U.S. patent application number 10/012169 was filed with the patent office on 2010-12-09 for prostate-specific membrane antigen and uses thereof.
This patent application is currently assigned to Sloan Kettering Institute for Cancer Research. Invention is credited to William R. Fair, Warren D.W. Heston, Ron S. Israeli, Ouathek Ouerfelli, John Pinto.
Application Number | 20100310452 10/012169 |
Document ID | / |
Family ID | 29782717 |
Filed Date | 2010-12-09 |
United States Patent
Application |
20100310452 |
Kind Code |
A1 |
Israeli; Ron S. ; et
al. |
December 9, 2010 |
PROSTATE-SPECIFIC MEMBRANE ANTIGEN AND USES THEREOF
Abstract
This invention provides an isolated nucleic acid molecule
encoding an alternatively spliced human prostate-specific membrane
antigen. This invention provides an isolated nucleic acid
comprising a promoter sequence normally associated with the
transcription of a gene encoding a human prostate-specific membrane
antigen. This invention provides an isolated polypeptide having the
biological activity of an alternatively spliced prostate-specific
membrane antigen. This invention provides a method of detecting a
nucleic acid encoding an alternatively spliced human
prostate-specific membrane antigen and a method of detecting a
prostate tumor cell in a subject. Lastly, this invention provides a
pharmaceutical composition comprising a compound in a
therapeutically effective amount and a pharmaceutically acceptable
carrier and a method of making prostate cells susceptible to a
cytotoxic chemotherapeutic agent.
Inventors: |
Israeli; Ron S.; (Staten
Island, NY) ; Heston; Warren D.W.; (New York, NY)
; Fair; William R.; (New York, NY) ; Ouerfelli;
Ouathek; (New York, NY) ; Pinto; John; (East
Norwalk, CT) |
Correspondence
Address: |
COOPER & DUNHAM LLP
30 Rockefeller Plaza, 20th Floor
NEW YORK
NY
10112
US
|
Assignee: |
Sloan Kettering Institute for
Cancer Research
|
Family ID: |
29782717 |
Appl. No.: |
10/012169 |
Filed: |
October 24, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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08705477 |
Aug 29, 1996 |
6569432 |
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10012169 |
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PCT/US96/02424 |
Feb 23, 1996 |
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08705477 |
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08394152 |
Feb 24, 1995 |
5935818 |
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PCT/US96/02424 |
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Current U.S.
Class: |
424/1.49 ;
424/9.1; 424/9.6; 435/7.23 |
Current CPC
Class: |
A61K 39/0011 20130101;
G01N 2800/342 20130101; A61K 39/001195 20180801; C12Q 1/6886
20130101; A61P 35/00 20180101; C12Q 2600/112 20130101; C12Q
2600/118 20130101; C07K 14/705 20130101 |
Class at
Publication: |
424/1.49 ;
424/9.1; 435/7.23; 424/9.6 |
International
Class: |
A61K 51/10 20060101
A61K051/10; A61K 49/16 20060101 A61K049/16; G01N 33/574 20060101
G01N033/574; A61P 35/00 20060101 A61P035/00 |
Goverment Interests
[0002] This invention disclosed herein was made in part with
Government support under Grants Nos. DK47650 and CA58192, CA-39203,
CA-29502, CA-08748-29 from the National Institute of Health, U.S.
Department of Health and Human Services. Accordingly, the U.S.
Government has certain rights in this invention.
Claims
1-44. (canceled)
45. A method of detecting cancerous kidney, colon or bladder cells
of a subject with cancer comprising: (A) contacting an endothelial
cell of the neovasculature of the kidney, colon or bladder of the
subject with an antibody or administering the antibody to the
subject, wherein the antibody binds to the extracellular domain of
prostate specific membrane antigen under conditions effective to
permit binding of the antibody to the endothelial cell so as to
form a complex and the antibody has bound thereto an imaging agent,
and (B) detecting the complex, wherein detection of the complex
indicates cancerous kidney, colon or bladder cells in the
subject.
46. (canceled)
47. (canceled)
48. (canceled)
49. (canceled)
50. (canceled)
52. The method of claim 45, wherein the subject is human.
53. The method of claim 45, wherein the imaging agent is a
radioactive, a colorimetric, a luminescent, or a fluorescent agent
or a standard pharmacophore.
54. The method of claim 45, wherein said administering is carried
out intravenously.
55. (canceled)
56. The method of claim 45, wherein the antibody is a monoclonal
antibody or a polyclonal antibody.
57. (canceled)
58. The method of claim 45, wherein the antibody is in a
composition further comprising a physiologically acceptable
carrier, excipient, or stabilizer.
59. The method of claim 45, wherein the antibody is in a
composition further comprising a pharmaceutically acceptable
carrier, excipient, or stabilizer.
60. The method of claim 45, wherein said contacting is carried out
in a sample of urine.
61. The method of claim 45, wherein said contacting is carried out
in a tissue sample.
Description
[0001] This application is a continuation-in-part of International
Application PCT/US96/02424, filed Jul. 19, 1996, which is a
continuation-in-part application of U.S. application Ser. No.
08/394,152, filed Feb. 24, 1995, the contents of which are hereby
incorporated by reference.
BACKGROUND OF THE INVENTION
[0003] Throughout this application various references are referred
to within parentheses. Disclosures of these publications in their
entireties are hereby incorporated by reference into this
application to more fully describe the state of the art to which
this invention pertains. Full bibliographic citation for these
references may be found at the end of each set of Examples in the
Experimental Details section.
[0004] Prostate cancer is among the most significant medical
problems in the United States, as the disease is now the most
common malignancy diagnosed in American males. In 1992 there were
over 132,000 new cases of prostate cancer detected with over 36,000
deaths attributable to the disease, representing a 17.3% increase
over 4 years (8). Five year survival rates for patients with
prostate cancer range from 88% for those with localized disease to
29% for those with metastatic disease. The rapid increase in the
number of cases appears to result in part from an increase in
disease awareness as well as the widespread use of clinical markers
such as the secreted proteins prostate-specific antigen (PSA) and
prostatic acid phosphatase (PAP) (7).
[0005] The prostate gland is a site of significant pathology
affected by conditions such as benign growth (BPH), neoplasia
(prostatic cancer) and infection (prostatitis). Prostate cancer
represents the second leading cause of death from cancer in man
(7). However prostatic cancer is the leading site for cancer
development in men. The difference between these two facts relates
to prostatic cancer occurring with increasing frequency as men age,
especially in the ages beyond 60 at a time when death from other
factors often intervenes. Also, the spectrum of biologic
aggressiveness of prostatic cancer is great, so that in some men
following detection the tumor remains a latent histologic tumor and
does not become clinically significant, whereas in other it
progresses rapidly, metastasizes and kills the man in a relatively
short 2-5 year period (7 and 59).
[0006] In prostate cancer cells, two specific proteins that are
made in very high concentrations are prostatic acid phosphatase
(PAP) and prostate specific antigen (PSA) (21, 47, and 65). These
proteins have been characterized and have been used to follow
response to therapy. With the development of cancer, the normal
architecture of the gland becomes altered, including loss of the
normal duct structure for the removal of secretions and thus the
secretions reach the serum. Indeed measurement of serum PSA is
suggested as a potential screening method for prostatic cancer.
Indeed, the relative amount of PSA and/or PAP in the cancer reduces
as compared to normal or benign tissue. PAP was one of the earliest
serum markers for detecting metastatic spread (47). PAP hydrolyses
tyrosine phosphate and has a broad substrate specificity. Tyrosine
phosphorylation is often increased with oncogenic transformation.
It has been hypothesized that during neoplastic transformation
there is less phosphatase activity available to inactivate proteins
that are activated by phosphorylation on tyrosine residues. In some
instances, insertion of phosphatases that have tyrosine phosphatase
activity has reversed the malignant phenotype.
[0007] PSA is a protease and it is not readily appreciated how loss
of its activity correlates with cancer development (21, and 65).
The proteolytic activity of PSA is inhibited by zinc. Zinc
concentrations are high in the normal prostate and reduced in
prostatic cancer. Possibly the loss of zinc allows for increased
proteolytic activity by PSA. As proteases are involved in
metastasis and some proteases stimulate mitotic activity, the
potentially increased activity of PSA could be hypothesized to play
a role in the tumors metastases and spread (39).
[0008] Both PSA and PAP are found in prostatic secretions. Both
appear to be dependent on the presence of androgens for their
production and are substantially reduced following androgen
deprivation.
[0009] Prostate-specific membrane antigen (PSM) which appears to be
localized to the prostatic membrane has been identified. This
antigen was identified as the result of generating monoclonal
antibodies to a prostatic cancer cell, LNCaP (22).
[0010] Dr. Horoszewicz established a cell line designated LNCaP
from the lymph node of a hormone refractory, heavily pretreated
patient (23). This line was found to have an aneuploid human male
karyotype. It maintained prostatic differentiation functionality in
that it produced both PSA and PAP. It possessed an androgen
receptor of high affinity and specificity. Mice were immunized with
LNCaP cells and hybridomas were derived from sensitized animals. A
monoclonal antibody was derived and was designated 7E11-C5 (22).
The antibody staining was consistent with a membrane location and
isolated fractions of LNCaP cell membranes exhibited a strongly
positive reaction with immunoblotting and ELISA techniques. This
antibody did not inhibit or enhance the growth of LNCaP cells in
vitro or in vivo. The antibody to this antigen was remarkably
specific to prostatic epithelial cells, as no reactivity was
observed in any other component. Immunohistochemical staining of
cancerous epithelial cells was more intense than that of normal or
benign epithelial cells.
[0011] Dr. Horoszewicz also reported detection of immunoreactive
material using 7E11-C5 in serum of prostatic cancer patients (22).
The immunoreactivity was detectable in nearly 60% of patients with
stage D-2 disease and in a slightly lower percentage of patients
with earlier stage disease, but the numbers of patients in the
latter group are small. Patients with benign prostatic hyperplasia
(BPH) were negative. Patients with no apparent disease were
negative, but 50-60% of patients in remission yet with active
stable disease or with progression demonstrated positive serum
reactivity. Patients with non prostatic tumors did not show
immunoreactivity with 7E11-C5.
[0012] The 7E11-C5 monoclonal antibody is currently in clinical
trials. The aldehyde groups of the antibody were oxidized and the
linker-chelator
glycol-tyrosyl-(n,.epsilon.-diethylenetriamine-pentacetic
acid)-lysine (GYK-DTPA) was coupled to the reactive aldehydes of
the heavy chain. The resulting antibody was designated CYT-356.
Immunohistochemical staining patterns were similar except that the
CYT-356 modified antibody stained skeletal muscle. The comparison
of CYT-356 with 7E11-C5 monoclonal antibody suggested both had
binding to type 2 muscle fibers. The reason for the discrepancy
with the earlier study, which reported skeletal muscle to be
negative, was suggested to be due to differences in tissue fixation
techniques. Still, the most intense and definite reaction was
observed with prostatic epithelial cells, especially cancerous
cells. Reactivity with mouse skeletal muscle was detected with
immunohistochemistry but not in imaging studies. The
Indium.sup.111-labeled antibody localized to LNCaP tumors grown in
nude mice with an uptake of nearly 30% of the injected dose per
gram tumor at four days. In-vivo, no selective retention of the
antibody was observed in antigen negative tumors such as PC-3 and
DU-145, or by skeletal muscle. Very little was known about the PSM
antigen. An effort at purification and characterization has been
described at meetings by Dr. George Wright and colleagues (14 and
64).
BRIEF DESCRIPTION OF THE FIGURES
[0013] FIGS. 1A-1C: [0014] Immunohistochemical detection of PSM
antigen expression in prostate cell lines. Top panel reveals
uniformly high level of expression in LNCaP cells; middle panel and
lower panel are DU-145 and PC-3 cells respectively, both
negative.
[0015] FIG. 2: Autoradiogram of protein gel revealing products of
PSM coupled in-vitro transcription/translation. Non-glycosylated
PSM polypeptide is seen at 84 kDa (lane 1) and PSM glycoprotein
synthesized following the addition of microsomes is seen at 100 kDa
(lane 2).
[0016] Western Blot analysis detecting PSM expression in
transfected non-PSM expressing PC-3 cells. 100 kDa PSM glycoprotein
species is clearly seen in LNCaP membranes (lane 1), LNCaP crude
lysate (lane 2), and PSM-transfected PC-3 cells (lane 4), but is
undetectable in native PC-3 cells (lane 3).
[0017] FIG. 4: Autoradiogram of ribonuclease protection gel
assaying for PSM mRNA expression in normal human tissues.
Radiolabeled 1 kb DNA ladder (Gibco-BRL) is shown in lane 1.
Undigested probe is 400 nucleotides (lane 2), expected protected
PSM band is 350 nucleotides, and tRNA control is shown (lane 3). A
strong signal is seen in human prostate (lane 11), with very faint,
but detectable signals seen in human brain (lane 4) and human
salivary gland (lane 12). No signal was detected in lane 5 kidney,
lane 6 liver, lane 7 lung, lane 8 mammary gland, lane 9 pancreas,
lane 10 placenta, lane 13 skeletal muscle, lane 14 spleen, and lane
15 testes.
[0018] FIG. 5: Autoradiogram of ribonuclease protection gel
assaying for PSM mRNA expression in LNCaP tumors grown in nude
mice, and in human prostatic tissues. .sup.32P-labeled 1 kb DNA
ladder is shown in lane 1. 298 nucleotide undigested probe is shown
(lane 2), and tRNA control is shown (lane 3). PSM mRNA expression
is clearly detectable in LNCaP cells (lane 4), orthotopically grown
LNCaP tumors in nude mice with and without matrigel (lanes 5 and
6), and subcutaneously implanted and grown LNCaP tumors in nude
mice (lane 7). PSM mRNA expression is also seen in normal human
prostate (lane 8), and in a moderately differentiated human
prostatic adenocarcinoma (lane 10). Very faint expression is seen
in a sample of human prostate tissue with benign hyperplasia (lane
9).
[0019] FIG. 6: Ribonuclease protection assay for PSM expression in
LNCaP cells treated with physiologic doses of various steroids for
24 hours. .sup.32P-labeled DNA ladder is shown in lane 1. 298
nucleotide undigested probe is shown (lane 2), and tRNA control is
shown (lane 3). PSM mRNA expression is highest in untreated LNCaP
cells in charcoal-stripped media (lane 4). Applicant see
significantly diminished PSM expression in LNCaP cells treated with
DHT (lane 5), Testosterone (lane 6), Estradiol (line 7), and
Progesterone (lane 8), with little response to Dexamethasone (lane
9).
[0020] FIG. 7: Data illustrating results of PSM DNA and RNA
presence in transfect Dunning cell lines employing Southern and
Northern blotting techniques
[0021] FIGS. 8A-8B: [0022] Figure A indicates the power of cytokine
transfected cells to teach unmodified cells. Administration was
directed to the parental flank or prostate cells. The results
indicate the microenvironment considerations. [0023] Figure B
indicates actual potency at a particular site. The tumor was
implanted in prostate cells and treated with immune cells at two
different sites.
[0024] FIGS. 9A-9B: [0025] Relates potency of cytokines in
inhibiting growth of primary tumors. Animals administered
un-modified parental tumor cells and administered as a vaccine
transfected cells. Following prostatectomy of rodent tumor results
in survival increase.
[0026] FIG. 10: PCR amplification with nested primers improved the
level of detection of prostatic cells from approximately one
prostatic cell per 10,000 MCF-7 cells to better than one cell per
million MCF-7 cells, using PSA.
[0027] FIG. 11: PCR amplification with nested primers improved the
level of detection of prostatic cells from approximately one
prostatic cell per 10,000 MCF-7 cells to better than one cell per
million MCF-7 cells, using PSM-derived primers.
[0028] FIG. 12: A representative ethidium stained gel photograph
for PSM-PCR. Samples run in lane A represent PCR products generated
from the outer primers and samples in lanes labeled B are products
of inner primer pairs.
[0029] FIG. 13: PSM Southern blot autoradiograph. The sensitivity
of the Southern blot analysis exceeded that of ethidium staining,
as can be seen in several samples where the outer product is not
visible, but is detectable by Southern blotting.
[0030] FIG. 14: Characteristics of the 16 patients analyzed with
respect to their clinical stage, treatment, serum PSA and PAP
values, and results of assay.
[0031] FIGS. 15A-15D: [0032] DNA sequence containing promoter
elements from nucleotide -1 to nucleotide -3017. -1 is upstream of
start site of PSM.
[0033] FIG. 16: Potential binding sites on the PSM promoter
fragment.
[0034] FIG. 17: Promoter activity of PSM up-stream fragment/CAT
gene chimera.
[0035] FIG. 18: Comparison between PSM and PSM' cDNA. Sequence of
the 5' end of PSM cDNA (32) is shown. Underlined region (beginning
at nucleotide 115 and continuing to nucleotide 380) denotes
nucleotides which are absent in PSM' cDNA but present in PSM cDNA.
Boxed region represents the putative transmembrane domain of PSM
antigen. * Asterisk denotes the putative translation initiation
site for PSM'.
[0036] FIG. 19: Graphical representation of PSM and PSM' cDNA
sequences and antisense PSM RNA probe (b). PSM cDNA sequence with
complete coding region (32). (a) PSM' cDNA sequence from this
study. (c) Cross hatched and open boxes denote sequences identity
in PSM and PSM'. Hatched box indicates sequence absent from PSM'.
Regions of cDNA sequence complementary to the antisense probe are
indicated by dashed lines between the sequences.
[0037] FIG. 20: RNase protection assay with PSM specific probe in
primary prostatic tissues. Total cellular RNA was isolated from
human prostatic samples: normal prostate, BPH, and CaP. PSM and
PSM' spliced variants are indicated with arrows at right. The left
lane is a DNA ladder. Samples from different patients are
classified as: lanes 3-6, CaP, carcinoma of prostate; BPH, benign
prostatic hypertrophy, lanes 7-9; normal, normal prostatic tissue,
lanes 10-12. Autoradiograph was exposed for longer period to read
lanes 5 and 9.
[0038] FIG. 21: Tumor Index, a quantification of the expression of
PSM and PSM'. Expression of PSM and PSM' was quantified by
densitometry and expressed as a ratio of PSM/PSM' on the Y-axis.
Three samples each were quantitated for primary CaP, BPH and normal
prostate tissues. Two samples were quantitated for LNCaP. Normal,
normal prostate tissue.
[0039] FIG. 22: Characterization of PSM membrane bound and PSM' in
the cytosol.
[0040] FIG. 23: Photograph of ethidium bromide stained gel
depicting representative negative and positive controls used in the
study. Samples 1-5 were from, respectively: male with prostatis, a
healthy female volunteer, a male with BPH, a control 1:1,000,000
dilution of LNCaP cells, and a patient with renal cell carcinoma.
Below each reaction is the corresponding control reaction performed
with beta-2-microglobulin primers to assure RNA integrity. No PCR
products were detected for any of these negative controls.
[0041] FIG. 24: Photograph of gel displaying representative
positive PCR results using PSM primers in selected patients with
either localized or disseminated prostate cancer. Sample 1-5 were
from respectively: a patient with clinically localized stage
T1.sub.c disease, a radical prostatectomy patient with organ
confined disease and a negative serum PSA, a radical prostatectomy
patient with locally advanced disease and a negative serum PSA, a
patient with treated stage D2 disease, and a patient with treated
hormone refractory disease.
[0042] FIG. 25: Chromosomal location of PSM based on in-situ
hybridization with cDNA and with genomic cosmids.
[0043] FIG. 26: Human monochromosomal somatic cell hybrid blot
showing that chromosome 11 contained the PSM genetic sequence by
Southern analysis. DNA panel digested with PstI restriction enzyme
and probed with PSM cDNA. Lanes M and H refer to mouse and hamster
DNAs. The numbers correspond to the human chromosomal DNA in that
hybrid.
[0044] FIG. 27: Ribonuclease protection assay using PSM
radiolabeled RNA probe revels an abundant PSM mRNA expression in
AT6.1-11 clone 1, but not in AT6.1-11 clone 2, thereby mapping PSM
to 11p11.2-13 region.
[0045] FIG. 28: Tissue specific expression of PSM RNA by Northern
blotting and RNAse protection assay.
[0046] FIG. 29: Mapping of the PSM gene to the 11p11.2-p13 region
of human chromosome 11 by southern blotting and in-situ
hybridization.
[0047] FIG. 30: Schematic of potential response elements.
[0048] FIG. 31: Schematic depiction of metastatic prostate cell
transfected with promoter for PSM which is driving expression of
prodrug activating enzyme cytosine deaminase. This allows for
prostate specific expression and tumor localized conversion of
non-toxic 5 fluorocytosine to 5 fluorouracil.
[0049] FIG. 32A-32C: [0050] Nucleic acid of PSM genomic DNA is read
5 prime away from the transcription start site: number on the
sequences indicates nucleotide upstream from the start site.
Therefore, nucleotide #121 is actually -121 using conventional
numbering system.
[0051] FIG. 33: Representation of NAAG 1, acividin, azotomycin, and
6-diazo-5-oxo-norleucine, DON.
[0052] FIG. 34: Representation of N-acetylaspartylglutamate (NAAG),
PALA, PALAGLU, phosphonate antagonist of glutamate receptor and
phosphonates of PALAGLU and NAAG.
[0053] FIG. 35: Synthesis of N-acetylaspartylglutamate, NAAG 1.
[0054] FIG. 36: Synthesis of
N-phosphonoacetylaspartyl-L-glutamate.
[0055] FIG. 37: Synthesis of 5-diethylphosphonon-2 amino
benzylvalerate intermediate.
[0056] FIG. 38: Synthesis of analog 4 and 5.
[0057] FIG. 39: Representation of DON, analogs 17-20.
[0058] FIG. 40: Substrates for targeted drug delivery, analog 21
and 22.
[0059] FIG. 41: Dynemycin A and its mode of action.
[0060] FIG. 42: Synthesis of analog 28.
[0061] FIG. 43: Synthesis for intermediate analog 28.
[0062] FIG. 44: Attachment points for PALA.
[0063] FIG. 45: Mode of action for substrate 21.
[0064] FIGS. 46A-46D: [0065] Intron 1F: Forward Sequence.
[0066] FIGS. 47A-47E: [0067] Intron 1R: Reverse Sequence
[0068] FIGS. 48A-48C: [0069] Intron 2F: Forward Sequence
[0070] FIGS. 49A-49C: [0071] Intron 2R: Reverse Sequence
[0072] FIGS. 50A-508: [0073] Intron 3F: Forward Sequence
[0074] FIGS. 51A-51B: [0075] Intron 3R: Reverse Sequence
[0076] FIGS. 52A-52C: [0077] Intron 4F: Forward Sequence
[0078] FIGS. 53A-53E: [0079] Intron 4RF: Reverse Sequence
[0080] FIG. 54: PSM genomic organization of the exon and 19 intron
junction sequences. The exon/intron junctions are as follows:
[0081] 1. Exon/intron 1 at by 389-390; [0082] 2. Exon/intron 2 at
by 490-491; [0083] 3. Exon/intron 3 at by 681-682; [0084] 4.
Exon/intron 4 at by 784-785; [0085] 5. Exon/intron 5 at by 911-912;
[0086] 6. Exon/intron 6 at by 1096-1097; [0087] 7. Exon/intron 7 at
by 1190-1191; [0088] 8. Exon/intron 8 at by 1289-1290; [0089] 9.
Exon/intron 9 at by 1375-1376; [0090] 10. Exon/intron 10 at by
1496-1497; [0091] 11. Exon/intron 11 at by 1579-1580; [0092] 12.
Exon/intron 12 at by 1643-1644; [0093] 13. Exon/intron 13 at by
1710-1711; [0094] 14. Exon/intron 14 at by 1803-1804; [0095] 15.
Exon/intron 15 at by 1894-1895; [0096] 16. Exon/intron 16 at by
2158-2159; [0097] 17. Exon/intron 17 at by 2240-2241; [0098] 18.
Exon/intron 18 at by 2334-2335; [0099] 19. Exon/intron 19 at by
2644-2645.
[0100] FIGS. 55A-55J: [0101] Alternatively spliced PSM
(PSM')nucleic acid sequence and amino acid sequence.
[0102] FIG. 56: PSM pteroyl (folate) hydrolase activity in LNCaP
membrane preparation. Time course of MTXglu.sub.3 hydrolysis
(-.box-solid.-) and concurrent formation of MTXglu.sub.2 (- -),
MTXglu.sub.1 (-.tangle-solidup.-), and MTX (- -), respectively.
Membrane fractions were prepared as described in Methods. Reaction
volume was 100 .mu.L containing 50 mM acetate/Triton buffer pH 4.5,
50 .mu.M MTXglu.sub.3, 10 .mu.g/mL protein. Values are x.+-.S.D.
from three separate LNCaP membrane preparations.
[0103] FIG. 57: PSM pteroyl (folate) hydrolase activity of
immunoprecipitated PSM antigen. Diagram shows typical capillary
electrophoretic separation patterns of MTXglu.sub.(n) derivatives
at 0, 30, 60 and 240 minute reaction times. Elution intervals for
MTXglu.sub.3, MTXglu.sub.2, MTXglu.sub.1, and MTX are 4.25, 3.95,
3.55, and 3.06 min, respectively. Total volume of reaction mixture
was 100 uL containing 50 uM MTXglu.sub.3.
[0104] FIG. 58: Effects of pH on gamma-glutamyl hydrolase (PSM
hydrolase) activity in LNCaP, PC-3 PSM-transfected (PC-3(+)) and
PSM non-transfected (PC-3(-)) cells. Enzymic activity is reported
as .mu.M MTXglu.sub.2 formed/mg protein. Each value represents the
mean of 3 reactions containing 50-60 .mu.g/mL protein. The
following buffers were used in 50 mM concentrations spanning a pH
range of 2 to 10: glycine-HCl, pH 2.2 to 3.6; acetate, pH 3.6 to
5.6; 2-(N-morpholino)ethanesulfonic acid (MES), pH 5.6 to 6.8;
Tris(hydroxymethyl)aminomethane (TRIS), pH 7 to 8.5; and
glycine-NaOH, pH 8.6 to 10.0.
[0105] FIG. 59: Comparison of pteroyl hydrolase activity in
membranes isolated from LNCaP, PC-3, TSU-Pr1, and Duke-145
adenocarcinoma cell lines. Membranes were isolated as described in
Methods. Each value represents the mean of triplicate reactions
normalized to 1 mg/mL protein.
[0106] FIG. 60A-60C: [0107] Immunohistochemical analysis of LNCaP
and PC-3 PSM-transfected and PSM-non-transfected cells. A 2.65 kb
PSM cDNA containing a hygromycin selection vector was cloned into
non PSM-antigen expressing PC-3 cells and maintained in regular
media supplemented with hygromycin B. As a control, PC-3 cells were
also transfected with the pREP7 vector alone (PC-3 PSM
non-transfected cells). Cells were permeabilized in
acetone/methanol (1:1 v/v) mixture, blocked with 5% bovine serum
albumin/Tris buffered saline (TBS) and the 7E11-C5 monoclonal PSM
antibody was added to cells. A secondary anti-mouse IgG.sub.1
antibody conjugated with alkaline phosphatase was added and
PSM-positive cell staining performed with bromochloroindolylphenol
phosphate. Panel A demonstrates intense immunoreactivity associated
with LNCaP cells using the monoclonal PSM antibody; In panel B,
comparable staining occurs in PC-3 cells transfected with PSM
expression vector. Panel C illustrates PC-3 cells expressing pREP7
hygromycin vector alone.
[0108] FIG. 61: Comparison of pteroyl (folate) hydrolase activity
in membranes isolated from PSM expressing PC-3 cells and PC-3 cells
expressing pREP7 hygromycin vector alone. Membranes were isolated
as described in Methods. Each value represents the mean of
triplicate reactions normalized to 1 mg/mL protein.
[0109] FIG. 62: Representation of N-acetylaspartylglutamate (NAAG),
folic acid, folate-gamma-polyglutamate, methotrexate,
methotrexate-gamma-polyglutamate, methotrexate-alpha-monoglutamate,
methotrexate-gamma-diglutamate, methotrexate-gamma-triglutamate,
methotrexate-gamma-tetraglutamate.
[0110] FIG. 63A-63B: [0111] Solid phase synthesis of methotrexate
alpha-polyglutamatae analogs.
[0112] FIG. 64: Sequence analysis of microsatellite instability in
PSM gene.
[0113] FIG. 65: PSM genomic organization.
[0114] FIG. 66: Location of microsatellite in PSM gene
SUMMARY OF THE INVENTION
[0115] This invention provides an isolated nucleic acid molecule
encoding an alternatively spliced human prostate-specific membrane
antigen. This invention provides an isolated nucleic acid
comprising a promoter sequence normally associated with the
transcription of a gene encoding a human prostate-specific membrane
antigen. This invention provides an isolated polypeptide having the
biological activity of an alternatively spliced prostate-specific
membrane antigen.
[0116] This invention provides a method of detecting a nucleic acid
encoding an alternatively spliced human prostate-specific membrane
antigen and a method of detecting a prostate tumor cell in a
subject.
[0117] Lastly, this invention provides a pharmaceutical composition
comprising a compound in a therapeutically effective amount and a
pharmaceutically acceptable carrier and a method of making prostate
cells susceptible to a cytotoxic agent.
DETAILED DESCRIPTION OF THE INVENTION
[0118] This invention provides an isolated nucleic acid encoding an
alternatively spliced human prostate-specific membrane (PSM')
antigen. As defined herein "nucleic acid encoding an alternatively
spliced prostate-specific membrane (PSM') antigen" means nucleic
acid encoding a prostate-specific membrane antigen which contains a
deletion in the DNA sequence encoding prostate specific membrane
antigen between nucleotide 115 and 380. In one embodiment the
isolated nucleic acid encodes the alternatively spliced human
prostate-specific membrane antigen as set forth in FIG. 55.
[0119] This invention further provides an isolated mammalian
genomic DNA molecule which encodes an alternatively spliced
prostate-specific membrane antigen. This invention further provides
an isolated mammalian DNA molecule of an isolated mammalian nucleic
acid molecule encoding an alternatively spliced prostate-specific
membrane antigen. This invention also provides an isolated
mammalian cDNA molecule encoding a mammalian alternatively spliced
prostate-specific membrane antigen. This invention provides an
isolated mammalian RNA molecule encoding a mammalian alternatively
spliced prostate-specific membrane antigen.
[0120] This invention also encompasses DNAs and cDNAs which encode
amino acid sequences which differ from those of PSM' antigen, but
which should not produce phenotypic changes. Alternatively, this
invention also encompasses DNAs and cDNAs which hybridize to the
DNA and cDNA of the subject invention. Hybridization methods are
well known to those of skill in the art.
[0121] This invention also provides a nucleic acid molecule of at
least 15 nucleotides capable of specifically hybridizing with a
sequence of a nucleic acid molecule encoding the prostate-specific
membrane antigen. This molecule may either be a DNA or RNA
molecule.
[0122] This invention provides a nucleic acid sequence of at least
15 nucleotides capable of specifically hybridizing to a sequence
within a DNA sequence encoding prostate specific membrane antigen
located between nucleotide 115 and nucleotide 380.
[0123] The nucleic acid molecule capable of specifically
hybridizing with a sequence of a nucleic acid molecule encoding the
prostate-specific membrane antigen can be used as a probe. Nucleic
acid probe technology is well known to those skilled in the art who
will readily appreciate that such probes may vary greatly in length
and may be labeled with a detectable label, such as a radioisotope
or fluorescent dye, to facilitate detection of the probe. DNA probe
molecules may be produced by insertion of a DNA molecule which
encodes PSM antigen into suitable vectors, such as plasmids or
bacteriophages, followed by transforming into suitable bacterial
host cells, replication in the transformed bacterial host cells and
harvesting of the DNA probes, using methods well known in the art.
Alternatively, probes may be generated chemically from DNA
synthesizers.
[0124] RNA probes may be generated by inserting the PSM antigen
molecule downstream of a bacteriophage promoter such as T3, T7 or
SP6. Large amounts of RNA probe may be produced by incubating the
labeled nucleotides with the linearized PSM antigen fragment where
it contains an upstream promoter in the presence of the appropriate
RNA polymerase.
[0125] For example, high stringent hybridization conditions are
selected at about 5.degree. C. lower than the thermal melting point
(Tm) for the specific sequence at a defined ionic strength and pH.
The Tm is the temperature (under defined ionic strength and pH) at
which 50% of the target sequence hybridizes to a perfectly matched
probe. Typically, stringent conditions will be those in which the
salt concentration is at least about 0.02 molar at pH 7 and the
temperature is at least about 60.degree. C. As other factors may
significantly affect the stringency of hybridization, including,
among others, base composition and size of the complementary
strands, the presence of organic solvents, ie. salt or formamide
concentration, and the extent of base mismatching, the combination
of parameters is more important than the absolute measure of any
one. For Example high stringency may be attained for example by
overnight hybridization at about 68.degree. C. in a 6.times.SSC
solution, washing at room temperature with 6.times.SSC solution,
followed by washing at about 68.degree. C. in a 6.times.SSC in a
0.6.times.SSX solution.
[0126] Hybridization with moderate stringency may be attained for
example by: 1) filter pre-hybridizing and hybridizing with a
solution of 3.times. sodium chloride, sodium citrate (SSC), 50%
formamide, 0.1M Tris buffer at Ph 7.5, 5.times.Denhardt's solution;
2.) pre-hybridization at 37.degree. C. for 4 hours; 3)
hybridization at 37.degree. C. with amount of labelled probe equal
to 3,000,000 cpm total for 16 hours; 4) wash in 2.times.SSC and
0.1% SDS solution; 5) wash 4.times. for 1 minute each at room
temperature at 4.times. at 60.degree. C. for 30 minutes each; and
6) dry and expose to film.
[0127] The DNA molecules described and claimed herein are useful
for the information which they provide concerning the amino acid
sequence of the polypeptide and as products for the large scale
synthesis of the polypeptide by a variety of recombinant
techniques. The molecule is useful for generating new cloning and
expression vectors, transformed and transfected prokaryotic and
eukaryotic host cells, and new and useful methods for cultured
growth of such host cells capable of expression of the polypeptide
and related products.
[0128] Moreover, the isolated mammalian nucleic acid molecules
encoding a mammalian prostate-specific membrane antigen and the
alternatively spliced PSM' are useful for the development of probes
to study the tumorigenesis of prostate cancer.
[0129] The nucleic acid molecules synthesized above may be used to
detect expression of a PSM' antigen by detecting the presence of
mRNA coding for the PSM antigen. Total mRNA from the cell may be
isolated by many procedures well known to a person of ordinary
skill in the art. The hybridizing conditions of the labelled
nucleic acid molecules may be determined by routine experimentation
well known in the art. The presence of mRNA hybridized to the probe
may be determined by gel electrophoresis or other methods known in
the art. By measuring the amount of the hybrid made, the expression
of the PSM and PSM' antigen by the cell can be determined. The
labeling may be radioactive. For an example, one or more
radioactive nucleotides can be incorporated in the nucleic acid
when it is made.
[0130] In one embodiment of this invention, nucleic acids are
extracted by precipitation from lysed cells and the mRNA is
isolated from the extract using an oligo-dT column which binds the
poly-A tails of the mRNA molecules. The mRNA is then exposed to
radioactively labelled probe on a nitrocellulose membrane, and the
probe hybridizes to and thereby labels complementary mRNA
sequences. Binding may be detected by luminescence autoradiography
or scintillation counting. However, other methods for performing
these steps are well known to those skilled in the art, and the
discussion above is merely an example.
[0131] The probes are also useful for in-situ hybridization or in
order to locate tissues which express this gene, or for other
hybridization assays for the presence of this gene or its mRNA in
various biological tissues. The in-situ hybridization using a
labelled nucleic acid molecule is well known in the art.
Essentially, tissue sections are incubated with the labelled
nucleic acid molecule to allow the hybridization to occur. The
molecule will carry a marker for the detection because it is
"labelled", the amount of the hybrid will be determined based on
the detection of the amount of the marker and so will the
expression of PSM antigen.
[0132] This invention further provides isolated PSM' antigen
nucleic acid molecule operatively linked to a promoter of RNA
transcription. The isolated PSM' antigen sequence can be linked to
vector systems. Various vectors including plasmid vectors, cosmid
vectors, bacteriophage vectors and other viruses are well known to
ordinary skilled practitioners. This invention further provides a
vector which comprises the isolated nucleic acid molecule encoding
for the PSM' antigen.
[0133] As an example to obtain these vectors, insert and vector DNA
can both be exposed to a restriction enzyme to create complementary
ends on both molecules which base pair with each other and are then
ligated together with DNA ligase. Alternatively, linkers can be
ligated to the insert DNA which correspond to a restriction site in
the vector DNA, which is then digested with the restriction enzyme
which cuts at that site. Other means are also available and known
to an ordinary skilled practitioner.
[0134] Plasmid, p55A-PSM, was deposited on Aug. 14, 1992 with the
American Type Culture Collection (ATCC), 12301 Parklawn Drive,
Rockville, Md. 20852, U.S.A. under the provisions of the Budapest
Treaty for the International Recognition of the Deposit of
Microorganism for the Purposes of Patent Procedure. Plasmid,
p55A-PSM, was accorded ATCC Accession Number 75294.
[0135] This invention further provides a host vector system for the
production of a polypeptide having the biological activity of the
alternatively spiced prostate-specific membrane antigen. These
vectors may be transformed into a suitable host cell to form a host
cell vector system for the production of a polypeptide having the
biological activity of PSM' antigen.
[0136] Regulatory elements required for expression include promoter
sequences to bind RNA polymerase and transcription initiation
sequences for ribosome binding. For example, a bacterial expression
vector includes a promoter such as the lac promoter and for
transcription initiation the Shine-Dalgarno sequence and the start
codon AUG. Similarly, a eukaryotic expression vector includes a
heterologous or homologous promoter for RNA polymerase II, a
downstream polyadenylation signal, the start codon AUG, and a
termination codon for detachment of the ribosome. Such vectors may
be obtained commercially or assembled from the sequences described
by methods well known in the art, for example the methods described
above for constructing vectors in general. Expression vectors are
useful to produce cells that express the PSM antigen.
[0137] This invention further provides an isolated DNA or cDNA
molecule described hereinabove wherein the host cell is selected
from the group consisting of bacterial cells (such as E. coli),
yeast cells, fungal cells, insect cells and animal cells. Suitable
animal cells include, but are not limited to Vero cells, HeLa
cells, Cos cells, CV1 cells and various primary mammalian
cells.
[0138] This invention provides an isolated polypeptide having the
biological activity of an alternatively spliced prostate-specific
membrane antigen.
[0139] This invention further provides a method of producing a
polypeptide having the biological activity of the prostate-specific
membrane antigen which comprising growing host cells of a vector
system containing the PSM' antigen sequence under suitable
conditions permitting production of the polypeptide and recovering
the polypeptide so produced.
[0140] This invention provides a mammalian cell comprising a DNA
molecule encoding a mammalian PSM' antigen, such as a mammalian
cell comprising a plasmid adapted for expression in a mammalian
cell, which comprises a DNA molecule encoding a mammalian PSM'
antigen and the regulatory elements necessary for expression of the
DNA in the mammalian cell so located relative to the DNA encoding
the mammalian PSM' antigen as to permit expression thereof.
[0141] Numerous mammalian cells may be used as hosts, including,
but not limited to, the mouse fibroblast cell NIH3T3, CHO cells,
HeLa cells, Ltk.sup.- cells, Cos cells, etc. Expression plasmids
such as that described supra may be used to transfect mammalian
cells by methods well known in the art such as calcium phosphate
precipitation, electroporation or DNA encoding the mammalian PSM
antigen may be otherwise introduced into mammalian cells, e.g., by
microinjection, to obtain mammalian cells which comprise DNA, e.g.,
cDNA or a plasmid, encoding a mammalian PSM antigen.
[0142] This invention further provides ligands bound to the
mammalian PSM' antigen.
[0143] This invention also provides a therapeutic agent comprising
a ligand identified by the above-described method and a cytotoxic
agent conjugated thereto. The cytotoxic agent may either be a
radioisotope or a toxin. Examples of radioisotopes or toxins are
well known to one of ordinary skill in the art.
[0144] This invention also provides a method of imaging prostate
cancer in human patients which comprises administering to the
patients at least one ligand identified by the above-described
method, capable of binding to the cell surface of the prostate
cancer cell and labelled with an imaging agent under conditions
permitting formation of a complex between the ligand and the cell
surface PSM' antigen. This invention further provides a composition
comprising an effective imaging agent of the PSM' antigen ligand
and a pharmaceutically acceptable carrier. Pharmaceutically
acceptable carriers are well known to one of ordinary skill in the
art. For an example, such a pharmaceutically acceptable carrier can
be physiological saline.
[0145] Also provided by this invention is a purified mammalian PSM'
antigen. As used herein, the term "purified alternatively spliced
prostate-specific membrane antigen" shall mean isolated
naturally-occurring prostate-specific membrane antigen or protein
(purified from nature or manufactured such that the primary,
secondary and tertiary conformation, and posttranslational
modifications are identical to naturally-occurring material) as
well as non-naturally occurring polypeptides having a primary
structural conformation (i.e. continuous sequence of amino acid
residues). Such polypeptides include derivatives and analogs.
[0146] This invention provides an isolated nucleic acid comprising
a promoter sequence normally associated with the transcription of a
gene encoding a human prostate-specific membrane antigen. In one
embodiment regulatory elements are set forth in FIG. 15. In another
embodiment the promoter is between nucleotide -1 to -641 of FIG.
15A.
[0147] This invention provides a method to identify such natural
ligand or other ligand which can bind to the PSM' antigen. A method
to identify the ligand comprises a) coupling the purified mammalian
PSM' antigen to a solid matrix, b) incubating the coupled purified
mammalian PSM' protein with the potential ligands under the
conditions permitting binding of ligands and the purified PSM'
antigen; c) washing the ligand and coupled purified mammalian PSM'
antigen complex formed in b) to eliminate the nonspecific binding
and impurities and finally d) eluting the ligand from the bound
purified mammalian PSM' antigen. The techniques of coupling
proteins to a solid matrix are well known in the art. Potential
ligands may either be deduced from the structure of mammalian PSM'
by other empirical experiments known by ordinary skilled
practitioners. The conditions for binding may also easily be
determined and protocols for carrying such experimentation are
known to those skilled in the art. The ligand-PSM' antigen complex
will be washed. Finally, the bound ligand is eluted and
characterized. Standard ligands characterization techniques are
well known in the art.
[0148] The above method may also be used to purify ligands from any
biological source. For purification of natural ligands in the cell,
cell lysates, serum or other biological samples will be used to
incubate with the mammalian PSM' antigen bound on a matrix.
Specific natural ligand will then be identified and purified as
above described.
[0149] With the protein sequence information, antigenic areas may
be identified and antibodies directed against these areas may be
generated and targeted to the prostate cancer for imaging the
cancer or therapies.
[0150] This invention provides an antibody directed against the
amino acid sequence of a mammalian PSM' antigen.
[0151] This invention provides a method to select specific regions
on the PSM' antigen to generate antibodies. The protein sequence
may be determined from the PSM' DNA sequence. Amino acid sequences
may be analyzed by methods well known to those skilled in the art
to determine whether they produce hydrophobic or hydrophilic
regions in the proteins which they build. In the case of cell
membrane proteins, hydrophobic regions are well known to form the
part of the protein that is inserted into the lipid bilayer of the
cell membrane, while hydrophilic regions are located on the cell
surface, in an aqueous environment. Usually, the hydrophilic
regions will be more immunogenic than the hydrophobic regions.
Therefore the hydrophilic amino acid sequences may be selected and
used to generate antibodies specific to mammalian PSM antigen. For
an example, hydrophilic sequences of the human PSM antigen shown in
hydrophilicity plot may be easily selected. The selected peptides
may be prepared using commercially available machines. As an
alternative, DNA, such as a cDNA or a fragment thereof, may be
cloned and expressed and the resulting polypeptide recovered and
used as an immunogen.
[0152] Polyclonal antibodies against these peptides may be produced
by immunizing animals using the selected peptides. Monoclonal
antibodies are prepared using hybridoma technology by fusing
antibody producing B cells from immunized animals with myeloma
cells and selecting the resulting hybridoma cell line producing the
desired antibody. Alternatively, monoclonal antibodies may be
produced by in vitro techniques known to a person of ordinary skill
in the art. These antibodies are useful to detect the expression of
mammalian PSM antigen in living animals, in humans, or in
biological tissues or fluids isolated from animals or humans.
[0153] In one embodiment, peptides Asp-Glu-Leu-Lys-Ala-Glu (SEQ ID
No.), Asn-Glu-Asp-Gly-Asn-Glu (SEQ ID No.) and
Lys-Ser-Pro-Asp-Glu-Gly (SEQ ID No.) of human PSM antigen are
selected.
[0154] This invention further provides polyclonal and monoclonal
antibody(ies) against peptides Asp-Glu-Leu-Lys-Ala-Glu (SEQ ID
No.), Asn-Glu-Asp-Gly-Asn-Glu (SEQ ID No.) and
Lys-Ser-Pro-Asp-Glu-Gly (SEQ ID No.).
[0155] This invention provides a method of imaging prostate cancer
in human patients which comprises administering to the patient the
monoclonal antibody directed against the peptide of the mammalian
PSM' antigen capable of binding to the cell surface of the prostate
cancer cell and labeled with an imaging agent under conditions
permitting formation of a complex between the monoclonal antibody
and the cell surface prostate-specific membrane antigen. The
imaging agent is a radioisotope such as Indium.sup.111.
[0156] This invention further provides a prostate cancer specific
imaging agent comprising the antibody directed against PSM' antigen
and a radioisotope conjugated thereto.
[0157] This invention also provides a composition comprising an
effective imaging amount of the antibody directed against the PSM'
antigen and a pharmaceutically acceptable carrier. The methods to
determine effective imaging amounts are well known to a skilled
practitioner. One method is by titration using different amounts of
the antibody.
[0158] In addition to the standard pharmacophores that can be added
to know structures, with the PSM transfectants one can identify
potential ligands from combinatorial libraries that might not have
been otherwise predicted such combinatorial libraries can be
synthetic, peptide, or RNA based.
[0159] This invention further provides an immunoassay for measuring
the amount of the prostate-specific membrane antigen in a
biological sample comprising steps of a) contacting the biological
sample with at least one antibody directed against the PSM' antigen
to form a complex with said antibody and the prostate-specific
membrane antigen, and b) measuring the amount of the
prostate-specific membrane antigen in said biological sample by
measuring the amount of said complex. One example of the biological
sample is a serum sample.
[0160] This invention provides a method to purify mammalian
prostate-specific membrane antigen comprising steps of a) coupling
the antibody directed against the PSM' antigen to a solid matrix;
b) incubating the coupled antibody of a) with lysate containing
prostate-specific membrane antigen under the condition which the
antibody and prostate membrane specific can bind; c) washing the
solid matrix to eliminate impurities and d) eluting the
prostate-specific membrane antigen from the coupled antibody.
[0161] This invention also provides a transgenic nonhuman mammal
which comprises the isolated nucleic acid molecule encoding a
mammalian PSM' antigen. This invention further provides a
transgenic nonhuman mammal whose genome comprises antisense DNA
complementary to DNA encoding a mammalian prostate-specific
membrane antigen so placed as to be transcribed into antisense mRNA
complementary to mRNA encoding the prostate-specific membrane
antigen and which hybridizes to mRNA encoding the prostate specific
antigen thereby reducing its translation.
[0162] Animal model systems which elucidate the physiological and
behavioral roles of mammalian PSM' antigen are produced by creating
transgenic animals in which the expression of the PSM' antigen is
either increased or decreased, or the amino acid sequence of the
expressed PSM antigen is altered, by a variety of techniques.
Examples of these techniques include, but are not limited to: 1)
Insertion of normal or mutant versions of DNA encoding a mammalian
PSM' antigen, by microinjection, electroporation, retroviral
transfection or other means well known to those skilled in the art,
into appropriate fertilized embryos in order to produce a
transgenic animal or 2) Homologous recombination of mutant or
normal, human or animal versions of these genes with the native
gene locus in transgenic animals to alter the regulation of
expression or the structure of these PSM' antigen sequences. The
technique of homologous recombination is well known in the art. It
replaces the native gene with the inserted gene and so is useful
for producing an animal that cannot express native PSM antigen but
does express, for example, an inserted mutant PSM antigen, which
has replaced the native PSM antigen in the animal's genome by
recombination, resulting in under expression of the transporter.
Microinjection adds genes to the genome, but does not remove them,
and so is useful for producing an animal which expresses its own
and added PSM antigens, resulting in over expression of the PSM
antigens.
[0163] One means available for producing a transgenic animal, with
a mouse as an example, is as follows: Female mice are mated, and
the resulting fertilized eggs are dissected out of their oviducts.
The eggs are stored in an appropriate medium such as Me medium
(16). DNA or cDNA encoding a mammalian PSM antigen is purified from
a vector by methods well known in the art. Inducible promoters may
be fused with the coding region of the DNA to provide an
experimental means to regulate expression of the trans-gene.
Alternatively or in addition, tissue specific regulatory elements
may be fused with the coding region to permit tissue-specific
expression of the trans-gene. The DNA, in an appropriately buffered
solution, is put into a microinjection needle (which may be made
from capillary tubing using a pipet puller) and the egg to be
injected is put in a depression slide. The needle is inserted into
the pronucleus of the egg, and the DNA solution is injected. The
injected egg is then transferred into the oviduct of a
pseudopregnant mouse (a mouse stimulated by the appropriate
hormones to maintain pregnancy but which is not actually pregnant),
where it proceeds to the uterus, implants, and develops to term. As
noted above, microinjection is not the only method for inserting
DNA into the egg cell, and is used here only for exemplary
purposes.
[0164] Another use of the PSM antigen sequence is to isolate
homologous gene or genes in different mammals. The gene or genes
can be isolated by low stringency screening of either cDNA or
genomic libraries of different mammals using probes from PSM
sequence. The positive clones identified will be further analyzed
by DNA sequencing techniques which are well known to an ordinary
person skilled in the art. For example, the detection of members of
the protein serine kinase family by homology probing.
[0165] This invention provides a method of suppressing or
modulating metastatic ability of prostate tumor cells, prostate
tumor growth or elimination of prostate tumor cells comprising
introducing a DNA molecule encoding an alternatively spliced
prostate specific membrane antigen operatively linked to a 5'
regulatory element into a tumor cell of a subject, in a way that
expression of the alternatively spliced prostate specific membrane
antigen is under the control of the regulatory element, thereby
suppressing or modulating metastatic ability of prostate tumor
cells, prostate tumor growth or elimination of prostate tumor
cells. The subject may be a mammal or more specifically a
human.
[0166] In one embodiment, the DNA molecule is operatively linked to
a 5' regulatory element forms part of a transfer vector which is
inserted into a cell or organism. In addition the vector is capable
or replication and expression of the alternatively spliced prostate
specific membrane antigen. The DNA molecule can be integrated into
a genome of a eukaryotic or prokaryotic cell or in a host cell
containing and/or expressing an alternatively spliced prostate
specific membrane antigen.
[0167] Further, the DNA molecule encoding alternatively spliced
prostate specific membrane antigen may be introduced by a
bacterial, viral, fungal, animal, or liposomal delivery vehicle.
Other means are also available and known to an ordinary skilled
practitioner.
[0168] Further, the DNA molecule encoding an alternatively spliced
prostate specific membrane antigen operatively linked to a promoter
or enhancer. A number of viral vectors have been described
including those made from various promoters and other regulatory
elements derived from virus sources. Promoters consist of short
arrays of nucleic acid sequences that interact specifically with
cellular proteins involved in transcription. The combination of
different recognition sequences and the cellular concentration of
the cognate transcription factors determines the efficiency with
which a gene is transcribed in a particular cell type.
[0169] Examples of suitable promoters include a viral promoter.
Viral promoters include: adenovirus promoter, an simian virus 40
(SV40) promoter, a cytomegalovirus (CMV) promoter, a mouse mammary
tumor virus (MMTV) promoter, a Malony murine leukemia virus
promoter, a murine sarcoma virus promoter, and a Rous sarcoma virus
promoter.
[0170] Further, another suitable promoter is a heat shock promoter.
Additionally, a suitable promoter is a bacteriophage promoter.
Examples of suitable bacteriophage promoters include but not
limited to, a T7 promoter, a T3 promoter, an SP6 promoter, a lambda
promoter, a baculovirus promoter.
[0171] Also suitable as a promoter is an animal cell promoter such
as an interferon promoter, a metallothionein promoter, an
immunoglobulin promoter. A fungal promoter is also a suitable
promoter. Examples of fungal promoters include but are not limited
to, an ADC1 promoter, an ARG promoter, an ADH promoter, a CYC1
promoter, a CUP promoter, an ENO1 promoter, a GAL promoter, a PHO
promoter, a PGK promoter, a GAPDH promoter, a mating type factor
promoter. Further, plant cell promoters and insect cell promoters
are also suitable for the methods described herein.
[0172] This invention provides a method of suppressing or
modulating metastatic ability of prostate tumor cells, prostate
tumor growth or elimination of prostate tumor cells, comprising
introducing a DNA molecule encoding an alternatively spliced
prostate specific membrane antigen operatively linked to a 5'
regulatory element coupled with a therapeutic DNA into a tumor cell
of a subject, thereby suppressing or modulating metastatic ability
of prostate tumor cells, prostate tumor growth or elimination of
prostate tumor cells. The subject may be a mammal or more
specifically a human.
[0173] Further, the therapeutic DNA which is coupled to the DNA
molecule encoding a prostate specific membrane antigen operatively
linked to a 5' regulatory element into a tumor cell may code for a
cytokine, viral antigen, or a pro-drug activating enzyme. Other
means are also available and known to an ordinary skilled
practitioner.
[0174] In addition, this invention provides a prostate tumor cell,
comprising a DNA molecule isolated from mammalian nucleic acid
encoding an alternatively spliced mammalian prostate-specific
membrane antigen under the control a 5' regulatory element.
[0175] As used herein, DNA molecules include complementary DNA
(cDNA), synthetic DNA, and genomic DNA.
[0176] This invention provides a therapeutic vaccine for preventing
human prostate tumor growth or stimulation of prostate tumor cells
in a subject, comprising administering an effective amount to the
prostate cell, and a pharmaceutical acceptable carrier, thereby
preventing the tumor growth or stimulation of tumor cells in the
subject. Other means are also available and known to an ordinary
skilled practitioner.
[0177] This invention provides a method of detecting hematogenous
micrometastic tumor cells of a subject, comprising (A) performing
nested polymerase chain reaction (PCR) on blood, bone marrow or
lymph node samples of the subject using the prostate specific
membrane antigen primers or alternatively spliced prostate specific
antigen primers, and (B) verifying micrometastases by DNA
sequencing and Southern analysis, thereby detecting hematogenous
micrometastic tumor cells of the subject. The subject may be a
mammal or more specifically a human.
[0178] The micrometastatic tumor cell may be a prostatic cancer and
the DNA primers may be derived from prostate specific antigen.
Further, the subject may be administered with simultaneously an
effective amount of hormones, so as to increase expression of
prostate specific membrane antigen. Further, growth factors or
cytokine may be administered in separately or in conjunction with
hormones. Cytokines include, but are not limited to: transforming
growth factor beta, epidermal growth factor (EGF) family,
fibroblast growth factors, hepatocyte growth factor, insulin-like
growth factors, B-nerve growth factor, platelet-derived growth
factor, vascular endothelial growth factor, interleukin 1, IL-1
receptor antagonist, interleukin 2, interleukin 3, interleukin 4,
interleukin 5, interleukin 6, IL-6 soluble receptor, interleukin 7,
interleukin 8, interleukin 9, interleukin 10, interleukin 11,
interleukin 12, interleukin 13, angiogenin, chemokines, colony
stimulating factors, granulocyte-macrophage colony stimulating
factors, erythropoietin, interferon, interferon gamma, leukemia
inhibitory factor, oncostatin M, pleiotrophin, secretory leukocyte
protease inhibitor, stem cell factor, tumor necrosis factors,
adhesion molecule, and soluble tumor necrosis factor (TNF)
receptors.
[0179] This invention provides a method of abrogating the mitogenic
response due to transferrin, comprising introducing a DNA molecule
encoding prostate specific membrane antigen operatively linked to a
5' regulatory element into a tumor cell, the expression of which
gene is directly associated with a defined pathological effect
within a multicellular organism, thereby abrogating mitogen
response due to transferrin. The tumor cell may be a prostate
cell.
[0180] This invention provides a method of determining prostate
cancer progression in a subject which comprises: a) obtaining a
suitable prostate tissue sample; b) extracting RNA from the
prostate tissue sample; c) performing a RNAse protection assay on
the RNA thereby forming a duplex RNA-RNA hybrid; d) detecting PSM
and PSM' amounts in the tissue sample; e) calculating a PSM/PSM'
tumor index, thereby determining prostate cancer progression in the
subject. In-situ hyribridization may be performed in conjunction
with the above detection method.
[0181] This invention provides a method of detecting prostate
cancer in a subject which comprises: (a) obtaining from a subject a
prostate tissue sample; (b) treating the tissue sample so as to
separately recover nucleic acid molecules present in the prostate
tissue sample; (c) contacting the resulting nucleic acid molecules
with multiple pairs of single-stranded labeled oligonucleotide
primers, each such pair being capable of specifically hybridizing
to the tissue sample, under hybridizing conditions; (d) amplifying
any nucleic acid molecules to which a pair of primers hybridizes so
as to obtain a double-stranded amplification product; (e) treating
any such double-stranded amplification product so as to obtain
single-stranded nucleic acid molecules therefrom; (f) contacting
any resulting single-stranded nucleic acid molecules with multiple
single-stranded labeled oligonucleotide probes, each such probe
containing the same label and being capable of specifically
hybridizing with such tissue sample, under hybridizing conditions;
(g) contacting any resulting hybrids with an antibody to which a
marker is attached and which is capable of specifically forming a
complex with the labeled-probe, when the probe is present in such a
complex, under complexing conditions; and (h) detecting the
presence of any resulting complexes, the presence thereof being
indicative of prostate cancer in a subject.
[0182] This invention provides a method of enhancing antibody based
targeting of PSM' in prostate tissue for diagnosis or therapy of
prostate cancer comprising administering to a patient b-FGF in
sufficient amount to cause upregulation of PSM' expression.
[0183] This invention provides a method of enhancing antibody based
targeting of PSM' in prostate tissue for diagnosis or therapy of
prostate cancer comprising administering to a patient TGF in
sufficient amount to cause upregulation of PSM expression or
PSM'.
[0184] This invention provides a method of enhancing antibody based
targeting of PSM' in prostate tissue for diagnosis or therapy of
prostate cancer comprising administering to a patient EGF in
sufficient amount to cause upregulation of PSM' expression.
[0185] This invention provides a method of detecting in a sample
the presence of a nucleic acid encoding an alternatively spliced
human prostate-specific membrane antigen which comprises: a)
obtaining a suitable sample; b) extracting RNA from the sample; c)
contacting the RNA with reverse transcriptase under suitable
conditions to obtain a cDNA; d) contacting the cDNA under
hybridizing conditions with two oligonucleotide primers, i) the
first primer being capable of specifically hybridizing to a
sequence within a DNA sequence encoding prostate specific membrane
antigen located immediately 3' of nucleotide 114 of such DNA
sequence, with the proviso that the 3' end of the primer does not
hybridize to any sequence located 5' of nucleotide 114, and ii) the
second primer being capable of specifically hybridizing to a
sequence within a DNA sequence encoding prostate specific membrane
antigen located immediately 5' of nucleotide 381 of such DNA
sequence, with the proviso that the 5' end of the primer does not
hybridize to any sequence located 3' of nucleotide 381; d)
amplifying any cDNA to which the primers hybridize to so as to
obtain amplification product; e) determining the size of the
amplification product; f) comparing the size of the amplification
product to the size of the amplification product known to be
obtained using the same primers with a non alternatively spliced
human prostate specific membrane antigen, wherein a smaller
amplification product is indicative of the presence of the
alternatively spliced prostate specific membrane antigen, so as to
thereby detect the presence of the alternatively spliced human
prostate-specific membrane antigen in the sample.
[0186] In one embodiment the suitable sample may be any bodily
tissue of fluid which includes but is not limited to: blood, bone
marrow, and lymph nodes.
[0187] In one embodiment the primers are at least 14-25 nucleotides
in length. In another embodiment the primers are at least 15
nucleotide in length. In another embodiment the primers are 15
nucleotides in length. In another embodiment multiple primers are
used. Construction of primers which hybridize and hybridizing
conditions are known to those skilled in the art. For example,
based on FIG. 18 one skilled in the art may construct primers which
hybridize to the prostate specific membrane antigen before
nucleotide 114 and after nucleotide 381.
[0188] Further, a method of determining the amount of the
amplification product or products (i.e. 2 or more bands) as well as
the ratio of each product is known to those skilled in the art. For
example, the amount of prostate specific membrane antigen or
alternatively spliced prostate specific membrane antigen may be
determined by density, binding radiolabled probes, autoradiography,
UV spectrography, spectrophotometer, optical scan, and
phospho-imaging.
[0189] This invention provides a method of detecting a prostate
tumor cell in a subject which comprises: which comprises: a)
obtaining a suitable sample; b) extracting RNA from the sample; c)
contacting the RNA with reverse transcriptase under suitable
conditions to obtain a cDNA; d) contacting the cDNA under
hybridizing conditions with two oligonucleotide primers, i) the
first primer being capable of specifically hybridizing to a
sequence within a DNA sequence encoding prostate specific membrane
antigen located immediately 3' of nucleotide 114 of such DNA
sequence, with the proviso that the 3' end of the primer does not
hybridize to any sequence located 5' of nucleotide 114, and ii) the
second primer being capable of specifically hybridizing to a
sequence within a DNA sequence encoding prostate specific membrane
antigen located immediately 5' of nucleotide 381 of such DNA
sequence, with the proviso that the 5' end of the primer does not
hybridize to any sequence located 3' of nucleotide 381; d)
amplifying any cDNA to which the primers hybridize to so as to
obtain amplification product; e) determining the amount of the
amplification product; f) comparing the amount of the amplification
product to the amount of the amplification product known to be
obtained using the same primers with a non alternatively spliced
human prostate specific membrane antigen, wherein a greater amount
of the prostate specific membrane antigen is indicative of a
prostate tumor cell in the subject, so as to thereby detect
prostate tumor cell in the subject.
[0190] In PCR techniques, oligonucleotide primers complementary to
the two 3' borders of the DNA of the prostate specific membrane
(PSM) antigen to be amplified are synthesized. The polymerase chain
reaction is then carried out using the two primers. See PCR
Protocols: A Guide to Methods and Applications. Hybridization of
PSM antigen DNA to the above nucleic acid probes can be performed
by a Southern blot under stringent hybridization conditions as
described herein.
[0191] Oligonucleotides for use as probes or PCR primers are
chemically synthesized according to the solid phase phosphoramidite
triester method first described by Beaucage and Carruthers using an
automated synthesizer, as described in Needham-VanDevanter.
Purification of oligonucleotides is by either native acrylamide gel
electrophoresis or by anion-exchange HPLC as described in Pearson,
J. D. and Regnier, F. E. The sequence of the synthetic
oligonucleotide can be verified using the chemical degradation
method of Maxam, A. M. and Gilbert, W.
[0192] Accepted means for conducting hybridization assays are known
and general overviews of the technology can be had from a review
of: Nucleic Acid Hybridization: A Practical Approach; Hybridization
of Nucleic Acids Immobilized on Solid Supports; Analytical
Biochemistry and Innis et al., PCR Protocols.
[0193] If PCR is used in conjunction with nucleic acid
hybridization, primers are designed to target a specific portion of
the nucleic acid of DNA of the PSM antigen. From the information
provided herein, those of skill in the art will be able to select
appropriate specific primers.
[0194] It will be apparent to those of ordinary skill in the art
that a convenient method for determining whether a probe is
specific for PSM antigen or PSM' antigen utilizes a Southern blot
(or Dot blot). Briefly, to identify a target specific probe DNA is
isolated from the PSM or PSM' antigen. Test DNA is transferred to a
solid (e.g., charged nylon) matrix. The probes are labelled
following conventional methods. Following denaturation and/or
prehybridization steps known in the art, the probe is hybridized to
the immobilized DNAs under stringent conditions. Stringent
hybridization conditions will depend on the probe used and can be
estimated from the calculated T.sub.m (melting temperature) of the
hybridized probe (see, e.g., Sambrook for a description of
calculation of the T.sub.m). For radioactively-labeled DNA or RNA
probes an example of stringent hybridization conditions is
hybridization in a solution containing denatured probe and
5.times.SSC at 65.degree. C. for 8-24 hours followed by washes in
0.1.times.SSC, 0.1% SDS (sodium dodecyl sulfate) at 50-65.degree.
C. In general, the temperature and salt concentration are chosen so
that the post hybridization wash occurs at a temperature that is
about 5.degree. C. below the T.sub.M of the hybrid. Thus for a
particular salt concentration the temperature may be selected that
is 5.degree. C. below the T.sub.M or conversely, for a particular
temperature, the salt concentration is chosen to provide a T.sub.M
for the hybrid that is 5.degree. C. warmer than the wash
temperature. Following stringent hybridization and washing, a probe
that hybridizes to the PSM antigen or PSM' antigen as evidenced by
the presence of a signal associated with the appropriate target and
the absence of a signal from the non-target nucleic acids, is
identified as specific. It is further appreciated that in
determining probe specificity and in utilizing the method of this
invention a certain amount of background signal is typical and can
easily be distinguished by one of skill from a specific signal. Two
fold signal over background is acceptable.
[0195] This invention provides a therapeutic agent comprising
antibodies or ligand(s) directed against PSM' antigen and a
cytotoxic agent conjugated thereto or antibodies linked enzymes
which activate prodrug to kill the tumor. The cytotoxic agent may
either be a radioisotope or toxin.
[0196] This invention provides a compound comprising a conjugate of
a cytotoxic agent and one or more amino acid residues, wherein each
amino acid residue is glutamate or aspartate. In one embodiment the
amino acid residues alternate.
[0197] Examples of cytotoxic chemotherapeutic agents or
antineolastic agents) include, but are not limited to the
following: Antimetaboloites: Denopterin, Edatrexate, Piritrexim,
Pteropterin, Tomudex, Tremetrexate, Cladribine, Fludarabine,
6-Mercaptopurine, Thiamiprine, Thioguanine, Ancitabine,
Azacitidine, 6-Azauridine, Carmofur, Cytarabine, Doxifluride,
Emitefur, Enocitabine, Floxuridine, Fluoroucit, Gemcitabine, and
Tegafur.
[0198] Alkaloids: Docetaxel, Etoposide, Irinotecan, Paclitaxel,
Teniposide, Topotecan, VinblastinE, Vincristine, and Vindesine.
[0199] Alkylating agents: Alkyl Sulfonates: Busulfan, Improsulfan,
Piposulfan, Aziridines, Benzodepa, Carboquone, Meuredepa, Uredepa,
Ethylenimines and Methylmelamines, Altretamine,
Triethylenemelamine, Triethylenophosphoramide,
Triethylenethiophosphoramide, Chlorambucil, Chlornaphazine,
Cyclophosphamide, Estramustine, Ifosfamide, Mechlorethamine,
Mechlorethamine Oxide Hydrochloride, Melphalan, Novembiechin,
Perfosfamide, Phenesterine, Prednimustine, Trofosfamide, Uracil
Mustard, Carmustine, Chlorozotocin, Fotemustine, Lomustine,
Nimustine, Ranimustine, Dacarbazine, Mannomustine, Mitbronitol,
Mitolactol, Pipobroman, Temozolomide, Antibiotics and Analogs:
Aclacinomycins, Actinomycin, Anthramycin, Azaserine, Bleomycins,
Cactinomycin, Carubicin, Carzinophilin, Chromomycins, Dactinomycin,
Caunorubicin, 6-Diazo-5-oxo-L-norleucine, Doxorubicin, Epirubicin,
Idarubicin, Menogaril, Mitomycins, Mycophenolic Acid, Nogalamycin,
Olivomycins, Peplomycin, Pirarubicin, Plicamycin, Porfiromycin,
Puromycin, Streptonigrin, Streptozocin, Tubercidin, Zinostatin,
Zorubicin, and L-Asparaginase.
[0200] Immunodulators: Interferon, Interferon-B, Interferon-Y,
Interleukin-2, Lentinan, Propagermanium, PSK, Roquinimex, Sizofran,
and Ubenimex. Platinum complexes: Carboplatin, Cisplatin,
Miboplatin, and Oxaliplatin.
[0201] Others: Aceglatone, Amsacrine, Bisantrene, Defoosfamide,
Demecolcine, Diaziqone, Eflornithine, Eliptinium Acetate,
Etoglucid, Fenertinide, Gallium Nitrate, Hydroxyurea, Lonidamine,
Miltefosine, Mitoguazone, Mitoxantrone, Mopidamol, Nitracirine,
Pentostatin, Phenamet, Podophyllinic Acid 2-Ethyl-hydrazide,
Procarbazine, Razoxane, Sobuzoxane, Spirogermanium, Tenuazonic
Acid, Triaziquone, Urethan, Calusterone, Dromostanolone,
Epitiostanol, Mepitiostane, Testolactone, Amiglutehimide, Mitotane,
Trilostane, Droloxifene, Tamoxifen, Toremifene, Aminoglutethimide,
Anastrozole, Fadrozole, Formestane, Letrozole, Fosfestrol,
Hexestrol, Polyestradiol Phosphate, Buserlin, Goserlin, Leuprolide,
Triptorelin, Chlormadinone Acetate, Medroxyprogesterone, Megerstrol
Acetate, Melengestrol, Porfimer Sodium, Americium, Chromic
Phosphate, Radioactive Cobalt, I-Ehtiodized Oil, Gold, Radioactive,
Colloidal, Iobenguane: Radium, Radon, Sodium Iodide, Sodium
Phosphate, Radioactive, Batimastat, Folinic Acid, Amifostine,
Etanidazole, Etamidozole, and Mesna.
[0202] This invention provides a compound, wherein the compound has
the structure:
##STR00001## [0203] wherein n is an integer from 1-10
inclusive.
[0204] In one embodiment glutamate may be in L or D to form either
4-amino-N.sup.10-methyl pteroyl-L-glutamate or
4-amino-N.sup.10-methyl pteroyl-D-glutamate. In another embodiment
aspartate may substitute the glutamate to form
4-amino-N.sup.10-methyl pteroyl-L-aspartate. In another embodiment
aspartate may substitute the glutamate to form
4-amino-N.sup.10-methyl pteroyl-D-aspartate. In another embodiment
the 4'-amino-N.sup.10-methyl pteroyl may have alternating glutamate
or aspartat moieties. The glutamate or aspartate are bound to the
methotrexate at the alpha carbon position of methotrexate.
[0205] This invention provides a compound, wherein the compound has
the structure:
##STR00002## [0206] wherein n is an integer from 1-10
inclusive.
[0207] In one embodiment glutamate may be in the L or D to form
either N-phosphonoacetyl-L-aspartyl (PALA)-glutamate or
N-phsophonoacetyl-D-aspartyl-glutamate. In another embodiment
aspartate may substitute the glutamate to form
N-phsophonoacetyl-L-aspartyl-aspartate. In another embodiment the
4-amino-N.sup.10-methyl pteroyl may have alternating glutamate or
aspartate moieties.
[0208] This invention provides a compound, wherein the compound has
the structure:
##STR00003## [0209] wherein n is an integer from 1-10
inclusive.
[0210] In one embodiment glutamate may be in the L or D to form
either 4-amino-10-ethyl-10-deazapteroyl (EDAM)-L-glutamate or
4-amino-10-ethyl-10-deazapteroyl-D-glutamate. In another embodiment
aspartate may substitute the glutamate to form
4-amino-10-ethyl-10-deazapteroyl-L-aspartate. In another embodiment
the 4-amino-10-ethyl-10-deazapteroyl may have alternating glutamate
or aspartat moieties.
[0211] This invention provides a pharmaceutical composition
comprising any of the above compounds in a therapeutically
effective amount and a pharmaceutically acceptable carrier.
[0212] This invention provides a method of making prostate cells
susceptible to a cytotoxic agent, which comprises contacting the
prostate cells with any of the above compounds in an amount
effective to render the prostate cells susceptible to the cytotoxic
chemotherapeutic agent.
[0213] This invention provides a pharmaceutical composition
comprising an effective amount the alternatively spliced. PSM' and
a carrier or diluent. Further, this invention provides a method for
administering to a subject, preferably a human, the pharmaceutical
composition. Further, this invention provides a composition
comprising an amount of the alternatively spliced PSM' and a
carrier or diluent. Specifically, this invention may be used as a
food additive.
[0214] The compositions are administered in a manner compatible
with the dosage formulation, and in a therapeutically effective
amount. Precise amounts of active ingredient required to be
administered depend on the judgment of the practitioner and are
peculiar to each subject.
[0215] In one embodiment the therapeutic effective amount is
100-10,000 mg/m.sup.2 IV with rescue. In another embodiment the
therapeutic effective amount is 300-1000 mg/m.sup.2 IV or
continuous infusion. In another embodiment the therapeutic
effective amount is 100 mg/m.sup.2 IV continuous infusion. In
another embodiment the therapeutic effective amount is 40-75
mg/m.sup.2 rapidly. In another embodiment the therapeutic effective
amount is 30 mg/m.sup.2 for 3 days by continuous IV.
[0216] Suitable regimes for initial administration and booster
shots are also variable, but are typified by an initial
administration followed by repeated doses at one or more hour
intervals by a subsequent injection or other administration.
[0217] As used herein administration means a method of
administering to a subject. Such methods are well known to those
skilled in the art and include, but are not limited to,
administration topically, parenterally, orally, intravenously,
intramuscularly, subcutaneously or by aerosol. Administration of
PSM may be effected continuously or intermittently.
[0218] The pharmaceutical formulations or compositions of this
invention may be in the dosage form of solid, semi-solid, or liquid
such as, e.g., suspensions, aerosols or the like. Preferably the
compositions are administered in unit dosage forms suitable for
single administration of precise dosage amounts. The compositions
may also include, depending on the formulation desired,
pharmaceutically-acceptable, non-toxic carriers or diluents, which
are defined as vehicles commonly used to formulate pharmaceutical
compositions for animal or human administration. The diluent is
selected so as not to affect the biological activity of the
combination. Examples of such diluents are distilled water,
physiological saline, Ringer's solution, dextrose solution, and
Hank's solution. In addition, the pharmaceutical composition or
formulation may also include other carriers, adjuvants; or
nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.
Effective amounts of such diluent or carrier are those amounts
which are effective to obtain a pharmaceutically acceptable
formulation in terms of solubility of components, or biological
activity, etc
[0219] This invention also provides a method of detecting a subject
with cancer comprising a) contacting a cell of the neo-vasculature
of a subject with a ligand which binds to the extraccelular domain
of the PSM antigen under conditions permitting formation of a
complex; and b) detecting the complex with a labelled imaging
agent, thereby detecting a subject with cancer.
[0220] In one embodiment the cancer is, but is not limited to:
kidney, colon, or bladder. In one embodiment the ligand is CYT-356.
In another embodiment the ligand is any antibody, monoclonal or
polyclonal which binds to the extracellular domain of PSM antigen.
In one embodiment the cells of endothelial cells of the
neo-vasculature of a subject with cancer.
[0221] This invention will be better understood from the
Experimental Details which follow. However, one skilled in the art
will readily appreciate that the specific methods and results
discussed are merely illustrative of the invention as described
more fully in the claims which follow thereafter.
EXPERIMENTAL DETAILS
Example 1
Expression of the Prostate Specific Membrane Antigen
[0222] A 2.65 kb complementary DNA encoding PSM was cloned.
Immunohistochemical analysis of the LNCaP, DU-145, and PC-3
prostate cancer cell lines for PSM expression using the 7E11-C5.3
antibody reveals intense staining in the LNCaP cells, with no
detectable expression in both the DU-145 and PC-3 cells. Coupled
in-vitro transcription/translation of the 2.65 kb full-length PSM
cDNA yields an 84 kDa protein corresponding to the predicted
polypeptide molecular weight of PSM. Post-translational
modification of this protein with pancreatic canine microsomes
yields the expected 100 kDa PSM antigen. Following transfection of
PC-3 cells with the full-length PSM cDNA in a eukaryotic expression
vector applicant's detect expression of the PSM glycoprotein by
Western analysis using the 7E11-C5.3 monoclonal antibody.
Ribonuclease protection analysis demonstrates that the expression
of PSM mRNA is almost entirely prostate-specific in human tissues.
PSM expression appears to be highest in hormone-deprived states and
is hormonally modulated by steroids, with DHT down regulating PSM
expression in the human prostate cancer cell line LNCaP by 8-10
fold, testosterone down regulating PSM by 3-4 fold, and
corticosteroids showing no significant effect. Normal and malignant
prostatic tissues consistently show high PSM expression, whereas
heterogeneous, and at times absent, from expression of PSM in
benign prostatic hyperplasia. LNCaP tumors implanted and grown both
orthotopically and subcutaneously in nude mice, abundantly express
PSM providing an excellent in-vivo model system to study the
regulation and modulation of PSM expression.
Materials and Methods:
[0223] Cells and Reagents: The LNCaP, DU-145, and PC-3 cell lines
were obtained from the American Type Culture Collection. Details
regarding the establishment and characteristics of these cell lines
have been previously published. Unless specified otherwise, LNCaP
cells were grown in RPMI 1640 media supplemented with L-glutamine,
nonessential amino acids, and 5% fetal calf serum (Gibco-BRL,
Gaithersburg, Md.) in a CO.sub.2 incubator at 37 C. DU-145 and PC-3
cells were grown in minimal essential medium supplemented with 10%
fetal calf serum. All cell media were obtained from the MSKCC Media
Preparation Facility. Restriction and modifying enzymes were
purchased from Gibco-BRL unless otherwise specified.
[0224] Immunohistochemical Detection of PSM: Avidin-biotin method
of detection was employed to analyze prostate cancer cell lines for
PSM antigen expression. Cell cytospins were made on glass slides
using 5.times.10.sup.4 cells/100 ul per slide. Slides were washed
twice with PBS and then incubated with the appropriate suppressor
serum for 20 minutes. The suppressor serum was drained off and the
cells were incubated with diluted 7E11-C5.3 (5 g/ml) monoclonal
antibody for 1 hour. Samples were then washed with PBS and
sequentially incubated with secondary antibodies for 30 minutes and
with avidin-biotin complexes for 30 minutes. Diaminobenzidine
served as the chromogen and color development followed by
hematoxylin counterstaining and mounting. Duplicate cell cytospins
were used as controls for each experiment. As a positive control,
the anti-cytokeratin monoclonal antibody CAM 5.2 was used following
the same procedure described above. Human EJ bladder carcinoma
cells served as a negative control.
[0225] In-Vitro Transcription/Translation of PSM Antigen: Plasmid
55A containing the full length 2.65 kb PSM cDNA in the plasmid
pSPORT 1 (Gibco-BRL) was transcribed in-vitro using the Promega TNT
system (Promega Corp. Madison, Wis.). T7 RNA polymerase was added
to the cDNA in a reaction mixture containing rabbit reticulocyte
lysate, an amino acid mixture lacking methionine, buffer, and
.sup.35S-Methionine (Amersham) and incubated at 30 C for 90
minutes. Post-translational modification of the resulting protein
was accomplished by the addition of pancreatic canine microsomes
into the reaction mixture (Promega Corp. Madison, Wis.). Protein
products were analyzed by electrophoresis on 10% SDS-PAGE gels
which were subsequently treated with Amplify autoradiography
enhancer (Amersham, Arlington Heights, Ill.) according to the
manufacturers instructions and dried at 80 C in a vacuum dryer.
Gels were autoradiographed overnight at -70 C using Hyperfilm MP
(Amersham).
[0226] Transfection of PSM into PC-3 Cells: The full length PSM
cDNA was subcloned into the pREP7 eukaryotic expression vector
(Invitrogen, San Diego, Calif.). Plasmid DNA was purified from
transformed DH5-alpha bacteria (Gibco-BRL) using Qiagen maxi-prep
plasmid isolation columns (Qiagen Inc., Chatsworth, Calif.).
Purified plasmid DNA (6-10 g) was diluted with 900 ul of Optimem
media (Gibco-BRL) and mixed with 30 ul of Lipofectin reagent
(Gibco-BRL) which had been previously diluted with 9001 of Optimem
media. This mixture was added to T-75 flasks of 40-50% confluent
PC-3 cells in Optimem media. After 24-36 hours, cells were
trypsinized and split into 100 mm dishes containing RPMI 1640 media
supplemented with 10% fetal calf serum and 1 mg/ml of Hygromycin B
(Calbiochem, La. Jolla, Calif.). The dose of Hygromycin B used was
previously determined by a time course/dose response cytotoxicity
assay. Cells were maintained in this media for 2-3 weeks with
changes of media and Hygromycin B every 4-5 days until discrete
colonies appeared. Colonies were isolated using 6 mm cloning
cylinders and expanded in the same media. As a control, PC-3 cells
were also transfected with the pREP7 plasmid alone. RNA was
isolated from the transfected cells and PSM mRNA expression was
detected by both RNase Protection analysis (described later) and by
Northern analysis.
[0227] Western Blot Detection of PSM Expression: Crude protein
lysates were isolated from LNCaP, PC-3, and PSM-transfected PC-3
cells as previously described. LNCaP cell membranes were also
isolated according to published methods. Protein concentrations
were quantitated by the Bradford method using the BioRad protein
reagent kit (BioRad, Richmond, Calif.). Following denaturation, 20
.mu.g of protein was electrophoresed on a 10% SDS-PAGE gel at 25 mA
for 4 hours. Gels were electroblotted onto Immobilon P membranes
(Millipore, Bedford, Mass.) overnight at 4 C. Membranes were
blocked in 0.15M NaCl/0.01M Tris-HCl (TS) plus 5% BSA followed by a
1 hour incubation with 7E11-C5.3 monoclonal antibody (10 .mu.g/ml).
Blots were washed 4 times with 0.15M NaCl/0.01M Tris-HCl/0.05%
Triton-X 100 (TS-X) and incubated for 1 hour with rabbit anti-mouse
IgG (Accurate Scientific, Westbury, N.Y.) at a concentration of 10
.mu.g/ml.
[0228] Blots were then washed 4 times with TS-X and labeled with
.sup.125I-Protein A (Amersham, Arlington Heights, Ill.) at a
concentration of 1 million cpm/ml. Blots were then washed 4 times
with TS-X and dried on Whatman 3MM paper, followed by overnight
autoradiography at -70 C using Hyperfilm MP (Amersham).
[0229] Orthotopic and Subcutaneous LNCaP Tumor Growth in Nude Mice:
LNCaP cells were harvested from sub-confluent cultures by a one
minute exposure to a solution of 0.25% trypsin and 0.02% EDTA.
Cells were resuspended in RPMI 1640 media with 5% fetal bovine
serum, washed and diluted in either Matrigel (Collaborative
Biomedical Products, Bedford, Mass.) or calcium and magnesium-free
Hank's balanced salt solution (HESS). Only single cell suspensions
with greater than 90% viability by trypan blue exclusion were used
for in vivo injection. Male athymic Swiss (nu/nu) nude mice 4-6
weeks of age were obtained from the Memorial Sloan-Kettering Cancer
Center Animal Facility. For subcutaneous tumor cell injection one
million LNCaP cells resuspended in 0.2 mls. of Matrigel were
injected into the hindlimb of each mouse using a disposable syringe
fitted with a 28 gauge needle. For orthotopic injection, mice were
first anesthetized with an intraperitoneal injection of
Pentobarbital and placed in the supine position. The abdomen was
cleansed with Betadine and the prostate was exposed through a
midline incision. 2.5 million LNCaP tumor cells in 0.1 ml. were
injected directly into either posterior lobe using a 1 ml
disposable syringe and a 28 gauge needle. LNCaP cells with and
without Matrigel were injected. Abdominal closure was achieved in
one layer using Autoclip wound clips (Clay Adams, Parsippany,
N.J.). Tumors were harvested in 6-8 weeks, confirmed histologically
by faculty of the Memorial Sloan-Kettering Cancer Center Pathology
Department, and frozen in liquid nitrogen for subsequent RNA
isolation.
[0230] RNA Isolation: Total cellular RNA was isolated from cells
and tissues by standard techniques (3 and 17) as well as by using
RMAzol B (Cinna/Biotecx, Houston, Tex.). RNA concentrations and
quality were assessed by UV spectroscopy on a Beckman DU 640
spectrophotometer and by gel analysis. Human tissue total RNA
samples were purchased from Clontech Laboratories, Inc., Palo Alto,
Calif.
[0231] Ribonuclease Protection Assays: A portion of the PSM cDNA
was subcloned into the plasmid vector pSPORT 1 (Gibco-BRL) and the
orientation of the cDNA insert relative to the flanking T7 and SP6
RNA polymerase promoters was verified by restriction analysis.
Linearization of this plasmid upstream of the PSM insert followed
by transcription with SP6 RNA polymerase yields a 400 nucleotide
antisense RNA probe, of which 350 nucleotides should be protected
from RNase digestion by PSM RNA. This probe was used in FIG. 20.
Plasmid IN-20, containing a 1 kb partial PSM cDNA in the plasmid
pCR II (Invitrogen) was also used for riboprobe synthesis. IN-20
linearized with Xmn I (Gibco-BRL) yields a 298 nucleotide
anti-sense RNA probe when transcribed using SP6 RNA polymerase, of
which 260 nucleotides should be protected from RNase digestion by
PSM mRNA. This probe was used in FIGS. 21 and 22. Probes were
synthesized using SP6 RNA polymerase (Gibco-BRL), rNTPs
(Gibco-BRL), RNAsin (Promega), and .sup.32P-rCTP (NEN, Wilmington,
Del.) according to published protocols (44). Probes were purified
over NENSORB 20 purification columns (NEN) and approximately 1
million cpm of purified, radiolabeled PSM probe was mixed with
10.mu. of each RNA and hybridized overnight at 45 C using buffers
and reagents from the RPA II kit (Ambion, Austin, Tex.). Samples
were processed as per manufacturer's instructions and analyzed on
5% polyacrilamide/7M urea denaturing gels using Seq ACRYL reagents
(ISS, Natick, Mass.). Gels were pre-heated to 55 C and run for
approximately 1-2 hours at 25 watts. Gels were then fixed for 30
minutes in 10% methanol/10% acetic acid, dried onto Whatman 3MM
paper at 80 C in a BioRad vacuum dryer and autoradiographed
overnight with Hyperfilm MP (Amersham). Quantitation of PSM
expression was determined by using a scanning laser densitometer
(LKB, Piscataway, N.J.).
[0232] Steroid Modulation Experiment: LNCaP cells (2 million) were
plated onto T-75 flasks in RPMI 1640 media supplemented with 5%
fetal calf serum and grown 24 hours until approximately 30-40%
confluent. Flasks were then washed several times with
phosphate-buffered saline and RPMI medium supplemented with 5%
charcoal-extracted serum was added. Cells were then grown for
another 24 hours, at which time dihydrotesterone, testosterone,
estradiol, progesterone, and dexamethasone (Steraloids Inc.,
Wilton, N.H.) were added at a final concentration of 2 nM. Cells
were grown for another 24 hours and RNA was then harvested as
previously described and PSM expression analyzed by ribonuclease
protection analysis.
Experimental Results
[0233] Immunohistochemical Detection of PSM: Using the 7E11-C5.3
anti-PSM monoclonal antibody, PSM expression is clearly detectable
in the LNCaP prostate cancer cell line, but not in the PC-3 and
DU-145 cell lines (FIGS. 17A-17C): All normal and malignant
prostatic tissues analyzed stained positively for PSM
expression.
[0234] In-Vitro Transcription/Translation of PSM Antigen: As shown
in FIG. 18, coupled in-vitro transcription/translation of the 2.65
kb full-length PSM cDNA yields an 84 kDa protein species in
agreement with the expected protein product from the 750 amino acid
PSM open reading frame. Following post-translational modification
using pancreatic canine microsomes were obtained a 100 kDa
glycosylated protein species consistent with the mature, native PSM
antigen.
[0235] Detection of PSM Antigen in LNCaP Cell Membranes and
Transfected PC-3 Cells: PC-3 cells transfected with the full length
PSM cDNA in the pREP7 expression vector were assayed for expression
of SM mRNA by Northern analysis. A clone with high PSM mRNA
expression was selected for PSM antigen analysis by Western
blotting using the 7E11-C5.3 antibody. In FIG. 19, the 100 kDa PSM
antigen is well expressed in LNCaP cell lysate and membrane
fractions, as well as in PSM-transfected PC-3 cells but not in
native PC-3 cells. This detectable expression in the transfected
PC-3 cells proves that the previously cloned 2.65 kb PSM cDNA
encodes the antigen recognized by the 7E11-C5.3 anti-prostate
monoclonal antibody.
[0236] PSM mRNA Expression: Expression of PSM mRNA in normal human
tissues was analyzed using ribonuclease protection assays. Tissue
expression of PSM appears predominantly within the prostate, with
very low levels of expression detectable in human brain and
salivary gland (FIG. 20). No detectable PSM mRNA expression was
evident in non-prostatic human tissues when analyzed by Northern
analysis. On occasion it is noted that detectable PSM expression in
normal human small intestine tissue, however this mRNA expression
is variable depending upon the specific riboprobe used. All samples
of normal human prostate and human prostatic adenocarcinoma assayed
have revealed clearly detectable PSM expression, whereas generally
decreased or absent expression of PSM in tissues exhibiting benign
hyperplasia (FIG. 21). In human LNCaP tumors grown both
orthotopically and subcutaneously in nude mice abundant PSM
expression with or without the use of matrigel, which is required
for the growth of subcutaneously implanted LNCaP cells was detected
(FIG. 21). PSM mRNA expression is distinctly modulated by the
presence of steroids in physiologic doses (FIG. 22). DHT
downregulated expression by 8-10 fold after 24 hours and
testosterone diminished PSM expression by 3-4 fold. Estradiol and
progesterone also downregulated PSM expression in LNCaP cells,
perhaps as a result of binding to the mutated androgen receptor
known to exist in the LNCaP cell. Overall, PSM expression is
highest in the untreated LNCaP cells grown in steroid-depleted
media, a situation that simulates the hormone-deprived (castrate)
state in-vivo. This experiment was repeated at steroid dosages
ranging from 2-200 nM and at time points from 6 hours to 7 days
with similar results; maximal downregulation of PSM mRNA was seen
with DHT at 24 hours at doses of 2-20 nM.
Experimental Discussion
[0237] Previous research has provided two valuable prostatic
bio-markers, PAP and PSA, both of which have had a significant
impact on the diagnosis, treatment, and management of prostate
malignancies. The present work describing the preliminary
characterization of the prostate-specific membrane antigen (PSM)
reveals it to be a gene with many interesting features. PSM is
almost entirely prostate-specific as are PAP and PSA, and as such
may enable further delineation of the unique functions and behavior
of the prostate. The predicted sequence of the PSM protein (30) and
its presence in the LNCaP cell membrane as determined by Western
blotting and immunohistochemistry, indicate that it is an integral
membrane protein. Thus, PSM provides an attractive cell surface
epitope for antibody-directed diagnostic imaging and cytotoxic
targeting modalities. The ability to synthesize the PSM antigen
in-vitro and to produce tumor xenografts maintaining high levels of
PSM expression provides us with a convenient and attractive model
system to further study and characterize the regulation and
modulation of PSM expression. Also, the high level of PSM
expression in the LNCaP cells provides an excellent in-vitro model
system. Since PSM expression is hormonally-responsive to steroids
and may be highly expressed in hormone-refractory disease. The
detection of PSM mRNA expression in minute quantities in brain,
salivary gland, and small intestine warrants further investigation,
although these tissues were negative for expression of PSM antigen
by immunohistochemistry using the 7E11-C5.3 antibody. In all of
these tissues, particularly small intestine, mRNA expression using
a probe corresponding to a region of the PSM cDNA near the 3' end,
whereas expression when using a 5' end PSM probe was not detected.
These results may indicate that the PSM mRNA transcript undergoes
alternative splicing in different tissues.
[0238] Applicants approach is based on prostate tissue specific
promotor: enzyme or cytokine chimeras. Promotor specific activation
of prodrugs such as non toxic gancyclovir which is converted to a
toxic metabolite by herpes simplex thymidine kinase or the prodrug
4-(bis(2chloroethyl)amino)benzoyl-1-glutamic acid to the benzoic
acid mustard alkylating agent by the pseudomonas carboxy peptidase
G2 was examined. As these drugs are activated by the enzyme
(chimera) specifically in the tumor the active drug is released
only locally in the tumor environment, destroying the surrounding
tumor cells. Promotor specific activation of cytokines such as
IL-12, IL-2 or GM-CSF for activation and specific antitumor
vaccination is examined. Lastly the tissue specific promotor
activation of cellular death genes may also prove to be useful in
this area.
[0239] Gene Therapy Chimeras: The establishment of "chimeric DNA"
for gene therapy requires the joining of different segments of DNA
together to make a new DNA that has characteristics of both
precursor DNA species involved in the linkage. In this proposal the
two pieces being linked involve different functional aspects of
DNA, the promotor region which allows for the reading of the DNA
for the formation of mRNA will provide specificity and the DNA
sequence coding for the mRNA will provide for therapeutic
functional DNA.
[0240] DNA-Specified Enzyme or Cytokine mRNA: When effective,
antitumor drugs can cause the regression of very large amounts of
tumor. The main requirements for antitumor drug activity is the
requirement to achieve both a long enough time (t) and high enough
concentration (c) (cxt) of exposure of the tumor to the toxic drug
to assure sufficient cell damage for cell death to occur. The drug
also must be "active" and the toxicity for the tumor greater than
for the hosts normal cells. The availability of the drug to the
tumor depends on tumor blood flow and the drugs diffusion ability.
Blood flow to the tumor does not provide for selectivity as blood
flow to many normal tissues is often as great or greater than that
to the tumor. The majority of chemotherapeutic cytotoxic drugs are
often as toxic to normal tissue as to tumor tissue. Dividing cells
are often more sensitive than non-dividing normal cells, but in
many slow growing solid tumors such as prostatic cancer this does
not provide for antitumor specificity.
[0241] Previously a means to increase tumor specificity of
antitumor drugs was to utilize tumor associated enzymes to activate
nontoxic prodrugs to cytotoxic agents. A problem with this approach
was that most of the enzymes found in tumors were not totally
specific in their activity and similar substrate active enzymes or
the same enzyme at only slightly lower amounts was found in other
tissue and thus normal tissues were still at risk for damage.
[0242] To provide absolute specificity and unique activity, viral,
bacterial and fungal enzymes which have unique specificity for
selected prodrugs were found which were not present in human or
other animal cells. Attempts to utilize enzymes such as herpes
simplex thymidine kinase, bacterial cytosine deaminase and
carboxypeptidase G-2 were linked to antibody targeting systems with
modest success. Unfortunately, antibody targeted enzymes limit the
number of enzymes available per cell. Also, most antibodies do not
have a high tumor target to normal tissue ratio thus normal tissues
are still exposed reducing the specificity of these unique enzymes.
Antibodies are large molecules that have poor diffusion properties
and the addition of the enzymes molecular weight further reduces
the antibodies diffusion.
[0243] Gene therapy could produce the best desired result if it
could achieve the specific expression of a protein in the tumor and
not normal tissue in order that a high local concentration of the
enzyme be available for the production in the tumor environment of
active drug.
Cytokines:
[0244] Results demonstrated that tumors such as the bladder and
prostate were not immunogenic, that is the administration of
irradiated tumor cells to the animal prior to subsequent
administration of non-irradiated tumor cells did not result in a
reduction of either the number of tumor cells to produce a tumor
nor did it reduce the growth rate of the tumor. But if the tumor
was transfected with a retrovirus and secreted large concentrations
of cytokines such as Il-2 then this could act as an antitumor
vaccine and could also reduce the growth potential of an already
established and growing tumor. IL-2 was the best, GM-CSF also had
activity whereas a number of other cytokines were much less active.
In clinical studies just using IL-2 for immunostimulation, very
large concentrations had to be given which proved to be toxic. The
key to the success of the cytokine gene modified tumor cell is that
the cytokine is produced at the tumor site locally and is not toxic
and that it stimulates immune recognition of the tumor and allows
specific and non toxic recognition and destruction of the tumor.
The exact mechanisms of how IL-2 production by the tumor cell
activates immune recognition is not fully understood, but one
explanation is that it bypasses the need for cytokine production by
helper T cells and directly stimulates tumor antigen activated
cytotoxic CD8 cells. Activation of antigen presenting cells may
also occur.
Tissue Promotor-Specific Chimera DNA Activation
Non-Prostatic Tumor Systems:
[0245] It has been observed in non-prostatic tumors that the use of
promotor specific activation can selectively lead to tissue
specific gene expression of the transfected gene. In melanoma the
use of the tyrosinase promotor which codes for the enzyme
responsible for melanin expression produced over a 50 fold greater
expression of the promotor driven reporter gene expression in
melanoma cells and not non melanoma cells. Similar specific
activation was seen in the melanoma cells transfected when they
were growing in mice. In that experiment no non-melanoma or
melanocyte cell expressed the tyrosinase drive reporter gene
product. The research group at Welcome Laboratories have cloned and
sequenced the promoter region of the gene coding for
carcinoembryonic antigen (CEA). CEA is expressed on colon and colon
carcinoma cells but specifically on metastatic. A gene chimera was
generated which cytosine deaminase. Cytosine deaminase which
converts 5 fluororocytosine into 5 fluorouracil and observed a
large increase in the ability to selectively kill CEA promotor
driven colon tumor cells but not normal liver cells. In vivo they
observed that bystander tumor cells which were not transfected with
the cytosine deaminase gene were also killed, and that there was no
toxicity to the host animal as the large tumors were regressing
following treatment. Herpes simplex virus, (HSV), thymidine kinase
similarly activates the prodrug gancyclovir to be toxic towards
dividing cancer cells and HSV thymidine kinase has been shown to be
specifically activatable by tissue specific promoters.
[0246] Prostatic Tumor Systems: The therapeutic key to effective
cancer therapy is to achieve specificity and spare the patient
toxicity. Gene therapy may provide a key part to specificity in
that non-essential tissues such as the prostate and prostatic
tumors produce tissue specific proteins, such as acid phosphatase
(PAP), prostate specific antigen (PSA), and a gene which was
cloned, prostate-specific membrane antigen (PSM). Tissues such as
the prostate contain selected tissue specific transcription factors
which are responsible for binding to the promoter region of the DNA
of these tissue specific mRNA. The promoter for PSA has been
cloned. Usually patients who are being treated for metastatic
prostatic cancer have been put on androgen deprivation therapy
which dramatically reduces the expression of mRNA for PSA. PSM on
the other hand increases in expression with hormone deprivation
which-means it would be even more intensely expressed on patients
being treated with hormone therapy.
Example 3
Cloning and Characterization of the Prostate Specific Membrane
Antigen (PSM) Promoter
[0247] The expression and regulation of the PSM gene is complex. By
immunostaining, PSM antigen was found to be expressed brilliantly
in metastasized tumor, and in organ confined tumor, less so in
normal prostatic tissue and more heterogenous in BPH. PSM is
strongly expressed in both anaplastic and hormone refractory
tumors. PSM mRNA has been shown to be down regulated by androgen.
Expression of PSM RNA is also modulated by a host of cytokines and
growth factors. Knowledge of the regulation of PSM expression
should aid in such diagnostic and therapeutic strategies as
immunoscintigraphic imaging of prostate cancer and
prostate-specific promoter-driven gene therapy.
[0248] Sequencing of a 3 kb genomic DNA clone revealed that two
stretches of about 300 B.P. (-260 to -600; and -1325 to -1625) have
substantial homology (79-87%) to known genes. The promoter lacks a
GC rich region, nor does it have a consensus TATA box. However, it
contains a TA-rich region from position -35 to -65.
[0249] Several consensus recognition sites for general
transcription factors such as AP1, AP2, NFkB, GRE and E2-RE were
identified. Chimeric constructs containing fragments of the
upstream region of the PSM gene fused to a promoterless
chloramphenicol acetyl transferase gene were transfected into, and
transiently expressed in LNCaP, PC-3, and SW620 (a colonic cell
line). With an additional SV40 enhancer, sequence from -565 to +76
exhibited promoter activity in LNCaP but not in PC-3 nor in
SW620.
Materials and Methods
[0250] Cell Lines. LNCaP and PC-3 prostatic carcinoma cell lines
(American Type Culture Collection) were cultured in RPMI and MEM
respectively, supplemented with 5% fetal calf serum at 37.degree.
C. and 5% CO.sub.2. SW620, a colonic cell line.
[0251] Polymerase Chain Reaction. The reaction was performed in a
50 l volume with a final concentration of the following reagents:
16.6 mM NH.sub.4SO.sub.4, 67 mM Tris-HCl pH 8.8, acetylated BSA 0.2
mg/ml, 2 mM MgCl.sub.2, 250 .mu.M dNTPs, 10 mM
.beta.-mercaptoethanol, and 1 U of the 111 Taq polymerase
(Boehringer Mannhiem, Calif.). A total of 25 cycles were completed
with the following profile: cycle 1, 94'C 4 min.; cycle 2 through
25, 94.degree. C. 1 min, 60.degree. C. 1 min, 72.degree. C. 1 min.
The final reaction was extended for 10 min at 72'C. Aliquots of the
reaction were electrophoresed on 1% agarose gels in 1.times.
Tris-acetate-EDTA buffer.
[0252] Cloning of PSM promoter. A bacteriophage P1 library of human
fibroblast genomic DNA (Genomic Systems, Inc., St. Louis, Mich.),
was screened using a PCR method of Pierce et al. Primers located at
the 5' end of PSM cDNA were used: 5'-CTCAAAAGGGGCCGGATTTCC-3' and
5'CTCTCAATCTCACTAATGCCTC-3'. A positive clone, p683, was digested
with XhoI restriction enzyme. Southern analysis of the restricted
fragments using a DNA probe from the extreme 5' to the Ava-1 site
of PSM cDNA confirmed that a 3 Kb fragment contains the 5'
regulatory sequence of the PSM gene. The 3 kb XhoI fragment was
subcloned into pKSBluescrpt vectors and sequenced using the dideoxy
method.
[0253] Functional Assay of PSM Promoter. Chloramphenicol Acetyl
Transferase, (CAT) gene plasmids were constructed from the
SmaI-HindIII fragments or subfragements (using either restriction
enzyme subfragments or PCR) by insertion into promoterless pCAT
basic or pCAT-enhancer vectors (Promega). pCAT-constructs were
cotransfected with pSV.beta.gal plasmid (5 .mu.g of each plasmid)
into cell lines in duplicates, using a calcium phosphate method
(Gibco-BRL, Gaithersburg, Md.). The transfected cells were
harvested 72 hours later and assayed (15 .mu.g of lysate) for CAT
activity using the LSC method and for .beta.gal activity (Promega).
CAT activities were standardized by comparision to that of the
.beta.gal activities.
Results
Sequence of the 5' End of the PSM Gene.
[0254] The DNA sequence of the 3 kb XhoI fragment of p683 which
includes 3017 by of DNA from the RNA start site was determined.
(FIG. 15) The sequence from the XhoI fragment displayed a
remarkable arrays of elements and motifs which are characteristic
of eukaryotic promoters and regulatory regions found in other genes
(FIG. 16).
Functional Analysis of Upstream PSM Genomic Elements for Promoter
Activity.
[0255] Various pCAT-PSM promoter constructs were tested for
promoter activities in two prostatic cell lines: LNCaP, PC-3 and a
colonic SW620 (FIG. 17). Induction of CAT activity was neither
observed in p1070-CAT which contained a 1070 by PSM 5' promoter
fragment, nor in p676-CAT which contained a 641 by PSM 5' promoter
fragment. However, with an additional SV-40 enhancer, sequence from
-641 to -1 (p676-CATE) exhibited promoter activity in LNCaP but not
in PC-3 nor in SW620.
[0256] Therefore, a LNCaP specific promoter fragment from -641 to
-1 has been isolated which can be used in PSM promoter-driven gene
therapy.
Example 4
Alternatively Spliced Variants of Prostate Specific Membrane
Antigen RNA: Ratio of Expression as a Potential Measurement of
Progression
Materials and Methods
[0257] Cell Lines. LNCaP and PC-3 prostatic carcinoma cell lines
were cultured in RPMI and MEM respectively, supplemented with 5%
fetal calf serum at 37.degree. C. and 5% CO.sub.2.
[0258] Primary tissues. Primary prostatic tissues were obtained
from MSKCC's in-house tumor procurement service. Gross specimen
were pathologically staged by MSKCC's pathology service.
[0259] RNA Isolation. Total RNA was isolated by a modified
guanidinium thiocynate/phenol/chloroform method using a RNAzol B
kit (Tel-Test, Friendswood, Tex.). RNA was stored in diethyl
pyrocarbonate-treated water at -80.degree. C. RNA was quantified
using spectrophometric absorption at 260 nm.
[0260] cDNA synthesis. Two different batches of normal prostate
mRNAs obtained from trauma-dead males (Clontech, Palo Alto, Calif.)
were denatured at 70.degree. C. for 10 min., then reverse
transcribed into cDNA using random hexamers and Superscript II
reverse transcriptase (GIBCO-BRL, Gaithersburg, Md.) at 50.degree.
C. for 30 min. followed by a 94.degree. C. incubation for 5
min.
[0261] Polymerase Chain Reaction. Oligonucleotide primers
(5'-CTCAAAAGGGGCCGGATTTCC-3' and 5'-AGGCTACTTCACTCAAAG-3'),
specific for the 5' and 3' ends of PSM cDNA were designed to span
the cDNA sequence. The reaction was performed in a 50 .mu.l volume
with a final concentration of the following reagents: 16.6 mM
NH.sub.4SO.sub.4, 67 mM Tris-HCl pH 8.8, acetylated BSA 0.2 mg/ml,
2 mM MgCl.sub.2, 250 .mu.M dNTPs, 10 mM .beta.-mercaptoethanol, and
1 U of rTth polymerase (Perkin Elmer, Norwalk, Conn.). A total of
25 cycles were completed with the following profile: cycle 1,
94.degree. C. 4 min.; cycle 2 through 25, 94.degree. C. 1 min,
60.degree. C. 1 min, 72.degree. C. 1 min. The final reaction was
extended for 10 min at 72.degree. C. Aliquots of the reaction were
electrophoresed on 1% agarose gels in 1.times. Tris-acetate-EDTA
buffer.
[0262] Cloning of PCR products. PCR products were cloned by the TA
cloning method into pCRII vector using a kit from Invitrogen (San
Diego, Calif.). Ligation mixture were transformed into competent
Escherichia coli Inv5.alpha..
[0263] Sequencing. Sequencing was done by the dideoxy method using
a sequenase kit from US Biochemical (Cleveland, Ohio). Sequencing
products were electrophoresed on a 5% polyacrylamide/7M urea gel at
52.degree. C.
[0264] RNase Protection Assays. Full length PSM cDNA clone was
digested with NgoM 1 and NheI. A 350 b.p. fragment was isolated and
subcloned into pSPORT1 vector (GIBCO-BRL, Gaithersburg, Md.). The
resultant plasmid, pSP350, was linearized, and the insert was
transcribed by SP6 RNA polymerase to yield antisense probe of 395
nucleotide long, of which 355 nucleotides and/or 210 nucleotides
should be protected from RNAse digestion by PSM RNA respectively.
Total celluar RNA (20 .mu.g) from different tissues were hybridized
to the aforementioned antisense RNA probe. Assays were performed as
described. tRNA was used as negative control. RPAs for LNCaP and
PC-3 were repeated.
Results
[0265] RT-PCR of mRNA from normal prostatic tissue. Two independent
RT-PCR of mRNA from normal prostates were performed as described in
Materials and Methods. Subsequent cloning and sequencing of the PCR
products revealed the presence of an alternatively spliced variant,
PSM'. PSM' has a shorter cDNA (2387 nucleotides) than PSM (2653
nucleotides). The results of the sequence analysis are shown in
FIG. 18. The cDNAs are identical except for a 266 nucleotide region
near the 5' end of PSM cDNA (nucleotide 114 to 380) that is absent
in PSM' cDNA. Two independent repetitions of RT-PCR of different
mRNA samples yielded identical results.
[0266] RNase Protection Assays. An RNA probe complementary to PSM
RNA and spanning the 3' splice junction of PSM' RNA was used to
measure relative expression of PSM and PSM' mRNAs (FIG. 19). With
this probe, both PSM and PSM' RNAs in LNCaP cells was detected and
the predominant form was PSM. Neither PSM nor PSM' RNA was detected
in PC-3 cells, in agreement with previous Northern and Western blot
data. FIG. 20 showed the presence of both splice variants in human
primary prostatic tissues. In primary prostatic tumor, PSM is the
dominant form. In contrast, normal prostate expressed more PSM'
than PSM. BPH samples showed about equal expression of both
variants.
[0267] Tumor Index. The relative expression of PSM and PSM' (FIG.
36) was quantified by densitometry and expressed as a tumor index
(FIG. 21). LNCaP has an index ranging from 9-11; CaP from 3-6; BPH
from 0.75 to 1.6; normal prostate has values from 0.075 to
0.45.
Discussion
[0268] Sequencing data of PCR products derived from human normal
prostatic mRNA with 5' and 3' end PSM oligonucleotide primers
revealed a second splice variant, PSM', in addition to the
previously described PSM cDNA.
[0269] PSM is a 750 a.a. protein with a calculated molecular weight
of 84,330. PSM was hypothesized to be a type II integral membrane
protein. A classic type II membrane protein is the transferrin
receptor and indeed PSM has a region that has modest homology with
the transferrin receptor. Analysis of the PSM amino acid sequence
by either the methods of Rao and Argos or Eisenburg et. al.
strongly predicted one transmembrane helix in the region from
a.a.#20 to #43. Both programs found other regions that could be
membrane associated but were not considered likely candidates for
being transmembrane regions.
[0270] PSM' antigen, on the other hand, is a 693 a.a. protein as
deduced from its mRNA sequence with a molecular weight of 78,000.
PSM' antigen lacks the first 57 amino acids present in PSM antigen
(FIG. 18). It is likely that PSM' antigen is cytosolic.
[0271] The function of PSM and PSM' are probably different. The
cellular location of PSM antigen suggests that it may interact with
either extra- or intra-cellular ligand(s) or both; while that of
PSM' implies that PSM' can only react with cytosolic ligand(s).
Furthermore, PSM antigen has 3 potential phosphorylation sites on
its cytosolic domain. These sites are absent in PSM' antigen. On
the other hand, PSM' antigen has 25 potential phosphorylation
sites, 10 N-myristoylation sites and 9 N-glycosylation sites. For
PSM antigen, all of these potential sites would be on the
extracellular surface. The modifications of these sites for these
homologous proteins would be different depending on their cellular
locations. Consequently, the function(s) of each form would depend
on how they are modified.
[0272] The relative differences in expression of PSM and PSM' by
RNase protection assays was analyzed. Results of expression of PSM
and PSM' in primary prostatic tissues strongly suggested a
relationship between the relative expression of these variants and
the status of the cell: either normal or cancerous. While it is
noted here that the sample size of the study is small (FIGS. 20 and
21), the consistency of the trend is evident. The samples used were
gross specimens from patients. The results may have been even more
dramatic if specimens that were pure in content of CaP, BPH or
normal had been used. Nevertheless, in these specimens, it is clear
that there is a relative increase of PSM over PSM' mRNA in the
change from normal to CaP. The Tumor Index (FIG. 21) could be
useful in measuring the pathologic state of a given sample. It is
also possible that the change in expression of PSM over PSM' may be
a reason for tumor progression. A more differentiated tumor state
may be restored by PSM' either by transfection or by the use of
differentiation agents.
Example 5
Enhanced Detection of Prostatic Hematogenous Micro-Metastases with
PSM Primers as Compared to PSA Primers Using a Sensitive Nested
Reverse Transcriptase-PCR Assay
[0273] 77 randomly selected samples were analyzed from patients
with prostate cancer and reveals that PSM and PSA primers detected
circulating prostate cells in 48 (62.3%) and 7 (9.1%) patients,
respectively. In treated stage D disease patients, PSM primers
detected cells in 16 of 24 (66.7%), while PSA primers detected
cells in 6 of 24 patients (25%). In hormone-refractory prostate
cancer (stage D3), 6 of 7 patients were positive with both PSA and
PSM primers. All six of these patients died within 2-6 months of
their assay, despite aggressive cytotoxic chemotherapy, in contrast
to the single patient that tested negatively in this group and is
alive 15 months after his assay, suggesting that PSA-PCR positivity
may serve as a predictor of early mortality. In post-radical
prostatectomy patients with negative serum PSA values, PSM primers
detected metastases in 21 of 31 patients (67.7%), while PSA primers
detected cells in only 1 of 33 (3.0%), indicating that
micrometastatic spread may be a relatively early event in prostate
cancer. The analysis of 40 individuals without known prostate
cancer provides evidence that this assay is highly specific and
suggests that PSM expression may predict the development of cancer
in patients without clinically apparent prostate cancer. Using PSM
primers, micrometastases were detected in 4 of 40 controls, two of
whom had known BPH by prostate biopsy and were later found to have
previously undetected prostate cancer following repeat prostate
biopsy performed for a rising serum PSA value. These results show
the clinical significance of detection of hematogenous
micrometastatic prostate cells using PSM primers and potential
applications of this molecular assay.
Example 6
Modulation of Prostate Specific Membrane Antigen (PSM) Expression
In Vitro by Cytokines and Growth Factors
[0274] The effectiveness of CYT-356 imaging is enhanced by
manipulating expression of PSM. PSM mRNA expression is
downregulated by steroids. This is consistent with the clinical
observations that PSM is strongly expressed in both anaplastic and
hormone refractory lesions. In contrast, PSA expression is
decreased following hormone withdrawal. In hormone refractory
disease, it is believed that tumor cells may produce both growth
factors and receptors, thus establishing an autocrine loop that
permits the cells to overcome normal growth constraints. Many
prostate tumor epithelial cells express both TGF.alpha. and its
receptor, epidermal growth factor receptor. Results indicate that
the effects of TGF.alpha. and other selected growth factors and
cytokines on the expression of PSM in-vitro, in the human prostatic
carcinoma cell line LNCaP.
[0275] 2.times.10.sup.6 LNCaP cells growing in androgen-depleted
media were treated for 24 to 72 hours with EGF, TGF.alpha.,
TNF.beta. or TNF.alpha. in concentrations ranging from 0.1 ng/ml to
100 ng/ml. Total RNA was extracted from the cells and PSM mRNA
expression was quantitated by Northern blot analysis and laser
densitometry. Both b-FGF and TGF.alpha. yielded a dose-dependent
10-fold upregulation of PSM expression, and EGF a 5-fold
upregulation, compared to untreated LNCaP. In contrast, other
groups have shown a marked downregulation in PSA expression induced
by these growth factors in this same in-vitro model. TNF.alpha.,
which is cytotoxic to LNCaP cells, and TNF.beta. downregulated PSM
expression 8-fold in androgen depleted LNCaP cells.
[0276] TGF.alpha. is mitogenic for aggressive prostate cancer
cells. There are multiple forms of PSM and only the membrane form
is found in association with tumor progression. The ability to
manipulate PSM expression by treatment with cytokines and growth
factors may enhance the efficacy of Cytogen 356 imaging, and
therapeutic targeting of prostatic metastases.
Example 7
Neoadjuvant Androgen-Deprivation Therapy (ADT) Prior to Radical
Prostatectomy Results in a Significantly Decreased Incidence of
Residual Micrometastatic Disease as Detected by Nested RT-PCT with
Primers.
[0277] Radical prostatectomy for clinically localized prostate
cancer is considered by many the "gold standard" treatment.
Advances over the past decade have served to decrease morbidity
dramatically. Improvements intended to assist clinicians in better
staging patients preoperatively have been developed, however the
incidence of extra-prostatic spread still exceeds 50%, as reported
in numerous studies. A phase III prospective randomized clinical
study designed to compare the effects of ADT for 3 months in
patients undergoing radical prostatectomy with similarly matched
controls receiving surgery alone was conducted. The previously
completed phase II study revealed a 10% margin positive rate in the
ADT group (N=69) as compared to a 33% positive rate (N=72) in the
surgery alone group.
[0278] Patients who have completed the phase III study were
analyzed to determine if there are any differences between the two
groups with respect to residual micrometastatic disease. A positive
PCR result in a post-prostatectomy patient identifies viable
metastatic cells in the circulation.
[0279] Nested RT-PCR was performed with PSM primers on 12 patients
from the ADT group and on 10 patients from the control group.
Micrometastatic cells were detected in 9/10 patients (90%) in the
control group, as compared to only 2/12 (16.7%) in the ADT group.
In the ADT group, 1 of 7 patients with organ-confined disease
tested positively, as compared to 3 of 3 patients in the control
group. In patients with extra-prostatic disease, 1 of 5 were
positive in the ADT group, as compared to 6 of 7 in the control
group. These results indicate that a significantly higher number of
patients may be rendered tumor-free, and potentially "cured" by the
use of neoadjuvant ADT.
Example 8
Sensitive Nested RT-PCR Detection of Circulation Prostatic Tumor
Cells Comparison of PSM and PSA-Based Assays
[0280] Despite the improved and expanded arsenal of modalities
available to clinician today, including sensitive serum PSA assays,
CT scan, transrectal ultrasonography, endorectal co.I MRI, etc.,
many patients are still found to have metastatic disease at the
time of pelvic lymph node dissection and radical prostatectomy. A
highly sensitive reverse transcription PCR assay capable of
detecting occult hematogenous micrometastatic prostatic cells that
would otherwise go undetected by presently available staging
modalities was developed. This assay is a modification of similar
PCR assays performed in patients with prostate cancer and other
malignancies. The assay employs PCR primers derived from the cDNA
sequences of prostate-specific antigen.sup.6 and the
prostate-specific membrane antigen recently cloned and
sequenced.
Materials and Methods
[0281] Cells and Reagents. LNCaP and MCF-7 cells were obtained from
the American Type Culture Collection (Rockville, Md.). Details
regarding the establishment and characteristics of these cell.
Cells grown in RPMI 1640 medium and supplemented with L-glutamine,
nonessential amino acids, and 5% fetal calf serum (Gibco-BRL,
Gaithersburg, Md.) In a 5% CO.sub.2 incubator at 37.degree. C. All
cell media was obtained from the MSKCC Media Preparation Facility.
Routine chemical reagents were of the highest grade possible and
were obtained from Sigma Chemical Company (St. Louis, Mo.).
[0282] Patient Blood Specimens. All blood specimens used in this
study were from patients seen in the outpatient offices of
urologists on staff at MSKCC. Two anti-coagulated tubes per patient
were obtained at the time of their regularly scheduled blood draws.
Specimens were obtained with informed consent of each patient, as
per a protocol approved by the MSKCC Institutional Review Board.
Samples were promptly brought to the laboratory for immediate
processing. Seventy-seven specimens from patients with prostate
cancer were randomly selected and delivered to the laboratory
"blinded" along with samples from negative controls for processing.
These included 24 patients with stage D disease (3 with D.sub.0, 3
with D.sup.1, 11 with D.sup.2, and 7 with D.sup.3), 31 patients who
had previously undergone radical prostatectomy and had undetectable
postoperative serum PSA levels (18 with pT2 lesions, 11 with pT3,
and 2 pT4), 2 patients with locally recurrent disease following
radical prostatectomy, 4 patients who had received either external
beam radiation therapy or interstitial l.sup.125 implants, 10
patients with untreated clinical stage T1-T2 disease, and 6
patients with clinical stage T3 disease on anti-androgen therapy.
The forty blood specimens used as negative controls were from 10
health males, 9 males with biopsy-proven BPH and elevated serum PSA
levels, 7 healthy females, 4 male patients with renal cell
carcinoma, 2 patients with prostatic intraepithelial neoplasia
(PIN), 2 patients with transitional cell carcinoma of the bladder
and a pathologically normal prostate, 1 patient with acute
prostatitis, 1 patient with acute promyelocytic leukemia, 1 patient
with testicular cancer, 1 female patient with renal cell carcinoma,
1 patient with lung cancer, and 1 patient with a cyst of the
testicle.
[0283] Blood Sample Processing/RNA Extraction. 4 ml of whole
anticoagulated venous blood was mixed with 3 ml of ice cold PBS and
then carefully layered atop 8 ml of Ficoll (Pharmacia, Uppsala,
Sweden) in a 14-ml polystyrene tube. Tubes were centrifuged at
200.times.g for 30 min. at 4.degree. C. The buffy coat layer
(approx. 1 ml.) was carefully removed and rediluted to 50 ml with
ice cold PBS in a 50 ml polypropylene tube. This tube was then
centrifuged at 2000.times.g for 30 min. at 4.degree. C. The
supernatant was carefully decanted and the pellet was allowed to
drip dry. One ml of RNazol B was then added to the pellet and total
RNA was isolated as per manufacturers directions (Cinna/Biotecx,
Houston, Tex.) RNA concentrations and purity were determined by UV
spectroscopy on a Beckman DU 640 spectrophotometer and by gel
analysis.
[0284] Determination of PCR Sensitivity. RNA was isolated from
LNCaP cells and from mixtures of LNCaP and MCF-7 cells at fixed
ratios (i.e. 1:100, 1:1,000, etc.) using RNAzol B. Nested PCR was
then performed as described below with both PSA and PSM primers in
order to determine the limit of detection for the assay.
LNCaP:MCF-7 (1:100,000) cDNA was diluted with distilled water to
obtain concentrations of 1:1,000,000. The human breast cancer cell
line MCF-7 was chosen because they had previously been tested by us
and shown not to express either PSM nor PSA by both
immunohistochemistry and conventional and nested PCR.
[0285] Polymerase Chain Reaction. The PSA outer primer sequences
are nucleotides 494-513 (sense) in exon 4 and nucleotides 960-979
(anti-sense) in exon 5 of the PSA cDNA. These primers yield a 486
by PCR product from PSA cDNA that can be distinguished from a
product synthesized from possible contaminating genomic DNA.
TABLE-US-00001 PSA-494 5'-TAC CCA CTG CAT CAG GAA CA-3' PSA-960
5'-CCT TGA AGC ACA CCA TTA CA-3'
[0286] The PSA inner upstream primer begins at nucleotide 559 and
the downstream primer at nucleotide 894 to yield a 355 by PCR
product.
TABLE-US-00002 PSA-559 5'-ACA CAG GCC AGG TAT TTC AG-3' PSA-894
5'-GTC CAG CGT CCA GCA CAC AG-3'
[0287] All primers were synthesized by the MSKCC Microchemistry
Core Facility. 5 .mu.g of total RNA was reverse-transcribed into
cDNA using random hexamer primers (Gibco-BRL) and Superscript II
reverse transcriptase (Gibco-BRL) according to the manufacturers
recommendations. 1 .mu.l of this cDNA served as the starting
template for the outer primer PCR reaction. The 20 .mu.l PCR mix
included: 0.5 U Taq polymerase (Promega) Promega reaction buffer,
1.5 mM MgCl.sub.2, 200 .mu.M dNTPs, and 1.0 .mu.M of each primer.
This mix was then transferred to a Perkin Elmer 9600 DNA thermal
cycler and incubated for 25 cycles. The PCR profile was as follows:
94.degree. C..times.15 sec., 60.degree. C..times.15 sec., and
72.degree. C. for 45 sec. After 25 cycles, samples were placed on
ice, and 1 .mu.l of this reaction mix served as the template for
another 25 cycles using the inner primers. The first set of tubes
were returned to the thermal cycler for 25 additional cycles. The
PSM outer upstream primer sequences are nucleotides 1368-1390 and
the downstream primers are nucleotides 1995-2015, yielding a 67 by
PCR product.
TABLE-US-00003 PSM-1368 5'-CAG ATA TGT CAT TCT GGG AGG TC-3'
PSM-2015 5'-AAC ACC ATC CCT CCT CGA ACC-3'
[0288] The PSM inner upstream primer span nucleotides 1689-1713 and
the downstream primer span nucleotides 1899-1923, yielding a 234 by
PCR product.
TABLE-US-00004 PSM-1689 5'-CCT AAC AAA AGA GCT GAA AAG CCC-3'
PSM-1923 5'-ACT GTG ATA CAG TGG ATA GCC GCT-3'
[0289] 2 .mu.l of cDNA was used as the starting DNA template in the
PCR assay. The 50 .mu.l PCR mix included: 1 U Taq polymerase
(Boehringer Mannheim), 250 .mu.M cNTPs, 10 mM
.beta.-mercaptoethanol, 2 mM MgCl.sub.2, and 5 .mu.l of a 10.times.
buffer mix containing: 166 mM NH.sub.4SO.sub.4, 670 mM Tris pH 8.8,
and 2 mg/ml of acetylated BSA. PCR was carried out in a Perkin
Elmer 480 DNA thermal cycler with the following parameters:
94.degree. C..times.4 minutes for 1 cycle, 94.degree. C..times.30
sec., 58.degree. C..times.1 minute, and 72.degree. C..times.1
minute for 25 cycles, followed by 72.degree. C..times.10 minutes.
Samples were then iced and 2.5 .mu.l of this reaction mix was used
as the template for another 25 cycles with a new reaction mix
containing the inner PSM primers. cDNA quality was verified by
performing control reactions using primers derived from the
.beta.-2-microglobulin gene sequence.sup.10 a ubiquitous
housekeeping gene. These primers span exons 2-4 and generate a 620
by PCR product. The sequences for these primers are:
TABLE-US-00005 B-2 (exon 2) 5'-AGC AGA GAA TGG AAA GTC AAA-3' B-2
(exon 4) 5'-TGT TGA TGT TGG ATA AGA GAA-3'
[0290] The entire PSA mix and 7-10 .mu.l of each PSM reaction mix
were run on 1.5-2% agarose gels, stained with ethidium bromide and
photographed in an Eage Eye Video Imaging System (Statagene, Torrey
Pines, Calif.). Assays were repeated at least twice to verify
results.
[0291] Cloning and Sequencing of PCR Products. PCR products were
cloned into the pCR II plasmid vector using the TA cloning system
(Invitrogen). These plasmids were transformed into competent E.
coli cells using standard methods.sup.11 and plasmid DNA was
isolated using Magic Minipreps (Promega) and screened by
restriction analysis. Double-stranded TA clones were then sequenced
by the dideoxy method using .sup.3SS-cCTP (NEN) and Sequenase (U.S.
Biochemical). Sequencing products were then analyzed on 6%
polyacrilamide/7M urea gels, which were fixed, dried, and
autoradiographed as described.
[0292] Southern Analysis. PCR products were transferred from
ethidium-stained agarose gels to Nytran nylon membranes (Schletcher
and Schuell) by pressure blotting with a Posi-blotter (Stratagene)
according to the manufacturer's instructions. DNA was cross-linked
to the membrane using a UV Stratalinker (Stratagene). Blots were
pre-hybridized at 65.degree. C. for 2 hours and subsequently
hybridized with denatured .sup.32P-labeled, random-primed cDNA
probes (either PSA or PSM)..sup.6,7 Blots were washed twice in
1.times.SSC/0.5% SDS at 42.degree. C. and twice in
0.1.times.SSC/0.1% SDS at 50.degree. C. for 20 minutes each.
Membranes were air-dried and autoradiographed for 1-3 hours at room
temperature with Hyperfilm MP (Amersham).
Results
[0293] PSA and PSM Nested PCR Assays: The application of nested PCR
increased the level of detection from an average of 1:10,000 using
outer primers alone, to better than 1:1,000,000. Dilution curves
demonstrating this added sensitivity are shown for PSA and PSM-PCR
in FIGS. 1 and 2 respectively. FIG. 1 shows that the 486 by product
of the PSA outer primer set is clearly detectable with ethidium
staining to 1:10,000 dilutions, whereas the PSA inner primer 355 by
product is clearly detectable in all dilutions shown. In FIG. 2 the
PSM outer primer 647 by product is also clearly detectable in
dilutions to only 1:10,000 with conventional PCR, in contrast to
the PSM inner nested PCR 234 by product which is detected in
dilutions as low as 1:1,000,000. Southern blotting was performed on
all controls and most of the patient samples in order to confirm
specificity. Southern blots of the respective dilution curves
confirmed the primer specificities but did not reveal any
significantly increased sensitivity.
[0294] PCR in Negative Controls: Nested PSA and PSM PCR was
performed on 40 samples from patients and volunteers as described
in the methods and materials section. FIG. 48 reveals results from
4 representtive negative control specimens, in addition to a
positive control. Each specimen in the study was also assayed with
the .beta.-2-midroglobulin control, as shown in the figure, in
order to verify RNA integrity. Negative results were obtained on 39
of these samples using the PSA primers, however PSM nested PCR
yielded 4 positive results. Two of these "false positives"
represented patients with elevated serum PSA values and an enlarged
prostate who underwent a transrectal prostate biopsy revealing
stromal and fibromuscular hyperplasia. In both of these patients
the serum PSA level continued to rise and a repeat prostate biopsy
performed at a later date revealed prostate cancer. One patient who
presented to the clinic with a testicular cyst was noted to have a
positive PSM nested PCR result which has been unable to explain.
Unfortunately, this patient never returned for follow up, and thus
have not been able to obtain another blood sample to repeat this
assay. Positive result were obtained with both PSA and PSM primers
in a 61 year old male patient with renal cell carcinoma. This
patient has a normal serum PSA level and a normal digital rectal
examination. Overall, if the two patients were excluded in whom a
positive PCR, but no other clinical test, accurately predicted the
presence of prostate cancer, 36/38 (94.7%) of the negative controls
were negative with PSM primers, and 39/40 (97.5%) were negative
using PSA primers.
[0295] Patient Samples: In a "blinded" fashion, in which the
laboratory staff were unaware of the nature of each specimen, 117
samples from 77 patients mixed randomly with 40 negative controls
were assayed. The patient samples represented a diverse and
heterogeneous group as described earlier. Several representative
patient samples are displayed in FIG. 49, corresponding to positive
results from patients with both localized and disseminated disease.
Patients 4 and 5, both with stage D prostate cancer exhibit
positive results with both the outer and inner primer pairs,
indicating a large circulating tumor cell burden, as compared to
the other samples. Although the PSM and PSA primers yielded similar
sensitivities in LNCaP dilution curves as previously shown, PSM
primers detected micrometastases in 62.3% of the patient samples,
whereas PSA primers only detected 9.1%. In patients with documented
metastatic prostate cancer (stages D.sub.0-D.sub.3) receiving
anti-androgen treatment, PSM primers detected micrometastases in
16/24 (66.7%), whereas PSA primers detected circulating cells in
only 6/24 (25%). In the study 6/7 patients with hormone-refractory
prostate cancer (stage D.sub.3) were positive. In the study, PSA
primers revealed micrometastatic cells in only 1/15 (6.7%) patients
with either pT3 or pT4 (locally-advanced) prostate cancer following
radical prostatectomy. PSM primers detected circulating cells in
9/15 (60%) of these patients. Interestingly, circulating cells
13/18 (72.2%) patients with pT2 (organ-confined) prostate cancer
following radical prostatectomy using PSM primers was detected.
None of these patient samples were positive by PSA-PCR.
[0296] Improved and more sensitive method for the detection of
minimal, occult micrometastic disease have been reported for a
number of malignancies by use of immunohistochemical methods, as
well as the polymerase chain reaction. The application of PCR to
detect occult hematogenous micrometastases in prostate cancer was
first described by Moreno, et al. using conventional PCR with
PSA-derived primers.
[0297] When human prostate tumors and prostate cancer cells
in-vitro were studied by immunohistochemistry and mRNA analysis,
PSM appeared to be highly expressed in anaplastic cells,
hormone-refractory cells, and bony metastases, in contrast to PSA.
If cells capable of hematogenous micrometastasis represent the more
aggressive and poorly-differentiated cells, they may express a
higher level of PSM per cell as compared to PSA, enhancing their
detectibility by RT-PCR.
[0298] Nested RT-PCR assays are both sensitive and specific.
Results have been reliably reproduced on repeated occasions. Long
term testing of both cDNA and RNA stability is presently underway.
Both assays are capable of detecting one prostatic cell in at least
one million non-prostatic cells of similar size. This confirms the
validity of the comparison of PSM vs. PSA primers. Similar levels
of PSM expression in both human prostatic cancer cells in-vivo and
LNCaP cells in-vitro resulted. The specificity of the PSM-PCR assay
was supported by the finding that two "negative control" patients
with positive PSM-PCR results were both subsequently found to have
prostate cancer. This suggests an exciting potential application
for this technique for use in cancer screening. In contrast to
recently published data, significant ability for PSA primers to
accurately detect micrometastatic cells in patients with
pathologically with pathologically organ-confined prostate cancer,
despite the sensitivity of the assay failed to result. Rather a
surprisingly high percentage of patients with localized prostate
cancer that harbor occult circulating prostate cells following
"curative" radical prostatectomy results which suggests that
micrometastasis is an early event in prostate cancer.
[0299] The application of this powerful new modality to potentially
stage and/or follow the response to therapy in patients with
prostate cancer certainly merits further investigation. In
comparison to molecular detection of occult tumor cells, present
clinical modalities for the detection of prostate cancer spread
appear inadequate.
[0300] Transition of prostate cancer from androgen dependent to
androgen independent state is a clinically important step which may
be caused or accompanied by genetic changes. Expression of prostate
specific membrane antigen (PSM) is most intense in LNCap cells, an
androgen dependent prostate carcinoma cell line: and is not
detectable in PC-3 nor in DU-145 cells, which are androgen
independent prostate carcinoma cell lines. A microsatellite repeat
of (TTTTG), (TTTG), has been found in the first intron of the PSM
gene. Our hypothesis is that this Microsatellite repeat could be a
cis-acting element in the regulation of PSM expression. A polymeric
chain reaction amplifying this repeat was used to look for any gene
alteration in several cell lines: LNCap, PC-3, PC-3M, DU-145 as
well as in 20 paired normal and early prostatic cancers (p12-4,
NO). In addition, immunohistochemistry (IHC) was used to analyze
PSM expression in patient samples. By IHC, no detectable expression
in DU-145, PC-3, and PC-3M was found, but all tumor expressed PSM.
Further sequencing data of the microsatellite repeat confirmed no
change in LNCap, and in contrast, an amplification in PC-3 and a
gross deletion in DU-145. Alteration of a T segment adjacent to the
microsatellite repeat was found in one tumor sample. These results
suggest that there is rarely alteration in the intronic
microsatellite sequence of the PSM gene in early prostate cancer.
The abnormal pattern in the absence of expression suggest genetic
instability in the more aggressive tumor lines such as the PC-3,
PC-3M and DU-145 cells.
Example 9
Chromosomal Localization of Cosmid Clones 194 and 683 by
Fluorescence In-Situ Hybridization
[0301] PSM was initially mapped as being located on chromosome
11p11.2-p13 (FIGS. 25-27). Further information from the cDNA
in-situ hybridizations experiments demonstrated as much
hybridization on the q as p arms. Much larger fragments of genomic
DNA was obtained as cosmids and two of these of about 60 kilobases
each one going 3' and the other 5' both demonstrated binding to
chromosome 11 p and q under low stringency. However under higher
stringency conditions only the binding at 11q14-q21 remained. This
result suggests that there is another gene on 11p that is very
similar to PSM because it is so strongly binding to nearly 120
kilobases of genomic DNA (FIG. 28).
[0302] Purified DNA from cosmid clones 194 and 683 was labelled
with biotin dUTP by nick translation. Labelled probes were combined
with sheared human DNA and independently hybridized to normal
metaphase chromosomes derived from PHA stimulated peripheral blood
lymphocytes in a solution containing 50% formamide, 10% dectran
sulfate, and 2.times.SSC. Specific hybridization signals were
detected by incubating the hybridized slides in fluoresein
conjugated avidin. Following signal detection the slides were
counterstained with propidium iodide and analyzed. These first
experiments resulted in the specific labelling of a group C
chromosome on both the long and short arms. This chromosome was
believed to be chromosome 11 on the basis of its size and
morphology. A second set of experiments were performed in which a
chromosome 11 centromere specific probe was cohybridized with the
cosmid clones. These experiments were carried out in 60% formamide
in an attempt to eliminate the cross reactive signal which was
observed when low stringency hybridizations were done. These
experiments resulted in the specific labelling of the centromere
and the long arm of chromosome 11. Measurements of 10 specifically
labelled chromosomes 11 demonstrated that the cosmid clones are
located at a position which is 44% of the distance from the
centromere to the telomere of chromosome arm 11q, an area that
corresponds to band 14q. A total of 160 metaphase cells were
examined with 153 cells exhibiting specific labelling.
[0303] Cloning of the 5' upstream and 3' downstream regions of the
PSM genomic DNA. A bacteriophage P1 library of human fibroblast
genomic DNA (Genomic Systems, St. Louis, Mich.) was screened using
the PCR method of Pierce et. al. Primer pairs located at either the
5' or 3' termini of PSM cDNA were used. Positive cosmid clones were
digested with restriction enzymes and confirmed by Southern
analysis using probes which were constructed from either the 5' or
3' ends of PSM cDNA. Positive clone p683 contains the 5' region of
PSM cDNA and about 60 kb upstream region. Clone -194 contains the
3' terminal of the PSM cDNA and about 60 kb downstream.
Example 10
Peptidase Enzymatic Activity
[0304] Prostate Specific Membrane Antigen has activity as a
carboxypeptidase and acts on both gamma linked or alpha linked
amino acids which have acidic amino acids such as glutamate in the
carboxy terminus.
[0305] Prostate specific membrane antigen is found in high
concentration in the seminal plasma. PSM antigen has enzymatic
activity with N-acetylaspartylglutamate as a substrate and
enzymatic action results in the release of, N-acetylaspartate and
glutamic acid. Because PSM action will release glutamate, and
because it is well known that the seminal fluid is highly enriched
in its content of glutamic acid, the action of PSM antigen of
endogenous protein/peptide substrates may be responsible for
generating the glutamic acid present.
[0306] It is also uncertain as to the role that seminal plasma
glutamic acid plays in fertility functions. It may be that
interruption of PSM antigen enzymatic activity may block the
generation of glutamate and could impact on seminal plasma glutamic
acid levels and its attendant fertility functions. Thus agents
which inhibit PSM antigen may prove to be useful in attenuating
male fertility.
Example 11
Ionotropicglutamate Receptors in Prostate Tissue
[0307] Prostate Specific Membrane antigen acts on
N-acetylaspartylglutamic acid to release glutamate and because a
homologous protein has been found in the rat brain which acts on
N-acetylyaspartylglutamate to free glutamate and N-acetylaspartate
and because these amino acids are considered to function as
neurotransmitters, the enzyme is considered to be potentially
important in modulating neurotransmitter excitatory amino acid
signalling as a neurocarboxypeptidase. This could be important in
the prostate as well, because of the neuroendocrine nature of a
subpopulation of cells in the prostate which are considered to be
important synthesizeing neuropeptide signaling molecules. PSM
antigen from the LNCaP cell was isolated and LNCaP cells can be
induced to exhibit a "neuron like" phenotype.
[0308] Excitatory neurotransmission in the central nervous system
(CNS) is mediated predominantly by glutamate receptors. Two types
of glutamate receptors have been identified in the human CNS:
metabotropic receptors, which serve G-protein coupled second
messenger signalling systems, and tonotropic receptors, which serve
as ligand gated ion channels. Ionotropic glutamate channels can
increase the inward flow of ions such as calcium ions. This can
result in the subsequent stimulation of nitric oxide, and nitric
oxide modulation of a number of signalling pathways. Nitric oxide
has been found to be a major signalling mechanism involved in cell
growth and death, response to inflammation, smooth muscle cell
contraction etc.
[0309] Methods: Detection of glutamate receptor expression was
performed using anti-gluR2/3 and anti-gluR4 polyclonal antibodies
and antibiotin immunohistochemical techniques in paraffin-embedded
human prostate tissues.
[0310] Results: Anti-gluR2/3 immunoreactivity was unique to
prostatic stroma and was absent in the prostatic epithelial
compartment. Strong anti-gluR4 immunoreactivity was observed in the
basal cells of the prostate. This implied a differential location
and function of glutamate receptors as defined by these
antibodies.
[0311] Discussion: Distribution of glutamate receptors in the
prostate has not been described. Basal cells are considered the
precursor cell for the prostatic acinar and neuroendocrine cells of
the prostate. Glutamate receptors may provide signalling functions
in their interactions with the prostate stroma and acinar cells,
and PSM may be involved in that interaction. Thus inhibition or
enhancement of PSM activity could serve to modulate activity of the
basal cells and prove to be a valuable aid for controlling basal
cell function in the prostate.
[0312] The finding of glutamate like receptors in the stroma is of
interest because a large part of the prostate volume is due to
stromal cells. Current observation have suggested that these
stromal cells have a smooth muscle cell phenotype and thus the
presence of glutamate receptors may play a role in their biologic
function and regulation of differentiation. A most common disease
in men is the abnormal benign growth of the prostate termed benign
prostatic hyperplasia, BPH.
[0313] In areas of BPH a decrease in the level of expression of PSM
antigen was observed. If PSM antigen activity is providing an
aspect of the signalling for normal stromal function then the
abnormal growth seen in BPH may be a response to that decreased
activity and agents to restore its function could play a role in
the treatment or prevention of BPH.
[0314] Altering PSM antigen function may have beneficial actions
outside the prostate. In the rat CNS a protein homology to PSM
antigen was discovered and provides a rational to consider prostate
specific membrane antigen as a neurocarboxypeptidase. Alterations
in its function may occur in neurotoxic disorders such as epilepsy,
or ALS, alzheimers, and multiple sclerosis.
Example 12
Identification of a Membrane-Bound Pteroylpolygammaglutamyl
Carboxypeptidase (Folate Hydrolase) that is Expressed in Human
Prostatic Carcinoma
[0315] As described PSM functions as a carboxypeptidase to
hydrolyze both alpha and gamma peptide linkages with amino acids
such as glutamate in the terminal carboxy position. The proximal
small intestine (duodenum-strong expression PSM) but not the distal
small intestine (ileum-absent PSM) was also very rich in expression
of message for prostate specific membrane antigen in RNase
protection assays. PSM antigen by immunohistochemistry was observed
in the brush border membranes of the duodenum. This location was
consistent with a hydrolase known as folate conjugase (folate
hydrolase as a carboxypeptidase, not an endopeptidase) that had
been described in the older literature, with the protein having
been partially purified from the human small intestine. No cloning
or sequencing of this gene had been done. There is a form of folate
hydrolase that is found in all cells in the lysosomes and it was
recently sequenced. There is no sequence relationship between the
lysosomal endopeptidase. Membrane fraction of the LNCaP cells was
very rich in folate hydrolase activity. The PSM specific monoclonal
could be used to immunoprecipitate the folate hydrolase activity.
This result always has the possibility that the folate hydrolase
activity is not the same as PSM antigen but is a coprecipitating
contaminant. Therefore PSM antigen was transfected into PC-3 cells.
PC-3 cells do not express PSM nor do they have membrane folate
hydrolase activity. In cells transfected with PSM antigen however
expression of folate hydrolase activity was observed in the
membranes. Thus PSM is a novel folate hydrolase, folate
carboxypeptidase, and is active in sequentially removing the
terminal gamma-linked glutamates. In the proximal small intestine
it is understandable why this enzyme would be in such a place, as
the majority of folate available from food is polygammglutamated
and this enzyme is responsible for its hydrolysis.
[0316] Materials: Methotrexate triglutamate
(4-NH.sub.2-10-CH.sub.3-PteGlu.sub.4 (MTXglu.sub.3)),
pteroylpentaglutamate (PteGlu.sub.5), and
para-aminobenzoylpentaglutamate, (pABAGlu.sub.5) were purchased
from Dr. B. Schircks Laboratories (Jona, Switzerland) and samples
were >98% pure when evaluated by HPLC.
N-acetyl-.alpha.-aspartylglutamate (NAAG) (40 Ci/mmol) was
purchased from New England Nuclear (Boston, Mass.). Protein A
Sepharose 4 Fast Flow was purchased from Pharmacia (Piscataway,
N.J.). The 7E11-C5 monoclonal antibody to prostate specific
membrane antigen was obtained from Cytogen Corporation, Princeton,
N.J. All other reagents (p-hydroxymercuribenzoate, homocysteine,
dithiothreitol (DTT), reduced glutathione) were of the highest
purity commercially available from Sigma Chemical Co. (St. Louis,
Mo.).
[0317] Culture and growth of human prostate adenocarcinoma cells
(LNCaP, PC-3, TSU-Pr1, and Duke-145): LNCaP cells were maintained
in defined culture medium, RPMI-1640 medium supplemented with
non-essential amino acids, 5 mM glutamine, and 5% heat-inactivated
fetal calf serum. Duke-145, PC-3, and TSU-Pr1 cells were grown in
minimal essential medium (MEM), Ham's F-12K, and MEM, respectively,
containing 5% fetal calf serum. No antibiotic was included in the
media. Cells (1.times.10.sup.6) were plated in T-75 tissue culture
flasks containing 15 mL of medium and incubated at 37.degree. C. in
a humidified atmosphere of 5% CO.sub.2. Cell numbers were
determined using a Model Z F Coulter Counter (Coulter Electronic,
Inc.). Prostate cells were harvested from plates by gentle scraping
at 4.degree. C. into phosphate buffered saline (136.9 mM NaCl, 2.68
mM KCl, 8.10 mM Na.sub.2HPO.sub.4, 1.47 mM KH.sub.2PO.sub.4, pH
7.34, PBS) and centrifuged at 500.times.g to obtain a cell pellet.
Sedimented cells were routinely rinsed twice with 15 mL volumes of
PBS.
[0318] Transfection of PSM into PC-3 Prostate Cell Line: The full
length 2.65 kb PSM cDNA was subcloned into a pREP7 eukaryotic
expression vector (Invitrogen, San Diego, Calif.) as previously
described. Plasmid DNA was purified from transfected DH5-.alpha.
(Gibco-BRL) using a Qiagen maxi prep plasmid isolation kit (Qiagen
Inc., Chatsworth, Calif.). Purified plasmid DNA (5 .mu.g) was
diluted with 300 .mu.L of serum free RPMI media and mixed with 45
.mu.L of lipofectamine (Gibco-BRL) which was previously diluted
with 300 .mu.L of serum free RPMI media to allow an DNA-liposome
complex to form. The mixture was kept at room temperature for 30
minutes, then added to a 60 mm petri dish containing 60-70%
confluent PC-3 cells in 2.4 mL serum free RPMI. The DNA-liposome
complex containing serum free media was mixed gently to ensure
uniform distribution and was then incubated for 6 h at 37.degree.
C. in a CO.sub.2 incubator. Following incubation, the media
containing liposome-DNA complex was aspirated and replaced with 6
mL of regular growth media (10% fetal bovine serum, 1%
penicillin-streptomycin, 1% glutamine). After 48 hours, cells were
trypsinized and split 1:3 into 60 mm dishes containing regular
media supplemented with 200 .mu.g/mL of hygromycin B (Calbiochem,
LaJolla, Calif.). Cells were maintained for 2 weeks with changes of
media containing hygromycin B every third day until discrete
colonies appeared. Colonies were isolated using a 6 mm cloning
cylinder and were expanded in the same media. As a control, PC-3
cells were also transfected with the pREP7 vector alone.
[0319] Immunohistochemistry: The 7E11-C5 monoclonal antibody to
prostate specific antigen was used. This antibody recognizes a
portion of carbohydrate-containing peptide epitope on the amino
terminal end of PSM that is located on the inner portion of the
cytosolic membrane. After permeabilization of LNCaP and PC-3
transfected and non-transfected cells with a mixture of acetone and
methanol (1:1 v/v) and blocking with 5% bovine serum albumin in 50
mM Tris buffered saline (TBS) pH 7.45, samples were incubated with
7E11-C5 antibody (20 .mu.g/mL) for 1 h at room temperature.
Negative controls were generated by substituting the same
concentration of mouse IgG2ak for the PSM antibody. Using a
secondary IgG.sub.1 anti-mouse antibody conjugated with alkaline
phosphatase, samples were re-incubated for 1 h, rinsed in TBS, and
stained with bromochloroindolylphenol phosphate in
2-amino-2-methyl-1-propanol buffer. Cells expressing PSM
demonstrate an intense blue staining.
[0320] Cell Membrane Preparation: Cell lysates were prepared by
sonicating approximately 6.times.10.sup.6 cells in 50 mM Tris pH
7.4 buffer (2.times.10 s pulses at 20 mWatts) in an ice-bath.
Membrane fractions were obtained by centrifuging lysates at
100,000.times.g for 30 mins. The supernatant fractions were saved
and pelleted membranes were re-suspended by gentle trituration and
re-sedimented at 100,000.times.g for 30 mins through 10 mL of cold
50 mM Tris pH 7.4 buffer. Washed membrane fractions were dissolved
in 50 mM Tris pH 7.4 buffer containing 0.1% Triton X-100
(Tris/Triton). Enzymatic activity and immunoprecipitation
preparations were performed using this membrane preparation.
[0321] Immunoprecipitation of PSM from Membrane: Membrane pellets
(.about.1 mg protein) solubilized in Tris/Triton buffer were
incubated at 4.degree. C. for 1 h in the presence of 7E11-C5
anti-prostate monoclonal antibody (6 ug protein). Protein A
Sepharose gel equilibrated in Tris/Triton buffer was added to the
immunocomplex. This preparation was subsequently incubated for an
additional hour at 4.degree. C. Sepharose beads were centrifuged at
500.times.g for 5 mins and rinsed twice with Tris/Triton buffer at
pH 7.4. Isolated beads were resuspended in 0.1 M glycine buffer pH
3.0, vortexed, and the supernatant fraction was assayed for
hydrolase activity using MTXglu.sub.3.
[0322] Pteroyl Gamma-Glutamyl Hydrolase Assay: Hydrolase activity
was determined using capillary electrophoresis. The standard assay
mixture contained 50 uM MTXGlu.sub.3, 50 mM acetate buffer (pH 4.5)
and enzyme to a final volume of 100 uL. A sample preparation
without enzyme was incubated concurrently with enzymatic assays and
reactions were conducted for times varying between 0 and 240 min at
37.degree. C. Activities were also determined in standard reaction
mixture at varied pHs for 60 min. Reactions were terminated in a
boiling water bath for 5 min and samples were stored frozen
(-20.degree. C.) until analysis. Following centrifugation
(7,000.times.g) to remove precipitated debris, capillary separation
of MTX glutamated analogues was performed with a Spectra Phoresis
1000 instrument (Thermo Separation, San Jose, Calif.) with a 75
.mu.m id.times.50 cm silica capillary (Polymicro Technology,
Phoenix, Ariz.). Separation of pteroyl(glutamate).sub.n derivatives
is achieved with an electrolyte of 20 mM sodium borate with 15 mM
sodium dodecylsulfate (pH 9.5) with +20 Kev at 25.degree. C.
Samples were applied hydrodynamically for 1-2 s and absorbance
monitored at 300 nm. Data were recorded with an IBM computer using
CE-1000 software (Thermo Separation).
[0323] Protein determination: Protein concentrations of isolated
membrane or supernatant fractions were determined by incubating
diluted aliquots with BCA reagent (Pierce Chemical Co., Rockford,
Ill.) at 37.degree. C. for 30 min. The spectrophotometric
quantitation of protein was conducted by determining the absorbance
at 562 nm against bovine serum albumin standard.
[0324] Statistical Analysis: Data were analyzed by using the
Statgraphics version 4.0 program (Statistical graphics Corporation,
Rockville, Md.) and where summarized are expressed as mean.+-.S.D.
Student's unpaired t test was used to determine significance of
differences.
Results:
[0325] Membrane fractions isolated from human prostate
adenocarcinoma cells (LNCaP) were incubated using primarily
MTXglu.sub.3 as substrate. The time course of hydrolysis of the
gamma-linked triglutamate derivatie and the subsequent appearance
of MTXglu.sub.2, MTXglu.sub.1, and MTX after 30, 60, 120, and 240
min of incubation are illustrated in FIG. 82. The semipurified PSM
antigen exhibits pteroyl poly gamma-glutamyl exopeptidase activity
that progressively liberates all of the possible glutamates from
MTXGlu.sub.3 with accumulation of MTX.
[0326] The PSM antigen was immunoprecipitated in the presence of
7E11-C5 anti-prostate monoclonal antibody and the PSM
antigen-antibody complex was adsorbed onto a Protein A Sepharose
Gel column. Following twice washing of the sepharose beads with 2
mL volumes of buffer and re-solubilization of the antigen-antibody
complex by adjusting the elution pH to 3.0, the supernatant
fraction was assayed for hydrolase activity. FIG. 55 shows the
capillary electrophoretic separation of successively cleaved
glutamyl moieties from MTXglu.sub.3 after 0, 30, 60 and 240 min
incubations. Results similar to these in FIG. 82 were obtained
using pteglu.sub.5 with formation of folate (pteglu.sub.1).
[0327] The optimum pH activity profiles of the immunoprecipitated
PSM hydrolase from LNCaP cells and of the membrane fractions from
PC-3 PSM-transfected and non-transfected (vector alone) cells are
shown in FIG. 57. The reaction was monitored as a function of pH
from 2 to 10 after an 1 h incubation with MTXglu.sub.3. The extent
of reaction was expressed as the concentration of MTXglu.sub.2
formed per mg protein. Although all reaction products were
detectable as illustrated in FIG. 56, MTXglu.sub.2 was the
predominant hydrolyzed species at incubation times ranging from 10
to 60 min. The pH profile of membrane fractions isolated from both
LNCaP and PC-3 PSM-transfected cells are identical and exhibit two
maxima of PSM hydrolase activity at pH 5 and 8 with no measurable
activity above pH 10.
[0328] To determine whether non-PSM expressing human adenocarcinoma
cell lines (PC-3, TSU-Pr1, and Duke-145) exhibit folate hydrolase
activity, isolated membrane preparations from these cell lines were
analyzed (FIG. 83). The less differentiated, hormone refractory
prostate cell lines (PC-3, TSU-Pr1, and Duke-145) exhibit no
appreciable activity after 2 h incubations. These results are in
agreement with previous findings that demonstrate neither a
presence of a mRNA for PSM nor antigen immunoreactivity with
7E11-C5 in these cells.
[0329] In further studies in which the cDNA for PSM was transfected
into non-PSM antigen expressing PC-3 cells, a close correlation
between PSM antigen immunoreactivity and hydrolase activity was
observed with MTXglu.sub.3 in membranes of LNCaP and PC-3
PSM-transfected cells (FIGS. 58 and 59). Immunohistochemical
analyses of LNCaP (FIG. 58) and PSM antigen expressing PC-3 (FIG.
85B) cells revealed distinct positive staining with 7E11-C5
anti-prostate monoclonal antibody. FIG. 85C illustrates no
immunoreactivity in PC-3 cells expressing the pREP7 hygromycin
vector alone. In preparations of negative controls, all three cell
lines were reacted with IgG2aK rather than with 7E11-C5 antibody.
No background staining resulted with the secondary antibody
conjugated with alkaline phosphatase.
[0330] To compare PSM hydrolase activity with that of other
gamma-glutamyl hydrolases that either reside within the lysosome or
are secreted as observed in several neoplastic cells, its
reactivity in the presence of thiol-containing reducing agents,
namely, reduced glutathione, homocysteine, and dithiothreitol
(DTT), and the thiol reagent, p-hydroxymercuribenzoate (PHMB), at
concentrations ranging from 0.05-0.5 mM was observed. Of the
reduced sulfhydryl derivatives, it was discovered that only DTT
(.gtoreq.0.2 mM) was slightly inhibitory (86.+-.3% of control).
Unlike gamma-linked peptide hydrolase retained within the lysosome,
PSM hydrolase activity was maintained in the presence of 0.5 mM
PHMB.
[0331] The reactivity of PSM hydrolase against an .alpha.-glutamate
dipeptide, N-acetyl-.alpha.-aspartylglutamate (NAAG), has been
investigated and that the PSM enzyme from either LNCaP or PSM
transfected PC-3 cell membranes hydrolyses NAAG producing
N-acetylaspartate and glutamate was observed. Furthermore,
MTXglu.sub.3, pteglu.sub.5, and pABAglu.sub.5 were potent
inhibitors of the PSM-mediated NAAG hydrolysis.
Discussion:
[0332] Membrane-bound PSM antigen has pteroyl poly gamma-glutamyl
carboxypeptidase (folate hydrolase) activity. Gamma-glutamyl
hydrolase activity is also present in lysosomes of cells and these
enzymes may be responsible for regulating the length of exogenous
and endogenous folyl polyglutamate chain lengths. A characteristic
difference between these two hydrolases is that the PSM enzyme
exhibits substantial activity at pH values 7.5 to 8.0 in addition
to having an acidic pH 4.5 to 5 optimum. Moderate levels of
hydrolase activity are present within LNCaP cytosolic compartment
and may represent the short intracellular fragment of this class II
enzyme. This reflects an interesting situation in these cells where
the majority of RNA codes for the membrane-bound enzyme that is
localized extracellularly. The ratio of the mRNAs in these samples
that code for the class II membrane and the cytosolic proteins is
ten to one. In normal prostate tissue, the mRNA coding for the
membrane protein is only one-tenth that of the cytosolic form.
[0333] It is clear from this study that the prostate specific
membrane antigen functions as a folate hydrolase and is unique in
that it has activity on both the gamma-linked as well as the alpha
linked peptide bonds. This is interesting for a number of reasons.
First in the normal prostate it was demonstrated that the majority
of the mRNA encodes a protein, PSM', that is likely to be cytosolic
and would imply that it may be that in the prostate that folates
could exists in the lesser glutamated species. If so then it means
that the folate in the prostate can readily leak out and that the
prostate may be subjected to "microenviromental folate
deficiencies" This may be related to the high worldwide incidence
of "microscopic prostate cancer" as folate deficiencies are
associated with carcinogenesis in a number of tissues.
[0334] Benign enlargement of the prostate and prostate cancer occur
in older men. It also occurs that the uptake of folate decreases
with aging. If folate uptake decreases with aging this may be due
to decreased PSM folate hydrolase activity in the proximal
intestine. To correct such a deficiency it might be possible to use
PSM folate hydrolase in foods to release the folate before
consumption or take it with foods as is done with lactase in
lactose intolerant individuals. If the prostate in men is
susceptible to folate depletion then nutritional supplementation
may help reduce the development of the microscopic lesion, indeed
in some cancers such as cancer of the colon, folate supplementation
was found to reduce cancer formation.
[0335] Why would the prostate cells prefer to have the lesser
glutamated forms of folate? It may be that methionine synthase
which is an enzyme key to folate uptake and folate utilization for
one carbon methyl transfer metabolism may utilize the nonglutamated
folate preferentially. In addition to folate deficiency, choline
and methionine deficiency is also associated with tumor
development. If shown to modulate one carbon transfers, it might be
useful to inhibit this enzyme as a means to inhibit cancer
development and thus serve as a chemopreventative agent. Again
modulation of PSM folate hydrolase may play a role in tumor
prevention and modulation of tumor growth.
[0336] A feature that cell biologists use in transfecting DNA into
cells often requires selection of the transfected gene and often
multiple transfections are performed. These are done with drugs
that are toxic to cells such as Hygromycin and use genes that code
for Hygromycin resistance which are bacterial. It may be that PSM
could be used as a selectable marker by growing the transfected
cells in folate free media and including polyglutamated folate
which would be able to rescue cells from folate deficiency if they
expressed PSM.
[0337] PSM folate hydrolase activity can possibly be used as a
prodrug converting enzyme. In the normal prostate PSM is
intracellular. In the transformed cell the majority of the protein
and its attendant enzymatic activity is extracellular in location.
It may be that as the enzymes associated with cell growth require
the polyglutamated forms the cancer finds a way to remove PSM
folate hydrolase from the interior by alternative splicing to an
extracellular enzyme. PSM is a membrane protein and is found to
predominate in cancer, but PSM' is likely a cytosolic protein which
predominates in the normal condition.
[0338] This implies that development of a prodrug that requires
metabolism before it can be taken up by the tumor cell could be
activated by the PSM folate hydrolase which is predominate in the
cancer.
[0339] Methotrexate triglutamate was one of the agents used to
identify the enzymatic activity of PSM antigen. Methotrexate
triglutamate would not be able to use the transport protein to be
taken into tumor cells, because there are specific structural
requirements for folate, or methotrexate transport. If one removes
the gamma-linked glutamates then methotrexate can be taken into
cells and can exerts its antifolate, antitumor growth action.
[0340] Therfore methotrexategammatriglutamate was used to examine
the action of this compound on the in vitro growth of PC-3 cells
transfected with a plasmid with a selectable marker versus a
plasmid with a selectable marker that expresses PSM antigen as
well. the PC-3 cells that were transfected with PSM were inhibited
85% in growth by day four by 10 uM methotrexate triglutamate, while
the PC-3 plasmid only transfectants did not exhibit any significant
inhibition of growth.
[0341] PSM's folate hydrolase activity hydrolyses down to the last
glutamate which is in alpha linked position but does not remove it.
Because it does not remove the last glutamate, PSM antigen's folate
hydrolase activity better serves the prodrug activation
requirements of such a prodrug. Also because it is a human enzyme
it is less likely than the carboxypeptidase G2 will cause an immune
response because PSM antigen is normally present in the body.
[0342] In addition PSM could also be used as part of a prodrug
strategy that utilized gene transfer and a tissue or tumor specific
promoter, say such that it would be linked to CEA promoter and PSM
expressed in colon tumors and the patients subsequently given the
prodrug such as methotrexate triglutamate. The same is also true
for the protein itself, either the whole protein or the components
of the active site or a modified version that would have increased
prodrug activating activity could be linked to a delivery vehicle
such as an antibody or other specific targeting ligand, delivered
to the tumor for localization and subsequent activation.
[0343] Methotrexate as a prodrug may be enhanced in specificity by
using alpha linked glutamates rather than gamma linked glutamates
because the ubiquitous lysosomal hydrolase enzyme is specific for
the gamma linked bond. A pro-drug with all alpha linked glutamates
would not be a substrate, but would be a substrate for the PSM
folate hydrolase.
[0344] In addition to methotrexate a number of potential enzyme
substrates can be employed as cytotoxic prodrugs. The synthesis of
potential prodrugs, PALAglu, and a number of other potential agents
are described.
[0345] Alpha-linked methotrexate material is synthesized by the
following Merifield solid phase scheme (see FIG. 88). The scheme is
based on a modification of the standard Merifield solid peptide
synthesis that was applied to the synthesis of methotrexate y
polyglutamates. In brief the N-Fmoc-4-terbutylglutamate is first
connected to the resin under standard coupling conditions using
diisoprpylazodicarboxylate as a coupling reagent. The Fmoc
protecting group is then removed with piperidine, and this cycle
would be reiterated for as many times as glutamates would be needed
to obtain the desired analog. For example say the pentaglutamate on
solid support is the intermediate required for the preparation of
methotrexate-alpha-tetraglutamate. It is deprotected at the
terminal nitrogen by treatment with piperidine, then coupled with
pteroic acid analogue under the same conditions used above. The
terbutyl and the resin are all removed in one step with 95%
trifluoroacetic acid (TPA) to provide the desired material. This
process is applied to every analog. The gamma linked material is
provided in a similar manner for use comparative studies with the
alpha-linked material (see FIG. 89). Because of the
carboxypeptidase activity a number of combination of alpha and
gamma linked acidic amino acid can be optimized for their
utilization of the enzyme and for in vivo activity. In addition to
the folate like antagonists, a number of amino acid analogs were
found in the past to have antitumor activity but lacked in vivo
specificity. These agents are targetable by attaching a glutamate
to the carboxy terminus of the amino acid as described and shown in
the figures.
[0346] PALA-Glutamate 3 and analog 5, was synthesized in a similar
manner with the addition to the introduction of a protected
phosphonoacetate moiety instead of a simple acetate. It is
compatible with the function of diethylphosphonoacetic acid which
allows the removal of the ethyl groups under relatively mild
conditions.
[0347] Commercially available diethylphosphonoacetic acid was
treated with perfluorophenyl acetate in pyridine at 0 deg.C to room
temperature for an hour to afford the corresponding
pentafluorophenyl ester in nearly quantitative yield after short
path column chromatography. This was then reacted with
gamma-benzylaspartate and HOAT in tetrahydrofuran for half an hour
at reflux temperature to give protected PALA 7
(N-phosphonoacetylaspartate) in 90% yield after flash column
chromatography. The free acid was then activated as its
pentafluorophenyl ester 8, then it was reacted with
delta-benzyl-L-glutamate and HOAT in a mixture of THF-DMF (9:1,
v/v) for 12 hours at reflux to give fully protected PALA-Glutamate
9 in 66% yield after column chromatography. Sequential removal of
the ethyl groups followed by the debenzylation was accomplished for
a one step deprotection of both the benzyl and ethyl groups. Hence
protected PALA-Glutamate was heated up to reflux in neat
trimethylsilylchloride for an overnight period. The resulting
bistrimethylsilylphosphonate ester 10 was submitted without
purification to hydrogenolysis (H.sub.2, 30 psi, 10% Pd/C,
ethylacetate). The desired material 3 was isolated after
purification by reverse phase column chromatography and ion
exchange resin.
[0348] Analogs 4 and 5 were synthesized by preparation of
phosphonoglutamate 14 from the alpha-carboxyl-protected
glutamate.
[0349] Commercially available alpha-benzyl-N-Boc-L-glutamate 11 was
treated at refluxing THF with neat boranedimethylsulfide complex to
afford the corresponding alcohol in 90% yield. This was transformed
into bromide 12 by the usual procedure (Pph.sub.3, CBr.sub.4).
[0350] The Michaelis-Arbuzov reaction using triethylphosphite to
give the corresponding diethylphosphonate 13 which would be
deprotected at the nitrogen with trifluoroacetic acid to give free
amine 14. The latter would be condensed separately with either
pentafluorophenylesters 6 or 8 to give 16 and 15 respectively,
under conditions similar to those described for 3. 15 and 16 would
be deprotected in the same manner as for 3 to yield desired analogs
4 and 5.
[0351] An inhibitor of the metabolism of purines and pyrimidine
like DON (6-diazo-5-oxo-norleucine) or its aspartate-like 17, and
glutamate-like 18 analogs would be added to the series of
substrates.
[0352] Analog 20 is transformed into compound 17 by treatment with
oxalyl chloride followed by diazomethane and deprotection under
known conditions to afford the desired analogs. In addition,
azotomycin is active only after in vivo conversion to DON which
will be released after action of PSM on analogs 17, 18, and 19.
[0353] Representative compounds, 21 and 22, were designed based on
some of the specific effects and properties of PSM, and the unique
features of some newly discovered cytotoxic molecules with now
known mode of action. The latter, referred to commonly as
enediynes, like dynemycin A 23 and or its active analogs. The
recent isolation of new natural products like Dynemycin A 23, has
generated a tremendous and rapidly growing interest in the medical
and chemical sciences. They have displayed cytotoxicities to many
cancer cell lines at the sub-nanomolar level. One problem is they
are very toxic, unstable, and non-selective. Although they have
been demonstrated, in vitro, to exert their activity through DNA
damage by a radical mechanism as described below, their high level
of toxicity might imply that they should be able to equally damage
anything in their path, from proteins to enzymes.
[0354] These molecules possess unusual structural features that
provide them with exceptional reactivities. Dynemycin A 23 is
relatively stable until the anthraquinone moiety is bioreduced into
hydroanthraquinone 24. This triggers a chain of events by which a
diradical species 25 is generated as a result of a Bergman
cycloaromatization.sup.F. Diradical species 25 is the ultimate
damaging edge of dynemycin A. It subtracts 2(two) protons from any
neighboring molecule or molecules (ie. DNA) producing radicals
therein. These radicals in turn combine with molecular oxygen to
give hydroperoxide intermediates that, in the case of DNA, lead to
single and double strand incision, and consequent cell death.
Another interesting feature was provided by the extensive work of
many organic chemists who not only achieved the total synthesis of
(+)-dynemycin A 23 and other enediynes. but also designed and
efficiently prepared simpler yet as active analogs like 26.
[0355] Enediyne 26 is also triggerable and acts by virtue of the
same mechanism as for 23. This aspect is very relevant to the
present proposed study in that 27 (a very close analog of 26) is
connected to NAAG such that the NAAG-27 molecule, 21, would be
inert anywhere in the body (blood, organs, normal prostate cells)
except in the vicinity of prostate cancer, and metastatic cells. In
this connection NAAG plays a multiple role: [0356] Solubilization
and transport: analogs of 26-type are hydrophobic and insoluble in
aqueous media, but with a water soluble dipeptide that is
indigenous to the body, substrate 21 should follow the ways by
which NAAG is transported and stored in the body. [0357]
Recognition, guidance, and selectivity: Homologs of PSM are located
in the small intestines and in the brain.
[0358] In the latter, a compound like 27 when attached to a
multiply charged dipeptide like NAAG, has no chance of crossing the
blood brain barrier. In the former case, PSM homolog concentration
in the small intestines is in the brush border and not likely to be
exposed to prodrugs in the serum. In addition, one could enhance
the selectivity of delivery of the prodrug by local injection in
the prostate.
[0359] 26 and its analogs are established active molecules that
portray the activity of dynemycin A. Their syntheses are described
in the literature. The total synthesis of optically active 27 has
been described. The synthetic scheme that for the preparation of 28
is almost the same as that of 27. However, they differ only at the
position of the methoxy group which is meta to the nitrogen in the
case of 28. This requires an intermediate of type 29 prepared by
modification of the Myers' method.
[0360] Since NAAG is optically pure, its combination with racemic
material sometimes complicates purification of intermediates. In
addition, to be able to modify the components of this system one at
a time, optically pure intermediates of the type 21 and 22 are
prepared. 27 was prepared in 17 steps starting from commercially
available material. Another interesting feature of 27 is
demonstrated in a very close analog 26, it possesses two (2)
triggers as shown by the arrows.
[0361] The oxygen and the nitrogen can both engender the Bergman
cycloaromatization and hence the desired damage. The simple
protection deprotection manipulation of either functionality should
permit the selective positioning of NAAG at the nitrogen or at the
oxygen centers. PSM should recognize the NAAG portion of 21 or 22,
then it would remove the glutamic acid moiety. This leaves 27
attached to N-acetylaspartate.
[0362] Intramolecular assisted hydrolysis of systems like
N-acetylaspartyle is well documented in the literature. The
aminoacid portion should facilitate the hydrolysis of such a
linkage. In the event this would not work when NAAG is placed on
the nitrogen, an alternative would be to attach NAAG to the oxygen
giving rise to phenolic ester 22 which is per se labile and
removable under milder conditions. PSM specific substrates can be
designed that could activate pro-drugs at the site of prostatic
tumor cells to kill those cells.
Example 13
Genomic Organization of PSM Exon/Intron Junction Sequences
[0363] RNA is synthesized and then processed by having variable
numbers of variable sized fragments cut out and remain in the
nucleus (introns) and the remaining fragments (exons) joined
together and transported out of the nucleus (mRNA) for use in
translation into protein in the cytoplasm. This mRNA is what make
the unique protein products of the cell, proteins of specialized
cells are often made in a great abundance as are their respective
coding mRNA's. These tissue specific mRNA's can be reverse
transcribed (RT) into DNA by reverse transcriptase and amplified
for detection by polymerase chain reaction (PCR) technology and
thus the technique is called RT-PCR. If DNA is a contaminant of the
MBNA fraction it would contain the message even though it was not
being transcribed.
[0364] Knowledge of the intron exon junctions allows for the
selection of primer pairs that cross an intron junction and thus
allow the determination of DNA contamination of the RNA
preparation, if present. If the intron junction were large it would
be unlikely to be amplified with primers, while if the intron
junction were small it would still produce a fragment that would be
much larger than the predicted fragment size which is based on the
cDNA sequence. Thus knowledge of the intron/exon junctions provides
a control to determine if the RT-PCR product is contaminated with
DNA. Another form of DNA that could also be amplified undesirably
if present as a contaminant are pseudo genes, which are intronless
forms of the mRNA that reside as DNA but are not expressed as RNA.
Thus, optimized primers for detection of PSM mRNA in samples would
preferably contain sequences hybridizing across the intro/exon
junctions which are as follows:
TABLE-US-00006 EXON 1 Intron 1 1F. strand CGGCTTCCTCTTCGG
cggcttcctcttcgg taggggggcgcctcgcggag . . . tatttttca 1R. strand . .
. ataaaaagtCACCAAA Exon 2 Intron 2 2F. strand ACATCAAGAAGTTCT
acatcaagaagttct caagtaagtccatactcgaag . . . 2R. strand . . .
caagtggtcATATATTAAAATG Exon 3 Intron 3 3F. strand GAAGATGGAAATGAG
gaagatggaaatgag gtaaaatataaataaataaataa... 3R. . . .
TAAAAGTTGTGTAGT Exon 4 Intron 4 4F. strand AAGGAATGCCAGAGG
aaggaatgccagagg taaaaacacagtgcaacaaa... 4R. strand . . .
agagttgCCGCTAGATCACA Exon 5 Intron 5 5F. strand CAGAGGAAATAAGGT
cagaggaaataaggt aggtaaaaattatctctttttt . . . 5R. strand . . .
gtgttttctATTTTTACGGGT Exon 6 Intron 6 6F. strand GTTACCCAGCAAATG
gttacccagcaatg gtgaatgatcaatccttgaat . . . 6R. strand . . .
aaaaaaagtTTATACGAATA Exon 7 Intron 7 7F. strand ACAGAAGCTCCTAGA
acagaagctcctaga gtaagtttgtaagaaaccargg . . . 7R. strand . . .
aaacacaggttatcTTTTTACCCA Exon 8 Intron 8 8F. strand AAACTTTTCTACACA
aaacttttctacaca gttaagagactatataaatttta . . . 8R. strand . . .
aaacgtaatcaTTTTCAGTTCTAC Exon 9 Intron 9 9F. strand AGCAGTGGAACCAG
agcagtggaaccag gtaaaggaatcgtttgctagca . . . 9R. strand . . .
aaagaTGTCTATACAGTAA Exon 10 Intron 10 10F. Strand CTGAAAAAGGAAGG
ctgaaaaaggaagg taatacaaacaaatagcaagaa . . . Exon 11 Intron 11 11F.
Strand TGAGTGGGCAGAGG agagg ttagttggtaatttgctataatata . . . Exon 12
Intron 12 12F. strand ATCTATAGAAGG gtagtttcct
gaaaaataagaaaagaatagat . . . Exon 13 Intron 13 13F. strand
CTAACAAAAGAG agggcttttcagct acacaaattaaaagaaaaaaag . . . Exon 14
Intron 14 14F. strand GTGGCATGCCCAGG gtggcatgcccagg
taaataaatgaatgaagtttcca . . . Exon 15 Intron 15 15F. strand
CTAAAAATTGGC aatttgtttgtttcc tacagaaaaaacaacaaaaca . . . Exon 16
Intron 16 16F. strand CAGTGTATCATTTG cagtgtatcatttg
gtatgttacccttcctttttcaaatt . . . 16R. strand . . . aaagtcTAAGTGAAAA
Exon 17 Intron 17 17F. strand TTTGACAAAAGCAA tttgacaaaagcaa
gtatgttctacatatatgtgcatat . . . 17R. strand . . .
aaagagtcGGGTTATCAT Exon 18 Intron 18 18F. strand GGCCTTTTTATAGG
ggcctttttatagg taaganaagaaaatatgactcct . . . Exon 19 Intron 19 19F.
strand GAATATTATATATA gaatattatatata gttatgtgagtgtttatatatgtgtgt .
. . Notes: F: Forward strand R: Reverse strand
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Sequence CWU 1
1
12812653DNAHomo sapiens 1ctcaaaaggg gccggatttc cttctcctgg
aggcagatgt tgcctctctc tctcgctcgg 60attggttcag tgcactctag aaacactgct
gtggtggaga aactggaccc caggtctgga 120gcgaattcca gcctgcaggg
ctgataagcg aggcattagt gagattgaga gagactttac 180cccgccgtgg
tggttggagg gcgcgcagta gagcagcagc acaggcgcgg gtcccgggag
240gccggctctg ctcgcgccga gatgtggaat ctccttcacg aaaccgactc
ggctgtggcc 300accgcgcgcc gcccgcgctg gctgtgcgct ggggcgctgg
tgctggcggg tggcttcttt 360ctcctcggct tcctcttcgg gtggtttata
aaatcctcca atgaagctac taacattact 420ccaaagcata atatgaaagc
atttttggat gaattgaaag ctgagaacat caagaagttc 480ttatataatt
ttacacagat accacattta gcaggaacag aacaaaactt tcagcttgca
540aagcaaattc aatcccagtg gaaagaattt ggcctggatt ctgttgagct
agcacattat 600gatgtcctgt tgtcctaccc aaataagact catcccaact
acatctcaat aattaatgaa 660gatggaaatg agattttcaa cacatcatta
tttgaaccac ctcctccagg atatgaaaat 720gtttcggata ttgtaccacc
tttcagtgct ttctctcctc aaggaatgcc agagggcgat 780ctagtgtatg
ttaactatgc acgaactgaa gacttcttta aattggaacg ggacatgaaa
840atcaattgct ctgggaaaat tgtaattgcc agatatggga aagttttcag
aggaaataag 900gttaaaaatg cccagctggc aggggccaaa ggagtcattc
tctactccga ccctgctgac 960tactttgctc ctggggtgaa gtcctatcca
gatggttgga atcttcctgg aggtggtgtc 1020cagcgtggaa atatcctaaa
tctgaatggt gcaggagacc ctctcacacc aggttaccca 1080gcaaatgaat
atgcttatag gcgtggaatt gcagaggctg ttggtcttcc aagtattcct
1140gttcatccaa ttggatacta tgatgcacag aagctcctag aaaaaatggg
tggctcagca 1200ccaccagata gcagctggag aggaagtctc aaagtgccct
acaatgttgg acctggcttt 1260actggaaact tttctacaca aaaagtcaag
atgcacatcc actctaccaa tgaagtgaca 1320agaatttaca atgtgatagg
tactctcaga ggagcagtgg aaccagacag atatgtcatt 1380ctgggaggtc
accgggactc atgggtgttt ggtggtattg accctcagag tggagcagct
1440gttgttcatg aaattgtgag gagctttgga acactgaaaa aggaagggtg
gagacctaga 1500agaacaattt tgtttgcaag ctgggatgca gaagaatttg
gtcttcttgg ttctactgag 1560tgggcagagg agaattcaag actccttcaa
gagcgtggcg tggcttatat taatgctgac 1620tcatctatag aaggaaacta
cactctgaga gttgattgta caccgctgat gtacagcttg 1680gtacacaacc
taacaaaaga gctgaaaagc cctgatgaag gctttgaagg caaatctctt
1740tatgaaagtt ggactaaaaa aagtccttcc ccagagttca gtggcatgcc
caggataagc 1800aaattgggat ctggaaatga ttttgaggtg ttcttccaac
gacttggaat tgcttcaggc 1860agagcacggt atactaaaaa ttgggaaaca
aacaaattca gcggctatcc actgtatcac 1920agtgtctatg aaacatatga
gttggtggaa aagttttatg atccaatgtt taaatatcac 1980ctcactgtgg
cccaggttcg aggagggatg gtgtttgagc tagccaattc catagtgctc
2040ccttttgatt gtcgagatta tgctgtagtt ttaagaaagt atgctgacaa
aatctacagt 2100atttctatga aacatccaca ggaaatgaag acatacagtg
tatcatttga ttcacttttt 2160tctgcagtaa agaattttac agaaattgct
tccaagttca gtgagagact ccaggacttt 2220gacaaaagca acccaatagt
attaagaatg atgaatgatc aactcatgtt tctggaaaga 2280gcatttattg
atccattagg gttaccagac aggccttttt ataggcatgt catctatgct
2340ccaagcagcc acaacaagta tgcaggggag tcattcccag gaatttatga
tgctctgttt 2400gatattgaaa gcaaagtgga cccttccaag gcctggggag
aagtgaagag acagatttat 2460gttgcagcct tcacagtgca ggcagctgca
gagactttga gtgaagtagc ctaagaggat 2520tctttagaga atccgtattg
aatttgtgtg gtatgtcact cagaaagaat cgtaatgggt 2580atattgataa
attttaaaat tggtatattt gaaataaagt tgaatattat atataaaaaa
2640aaaaaaaaaa aaa 26532750PRTHomo sapiens 2Met Trp Asn Leu Leu His
Glu Thr Asp Ser Ala Val Ala Thr Ala Arg1 5 10 15Arg Pro Arg Trp Leu
Cys Ala Gly Ala Leu Val Leu Ala Gly Gly Phe 20 25 30Phe Leu Leu Gly
Phe Leu Phe Gly Trp Phe Ile Lys Ser Ser Asn Glu 35 40 45Ala Thr Asn
Ile Thr Pro Lys His Asn Met Lys Ala Phe Leu Asp Glu 50 55 60Leu Lys
Ala Glu Asn Ile Lys Lys Phe Leu Tyr Asn Phe Thr Gln Ile65 70 75
80Pro His Leu Ala Gly Thr Glu Gln Asn Phe Gln Leu Ala Lys Gln Ile
85 90 95Gln Ser Gln Trp Lys Glu Phe Gly Leu Asp Ser Val Glu Leu Ala
His 100 105 110Tyr Asp Val Leu Leu Ser Tyr Pro Asn Lys Thr His Pro
Asn Tyr Ile 115 120 125Ser Ile Ile Asn Glu Asp Gly Asn Glu Ile Phe
Asn Thr Ser Leu Phe 130 135 140Glu Pro Pro Pro Pro Gly Tyr Glu Asn
Val Ser Asp Ile Val Pro Pro145 150 155 160Phe Ser Ala Phe Ser Pro
Gln Gly Met Pro Glu Gly Asp Leu Val Tyr 165 170 175Val Asn Tyr Ala
Arg Thr Glu Asp Phe Phe Lys Leu Glu Arg Asp Met 180 185 190Lys Ile
Asn Cys Ser Gly Lys Ile Val Ile Ala Arg Tyr Gly Lys Val 195 200
205Phe Arg Gly Asn Lys Val Lys Asn Ala Gln Leu Ala Gly Ala Lys Gly
210 215 220Val Ile Leu Tyr Ser Asp Pro Ala Asp Tyr Phe Ala Pro Gly
Val Lys225 230 235 240Ser Tyr Pro Asp Gly Trp Asn Leu Pro Gly Gly
Gly Val Gln Arg Gly 245 250 255Asn Ile Leu Asn Leu Asn Gly Ala Gly
Asp Pro Leu Thr Pro Gly Tyr 260 265 270Pro Ala Asn Glu Tyr Ala Tyr
Arg Arg Gly Ile Ala Glu Ala Val Gly 275 280 285Leu Pro Ser Ile Pro
Val His Pro Ile Gly Tyr Tyr Asp Ala Gln Lys 290 295 300Leu Leu Glu
Lys Met Gly Gly Ser Ala Pro Pro Asp Ser Ser Trp Arg305 310 315
320Gly Ser Leu Lys Val Pro Tyr Asn Val Gly Pro Gly Phe Thr Gly Asn
325 330 335Phe Ser Thr Gln Lys Val Lys Met His Ile His Ser Thr Asn
Glu Val 340 345 350Thr Arg Ile Tyr Asn Val Ile Gly Thr Leu Arg Gly
Ala Val Glu Pro 355 360 365Asp Arg Tyr Val Ile Leu Gly Gly His Arg
Asp Ser Trp Val Phe Gly 370 375 380Gly Ile Asp Pro Gln Ser Gly Ala
Ala Val Val His Glu Ile Val Arg385 390 395 400Ser Phe Gly Thr Leu
Lys Lys Glu Gly Trp Arg Pro Arg Arg Thr Ile 405 410 415Leu Phe Ala
Ser Trp Asp Ala Glu Glu Phe Gly Leu Leu Gly Ser Thr 420 425 430Glu
Trp Ala Glu Glu Asn Ser Arg Leu Leu Gln Glu Arg Gly Val Ala 435 440
445Tyr Ile Asn Ala Asp Ser Ser Ile Glu Gly Asn Tyr Thr Leu Arg Val
450 455 460Asp Cys Thr Pro Leu Met Tyr Ser Leu Val His Asn Leu Thr
Lys Glu465 470 475 480Leu Lys Ser Pro Asp Glu Gly Phe Glu Gly Lys
Ser Leu Tyr Glu Ser 485 490 495Trp Thr Lys Lys Ser Pro Ser Pro Glu
Phe Ser Gly Met Pro Arg Ile 500 505 510Ser Lys Leu Gly Ser Gly Asn
Asp Phe Glu Val Phe Phe Gln Arg Leu 515 520 525Gly Ile Ala Ser Gly
Arg Ala Arg Tyr Thr Lys Asn Trp Glu Thr Asn 530 535 540Lys Phe Ser
Gly Tyr Pro Leu Tyr His Ser Val Tyr Glu Thr Tyr Glu545 550 555
560Leu Val Glu Lys Phe Tyr Asp Pro Met Phe Lys Tyr His Leu Thr Val
565 570 575Ala Gln Val Arg Gly Gly Met Val Phe Glu Leu Ala Asn Ser
Ile Val 580 585 590Leu Pro Phe Asp Cys Arg Asp Tyr Ala Val Val Leu
Arg Lys Tyr Ala 595 600 605Asp Lys Ile Tyr Ser Ile Ser Met Lys His
Pro Gln Glu Met Lys Thr 610 615 620Tyr Ser Val Ser Phe Asp Ser Leu
Phe Ser Ala Val Lys Asn Phe Thr625 630 635 640Glu Ile Ala Ser Lys
Phe Ser Glu Arg Leu Gln Asp Phe Asp Lys Ser 645 650 655Asn Pro Ile
Val Leu Arg Met Met Asn Asp Gln Leu Met Phe Leu Glu 660 665 670Arg
Ala Phe Ile Asp Pro Leu Gly Leu Pro Asp Arg Pro Phe Tyr Arg 675 680
685His Val Ile Tyr Ala Pro Ser Ser His Asn Lys Tyr Ala Gly Glu Ser
690 695 700Phe Pro Gly Ile Tyr Asp Ala Leu Phe Asp Ile Glu Ser Lys
Val Asp705 710 715 720Pro Ser Lys Ala Trp Gly Glu Val Lys Arg Gln
Ile Tyr Val Ala Ala 725 730 735Phe Thr Val Gln Ala Ala Ala Glu Thr
Leu Ser Glu Val Ala 740 745 75038PRTHomo
sapiensMISC_FEATURE(6)..(6)Xaa=any amino acid 3Ser Leu Tyr Glu Ser
Xaa Thr Lys1 5415PRTHomo sapiensMISC_FEATURE(1)..(1)Xaa=any amino
acid 4Xaa Tyr Pro Asp Gly Xaa Asn Leu Pro Gly Gly Xaa Val Gln Arg1
5 10 1557PRTHomo sapiens 5Phe Tyr Asp Pro Met Phe Lys1 569PRTHomo
sapiens 6Ile Tyr Asn Val Ile Gly Thr Leu Lys1 5722PRTHomo
sapiensMISC_FEATURE(4)..(5)Xaa=any amino acid 7Phe Leu Tyr Xaa Xaa
Thr Gln Ile Pro His Leu Ala Gly Thr Glu Gln1 5 10 15Asn Phe Gln Leu
Ala Lys 20817PRTHomo sapiens 8Gly Val Ile Leu Tyr Ser Asp Pro Ala
Asp Tyr Phe Ala Pro Asp Val1 5 10 15Lys917PRTHomo sapiens 9Pro Val
Ile Leu Tyr Ser Asp Pro Ala Asp Tyr Phe Ala Pro Gly Val1 5 10
15Lys1015PRTHomo sapiens 10Ala Phe Ile Asp Pro Leu Gly Leu Pro Asp
Arg Pro Phe Tyr Arg1 5 10 151119PRTHomo sapiens 11Tyr Ala Gly Glu
Ser Phe Pro Gly Ile Tyr Asp Ala Leu Phe Asp Ile1 5 10 15Glu Ser
Lys1222PRTHomo sapiensMISC_FEATURE(7)..(7)Xaa=any amino acid 12Thr
Ile Leu Phe Ala Ser Xaa Asp Ala Glu Glu Phe Gly Xaa Xaa Xaa1 5 10
15Ser Thr Glu Glu Ala Glu 201317DNAHomo
sapiensmisc_feature(12)..(12)n=any nucleotide 13ttytaygayc cnatgtt
171417DNAHomo sapiensmisc_feature(6)..(6)n=any nucleotide
14aacatnggrt crtaraa 171517DNAHomo
sapiensmisc_feature(12)..(12)n=any nucleotide 15athtayaayg tnathgg
171617DNAHomo sapiensmisc_feature(6)..(6)n=any nucleotide
16ccdatnacrt trtadat 171717DNAHomo sapiensmisc_feature(3)..(3)n=any
nucleotide 17ccngcngayt ayttygc 171817DNAHomo
sapiensmisc_feature(12)..(12)n=any nucleotide 18gcraartart cngcngg
171920DNAHomo sapiensmisc_feature(3)..(3)n=any nucleotide
19acngarcara ayttycarct 202020DNAHomo
sapiensmisc_feature(18)..(18)n=any nucleotide 20agytgraart
tytgytcngt 202117DNAHomo sapiens 21garcaraayt tycarct 172217DNAHomo
sapiens 22agytgraart tytgytc 172320DNAHomo
sapiensmisc_feature(9)..(9)n=any nucleotide 23tgggaygcng argarttygg
202420DNAHomo sapiensmisc_feature(12)..(12)n=any nucleotide
24ccraaytcyt cngcrtccca 202517DNAHomo
sapiensmisc_feature(9)..(9)n=any nucleotide 25tgggaygcng argartt
172617DNAHomo sapiensmisc_feature(9)..(9)n=any nucleotide
26aaytcytcng crtccca 1727780DNAHomo
sapiensmisc_feature(82)..(84)n=any nucleotide 27tacacttatc
ccattcggac atgcccacct tggaactgga gacccttaca ccccaggctt 60cccttcgttc
aaccacaccc annngtttcc accagttgaa tcttcaggac taccccacat
120tgctgttcag accatctcta gcagtgcagc agccaggctg ttcagcaaaa
tggatggaga 180cacatgctct ganagnngtt ggaaaggtgc gatccannnt
tcctgtaagg tnngacnnaa 240caaagcagga gannnngcca gantaatggt
gaaactagat gtgaacaatt ccatgaaaga 300caggaagatt ctgaacatct
tcggtgctat ccagggattt gaagaacctg atcggtatgt 360tgtgattgga
gcccagagag actcctgggg cccaggagtg gctaaagctg gcactggaac
420tgctatattg ttggaacttg cccgtgtgat ctcagacata gtgaaaaacg
agggctacaa 480accgaggcga agcatcatct ttgctagctg gagtgcagga
gactacggag ctgtgggtgc 540tactgaatgg ctggaggggt actctgccat
gctgcatgcc aaagctttca cttacatcan 600ngcttggatg ctccagtcct
gggagcaagc catgtcaaga tttctgccag ccccttgctg 660tatatgctgc
tggggagtat tatgaagggg gtgaagaatc cagcagcagt ctcagagagc
720nnnnctctat aacagacttg gcccagactg ggtaaaagca gttgttcctc
ttggcctgga 78028660DNAHomo sapiensmisc_feature(224)..(224)n=any
nucleotide 28tgcagaaaag ctattcaaaa acatggaagg aaactgtcct cctagttgga
atatagattc 60ctcatgtaag ctggaacttt cacagaatca aaatgtgaag ctcactgtga
acaatgtact 120gaaagaaaca agaatactta acatctttgg cgttattaaa
ggctatgagg aaccagaccg 180ctacattgta gtaggagccc agagagacgc
ttggggccct ggtngttgcg aagtccagtg 240tgggaacagg tcttnctgtt
gaaacttgcc caagtattct cagatatgat ttcaaaagat 300ggatttagac
ccagcaggag tattatcttt gccagctgga ctgcaggaga ctatggagct
360gttggtccga ctgagtggct ggaggggtac ctttcatctt tgcatctaaa
gnnngctttc 420acttacatta atnctggata aagtcgtcct gggtactagc
aacttcaagg tttctgccag 480ccccctatta tatacactta tggggaagat
aatgcaggan ncgtaaagca tccgannnnn 540nnnttgatgg aaaatatcta
tatcgaaaca gtaattggat tagcaaaatt gaggaacttt 600ccttggacaa
tgctgcattc ccttttcttg catattcagg aatcccagca gtttctttct
66029540DNAHomo sapiensmisc_feature(214)..(214)n=any nucleotide
29tatggaagga gactgtccct ctgactggaa aacagactct acatgtagga tggtaacctc
60agaaagcaag aatgtgaagc tcactgtgag caatgtgctg aaagagataa aaattcttaa
120catctttgga gttattaaag gctttgtaga accagatcac tatgttgtag
ttggggccca 180gagagatgca tggggccctg gagctgcaaa atcncggtgt
aggcacagct ctcctattga 240aacttgccca gatgttctca gatatggtct
taaaagatgg gtttcagccc agcagaagca 300ttatctttgc cagttggagt
gctggagact ttggatcggt tggtgccact gaatggctag 360agggatacct
ttcgtcncct gcatttaaag gctttcactt atattaatct ggataaagcg
420gttcttggta ccagcaactt caaggtttct gccagcccac tgttgtatac
gcttattgag 480aaaacaatgc aaaatgtgaa gcatccggtt actgggcaat
ttctatatca ggacagcaac 5403027DNAHomo sapiens 30acggagcaaa
actttcagct tgcaaag 27319PRTHomo sapiens 31Thr Glu Gln Asn Phe Gln
Leu Ala Lys1 53236DNAHomo sapiens 32ctcttcggca tcccagcttg
caaacaaaat tgttct 363336DNAHomo sapiens 33agaacaattt tgtttgcaag
ctgggatgcc aaggag 363412PRTHomo sapiens 34Arg Thr Ile Leu Phe Ala
Ser Trp Asp Ala Glu Glu1 5 10356PRTHomo sapiens 35Asp Glu Leu Lys
Ala Glu1 5366PRTHomo sapiens 36Asn Glu Asp Gly Asn Glu1 5376PRTHomo
sapiens 37Lys Ser Pro Asp Glu Gly1 53817PRTHomo sapiens 38Ala Gly
Ala Leu Val Leu Ala Gly Gly Phe Phe Leu Leu Gly Phe Leu1 5 10
15Phe393017DNAHomo sapiens 39aagggtgctc cttaggctga atgcttgcag
acaggatgct tggttacaga tgggctgtga 60ctcgagtgga gttttataag ggtgctcctt
aggctgaatg cttgcagaca ggatgcttgg 120ttacagatgg gctgtgagct
gggtgcttgt aagaggatgc ttgggtgcta agtgagccat 180ttgcagttga
ccctattctt ggaacattca ttcccctcta cccctgtttc tgttcctgcc
240agctaagccc atttttcatt tttcttttaa ctccttagcg ctccgcaaaa
cttaatcaat 300ttctttaaac ctcagttttc ttatctgtaa aaggtaaata
ataatacagg gtgcaacaga 360aaaatctagt gtggtttaca taatcacctg
ttagagattt taaattattt caggataagt 420catgataatt aaatgaaata
atgcacataa agcacatagt gtggtgtcct ccatatagaa 480aatgctcagt
atattggtta ttaactactt gttgaaggtt tatcttctcc actaaactgt
540aagttccaca agccttacaa tatgtgacag atattcattc attgtctgaa
ttcttcaaat 600acatcctctt caccatagcg tcttattaat tgaattatta
attgaataaa ttctattgtt 660caaaaatcac ttttatattt aactgaaatt
tgcttactta taatcacatc taaccttcaa 720agaaaacaca ttaaccaact
gtactgggta atgttactgg gtgatcccac gttttacaaa 780tgagaagata
tattctggta agttgaatac ttagcaccca ggggtaatca gcttggacag
840gaccaggtcc aaagactgtt aagagtcttc tgactccaaa ctcagtgctc
cctccagtgc 900cacaagcaaa ctccataaag gtatcctgtg ctgaatagag
actgtagagt ggtacaaagt 960aagacagaca ttatattaag tcttagcttt
gtgacttcga atgacttacc taatctagct 1020aaatttcagt tttaccatgt
gtaaatcagg aagagtaata gaacaaacct tgaagggtcc 1080caatggtgat
taaatgaggt gatgtacata acatgcatca ctcataataa gtgctcttta
1140aatattagtc actattatta gccatctctg attagatttg acaataggaa
cattaggaaa 1200gatatagtac attcaggatt ttgttagaaa gagatgaaga
aattcccttc cttcctgccc 1260taggtcatct aggagttgtc atggttcatt
gttgacaaat taattttccc aaatttttca 1320ctttgctcag aaagtctaca
tcgaagcacc caagactgta caatctagtc catctttttc 1380cacttaactc
atactgtgct ctccctttct caaagcaaac tgtttgctat tccttgaata
1440cactctgagt tttctgcctt tgcctactca gctggcccat ggcccctaat
gtttcttctc 1500atctccactg ggtcaaatcc tacctgtacc ttatggttct
gttaaaagca gtgcttccat 1560aaagtactcc tagcaaatgc acggcctctc
tcacggatta taagaacaca gtttatttta 1620taaagcatgt agctattctc
tccctcgaaa tacgattatt attattaaga atttatagca 1680gggatataat
tttgtatgat gattcttctg gttaatccaa ccaagattga ttttatatct
1740attacgtaag acagtagcca gacatagccg ggatatgaaa ataaagtctc
tgccttcaac 1800aagttccagt attcttttct ttcctcccct cccctcccct
cccttcccct ccccttcctt 1860ccctttccct tcccttcctt tctttcttga
gggagtctca ctctgtcacc aggctccagt 1920gcagtggcgc tatcttggct
gactgcaacc tccgcctccc cggttcaagc gattctcctg 1980cctcagcctc
ctgagtagct gggactacag gagcccgcca ccacgcccag ctaatttttg
2040tatttttagt agagatgggg tttcaccatg ttggccagga tggtctcgat
ttctcgactt 2100cgtgatccgc ctgtctgggc ctcccaaagt gctgggatta
caggcgtgag ccaccacgcc
2160cggctttaaa aaatggtttt gtaatgtaag tggaggataa taccctacat
gtttattaat 2220aacaataata ttctttagga aaaagggcgc ggtggtgatt
tacactgatg acaagcattc 2280ccgactatgg aaaaaaagcg cagctttttc
tgctctgctt ttattcagta gagtattgta 2340gagattgtat agaatttcag
agttgaataa aagttcctca taattatagg agtggagaga 2400ggagagtctc
tttcttcctt tcatttttat atttaagcaa gagctggaca ttttccaaga
2460aagttttttt tttttaaggc gcctctcaaa aggggccgga tttccttctc
ctggaggcag 2520atgttgcctc tctctctcgc tcggattggt tcagtgcact
ctagaaacac tgctgtggtg 2580gagaaactgg accccaggtc tggagcgaat
tccagcctgc agggctgata agcgaggcat 2640tagtgagatt gagagagact
ttaccccgcc gtggtggttg gagggcgcgc agtagagcag 2700cagcacaggc
gcgggtcccg ggaggccggc tctgctcgcg ccgagatgtg gaatctcctt
2760cacgaaaccg actcggctgt ggccaccgcg cgccgcccgc gctggctgtg
cgctggggcg 2820ctggtgctgg cgggtggctt ctttctcctc ggcttcctct
tcggtagggg ggcgcctcgc 2880ggagcaaacc tcggagtctt ccccgtggtg
ccgcggtgct gggactcgcg ggtcagctgc 2940cgagtgggat cctgttgctg
gtcttcccca ggggcggcga ttagggtcgg ggtaatgtgg 3000ggtgagcacc cctcgag
30174015DNAHomo sapiens 40cggcttcctc ttcgg 154144DNAHomo sapiens
41cggcttcctc ttcggtaggg gggcgcctcg cggagtattt ttca 444216DNAHomo
sapiens 42ataaaaagtc accaaa 164315DNAHomo sapiens 43acatcaagaa
gttct 154436DNAHomo sapiens 44acatcaagaa gttctcaagt aagtccatac
tcgaag 364522DNAHomo sapiens 45caagtggtca tatattaaaa tg
224615DNAHomo sapiens 46gaagatggaa atgag 154738DNAHomo sapiens
47gaagatggaa atgaggtaaa atataaataa ataaataa 384815DNAHomo sapiens
48taaaagttgt gtagt 154915DNAHomo sapiens 49aaggaatgcc agagg
155035DNAHomo sapiens 50aaggaatgcc agaggtaaaa acacagtgca acaaa
355120DNAHomo sapiens 51agagttgccg ctagatcaca 205215DNAHomo sapiens
52cagaggaaat aaggt 155337DNAHomo sapiens 53cagaggaaat aaggtaggta
aaaattatct ctttttt 375421DNAHomo sapiens 54gtgttttcta tttttacggg t
215515DNAHomo sapiens 55gttacccagc aaatg 155635DNAHomo sapiens
56gttacccagc aatggtgaat gatcaatcct tgaat 355720DNAHomo sapiens
57aaaaaaagtt tatacgaata 205815DNAHomo sapiens 58acagaagctc ctaga
155937DNAHomo sapiens 59acagaagctc ctagagtaag tttgtaagaa accargg
376024DNAHomo sapiens 60aaacacaggt tatcttttta ccca 246115DNAHomo
sapiens 61aaacttttct acaca 156238DNAHomo sapiens 62aaacttttct
acacagttaa gagactatat aaatttta 386324DNAHomo sapiens 63aaacgtaatc
attttcagtt ctac 246414DNAHomo sapiens 64agcagtggaa ccag
146536DNAHomo sapiens 65agcagtggaa ccaggtaaag gaatcgtttg ctagca
366619DNAHomo sapiens 66aaagatgtct atacagtaa 196714DNAHomo sapiens
67ctgaaaaagg aagg 146836DNAHomo sapiens 68ctgaaaaagg aaggtaatac
aaacaaatag caagaa 366914DNAHomo sapiens 69tgagtgggca gagg
147030DNAHomo sapiens 70agaggttagt tggtaatttg ctataatata
307112DNAHomo sapiens 71atctatagaa gg 127232DNAHomo sapiens
72gtagtttcct gaaaaataag aaaagaatag at 327312DNAHomo sapiens
73ctaacaaaag ag 127436DNAHomo sapiens 74agggcttttc agctacacaa
attaaaagaa aaaaag 367514DNAHomo sapiens 75gtggcatgcc cagg
147637DNAHomo sapiens 76gtggcatgcc caggtaaata aatgaatgaa gtttcca
377712DNAHomo sapiens 77ctaaaaattg gc 127836DNAHomo sapiens
78aatttgtttg tttcctacag aaaaaacaac aaaaca 367914DNAHomo sapiens
79cagtgtatca tttg 148040DNAHomo sapiens 80cagtgtatca tttggtatgt
tacccttcct ttttcaaatt 408116DNAHomo sapiens 81aaagtctaag tgaaaa
168214DNAHomo sapiens 82tttgacaaaa gcaa 148339DNAHomo sapiens
83tttgacaaaa gcaagtatgt tctacatata tgtgcatat 398418DNAHomo sapiens
84aaagagtcgg gttatcat 188514DNAHomo sapiens 85ggccttttta tagg
148637DNAHomo sapiensmisc_feature(20)..(20)n=any nucleotide
86ggccttttta taggtaagan aagaaaatat gactcct 378720DNAHomo sapiens
87aatagttggt acagtagata 208814DNAHomo sapiens 88gaatattata tata
148941DNAHomo sapiens 89gaatattata tatagttatg tgagtgttta tatatgtgtg
t 41903077DNAHomo sapiens 90gcgccttaaa aaaaaaaaac tttcttggaa
aatgtccagc tcttgcttaa atataaaaat 60gaaaggaaga aagagactct cctctctcca
ctcctataat tatgaggaac ttttattcaa 120ctctgaaatt ctatacaatc
tctacaatac tctactgaat aaaagcagag cagaaaaagc 180tgcgcttttt
ttccatagtc gggaatgctt gtcatcagtg taaatcacca ccgcgccctt
240tttcctaaag aatattattg ttattaataa acatgtaggg tattatcctc
cacttacatt 300acaaaaccat tttttaaagc cgggcgtggt ggctcacgcc
tgtaatccca gcactttggg 360aggcccagac aggcggatca cgaagtcgag
aaatcgagac catcctggcc aacatggtga 420aaccccatct ctactaaaaa
tacaaaaatt agctgggcgt ggtggcgggc tcctgtagtc 480ccagctactc
aggaggctga ggcaggagaa tcgcttgaac cggggaggcc gaggttgcag
540tcagccaaga tagcgccact gcactggagc ctggtgacag agtgagactc
cctcaagaaa 600gaaaggaagg gaagggaaag ggaaggaagg ggaggggaag
ggaggggagg ggaggggagg 660aaagaaaaga atactggaac ttgttgaagg
cagagacttt attttcatat cccggctatg 720tctggctact gtcttacgta
atagatataa aatcaatctt ggttggatta accagaagaa 780tgagaagata
tattctggta agttgaatac ttagcaccca ggggtaatca gcttggacag
840gaccaggtcc aaagactgtt aagagtcttc tgactccaaa ctcagtgctc
cctccagtgc 900cacaagcaaa ctccataaag gtatcctgtg ctgaatagag
actgtagagt ggtacaaagt 960aagacagaca ttatattaag tcttagcttt
gtgacttcga atgacttacc taatctagct 1020aaatttcagt tttaccatgt
gtaaatcagg aagagtaata gaacaaacct tgaagggtcc 1080caatggtgat
taaatgaggt gatgtacata acatgcatca ctcataataa gtgctcttta
1140aatattagtc actattatta gccatctctg attagatttg acaataggaa
cattaggaaa 1200gatatagtac attcaggatt ttgttagaaa gagatgaaga
aattcccttc cttcctgccc 1260taggtcatct aggagttgtc atggttcatt
gttgacaaat taattttccc aaatttttca 1320ctttgctcag aaagtctaca
tcgaagcacc caagactgta caatctagtc catctttttc 1380cacttaactc
atactgtgct ctccctttct caaagcaaac tgtttgctat tccttgaata
1440cactctgagt tttctgcctt tgcctactca gctggcccat ggcccctaat
gtttcttctc 1500atctccactg ggtcaaatcc tacctgtacc ttatggttct
gttaaaagca gtgcttccat 1560aaagtactcc tagcaaatgc acggcctctc
tcacgcatta taagaacaca gtttatttta 1620tttcatgagg atcgtttacg
tgccggagag agtgcctaat attcttgtgt caaataaaat 1680taaagcatgt
agctattctc tccctcgaaa tacgattatt attattaaga atttatagca
1740gggatataat tttgtatgat gattcttctg gttaatccaa ccaagattga
ttttatatct 1800attacgtaag acagtagcca gacatagccg ggatatgaaa
ataaagtctc tgccttcaac 1860aagttccagt attcttttct ttcctcccct
cccctcccct cccttcccct ccccttcctt 1920ccctttccct tcccttcctt
tctttcttga gggagtctca ctctgtcacc aggctccagt 1980gcagtgccgc
tatcttggct gactgcaacc tccgcctccc cggttcaagc gattctcctg
2040cctcagcctc ctgagtagct ggcactacag gagcccgcca ccacgcccag
ctaatttttg 2100tatttttagt agagatgggg tttcaccatg ttggccagga
tggtctcgat ttctcgactt 2160cgtgatccgc ctgtctgggc ctcccaaagt
gctgggatta caggcgtgag ccaccacgcc 2220cgcctttaaa aaatggtttt
gtaatgtaag tggaggataa taccctacat gtttattaat 2280aacaataata
ttctttagga aaaaggccgc ggtggtgatt tacactgatg acaagcattc
2340ccgactatgg aaaaaaagcg cagctttttc tgctctgctt ttattcagta
gagtattgta 2400gagattgtat agaatttcac agttgaataa aagttcctca
taattatagg agtggagaga 2460ggagagtctc tttcttcctt tcatttttat
atttaagcaa gagctggaca ttttccaaga 2520aagttttttt tttttaaggc
gcctctcaaa aggggccgga tttccttctc ctggaggcag 2580atgttgcctc
tctctctcgc tcggattggt tcagtgcact ctagaaacac tgctgtggtg
2640gagaaactgg accccaggtc tgcagcgaat tccagcctgc agggctgata
agcgaggcat 2700tagtgagatt gagagagact ttaccccgcc gtggtggttg
gagggcgcgc agtagagcag 2760cagcacaggc gcgggtcccg ggaggccggc
tctgctcgcg ccgagatgtg gaatctcctt 2820cacgaaaccg actcggctgt
ggccaccgcg cgccgcccgc gctggctgtg cgctggggcg 2880ctggtgctgg
cgggtggctt ctttctcctc ggcttcctct tcggtagggg ggcgcctcgc
2940ggagcaaacc tcggagtctt ccccgtggtg ccgcgctgct gggactcgcg
ggtcagctgc 3000cgagtgggat cctgttgctg gtcttcccca ggggcggcga
ttagggtcgg ggtaatgtgg 3060ggtgagcacc cctcgag 307791519DNAHomo
sapiens 91ctcaaaaggg gccggatttc cttctcctgg aggcagatgt tgcctctctc
tctcgctcgg 60attggttcag tgcactctag aaacactgct gtggtggaga aactggaccc
caggtctgga 120gcgaattcca gcctgcaggg ctgataagcg aggcattagt
gagattgaga gagactttac 180cccgccgtgg tggttggagg gcgcgcagta
gagcagcagc acaggcgcgg gtcccgggag 240gccggctctg ctcgcgccga
gatgtggaat ctccttcacg aaaccgactc ggctgtggcc 300accgcgcgcc
gcccgcgctg gctgtgcgct ggggcgctgg tgctggcggg tggcttcttt
360ctcctcggct tcctcttcgg atggtttata aaatcctcca atgaagctac
taacattact 420ccaaagcata atatgaaagc atttttggat gaatggaaag
ctgagaacat caagaagttc 480ttatataatt ttacacagat accacattta gcaggaaca
519922125DNAHomo sapiens 92taggggggcg cctcgcggag aaacctcgga
gtcttccccg tggtgccgcg gtgctgggac 60tcgcgggtca gctgccgagt gggatcctgt
tgctggtctt ccccaggggc ggcgattagg 120gtcggggtaa tgtggggtga
gcacccctcg agttaggagg agggtagctg ggaacggtgc 180agggctgagt
tctcgacaag ctgctggtag gacagtcact caggttgagg gtagaactga
240gagaacctga aactgggcgt aggaaggttc caagtgctgg agccctgcaa
gacagaggaa 300gttttttttt tgcttttgtt ttgttttgtt ttgttttgtt
ttgttttgtt tgtttgtttg 360tttttttacc tctctgtgca ttctttcttc
cttggaagta acagaggcaa gcttgggaac 420tgtgtgaacc aggtcaccaa
tctcgacagg tctttaccag cgggtctttt gctgtttttc 480ctgggtactg
atttgcagac ttgatccaac tttctaagaa aagcagaacc acacaggcaa
540gctcagactc ttttattaaa ttccagtttt gactttgcca cttcttagtg
gccttgaaca 600agttaccgag tccctctcag cgttagttac cctattttat
gatgaggata atattatctg 660caaattattg gtaatagtaa ataatatagc
atgtaaatct cctagcacag tactgggatt 720ttcgccactt tatttcttct
tttaccaaga tactcctcat tggactttaa tacacaggac 780tagtctaagg
tatcaccagg tagtccactc ctgctcggaa ttcttgaccc tctttcggga
840tttagaagaa tagggcatgg accagatggg tttaaacaaa ttcaatatct
tccactagct 900tcaccttggg gttgttaaaa gatttttgaa ccacacactg
tgctcataac aatcttcatc 960tcttaaaagg attttattct tcctggtatt
gccctcactc tcatccctgt attccgtgct 1020cagtggctga cacagaagag
ttctttattg atgtccgccc cccacccact aggattctct 1080gctctcccct
ccccctacag gcctccatcc tcttcatcct gttcattttt cagatctcag
1140ttcaagcatc tcgtcctcag tgtggtgttt cctgatccct cactctaatc
caagtctttc 1200tgttttatgc acaggtggaa tcttatttcc gtttgcgtcc
aatcatgtat tttaatatgc 1260atgtatatat gtatgtgcat ttgtatgcat
gcgattaaga actagaataa ttaataattg 1320gaaagctcca tgaaagctgg
ttggggacta attttgtaac tactttattc ccacatcctg 1380taatttctct
aaataaaccc tggaatcttg ccttatctcc ttcaggttaa aagccaactg
1440caaggtctaa tgactgcagg atctagctat ccattgtttc tggccgccta
tgcgtgcact 1500gggtgtctgg cagagaggct gggtaaattg tagtttcatt
gtagctgtct gacttggatt 1560tctcacgcct acttcactgg aaacgcaaac
tctcacagca ttttctttta gtttcagaat 1620cagagcaaat tagaagtctg
aatttccttc aacacttgga aataatttat ttatttgaaa 1680tatattcata
attaattcgt tataaaaatg tattaaatgc ttatttgagt cagcagagga
1740agatagaaac tttatgaaag tagaaggtgg atctcctttt tgccttcatt
ttcagaacat 1800ctcgtttaca cccattagtt gaaacattaa tgtcatttta
ttttcgtcct gattatctca 1860taaaacattt cttagaataa cagcaatacc
tatcattgaa gttggataag aaatattttg 1920caattggttt gcaacttaaa
aatctgtttg catgactctt tttcagtgaa agtaggcaag 1980agaaattaaa
attcagaaat atgtgaccta atgtcagagg taatattgat aatttgtgtt
2040ttacaaataa tacatacaac aataatgaaa aataagtcct atctataggc
tcgtatctca 2100tgcctatttt tggatgtatt tttca 2125931896DNAHomo
sapiensmisc_feature(634)..(650)n=any nucleotide 93tgaaaaatag
atgaaaaata ggcatgagat acgagcctat agataggact tattttttat 60tattgttgta
tgtattattt gtaaaacaca aattatcaat attacctctg acattaggtg
120agatattctg aattttaatt tctcttgcct actttcactg aaaaagagtc
atgcaaacag 180atttttaagt tgcaaaccaa ttgcaaaata tttttttatc
caacttcaat gataggtatt 240gctgttaatt ctaagatatg cattaattgt
ttcaactaat gggtgtcaaa cgagatgttc 300tgaaaatgaa ggcaaaaagg
agatccacct tctactttca taaagtttct atcttcctct 360gctgactcaa
ataagcattt aatacatttt ataacgaatt aattatgaat atatttcaaa
420taaataaatt atttccaagt gttcaaggaa attcagactt ctaatttgct
ctgattctga 480aactaaaaca aatgctctgt gagagtttgc gtttccagtg
aagtagcgtg agaaatccaa 540gtcagacagc tacatgaaac tacatttacc
agctctctgc cagacaccag tgcacgatag 600cgcagaacat gtagctagat
ctcagtcata gctnnnnnnn nnnnnnnnnn agaccttgca 660gttggctttt
aacctgaagg agataaggca agattccagg gtttatttag agaaattaca
720ggatctggga ataaagtagt tacaaaatta gtccccaacc agctttcatg
gagctttcaa 780ttattaatta ttctagttct taatcgcatg catacaatgc
acatacatat atacatgcat 840attaaaatac atgattggac gcaaacggaa
ataacattgg acctgtgcat aaaacagaaa 900gacttggtta gagtgaggga
tcaggaaaca ccacactgag gacgagatgn nnnnnnnnnn 960ntagtgggtg
gggggcggac atcaataaag aactcttctg tgtcagccac tgagcacgga
1020ataaagggat gagagtgagg gcaantacca gaagaataaa atccttttaa
gagatgaaga 1080ttgttatgag cacagtgtgt ggnttcaaaa atcttttaac
aaccccaagg tgaagctagt 1140tggaagatat ttgaatttgt ttaaacccat
ctggtggtag ccctattctt tgaatcccga 1200aagagggtca agaattccga
gcaggagtgg actacctggt gataccttag actagtcctg 1260tgtattaaag
tccaatgagg agtatcttgg taaaataata aataaagtcc cgaaaatccc
1320agtactctgc taggagattt acatgctata ttatttacta tnnnnhnnnt
aatttgcaga 1380taatattatc ctcatcataa aatagggtaa ctaacgctga
gagggactcg gtaacttgtt 1440caaggccact aagaagtggc aaagtcaaaa
ctggaatttt aataaaagag tctagcttgc 1500ctgtgtggtt ctgcttttct
tagaaagttg gannaagtct camatcagta cccaggaaaa 1560acagcaaaag
acccgctggt aaagacctgt ccagattgct gacctggttc acacanntcc
1620aagcttgcct ctgttacttc caaggaacaa agaatgcaca gagaggtaaa
aaaacaaaca 1680aaccaaacaa aacaaaacaa aacaaaacaa aacaaaacaa
aagcaaaaaa aaacttcctc 1740tgtcttgcag ggctccagca cttggaacct
tcctacgtcc tantttcagg ttctctcagt 1800tctaccctca acctgagtga
ctgtcctacc agcagcttgt cgagaactca gccctgcacc 1860gttcccagct
accctcctcc taactcgagg ggtgct 1896941280DNAHomo
sapiensmisc_feature(315)..(320)n=any nucleotide 94ggattctgtt
gagccctagc tcattatgat gtcctgttgt cctacccaaa taagactcat 60cccaactaca
tctcaataat taatgaagat ggaaatgagg taaaaaataa ataaataaat
120aaaagaaaca ttccccccca tttattattt tttcaaatac cttctatgaa
ataatgttct 180atccctctct aaatattaat agaaatcaat attattgcaa
ctgtgaatac ctttaatatc 240tcattatccg gtgtcaacta ctttcctatg
atgttgagtt actgggttta gaagtcggga 300aataatgctg taaannnnnn
agttagtcta cacaccaata tcaaatatga tatacttgta 360aacctccaag
cataaaaaga gatactttat aaaagaggtt ctttttttct tttttttttt
420tccagatgga gtttcactcc tgtcaggcag gcngagtgca gtggtgccat
ctcggctcac 480tgcaacctcc acctcccatg ttcaagggat tctccttcct
cagtctcctg agtagctggg 540attacaggtg tgcaccacca cacccagcta
atttttgtat ttttaataga gacagggttt 600catcgatgtt ggccaggcta
gtctcgaact cctgacctct aggtgatcca cccgcctcag 660cctcccaaag
ttctacaatt acacgtgtga ggcactgctc tggccaggag atacattttt
720gataggttta atttataaag acactgcaca gatttggagt tgctgggaaa
tcacgatcca 780gtatgcattt gacccagcaa tttttattgg tacttaatga
ttatatctca attgatcagg 840ttgaactctg tgcgaagaat ttgtgtgtgg
acatttgaga ggacagtttg gaggcaaggt 900attttagtag atttaaagaa
tttgaatctt gtttgcaagt tggggcatat actgagaaag 960agaagacaat
gcagataaat tgatatattt attatgatgt atgttcaata tgaaagatca
1020caaaatataa catacatnna tcttacttaa catacctcag ttttagagct
accgtatgta 1080gaagagtcca tttctattta ggtaagttcc tttagtcctt
ttattactgg gcactcttaa 1140ttacatgtag cttgaaatat gtccagtttg
agcagtgaac tgaaaatgtc atgtgattaa 1200gtacatatat aatttttttt
catagtaggt caataacctc cttttattgt ctaatgaatc 1260agttctctaa
tgattatacg 1280951242DNAHomo sapiensmisc_feature(381)..(388)n=any
nucleotide 95aatcaaaata aaacagttaa agtttgatta ctataatcaa acacaaaaaa
aatgaatatt 60atcttttatg tcagtagagg gtgaatgaat ccttcaggat tttgatgata
gtatcagata 120cccagcacta tgctagaagt tgtgaagaat tcacgagatg
aataaatcac agattctgtc 180ctcaaaatgg ttagatctat tcaggaaaca
aagctaaaaa aaccccacca ataactaaaa 240atcaaccaaa tgaaaaacaa
caatcataaa ataagtaagt acctatagaa agaaaagctc 300agaggaggta
aaaagataac tcttccaaaa ggaatactat atactgtaaa ctgtgtactg
360atagaaggaa gaattagaaa nnnnnnnntg taagtggcat acatactaag
ctagtgtgaa 420cacaagccta aatatgtagt tgcttcacag aaggttagaa
gtaaattaac ctcatgaatt 480tcttgagaga acttgtaagg actaagcttt
cgattttgga gaaagatttt aataccaaat 540aaaaagtacc tttgtttggt
aatctcaatc attataatag tgcttagata atacctagga 600acaaattaaa
tattaaattt actttaaaaa aaagtacatg attggggaat cacaacaggc
660cttactagat tctctnnnnn natatgcact gaaaagaatg aaaaacactg
aaccaaatat 720ntgttttttt aagtttaaaa ttaaattgga aaaaaatagt
aaggaatatc agaagcaaaa 780aaataaaatg aaagcaagaa tcctcagagg
tagcacgaaa tttggctttg cttagatgga
840tctatcaaag ctatggccca tgaaaaggat tcaggagtta gtttaaagct
ggttcacata 900atggaatcta gcagaagact gtgcataaag gtggtctaag
aacaacaata tcctgaccag 960gtgagggggc tcacnctnaa tnccagcact
ttgggagccc aaggtgggtg gatcacgagg 1020tcaggagttt gagaccagcc
tgaccaacat ggtgaaaccg cgtctctact aaaaatagaa 1080aaattagccg
ngcctacgtg cttctaatcc cagctgaact caggagactg agacaggaga
1140ataccttcaa cccagcatgc aagcttnnnn nngccactgc actccagcct
agggtgcaaa 1200aaaaaaaaaa angacacatt actcaggtaa ggtaatcaat aa
124296783DNAHomo sapiens 96aaggtaaaaa ttatctcttt ttttctctcc
cccaatgtaa aaagttatag tgggttttac 60atgtgtagaa tcattttctt aaaactttat
gaataccatt attttcttgt attctgtgac 120atgcccacct tacagagagg
acacatttac taggttatat cccggggtta aattcgagga 180ttggaatttg
gccagtgtag atgtttagag tgaacagaac aaatttttct gtgcttacag
240gttatggctg tggcctacaa gaagcatgca ctgggtttat tattaacttt
cagtatcttt 300gttttaaata ttttctacaa aaatgtttac taaattaaat
tgtagtatga attgttataa 360ataatgaggg aaaacaattt acacatagca
aatttaaaaa ttactgtcat ttgatttgtt 420aatatatttt tctctttagt
gggaaattaa attttaaaaa attccctttc gactgtagaa 480caaataggaa
tttggcctgt ggggtctact tgcttattat atttgtaagc tagtggtagg
540aaatagcaaa tgctcactac cactaataag aacatttcta aatctgatgt
tctgaggatt 600tttagagctt atagtagcaa aaagaaaagg gaaattctat
ccgagatgtc ctttgttgta 660ggcctaatga gaaaaggttg aagataaagt
tctggtactc atttaagtgt aatattgaaa 720attgatatta ccgaatctgg
aacaaccaat ttaaaataag gaaagaaaga cactgtgttt 780tct 78397782DNAHomo
sapiensmisc_feature(505)..(505)n=any nucleaotide 97agaaaacaca
gtgtctttct ttccttattt taaattggtt gttccagatt cggtaatatc 60aattttcaat
attacactta aatgagtacc agaactttat cttcaacctt ttctcattag
120gcctacaaca aaggacatct cggatagaat ttcccttttc tttttgctac
tataagctct 180aaaaatcctc agaacatcag atttagaaat gttcttatta
gtggtagtga gcatttgcta 240tttcctacca ctagcttaca aatataataa
gcaagtagac cccacaggcc aaattcctat 300ttgttctaca gtcgaaaggg
aattttttaa aatttaattt cccactaaag agaaaaatat 360attaacaaat
caaatgacag taatttttaa atttcgtatg tgtaaattgt tttccctcat
420tatttataac aattcatact acaatttaat ttagtaaaca tttttgtaga
aaatatttaa 480aacaaagata ctgaaagtta atatnaaacc cagtgcatgc
ttcttgtagg ccacagccat 540aacctgtaag cacagaaaaa tttgttctgt
tactctaaac atctacactg gccaaattcc 600aatgctcgaa tttaaccccg
ggatataacc tagtaaatgt gtcctctctg taaggtggcc 660atgtcacaga
atacaagaaa ataatggtat tcataaagtt ttaagaaaat gattctacac
720atgtaaaacc cactataact ttttacattg ggggagagaa aaaaagagat
aatttttacc 780tt 782981079DNAHomo
sapiensmisc_feature(262)..(268)n=any nucelotide 98gatgctattt
gggcaatttc ttattgacag ttttgaaatg ttaggctttt atctccattt 60tttagtactt
aaattttcca acatgggtgt tgcttgttat tttgtcagta taaaatagaa
120gagtggttct gttctggaat ttagtatata catgagtatc tagtgtatgt
cagccatgaa 180aatgaacctt tcagatgttt aacttcaggg aacctaattg
agtcattgct ccagacattg 240ttgctttgaa cccactatat tnnnnnnnct
cgggcaatga ctcagtgtgg caaggatact 300actgcaggcc tgtttctgga
aggcactgga ctcctctgat gcaaactttg gccagggact 360ccttgatagc
tgttaaatag atgctgcacc aacactctct ttcttttctc tctttttctt
420tattcaatat tagactacaa gcagtctaag gacttctcag ggtttctagc
tctctctcat 480ttcacacatg ctttcctagt aatctctact catatatctt
actgctacgc tggggccaga 540taacnnnnnn cttccatttt gtttttatct
ctattcttct tccccttctg ctttcattat 600tgaaactttc tgctttcatt
attgaaactt tcccagattt gttctgctta acctggcatt 660ggaactgttt
cctcttccct gtgctgcttt ctcccattgc catgtccttt tttttttttt
720tttttttttt tgagacagtg tcactctgtt gcccaggctg gagtgcaatg
gtgcaatctt 780ggccactgca acccccgcct cccgggttca agtgattctc
ctgcctcagc ctcctgagta 840gctgggatta caggtgccca ccactatgcc
cggctgattt ttgtattttt agtagagatn 900nnnnnnnttt caccatngct
gatcaggctg gtctcgaact cctgaccgca gtgantccgc 960cctccttggc
ctcccaaagt gctgagatta caggcatgag tcactgcgnc cagccaccat
1020tattctctag aggtgagaga acactggctc ttctaacaag ttgaaatttg
atagagacc 1079991977DNAHomo sapiensmisc_feature(840)..(843)n=any
nucleotide 99cacaaaaaaa gattattagc cacaaaaaaa ccttgaagta acgcattaaa
atgttaatgg 60attcacttta ttgagcatct gctcataata ctttaatgag tgcaaagtgc
tttgaatata 120atacgtcatt taaaccttac cataattctg aggaattgct
acctccactt cacagatggg 180gcacaggagg cttagataac atgcccaaag
tcatgcttct agtaaatgga tataattaag 240attcaaatta ttgataagaa
tttgatctgc cttaccagta tctagtagta aatctaaaag 300cgctttccag
agcatgtgct gttgatagag cttgatgtct aactctctga aattttccat
360tcttatttgt ctcactggta tatagttatt ttttactact ttcatacacc
tactaagaag 420acaggaggat caaagatagg atttcattta gaatgcctaa
agcttcacgt attttaattc 480agaataagat tcaggcagac caccagtata
tgccatggtc cctggttatc tttcagcagg 540tgaccgagaa agaaaacatg
gtaatgttta tgaaatggtg ggttcttgta gtttcacttc 600aacatatctg
cctttactgt attaagatga tggattaact tattcttgat atgggcatgt
660aaaacaatat acttttacta aacagctaca gagagacaaa tgtgtttcca
gacaaactta 720agagactgag tgttcaaact gaataatctc gaccttaatt
gtaactatat tttatgaaat 780ccagctgtaa ggcaaaaaca gacttctttg
ggcctaccac gggcattttg ttcctgttan 840nnntactcca aaccttaaac
ccacgtccac ttaaataatg gcctggaaat aaatgtcaat 900atctgatatt
atactgagat gtttagttat gaaatcaaaa gtggagaatt tcaatctgtc
960ctgtaagctt tctctgcggt cacgaccctc atgcactcag gctgtgcggt
gcagcatgct 1020ctgtcatgtc tgttttcttc tgcctgtaca cgggtggttg
ttcctgtcta cctgtttgag 1080gaaatatgaa tacgtnnnnn nctagaatct
actgcacatg caataaggaa acaatcagta 1140agaatcactt tctcgtggaa
aattcattag aattaacatc tcgttttaaa atgctctatc 1200aaagtgtaaa
taattcctct ctcttttccc tttttcacta aggagtttgt atattaaaca
1260gaatttcaag taatgtatta taaatttatt taanntattt acaataaaat
gccacgtata 1320agcatcaagc aacatgannn nnncattggt agaaagcaca
atacatagtc aaaacagcag 1380agtattaaat aaacagaaaa tttgcaaaag
gcaagtaaag aatatacata tacttaatta 1440tacataaaat attgatacag
gaggtagaaa gaaatttagt aagcagataa tgggggcaac 1500agagtcctca
gcagagcttc ccttctaaca aaaagcagcc caataaatta tttttttttt
1560ctaacaaaaa gcagcctgaa aaatcgagct gcaaacatag attagcaatc
ggctgaaagt 1620gcgggagaat gctggcagct gtgccaatag taaagggcta
cctggagccg ggcgcgtggc 1680tcacgctgta atcccagcac tttgggaggg
cgaggcaacg cggatcacct gaggtcggga 1740gtttgagatc agcccgacca
acatggagaa accccgtctc tactaaaaaa aaaaaaaaaa 1800aaaggcaaaa
aatgagccgg gcatggtggc acatgccttg cacatcccag ctgaggcagg
1860agaattcact tgaacctggg aggtagagat tgcggtgaag cgagatcacg
tcattgcact 1920ccagcctggg caaaaagagc aaaacttagt ctcaaaaaaa
aaaanncaaa gaaaaaa 19771002387DNAHomo sapiens 100ctcaaaaggg
gccggatttc cttctcctgg aggcagatgt tgcctctctc tctcgctcgg 60attggttcag
tgcactctag aaacactgct gtggtggaga aactggaccc cagggtggtt
120tataaaatcc tccaatgaag ctactaacat tactccaaag cataatatga
aagcattttt 180ggatgaattg aaagctgaga acatcaagaa gttcttatat
aattttacac agataccaca 240tttagcagga acagaacaaa actttcagct
tgcaaagcaa attcaatccc agtggaaaga 300atttggcctg gattctgttg
agctagcaca ttatgatgtc ctgttgtcct acccaaataa 360gactcatccc
aactacatct caataattaa tgaagatgga aatgagattt tcaacacatc
420attatttgaa ccacctcctc caggatatga aaatgtttcg gatattgtac
cacctttcag 480tgctttctct cctcaaggaa tgccagaggg cgatctagtg
tatgttaact atgcacgaac 540tgaagacttc tttaaattgg aacgggacat
gaaaatcaat tgctctggga aaattgtaat 600tgccagatat gggaaagttt
tcagaggaaa taaggttaaa aatgcccagc tggcaggggc 660caaaggagtc
attctctact ccgaccctgc tgactacttt gctcctgggg tgaagtccta
720tccagatggt tggaatcttc ctggaggtgg tgtccagcgt ggaaatatcc
taaatctgaa 780tggtgcagga gaccctctca caccaggtta cccagcaaat
gaatatgctt ataggcgtgg 840aattgcagag gctgttggtc ttccaagtat
tcctgttcat ccaattggat actatgatgc 900acagaagctc ctagaaaaaa
tgggtggctc agcaccacca gatagcagct ggagaggaag 960tctcaaactg
ccctacaatg ttggacctgg ctttagtgga aacttttcta cacaaaaact
1020caagatgcac atccactcta gcaatgaact gacaagaatt tacaatgtga
taggtagtct 1080cagaggagca gtggaaccag acagatatgt cattctggga
ggtcaccggg actcatgggt 1140gtttggtggt attgaccctc agagtggagc
agctgttgtt catgaaattg tgaggagctt 1200tggaacactg aaaaaggaag
ggtggagacc tagaagaaca attttgtttg caagctggga 1260tgcagaagaa
tttggtcttc ttggttctac tgagtgggca gaggagaatt caagactcct
1320tcaagagcgt ggcgtggctt atattaatgc tgactcatct atagaaggaa
actacactct 1380gagacttgat tgtacaccgc tgatgtacag cttggtacac
aacctaacaa aagagctgaa 1440aagccctgat gaaggctttg aaggcaaatc
tctttatgaa agttggacta aaaaaagtcc 1500ttccccagag ttcagtggca
tgcccaggat aagcaaattg ggatctggaa atgattttga 1560ggtgttcttc
caacgacttg gaattgcttc aggcagagca cggtatagta aaaattggga
1620aacaaacaaa ttcagcggct atccactgta tcacagtgtc tatgaaacat
atgagttggt 1680ggaaaagttt tatgatccaa tgtttaaata tcacctcact
gtggcccagg ttcgaggagg 1740gatggtgttt gagctagcca attccatact
gctccctttt gattgtcgag attatgctgt 1800acttttaaga aagtatgctg
acaaaatcta cagtatttct atgaaacatc cacaggaaat 1860gaagacatac
agtgtatcat ttgattcact tttttctgca ctaaagaatt ttacagaaat
1920tgcttccaag ttcagtgaga gactccagga ctttgacaaa agcaacccaa
tactattaag 1980aatgatgaat gatcaactca tgtttctgga aagagcattt
attgatccat tagggttacc 2040agacaggcct ttttataggc atgtcatcta
tgctccaagc agccacaaca agtatgcagg 2100ggagtcattc ccaggaattt
atgatgctct gtttgatatt gaaagcaaac tggacccttc 2160caaggcctgg
ggagaagtga agagacagat ttatgttgca gccttcacag tgcaggcagc
2220tgcagagact ttgagtgaag tagcctaaga ggattcttta gagaatccgt
attgaatttg 2280tgtggtatgt cactcagaaa gaatcgtaat gggtatattg
ataaatttta aaattggtat 2340atttgaaata aagttgaata ttatatataa
aaaaaaaaaa aaaaaaa 2387101693PRTHomo Sapiens 101Met Lys Ala Phe Leu
Asp Glu Leu Lys Ala Glu Asn Ile Lys Lys Phe1 5 10 15Leu Tyr Asn Phe
Thr Gln Ile Pro His Leu Ala Gly Thr Glu Gln Asn 20 25 30Phe Gln Leu
Ala Lys Gln Ile Gln Ser Gln Trp Lys Glu Phe Gly Leu 35 40 45Asp Ser
Val Glu Leu Ala His Tyr Asp Val Leu Leu Ser Tyr Pro Asn 50 55 60Lys
Thr His Pro Asn Tyr Ile Ser Ile Ile Asn Glu Asp Gly Asn Glu65 70 75
80Ile Phe Asn Thr Ser Leu Phe Glu Pro Pro Pro Pro Gly Tyr Glu Asn
85 90 95Val Ser Asp Ile Val Pro Pro Phe Ser Ala Phe Ser Pro Gln Gly
Met 100 105 110Pro Glu Gly Asp Leu Val Tyr Val Asn Tyr Ala Arg Thr
Glu Asp Phe 115 120 125Phe Lys Leu Glu Arg Asp Met Lys Ile Asn Cys
Ser Gly Lys Ile Val 130 135 140Ile Ala Arg Tyr Gly Lys Val Phe Arg
Gly Asn Lys Val Lys Asn Ala145 150 155 160Gln Leu Ala Gly Ala Lys
Ala Val Ile Leu Tyr Ser Asp Pro Ala Asp 165 170 175Tyr Phe Ala Pro
Gly Val Lys Ser Tyr Pro Asp Gly Trp Asn Leu Pro 180 185 190Gly Gly
Gly Val Gln Arg Gly Asn Ile Leu Asn Leu Asn Gly Ala Gly 195 200
205Asp Pro Leu Thr Pro Gly Tyr Pro Ala Asn Glu Tyr Ala Tyr Arg Arg
210 215 220Gly Ile Ala Glu Ala Val Gly Leu Pro Ser Ile Pro Val His
Pro Ile225 230 235 240Gly Tyr Tyr Asp Ala Gln Lys Leu Leu Glu Lys
Met Gly Gly Ser Ala 245 250 255Pro Pro Asp Ser Ser Trp Arg Gly Ser
Leu Lys Val Pro Tyr Asn Val 260 265 270Gly Pro Gly Phe Thr Gly Asn
Phe Ser Thr Gln Lys Val Lys Met His 275 280 285Ile His Ser Thr Asn
Glu Val Thr Arg Ile Tyr Asn Val Ile Gly Thr 290 295 300Leu Arg Gly
Ala Val Glu Pro Asp Arg Tyr Val Ile Leu Gly Gly His305 310 315
320Arg Asp Ser Trp Val Phe Gly Gly Ile Asp Pro Gln Ser Gly Ala Ala
325 330 335Val Val His Glu Ile Val Arg Ser Phe Gly Thr Leu Lys Lys
Glu Gly 340 345 350Trp Arg Pro Arg Arg Thr Ile Leu Phe Ala Ser Trp
Asp Ala Glu Glu 355 360 365Phe Gly Leu Leu Gly Ser Thr Glu Trp Ala
Glu Glu Asn Ser Arg Leu 370 375 380Leu Gln Glu Arg Gly Val Ala Tyr
Ile Asn Ala Asp Ser Ser Ile Glu385 390 395 400Gly Asn Tyr Thr Leu
Arg Val Asp Cys Thr Pro Leu Met Tyr Ser Leu 405 410 415Val His Asn
Leu Thr Lys Glu Leu Lys Ser Pro Asp Glu Gly Phe Glu 420 425 430Gly
Lys Ser Leu Tyr Glu Ser Trp Thr Lys Lys Ser Pro Ser Pro Glu 435 440
445Phe Ser Gly Met Pro Arg Ile Ser Lys Leu Gly Ser Gly Asn Asp Phe
450 455 460Glu Val Phe Phe Gln Arg Leu Gly Ile Ala Ser Gly Arg Ala
Arg Tyr465 470 475 480Thr Lys Asn Trp Glu Thr Asn Lys Phe Ser Gly
Tyr Pro Leu Tyr His 485 490 495Ser Val Tyr Glu Thr Tyr Glu Leu Val
Glu Gly Phe Tyr Asp Pro Met 500 505 510Phe Lys Tyr His Leu Thr Val
Ala Gln Val Arg Gly Gly Met Val Phe 515 520 525Glu Leu Ala Asn Ser
Ile Val Leu Pro Phe Asp Cys Arg Asp Tyr Ala 530 535 540Val Val Leu
Arg Lys Tyr Ala Asp Lys Ile Tyr Ser Ile Ser Met Lys545 550 555
560His Pro Gln Glu Met Lys Thr Tyr Ser Val Ser Phe Asp Ser Leu Phe
565 570 575Ser Ala Val Lys Asn Phe Thr Glu Ile Ala Ser Lys Phe Ser
Glu Arg 580 585 590Leu Gln Asp Phe Asp Lys Ser Asn Pro Ile Val Leu
Arg Met Met Asn 595 600 605Asp Gln Leu Met Phe Leu Glu Arg Ala Phe
Ile Asp Pro Leu Gly Leu 610 615 620Pro Asp Arg Pro Phe Tyr Arg His
Val Ile Tyr Ala Pro Ser Ser His625 630 635 640Asn Lys Tyr Ala Gly
Glu Ser Phe Pro Gly Ile Tyr Asp Ala Leu Phe 645 650 655Asp Ile Glu
Ser Lys Val Asp Pro Ser Lys Ala Trp Gly Glu Val Lys 660 665 670Arg
Gln Ile Tyr Val Ala Ala Phe Thr Val Gln Ala Ala Ala Glu Thr 675 680
685Leu Ser Glu Val Ala 69010286PRTHomo Sapiens 102Met Trp Asn Leu
Leu His Glu Thr Asp Ser Ala Val Ala Ala Ala Arg1 5 10 15Arg Pro Arg
Trp Leu Cys Ala Gly Ala Leu Val Leu Ala Gly Gly Phe 20 25 30Phe Leu
Leu Gly Phe Leu Phe Gly Trp Phe Ile Lys Ser Ser Asn Glu 35 40 45Ala
Thr Asn Ile Thr Pro Lys His Asn Met Lys Ala Phe Leu Asp Glu 50 55
60Leu Lys Ala Glu Asn Ile Lys Lys Phe Leu Tyr Asn Phe Thr Gln Ile65
70 75 80Pro Ile Leu Ala Gly Thr 851037DNAHomo sapiens 103tkagtca
710412DNAHomo sapiensmisc_feature(4)..(9)n=any nucleotide
104accnnnnnng gt 1210511DNAHomo sapiensmisc_feature(1)..(3)n=any
nucleotide 105nnntaaatnn n 111068DNAHomo
sapiensmisc_feature(4)..(4)n=any nucleotide 106gggnggrr
810710DNAHomo sapiens 107gggrhtyyhc 101088DNAHomo sapiens
108ryywsgtg 810913DNAHomo sapiensmisc_feature(6)..(6)n=any
nucleotide 109aawaangaaa ggr 1311061DNAHomo sapiens 110tttttttttg
ccttttgttt tgttttgttt tgttttgttt tgtttgtttg tttgtttttt 60t
6111169DNAHomo sapiens 111tttttttttg cttttgtttt gttttgtttt
gttttgtttt gttttgtttt gtttgtttgt 60ttgtttttt 6911252DNAHomo sapiens
112tttttttttt gcttttgttt tgttttgttt tgttttgttt gtttgttttt tt
5211361DNAHomo sapiens 113tttttttttt gcttttgttt tgttttgttt
tgttttgttt tgtttgtttg tttgtttttt 60t 6111460DNAHomo sapiens
114tttttttttg cttttgtttt gttttgtttt gttttgtttt gtttgtttgt
ttgttttttt 6011559DNAHomo sapiens 115tttttttttg cttttgtttt
gttttgtttt gttttgtttt gtttgtttgt ttgtttttt 5911621DNAHomo sapiens
116ctcaaaaggg gccggatttc c 2111721DNAHomo sapiens 117ctctcaatct
actaatgcct c 2111820DNAHomo sapiens 118tacccactgc atcaggaaca
2011920DNAHomo sapiens 119ccttgaagca caccattaca 2012020DNAHomo
sapiens 120acacaggcca cctatttcag 2012120DNAHomo sapiens
121gtccagcgtc cagcacacag 2012223DNAHomo sapiens 122cagatatgtc
attctgggag gtc 2312321DNAHomo sapiens 123aacaccatcc ctcctcgaac c
2112424DNAHomo sapiens 124cctaacaaaa gagctgaaaa gccc 2412524DNAHomo
sapiens 125actgtgatac agtggatagc cgct 2412621DNAHomo sapiens
126agcagagaat ggaaagtcaa a 2112721DNAHomo sapiens 127tgttgatgtt
ggataagaga a 211283017DNAHomo sapiens 128gcgccttaaa aaaaaaaaac
tttcttggaa aatgtccagc tcttgcttaa atataaaaat 60gaaaggaaga aagagactct
cctctctcca ctcctataat tatgaggaac ttttattcaa 120ctctgaaatt
ctatacaatc tctacaatac
tctactgaat aaaagcagag cagaaaaagc 180tgcgcttttt ttccatagtc
gggaatgctt gtcatcagtg taaatcacca ccgcgccctt 240tttcctaaag
aatattattg ttattaataa acatgtaggg tattatcctc cacttacatt
300acaaaaccat tttttaaagc cgggcgtggt ggctcacgcc tgtaatccca
gcactttggg 360aggcccagac aggcggatca cgaagtcgag aaatcgagac
catcctggcc aacatggtga 420aaccccatct ctactaaaaa tacaaaaatt
agctgggcgt ggtggcgggc tcctgtagtc 480ccagctactc aggaggctga
ggcaggagaa tcgcttgaac cggggaggcg gaggttgcag 540tcagccaaga
tagcgccact gcactggagc ctggtgacag agtgagactc cctcaagaaa
600gaaaggaagg gaagggaaag ggaaggaagg ggaggggaag ggaggggagg
ggaggggagg 660aaagaaaaga atactggaac ttgttgaagg cagagacttt
attttcatat cccggctatg 720tctggctact gtcttacgta atagatataa
aatcaatctt ggttggatta accagaagaa 780tgagaagata tattctggta
agttgaatac ttagcaccca ggggtaatca gcttggacag 840gaccaggtcc
aaagactgtt aagagtcttc tgactccaaa ctcagtgctc cctccagtgc
900cacaagcaaa ctccataaag gtatcctgtg ctgaatagag actgtagagt
ggtacaaagt 960aagacagaca ttatattaag tcttagcttt gtgacttcga
atgacttacc taatctagct 1020aaatttcagt tttaccatgt gtaaatcagg
aagagtaata gaacaaacct tgaagggtcc 1080caatggtgat taaatgaggt
gatgtacata acatgcatca ctcataataa gtgctcttta 1140aatattagtc
actattatta gccatctctg attagatttg acaataggaa cattaggaaa
1200gatatagtac attcaggatt ttgttagaaa gagatgaaga aattcccttc
cttcctgccc 1260taggtcatct aggagttgtc atggttcatt gttgacaaat
taattttccc aaatttttca 1320ctttgctcag aaagtctaca tcgaagcacc
caagactgta caatctagtc catctttttc 1380cacttaactc atactgtgct
ctccctttct caaagcaaac tgtttgctat tccttgaata 1440cactctgagt
tttctgcctt tgcctactca gctggcccat ggcccctaat gtttcttctc
1500atctccactg ggtcaaatcc tacctgtacc ttatggttct gttaaaagca
gtgcttccat 1560aaagtactcc tagcaaatgc acggcctctc tcacggatta
taagaacaca gtttatttta 1620taaagcatgt agctattctc tccctcgaaa
tacgattatt attattaaga atttatagca 1680gggatataat tttgtatgat
gattcttctg gttaatccaa ccaagattga ttttatatct 1740attacgtaag
acagtagcca gacatagccg ggatatgaaa ataaagtctc tgccttcaac
1800aagttccagt attcttttct ttcctcccct cccctcccct cccttcccct
ccccttcctt 1860ccctttccct tcccttcctt tctttcttga gggagtctca
ctctgtcacc aggctccagt 1920gcagtggcgc tatcttggct gactgcaacc
tccgcctccc cggttcaagc gattctcctg 1980cctcagcctc ctgagtagct
gggactacag gagcccgcca ccacgcccag ctaatttttg 2040tatttttagt
agagatgggg tttcaccatg ttggccagga tggtctcgat ttctcgactt
2100cgtgatccgc ctgtctgggc ctcccaaagt gctgggatta caggcgtgag
ccaccacgcc 2160cggctttaaa aaatggtttt gtaatgtaag tggaggataa
taccctacat gtttattaat 2220aacaataata ttctttagga aaaagggcgc
ggtggtgatt tacactgatg acaagcattc 2280ccgactatgg aaaaaaagcg
cagctttttc tgctctgctt ttattcagta gagtattgta 2340gagattgtat
agaatttcag agttgaataa aagttcctca taattatagg agtggagaga
2400ggagagtctc tttcttcctt tcatttttat atttaagcaa gagctggaca
ttttccaaga 2460aagttttttt tttttaaggc gcctctcaaa aggggccgga
tttccttctc ctggaggcag 2520atgttgcctc tctctctcgc tcggattggt
tcagtgcact ctagaaacac tgctgtggtg 2580gagaaactgg accccaggtc
tggagcgaat tccagcctgc agggctgata agcgaggcat 2640tagtgagatt
gagagagact ttaccccgcc gtggtggttg gagggcgcgc agtagagcag
2700cagcacaggc gcgggtcccg ggaggccggc tctgctcgcg ccgagatgtg
gaatctcctt 2760cacgaaaccg actcggctgt ggccaccgcg cgccgcccgc
gctggctgtg cgctggggcg 2820ctggtgctgg cgggtggctt ctttctcctc
ggcttcctct tcggtagggg ggcgcctcgc 2880ggagcaaacc tcggagtctt
ccccgtggtg ccgcggtgct gggactcgcg ggtcagctgc 2940cgagtgggat
cctgttgctg gtcttcccca ggggcggcga ttagggtcgg ggtaatgtgg
3000ggtgagcacc cctcgag 3017
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