotting. In certain embodiments, the PAX2-to-DEFB1 expression ratio is
determined by determining the expression level of PAX2 gene relative to
the expression level of a control gene, determining the expression level
of DEFB1 gene relative to the expression level of the same control gene,
and calculating the PAX2-to-DEFB1 expression ratio based on the
expression levels of PAX2 and DEFB1. In one embodiment, the control gene
is the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene.
Immunoassays
[0136] Immunoassays, in their most simple and direct sense, are binding
assays involving binding between antibodies and antigen. Many types and
formats of immunoassays are known and all are suitable for detecting the
disclosed biomarkers. Examples of immunoassays are enzyme linked
immunosorbent assays (ELISAs), radioimmunoassays (RIA), radioimmune
precipitation assays (RIPA), immunobead capture assays, Western blotting,
dot blotting, gel-shift assays, Flow cytometry, protein arrays,
multiplexed bead arrays, magnetic capture, in vivo imaging, fluorescence
resonance energy transfer (FRET), and fluorescence recovery/localization
after photobleaching (FRAP/FLAP).
[0137] In general, immunoassays involve contacting a sample suspected of
containing a molecule of interest (such as the disclosed biomarkers) with
an antibody to the molecule of interest or contacting an antibody to a
molecule of interest (such as antibodies to the disclosed biomarkers)
with a molecule that can be bound by the antibody, as the case may be,
under conditions effective to allow the formation of immunocomplexes. In
many forms of immunoassay, the sample-antibody composition, such as a
tissue section, ELISA plate, dot blot or Western blot, can then be washed
to remove any non-specifically bound antibody species, allowing only
those antibodies specifically bound within the primary immune complexes
to be detected.
[0138] Radioimmune Precipitation Assay (RIPA) is a sensitive assay using
radiolabeled antigens to detect specific antibodies in serum. The
antigens are allowed to react with the serum and then precipitated using
a special reagent such as, for example, protein A sepharose beads. The
bound radiolabeled immunoprecipitate is then commonly analyzed by gel
electrophoresis. Radioimmunoprecipitation assay (RIPA) is often used as a
confirmatory test for diagnosing the presence of HIV antibodies. RIPA is
also referred to in the art as Farr Assay, Precipitin Assay, Radioimmune
Precipitin Assay; Radioimmunoprecipitation Analysis;
Radioimmunoprecipitation Analysis, and Radioimmunoprecipitation Analysis.
[0139] Also contemplated are immunoassays wherein the protein or antibody
specific for the protein is bound to a solid support (e.g., tube, well,
bead, or cell) to capture the antibody or protein of interest,
respectively, from a sample, combined with a method of detecting the
protein or antibody specific for the protein on the support. Examples of
such immunoassays include Radioimmunoassay (RIA), Enzyme-Linked
Immunosorbent Assay (ELISA), Flow cytometry, protein array, multiplexed
bead assay, and magnetic capture.
[0140] Protein arrays are solid-phase ligand binding assay systems using
immobilized proteins on surfaces which include glass, membranes,
microtiter wells, mass spectrometer plates, and beads or other particles.
The assays are highly parallel (multiplexed) and often miniaturized
(microarrays, protein chips). Their advantages include being rapid and
automatable, capable of high sensitivity, economical on reagents, and
giving an abundance of data for a single experiment. Bioinformatics
support is important; the data handling demands sophisticated software
and data comparison analysis. However, the software can be adapted from
that used for DNA arrays, as can much of the hardware and detection
systems.
[0141] Capture arrays form the basis of diagnostic chips and arrays for
expression profiling. They employ high affinity capture reagents, such as
conventional antibodies, single domains, engineered scaffolds, peptides
or nucleic acid aptamers, to bind and detect specific target ligands in
high throughput manner. Antibody arrays are available commercially. In
addition to the conventional antibodies, Fab and scFv fragments, single
V-domains from camelids or engineered human equivalents (Domantis,
Waltham, Mass.) may also be useful in arrays.
[0142] Nonprotein capture molecules, notably the single-stranded nucleic
acid aptamers which bind protein ligands with high specificity and
affinity, are also used in arrays (SomaLogic, Boulder, Colo.). Aptamers
are selected from libraries of oligonucleotides by the Selex.TM.
procedure and their interaction with protein can be enhanced by covalent
attachment, through incorporation of brominated deoxyuridine and
UV-activated crosslinking (photoaptamers). Photocrosslinking to ligand
reduces the crossreactivity of aptamers due to the specific steric
requirements. Aptamers have the advantages of ease of production by
automated oligonucleotide synthesis and the stability and robustness of
DNA; on photoaptamer arrays, universal fluorescent protein stains can be
used to detect binding.
[0143] An alternative to an array of capture molecules is one made through
`molecular imprinting` technology, in which peptides (e.g., from the
C-terminal regions of proteins) are used as templates to generate
structurally complementary, sequence-specific cavities in a polymerizable
matrix; the cavities can then specifically capture (denatured) proteins
that have the appropriate primary amino acid sequence (ProteinPrint.TM.,
Aspira Biosystems, Burlingame, Calif.).
[0144] Another methodology which can be used diagnostically and in
expression profiling is the ProteinChip.RTM. array (Ciphergen, Fremont,
Calif.), in which solid phase chromatographic surfaces bind proteins with
similar characteristics of charge or hydrophobicity from mixtures such as
plasma or tumor extracts, and SELDI-TOF mass spectrometry is used to
detection the retained proteins.
[0145] Other useful methodology includes large-scale functional chips
constructed by immobilizing large numbers of purified proteins on a chip,
and multiplexed bead assays.
Antibodies
[0146] The term "antibodies" is used herein in a broad sense and includes
both polyclonal and monoclonal antibodies. In addition to intact
immunoglobulin molecules, also included in the term "antibodies" are
fragments or polymers of those immunoglobulin molecules, and human or
humanized versions of immunoglobulin molecules or fragments thereof, as
long as they are chosen for their ability to interact with, for example,
PAX2 or DEFB1, such that PAX2 is inhibited from interacting with DEFB1.
Antibodies that bind the disclosed regions of PAX2 or DEFB1 involved in
the interaction between PAX2 and DEFB1 are also disclosed. The antibodies
can be tested for their desired activity using the in vitro assays
described herein, or by analogous methods, after which their in vivo
therapeutic and/or prophylactic activities are tested according to known
clinical testing methods.
[0147] The monoclonal antibodies herein specifically include "chimeric"
antibodies in which a portion of the heavy and/or light chain is
identical with or homologous to corresponding sequences in antibodies
derived from a particular species or belonging to a particular antibody
class or subclass, while the remainder of the chain(s) is identical with
or homologous to corresponding sequences in antibodies derived from
another species or belonging to another antibody class or subclass, as
well as fragments of such antibodies, as long as they exhibit the desired
antagonistic activity (See, U.S. Pat. No. 4,816,567 and Morrison et al.,
Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).
[0148] As used herein, the term "antibody" or "antibodies" can also refer
to a human antibody and/or a humanized antibody. Many non-human
antibodies (e.g., those derived from mice, rats, or rabbits) are
naturally antigenic in humans, and thus can give rise to undesirable
immune responses when administered to humans. Therefore, the use of human
or humanized antibodies in the methods serves to lessen the chance that
an antibody administered to a human will evoke an undesirable immune
response. Methods for humanizing non-human antibodies are well known in
the art.
DNA Arrays
[0149] A DNA or oligonucleotide microarray consists of an arrayed series
of a plurality of microscopic spots of oligonucleotides, called features,
each containing a small amount (typically in the range of picomoles) of a
specific oligonucleotide sequence. The specific oligonucleotide sequence
can be a short section of a gene or other oligonucleotide element that
are used as probes to hybridize a cDNA or cRNA sample under
high-stringency conditions. Probe-target hybridization is usually
detected and quantified by fluorescence-based detection of
fluorophore-labeled targets to determine relative abundance of nucleic
acid sequences in the target.
[0150] The probes are typically attached to a solid surface by a covalent
bond to a chemical matrix (via epoxy-silane, amino-silane, lysine,
polyacrylamide or others). The solid surface can be glass or a silicon
chip or microscopic beads. Oligonucleotide arrays are different from
other types of microarray only in that they either measure nucleotides or
use oligonucleotide as part of its detection system.
[0151] To detect gene expression in target tissue or cells using an
oligonucleotide array, nucleic acid of interest is purified from the
target tissue or cells. The nucleotide can be all RNA for expression
profiling, DNA for comparative hybridization, or DNA/RNA bound to a
particular protein which is immunoprecipitated (ChIP-on-chip) for
epigenetic or regulation studies.
[0152] In one embodiment, total RNA is isolated (total as it is nuclear
and cytoplasmic) by guanidinium thiocyanate-phenol-chloroform extraction
(e.g. Trizol). The purified RNA may be analyzed for quality (e.g., by
capillary electrophoresis) and quantity (e.g., by using a nanodrop
spectrometer. The total RNA is RNA is reverse transcribed into DNA with
either polyT primers or random primers. The DNA products may be
optionally amplified by PCR. A label is added to the amplification
product either in the RT step or in an additional step after
amplification if present. The label can be a fluorescent label or
radioactive labels. The labeled DNA products are then hybridized to the
microarray. The microarray is then washed and scanned. The expression
level of the gene of interest is determined based on the hybridization
result using method well known in the art.
Pharmacogenomics
[0153] In another embodiment, the PAX2 and/or DEFB1 expression profiles
are used for determine pharmacogenomics of breast cancer.
Pharmacogenomics refers to the relationship between an individual's
genotype and that individual's response to a foreign compound or drug.
Differences in metabolism of therapeutics can lead to severe toxicity or
therapeutic failure by altering the relation between dose and blood
concentration of the pharmacologically active drug. Thus, a physician or
clinician may consider applying knowledge obtained in relevant
pharmacogenomics studies in determining whether to administer an
anti-cancer drug, as well as tailoring the dosage and/or therapeutic
regimen of treatment with the anti-cancer drug.
[0154] Pharmacogenomics deals with clinically significant hereditary
variations in the response to drugs due to altered drug disposition and
abnormal action in affected persons. In general, two types of
pharmacogenetic conditions can be differentiated. Genetic conditions
transmitted as a single factor altering the way drugs act on the body
(altered drug action) or genetic conditions transmitted as single factors
altering the way the body acts on drugs (altered drug metabolism). These
pharmacogenetic conditions can occur either as rare genetic defects or as
naturally-occurring polymorphisms. For example, glucose-6-phosphate
dehydrogenase deficiency (G6PD) is a common inherited enzymopathy in
which the main clinical complication is hemolysis after ingestion of
oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and
consumption of fava beans.
[0155] One pharmacogenomics approach to identifying genes that predict
drug response, known as "a genome-wide association," relies primarily on
a high-resolution map of the human genome consisting of already known
gene-related sites (e.g., a "bi-allelic" gene marker map which consists
of 60,000-100,000 polymorphic or variable sites on the human genome, each
of which has two variants). Such a high-resolution genetic map can be
compared to a map of the genome of each of a statistically substantial
number of subjects taking part in a Phase II/III drug trial to identify
genes associated with a particular observed drug response or side effect.
Alternatively, such a high resolution map can be generated from a
combination of some ten-million known single nucleotide polymorphisms
(SNPs) in the human genome. As used herein, an "SNP" is a common
alteration that occurs in a single nucleotide base in a stretch of DNA.
For example, an SNP may occur once per every 1,000 bases of DNA. An SNP
may be involved in a disease process. However, the vast majority of SNPs
may not be disease associated. Given a genetic map based on the
occurrence of such SNPs, individuals can be grouped into genetic
categories depending on a particular pattern of SNPs in their individual
genome. In such a manner, treatment regimens can be tailored to groups of
genetically similar individuals, taking into account traits that may be
common among such genetically similar individuals. Thus, mapping of the
PAX2 and/or DEFB1 to SNP maps of breast patients may allow easier
identification of these genes according to the genetic methods described
herein.
[0156] Alternatively, a method termed the "candidate gene approach," can
be utilized to identify genes that predict drug response. According to
this method, if a gene that encodes a drug target is known, all common
variants of that gene can be fairly easily identified in the population
and it can be determined if having one version of the gene versus another
is associated with a particular drug response.
[0157] As an illustrative embodiment, the activity of drug metabolizing
enzymes is a major determinant of both the intensity and duration of drug
action. The discovery of genetic polymorphisms of drug metabolizing
enzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes
CYP2D6 and CYPZC19) has provided an explanation as to why some subjects
do not obtain the expected drug effects or show exaggerated drug response
and serious toxicity after taking the standard and safe dose of a drug.
These polymorphisms are expressed in two phenotypes in the population,
the extensive metabolizer and poor metabolizer. The prevalence of poor
metabolizer phenotypes is different among different populations. For
example, the gene coding for CYP2D6 is highly polymorphic and several
mutations have been identified in poor metabolizers, which all lead to
the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19
quite frequently experience exaggerated drug response and side effects
when they receive standard doses. If a metabolite is the active
therapeutic moiety, poor metabolizers show no therapeutic response, as
demonstrated for the analgesic effect of codeine mediated by its
CYP2D6-formed metabolite morphine. The other extreme are the so called
ultra-rapid metabolizers who do not respond to standard doses. Recently,
the molecular basis of ultra-rapid metabolism has been identified to be
due to CYP2D6 gene amplification.
[0158] Alternatively, a method termed the "gene expression profiling" can
be utilized to identify genes that predict drug response. For example,
the gene expression of an animal dosed with a drug can give an indication
whether gene pathways related to toxicity have been turned on.
[0159] Information generated from more than one of the above
pharmacogenomics approaches can be used to determine appropriate dosage
and treatment regimens for prophylactic or therapeutic treatment an
individual. This knowledge, when applied to dosing or drug selection, can
avoid adverse reactions or therapeutic failure and thus enhance
therapeutic or prophylactic efficiency when treating a subject with a
breast condition.
[0160] In one embodiment, the PAX2 and/or DEFB1 expression profiles, as
well as the ER/PR status, in a subject are used to determine the
appropriate treatment regimens for an individual with a breast condition.
[0161] In another embodiment, the PAX2 expression level (typically
determine in reference to a control gene as actin gene or GAPDH gene) is
used in patients with triple negative breast cancer (i.e., oestrogen
receptor (ER) negative, progesterone receptor (PR) negative, human
epidermal growth factor receptor 2 (HER2) negative) to measure of the
effectiveness of cancer therapy, to determine treatment course, or to
monitor cancer recurrence.
[0162] The present invention is further illustrated by the following
examples which should not be construed as limiting. The contents of all
references, patents and published patent applications cited throughout
this application, as well as the Figures and Tables are incorporated
herein by reference.
EXAMPLE 1
Human Beta Defensin-1 is Cytotoxic to Late-Stage Prostate Cancer and
Plays a Role in Prostate Cancer Tumor Immunity
[0163] In this example, DEFB1 was cloned into an inducible expression
system to examine what effect it had on normal prostate epithelial cells,
as well as androgen receptor positive (AR+) and androgen receptor
negative (AR-) prostate cancer cell lines. Induction of DEFB1 expression
resulted in a decrease in cellular growth in AR- cells DU145 and PC3, but
had no effect on the growth of the AR+ prostate cancer cells LNCaP. DEFB1
also caused rapid induction of caspase-mediated apoptosis. Data presented
here are the first to provide evidence of its role in innate tumor
immunity and indicate that its loss contributes to tumor progression in
prostate cancer.
Materials and Methods
[0164] Cell Lines: The cell lines DU145 were cultured in DMEM medium, PC3
were grown in F12 medium, and LNCaP were grown in RPMI medium (Life
Technologies, Inc., Grand Island, N.Y.). Growth media for all three lines
was supplemented with 10% (v/v) fetal bovine serum (Life Technologies).
The hPrEC cells were cultured in prostate epithelium basal media (Cambrex
Bio Science, Inc., Walkersville, Md.). All cell lines were maintained at
37.degree. C. and 5% CO2.
[0165] Tissue Samples and Laser Capture Microdissection: Prostate tissues
obtained from consented patients that underwent radical prostatectomy
were acquired through the Hollings Cancer Center tumor bank in accordance
with an Institutional Review Board-approved protocol. This included
guidelines for the processing, sectioning, histological characterization,
RNA purification and PCR amplification of samples. Following pathologic
examination of frozen tissue sections, laser capture microdissection
(LCM) was performed to ensure that the tissue samples assayed consisted
of pure populations of benign prostate cells. For each tissue section
analyzed, LCM was performed at three different regions containing benign
tissue and the cells collected were then pooled.
[0166] Prostate tissues were obtained from patients who provided informed
consent prior to undergoing radical prostatectomy. Samples were acquired
through the Hollings Cancer Center tumor bank in accordance with an
Institutional Review Board-approved protocol. This included guidelines
for the processing, sectioning, histological characterization, RNA
purification and PCR amplification of samples. Prostate specimens
received from the surgeons and pathologists were immediately frozen in
OCT compound. Each OCT block was cut to produce serial sections which
were stained and examined. Areas containing benign cells, prostatic
intraepithelial neoplasia (PIN), and cancer were identified and used to
guide our selection of regions from unstained slides using the Arcturus
PixCell II System (Sunnyvale, Calif.). Caps containing captured material
were exposed to 20 .mu.l of lysate from the Arcturus Pico Pure RNA
Isolation Kit and processed immediately. RNA quantity and quality was
evaluated using sets of primers that produce 5' amplicons. The sets
include those for the ribosomal protein L32 (the 3' amplicon and the 5'
amplicon are 298 bases apart), for the glucose phosphate isomerase (391
bases apart), and for the glucose phosphate isomerase (842 bases apart).
Ratios of 0.95 to 0.80 were routinely obtained for these primer sets
using samples from a variety of prepared tissues. Additional tumor and
normal samples were grossly dissected by pathologists, snap frozen in
liquid nitrogen and evaluated for hBD-1 and cMYC expression.
[0167] Cloning of DEFB1 Gene: DEFB I cDNA was generated from RNA by
reverse transcription-PCR. The PCR primers were designed to contain ClaI
and KpnI restriction sites. DEFB1 PCR products were restriction digested
with ClaI and KpnI and ligated into a TA cloning vector. The TA/DEFB1
vector was then transfected into E. coli by heat shock and individual
clones were selected and expanded. Plasmids were isolated by Cell Culture
DNA Midiprep (Qiagen, Valencia, Calif.) and sequence integrity verified
by automated sequencing. The DEFB1 gene fragment was then ligated into
the pTRE2 digested with ClaI and KpnI, which served as an intermediate
vector for orientation purposes. Then the pTRE2/DEFB1 construct was
digested with ApaI and KpnI to excise the DEFB1 insert, which was ligated
into pIND vector of the Ecdysone Inducible Expression System (Invitrogen,
Carlsbad, Calif.) also double digested with ApaI and KpnI. The construct
was again transfected into E. coli and individual clones were selected
and expanded. Plasmids were isolated and sequence integrity of pIND/DEFB1
was again verified by automated sequencing.
[0168] Transfection: Cells (1.times.10.sup.6) were seeded onto 100-mm
Petri dishes and grown overnight. Then the cells were co-transfected
using Lipofectamine 2000 (Invitrogen, Carlsbad, Calif.) with 1 .mu.g of
pVgRXR plasmid, which expresses the heterodimeric ecdysone receptor, and
1 .mu.g of the pIND/DEFB1 vector construct or empty pIND control vector
in Opti-MEM media (Life Technologies, Inc., Grand Island, N.Y.).
[0169] RNA Isolation and Quantitative RT-PCR: In order to verify DEFB1
protein expression in the cells transfected with DEFB1 construct, RNA was
collected after a 24 hour induction period with Ponasterone A (Pon A).
Briefly, total RNA was isolated using the SV Total RNA Isolation System
(Promega, Madison, Wis.) from approximately 1.times.10.sup.6 cells
harvested by trypsinizing. Here, cells were lysed and total RNA was
isolated by centrifugation through spin columns. For cells collected by
LCM, total RNA was isolated using the PicoPure RNA Isolation Kit
(Arcturus Biosciences, Mt. View, Calif.) following the manufacturer's
protocol. Total RNA (0.5 .mu.g per reaction) from both sources was
reverse transcribed into cDNA utilizing random primers (Promega). AMV
Reverse Transcriptase II enzyme (500 units per reaction; Promega) was
used for first strand synthesis and TfI DNA Polymerase for second strand
synthesis (500 units per reaction; Promega) as per the manufacturer's
protocol. In each case, 50 pg of cDNA was used per ensuing PCR reaction.
Two-step QRT-PCR was performed on cDNA generated using the MultiScribe
Reverse Transcripatase from the TaqMan Reverse Transcription System and
the SYBR.RTM. Green PCR Master Mix (Applied Biosystems).
[0170] The primer pair for DEFB1 was generated from the published DEFB1
sequence (GenBank Accession No. U50930). The primer sequences are:
TABLE-US-00005
Sense (5'-3')
SEQ ID NO: 51
.beta.-actin 5'-CCTGGCACCCAGCACAAT-3'
SEQ ID NO: 53
DEFB1 5'-GTTGCCTGCCAGTCGCCATGAGAACTTCCTAC-3'
Antisense (5'-3')
SEQ ID NO: 52
.beta.-actin 5'-GCCGATCCACACGGAGTACT-3'
SEQ ID NO: 54
DEFB1 5'-TGGCCTTCCCTCTGTAACAGGTGCCTTGAATT-3'
[0171] Forty cycles of PCR were performed under standard conditions using
an annealing temperature of 56.degree. C. In addition, .beta.-actin
(Table 2) was amplified as a housekeeping gene to normalize the initial
content of total cDNA. DEFB1 expression was calculated as the relative
expression ratio between DEFB1 and .beta.-actin and was compared in cells
lines induced and uninduced for DEFB1 expression, as well as LCM benign
prostatic tissue. As a negative control, QRT-PCR reactions without cDNA
template were also performed. All reactions were run three times in
triplicate.
[0172] MIT Cell Viability Assay: To examine the effects of DEFB1 on cell
growth, metabolic 3-[4,5-dimethylthiazol-2yl]-2,5 diphenyl tetrazolium
bromide (MTT) assays were performed. PC3, DU145 and LNCaP cells
co-transfected with pVgRXR plasmid and pIND/DEFB1 construct or empty pIND
vector were seeded onto a 96-well plate at 1-5.times.10.sup.3 cells per
well. Twenty-four hours after seeding, fresh growth medium was added
containing 10 .mu.M Ponasterone A daily to induce DEFB1 expression for
24-, 48- and 72 hours after which the MTT assay was performed according
to the manufacturer's instructions (Promega). Reactions were performed
three times in triplicate.
[0173] Flow Cytometry: PC3 and DU145 cells co-transfected with the DEFB1
expression system were grown in 60-mm dishes and induced for 12, 24, and
48 hours with 10 .mu.M Ponasterone A. Following each incubation period,
the medium was collected from the plates (to retain any detached cells)
and combined with PBS used to wash the plates. The remaining attached
cells were harvested by trypsinization and combined with the detached
cells and PBS. The cells were then pelleted at 4oC (500.times.g) for 5
min, washed twice in PBS, and resuspended in 100 ul of 1.times. Annexin
binding buffer (0.1 M Hepes/NaOH at pH 7.4, 1.4 M NaCl, 25 mM CaCl.sub.2)
containing 5 .mu.l of Annexin V-FITC and 5 .mu.l of PI. The cells were
incubated at RT for 15 min in the dark, then diluted with 400 .mu.l of
1.times. Annexin binding buffer and analyzed by FACscan (Becton
Dickinson, San Jose, Calif.). All reactions were performed three times.
[0174] Microscopic Analysis: Cell morphology was analyzed by phase
contrast microscopy. DU145, PC3 and LNCaP cells containing no vector,
empty plasmid or DEFB1 plasmid were seeded onto 6 well culture plates (BD
Falcon, USA). The following day plasmid-containing cells were induced for
a period of 48 h with media containing 10 .mu.M Ponasterone A, while
control cells received fresh media. The cells were then viewed under an
inverted Zeiss IM 35 microscope (Carl Zeiss, Germany). Phase contrast
pictures of a field of cells were obtained using the SPOT Insight Mosaic
4.2 camera (Diagnostic Instruments, USA). Cells were examined by phase
contrast microscopy under 32.times. magnification and digital images were
stored as uncompressed TIFF files and exported into Photoshop CS software
(Adobe Systems, San Jose, Calif.) for image processing and hard copy
presentation.
[0175] Caspase Detection: Detection of caspase activity in the prostate
cancer cell lines was performed using APO LOGIX.TM. Carboxyfluorescin
Caspase detection kit (Cell Technology, Mountain View, Calif.). Active
caspases were detected through the use of a FAM-VAD-FMK inhibitor that
irreversibly binds to active caspases. Briefly, DU145 and PC3 cells
(1.5-3.times.10.sup.5) containing the DEFB1 expression system were plated
in 35 mm glass bottom microwell dishes (Matek, Ashland, Mass.) and
treated for 24 hours with media only or with media containing PonA as
previously described. Next, 10 .mu.l of a 30.times. working dilution of
carboxyfluorescein labeled peptide fluoromethyl ketone (FAM-VAD-FMK) was
added to 300 .mu.l of media and added to each 35 mm dish. Cells were then
incubated for 1 hour at 37.degree. C. under 5% CO2. Then, the medium was
aspirated and the cells were washed twice with 2 ml of a 1.times. Working
dilution Wash Buffer. Cells were viewed under differential interference
contrast (DIC) or under laser excitation at 488 nm. The fluorescent
signal was analyzed using a confocal microscope (Zeiss LSM 5 Pascal) and
a 63.times. DIC oil lens with a Vario 2 RGB Laser Scanning Module.
[0176] Statistical Analysis: Statistical differences were evaluated using
the Student's t-test for unpaired values. P values were determined by a
two-sided calculation, and a P value of less than 0.05 was considered
statistically significant.
Results
[0177] DEFB1 Expression in Prostate Tissue and Cell Lines: DEFB1
expression levels were measured by QRT-PCR in benign and malignant
prostatic tissue, hPrEC prostate epithelial cells and DU145, PC3 and
LNCaP prostate cancer cells. DEFB1 expression was detected in all of the
benign clinical samples. The average amount of DEFB1 relative expression
was 0.0073. In addition, DEFB1 relative expression in hPrEC cells was
0.0089. There was no statistical difference in DEFB1 expression detected
in the benign prostatic tissue samples and hPrEC (FIG. 1A). Analysis of
the relative DEFB1 expression levels in the prostate cancer cell lines
revealed significantly lower levels in DU145, PC3 and LNCaP. As a further
point of reference, relative DEFB1 expression was measured in the
adjacent malignant section of prostatic tissue from patient #1215. There
were no significant differences in the level of DEFB1 expression observed
in the three prostate cancer lines compared to malignant prostatic tissue
from patient #1215 (FIG. 1B). In addition, expression levels in all four
samples were close to the no template negative controls which confirmed
little to no endogenous DEFB1 expression (data not shown). QRT-PCR was
also performed on the prostate cancer cell lines transfected with the
DEFB1 expression system. Following a 24 hour induction period, relative
expression levels were 0.01360 in DU145, 0.01503 in PC3 and 0.138 in
LNCaP. Amplification products were verified by gel electrophoresis.
[0178] QRT-PCR was performed on LCM tissues regions containing benign, PIN
and cancer. DEFB1 relative expression was 0.0146 in the benign region
compared to 0.0009 in the malignant region (FIG. 1C). This represents a
94% decrease which again demonstrates a significant down-regulation of
expression. Furthermore, analysis of PIN revealed that DEFB1 expression
level was 0.044 which was a 70% decrease. Comparing expression in patient
#1457 to the average expression level found in benign regions of six
other patients (FIG. 1A) revealed a ratio of 1.997 representing almost
twice as much expression (FIG. 1D). However, the expression ratio was
0.0595 in PIN and was 0.125 in malignant tissue compared to average
expression levels in benign tissue.
[0179] DEFB1 Causes Cell Membrane Permeability and Ruffling: Induction of
DEFB1 in the prostate cancer cell lines resulted in a significant
reduction in cell number in DU145 and PC3, but had no effect on cell
proliferation in LNCaP (FIG. 2). As a negative control, cell
proliferation was monitored in all three lines containing empty plasmid.
There were no observable changes in cell morphology in DU145, PC3 or
LNCaP cells following the addition of PonA. In addition, DEFB1 induction
resulted in morphological changes in both DU 145 and PC3. Here cells
appeared more rounded and exhibited membrane ruffling indicative of cell
death. Apoptotic bodies were also present in both lines.
[0180] Expression of DEFB1 Results in Decreased Cell Viability: The MTT
assay showed a reduction in cell viability by DEFB1 in PC3 and DU145
cells, but no significant effect on LNCaP cells (FIG. 3). After 24 hours,
relative cell viability was 72% in DU145 and 56% in PC3. Analysis 48
hours after induction revealed 49% cell viability in DU145 and 37% cell
viability in PC3. After 72 hours of DEFB1 expression resulted in 44% and
29% relative cell viability in DU145 and PC3 cells, respectively.
[0181] DEFB1 Causes Rapid Caspase-mediated Apoptosis in Late-stage
Prostate Cancer Cells: In order to determine whether the effects of DEFB1
on PC3 and DU145 were cytostatic or cytotoxic, FACS analysis was
performed. Under normal growth conditions, more than 90% of PC3 and DU145
cultures were viable and non-apoptotic (lower left quadrant) and did not
stain with annexin V or PI. After inducing DEFB1 expression in PC3 cells,
the number of apoptotic cells (lower and upper right quadrants) totaled
10% at 12 hours, 20% at 24 hours, and 44% at 48 hours (FIG. 4B). For
DU145 cells, the number of apoptotic cells totaled 12% after 12 hours,
34% at 24 hours, and 59% after 48 hours of induction (FIG. 4A). There was
no increase in apoptosis observed in cells containing empty plasmid
following induction with PonA (data not shown).
[0182] Caspase activity was determined by confocal laser microscopic
analysis (FIG. 5). DU145 and PC3 cell were induced for DEFB1 expression
and activity was monitored based on the binding of green fluorescing
FAM-VAD-FMK to caspases in cells actively undergoing apoptosis. Analysis
of cells under DIC showed the presence of viable control DU145 (panel A),
PC3 (panel E) and LNCaP (panel I) cells at 0 hours. Excitation by the
confocal laser at 488 nm produced no detectable green staining which
indicates no caspase activity in DU145 (panel B), PC3 (panel F) or LNCaP
(panel J). Following induction for 24 hours, DU145 (panel C), PC3 (panel
G) and LNCaP (panel K) cells were again visible under DIC. Confocal
analysis under fluorescence revealed green staining in DU145 (panel D)
and PC3 (panel H) cell indicating caspase activity. However, there was no
green staining in LNCaP (panel L), indicating no induction of apoptosis
by DEFB1.
[0183] In conclusion, this study provides the functional role of DEFB1 in
prostate cancer. Furthermore, these findings show that DEFB1 is part of
an innate immune system involved in tumor immunity. Data presented here
demonstrate that DEFB1 expressed at physiological levels is cytotoxic to
AR- hormone refractory prostate cancer cells, but not to AR+ hormone
sensitive prostate cancer cell nor to normal prostate epithelial cells.
Given that DEFB1 is constitutively expressed in normal prostate cells
without cytotoxicity, it may be that late-stage AR- prostate cancer cells
possess distinct phenotypic characteristics that render them sensitive to
DEFB1 cytotoxicity. Thus, DEFB1 is a viable therapeutic agent for the
treatment of late-stage prostate cancer, and potentially other cancers as
well.
EXAMPLE 2
SiRNA Mediated Knockdown of PAX2 Expression Results in Prostate Cancer
Cell Death Independent of P53 Status
[0184] This example examines the effects of inhibiting PAX2 expression by
RNA interference in prostate cancer cells which differ in p53 gene
status. The results demonstrate that the inhibition of PAX2 results in
cell death irrespective of p53 status, indicating that there are
additional tumor suppressor genes or cell death pathways inhibited by
PAX2 in prostate cancer.
Materials and Methods
[0185] siRNA Silencing of PAX2: In order to achieve efficient gene
silencing, a pool of four complementary short interfering ribonucleotides
(siRNAs) targeting human PAX2 mRNA (Accession No. NM.sub.--003989.1),
were synthesized (Dharmacon Research, Lafayette, Colo., USA). A second
pool of four siRNAs were used as an internal control to test for the
specificity of PAX2 siRNAs. Two of the sequences synthesized target the
GL2 luciferase mRNA (Accession No. X65324), and two were
non-sequence-specific (Table 3). For annealing of siRNAs, 35 M of single
strands were incubated in annealing buffer (100 mM potassium acetate, 30
mM HEPES-KOH at pH 7.4, 2 mM magnesium acetate) for 1 min at 90.degree.
C. followed by 1 h incubation at 37.degree. C.
TABLE-US-00006
TABLE 3
PAX2 siRNA Sequences.
A pool of four siRNA was utilized to inhibit PAX2 protein expression.
Sense (5'-3')
Sequence A 5'-GAAGUCAAGUCGAGUCUAUUU-3' SEQ ID NO: 7
Sequence B 5'-GAGGAAACGUGAUGAAGAUUU-3' SEQ ID NO: 8
Sequence C 5'-GGACAAGAUUGCUGAAUACUU-3' SEQ ID NO: 9
Sequence D 5'-CAUCAGAGCACAUCAAAUCUU-3' SEQ ID NO: 10
Antisense (5'-3')
Sequence A 5'-AUAGACUCGACUUGACUUCUU-3' SEQ ID NO: 3
Sequence B 5'-AUCUUCAUCACGUUUCCUCUU-3' SEQ ID NO: 4
Sequence C 5'-GUAUUCAGCAAUCUUGUCCUU-3' SEQ ID NO: 5
Sequence D 5'-GAUUUGAUGUGCUCUGAUGUU-3' SEQ ID NO: 6
[0186] Western Analysis: Briefly, cells were harvested by trypsinization
and washed twice with PBS. Lysis buffer was prepared according to the
manufacturer's instructions (Sigma), and was then added to the cells.
Following a 15 minute incubation period at 4.degree. C. on an orbital
shaker, cell lysate were then collected and centrifuged for 10 minutes at
12000.times.g to pellet cellular debris. The protein-containing
supernatant were then collected and quantitated. Next, 25 .mu.g protein
extract was loaded onto an 8-16% gradient SDS-PAGE (Novex). Following
electrophoresis, proteins were transferred to PVDF membranes, and then
blocked with 5% nonfat dry milk in TTBS (0.05% Tween 20 and 100mM
Tris-Cl) for 1 hour. Blots were then probed with rabbit anti-PAX2 primary
antibody (Zymed, San Francisco, Calif.) at a 1:2000 dilution. After
washing, the membranes were incubated with anti-rabbit antibody
conjugated to horseradish peroxidase (HRP) (dilution 1:5000; Sigma), and
signal detection was visualized using chemilluminescence reagents
(Pierce) on an Alpha Innotech Fluorchem 8900. As a control, blots were
stripped and reprobed with mouse anti-.beta.-actin primary antibody
(1:5000; Sigma-Aldrich) and HRP-conjugated anti-mouse secondary antibody
(1:5000; Sigma-Aldrich) and signal detection was again visualized.
[0187] Phase Contrast Microscopy: The effect of PAX2 knock-down on cell
growth was analyzed by phase contrast microscopy as described in Example
1.
[0188] MIT Cytotoxicity Assay: DU145, PC3 and LNCaP cells
(1.times.10.sup.5) were transfected with 0.5 .mu.g of the PAX2 siRNA pool
or control siRNA pool using Codebreaker transfection reagent according to
the manufacturer's protocol (Promega). Next, cell suspensions were
diluted and seeded onto a 96-well plate at 1-5.times.10.sup.3 cells per
well and allowed to grow for 2-, 4- or 6 days. After culture, cell
viability was determined by measuring the conversion of
3-[4,5-dimethylthiazol-2yl]-2,5 diphenyl tetrazolium bromide, MTT
(Promega), to a colored formazan product. Absorbance was read at 540 nm
on a scanning multiwell spectrophotometer.
[0189] Pan-Caspase Detection: Detection of caspase activity in the
prostate cancer cell lines was performed s described in Example 1.
[0190] Quantitative Real-time RT-PCR: Quantitative real-time RT-PCR was
performed as described in Example 1 in order to verify gene expression
after PAX2 siRNA treatment in PC3, DU145 and LNCaP cell lines. The primer
pairs for GAPDH (control gene), BAX, BID and BAD are:
TABLE-US-00007
Sense (5'-3')
GAPDH
5'-CCACCCATGGCAAATTCCATGGCA-3' SEQ ID NO: 55
BAD
5'-CTCAGGCCTATGCAAAAAGAGGA-3' SEQ ID NO: 57
BID
5'-AACCTACGCACCTACGTGAGGAG-3' SEQ ID NO: 59
BAX
5'-GACACCTGAGCTGACCTTGG-3' SEQ ID NO: 61
Antisense (5'-3')
GAPDH
5'-TCTAGACGGCAGGTCAGGTCAACC-3' SEQ ID NO: 56
BAD
5'-GCCCTCCCTCCAAAGGAGAC-3' SEQ ID NO: 58
BID
5'-CGTTCAGTCCATCCCATTTCTG-3' SEQ ID NO: 60
BAX
5'-GAGGAAGTCCAGTGTCCAGC-3' SEQ ID NO: 62
[0191] Reactions were performed in MicroAmp Optical 96-well Reaction Plate
(PE Biosystems). Forty cycles of PCR were performed under standard
conditions using an annealing temperature of 60.degree. C. Quantification
was determined by the cycle number where exponential amplification began
(threshold value) and averaged from the values obtained from the
triplicate repeats. There was an inverse relationship between message
level and threshold value. In addition, GAPDH was used as a housekeeping
gene to normalize the initial content of total cDNA. Gene expression was
calculated as the relative expression ratio between the pro-apoptotic
genes and GAPDH. All reactions were carried out in triplicate.
Results
[0192] siRNA Inhibition of PAX2 Protein: In order to confirm that the
siRNA effective targeted the PAX2 mRNA, Western Analysis was performed to
monitor PAX2 protein expression levels over a six day treatment period.
Cells were given a single round of transfection with the pool of PAX2
siRNA. The results confirmed specific targeting of PAX2 mRNA by showing
knock-down of PAX2 protein by day four in DU145 (FIG. 6, panel A) and by
day six in PC3 (FIG. 6, panel B).
[0193] Knock-down PAX2 inhibit Prostate Cancer Cell Growth: Cells were
analyzed following a six day treatment period with media only, negative
control non-specific siRNA or PAX2 siRNA (FIG. 7). DU145 (panel A), PC3
(panel D) and LNCaP (panel G) cells all reached at least 90% confluency
in the culture dishes containing media only. Treatment of DU145 (panel
B), PC3 (panel E) and LNCaP (panel H) with negative control non-specific
siRNA had no effect on cell growth, and cells again reached confluency
after six days. However, treatment with PAX2 siRNA resulted in a
significant decrease in cell number. DU145 cells were approximately 15%
confluent (panel C) and PC3 cells were only 10% confluent (panel F).
LNCaP cell were 5% confluent following siRNA treatment.
[0194] Cytotoxicity Assays: Cell viability was measured after two-, four-,
and six-day exposure times, and is expressed as a ratio of the 570-630 nm
absorbance of treated cells divided by that of the untreated control
cells (FIG. 8). Relative cell viability following 2 days of treatment was
77% in LNCaP, 82% in DU145 and 78% in PC3. After four days, relative cell
viability was 46% in LNCaP, 53% in DU145 and 63% in PC3. After six days
of treatment, relative cell viability decreased to 31% in LNCaP, 37% in
PC3, and was 53% in DU145. As negative controls, cell viability was
measured in after a six day treatment period with negative control
non-specific siRNA or transfection reagent alone. For both conditions,
there was no statistically significant change in cell viability compared
to normal growth media.
[0195] Pan-Caspase Detection: Caspase activity was detected by confocal
laser microscopic analysis. DU145, PC3 and LNCaP cells were treated with
PAX2 siRNA and activity was monitored based on the binding of FAM-labeled
peptide to caspases in cells actively undergoing apoptosis which will
fluoresce green. Analysis of cells with media only under DIC shows the
presence of viable DU145 (A), PC3 (E) and LNCaP (I) cells at 0 hours
(FIG. 9). Excitation by the confocal laser at 488 nm produced no
detectable green staining which indicates no caspase activity in
untreated DU145 (B), PC3 (F) or LNCaP (J). Following four days of
treatment with PAX2 siRNA, DU145 (C), PC3 (G) and LNCaP (K) cells were
again visible under DIC. Under fluorescence, the treated DU145 (D), PC3
(H) and LNCaP (L) cells presented green staining indicating caspase
activity.
[0196] Effect of PAX2 Inhibition on Pro-apoptotic Factors: DU145, PC3 and
LNCaP cells were treated with siRNA against PAX2 for six days and
expression of pro-apoptotic genes dependent and independent of p53
transcription regulation were measured to monitor cell death pathways.
For BAX, there was a 1.81-fold increase in LNCaP, a 2.73-fold increase in
DU145, and a 1.87-fold increase in PC3 (FIG. 10, panel A). Expression
levels of BID increased by 1.38-fold in LNCaP and 1.77-fold in DU145
(FIG. 10, panel B). However, BID expression levels decreased by 1.44-fold
in PC3 following treatment (FIG. 10, panel C). Analysis of BAD revealed a
2.0-fold increase in expression in LNCaP, a 1.38-fold increase in DU145,
and a 1.58-fold increase in PC3.
[0197] These results demonstrate dependency of prostate cancer cell
survival on PAX2 expression. Following p53 activation as a result of PAX2
knock-down in the p53-expressing cell line LNCaP, the p53-mutated line
DU145, and the p53-null line PC3, caspase activity was detected in all
three lines, indicating of the initiation of programmed cell death. BAX
expression was upregulated in all three cell lines independent of p53
status. The expression of pro-apoptotic factor BAD was also increased in
all three lines following PAX2 inhibition. Following treatment with PAX2
siRNA, BID expression was increased in LNCaP and DU145, but actually
decreased in PC3. These results indicate that cell death observed in
prostate cancer is influenced by but is not dependent on p53 expression.
The initiation of apoptosis in prostate cancer cells through different
cell death pathways irrespective of p53 status indicates that PAX2
inhibits other tumor suppressors.
EXAMPLE 3
Inhibition of PAX2 Oncogene Results in DEFB1-Mediated Death of Prostate
Cancer Cells
[0198] The identification of tumor-specific molecules that serve as
targets for the development of new cancer drugs is considered to be a
major goal in cancer research. Example 1 demonstrated that there is a
high frequency of DEFB1 expression loss in prostate cancer, and that
induction of DEFB1 expression results in rapid apoptosis in androgen
receptor negative-stage prostate cancer. These data show that DEFB1 plays
a role in prostate tumor suppression. In addition, given that it is a
naturally occurring component of the immune system of normal prostate
epithelium, DEFB1 is expected to be a viable therapeutic agent with
little to no side effects. Example 2 demonstrated that inhibition of PAX2
expression results in prostate cancer cell death independent of p53.
These data indicate that there is an addition pro-apoptotic factor or
tumor suppressor that is inhibited by PAX2. In addition, the data show
that the oncogenic factor PAX2, which is over-expressed in prostate
cancer, is a transcriptional repressor of DEFB1. The purpose of this
study is to determine if loss of DEFB1 expression is due to aberrant
expression of the PAX2 oncogene, and whether inhibiting PAX2 results in
expression of DEFB1 and DEFB1-mediated cell death (FIG. 11).
Materials and Methods
[0199] RNA Isolation and Quantitative RT-PCR: RNA isolation and
quantitative RT-PCR of DEFB1 were performed as described in Example 1.
[0200] Generation of the DEFB1 Reporter Construct: The pGL3 luciferase
reporter plasmid was used to monitor DEFB1 reporter activity. Here, a
region 160 bases upstream of the DEFB1 transcription initiation site and
included the DEFB1 TATA box. The region also included the CCTTG (SEQ ID
NO: 1) sequence which is necessary for PAX2 binding. The PCR primers were
designed to contain KpnI and NheI restriction sites. The DEFB1 promoter
PCR products were restriction digested Kpn I and NheI and ligated into a
similary restriction digested pGL3 plasmid (FIG. 12). The constructs were
transfected into E. coli and individual clones were selected and
expanded. Plasmids were isolated and sequence integrity of the DEFB1/pGL3
construct was verified by automated sequencing.
[0201] Luciferase Reporter Assay: Here, 1 .mu.g of the DEFB1 reporter
construct or the control pGL3 plasmid was transfected into
1.times.10.sup.6 DU145 cells. Next, 0.5.times.103 cells were seeded onto
each well of a 96-well plate and allowed to grow overnight. Then fresh
medium was added containing PAX2 siRNA or media only and the cells were
incubated for 48 hours. Luciferase was detected by the BrightGlo kit
according to the manufacturer's protocol (Promega) and the plates were
read on a Veritas automated 96-well luminometer. Promoter activity was
expressed as relative luminescence.
[0202] Analysis of Membrane Permeability: Acridine orange (AO)/ethidium
bromide (EtBr) dual staining was performed to identify changes in cell
membrane integrity, as well as apoptotic cells by staining the condensed
chromatin. AO stains viable cells as well as early apoptotic cells,
whereas EtBr stains late stage apoptotic cells that have lost membrane
permeability. Briefly, cells were seeded into 2 chamber culture slides
(BD Falcon, USA). Cells transfected with empty pIND plasmid/pvgRXR or
pIND DEFB1/pvgRXR were induced for 24 or 48 h with media containing 10
.mu.M Ponasterone A. Control cells were provided fresh media at 24 and
48h. In order to determine the effect of PAX2 inhibition on membrane
integrity, separate culture slides containing DU145, PC3 and LNCaP were
treated with PAX2 siRNA and incubated for 4 days. Following this, cells
were washed once with PBS and stained with 2 ml of a mixture (1:1) of AO
(Sigma, USA) and EtBr (Promega, USA) (5 ug/ml) solution for 5 min.
Following staining, the cells were again washed with PBS. Fluorescence
was viewed by a Zeiss LSM 5 Pascal Vario 2 Laser Scanning Confocal
Microscope (Carl Zeiss Jena, Germany). The excitation color wheel contain
BS505-530 (green) and LP560 (red) filter blocks which allowed for the
separation of emitted green light from AO into the green channel and red
light from EtBr into the red channel. The laser power output and gain
control settings within each individual experiment were identical between
control and DEFB1 induced cells. The excitation was provided by a Kr/Ar
mixed gas laser at wavelengths of 543 nm for AO and 488 nm for EtBr.
Slides were analyzed under 40.times. magnification and digital images
were stored as uncompressed TIFF files and exported into Photoshop CS
software (Adobe Systems, San Jose, Calif.) for image processing and hard
copy presentation.
[0203] ChIP Analysis of PAX2: Chromatin immunoprecipitation (ChIP) allows
the identification of binding sites for DNA-binding proteins based upon
in vivo occupancy of a promoter by a transcription factor and enrichment
of transcription factor bound chromatin by immunoprecipitation. A
modification of the protocol described by the Farnham laboratory was
used; also on line at http://mcardle.oncology.wisc.edu/farnham/). The
DU145 and PC3 cell lines over-expresses the PAX2 protein, but does not
express DEFB1. Cells were incubated with PBS containing 1.0% formaldehyde
for 10 minutes to crosslink proteins to DNA. Samples were then sonicated
to yield DNA with an average length of 600 bp. Sonicated chromatin
precleared with Protein A Dynabeads was incubated with PAX2-specific
antibody or "no antibody" control [isotype-matched control antibodies].
Washed immunoprecipitates were then collected. After reversal of the
crosslinks, DNA was analyzed by PCR using promoter-specific primers to
determine whether DEFB1 is represented in the PAX2-immunoprecipitated
samples. Primers were designed to amplify the 160 bp region immediately
upstream of the DEFB1 mRNA start site which contained the DEFB1 TATA box
and the functional CCTTG (SEQ ID NO: 1) PAX2 recognition site. For these
studies, positive controls included PCR of an aliquot of the input
chromatin (prior to immunoprecipitation, but crosslinks reversed). All
steps were performed in the presence of protease inhibitors.
Results
[0204] siRNA Inhibition of PAX2 Increases DEFB1 Expression: QRT-PCR
analysis of DEFB1 expression before siRNA treatment revealed relative
expression levels of 0.00097 in DU145, 0.00001 in PC3, and 0.00004 LNCaP
(FIG. 13). Following siRNA knock-down of PAX2, relative expression was
0.03294 (338-fold increase) in DU145, 0.00020 (22.2-fold increase) in PC3
and 0.00019 (4.92-fold increase) in LNCaP. As a negative control, the
human prostate epithelial cell line (hPrEC) which is PAX2 null, revealed
expression levels at 0.00687 before treatment and 0.00661 following siRNA
treatment confirming no statistical change in DEFB1 expression.
[0205] siRNA Inhibition of PAX2 Increases DEFB1 Promoter Activity: FIG. 14
shows that inhibition of PAX2 results in increased DEFB1 promoter
activity. PC3 promoter/pGL3 and DU145 promoter/pGL3 construct were
generated and were transfected into PC3 and DU145 cells, respectively.
Promoter activity was compared before and after PAX2 inhibition by siRNA
treatment. DEFB1 promoter activity increased 2.65-fold in DU145 and 3.78
fold in PC3 following treatment.
[0206] DEFB1 Causes Cell Membrane Permeability: Membrane integrity was
monitored by confocal analysis. As shown in FIG. 15, intact cells stain
green due to AO which is membrane permeable. In addition, cells with
compromised plasma membranes would stain red by EtBr which is membrane
impermeable. Here, uninduced DU145 (A) and PC3 (D) cells stained
positively with AO and emitted green color, but did not stain with EtBr.
However, DEFB1 induction in both DU145 (B) and PC3 (E) resulted in the
accumulation of EtBr in the cytoplasm at 24 hours indicated by the red
staining. By 48 hours, DU145 (C) and PC3 (F) possessed condensed nuclei
and appeared yellow, which was due to the presence of both green and red
staining resulting from the accumulation of AO and EtBr, respectively.
[0207] Inhibition of PAX2 Results in Membrane Permeability: Cells were
treated with PAX2 siRNA for 4 days and membrane integrity was monitored
again by confocal analysis. As shown in FIG. 16, both DU145 and PC3
possessed condensed nuclei and appeared yellow. However, LNCaP cells'
cytoplasm and nuclei remained green following siRNA treatment. Also red
staining at the cell periphery indicates the maintenance of cell membrane
integrity. These findings indicate that the inhibition of PAX2 results in
specifically DEFB1-mediated cell death in DU1145 and PC3, but not LNCaP
cells. Death observed in LNCaP is due to the transactivation of the
existing wild-type p53 in LNCap following PAX2 inhibition.
[0208] PAX2 Binds to the DEFB1 Promoter: ChIP analysis was performed on
DU145 and PC3 cells to determine if the PAX2 transcriptional repressor is
bound to the DEFB1 promoter (FIG. 17). Lane 1 contains a 100 by molecular
weight marker. Lane 2 is a positive control representing 160 by region of
the DEFB1 promoter amplified from DU145 before cross-linking and
immunoprecipitation. Lane 3 is a negative control representing PCR
performed without DNA. Lanes 4 and 5 are negative controls representing
PCR from immunoprecipitations performed with IgG from cross-linked DU145
and PC3, respectively. PCR amplification of 25 pg of DNA (lane 6 and 8)
and 50 pg of DNA (lane 7 and 9) immunoprecitipated with anti-PAX2
antibody after crosslinking show 160 by promoter fragment in DU145 and
PC3, respectively.
[0209] FIG. 18 shows predicted structure of the PrdPD and PrdHD with DNA.
The coordinates of the structures of the PrdPD bound to DNA (Xu et al.,
1995) and the PrdHD bound to DNA (Wilson et al., 1995) were used to
construct a model of the two domains as they bound to a PHO site. The
individual binding sites are abutted next to each other with a specific
orientation as indicated. The RED domain is oriented based on the PrdPD
crystal structure.
[0210] FIG. 19 shows comparison of consensus sequences of different paired
domains. At the top of the Figure is drawn a schematic representation of
protein.+-.DNA contacts described in the crystallographic analysis of the
Prd-paired-domain.+-.DNA complex. Empty boxes indicate a-helices, shaded
boxes indicates b-sheets and a thick line indicate a b-turn. Contacting
amino acids are shown by single-letter code. Only direct amino
acid.+-.base contacts are shown. Empty circles indicate major groove
contacts while red arrows indicate minor groove contacts. This scheme is
aligned to all known consensus sequences for paired-domain proteins (top
strands only are shown). Vertical lines between consensus sequences
indicate conserved base-pairs. Numbering of the positions is shown at the
bottom of the Figure.
[0211] These results demonstrate that the oncogenic factor PAX2 suppresses
DEFB1 expression. The suppression occurs at the transcriptional level.
Furthermore, computational analysis of the DEFB1 promoter revealed the
presence of a CCTTG (SEQ ID NO: 1) DNA binding site for the PAX2
transcriptional repressor next to the DEFB1 TATA box (FIG. 1). One of the
hallmarks of defensin cytotoxicity is the disruption of membrane
integrity. These results show that ectopic expression of DEFB1 in
prostate cancer cells results in a loss of membrane potential due to
compromised cell membranes. The same phenomenon is observed after
inhibiting PAX2 protein expression. Therefore, suppression of PAX2
expression or function, results in the re-establishment of DEFB1
expression and subsequently DEFB1-mediated cell death. Also, the present
results establish the utility of DEFB1 as a directed therapy for prostate
cancer treatment, and potentially other cancer treatments, through innate
immunity.
EXAMPLE 4
Effect of DEFB1 Expression in Implanted Tumor Cells
[0212] The anti-tumoral ability of DEFB1 is evaluated by injecting tumor
cells that overexpress DEFB1 into nude mice. DEFB1 is cloned into
pBI-EGFP vector, which has a bidirectional tetracycline responsible
promoter. Tet-Off Cell lines are generated by transfecting pTet-Off into
DU145, PC3 and LNCaP cells and selecting with G418. The pBI-EGFP-DEFB1
plasmid is co-transfected with pTK-Hyg into the Tet-off cell lines and
selected with hygromycin. Only single-cell suspensions with a viability
of >90% are used. Each animal receives approximately 500,000 cells
administered subcutaneously into the right flank of female nude mice.
There are two groups, a control group injected with vector only clones
and a group injected with the DEFB1 over-expressing clones. 35 mice are
in each group as determined by a statistician. Animals are weighed twice
weekly, tumor growth monitored by calipers and tumor volumes determined
using the following formula: volume=0.5.times.(width)2.times.length. All
animals are sacrificed by CO2 overdose when tumor size reaches 2 mm3 or 6
months following implantation; tumors are excised, weighed and stored in
neutral buffered formalin for pathological examination. Differences in
tumor growth between the groups are descriptively characterized through
summary statistics and graphical displays. Statistical significance is
evaluated with either the t-test or non-parametric equivalent.
EXAMPLE 5
Effect of PAX2 siRNA on Implanted Tumor Cells
[0213] Hairpin PAX2 siRNA template oligonucleotides utilized in the in
vitro studies are utilized to examine the effect of the up-regulation of
DEFB1 expression in vivo. The sense and antisense strand (see Table 3)
are annealed and cloned into pSilencer 2.1 U6 hygro siRNA expression
vector (Ambion) under the control of the human U6 RNA pol III promoter.
The cloned plasmid is sequenced, verified and transfected into PC3,
Du145, and LNCap cell lines. Scrambled shRNA is cloned and used as a
negative control in this study. Hygromycin resistant colonies are
selected, cells are introduced into the mice subcutaneously and tumor
growth is monitored as described above.
EXAMPLE 6
Effect of Small Molecule Inhibitors of PAX2 Binding on Implanted Tumor
Cells
[0214] The DNA recognition sequence for PAX2 binding resides in the DEFB1
promoter between nucleotides -75 and -71 (+1 refers to the
transcriptional start site). Short oligonucleotides complementary to the
PAX2 DNA-binding domain are provided. Examples of such oligonucleotides
include the 20-mer and 40-mer oligonucleotides containing the CCTTG (SEQ
ID NO: 1) recognition sequence provided below. These lengths were
randomly selected, and other lengths are expected to be effective as
blockers of binding. As a negative control, oligonicleotides with a
scrambled sequence (CTCTG)-(SEQ ID NO: 22) were designed to verify
specificity. The oligonucleotides are transfected into the prostate
cancer cells and the HPrEC cells with lipofectamine reagent or
Codebreaker transfection reagent (Promega, Inc). In order to confirm
DNA-protein interactions, double stranded oligonucleotides will be
labeled with [.sup.32P] dCTP and electrophoretic mobility shift assays
are performed. In addition, DEFB1 expression is monitored by QRT-PCR and
Western analysis following treatment with oligonucleotides. Finally, cell
death is detected by MTT assay and flow cytometry as previously
described.
TABLE-US-00008
Recognition Sequence #1:
(SEQ ID NO: 18)
CTCCCTTCAGTTCCGTCGAC
Recognition Sequence #2:
(SEQ ID NO: 19)
CTCCCTTCACCTTGGTCGAC
Scramble Sequence #1:
(SEQ ID NO: 23)
CTCCCTTCACTCTGGTCGAC
Recognition Sequence #3:
(SEQ ID NO: 20)
ACTGTGGCACCTCCCTTCAGTTCCGTCGACGAGGTTGTGC
Recognition Sequence #4:
(SEQ ID NO: 21)
ACTGTGGCACCTCCCTTCACCTTGGTCGACGAGGTTGTGC
Scramble Sequence #2:
(SEQ ID NO: 24)
ACTGTGGCACCTCCCTTCACTCTGGTCGACGAGGTTGTGC
Further examples of oligonucleotides of the
invention include:
Recognition Sequence #1:
(SEQ ID NO: 25)
5'-AGAAGTTCACCCTTGACTGT-3'
Recognition Sequence #2:
(SEQ ID NO: 26)
5'-AGAAGTTCACGTTCCACTGT-3'
Scramble Sequence #1:
(SEQ ID NO: 27)
5'-AGAAGTTCACGCTCTACTGT-3'
Recognition Sequence #3:
(SEQ ID NO: 28)
5'-TTAGCGATTAGAAGTTCACCCTTGACTGTGGCACCTCCC-3'
Recognition Sequence #4:
(SEQ ID NO: 29)
5'-GTTAGCGATTAGAAGTTCACGTTCCACTGTGGCACCTCCC-3'
Scramble Sequence #2:
(SEQ ID NO: 30)
5'-GTTAGCGATTAGAAGTTCACGCTCTACTGTGGCACCTCCC-3'
[0215] This set of alternative inhibitory oligonucleotides represents the
recognition sequence for the PAX2 binding domain and homeobox. These
include actual sequences from the DEFB1 promoter.
[0216] The PAX2 gene is required for the growth and survival of various
cancer cells including prostate. In addition, the inhibition of PAX2
expression results in cell death mediated by the innate immunity
component DEFB1. Suppression of DEFB1 expression and activity is
accomplished by binding of the PAX2 protein to a CCTTG (SEQ ID NO: 1)
recognition site in the DEFB1 promoter. Therefore, this pathway provides
a viable therapeutic target for the treatment of prostate cancer. In this
method, the sequences bind to the PAX2 DNA binding site and block PAX2
binding to the DEFB1 promoter thus allowing DEFB1 expression and
activity. The oligonucleotide sequences and experiment described above
are examples of and demonstrate a model for the design of additional PAX2
inhibitor drugs.
[0217] Given that the CCTTG (SEQ ID NO: 1) sequence exists in
interleukin-3, interleukin-4, the insulin receptor and others, PAX2
regulates their expression and activity as well. Therefore the PAX2
inhibitors disclosed herein have utility in a number of other diseases
including those directed related to inflammation including prostatitis
and benign prostatic hypertrophy (BPH).
EXAMPLE 7
Loss of DEFB1 Expression Results in Increased Tumor
[0218] Generation of Loss of Function Mice: The Cre/loxP system has been
useful in elucidating the molecular mechanisms underlying prostate
carcinogenesis. Here a DEFB1 Cre conditional KO is used for inducible
disruption within the prostate. The DEFB1 Cre conditional KO involves the
generation of a targeting vector containing loxP sites flanking DEFB1
coding exons, targeted ES cells with this vector and the generation of
germline chimeric mice from these targeted ES cells. Heterozygotes are
mated to prostate-specific Cre transgenics and heterozygous intercross is
used to generate prostate-specific DEFB1 KO mice. Four genotoxic chemical
compounds have been found to induce prostate carcinomas in rodents:
N-methyl-N-nitrosourea (MNU), N-nitrosobis 2-oxopropyl amine (BOP),
3,2X-dimethyl-4-amino-biphenyl (MAB) and
2-amino-1-methyl-6-phenylimidazow 4,5-oxpyridine (PhIP). DEFB1-transgenic
mice are treated with these carcinogenic compounds via intra-gastric
administration or i.v. injection for prostate adenoma and adenocarcinoma
induction studies. Prostate samples are studied for differences in tumor
growth and changes gene expression though histological,
immunohistological, mRNA and protein analyses.
[0219] Generation of GOF mice: For PAX2 inducible GOF mice, PAX2 GOF
(bi-transgenic) and wild-type (mono-transgenic) littermates are
administered doxycycline (Dox) from 5 weeks of age to induce
prostate-specific PAX2 expression. Briefly, PROBASIN-rtTA mono-transgenic
mice (prostate cell-specific expression of tet-dependent rtTA inducer)
are crossed to our PAX2 transgenic responder lines. For induction,
bi-transgenic mice are fed Dox via the drinking water (500 mg/L freshly
prepared twice a week). Initial experiments verify low background levels,
good inducibility and cell-type specific expression of PAX2 and the EGFP
reporter using transgenic founder line in bi-transgenic mice. Regarding
experimental group sizes, 5-7 age- and sex-matched individuals in each
group (wild-type and GOF) allow for statistical significance. For all
animals in this study, prostate tissues are collected initially at weekly
intervals for analysis and comparison, to determine carcinogenic time
parameters.
[0220] PCR Genotyping, RT-PCR and qPCR: PROBASIN-rtTA transgenic mice are
genotyped using the following PCR primers and conditions:
TABLE-US-00009
PROBASIN5 (forward)
5'-ACTGCCCATTGCCCAAACAC-3'; (SEQ ID NO: 31)
RTTA3 (reverse)
5'-AAAATCTTGCCAGCTTTCCCC-3'; (SEQ ID NO: 32)
95.degree. C. denaturation for 5 min, followed by 30 cycles of 95.degree.
C. for 30 sec, 57.degree. C. for 30 sec, 72.degree. C. for 30 sec,
followed by a 5 min extension at 72.degree. C., yielding a 600 bp
product. PAX2 inducible transgenic mice are genotyped using the following
PCR primers and conditions: PAX2For 5'-GTCGGTTACGGAGCGGACCGGAG-3' (SEQ ID
NO: 33); Rev5'IRES 5'-TAACATATAGACAAACGCACACCG-3' (SEQ ID NO: 34);
95.degree. C. denaturation for 5 min, followed by 34 cycles of 95.degree.
C. for 30 sec, 63.degree. C. for 30 sec, 72.degree. C. for 30 sec,
followed by a 5 min extension at 72.degree. C., yielding a 460 bp
product.
[0221] Immortomouse hemizygotes are be genotyped using the following PCR
primers and conditions: Immol1, 5'-GCGCTTGTGTC GCCATTGTATTC-3' (SEQ ID
NO: 35); Immol2,5'-GTCACACCACAGAAGTAAGGTTCC-3' (SEQ ID NO: 36);
94.degree. C. 30 sec, 58.degree. C. 1 min, 72.degree. C. 1 min 30 sec, 30
cycles to yield a .about.1 kb transgene band. For genotyping PAX2
knockout mice, the following PCR primers and conditions are used: PAX2
For 5'-GTCGGTTACGGAGCGGACCGGAG-3' (SEQ ID NO: 37); PAX2Rev
5'-CACAGAGCATTGGCGATCTCGATGC-3' (SEQ ID NO: 38); 94.degree. C. 1 min,
65.degree. C. 1 min, 72.degree. C. 30 sec, 36 cycles to yield a 280 bp
band.
[0222] DEFB1 Peptide Animal Studies: Six-week-old male athymic (nude) mice
purchased from Charles River Laboratories are injected sub-cutaneously
over the scapula with 10.sup.6 viable PC3 cells. One week after
injection, the animals are randomly allocated to one of three
groups--group I: control; group II: intraperitoneal injections of DEFB1,
100 .mu.g/day, 5 days a week, for weeks 2-14; group III: intraperitoneal
injections of DEFB1, 100 mg/day, 5 days a week, for weeks 8-14. Animals
are maintained in sterile housing, four animals to a cage, and observed
on a daily basis. At 10-day intervals, the tumors are measured by using
calipers, and the volumes of the tumors are calculated by using
V=(L.times.W2)/2.
EXAMPLE 8
Targeting PAX2 Expression for the Chemoprevention of Intraepithelial
Neoplasia and Cancer
[0223] Cancer chemoprevention is defined as the prevention of cancer or
treatment at the pre-cancer state or even earlier. The long period of
progression to invasive cancer is a major scientific opportunity but also
an economic obstacle to showing the clinical benefit of candidate
chemopreventive drugs. Therefore, an important component of
chemopreventive agent development research in recent years has been to
identify earlier (than cancer) end points or biomarkers that accurately
predict an agent's clinical benefit or cancer incidence-reducing effect.
In many cancers, IEN is an early end point such as in prostate cancer.
Given that the PAX2/DEFB1 pathway is deregulated during IEN and perhaps
at even an earlier histopathological state makes it a powerful predictive
biomarker and an excellent target for chemoprevention of cancer. Shown
are a number of compounds that suppress PAX2 and increases DEFB1
expression that may have utility as chemoprevention agents for prostate
cancer.
[0224] As shown in Table 1, the PAX2 gene is expressed in a number of
cancers. In addition, several cancers have been shown to have aberrant
PAX2 expression (FIG. 20). Angiotensin II (AngII) is a major regulator of
blood pressure and cardiovascular homeostasis and is recognized as a
potent mitogen. AngII mediates its biological effects through binding to
two subtypes of receptors, Angiotensin Type I receptor (AT1R) and
Angiotensin Type II receptor (AT2R) which belong to the super-family of
G-protein-coupled receptors but have different tissue distribution and
intracellular signaling pathways. In addition to its effects on blood
pressure, AngII has been shown to play a role in various pathological
situations involving tissue remodeling, such as wound healing, cardiac
hypertrophy and development. In fact, recent studies have revealed local
expression of several components of the Renin-Angiotensin System (RAS) in
various cancer cells and tissues including the prostate. Upregulation of
AT1R provides a considerable advantage to cancer cells that have learn to
evade apoptosis and growth regulatory elements. To date a number of
cancers have been shown to aberrantly express PAX2. Chemoprevention via
target PAX2 expression may have a significant impact on cancer related
deaths
Materials and Methods
[0225] Cell Culture: The cell lines DU145, LnCap and PC3 were cultured as
described in Example 1. The hPrEC cells were cultured in prostate
epithelium basal media (Cambrex Bio Science, Inc., Walkersville, Md.) and
maintained at 37.degree. C. and 5% CO2.
[0226] Reagents and Treatments: Cells were treated with 5 or 10 uM of
AngII, 5 uM of the ATR1 antagonist Los, 5 uM of the ATR2 antagonist
PD123319, 25 uM of the MEK inhibitor U0126, 20 uM of the MEK/ERK
inhibitor PD98059 or 250 .mu.M of the AMP kinase inducer AICAR.
[0227] Western Analysis: Western blot was performed as described in
Example 2. Blots were then probed with primary antibody (anti-PAX2,
-phospho-PAX2, -JNK, -phospho-JNK, -ERK1/2, or -phospho-ERK1/2) (Zymed,
San Francisco, Calif.) at 1:1000-2000 dilutions. After washing, the
membranes were incubated with anti-rabbit antibody conjugated to
horseradish peroxidase (HRP) (dilution 1:5000; Sigma), and signal
detection was visualized using chemilluminescence reagents (Pierce) on an
Alpha Innotech Fluorchem 8900. As a control, blots were stripped and
re-probed with mouse anti-.beta.-actin primary antibody (1:5000;
Sigma-Aldrich) and HRP-conjugated anti-mouse secondary antibody (1:5000;
Sigma-Aldrich), and signal detection was again visualized.
[0228] QRT-PCR Analysis: Quantitative real-time RT-PCR was performed as
described in Example 1 to verify changes in gene expression following
PAX2 knockdown in PC3 and DU 145 prostate cancer cell lines and the hPrEC
normal prostate epithelial cells. Forty cycles of PCR were performed
under standard conditions using an annealing temperature of 60.degree. C.
Quantification was determined by the cycle number where exponential
amplification began (threshold value) and averaged from the values
obtained from the triplicate repeats. There was an inverse relationship
between message level and threshold value. In addition, GAPDH was used as
a housekeeping gene to normalize the initial content of total cDNA.
Relative expression was calculated as the ratio between each genes and
GAPDH. All reactions were carried out in triplicate.
[0229] Thymidine Incorporation: Proliferation of cells was determined by
[.sup.3H] thymidine ribotide ([3H] TdR) incorporation into DNA.
0.5.times.106 cells/well of suspension DU145 cells were plated in their
appropriate media. Cells were incubated for 72 h with or without the
presence of AngII at the indicated concentrations. Cells were exposed to
37 kBq/ml [methyl-3H] thymidine in the same medium for 6 h. The adherent
cells were fixed by 5% trichloroacetic acid and lysed in SDS/NaOH lysis
buffer overnight. Radioactivity was measured by Beckman LS3801 liquid
scintillation counter (Canada). Suspension cell culture was harvested by
cell harvester (Packard instrument Co., Meriden, Conn.), and
radioactivity was measured by 1450 microbeta liquid scintillation counter
(PerkinElmer Life Sciences).
Results
[0230] To investigate the effect of AngII on PAX2 expression in DU145
prostate cancer cells, PAX2 expression was examined following treatment
with AngII over a 30 min to 48 hour period. As shown in FIG. 21, PAX2
expression progressively increased over time following AngII treatment.
Blocking RAS signaling by treating DU145 with Los significantly reduced
PAX2 expression. Here, PAX2 expression was 37% after 48 hours and was 50%
after 72 hours of Los treatment compared to untreated control DU145 cells
(FIG. 22A). It is known that the AT2R receptor oppose the action of the
AT 1 R. Therefore, the effect of blocking the AT2R receptor on PAX2
expression was examined. Treatment of DU145 with the AT2R blocker
PD123319 resulted in a 7-fold increase in PAX2 expression after 48 hours
and an 8-fold increase after 96 hours of treatment (FIG. 22B).
Collectively, these findings demonstrate that PAX2 expression is
regulated by the ATR1 receptor.
[0231] It is known that AngII directly affects the proliferation of
prostate cancer cells through AT1R-mediated activation of MAPK and STAT3
phosphorylation. Treatment of DU145 with AngII resulted in a two- to
three-fold increase in proliferation rate (FIG. 23). However, treatment
with Los decreased proliferated rates by 50%. In addition, blocking the
AT1R receptor by pre-treating with Los for 30 min suppressed the effect
of AngII on proliferation.
[0232] To further examine the role of the AT1R signaling in the regulation
of PAX2 expression and activation, the effect of blocking various
components of the MAP kinase signaling pathway on PAX2 expression was
examined. Here, DU145 cells treated with the MEK inhibitor U0126 resulted
in a significant reduction of PAX2 expression (FIG. 24). Furthermore,
treatment with MEK/ERK inhibitor PD98059 also resulted in decreased PAX2.
Treatment of DU145 cells with Los had no effect on ERK protein levels,
but reduced the amount of phospho-ERK (FIG. 25A). However, treatment of
DU145 with Los resulted in a significant reduction of PAX2 expression.
Similar results were observed with U0126 and PD98059 (FIG. 25B). It is
also known that PAX2 expression is regulated by STAT3 which is a
down-stream target of ERK. Treatment of DU145 with Los, U0126, and
PD98059 reduced phospho-STAT3 protein levels (FIG. 25C). These results
demonstrate that PAX2 is regulated via AT1R in prostate cancer cells.
[0233] In addition, the effect of AT1R signaling on PAX2 activation by JNK
was examined. Treatment of DU145 with Los, U0126, and PD98059 all
resulted in a significant decrease or suppression of phospho-PAX2 protein
levels (FIG. 26A). However, Los and U0126 did not decrease phospho-JNK
protein levels (FIG. 26B). Therefore, the decrease in phospho-PAX2
appears to be due to decreased PAX2 levels, but not decreased
phosphorylation.
[0234] 5-Aminoimidazole-4-carboxamide-1-.beta.-4-ribofuranoside (AICAR) is
widely used as an AMP-kinase activator, which regulates energy
homeostasis and response to metabolic stress. Recent reports have
indicated anti-proliferative and pro-apoptotic action of activated AMPK
using pharmacological agents or AMPK overexpression. AMPK activation has
been shown to induce apoptosis in human gastric cancer cells, lung cancer
cells, prostate cancer, pancreatic cells, and hepatic carcinoma cells and
enhance oxidative stress induced apoptosis in mouse neuroblastoma cells,
by various mechanisms that include inhibition of fatty acid synthase
pathway and induction of stress kinases and caspase 3. In addition,
treatment of PC3 prostate cancer cells increased expression of p21, p27,
and p53 proteins and inhibition of PI3K-Akt pathway. All of these
pathways are directly or indirectly regulated by PAX2. Treatment of
prostate cancer cells with AICAR resulted in the suppression of PAX2
pression expression (FIG. 25B) as well as its activated form
phosphor-PAX2 (FIG. 26A). In addition, phospho-STAT3 which regulated PAX2
expression was also suppressed (FIG. 25C).
[0235] Finally, it was hypothesized that aberrant RAS signaling which
leads to upregulation and overexpression of PAX2 suppresses the
expression of the DEFB1 tumor suppressor gene. To investigate this, the
normal prostate epithelial primary culture hPrEC was treated with AngII
and examined both PAX2 and DEFB1 expression levels. An inverse
relationship between DEFB1 and PAX2 expression was discovered in normal
prostate cells versus prostate cancer cells. As shown in FIG. 27,
untreated hPrEC exhibited 10% relative PAX2 expression compared to
expression in PC3 prostate cancer cells. Conversely, untreated PAX2
exhibited only 2% relative DEFB1 expression compared to expression in
hPrEC. Following 72 hours of treatment with 10 uM of AngII, there was a
35% decrease in DEFB1 expression compared to untreated hPrEC, and by 96
hours there was a 50% decrease in DEFB1 expression compared to untreated
hPrEC cells. However, there was 66% increase in PAX2 expression at 72
hours, and by 96 hours there was a 79% increase in PAX2 expression
compared to untreated hPrEC cells. Furthermore, the increase in PAX2
expression in hPrEC after 72 hours was 77% of PAX2 levels observed in PC3
prostate cancer cells. After 96 hours of AngII treatment PAX2 expression
was 89% of PAX2 expression in PC3. These results demonstrate that
deregulated RAS signaling suppresses DEFB1 expression via the
upregulation of PAX2 expression in prostate cells.
[0236] Inhibition of apoptosis is a critical pathophysiological factor
that contributes to the development of cancer. Despite significant
advances in cancer therapeutics, little progress has been made in the
treatment of advanced disease. Given that carcinogenesis is a multiyear,
multistep, multipath disease of progression, chemoprevention through the
use of drug or other agents to inhibit, delay, or reverse this process
has been recognized as a very promising area of cancer research.
Successful drug treatment for the chemoprevention of prostate cancer
requires the use of therapeutics with specific effects on target cells
while maintaining minimal clinical effects on the host with the overall
goal of suppressing cancer development. Therefore, understanding the
mechanisms in early stage carcinogenesis is critical in determining the
efficacy of a specific treatment. The significance of aberrant PAX2
expression and its abrogation of apoptosis, with subsequent contribution
to tumor formation, suggest that it may be a suitable target for prostate
cancer treatment. PAX2 was regulated by the AT1R in prostate cancer (FIG.
28). In this, deregulated RAS signaling resulted in increased PAX2
oncogene expression, and a decrease in the expression of DEFB1 tumor
suppressor. Therefore, the use of AT1R antagonists decreases PAX2
expression and results in increased prostate cancer cell death via
re-expression of DEFB1 (FIG. 29). These results offer a novel finding
that targeting PAX2 expression via the Renin-Angiotensin signaling
pathway, the AMP Kinase pathway, or other methods involving the
inactivation of the PAX2 protein (i.e. anti-PAX2 antibody vaccination)
may be a viable target for cancer prevention (Table 4).
TABLE-US-00010
TABLE 4
Compounds Utilized to Inhibit PAX2 Expression for Chemoprevention
NAME Drug Class
Drug 1 Losartan Angiotensin Type 1 Receptor blocker
Drug 2 PD123319 Angiotensin Type 2 Receptor blocker
Drug 3 U0126 MEK inhibitor
Drug 4 PD98059 MEK/ERK inhibitor
Drug 5 AICAR AMP kinase inducer
Target Drug Function
Drug A Anti-PAX2 Antibody PAX2 Vaccine
Drug B Angiotensinogen Renin-AngII pathway inhibitor
Drug C Angiotensin Converting Renin-AngII pathway inhibitor
Enzyme
[0237] This study demonstrates that the upregulation of the PAX2 oncogene
in prostate cancer is due to deregulated RAS signaling. PAX2 expression
is regulated by the ERK 1/2 signaling pathway which is mediated by the
Angiotensin type I receptor. In addition, blocking the AT1R with Losartan
(Los) suppresses PAX2 expression. In addition, AICAR which is an AMPK
activator has also shown promise as a potential PAX2 inhibitor.
Collectively, these studies strongly implicate these classes of drugs as
potential suppressors of PAX2 expression and may ultimately serve as
novels chemoprevention agents.
EXAMPLE 9
PAX2-DEEB1 Expression Level as a Grading Tool for Prostate Tissue and
Predictor of Prostate Cancer Development
Materials and Methods
[0238] QRT-PCR Analysis: Prostate sections were collected from patients
that underwent radical prostatectomies. Following pathological
examination, laser capture microdisection was performed to isolate areas
of Normal, Proliferative Intraepithelial Neoplasia (PIN) and Cancerous
tissue. QRT-PCR was performed as previously described to assess
expression. DEFB land PAX2 expression in each region and GAPDH was used
as an internal control.
[0239] Blood collection and RNA isolation: For QRT-PCR, blood (2.5 ml)
from each individual was colleted into a PAXgene.TM. Blood RNA tube
(QIAGEN) following the manufacturer's protocol. Whole blood was
thoroughly mixed with PAXgene stabilization reagent and stored at room
temperature for 6 hours prior to RNA extraction. Total RNA was then
extracted using the PAXgene.TM. Blood RNA kit according to the
manufacturer's directions (QIAGEN). In order to remove contaminating
genomic DNA, total RNA samples absorbed to the PAXgene.TM. Blood RNA
System spin column was incubated with DNase I (QIAGEN) at 25.degree. C.
for 20 min to remove genomic DNA. Total RNA was eluted, quantitated, and
QRT-PCR is performed as previously mentioned to compare PAX2 and DEFB1
expression ratios.
Results
[0240] QRT-PCR analysis of LCM normal tissue demonstrated that patients
with relative DEFB1 expression levels greater than 0.005 have a lower
Gleason Score compared to those with expression levels lower than 0.005
(FIG. 30). Thus, there is an inverse relationship between DEFB1
expression and Gleason score. Conversely, there was a positive
correlation between PAX2 expression and Gleason score in malignant
prostate tissue and PIN (FIG. 30, panel B).
[0241] The PAX2 and DEFB1 expression levels in normal, PIN and cancerous
tissues from separate patients were calculated and compared (FIGS. 31A
and 31B). Overall, PAX2 expression levels relative to GAPDH internal
control ranged between 0 and 0.2 in normal (benign) tissue, 0.2 and 0.3
in PIN, and between 0.3 and 0.5 in cancerous (malignant) tissue (FIG.
32). For DEFB1 there was an inverse relationship compared to PAX2. Here,
DEFB1 expression levels relative to GAPDH internal control ranged between
0.06 and 0.005 in normal (benign) tissue, 0.005 and 0.003 in PIN, and
between 0.003 and 0.001 in cancerous (malignant) tissue. Therefore,
disclosed is a predictive scale, designted as Donld Predictive Factor
(DPF), which utilizes the PAX2-DEFB1 expression ratio as a prognosticator
of benign, precancerous (PIN) and malignant prostate tissue. Tissues with
PAX2-DEFB1 ratios between 0 and 39 based on the DPF will represent normal
(pathologically benign). Tissue with a PAX2-DEFB1 ratio between 40 and 99
will represent PIN (pre-cancerous) based on the DPF scale. Finally,
tissue with a PAX2-DEFB1 ratio between 100 and 500 will be malignant (low
to high grade cancer).
[0242] There currently is a critical need for predictive biomarkers for
prostate cancer development. It is known that the onset of prostate
cancer occurs long before the disease is detectable by current screening
methods such as the PSA test or the digital rectal exam. It is thought
that a reliable test which could monitor the progression and early onset
of prostate cancer would greatly reduce the mortality rate through more
effective disease management. Disclosed herein is a predictive index to
allow physicians to know well in advance the pathological state of the
prostate. The DPF measures the decrease in the PAX2-DEFB1 expression
ratio associated with prostate disease progression. This powerful measure
can not only predict the likelihood of a patient developing prostate
cancer, but also may pinpoint the early onset of pre-malignant cancer.
Ultimately, this tool can allow physicians to segregate which patients
have more aggressive disease from those which do not.
[0243] The identification of cancer-specific markers has been utilized to
help identify circulating tumor cells (CTCs). There is also emerging
evidence which demonstrates that detection of tumor cells disseminated in
peripheral blood can provide clinically important data for tumor staging,
prognostication, and identification of surrogate markers for early
assessment of the effectiveness of adjuvant therapy. Furthermore, by
comparing gene expression profiling of all circulating cells, one can
examine the expression of the DEFB1 and PAX2 genes which play a role in
"immunosurveillance" and "cancer survival", respectively as a
prognosticator for the early detection of prostate cancer.
EXAMPLE 10
Functional Analysis of the Host Defense Peptide Human Beta Defensin-1:
New Insight Into its Potential Role in Cancer
Materials and Methods
[0244] Cell culture: The prostate cancer cell lines were cultured as
described in Example 1. The hPrEC primary culture was obtained from
Cambrex Bio Science, Inc. (Walkersville, Md.) and cells were grown in
prostate epithelium basal media.
[0245] Tissue samples and laser capture microdissection: Prostate tissues
were obtained from patients who provided informed consent prior to
undergoing radical prostatectomy. Samples were acquired through the
Hollings Cancer Center tumor bank in accordance with an Institutional
Review Board-approved protocol. This included guidelines for the
processing, sectioning, histological characterization, RNA purification
and PCR amplification of samples. Prostate specimens received from the
surgeons and pathologists were immediately frozen in OCT compound. Each
OCT block was cut to produce serial sections which were stained and
examined. Areas containing benign cells, prostatic intraepithelial
neoplasia (PIN), and cancer were identified and used to guide our
selection of regions from unstained slides using the Arcturus PixCell II
System (Sunnyvale, Calif.). Caps containing captured material were
exposed to 20 .mu.l of lysate from the Arcturus Pico Pure RNA Isolation
Kit and processed immediately. RNA quantity and quality was evaluated
using sets of primers that produce 5' amplicons. The sets include those
for the ribosomal protein L32 (the 3' amplicon and the 5' amplicon are
298 bases apart), for the glucose phosphate isomerase (391 bases apart),
and for the glucose phosphate isomerase (842 bases apart). Ratios of 0.95
to 0.80 were routinely obtained for these primer sets using samples from
a variety of prepared tissues. Additional tumor and normal samples were
grossly dissected by pathologists, snap frozen in liquid nitrogen and
evaluated for hBD-1 and cMYC expression.
[0246] Cloning of hBD-1 gene: hBD-1 cDNA was generated from RNA by reverse
transcription-PCR using primers generated from the published hBD-1
sequence (accession no. U50930) (Ganz, 2004). The PCR primers were
designed to contain ClaI and KpnI restriction sites. hBD-1 PCR products
were restriction digested with ClaI and KpnI and ligated into a TA
cloning vector. The TA/hBD1 vector was then transfected into the XL-1
Blue strain of E. coli by heat shock and individual clones were selected
and expanded. Plasmids were isolated by Cell Culture DNA Midiprep
(Qiagen, Valencia, Calif.) and sequence integrity verified by automated
sequencing. The hBD-1 gene fragment was then ligated into the pTRE2
digested with ClaI and KpnI, which served as an intermediate vector for
orientation purposes. The pTRE2/hBD-1 construct was digested with ApaI
and KpnI to excise the hBD-1 insert. The insert was ligated into pIND
vector of the Ecdysone Inducible Expression System (Invitrogen, Carlsbad,
Calif.) also double digested with ApaI and KpnI. The construct was
transfected into E. coli and individual clones were selected and
expanded. Plasmids were isolated and sequence integrity of pIND/hBD-1 was
again verified by automated sequencing.
[0247] Transfection: Cells (1.times.10.sup.6) were seeded onto 100-mm
Petri dishes and grown overnight. Next, the cells were co-transfected
using Lipofectamine 2000 (Invitrogen) with 1 .mu.g of pvgRXR plasmid,
which expresses the heterodimeric ecdysone receptor, and 1 .mu.g of the
pIND/hBD-1 vector construct or pIND/.beta.-galactosidase (.beta.-gal)
control vector in Opti-MEM media (Life Technologies, Inc.). Transfection
efficiency was determined by inducing .beta.-gal expression with
Ponasterone A (PonA) and staining cells with a .beta.-galactosidase
detection kit (Invitrogen). Assessment of transfection efficiency by
counting positive staining (blue) colonies which demonstrated that 60-85%
of cells expressed .beta.-galactosidase for the cell lines.
[0248] Immunocytochemistry: In order to verify hBD-1 protein expression,
DU145 and hPrEC cells were seeded onto 2-chamber culture slides (BD
Falcon, USA) at 1.5-2.times.10.sup.4 cells per chamber. DU145 cells
transfected with pvgRXR alone (control) or with the hBD-1 plasmid were
induced for 18 h with media containing 10 .mu.M Pon A, while
untransfected cells received fresh growth media. Following induction,
cells were washed in 1.times. PBS and fixed for 1 h at room temperature
with 4% paraformaldehyde. Cells were then washed six times with 1.times.
PBS and blocked in 1.times. PBS supplemented with 2% BSA, 0.8% normal
goat serum (Vector Laboratories, Inc., Burlingame, Calif.) and 0.4%
Triton-X 100 for 1 h at room temperature. Next, cells were incubated
overnight in primary rabbit anti-human BD-1 polyclonal antibody
(PeproTech Inc., Rocky Hill, N.J.) diluted 1:1000 in blocking solution.
Following this, cells were washed six times with blocking solution and
incubated for 1 h at room temperature in Alexa Fluor 488 goat anti-rabbit
IgG (H+L) secondary antibody at a dilution of 1:1000 in blocking
solution. After washing cells with blocking solution six times,
coverslips were mounted with Gel Mount (Biomeda, Foster City, Calif.).
Finally, cells were viewed under differential interference contrast (DIC)
and under laser excitation at 488 nm. The fluorescent signal was analyzed
by confocal microscopy (Zeiss LSM 5 Pascal) using a 63.times. DIC oil
lens with a Vario 2 RGB Laser Scanning Module. The digital images were
exported into Photoshop CS Software (Adobe Systems) for image processing
and hard copy presentation.
[0249] RNA isolation and quantitative RT-PCR: QRT-PCR was performed as
previously described (Gibson et al., 2007). Briefly, total RNA (0.5 .mu.g
per reaction) from tissue sections were reverse transcribed into cDNA
utilizing random primers (Promega). Two-step QRT-PCR was performed on
cDNA generated using the MultiScribe Reverse Transcriptase from the
TaqMan Reverse Transcription System and the SYBR Green PCR Master Mix
(Applied Biosystems, Foster City, Calif.). The primer pairs for hBD-1 and
c-MYC were generated from the published sequences (Table 5). Forty cycles
of PCR were performed under standard conditions using an annealing
temperature of 56.4.degree. C. for hBD-1 and c-MYC and 55.degree. C. for
PAX2. In addition, .beta.-actin (Table 5) was amplified as a housekeeping
gene to normalize the initial content of total cDNA. Gene expression in
benign prostate tissue samples was calculated as the expression ratio
compared to .beta.-actin. Levels of hBD-1 expression in malignant
prostate tissue, hPREC prostate primary culture, and prostate cancer cell
lines before and after induction were calculated relative to the average
level of hBD-1 expression in hPrEC cells. As a negative control, QRT-PCR
reactions without cDNA template were also performed. All reactions were
run a minimum of three times.
TABLE-US-00011
TABLE 5
Sequences of QRT-PCR primers
Sense (5'-3') Antisense (5'-3')
.beta.- CCTGGCACCCAGCACAAT GCCGATCCACACGGAGTACT
Actin (SEQ ID NO: 51) (SEQ ID NO: 52)
hBD-1 TCAGCAGTGGAGGGCAATG CCTCTGTAACAGGTGCCTTGAAT
(SEQ ID NO: 65) (SEQ ID NO: 66)
cMYC ACAGCAAACCTCCTCACAGCC TGGAGACGTGGCACCTCTTG
(SEQ ID NO: 67) (SEQ ID NO: 68)
[0250] MTT cell viability assay: To examine the effects of hBD-1 on cell
growth, metabolic 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl tetrazolium
bromide (MTT) assay was performed. DU145, LNCaP, PC3 and PC3/AR+ cells
co-transfected with pvgRXR plasmid and pIND/hBD-1 construct or control
pvgRXR plasmid were seeded onto a 96-well plate at 1-5.times.10.sup.3
cells per well. Twenty-four hours after seeding, fresh growth medium was
added containing 10 .mu.M. Pon A daily to induce hBD-1 expression for 24,
48 and 72 h after which the MTT assay was performed according to the
manufacturer's instructions (Promega). Reactions were performed three
times in triplicate.
[0251] Analysis of membrane integrity: Acridine orange (AO)/ethidium
bromide (EtBr) dual staining was performed to identify changes in cell
membrane integrity, as well as apoptotic cells by staining the condensed
chromatin. AO stains viable cells and early apoptotic cells, whereas EtBr
stains late stage apoptotic cells that have compromised membranes.
Briefly, PC3, DU145 and LNCaP cells were seeded into 2-chamber culture
slides (BD Falcon). Cells transfected with empty plasmid or hBD-1 plasmid
were induced for 24 or 48 h with media containing 10 .mu.M Pon A, while
control cells received fresh growth media at each time point. After
induction, cells were washed once with PBS and stained with 2 ml of a
mixture (1:1) of AO (Sigma, St. Louis, Mo.) and EtBr (Promega) (5
.mu.g/ml) solution for 5 min and were again washed with PBS.
[0252] Fluorescence was viewed by a Zeiss LSM 5 Pascal Vario 2 Laser
Scanning Confocal Microscope (Carl Zeiss). The excitation color wheel
contains BS505-530 (green) and LP560 (red) filter blocks which allowed
for the separation of emitted green light from AO into the green channel
and red light from EtBr into the red channel. The laser power output and
gain control settings within each individual experiment were identical
between control and hBD-1 induced cells. The excitation was provided by a
Kr/Ar mixed gas laser at wavelengths of 543 nm for AO and 488 nm for
EtBr. Slides were analyzed under 40.times. magnification and digital
images were stored as uncompressed TIFF files and exported into Photoshop
CS software (Adobe Systems) for image processing and hard copy
presentation.
[0253] Flow cytometry: PC3 and DU145 cells transfected with the hBD-1
expression system were grown in 60-mm dishes and induced for 12, 24, and
48 h with 10 .mu.M Pon A. The cells were harvested and analyzed by flow
cytometry as described in Example 1.
[0254] Caspase detection: Detection of caspase activity in the prostate
cancer cell lines was performed described in Example 1.
[0255] siRNA silencing of PAX2: SiRNA knock-down and verification was
performed as described in Example 2.
Results
[0256] hBD-1 expression in prostate tissue: 82% of prostate cancer frozen
tissue sections analyzed exhibited little or no expression of hBD-1
(Donald et al., 2003). To compare hBD-1 expression levels, QRTPCR
analysis was performed on normal prostate tissue obtained by gross
dissection or LCM of normal prostate tissue adjacent to malignant regions
which were randomly chosen. Here, hBD-1 was detected in all of the gross
dissected normal clinical samples with a range of expression that
represents approximately a 6.6-fold difference in expression levels (FIG.
33A). LCM captured normal tissue samples expressed hBD-1 at levels in a
range that represents a 32-fold difference in expression (FIG. 33B).
Matching sample numbers to corresponding patient profiles revealed that
in most cases, the hBD-1 expression level was higher in patient samples
with a Gleason score of 6 than in patient samples with a Gleason score of
7. In addition, a comparison of hBD-1 expression levels in tissue
obtained by gross dissection and LCM from the same patient, #1343,
demonstrated an 854-fold difference in expression between the two
isolation techniques. Therefore, these results indicate that LCM provides
a more sensitive technique to assess hBD-1 expression in prostate tissue.
[0257] hBD-1 expression in prostate cell lines: To verify upregulation of
hBD-1 in the prostate cancer cell lines after transfection with the hBD-1
expression system, QRTPCR was performed. In addition, no template
negative controls were also performed, and amplification products were
verified by gel electrophoresis. Here, hBD-1 expression was significantly
lower in the prostate cancer cell lines compared to hPrEC cells.
Following a 24 h induction period, relative expression levels of hBD-1
significantly increased in DU145, PC3 and LNCaP as compared to the cell
lines prior to hBD-1 induction (FIG. 34A).
[0258] Next, protein expression of hBD-1 in was verified DU145 cells
transfected with the hBD-1 expression system after induction with Pon A
by immunocytochemistry. As a positive control, hBD-1 expressing hPrEC
prostate epithelial cells were also examined. Cells were stained with
primary antibody against hBD-1 and protein expression was monitored based
on the green fluorescence of the secondary antibody (FIG. 34B). Analysis
of cells under DIC verify the presence of hPrEC cells and DU145 cells
induced for hBD-1 expression at 18 h. Excitation by the confocal laser at
488 nm produced revealed green fluorescence indicating the presence of
hBD-1 protein in hPrEC as a positive control. However, there was no
detectable green fluorescence in control DU145 cells and empty plasmid
induced DU145 cells demonstrating no hBD-1 expression. Confocal analysis
of DU145 cells induced for hBD-1 expression revealed green fluorescence
indicating the presence of hBD-1 protein following induction with Pon A.
[0259] Expression of hBD-1 results in decreased cell viability: MTT assay
was performed to assess the effect of hBD-1 expression on relative cell
viability in DU145, PC3, PC3/AR+ and LNCaP prostate cancer cell lines.
MTT analysis with empty vector exhibited no statistical significant
change in cell viability. Twenty-four hours following hBD-1 induction,
relative cell viability was 72% in DU145 and 56% in PC3 cells, and after
48 h cell viability was reduced to 49% in DU145 and 37% in PC3 cells
(FIG. 35). Following 72 h of hBD-1 induction, relative cell viability
decreased further to 44% in DU145 and 29% PC3 cells. Conversely, there
was no significant effect on the viability of LNCaP cells. In order to
assess whether the resistance to hBD-1 cytotoxicity observed in LNCaP was
due to the presence of the androgen receptor (AR), the hBD-1 cytotoxicity
in PC3 cells was examined with ectopic AR expression (PC3/AR+). Here,
there was no difference between PC3/AR+ and PC3 cells. Therefore, the
data indicates that hBD-1 is cytotoxic specifically to late-stage
prostate cancer cells.
[0260] In order to determine whether the effects of hBD-1 on PC3 and DU145
were cytostatic or cytotoxic, FACS analysis was performed to measure cell
death. Under normal growth conditions, more than 90% of PC3 and DU145
cultures were viable and non-apoptotic (lower left quadrant) and did not
stain with annexin V or PI. After inducing hBD-1 expression in PC3 cells,
the number of cells undergoing early apoptosis and late
apoptosis/necrosis (lower and upper right quadrants, respectively)
totaled 10% at 12 h, 20% at 24 h, and 44% at 48 h (FIG. 4B). For DU145
cells, the number of cells undergoing early apoptosis and late
apoptosis/necrosis totaled 12% after 12 h, 34% at 24 h, and 59% after 48
h of induction (FIG. 4A). No increase in apoptosis was observed in cells
containing empty plasmid following induction with Pon A. Annexin V and
propidium iodide uptake studies have demonstrated that hBD-1 has
cytotoxic activity against DU 145 and PC3 prostate cancer cells and
results indicate apoptosis as a mechanism of cell death.
[0261] hBD-1 causes alterations in membrane integrity and caspase
activation: It was investigated whether the cell death observed in
prostate cancer cells after hBD-1 induction is caspase-mediated
apoptosis. To better understand the cellular mechanisms involved in hBD-1
expression, confocal laser microscopic analysis was performed (FIG. 5) on
DU145 and LNCaP cells induced for hBD-1 expression. Pan-caspase
activation was monitored based on the binding and cleavage of green
fluorescing FAM-VAD-FMK to caspases in cells actively undergoing
apoptosis. Analysis of cells under DIC showed the presence of viable
control DU145 (panel A) and LNCaP (panel E) cells at 0 h. Excitation by
the confocal laser at 488 nm produced no detectable green staining which
indicates no caspase activity in DU145 (panel B) or LNCaP (panel F)
control cells. Following induction for 24 h, DU145 (panel C) and LNCaP
(panel G) cells were again visible under DIC. Confocal analysis under
fluorescence revealed green staining in DU145 (panel D) cells indicating
pan-caspase activity after the induction of hBD-1 expression. However,
there was no green staining in LNCaP (panel H) cells induced for hBD-1
expression. Therefore, cell death observed following induction of hBD-1
is caspase-mediated apoptosis.
[0262] The proposed mechanism of antimicrobial activity of defensin
peptides is the disruption of the microbial membrane due to pore
formation (Papo and Shai, 2005). In order to determine if hBD-1
expression altered membrane integrity EtBr uptake was examined by
confocal analysis. Intact cells were stained green due to AO which is
membrane permeable, while only cells with compromised plasma membranes
stained red due to incorporation of membrane impermeable EtBr. Control
DU145 and PC3 cells stained positively with AO and emitted green color,
but did not stain with EtBr. However, hBD-1 induction in both DU145 and
PC3 resulted in the accumulation of EtBr in the cytoplasm at 24 as
indicated by the red staining. By 48 h, DU145 and PC3 possessed condensed
nuclei and appeared yellow due to the colocalization of green and red
staining from AO and EtBr, respectively. Conversely, there were no
observable alterations to membrane integrity in LNCaP cells after 48 h of
induction as indicated by positive green fluorescence with AO, but lack
of red EtBr fluorescence. This finding indicates that alterations to
membrane integrity and permeabilization in response to hBD-1 expression
differ between early- and late-stage prostate cancer cells.
[0263] Comparison of hBD-1 and cMYC expression levels: QRT-PCR analysis
was performed on LCM prostate tissue sections from three patients (FIG.
34). In patient #1457, hBD-1 expression exhibited a 2.7-fold decrease
from normal to PIN, a 3.5-fold decrease from PIN to tumor and a 9.3-fold
decrease from normal to tumor (FIG. 36A). Likewise, cMYC expression
followed a similar expression pattern in patient #1457 where expression
decreased by 1.7-fold from normal to PIN, 1.7-fold from PIN to tumor and
2.8-fold from normal to tumor (FIG. 36B). In addition, there was a
statistically significant decrease in cMYC expression in the other two
patients. Patient #1569 had a 2.3-fold decrease from normal to PIN, while
in patient #1586 there was a 1.8-fold decrease from normal to PIN, a
4.3-fold decrease from PIN to tumor and a 7.9-fold decrease from normal
to tumor.
[0264] Induction of hBD-1 expression following PAX2 inhibition: To further
examine the role of PAX2 in regulating hBD-1 expression, siRNA was
utilized to knockdown PAX2 expression and QRT-PCR performed to monitor
hBD-1 expression. Treatment of hPrEC cells with PAX2 siRNA exhibited no
effect on hBD-1 expression (FIG. 37). However, PAX2 knockdown resulted in
a 42-fold increase in LNCaP, a 37-fold increase in PC3 and a 1026-fold
increase in DU145 expression of hBD-1 compared to untreated cells. As a
negative control, cells were treated with non-specific siRNA which had no
significant effect on hBD-1 expression.
EXAMPLE 11
Inhibition of PAX2 Expression Results in Alternate Cell Death Pathways in
Prostate Cancer Cells Differeing in P53 Status
Materials and Methods
[0265] Cell lines: The cancer cell lines PC3, DU145 and LNCaP, which all
differ in p53 mutational status (Table 6), were cultured as described in
Example 1. The prostate epithelial cell line HPrEC was obtained from
Cambrex Bio Science, Inc., (Walkersville, Md.) and were cultured in
prostate epithelium basal media. Cells were maintained at 37.degree. C.
in 5% CO2.
TABLE-US-00012
TABLE 6
p53 gene mutation in prostate cancer cell lines
Nucleotide Amino acid
change change Gene status Reference
CCT-CTT Pro-Leu Gain/loss- Tapper et al. 2005;
of-function Bodhoven et al. 2003
GTT-TTT Val-Phe
Deleted a C, Frame-shift No activity Isaacs et al. 1991
GCC-GC
No deletion, -- Normal Carroll et al. 1993
wild-type function
[0266] siRNA silencing of PAX2: siRNA silencing of PAX2 was performed s
described in Example 2.
[0267] Western analysis: Western blot was performed as described in
Example 2. Blots were then probed with rabbit anti-PAX2 primary antibody
(Zymed, San Francisco, Calif.) at a 1:1000 dilution. After washing, the
membranes were incubated with anti-rabbit antibody conjugated to
horseradish peroxidase (HRP) (dilution 1:5000; Sigma), and signal
detection was visualized using chemiluminescence reagents (Pierce) on an
Alpha Innotech Fluorchem 8900. As a control, blots were stripped and
reprobed with mouse anti-.beta.-actin primary antibody (1:5000;
Sigma-Aldrich) and HRP-conjugated anti-mouse secondary antibody (1:5000;
Sigma-Aldrich), and signal detection was again visualized.
[0268] Phase contrast microscopy: The effect of PAX2 knockdown on cell
number was analyzed by phase contrast microscopy as described in Example
1.
[0269] MIT cytotoxicity assay: MTT cytotoxicity assay was performed as
described in Example 1.
[0270] Pan-caspase detection: Detection of caspase activity in the
prostate cancer cell lines was performed as described in Example 1.
[0271] Quantitative real-time RT-PCR: To verify changes in gene expression
following PAX2 knockdown in PC3, DU145 and LNCaP cell lines, quantitative
real-time RT-PCR was performed as described in Example 1. The primer
pairs for BAX, BID, BCL-2, AKT and BAD were generated from the published
sequences (Table 7). Reactions were performed in MicroAmp Optical 96-well
Reaction Plate (PE Biosystems). Forty cycles of PCR were performed under
standard conditions using an annealing temperature of 60.degree. C.
Quantification was determined by the cycle number where exponential
amplification began (threshold value) and averaged from the values
obtained from the triplicate repeats. There was an inverse relationship
between message level and threshold value. In addition, GAPDH was used as
a housekeeping gene to normalize the initial content of total cDNA.
Relative expression was calculated as the ratio between each genes and
GAPDH. All reactions were carried out in triplicate.
TABLE-US-00013
TABLE 10
Quantitative RT-PCR primers
Sense (5'-3') Antisense (5'-3')
GAPDH CCACCCATGGCAAATTCCATGGCA TCTAGACGGCAGGTCAGGTCAACC
(SEQ ID NO: 55) (SEQ ID NO: 56)
BAD CTCAGGCCTATGCAAAAAGAGGA GCCCTCCCTCCAAAGGAGAC
(SEQ ID NO: 57) (SEQ ID NO: 58)
BID AACCTACGCACCTACGTGAGGAG CGTTCAGTCCATCCCATTTCTG
(SEQ ID NO: 59) (SEQ ID NO: 60)
BAX GACACCTGAGCTGACCTTGG GAGGAAGTCCAGTGTCCAGC
(SEQ ID NO: 61) (SEQ ID NO: 62)
BCL-2 TATGATACCCGGGAGATCGTGATC GTGCAGATGCCGGTTCAGGTACTC
(SEQ ID NO: 69) (SEQ ID NO: 70)
AKT TCAGCCCTGGACTACCTGCA GAGGTCCCGGTACACCACGT
(SEQ ID NO: 71) (SEQ ID NO: 72)
Membrane permeability assay: Membrane permeability assay was performed s
described in Example 3.
Results
[0272] Analysis of PAX2 protein expression in prostate cells: PAX2 protein
expression was examined by Western analysis in HPrEC prostate primary
culture and in LNCaP, DU145 and PC3 prostate cancer cell lines. Here,
PAX2 protein was detected in all of the prostate cancer cell lines (FIG.
38A). However, no PAX2 protein was detectable in HPrEC. Blots were
stripped and re-probed for .beta.-actin as internal control to ensure
equal loading. PAX2 protein expression was also monitored after selective
targeting and inhibition by PAX2 specific siRNA in DU145, PC3 and LNCaP
prostate cancer cell lines. Cells were given a single round of
transfection with the pool of PAX2 siRNA over a 6-day treatment period.
PAX2 protein was expressed in control cells treated with media only.
Specific targeting of PAX2 mRNA was confirmed by observing knockdown of
PAX2 protein in all three cell lines (FIG. 38B).
[0273] Effect of PAX2 knockdown on prostate cancer cell growth: The effect
of PAX2 siRNA on cell number and cell viability was analyzed using light
microscopy and MTT analysis. To examine the effect of PAX2 siRNA on cell
number, PC3, DU145 and LNCaP cell lines were transfected with media only,
non-specific siRNA or PAX2 siRNA over a period of 6 days. Each of the
cell lines reached a confluency of 80-90% in 60 mm culture dishes
containing media only. Treatment of HPrEC, DU145, PC3 and LNCaP cells
with non-specific siRNA appeared to have little to no effect on cell
growth compared to cell treated with media only (FIG. 39, panels A, C and
E, respectively). Treatment of the PAX2-null cell line HPrEC with PAX2
siRNA appeared to have no significant effect on cell growth (FIG. 39,
panel B). However, treatment of the prostate cancer cell lines DU145, PC3
and LNCaP with PAX2 siRNA resulted in a significant decrease in cell
number (FIG. 39, panels D, F and H, respectively).
[0274] Effect of PAX2 knockdown on prostate cancer cell viability: Cell
viability was measured after 2-, 4-, and 6-day exposure times. Percent
viability was calculated as the ratio of the 570-630 nm absorbance of
cell treated with PAX2 siRNA divided by untreated control cells. As
negative controls, cell viability was measured after each treatment
period with negative control non-specific siRNA or transfection with
reagent alone. Relative cell viability was calculated by dividing percent
viability following PAX2 siRNA treatment by percent viability following
treatment with non-specific siRNA (FIG. 40). After 2 days of treatment,
relative viability was 116% in DU145, 81% in PC3 and 98% in LNCaP. After
4 days of treatment, relative cell viability decreased to 69% in DU145,
79% in PC3, and 80% in LNCaP. Finally, by 6 days relative viability was
63% in DU145, 43% in PC3 and 44% in LNCaP. In addition, cell viability
was also measured following treatment with transfection reagent alone.
Here, each cell line exhibited no significant decrease in cell viability.
[0275] Detection of pan-caspase activity: Caspase activity was detected by
confocal laser microscopic analysis. LNCaP, DU145 and PC3 cells were
treated with PAX2 siRNA and activity was monitored based on the binding
of FAM-labeled peptide to caspases in cells actively undergoing apoptosis
which will fluoresce green. Analysis of cells with media only shows the
presence of viable LNCaP, DU145 and PC3 cells, respectively. Excitation
by the confocal laser at 488 nm produced no detectable green staining
which indicates no caspase activity in the untreated cells (FIG. 41,
panels A, C and E, respectively). Following 4 days of treatment with PAX2
siRNA, LNCaP, DU145 and PC3 cells under fluorescence presented green
staining indicating caspase activity (FIG. 41, panels B, D and F,
respectively).
[0276] Effect of PAX2 inhibition on apoptotic factors: LNCaP, DU145 and
PC3 cells were treated with siRNA against PAX2 for 4 days and expression
of both pro- and anti-apoptotic factors were measured by QRTPCR.
Following PAX2 knockdown, analysis of BAD revealed a 2-fold in LNCaP,
1.58-fold in DU145 and 1.375 in PC3 (FIG. 42A). Expression levels of BID
increased by 1.38-fold in LNCaP and a 1.78-fold increase in DU145, but
there was no statistically significant difference in BID observed in PC3
after suppressing PAX2 expression (FIG. 42B). Analysis of the
anti-apoptotic factor AKT revealed a 1.25-fold decrease in expression in
LNCaP and a 1.28-fold decrease in DU145 following treatment, but no
change was observed in PC3 (FIG. 42C).
[0277] Analysis of membrane integrity and necrosis: Membrane integrity was
monitored by confocal analysis in LNCaP, DU145 and PC3 cells. Here,
intact cells stained green due to AO which is membrane permeable, while
cells with compromised plasma membranes would stained red due to
incorporation of membrane impermeable EtBr into the cytoplasm, and yellow
due to co-localization of AO and EtBr in the nuclei. Untreated LNCaP,
DU145 and PC3 cells stained positively with AO and emitted green color,
but did not stain with EtBr. Following PAX2 knockdown, there were no
observable alterations to membrane integrity in LNCaP cells as indicated
by positive green fluorescence with AO and absence of red EtBr
fluorescence. These finding further indicate that LNCaP cells can be
undergoing apoptotic, but not necrotic cell death following PAX2
knockdown. Conversely, PAX2 knockdown in DU145 and PC3 resulted in the
accumulation of EtBr in the cytoplasm as indicated by the red staining.
In addition, both DU145 and PC3 possessed condensed nuclei which appeared
yellow due to the co-localization of green and red staining from AO and
EtBr, respectively. These results indicate that DU145 and PC3 are
undergoing an alternate cell death pathway involving necrotic cell death
compared to LNCaP.
EXAMPLE 12
PAX2 and DEFB-1 Expression in Breast Cancer Cell Lines and Mammary
Tissues with Ductal or Lobular Intraepithelial Neoplasia
[0278] PAX2 and DEFB-1 expression will be determined in breast biopsy
samples of ductal or lobular intraepithelial neoplasia, and in the
following breast cancer cell lines:
[0279] BT-20: Isolated from a primary invasive ductal carcinoma; cell
express E-cadherin, ER, EGFR and uPA.
[0280] BT-474: Isolated from a primary invasive ductal carcinoma; cell
express E-cadherin, ER, PR, and have amplified HER2/neu.
[0281] Hs578T: Isolated from a primary invasive ductal carcinoma; a cell
line was also established from normal adjacent tissue, termed Hs578Bst.
[0282] MCF-7: Established from a pleural effusion. The cells express ER
and are the most common example of estrogen-responsive breast cancer
cells.
[0283] MDA-MB-231: Established from a pleural effusion. The cells are
ER-negative, E-cadherin negative and highly invasive in in vitro assays.
[0284] MDA-MB-361: Established from a brain metastasis. The cells express
ER, PR, EGFR and HER2/neu.
[0285] MDA-MB-435: Established from a pleural effusion. The cells are
ER-negative, E-cadherin negative, and are highly invasive and metastatic
in immunodeficient mice.
[0286] MDA-MB-468: Established from a pleural effusion. The cells have
amplified EGFR and are ER-negative.
[0287] SK-BR-3: Established from a pleural effusion. The cells have
amplified HER/2neu, express EGFR and are ER-negative.
[0288] T-47D: established from a pleural effusion. The cells retain
expression of E-cadherin, ER and PR.
[0289] ZR-75-1: Established from ascites fluid. The cells express ER,
E-cadherin, HER2/neu and VEGF.
[0290] The PAX2-to-DEFB-1 expression ratio will be determined using the
methods described in Example 9.
EXAMPLE 13
Expression of DEFB1 in Breast Cancer Cells
[0291] DEFB1 will be expressed in breast cancer cells using methods
described in Example 1. The cell viability and caspase activity will be
determined as described in Example 1.
EXAMPLE 14
Inhibition of PAX2 Expression in Breast Cancer Cells
[0292] PAX2 expression in breast cancer cells will be inhibited using the
siRNA described in Example 2. The expression levels of pro-apoptotic
genes such as BAX, BID and BAD, the cell viability and caspase activity
will be determined as described in Example 2.
EXAMPLE 15
Effect of DEFB1 Expression on Tumor Growth In Vivo
[0293] The anti-tumoral ability of DEFB1 will be evaluated by injecting
breast cancer cells that overexpress DEFB1 into nude mice. Breast cancer
cells will be transfected with an expression vector carrying the DEFB1
gene. Cells expressing the exogenous DEFB1 gene will be selected and
cloned. Only single-cell suspensions with a viability of >90% are
used. Each animal receives approximately 500,000 cells administered
subcutaneously into the right flank of female nude mice. There are two
groups, a control group injected with vector only clones and a group
injected with the DEFB1 over-expressing clones. 35 mice are in each group
as determined by a statistician. Animals are weighed twice weekly, tumor
growth monitored by calipers and tumor volumes determined using the
following formula: volume=0.5.times.(width)2.times.length. All animals
are sacrificed by CO2 overdose when tumor size reaches 2 mm3 or 6 months
following implantation; tumors are excised, weighed and stored in neutral
buffered formalin for pathological examination. Differences in tumor
growth between the groups are descriptively characterized through summary
statistics and graphical displays. Statistical significance is evaluated
with either the t-test or non-parametric equivalent.
EXAMPLE 16
Effect of PAX2 siRNA on Tumor Growth In Vivo
[0294] Hairpin PAX2 siRNA template oligonucleotides utilized in the in
vitro studies are utilized to examine the effect of the up-regulation of
DEFB1 expression in vivo. The sense and antisense strand (see Table 3)
are annealed and cloned into pSilencer 2.1 U6 hygro siRNA expression
vector (Ambion) under the control of the human U6 RNA pol III promoter.
The cloned plasmid is sequenced, verified and transfected into breast
cancer cell lines. Scrambled shRNA is cloned and used as a negative
control in this study. Hygromycin resistant colonies are selected, cells
are introduced into the mice subcutaneously and tumor growth is monitored
as described above.
EXAMPLE 17
Effect of Small Molecule Inhibitors of PAX2 Binding on Breast Cancer
Cells
[0295] The alternative inhibitory oligonucleotides described in Example 6
will be transfected into the breast cancer cells with lipofectamine
reagent or Codebreaker transfection reagent (Promega, Inc). In order to
confirm DNA-protein interactions, double stranded oligonucleotides will
be labeled with [32P] dCTP and electrophoretic mobility shift assays are
performed DEFB1 expression will be monitored by QRT-PCR and Western
analysis following treatment with oligonucleotides. Finally, cell death
will be detected by MTT assay and flow cytometry as previously described.
[0296] The above description is for the purpose of teaching the person of
ordinary skill in the art how to practice the present invention, and it
is not intended to detail all those obvious modifications and variations
of it which will become apparent to the skilled worker upon reading the
description. It is intended, however, that all such obvious modifications
and variations be included within the scope of the present invention,
which is defined by the following claims. The claims are intended to
cover the claimed components and steps in any sequence which is effective
to meet the objectives there intended, unless the context specifically
indicates the contrary.
Sequence CWU
1
7215DNAHomo sapiens 1ccttg
525DNAHomo sapiens 2caagg
5321RNAHomo sapiens 3auagacucga
cuugacuucu u 21421RNAHomo
sapiens 4aucuucauca cguuuccucu u
21521RNAHomo sapiens 5guauucagca aucuuguccu u
21621RNAHomo sapiens 6gauuugaugu gcucugaugu u
21721RNAHomo sapiens 7gaagucaagu
cgagucuauu u 21821RNAHomo
sapiens 8gaggaaacgu gaugaagauu u
21921RNAHomo sapiens 9ggacaagauu gcugaauacu u
211021RNAHomo sapiens 10caucagagca caucaaaucu u
211120DNAHomo sapiens
11acccgactat gttcgcctgg
201221DNAHomo sapiens 12aagctctgga tcgagtcttt g
211320DNAHomo sapiens 13atgtgtcagg cacacagacg
201421RNAHomo sapiens
14gucgagucua ucugcauccu u
211521RNAHomo sapiens 15ggaugcagau agacucgacu u
211619DNAHomo sapiens 16aagttcaccc ttgactgtg
19175DNAHomo sapiens 17ccttg
51820DNAHomo
sapiens 18ctcccttcag ttccgtcgac
201920DNAHomo sapiens 19ctcccttcac cttggtcgac
202040DNAHomo sapiens 20actgtggcac ctcccttcag
ttccgtcgac gaggttgtgc 402140DNAHomo sapiens
21actgtggcac ctcccttcac cttggtcgac gaggttgtgc
40225DNAHomo sapiens 22ctctg
52320DNAHomo sapiens 23ctcccttcac tctggtcgac
202440DNAHomo sapiens 24actgtggcac
ctcccttcac tctggtcgac gaggttgtgc 402520DNAHomo
sapiens 25agaagttcac ccttgactgt
202620DNAHomo sapiens 26agaagttcac gttccactgt
202720DNAHomo sapiens 27agaagttcac gctctactgt
202839DNAHomo sapiens
28ttagcgatta gaagttcacc cttgactgtg gcacctccc
392940DNAHomo sapiens 29gttagcgatt agaagttcac gttccactgt ggcacctccc
403040DNAHomo sapiens 30gttagcgatt agaagttcac
gctctactgt ggcacctccc 403120DNAHomo sapiens
31actgcccatt gcccaaacac
203221DNAHomo sapiens 32aaaatcttgc cagctttccc c
213323DNAHomo sapiens 33gtcggttacg gagcggaccg gag
233424DNAHomo sapiens
34taacatatag acaaacgcac accg
243523DNAHomo sapiens 35gcgcttgtgt cgccattgta ttc
233624DNAHomo sapiens 36gtcacaccac agaagtaagg ttcc
243723DNAHomo sapiens
37gtcggttacg gagcggaccg gag
233825DNAHomo sapiens 38cacagagcat tggcgatctc gatgc
2539135PRTHomo sapiens 39Met Asp Met His Cys Lys Ala
Asp Pro Phe Ser Ala Met His Arg His1 5 10
15Gly Gly Val Asn Gln Leu Gly Gly Val Phe Val Asn Gly
Arg Pro Leu 20 25 30Pro Asp
Val Val Arg Gln Arg Ile Val Glu Leu Ala His Gln Gly Val 35
40 45Arg Pro Cys Asp Ile Ser Arg Gln Leu Arg
Val Ser His Gly Cys Val 50 55 60Ser
Lys Ile Leu Gly Arg Tyr Tyr Glu Thr Gly Ser Ile Lys Pro Gly65
70 75 80Val Ile Gly Gly Ser Lys
Pro Lys Val Ala Thr Pro Lys Val Val Asp 85
90 95Lys Ile Ala Glu Tyr Lys Arg Gln Asn Pro Thr Met
Phe Ala Trp Glu 100 105 110Ile
Arg Asp Arg Leu Leu Ala Glu Gly Ile Cys Asp Asn Asp Thr Val 115
120 125Pro Ser Val Ser Ser Ile Asn130
135407331DNAHomo sapiensmisc_feature(1)..(7331)n= a, c, g, or t
40ttcccccttt ccangagggc ctaatccgtt gcgcgcgcgc acgcggacac acacacacac
60acacacacac acacacacac acacacggcc cccatagcca ccgcaactct cagcagcagn
120ncctagctcc tctgacccga ggccccaaga cggcgggcac aggaacccct gggacgtcct
180ggctccaggc tggacgtagg cggaggtggc aggagtggac aaacccaggc gggtcccacg
240acgccccttt cctcgggtct ctccttgttt cagccagccg ctctcgcccc tggtcccctc
300ttccctgcgt tagggtcctt tgtctccagc cacctcgcag cctgtccccg cctcggcggc
360cctgcccttt gggcctccca gatctctctg gcgggtcccc ctgccttacc agctcccggc
420tgtggcgcgc tcttcgcctg ctcctcacat ncacacagct gctgggagag gaggaaggaa
480aggcggncgc gccgcggatg gatccgagac ggtagatttg gtgccggctc gcaaactctg
540ggaaacttaa ngccggttct tccgcccctc tncaactatg nccagcgcgg cccggtcgcg
600cgcgctcacc ccgcggggac cctttccttt tcctgtattt cggctgcggc tgtttcgctt
660cctctggtct cccagccttt ggagtggctt ccctggccct gcactccgtt ccctttcggc
720cgcccccggc tgtcgcctgc ccccaccctc cgcaggtccc acggtcgcgg cggcgatgac
780tgtggaggta acgccgggga cgtcctgggt cagcctgcac cgtctccctc gaccacagcc
840cgatgaggcc gcgggctccg ggccggctgc taagagagtt aatcattact tcgccagcga
900cactcagcct ccccttccga ctctctcgcc cggcctaggg gaggagggga ggggacagct
960ggccaggtgg ggacttcggc ttcgcacaaa ccagcctctt caggcctccc agagacaggt
1020ggtggcttct cagttccctc ggcaactctc taaggtcctc tttcttcccc tcctgtctct
1080ccctccttcg agcctcctcc cagccaggcc tctccccacc gtctcctgtc cgctctggct
1140ttgactgatt aactgcaggt cctgggagaa ccaactttct ttgtttggaa ccggaccgga
1200cgggatttcc ttccctaggt ctccgccaat gggccagctc ctcccgacgg ttttggcgga
1260ctggctgaag aggaccgcgc ctgaggccac aattaacccg gctgttggtg gtggtggttg
1320gggggtgggc agtgaggaat ttaaccgatc ctctagcagc tgcgctggtg cagttgggag
1380gggggtgcag gaagtgggaa tggaggagtg gcaggaggta tagacagagg gaagaacgat
1440aaacctggac aggtgtggca tagccaatag aaggggaaac aaaataaaac aggaaggcgg
1500cgcggggagg aatccccagt aacctttata ggattgaagt tgggtggaaa acgccacctc
1560ctgccctacc ttagcactca gatccctcct ttacctcttt gtgaaagggt aagagttcag
1620aaagctggcc atttactcca taatctacta gagaaatgtc tgggtttgca aaatgcctat
1680tgattagctc catggagtag acaagacagg cgtaattatc cccattttac aggtgagaaa
1740actgagtctc aaagaagcaa agggactgtg tatgtagtgg ctgtcacttt ttcctgtagg
1800ctgtggggtg agtggcccct ttagctgtgc agaggtccat gggtatctag ggaggcggta
1860caggctgtgt ccaggtctga gccagaagta ccagggcctc acggggctcc tagccctttt
1920agcttgttct ctgttggaca ggaccttcac tcttactctc tagacctgct ggctgggttt
1980ctcccagctt cgctattttt tcagttccct agtagagtgg cccatgggcg gtagccacct
2040ggctggcccg tgccactaag aggcagcttt ggtggccaag tggcttgcat tgttgttgct
2100cctcaaaggg cctgtgaagg gctgggcagg tcgcaaagac ctcttgtgag gggaaagcta
2160gattaaaggg ggtaaggatc ctggaggata aaggccaagc acgtgcgcct ggactccaca
2220ggaccaacag accgagcggg cggggccngc tgggagtcag gccccccggg cttcacgcag
2280ggagcccaaa tattgggaac aaaagcagga aaagaagagt gagagcagga gggagggagg
2340gagcgaggaa gcagaaatta gggggtctta gatgaaaaaa aaaagaaagt agctttaggg
2400ggaatgtgct gtggagtgtg aaattgcagc ccatggtgct ccatattgta ccagaagctc
2460ttccaaaaaa aaaaaaaaaa accatcctcc aacgtgacca gagggccagg cagggggaag
2520ggcggggaga gaatggggag gaggaggggg aaaggccggg caggagccgg tcaggccttt
2580ctgcggaagg ggctggggtg taagtttcgg ctccctggga tctgacagcc gagggtatgc
2640gccctggggt gcgccgggac ccagagggcg agtgagcctc ggttggtcgg ctctggagtt
2700cggttgtcag aagaactttt atttttcttt ttggtggtga cttctaaaag tgggaataat
2760ccagaaatga agctcagctg cggagctgca gctctgttct ccctctctcc cctgcctttc
2820tgcttctctt cccttcggac tacttttctc cccttggttc taaatagctt tttcccctct
2880gaactttaat gcatttaatt tggtccgcgc tgtggggagc atttcctggg gagatgcatt
2940taatttcgga atttctaatc ccctccctca gaccccggtc ctagctcccc tagccgctcc
3000ccgggaagtg gaaggaggaa ggcaggtccc ggccacgggg gaggggcgcg gctgggatgc
3060tcccgcggcc ccctccgtct caccaaggct cagccgcctt cccaagctac tggaggccgg
3120gcgcctgggc cccgggtcag ggccctgcan gaagaagaga ggcaaccccc gctttctgcc
3180ttttcttcgc ctgggcaaga aaacgctggg ccagggaact ggaaaccgga aaacaggaga
3240aagggtttnt ggaaggcanc gggagcgggt ggcagncggg gcancgggca ntggactagg
3300tctacaccgg cacttcactt ttgcacaaca tgcccagaaa cgcatttgag agccctggag
3360tcgcgcttgg cttggcttgg ggcgccggtg cgtgggtaca ctcgaggtcg gggtgcctat
3420ccgccacccc gacacctaca cccagtgcag agcaggcgcg gcccagccag acaaccaggc
3480cggcagtagc tcggcctgga gggcggaggc aaggttgggg gccgccaggc gcctgggcaa
3540gcctggcagg gaagggagcc gagaaggcaa aggagccgag atccacaagg aagattnntt
3600gggcagatca gatgcacaga ggcggctaat gaagcaaatc ccgagatggg tttcagagca
3660actccccaaa agtttatttt gcctttaaat ttccgcaggg aggcgggctc cttgtttgaa
3720gtgtaaatgc ccctaggttg gggggtggaa gggccgcttt gaaaacacca gagagaaaag
3780gttcatttag aggcggacgg gaaaagcaac caaccctgac aggtcggagc ccgggtagtg
3840tttggggttg ggtngttttc tttctttctc tttcttttcc cctttcctct tctttcttcc
3900cttttgtgnn ttttnnttgt tttttttntn ttntttttnt ttaantggct ttcttgcttc
3960cccccacccc tctactagac tctatagaag aaagagaaca gaaaaggggg agtcagagga
4020gcggccagtg actggatgaa ggccagccct tcatcctgga gccccaggag aaggcagagc
4080tttggagaaa aggggttcct aatctccagg gagcattact ctttgactct ctagacccag
4140gaatgggctg gacgctaatg gggaagcggc caggaacccg gcctggcgga agagtgagtg
4200tccagctagt gcagtgctgg gaagacgatc ccaggagcag gggggactct caggggctac
4260ctgggaatgg gactatcaga agggtcttta ctcctcanaa ggtgcatgtg aaggacaggt
4320gtgtgaggac aacttccagc acacttggcg cattaagtcc ccttctctac aaaatggaaa
4380atccttctcg cccaacatgt gaaaatgctt gttgtgggca cccacatttc atggtacttg
4440taacatagga catgtctagc tggttctaga aaaatctgtg tctgtgtgga aggggggggg
4500tttactcaca gctttcttcc ttcaatagtt cacacacccc gagacaaatt cctggatgac
4560caacttggag agacctgggg caaaggttac tttagttctg agctcctcta aataaggacc
4620ctttctcaac gttcctttca ccccagttct gggttaatta cttccagtta gtgcgtgttc
4680gtggggttgt gaggccaaag caaacccggg agcgccatct gcaggcctca agaggaagag
4740actgacctta gaggctaggc cctgcgtctt caacctctag cccaagggaa ccaacctgcc
4800tagccaccca agggaagtgg gataggggct gggaggggca ggcggtgagg agtgttttcc
4860tcccagactt taccccgcag gtggattaag cttattgggc tctggaggat acaggaggga
4920gggcaaatgc caggatccca gcggacccag gccccacagg agtgagaggc tcagaacctc
4980gtcccgctga gcctggcctg agctcctcct gaggaataag ggcatcccaa aaacccgggt
5040acaagacgcc cagtagtagt agttaggctg agtcaggcag gtgcatctct ccccatggta
5100tctgccgccc aggctccggc cagagggagg ggagcgcgag tccgcggcgc ttccgcgggg
5160cgcccggaac tgcagacggg ggctggagga atctcggatt cgggctgcaa gagcgctgcg
5220caagcttcgc cgagccgccc tttcgcagac ccagggaagc ggggggaggg agcgaaggag
5280ggagagagag ttaaaacatc agcttgaaag tgcccaagat gattttatta agaccgaggg
5340gaaaattatt ttcatgaaag attctccccg gaatatttct tgtacttaac ccagttagga
5400agacaaaggg cttctttctg cctggtgcgg tgcgagcgga ccccagcgag caagggagct
5460agtgccaaag agaactgcgg aggctccggc aggagtgggg acgtccccgt ggttgcgcct
5520cctgcgctcg ccccggatcc accgagctag cagcgggcgg cgctcagccg cgtccgcagc
5580ctcctcttct ccccagccgg ggagagccag cctcgtctcc cacatcctct gccgccagcg
5640acctgcagct ccgcactgtt tccctcccct gtaccccctt cccagtcacc cgagggttca
5700gaaaccaagt cccccggctc tcccgccatc cgctgggtcc caccgaggca ggtgggtact
5760cgccggaggt cttcagctcg attctgaacc aagcgttctg gactgcccag acccggtggg
5820caaggggact ggggaggccc tgcgcacagt cgcgtggaac gggaggggac aagacaaact
5880gctggacact tttccgtgga atgagaagtg gggggtgcgt gggtgggaag gtacctccgg
5940agggaaaggc caaagggaag gaccagaaag agaggaagga agagccggga aggaacggaa
6000gggaactcag agccgagggt ggtggggttg gggctaggga tgcgcactgg gcccggggcc
6060gcgcggccca ggcgggcact ggccagtgga tggcagggct gggcgagtta gaactgagag
6120cccggcttca cagcgcagcg cgctccgagg ccctctgtcg ttacctgaat attcattaga
6180ctgaccgctc tttatcctta tctaacgttt atcttatcgg cgagtttcgt ttctcagtgt
6240agttttaatc ccgggctccc attccccctc ccccggtccg ctcccctccc tccctcttcc
6300ttcgccggct gctccctccc tccctccctc ccatttctcc ctcccctgcc ctccccttgc
6360cggcaccgga gtgacaggct cggggccctc ctcgccgaag ctcggggctc cagcgctggc
6420gaatcacaga gtggtggaat ctattgcctt tgtctgacaa gtcatccatc tcccggcgcg
6480gggaggggga ggaggtctgg agggggcttt gcagctttta gagagacaca caccgggagc
6540cgaggctcca gtctccggcc gagtcttcta gcagccgcaa cccacctggg gccagcccag
6600agctgccagc gccgctcggc tccctccctc cctcccggcc cttcggccgc ggcggcgtgc
6660gcctgccttt tccgggggcg ggggcctggc ccgcgcgctc ccctcccgca ggcgccacct
6720cggacatccc cgggattgct acttctctgc caacttcgcc aactcgccag cacttggaga
6780ggcccggctc ccctcccggc gccctctgac cgcccccgcc ccgcgcgctc tccgaccacc
6840gcctctcgga tgaacaggtt ccaggggagc tgagcgagtc gcctcccccg cccagcttca
6900gccctggctg cagctgcagc gcgagccatg cgcccccagt gcaccccggc ccggcccacc
6960gccccggggc cattctgctg accgcccagc cccgagcccc gacagtggca agttgcggct
7020actgcggttg caagctccgg ccaacccgga ggagccccag cggggagcgc agtgttgcgc
7080cccccgcccc cgcgcgcgcc gcagcagccg ggcgttcact catcctccct cccccaccgt
7140ccctcccttt tctcctcaag tcctgaagtt gagtttgaga ggcgacacgg cggcggcggc
7200cgcgctgctc ccgctcctct gcctccccat ggatatgcac tgcaaagcag accccttctc
7260cgcgatgcac cgtgagtacc cgcgcccggc tcctgtcccg gctcgggctc tccgtcccaa
7320ccctgtccag t
733141416PRTHomo sapiens 41Met Asp Met His Cys Lys Ala Asp Pro Phe Ser
Ala Met His Pro Gly1 5 10
15His Gly Gly Val Asn Gln Leu Gly Gly Val Phe Val Asn Gly Arg Pro
20 25 30Leu Pro Asp Val Val Arg Gln
Arg Ile Val Glu Leu Ala His Gln Gly 35 40
45Val Arg Pro Cys Asp Ile Ser Arg Gln Leu Arg Val Ser His Gly
Cys 50 55 60Val Ser Lys Ile Leu Gly
Arg Tyr Tyr Glu Thr Gly Ser Ile Lys Pro65 70
75 80Gly Val Ile Gly Gly Ser Lys Pro Lys Val Ala
Thr Pro Lys Val Val 85 90
95Asp Lys Ile Ala Glu Tyr Lys Arg Gln Asn Pro Thr Met Phe Ala Trp
100 105 110Glu Ile Arg Asp Arg Leu
Leu Ala Glu Gly Ile Cys Asp Asn Asp Thr 115 120
125Val Pro Ser Val Ser Ser Ile Asn Arg Ile Ile Arg Thr Lys
Val Gln 130 135 140Gln Pro Phe His Pro
Thr Pro Asp Gly Ala Gly Thr Gly Val Thr Ala145 150
155 160Pro Gly His Thr Ile Val Pro Ser Thr Ala
Ser Pro Pro Val Ser Ser 165 170
175Ala Ser Asn Asp Pro Val Gly Ser Tyr Ser Ile Asn Gly Ile Leu Gly
180 185 190Ile Pro Arg Ser Asn
Gly Glu Lys Arg Lys Arg Asp Glu Val Glu Val 195
200 205Tyr Thr Asp Pro Ala His Ile Arg Gly Gly Gly Gly
Leu His Leu Val 210 215 220Trp Thr Leu
Arg Asp Val Ser Glu Gly Ser Val Pro Asn Gly Asp Ser225
230 235 240Gln Ser Gly Val Asp Ser Leu
Arg Lys His Leu Arg Ala Asp Thr Phe 245
250 255Thr Gln Gln Gln Leu Glu Ala Leu Asp Arg Val Phe
Glu Arg Pro Ser 260 265 270Tyr
Pro Asp Val Phe Gln Ala Ser Glu His Ile Lys Ser Glu Gln Gly 275
280 285Asn Glu Tyr Ser Leu Pro Ala Leu Thr
Pro Gly Leu Asp Glu Val Lys 290 295
300Ser Ser Leu Ser Ala Ser Thr Asn Pro Glu Leu Gly Ser Asn Val Ser305
310 315 320Gly Thr Gln Thr
Tyr Pro Val Val Thr Gly Arg Asp Met Ala Ser Thr 325
330 335Thr Leu Pro Gly Tyr Pro Pro His Val Pro
Pro Thr Gly Gln Gly Ser 340 345
350Tyr Pro Thr Ser Thr Leu Ala Gly Met Val Pro Gly Ser Glu Phe Ser
355 360 365Gly Asn Pro Tyr Ser His Pro
Gln Tyr Thr Ala Tyr Asn Glu Ala Trp 370 375
380Arg Phe Ser Asn Pro Ala Leu Leu Ser Ser Pro Tyr Tyr Tyr Ser
Ala385 390 395 400Ala Pro
Arg Ser Ala Pro Ala Ala Ala Ala Ala Ala Tyr Asp Arg His
405 410 415424276DNAHomo sapiens
42aggctccagt ctccggccga gtcttctcgc agccgcaacc cacctggggc cagcccagag
60ctgccagcgc cgctcggctc cctccctccc tcccggccct tcggccgcgg cggcgtgcgc
120ctgccttttc cgggggcggg ggcctggccc gcgcgctccc ctcccgcagg cgccacctcg
180gacatccccg ggattgctac ttctctgcca acttcgccaa ctcgccagca cttggagagg
240cccggctccc ctcccggcgc cctctgaccg cccccgcccc gcgcgctctc cgaccaccgc
300ctctcggatg accaggttcc aggggagctg agcgagtcgc ctcccccgcc cagcttcagc
360cctggctgca gctgcagcgc gagccatgcg cccccagtgc accccggccc ggcccaccgc
420cccggggcca ttctgctgac cgcccagccc cgagccccga cagtggcaag ttgcggctac
480tgcagttgca agctccggcc aacccggagg agccccagcg gggagcgcag tgttgcgccc
540cccgcccccg cgcgccccgc agcagccggg cgttcactca tcctccctcc cccaccgtcc
600ctcccttttc tcctcaagtc ctgaagttga gtttgagagg cgacacggcg gcggcggccg
660cgctgctccc gctcctctgc ctccccatgg atatgcactg caaagcagac cccttctccg
720cgatgcaccc agggcacggg ggtgtgaacc agctcggggg ggtgtttgtg aacggccggc
780ccctacccga cgtggtgagg cagcgcatcg tggagctggc ccaccagggt gtgcggccct
840gtgacatctc ccggcagctg cgggtcagcc acggctgtgt cagcaaaatc ctgggcaggt
900actacgagac cggcagcatc aagccgggtg tgatcggtgg ctccaagccc aaagtggcga
960cgcccaaagt ggtggacaag attgctgaat acaaacgaca gaacccgact atgttcgcct
1020gggagattcg agaccggctc ctggccgagg gcatctgtga caatgacaca gtgcccagcg
1080tctcttccat caacagaatc atccggacca aagttcagca gcctttccac ccaacgccgg
1140atggggctgg gacaggagtg accgcccctg gccacaccat tgttcccagc acggcctccc
1200ctcctgtttc cagcgcctcc aatgacccag tgggatccta ctccatcaat gggatcctgg
1260ggattcctcg ctccaatggt gagaagagga aacgtgatga agttgaggta tacactgatc
1320ctgcccacat tagaggaggt ggaggtttgc atctggtctg gactttaaga gatgtgtctg
1380agggctcagt ccccaatgga gattcccaga gtggtgtgga cagtttgcgg aagcacttgc
1440gagctgacac cttcacccag cagcagctgg aagctttgga tcgggtcttt gagcgtcctt
1500cctaccctga cgtcttccag gcatcagagc acatcaaatc agaacagggg aacgagtact
1560ccctcccagc cctgacccct gggcttgatg aagtcaagtc gagtctatct gcatccacca
1620accctgagct gggcagcaac gtgtcaggca cacagacata cccagttgtg actggtcgtg
1680acatggcgag caccactctg cctggttacc cccctcacgt gccccccact ggccagggaa
1740gctaccccac ctccaccctg gcaggaatgg tgcctgggag cgagttctcc ggcaacccgt
1800acagccaccc ccagtacacg gcctacaacg aggcttggag attcagcaac cccgccttac
1860taagttcccc ttattattat agtgccgccc cccggtccgc ccctgccgct gctgccgctg
1920cctatgaccg ccactagtta ccgcggggac cacatcaagc ttcaggccga cagcttcggc
1980ctccacatcg tccccgtctg accccacccc ggagggaggg aggaccgacg cgacgcgatg
2040cctcccggcc accgccccag cctcacccca tcccacgacc cccgcaaccc ttcacatcac
2100ccccctcgaa ggtcggacag gacgggtgga gccgtgggcg ggaccctcag gcccgggccc
2160gccgccccca gccccgcctg ccgcccctcc ccgcctgcct ggactgcgcg gcgccgtgag
2220ggggattcgg cccagctcgt cccggcctcc accaagccag ccccgaagcc cgccagccac
2280cctgccggac tcgggcgcga cctgctggcg cgcgccggat gtttctgtga cacacaatca
2340gcgcggaccg cagcgcggcc cagccccggg cacccgcctc ggacgctcgg gcgccaggag
2400gcttcgctgg aggggctggg ccaaggagat taagaagaaa acgactttct gcaggaggaa
2460gagcccgctg ccgaatccct gggaaaaatt cttttccccc agtgccagcc ggactgccct
2520cgccttccgg gtgtgccctg tcccagaaga tggaatgggg gtgtgggggt ccggctctag
2580gaacgggctt tgggggcgtc aggtctttcc aaggttggga cccaaggatc ggggggccca
2640gcagcccgca ccgatcgagc cggactctcg gctcttcact gctcctcctg gcctgcctag
2700ttccccaggg cccggcacct cctgctgcga gacccggctc tcagccctgc cttgccccta
2760cctcagcgtc tcttccacct gctggcctcc cagtttcccc tcctgccagt ccttcgcctg
2820tcccttgacg ccctgcatcc tcctccctga ctcgcagccc catcggacgc tctcccggga
2880ccgccgcagg accagtttcc atagactgcg gactggggtc ttcctccagc agttacttga
2940tgccccctcc cccgacacag actctcaatc tgccggtggt aagaaccggt tctgagctgg
3000cgtctgagct gctgcggggt ggaagtgggg ggctgcccac tccactcctc ccatcccctc
3060ccagcctcct cctccggcag gaactgaaca gaaccacaaa aagtctacat ttatttaata
3120tgatggtctt tgcaaaaagg aacaaaacaa cacaaaagcc caccaggctg ctgctttgtg
3180gaaagacggt gtgtgtcgtg tgaaggcgaa acccggtgta cataacccct ccccctccgc
3240cccgccccgc ccggccccgt agagtccctg tcgcccgccg gccctgcctg tagatacgcc
3300ccgctgtctg tgctgtgaga gtcgccgctc gctggggggg aaggggggga cacagctaca
3360cgcccattaa agcacagcac gtcctggggg aggggggcat tttttatgtt acaaaaaaaa
3420attacgaaag aaaagaaatc tctatgcaaa atgacgaaca tggtcctgtg gactcctctg
3480gcctgttttg ttggctcttt ctctgtaatt ccgtgttttc gctttttcct ccctgcccct
3540ctctccctct gcccctctct cctctccgct tctctccccc tctgtctctg tctctctccg
3600tctctgtcgc tcttgtctgt ctgtctctgc tctttcctcg gcctctctcc ccagacctgg
3660cccggccgcc ctgtctccgc aggctagatc cgaggtggca gctccagccc ccgggctcgc
3720cccctcgcgg gcgtgccccg cgcgccccgg gcggccgaag gccgggccgc cccgtcccgc
3780cccgtagttg ctctttcggt agtggcgatg cgccctgcat gtctcctcac ccgtggatcg
3840tgacgactcg aaataacaga aacaaagtca ataaagtgaa aataaataaa aatccttgaa
3900caaatccgaa aaggcttgga gtcctcgccc agatctctct cccctgcgag ccctttttat
3960ttgagaagga aaaagagaaa agagaatcgt ttaagggaac ccggcgccca gccaggctcc
4020agtggcccga acggggcggc gagggcggcg agggcgccga ggtccggccc atcccagtcc
4080tgtggggctg gccgggcaga gaccccggac ccaggcccag gcctaacctg ctaaatgtcc
4140ccggacggtt ctggtctcct cggccacttt cagtgcgtcg gttcgttttg attctttttc
4200ttttgtgcac ataagaaata aataataata ataaataaag aataaaattt tgtatgtcaa
4260aaaaaaaaaa aaaaaa
427643393PRTHomo sapiens 43Met Asp Met His Cys Lys Ala Asp Pro Phe Ser
Ala Met His Pro Gly1 5 10
15His Gly Gly Val Asn Gln Leu Gly Gly Val Phe Val Asn Gly Arg Pro
20 25 30Leu Pro Asp Val Val Arg Gln
Arg Ile Val Glu Leu Ala His Gln Gly 35 40
45Val Arg Pro Cys Asp Ile Ser Arg Gln Leu Arg Val Ser His Gly
Cys 50 55 60Val Ser Lys Ile Leu Gly
Arg Tyr Tyr Glu Thr Gly Ser Ile Lys Pro65 70
75 80Gly Val Ile Gly Gly Ser Lys Pro Lys Val Ala
Thr Pro Lys Val Val 85 90
95Asp Lys Ile Ala Glu Tyr Lys Arg Gln Asn Pro Thr Met Phe Ala Trp
100 105 110Glu Ile Arg Asp Arg Leu
Leu Ala Glu Gly Ile Cys Asp Asn Asp Thr 115 120
125Val Pro Ser Val Ser Ser Ile Asn Arg Ile Ile Arg Thr Lys
Val Gln 130 135 140Gln Pro Phe His Pro
Thr Pro Asp Gly Ala Gly Thr Gly Val Thr Ala145 150
155 160Pro Gly His Thr Ile Val Pro Ser Thr Ala
Ser Pro Pro Val Ser Ser 165 170
175Ala Ser Asn Asp Pro Val Gly Ser Tyr Ser Ile Asn Gly Ile Leu Gly
180 185 190Ile Pro Arg Ser Asn
Gly Glu Lys Arg Lys Arg Asp Glu Asp Val Ser 195
200 205Glu Gly Ser Val Pro Asn Gly Asp Ser Gln Ser Gly
Val Asp Ser Leu 210 215 220Arg Lys His
Leu Arg Ala Asp Thr Phe Thr Gln Gln Gln Leu Glu Ala225
230 235 240Leu Asp Arg Val Phe Glu Arg
Pro Ser Tyr Pro Asp Val Phe Gln Ala 245
250 255Ser Glu His Ile Lys Ser Glu Gln Gly Asn Glu Tyr
Ser Leu Pro Ala 260 265 270Leu
Thr Pro Gly Leu Asp Glu Val Lys Ser Ser Leu Ser Ala Ser Thr 275
280 285Asn Pro Glu Leu Gly Ser Asn Val Ser
Gly Thr Gln Thr Tyr Pro Val 290 295
300Val Thr Gly Arg Asp Met Ala Ser Thr Thr Leu Pro Gly Tyr Pro Pro305
310 315 320His Val Pro Pro
Thr Gly Gln Gly Ser Tyr Pro Thr Ser Thr Leu Ala 325
330 335Gly Met Val Pro Gly Ser Glu Phe Ser Gly
Asn Pro Tyr Ser His Pro 340 345
350Gln Tyr Thr Ala Tyr Asn Glu Ala Trp Arg Phe Ser Asn Pro Ala Leu
355 360 365Leu Ser Ser Pro Tyr Tyr Tyr
Ser Ala Ala Pro Arg Ser Ala Pro Ala 370 375
380Ala Ala Ala Ala Ala Tyr Asp Arg His385
390444207DNAHomo sapiens 44aggctccagt ctccggccga gtcttctcgc agccgcaacc
cacctggggc cagcccagag 60ctgccagcgc cgctcggctc cctccctccc tcccggccct
tcggccgcgg cggcgtgcgc 120ctgccttttc cgggggcggg ggcctggccc gcgcgctccc
ctcccgcagg cgccacctcg 180gacatccccg ggattgctac ttctctgcca acttcgccaa
ctcgccagca cttggagagg 240cccggctccc ctcccggcgc cctctgaccg cccccgcccc
gcgcgctctc cgaccaccgc 300ctctcggatg accaggttcc aggggagctg agcgagtcgc
ctcccccgcc cagcttcagc 360cctggctgca gctgcagcgc gagccatgcg cccccagtgc
accccggccc ggcccaccgc 420cccggggcca ttctgctgac cgcccagccc cgagccccga
cagtggcaag ttgcggctac 480tgcagttgca agctccggcc aacccggagg agccccagcg
gggagcgcag tgttgcgccc 540cccgcccccg cgcgccccgc agcagccggg cgttcactca
tcctccctcc cccaccgtcc 600ctcccttttc tcctcaagtc ctgaagttga gtttgagagg
cgacacggcg gcggcggccg 660cgctgctccc gctcctctgc ctccccatgg atatgcactg
caaagcagac cccttctccg 720cgatgcaccc agggcacggg ggtgtgaacc agctcggggg
ggtgtttgtg aacggccggc 780ccctacccga cgtggtgagg cagcgcatcg tggagctggc
ccaccagggt gtgcggccct 840gtgacatctc ccggcagctg cgggtcagcc acggctgtgt
cagcaaaatc ctgggcaggt 900actacgagac cggcagcatc aagccgggtg tgatcggtgg
ctccaagccc aaagtggcga 960cgcccaaagt ggtggacaag attgctgaat acaaacgaca
gaacccgact atgttcgcct 1020gggagattcg agaccggctc ctggccgagg gcatctgtga
caatgacaca gtgcccagcg 1080tctcttccat caacagaatc atccggacca aagttcagca
gcctttccac ccaacgccgg 1140atggggctgg gacaggagtg accgcccctg gccacaccat
tgttcccagc acggcctccc 1200ctcctgtttc cagcgcctcc aatgacccag tgggatccta
ctccatcaat gggatcctgg 1260ggattcctcg ctccaatggt gagaagagga aacgtgatga
agatgtgtct gagggctcag 1320tccccaatgg agattcccag agtggtgtgg acagtttgcg
gaagcacttg cgagctgaca 1380ccttcaccca gcagcagctg gaagctttgg atcgggtctt
tgagcgtcct tcctaccctg 1440acgtcttcca ggcatcagag cacatcaaat cagaacaggg
gaacgagtac tccctcccag 1500ccctgacccc tgggcttgat gaagtcaagt cgagtctatc
tgcatccacc aaccctgagc 1560tgggcagcaa cgtgtcaggc acacagacat acccagttgt
gactggtcgt gacatggcga 1620gcaccactct gcctggttac ccccctcacg tgccccccac
tggccaggga agctacccca 1680cctccaccct ggcaggaatg gtgcctggga gcgagttctc
cggcaacccg tacagccacc 1740cccagtacac ggcctacaac gaggcttgga gattcagcaa
ccccgcctta ctaagttccc 1800cttattatta tagtgccgcc ccccggtccg cccctgccgc
tgctgccgct gcctatgacc 1860gccactagtt accgcgggga ccacatcaag cttcaggccg
acagcttcgg cctccacatc 1920gtccccgtct gaccccaccc cggagggagg gaggaccgac
gcgacgcgat gcctcccggc 1980caccgcccca gcctcacccc atcccacgac ccccgcaacc
cttcacatca cccccctcga 2040aggtcggaca ggacgggtgg agccgtgggc gggaccctca
ggcccgggcc cgccgccccc 2100agccccgcct gccgcccctc cccgcctgcc tggactgcgc
ggcgccgtga gggggattcg 2160gcccagctcg tcccggcctc caccaagcca gccccgaagc
ccgccagcca ccctgccgga 2220ctcgggcgcg acctgctggc gcgcgccgga tgtttctgtg
acacacaatc agcgcggacc 2280gcagcgcggc ccagccccgg gcacccgcct cggacgctcg
ggcgccagga ggcttcgctg 2340gaggggctgg gccaaggaga ttaagaagaa aacgactttc
tgcaggagga agagcccgct 2400gccgaatccc tgggaaaaat tcttttcccc cagtgccagc
cggactgccc tcgccttccg 2460ggtgtgccct gtcccagaag atggaatggg ggtgtggggg
tccggctcta ggaacgggct 2520ttgggggcgt caggtctttc caaggttggg acccaaggat
cggggggccc agcagcccgc 2580accgatcgag ccggactctc ggctcttcac tgctcctcct
ggcctgccta gttccccagg 2640gcccggcacc tcctgctgcg agacccggct ctcagccctg
ccttgcccct acctcagcgt 2700ctcttccacc tgctggcctc ccagtttccc ctcctgccag
tccttcgcct gtcccttgac 2760gccctgcatc ctcctccctg actcgcagcc ccatcggacg
ctctcccggg accgccgcag 2820gaccagtttc catagactgc ggactggggt cttcctccag
cagttacttg atgccccctc 2880ccccgacaca gactctcaat ctgccggtgg taagaaccgg
ttctgagctg gcgtctgagc 2940tgctgcgggg tggaagtggg gggctgccca ctccactcct
cccatcccct cccagcctcc 3000tcctccggca ggaactgaac agaaccacaa aaagtctaca
tttatttaat atgatggtct 3060ttgcaaaaag gaacaaaaca acacaaaagc ccaccaggct
gctgctttgt ggaaagacgg 3120tgtgtgtcgt gtgaaggcga aacccggtgt acataacccc
tccccctccg ccccgccccg 3180cccggccccg tagagtccct gtcgcccgcc ggccctgcct
gtagatacgc cccgctgtct 3240gtgctgtgag agtcgccgct cgctgggggg gaaggggggg
acacagctac acgcccatta 3300aagcacagca cgtcctgggg gaggggggca ttttttatgt
tacaaaaaaa aattacgaaa 3360gaaaagaaat ctctatgcaa aatgacgaac atggtcctgt
ggactcctct ggcctgtttt 3420gttggctctt tctctgtaat tccgtgtttt cgctttttcc
tccctgcccc tctctccctc 3480tgcccctctc tcctctccgc ttctctcccc ctctgtctct
gtctctctcc gtctctgtcg 3540ctcttgtctg tctgtctctg ctctttcctc ggcctctctc
cccagacctg gcccggccgc 3600cctgtctccg caggctagat ccgaggtggc agctccagcc
cccgggctcg ccccctcgcg 3660ggcgtgcccc gcgcgccccg ggcggccgaa ggccgggccg
ccccgtcccg ccccgtagtt 3720gctctttcgg tagtggcgat gcgccctgca tgtctcctca
cccgtggatc gtgacgactc 3780gaaataacag aaacaaagtc aataaagtga aaataaataa
aaatccttga acaaatccga 3840aaaggcttgg agtcctcgcc cagatctctc tcccctgcga
gcccttttta tttgagaagg 3900aaaaagagaa aagagaatcg tttaagggaa cccggcgccc
agccaggctc cagtggcccg 3960aacggggcgg cgagggcggc gagggcgccg aggtccggcc
catcccagtc ctgtggggct 4020ggccgggcag agaccccgga cccaggccca ggcctaacct
gctaaatgtc cccggacggt 4080tctggtctcc tcggccactt tcagtgcgtc ggttcgtttt
gattcttttt cttttgtgca 4140cataagaaat aaataataat aataaataaa gaataaaatt
ttgtatgtca aaaaaaaaaa 4200aaaaaaa
420745396PRTHomo sapiens 45Met Asp Met His Cys Lys
Ala Asp Pro Phe Ser Ala Met His Pro Gly1 5
10 15His Gly Gly Val Asn Gln Leu Gly Gly Val Phe Val
Asn Gly Arg Pro 20 25 30Leu
Pro Asp Val Val Arg Gln Arg Ile Val Glu Leu Ala His Gln Gly 35
40 45Val Arg Pro Cys Asp Ile Ser Arg Gln
Leu Arg Val Ser His Gly Cys 50 55
60Val Ser Lys Ile Leu Gly Arg Tyr Tyr Glu Thr Gly Ser Ile Lys Pro65
70 75 80Gly Val Ile Gly Gly
Ser Lys Pro Lys Val Ala Thr Pro Lys Val Val 85
90 95Asp Lys Ile Ala Glu Tyr Lys Arg Gln Asn Pro
Thr Met Phe Ala Trp 100 105
110Glu Ile Arg Asp Arg Leu Leu Ala Glu Gly Ile Cys Asp Asn Asp Thr
115 120 125Val Pro Ser Val Ser Ser Ile
Asn Arg Ile Ile Arg Thr Lys Val Gln 130 135
140Gln Pro Phe His Pro Thr Pro Asp Gly Ala Gly Thr Gly Val Thr
Ala145 150 155 160Pro Gly
His Thr Ile Val Pro Ser Thr Ala Ser Pro Pro Val Ser Ser
165 170 175Ala Ser Asn Asp Pro Val Gly
Ser Tyr Ser Ile Asn Gly Ile Leu Gly 180 185
190Ile Pro Arg Ser Asn Gly Glu Lys Arg Lys Arg Asp Glu Asp
Val Ser 195 200 205Glu Gly Ser Val
Pro Asn Gly Asp Ser Gln Ser Gly Val Asp Ser Leu 210
215 220Arg Lys His Leu Arg Ala Asp Thr Phe Thr Gln Gln
Gln Leu Glu Ala225 230 235
240Leu Asp Arg Val Phe Glu Arg Pro Ser Tyr Pro Asp Val Phe Gln Ala
245 250 255Ser Glu His Ile Lys
Ser Glu Gln Gly Asn Glu Tyr Ser Leu Pro Ala 260
265 270Leu Thr Pro Gly Leu Asp Glu Val Lys Ser Ser Leu
Ser Ala Ser Thr 275 280 285Asn Pro
Glu Leu Gly Ser Asn Val Ser Gly Thr Gln Thr Tyr Pro Val 290
295 300Val Thr Gly Arg Asp Met Ala Ser Thr Thr Leu
Pro Gly Tyr Pro Pro305 310 315
320His Val Pro Pro Thr Gly Gln Gly Ser Tyr Pro Thr Ser Thr Leu Ala
325 330 335Gly Met Val Pro
Glu Ala Ala Val Gly Pro Ser Ser Ser Leu Met Ser 340
345 350Lys Pro Gly Arg Lys Leu Ala Glu Val Pro Pro
Cys Val Gln Pro Thr 355 360 365Gly
Ala Ser Ser Pro Ala Thr Arg Thr Ala Thr Pro Ser Thr Arg Pro 370
375 380Thr Thr Arg Leu Gly Asp Ser Ala Thr Pro
Pro Tyr385 390 395464290DNAHomo sapiens
46aggctccagt ctccggccga gtcttctcgc agccgcaacc cacctggggc cagcccagag
60ctgccagcgc cgctcggctc cctccctccc tcccggccct tcggccgcgg cggcgtgcgc
120ctgccttttc cgggggcggg ggcctggccc gcgcgctccc ctcccgcagg cgccacctcg
180gacatccccg ggattgctac ttctctgcca acttcgccaa ctcgccagca cttggagagg
240cccggctccc ctcccggcgc cctctgaccg cccccgcccc gcgcgctctc cgaccaccgc
300ctctcggatg accaggttcc aggggagctg agcgagtcgc ctcccccgcc cagcttcagc
360cctggctgca gctgcagcgc gagccatgcg cccccagtgc accccggccc ggcccaccgc
420cccggggcca ttctgctgac cgcccagccc cgagccccga cagtggcaag ttgcggctac
480tgcagttgca agctccggcc aacccggagg agccccagcg gggagcgcag tgttgcgccc
540cccgcccccg cgcgccccgc agcagccggg cgttcactca tcctccctcc cccaccgtcc
600ctcccttttc tcctcaagtc ctgaagttga gtttgagagg cgacacggcg gcggcggccg
660cgctgctccc gctcctctgc ctccccatgg atatgcactg caaagcagac cccttctccg
720cgatgcaccc agggcacggg ggtgtgaacc agctcggggg ggtgtttgtg aacggccggc
780ccctacccga cgtggtgagg cagcgcatcg tggagctggc ccaccagggt gtgcggccct
840gtgacatctc ccggcagctg cgggtcagcc acggctgtgt cagcaaaatc ctgggcaggt
900actacgagac cggcagcatc aagccgggtg tgatcggtgg ctccaagccc aaagtggcga
960cgcccaaagt ggtggacaag attgctgaat acaaacgaca gaacccgact atgttcgcct
1020gggagattcg agaccggctc ctggccgagg gcatctgtga caatgacaca gtgcccagcg
1080tctcttccat caacagaatc atccggacca aagttcagca gcctttccac ccaacgccgg
1140atggggctgg gacaggagtg accgcccctg gccacaccat tgttcccagc acggcctccc
1200ctcctgtttc cagcgcctcc aatgacccag tgggatccta ctccatcaat gggatcctgg
1260ggattcctcg ctccaatggt gagaagagga aacgtgatga agatgtgtct gagggctcag
1320tccccaatgg agattcccag agtggtgtgg acagtttgcg gaagcacttg cgagctgaca
1380ccttcaccca gcagcagctg gaagctttgg atcgggtctt tgagcgtcct tcctaccctg
1440acgtcttcca ggcatcagag cacatcaaat cagaacaggg gaacgagtac tccctcccag
1500ccctgacccc tgggcttgat gaagtcaagt cgagtctatc tgcatccacc aaccctgagc
1560tgggcagcaa cgtgtcaggc acacagacat acccagttgt gactggtcgt gacatggcga
1620gcaccactct gcctggttac ccccctcacg tgccccccac tggccaggga agctacccca
1680cctccaccct ggcaggaatg gtgcctgagg ctgcagttgg tccctcatcc tccctcatga
1740gcaagccggg gaggaagctt gcagaagtgc ccccttgtgt gcaacccact ggagcgagtt
1800ctccggcaac ccgtacagcc acccccagta cacggcctac aacgaggctt ggagattcag
1860caaccccgcc ttactaagtt ccccttatta ttatagtgcc gccccccggt ccgcccctgc
1920cgctgctgcc gctgcctatg accgccacta gttaccgcgg ggaccacatc aagcttcagg
1980ccgacagctt cggcctccac atcgtccccg tctgacccca ccccggaggg agggaggacc
2040gacgcgacgc gatgcctccc ggccaccgcc ccagcctcac cccatcccac gacccccgca
2100acccttcaca tcacccccct cgaaggtcgg acaggacggg tggagccgtg ggcgggaccc
2160tcaggcccgg gcccgccgcc cccagccccg cctgccgccc ctccccgcct gcctggactg
2220cgcggcgccg tgagggggat tcggcccagc tcgtcccggc ctccaccaag ccagccccga
2280agcccgccag ccaccctgcc ggactcgggc gcgacctgct ggcgcgcgcc ggatgtttct
2340gtgacacaca atcagcgcgg accgcagcgc ggcccagccc cgggcacccg cctcggacgc
2400tcgggcgcca ggaggcttcg ctggaggggc tgggccaagg agattaagaa gaaaacgact
2460ttctgcagga ggaagagccc gctgccgaat ccctgggaaa aattcttttc ccccagtgcc
2520agccggactg ccctcgcctt ccgggtgtgc cctgtcccag aagatggaat gggggtgtgg
2580gggtccggct ctaggaacgg gctttggggg cgtcaggtct ttccaaggtt gggacccaag
2640gatcgggggg cccagcagcc cgcaccgatc gagccggact ctcggctctt cactgctcct
2700cctggcctgc ctagttcccc agggcccggc acctcctgct gcgagacccg gctctcagcc
2760ctgccttgcc cctacctcag cgtctcttcc acctgctggc ctcccagttt cccctcctgc
2820cagtccttcg cctgtccctt gacgccctgc atcctcctcc ctgactcgca gccccatcgg
2880acgctctccc gggaccgccg caggaccagt ttccatagac tgcggactgg ggtcttcctc
2940cagcagttac ttgatgcccc ctcccccgac acagactctc aatctgccgg tggtaagaac
3000cggttctgag ctggcgtctg agctgctgcg gggtggaagt ggggggctgc ccactccact
3060cctcccatcc cctcccagcc tcctcctccg gcaggaactg aacagaacca caaaaagtct
3120acatttattt aatatgatgg tctttgcaaa aaggaacaaa acaacacaaa agcccaccag
3180gctgctgctt tgtggaaaga cggtgtgtgt cgtgtgaagg cgaaacccgg tgtacataac
3240ccctccccct ccgccccgcc ccgcccggcc ccgtagagtc cctgtcgccc gccggccctg
3300cctgtagata cgccccgctg tctgtgctgt gagagtcgcc gctcgctggg ggggaagggg
3360gggacacagc tacacgccca ttaaagcaca gcacgtcctg ggggaggggg gcatttttta
3420tgttacaaaa aaaaattacg aaagaaaaga aatctctatg caaaatgacg aacatggtcc
3480tgtggactcc tctggcctgt tttgttggct ctttctctgt aattccgtgt tttcgctttt
3540tcctccctgc ccctctctcc ctctgcccct ctctcctctc cgcttctctc cccctctgtc
3600tctgtctctc tccgtctctg tcgctcttgt ctgtctgtct ctgctctttc ctcggcctct
3660ctccccagac ctggcccggc cgccctgtct ccgcaggcta gatccgaggt ggcagctcca
3720gcccccgggc tcgccccctc gcgggcgtgc cccgcgcgcc ccgggcggcc gaaggccggg
3780ccgccccgtc ccgccccgta gttgctcttt cggtagtggc gatgcgccct gcatgtctcc
3840tcacccgtgg atcgtgacga ctcgaaataa cagaaacaaa gtcaataaag tgaaaataaa
3900taaaaatcct tgaacaaatc cgaaaaggct tggagtcctc gcccagatct ctctcccctg
3960cgagcccttt ttatttgaga aggaaaaaga gaaaagagaa tcgtttaagg gaacccggcg
4020cccagccagg ctccagtggc ccgaacgggg cggcgagggc ggcgagggcg ccgaggtccg
4080gcccatccca gtcctgtggg gctggccggg cagagacccc ggacccaggc ccaggcctaa
4140cctgctaaat gtccccggac ggttctggtc tcctcggcca ctttcagtgc gtcggttcgt
4200tttgattctt tttcttttgt gcacataaga aataaataat aataataaat aaagaataaa
4260attttgtatg tcaaaaaaaa aaaaaaaaaa
429047408PRTHomo sapiens 47Met Asp Met His Cys Lys Ala Asp Pro Phe Ser
Ala Met His Pro Gly1 5 10
15His Gly Gly Val Asn Gln Leu Gly Gly Val Phe Val Asn Gly Arg Pro
20 25 30Leu Pro Asp Val Val Arg Gln
Arg Ile Val Glu Leu Ala His Gln Gly 35 40
45Val Arg Pro Cys Asp Ile Ser Arg Gln Leu Arg Val Ser His Gly
Cys 50 55 60Val Ser Lys Ile Leu Gly
Arg Tyr Tyr Glu Thr Gly Ser Ile Lys Pro65 70
75 80Gly Val Ile Gly Gly Ser Lys Pro Lys Val Ala
Thr Pro Lys Val Val 85 90
95Asp Lys Ile Ala Glu Tyr Lys Arg Gln Asn Pro Thr Met Phe Ala Trp
100 105 110Glu Ile Arg Asp Arg Leu
Leu Ala Glu Gly Ile Cys Asp Asn Asp Thr 115 120
125Val Pro Ser Val Ser Ser Ile Asn Arg Ile Ile Arg Thr Lys
Val Gln 130 135 140Gln Pro Phe His Pro
Thr Pro Asp Gly Ala Gly Thr Gly Val Thr Ala145 150
155 160Pro Gly His Thr Ile Val Pro Ser Thr Ala
Ser Pro Pro Val Ser Ser 165 170
175Ala Ser Asn Asp Pro Val Gly Ser Tyr Ser Ile Asn Gly Ile Leu Gly
180 185 190Ile Pro Arg Ser Asn
Gly Glu Lys Arg Lys Arg Asp Glu Asp Val Ser 195
200 205Glu Gly Ser Val Pro Asn Gly Asp Ser Gln Ser Gly
Val Asp Ser Leu 210 215 220Arg Lys His
Leu Arg Ala Asp Thr Phe Thr Gln Gln Gln Leu Glu Ala225
230 235 240Leu Asp Arg Val Phe Glu Arg
Pro Ser Tyr Pro Asp Val Phe Gln Ala 245
250 255Ser Glu His Ile Lys Ser Glu Gln Gly Asn Glu Tyr
Ser Leu Pro Ala 260 265 270Leu
Thr Pro Gly Leu Asp Glu Val Lys Ser Ser Leu Ser Ala Ser Thr 275
280 285Asn Pro Glu Leu Gly Ser Asn Val Ser
Gly Thr Gln Thr Tyr Pro Val 290 295
300Val Thr Gly Arg Asp Met Ala Ser Thr Thr Leu Pro Gly Tyr Pro Pro305
310 315 320His Val Pro Pro
Thr Gly Gln Gly Ser Tyr Pro Thr Ser Thr Leu Ala 325
330 335Gly Met Val Pro Gly Ser Glu Phe Ser Gly
Asn Pro Tyr Ser His Pro 340 345
350Gln Tyr Thr Ala Tyr Asn Glu Ala Trp Arg Phe Ser Asn Pro Ala Leu
355 360 365Leu Met Pro Pro Pro Gly Pro
Pro Leu Pro Leu Leu Pro Leu Pro Met 370 375
380Thr Ala Thr Ser Tyr Arg Gly Asp His Ile Lys Leu Gln Ala Asp
Ser385 390 395 400Phe Gly
Leu His Ile Val Pro Val 405484188DNAHomo sapiens
48aggctccagt ctccggccga gtcttctcgc agccgcaacc cacctggggc cagcccagag
60ctgccagcgc cgctcggctc cctccctccc tcccggccct tcggccgcgg cggcgtgcgc
120ctgccttttc cgggggcggg ggcctggccc gcgcgctccc ctcccgcagg cgccacctcg
180gacatccccg ggattgctac ttctctgcca acttcgccaa ctcgccagca cttggagagg
240cccggctccc ctcccggcgc cctctgaccg cccccgcccc gcgcgctctc cgaccaccgc
300ctctcggatg accaggttcc aggggagctg agcgagtcgc ctcccccgcc cagcttcagc
360cctggctgca gctgcagcgc gagccatgcg cccccagtgc accccggccc ggcccaccgc
420cccggggcca ttctgctgac cgcccagccc cgagccccga cagtggcaag ttgcggctac
480tgcagttgca agctccggcc aacccggagg agccccagcg gggagcgcag tgttgcgccc
540cccgcccccg cgcgccccgc agcagccggg cgttcactca tcctccctcc cccaccgtcc
600ctcccttttc tcctcaagtc ctgaagttga gtttgagagg cgacacggcg gcggcggccg
660cgctgctccc gctcctctgc ctccccatgg atatgcactg caaagcagac cccttctccg
720cgatgcaccc agggcacggg ggtgtgaacc agctcggggg ggtgtttgtg aacggccggc
780ccctacccga cgtggtgagg cagcgcatcg tggagctggc ccaccagggt gtgcggccct
840gtgacatctc ccggcagctg cgggtcagcc acggctgtgt cagcaaaatc ctgggcaggt
900actacgagac cggcagcatc aagccgggtg tgatcggtgg ctccaagccc aaagtggcga
960cgcccaaagt ggtggacaag attgctgaat acaaacgaca gaacccgact atgttcgcct
1020gggagattcg agaccggctc ctggccgagg gcatctgtga caatgacaca gtgcccagcg
1080tctcttccat caacagaatc atccggacca aagttcagca gcctttccac ccaacgccgg
1140atggggctgg gacaggagtg accgcccctg gccacaccat tgttcccagc acggcctccc
1200ctcctgtttc cagcgcctcc aatgacccag tgggatccta ctccatcaat gggatcctgg
1260ggattcctcg ctccaatggt gagaagagga aacgtgatga agatgtgtct gagggctcag
1320tccccaatgg agattcccag agtggtgtgg acagtttgcg gaagcacttg cgagctgaca
1380ccttcaccca gcagcagctg gaagctttgg atcgggtctt tgagcgtcct tcctaccctg
1440acgtcttcca ggcatcagag cacatcaaat cagaacaggg gaacgagtac tccctcccag
1500ccctgacccc tgggcttgat gaagtcaagt cgagtctatc tgcatccacc aaccctgagc
1560tgggcagcaa cgtgtcaggc acacagacat acccagttgt gactggtcgt gacatggcga
1620gcaccactct gcctggttac ccccctcacg tgccccccac tggccaggga agctacccca
1680cctccaccct ggcaggaatg gtgcctggga gcgagttctc cggcaacccg tacagccacc
1740cccagtacac ggcctacaac gaggcttgga gattcagcaa ccccgcctta ctaatgccgc
1800cccccggtcc gcccctgccg ctgctgccgc tgcctatgac cgccactagt taccgcgggg
1860accacatcaa gcttcaggcc gacagcttcg gcctccacat cgtccccgtc tgaccccacc
1920ccggagggag ggaggaccga cgcgacgcga tgcctcccgg ccaccgcccc agcctcaccc
1980catcccacga cccccgcaac ccttcacatc acccccctcg aaggtcggac aggacgggtg
2040gagccgtggg cgggaccctc aggcccgggc ccgccgcccc cagccccgcc tgccgcccct
2100ccccgcctgc ctggactgcg cggcgccgtg agggggattc ggcccagctc gtcccggcct
2160ccaccaagcc agccccgaag cccgccagcc accctgccgg actcgggcgc gacctgctgg
2220cgcgcgccgg atgtttctgt gacacacaat cagcgcggac cgcagcgcgg cccagccccg
2280ggcacccgcc tcggacgctc gggcgccagg aggcttcgct ggaggggctg ggccaaggag
2340attaagaaga aaacgacttt ctgcaggagg aagagcccgc tgccgaatcc ctgggaaaaa
2400ttcttttccc ccagtgccag ccggactgcc ctcgccttcc gggtgtgccc tgtcccagaa
2460gatggaatgg gggtgtgggg gtccggctct aggaacgggc tttgggggcg tcaggtcttt
2520ccaaggttgg gacccaagga tcggggggcc cagcagcccg caccgatcga gccggactct
2580cggctcttca ctgctcctcc tggcctgcct agttccccag ggcccggcac ctcctgctgc
2640gagacccggc tctcagccct gccttgcccc tacctcagcg tctcttccac ctgctggcct
2700cccagtttcc cctcctgcca gtccttcgcc tgtcccttga cgccctgcat cctcctccct
2760gactcgcagc cccatcggac gctctcccgg gaccgccgca ggaccagttt ccatagactg
2820cggactgggg tcttcctcca gcagttactt gatgccccct cccccgacac agactctcaa
2880tctgccggtg gtaagaaccg gttctgagct ggcgtctgag ctgctgcggg gtggaagtgg
2940ggggctgccc actccactcc tcccatcccc tcccagcctc ctcctccggc aggaactgaa
3000cagaaccaca aaaagtctac atttatttaa tatgatggtc tttgcaaaaa ggaacaaaac
3060aacacaaaag cccaccaggc tgctgctttg tggaaagacg gtgtgtgtcg tgtgaaggcg
3120aaacccggtg tacataaccc ctccccctcc gccccgcccc gcccggcccc gtagagtccc
3180tgtcgcccgc cggccctgcc tgtagatacg ccccgctgtc tgtgctgtga gagtcgccgc
3240tcgctggggg ggaagggggg gacacagcta cacgcccatt aaagcacagc acgtcctggg
3300ggaggggggc attttttatg ttacaaaaaa aaattacgaa agaaaagaaa tctctatgca
3360aaatgacgaa catggtcctg tggactcctc tggcctgttt tgttggctct ttctctgtaa
3420ttccgtgttt tcgctttttc ctccctgccc ctctctccct ctgcccctct ctcctctccg
3480cttctctccc cctctgtctc tgtctctctc cgtctctgtc gctcttgtct gtctgtctct
3540gctctttcct cggcctctct ccccagacct ggcccggccg ccctgtctcc gcaggctaga
3600tccgaggtgg cagctccagc ccccgggctc gccccctcgc gggcgtgccc cgcgcgcccc
3660gggcggccga aggccgggcc gccccgtccc gccccgtagt tgctctttcg gtagtggcga
3720tgcgccctgc atgtctcctc acccgtggat cgtgacgact cgaaataaca gaaacaaagt
3780caataaagtg aaaataaata aaaatccttg aacaaatccg aaaaggcttg gagtcctcgc
3840ccagatctct ctcccctgcg agcccttttt atttgagaag gaaaaagaga aaagagaatc
3900gtttaaggga acccggcgcc cagccaggct ccagtggccc gaacggggcg gcgagggcgg
3960cgagggcgcc gaggtccggc ccatcccagt cctgtggggc tggccgggca gagaccccgg
4020acccaggccc aggcctaacc tgctaaatgt ccccggacgg ttctggtctc ctcggccact
4080ttcagtgcgt cggttcgttt tgattctttt tcttttgtgc acataagaaa taaataataa
4140taataaataa agaataaaat tttgtatgtc aaaaaaaaaa aaaaaaaa
418849431PRTHomo sapiens 49Met Asp Met His Cys Lys Ala Asp Pro Phe Ser
Ala Met His Pro Gly1 5 10
15His Gly Gly Val Asn Gln Leu Gly Gly Val Phe Val Asn Gly Arg Pro
20 25 30Leu Pro Asp Val Val Arg Gln
Arg Ile Val Glu Leu Ala His Gln Gly 35 40
45Val Arg Pro Cys Asp Ile Ser Arg Gln Leu Arg Val Ser His Gly
Cys 50 55 60Val Ser Lys Ile Leu Gly
Arg Tyr Tyr Glu Thr Gly Ser Ile Lys Pro65 70
75 80Gly Val Ile Gly Gly Ser Lys Pro Lys Val Ala
Thr Pro Lys Val Val 85 90
95Asp Lys Ile Ala Glu Tyr Lys Arg Gln Asn Pro Thr Met Phe Ala Trp
100 105 110Glu Ile Arg Asp Arg Leu
Leu Ala Glu Gly Ile Cys Asp Asn Asp Thr 115 120
125Val Pro Ser Val Ser Ser Ile Asn Arg Ile Ile Arg Thr Lys
Val Gln 130 135 140Gln Pro Phe His Pro
Thr Pro Asp Gly Ala Gly Thr Gly Val Thr Ala145 150
155 160Pro Gly His Thr Ile Val Pro Ser Thr Ala
Ser Pro Pro Val Ser Ser 165 170
175Ala Ser Asn Asp Pro Val Gly Ser Tyr Ser Ile Asn Gly Ile Leu Gly
180 185 190Ile Pro Arg Ser Asn
Gly Glu Lys Arg Lys Arg Asp Glu Val Glu Val 195
200 205Tyr Thr Asp Pro Ala His Ile Arg Gly Gly Gly Gly
Leu His Leu Val 210 215 220Trp Thr Leu
Arg Asp Val Ser Glu Gly Ser Val Pro Asn Gly Asp Ser225
230 235 240Gln Ser Gly Val Asp Ser Leu
Arg Lys His Leu Arg Ala Asp Thr Phe 245
250 255Thr Gln Gln Gln Leu Glu Ala Leu Asp Arg Val Phe
Glu Arg Pro Ser 260 265 270Tyr
Pro Asp Val Phe Gln Ala Ser Glu His Ile Lys Ser Glu Gln Gly 275
280 285Asn Glu Tyr Ser Leu Pro Ala Leu Thr
Pro Gly Leu Asp Glu Val Lys 290 295
300Ser Ser Leu Ser Ala Ser Thr Asn Pro Glu Leu Gly Ser Asn Val Ser305
310 315 320Gly Thr Gln Thr
Tyr Pro Val Val Thr Gly Arg Asp Met Ala Ser Thr 325
330 335Thr Leu Pro Gly Tyr Pro Pro His Val Pro
Pro Thr Gly Gln Gly Ser 340 345
350Tyr Pro Thr Ser Thr Leu Ala Gly Met Val Pro Gly Ser Glu Phe Ser
355 360 365Gly Asn Pro Tyr Ser His Pro
Gln Tyr Thr Ala Tyr Asn Glu Ala Trp 370 375
380Arg Phe Ser Asn Pro Ala Leu Leu Met Pro Pro Pro Gly Pro Pro
Leu385 390 395 400Pro Leu
Leu Pro Leu Pro Met Thr Ala Thr Ser Tyr Arg Gly Asp His
405 410 415Ile Lys Leu Gln Ala Asp Ser
Phe Gly Leu His Ile Val Pro Val 420 425
430504257DNAHomo sapiens 50aggctccagt ctccggccga gtcttctcgc
agccgcaacc cacctggggc cagcccagag 60ctgccagcgc cgctcggctc cctccctccc
tcccggccct tcggccgcgg cggcgtgcgc 120ctgccttttc cgggggcggg ggcctggccc
gcgcgctccc ctcccgcagg cgccacctcg 180gacatccccg ggattgctac ttctctgcca
acttcgccaa ctcgccagca cttggagagg 240cccggctccc ctcccggcgc cctctgaccg
cccccgcccc gcgcgctctc cgaccaccgc 300ctctcggatg accaggttcc aggggagctg
agcgagtcgc ctcccccgcc cagcttcagc 360cctggctgca gctgcagcgc gagccatgcg
cccccagtgc accccggccc ggcccaccgc 420cccggggcca ttctgctgac cgcccagccc
cgagccccga cagtggcaag ttgcggctac 480tgcagttgca agctccggcc aacccggagg
agccccagcg gggagcgcag tgttgcgccc 540cccgcccccg cgcgccccgc agcagccggg
cgttcactca tcctccctcc cccaccgtcc 600ctcccttttc tcctcaagtc ctgaagttga
gtttgagagg cgacacggcg gcggcggccg 660cgctgctccc gctcctctgc ctccccatgg
atatgcactg caaagcagac cccttctccg 720cgatgcaccc agggcacggg ggtgtgaacc
agctcggggg ggtgtttgtg aacggccggc 780ccctacccga cgtggtgagg cagcgcatcg
tggagctggc ccaccagggt gtgcggccct 840gtgacatctc ccggcagctg cgggtcagcc
acggctgtgt cagcaaaatc ctgggcaggt 900actacgagac cggcagcatc aagccgggtg
tgatcggtgg ctccaagccc aaagtggcga 960cgcccaaagt ggtggacaag attgctgaat
acaaacgaca gaacccgact atgttcgcct 1020gggagattcg agaccggctc ctggccgagg
gcatctgtga caatgacaca gtgcccagcg 1080tctcttccat caacagaatc atccggacca
aagttcagca gcctttccac ccaacgccgg 1140atggggctgg gacaggagtg accgcccctg
gccacaccat tgttcccagc acggcctccc 1200ctcctgtttc cagcgcctcc aatgacccag
tgggatccta ctccatcaat gggatcctgg 1260ggattcctcg ctccaatggt gagaagagga
aacgtgatga agttgaggta tacactgatc 1320ctgcccacat tagaggaggt ggaggtttgc
atctggtctg gactttaaga gatgtgtctg 1380agggctcagt ccccaatgga gattcccaga
gtggtgtgga cagtttgcgg aagcacttgc 1440gagctgacac cttcacccag cagcagctgg
aagctttgga tcgggtcttt gagcgtcctt 1500cctaccctga cgtcttccag gcatcagagc
acatcaaatc agaacagggg aacgagtact 1560ccctcccagc cctgacccct gggcttgatg
aagtcaagtc gagtctatct gcatccacca 1620accctgagct gggcagcaac gtgtcaggca
cacagacata cccagttgtg actggtcgtg 1680acatggcgag caccactctg cctggttacc
cccctcacgt gccccccact ggccagggaa 1740gctaccccac ctccaccctg gcaggaatgg
tgcctgggag cgagttctcc ggcaacccgt 1800acagccaccc ccagtacacg gcctacaacg
aggcttggag attcagcaac cccgccttac 1860taatgccgcc ccccggtccg cccctgccgc
tgctgccgct gcctatgacc gccactagtt 1920accgcgggga ccacatcaag cttcaggccg
acagcttcgg cctccacatc gtccccgtct 1980gaccccaccc cggagggagg gaggaccgac
gcgacgcgat gcctcccggc caccgcccca 2040gcctcacccc atcccacgac ccccgcaacc
cttcacatca cccccctcga aggtcggaca 2100ggacgggtgg agccgtgggc gggaccctca
ggcccgggcc cgccgccccc agccccgcct 2160gccgcccctc cccgcctgcc tggactgcgc
ggcgccgtga gggggattcg gcccagctcg 2220tcccggcctc caccaagcca gccccgaagc
ccgccagcca ccctgccgga ctcgggcgcg 2280acctgctggc gcgcgccgga tgtttctgtg
acacacaatc agcgcggacc gcagcgcggc 2340ccagccccgg gcacccgcct cggacgctcg
ggcgccagga ggcttcgctg gaggggctgg 2400gccaaggaga ttaagaagaa aacgactttc
tgcaggagga agagcccgct gccgaatccc 2460tgggaaaaat tcttttcccc cagtgccagc
cggactgccc tcgccttccg ggtgtgccct 2520gtcccagaag atggaatggg ggtgtggggg
tccggctcta ggaacgggct ttgggggcgt 2580caggtctttc caaggttggg acccaaggat
cggggggccc agcagcccgc accgatcgag 2640ccggactctc ggctcttcac tgctcctcct
ggcctgccta gttccccagg gcccggcacc 2700tcctgctgcg agacccggct ctcagccctg
ccttgcccct acctcagcgt ctcttccacc 2760tgctggcctc ccagtttccc ctcctgccag
tccttcgcct gtcccttgac gccctgcatc 2820ctcctccctg actcgcagcc ccatcggacg
ctctcccggg accgccgcag gaccagtttc 2880catagactgc ggactggggt cttcctccag
cagttacttg atgccccctc ccccgacaca 2940gactctcaat ctgccggtgg taagaaccgg
ttctgagctg gcgtctgagc tgctgcgggg 3000tggaagtggg gggctgccca ctccactcct
cccatcccct cccagcctcc tcctccggca 3060ggaactgaac agaaccacaa aaagtctaca
tttatttaat atgatggtct ttgcaaaaag 3120gaacaaaaca acacaaaagc ccaccaggct
gctgctttgt ggaaagacgg tgtgtgtcgt 3180gtgaaggcga aacccggtgt acataacccc
tccccctccg ccccgccccg cccggccccg 3240tagagtccct gtcgcccgcc ggccctgcct
gtagatacgc cccgctgtct gtgctgtgag 3300agtcgccgct cgctgggggg gaaggggggg
acacagctac acgcccatta aagcacagca 3360cgtcctgggg gaggggggca ttttttatgt
tacaaaaaaa aattacgaaa gaaaagaaat 3420ctctatgcaa aatgacgaac atggtcctgt
ggactcctct ggcctgtttt gttggctctt 3480tctctgtaat tccgtgtttt cgctttttcc
tccctgcccc tctctccctc tgcccctctc 3540tcctctccgc ttctctcccc ctctgtctct
gtctctctcc gtctctgtcg ctcttgtctg 3600tctgtctctg ctctttcctc ggcctctctc
cccagacctg gcccggccgc cctgtctccg 3660caggctagat ccgaggtggc agctccagcc
cccgggctcg ccccctcgcg ggcgtgcccc 3720gcgcgccccg ggcggccgaa ggccgggccg
ccccgtcccg ccccgtagtt gctctttcgg 3780tagtggcgat gcgccctgca tgtctcctca
cccgtggatc gtgacgactc gaaataacag 3840aaacaaagtc aataaagtga aaataaataa
aaatccttga acaaatccga aaaggcttgg 3900agtcctcgcc cagatctctc tcccctgcga
gcccttttta tttgagaagg aaaaagagaa 3960aagagaatcg tttaagggaa cccggcgccc
agccaggctc cagtggcccg aacggggcgg 4020cgagggcggc gagggcgccg aggtccggcc
catcccagtc ctgtggggct ggccgggcag 4080agaccccgga cccaggccca ggcctaacct
gctaaatgtc cccggacggt tctggtctcc 4140tcggccactt tcagtgcgtc ggttcgtttt
gattcttttt cttttgtgca cataagaaat 4200aaataataat aataaataaa gaataaaatt
ttgtatgtca aaaaaaaaaa aaaaaaa 42575118DNAHomo sapiens 51cctggcaccc
agcacaat 185220DNAHomo
sapiens 52gccgatccac acggagtact
205332DNAHomo sapiens 53gttgcctgcc agtcgccatg agaacttcct ac
325432DNAHomo sapiens 54tggccttccc tctgtaacag
gtgccttgaa tt 325524DNAHomo sapiens
55ccacccatgg caaattccat ggca
245624DNAHomo sapiens 56tctagacggc aggtcaggtc aacc
245723DNAHomo sapiens 57ctcaggccta tgcaaaaaga gga
235820DNAHomo sapiens
58gccctccctc caaaggagac
205923DNAHomo sapiens 59aacctacgca cctacgtgag gag
236022DNAHomo sapiens 60cgttcagtcc atcccatttc tg
226120DNAHomo sapiens
61gacacctgag ctgaccttgg
206220DNAHomo sapiens 62gaggaagtcc agtgtccagc
206368PRTHomo sapiens 63Met Arg Thr Ser Tyr Leu Leu
Leu Phe Thr Leu Cys Leu Leu Leu Ser1 5 10
15Glu Met Ala Ser Gly Gly Asn Phe Leu Thr Gly Leu Gly
His Arg Ser 20 25 30Asp His
Tyr Asn Cys Val Ser Ser Gly Gly Gln Cys Leu Tyr Ser Ala 35
40 45Cys Pro Ile Phe Thr Lys Ile Gln Gly Thr
Cys Tyr Arg Gly Lys Ala 50 55 60Lys
Cys Cys Lys6564914DNAHomo sapiensmisc_feature(1)..(914)n= a, c, g, or t
64ctgcagggtg ggcccaggct gggccnagac cctcaccctc caagggccac actgggggct
60cactttctga ggagtgccct ttggaaacgt cccaggaaca cgtctagtgg gaaaagagaa
120aagttggtcc atcgaggaga gtgttctgca taaggggaga gatgagaagg tagccttggc
180cagaggaaga aacttcatta caaccagctc tccttctsca agggaagagg gtgaagtttg
240agtttgtctt gcaggaagac aatcaaacta aagaggccaa caccagctta gagccgagcg
300gccccctgct cagagcttcc ctgtggctct cctccatgtg atccagaagg agggactcca
360gtgtgaactg cctgttccag aaaccccatc agaactgcct aacctagaaa accaaacagg
420aggagctggc accagggctc caggctgaaa gctaaatcca gcggcagcca gatggagaca
480atgtgccatg tgactgctga ctgctcaggg caaatgacac caggggttag cgattagaag
540ttcacccttg actgtggcac ctcccttcag ttccgtcgac gaggttgtgc aatccaccag
600tcttataaat acagtgacgc tccagcctct ggaagcctct gtcagctcag cctccaaagg
660agccagcctc tccccagttc ctgaaatcct gagtgttgcc tgccagtcgc catgagaact
720tcctaccttc tgctgtttac tctctgctta cttttgtctg agatggcctc aggtaagctc
780tggtacctgc tagagtttcc catccccagg gctggggaca atggggctga tgtgagtctc
840ggatggctgc ctccgtgtcc caagggacga ggaacaagca gcaggaaagc atcccgtggt
900tgagtggcct gcag
9146519DNAHomo sapiens 65tcagcagtgg agggcaatg
196623DNAHomo sapiens 66cctctgtaac aggtgccttg aat
236721DNAHomo sapiens
67acagcaaacc tcctcacagc c
216820DNAHomo sapiens 68tggagacgtg gcacctcttg
206924DNAHomo sapiens 69tatgataccc gggagatcgt gatc
247024DNAHomo sapiens
70gtgcagatgc cggttcaggt actc
247120DNAHomo sapiens 71tcagccctgg actacctgca
207220DNAHomo sapiens 72gaggtcccgg tacaccacgt
20
* * * * *