U.S. patent application number 09/870725 was filed with the patent office on 2002-01-24 for methods for detecting and classifying bladder cancer via human uroplakin genes.
This patent application is currently assigned to NEW YORK UNIVERSITY. Invention is credited to Sun, Tung-Tien, Wu, Xue-Ru.
Application Number | 20020009745 09/870725 |
Document ID | / |
Family ID | 25515420 |
Filed Date | 2002-01-24 |
United States Patent
Application |
20020009745 |
Kind Code |
A1 |
Sun, Tung-Tien ; et
al. |
January 24, 2002 |
Methods for detecting and classifying bladder cancer via human
uroplakin genes
Abstract
The human gene for uroplakin II is identified and sequenced.
Using this gene, oligonucleotide primers were constructed which
were then used to identify bladder cancer cells in blood and
tissue.
Inventors: |
Sun, Tung-Tien; (Scarsdale,
NY) ; Wu, Xue-Ru; (New York, NY) |
Correspondence
Address: |
BROWDY AND NEIMARK, P.L.L.C.
PATENT AND TRADEMARK CAUSES
SUITE 300
624 NINTH STREET, N.W.
WASHINGTON
DC
20001-5303
US
|
Assignee: |
NEW YORK UNIVERSITY
New York
NY
|
Family ID: |
25515420 |
Appl. No.: |
09/870725 |
Filed: |
June 1, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09870725 |
Jun 1, 2001 |
|
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08969317 |
Nov 13, 1997 |
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6277968 |
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Current U.S.
Class: |
435/6.14 ;
435/91.2 |
Current CPC
Class: |
C07K 14/705 20130101;
C12Q 1/6886 20130101; C12Q 2600/112 20130101 |
Class at
Publication: |
435/6 ;
435/91.2 |
International
Class: |
C12Q 001/68; C12P
019/34 |
Claims
What is claimed is:
1. A method for identifying human bladder cancer cells, comprising:
a) extracting total RNA from human blood or tissue cells; b)
reverse transcribing the extracted total RNA; c) amplifying the
reverse transcribed RNA by polymerase chain reaction using
oligonucleotide primers complementary to a human uroplakin gene
selected from the group consisting of uroplakin Ia, uroplakin Ib,
uroplakin II, and uroplakin III; and d) detecting the presence of
RNA from the human uroplakin gene in the cell so that human bladder
cancer cells are identified.
2. The method according to claim 1, wherein said RNA extracting
step extracts RNA from human blood cells.
3. The method according to claim 1, wherein said RNA extracting
step extracts RNA from human tissue cells.
4. A method for detecting the presence of mutations in a human
uroplakin gene, comprising detecting in a DNA sample, via
polymerase chain reaction-single strand conformation polymorphism
methods that employ oligonucleotide primers complementary to a
human uroplakin gene selected from the group consisting of
uroplakin Ia, uroplakin Ib, uroplakin II, and uroplakin III,
mobility-shifted bands, wherein mobility-shifted bands are
indicative of a mutation in the human uroplakin gene.
Description
BACKGROUND OF THE INVENTION
[0001] Histological differentiation markers are useful in the
diagnosis of carcinoma metastases where the location of the primary
tumor is uncertain or unknown. Unfortunately, markers specific for
a single epithelium or organ are currently available for a only few
types of carcinoma, e.g., prostate-specific antigen for prostate
carcinomas and thyroglobulin for thyroid carcinomas.
[0002] Less specific markers of transitional cell carcinomas have
been identified and associated with malignant transformation, tumor
progression and the prognosis. Many of these markers are epithelial
membrane antigen (EMA) or oncogene/tumor suppressor gene products.
For example, Summerhayes et al. (1985. JNCI 75:1025-1038) have
described a series of monoclonal antibodies (group III), directed
against the urothelium which produce luminal-membrane staining of
normal superficial (umbrella) cells of the urothelium. Other
markers are expressed in superficial bladder tumors but disappear
in invasive and metastatic transitional cell carcinomas. All of
these markers are antibodies most of which stain non-urinary
epithelia and carcinomas too. Certain antigens such as involucrin,
E48 antigen and SCC antigen are markers shared by both transitional
and stratified squamous epithelia (of skin, esophagus, cervix,
etc.) and their carcinomas. However, no differentiation or lineage
marker specific for transitional cell carcinomas and their
metastases has been identified to date.
[0003] Normal urothelium contains tissue-specific differentiation
products that have been well characterized morphologically and
biochemically. It has been found that large numbers of urothelial
plaques are present in the superficial plasma membrane of
urothelial superficial or umbrella cells. These plaques are
characterized by a highly unusual membrane structure, i.e., the
asymmetric unit membrane (AUM), whose luminal leaflet is twice as
thick as its cytoplasmic leaflet. The thickening of the luminal
leaflet is due to the presence of particles exhibiting a
semi-crystalline organization. The molecular constituents
principally comprise four transmembrane proteins: uroplakin (UP) Ia
(27 kDa); UP Ib (28 kDa); UP II (15 kDa) and UP III (47 kDa). These
UPs, particularly UP Ia, Ib, and II, are characterized by their
markedly asymmetric mass distribution, with the extracellular
domain being considerably larger than the intracellular one. This
accounts for the clearly visible ultrastructural thickening of the
luminal leaflet of the unit membrane. UP III is believed to play a
role in the formation of the urothelial glycocalyx and may
interact, via its cytoplasmic portion, with the cytoskeleton.
[0004] The identification and characterization of the uroplakins
and their role as molecular markers has been described by Yu et al.
1990. J. Cell Biol. 111:1207-1216; Wu et al. 1990. J. Biol. Chem.
265:19170-19179; Wu et al. 1993. J. Cell Sci. 106:31-43; Wu et al.
1994. J. Biol. Chem. 269:13716-13724; Yu et al. 1994. J. Cell Biol.
125:171-182; and Lin et al. 1994. J. Biol. Chem. 269:1775-1784.
Further, Ryan et al. (1993. Mammalian Genome 4:656-661) describe
mapping the chromosomal locations of mouse and bovine UP Ia and UP
Ib and suggest likely locations for human genes. The locations of
human UP II, UP IIIa and UP IIIb genes are also predicted. Ryan et
al. suggest that uroplakin genes could be involved in chronic
urinary tract diseases and also suggests diagnostic uses (e.g.,
interstitial cystitis). Mono-specific antibodies to UP Ia, UP Ib,
UP II and UP III were used to show that uroplakin expression is
confined to the urinary bladder and to determine the location of UP
Ia, UP Ib, UP II and UP III within the asymmetric unit membrane.
However, identification, sequencing, or chromosomal location of the
human gene for any of the uroplakins has not yet been
disclosed.
[0005] Yu et al. (1992. Epith. Cell Biol. 1:4-12) describe the use
of specific antibodies to AE31 and AE32 for analysis of the
differentiation state of bovine urothelial cells. The diagnostic
use of antibodies to uroplakins to positively identify metastatic
bladder carcinomas has also been described by Moll, et al. 1993.
Verh. Deutsc. Ges. Path. 77 and Moll, R. et al. 1995. Am. J.
Pathol. 147:1383-1397.
[0006] The human gene for uroplakin II has now been isolated and
sequenced. Using the knowledge of the human uroplakin gene sequence
a highly specific uroplakin-based reverse transcriptase
(RT)-polymerase chain reaction (PCR) assay has now been developed
which is useful in the detection of circulating bladder cancer
cells in metastatic bladder cancer patients.
SUMMARY OF THE INVENTION
[0007] UP Ia, Ib, II and III are the first molecular markers that
have been found to be specific for urothelial differentiation. The
human gene sequence of UP II has now been determined. Knowledge of
this sequence has been used to develop molecular probes, primer
sequences, that are used in a RT-PCR assay to detect the presence
of bladder cancer cells in blood and tissues. Accordingly, the
assay of the present invention is useful in identifying and
diagnosing metastatic bladder cancer cells. Further, the RT-PCR
assay can be used to identify mutations in the uroplakin genes.
DETAILED DESCRIPTION OF THE INVENTION
[0008] UPs Ia, Ib, II and III are specialized membrane proteins of
the urothelial plaque constituting the AUM and represent the first
specific molecular markers of urothelial differentiation. UPs are
widely conserved with respect to their structural organization and
amino acid sequence among all mammals. UP II and III have been
found to be immunohistochemically detectable in routinely prepared
paraffin sections of human urothelium. Extensive UP III screening
of a variety of normal tissues revealed that the
urothelium-specificity of this glycoprotein, which until now has
been documented only in bovine tissues, is also valid for human
tissues. Unlike UP Ia and UP Ib which have 4 putative transmembrane
domains (Yu, J. et al. 1994. J. Cell Biol. 125:171-182), UP II and
UP III have only one transmembrane domain (Lin, J. H. et al. 1994.
J. Biol. Chem. 269:1775-1784; Wu, X. R. and T. T. Sun. 1993. J.
Cell Sci. 106:31-43). Recent data indicate that UP II and UP III
are preferentially crosslinked to UP Ia and UP Ib, respectively,
suggesting the existence of two types of 16 nm AUM particles
consisting of UP II/UP Ia and UP III/UP Ib (Wu, X. R. et al. 1995.
J. Biol. Chem. 270:29752-29759).
[0009] Mouse and bovine UP II genes have been sequenced. The
cDNA-derived amino acid sequences of the mouse and bovine UP II are
83% identical thus indicating a high degree of structural and
possibly functional conservation (Wu, X. R. et al. 1995. J. Biol.
Chem. 270:29752-29759).
[0010] The human UP II gene has also now been sequenced. In these
experiments, a human genomic library in lambda Fix-II phage
(Stratagene, La Jolla, Calif.) was screened with a .sup.32P-labeled
bovine uroplakin II cDNA (Lin, J. H. et al. 1994. J. Biol. Chem.
269:1775-1784). Two positive clones were identified. The clone
having a longer 5'-flanking sequence was then further
characterized. Two Sac I fragments (3.3 kb and 5 kb), that together
contained the entire coding region of human uroplakin II gene, were
subcloned and sequenced. This sequence is shown as SEQ ID NO: 1.
The gene contains 5 exons spanning approximately 2 kb, similar to
the mouse and bovine genes. Its deduced amino acid sequence, shown
as SEQ ID NO: 2, is 79% identical to those of the mouse and bovine
analogues, consisting of a hydrophobic N-terminal signal peptide
(approximately 25 amino acid residues) and a prosequence
(approximately 59 amino acid residues) harboring 3 potential
N-glycosylation sites, and ending with a RGRR cleavage site for
furin, which may be involved in UP II processing and maturation,
and a mature protein (100 residues) with a C-terminal hydrophobic
potential transmembrane domain (approximately 25 residues).
[0011] The chromosomal location of human UP II gene has also now
been determined. In these experiments, a 21 kb fragment of the
human UP II gene containing all five exons was labeled with
digoxigenin and used as a probe to hybridize to metaphase
chromosomes of human lymphocytes. The initial experiment resulted
in specific labeling of the long arm of group C chromosome, which
was believed to be chromosome 11 on the basis of DAPI staining.
Additional experiments were conducted wherein a biotin-labeled
probe from the cyclin D1 locus, which has previously been mapped to
11p13, was co-hybridized with the uroplakin II probe. Specific
labeling of the proximal and distal long arm of chromosome 11 was
observed. A total of 80 metaphase cells were analyzed, with 59 of
these exhibiting specific labeling. Measurements of 10 specifically
hybridized chromosomes demonstrated that uroplakin II gene is
located at a position which is 82% of the distance from the
centromere to the telemere of the long arm of chromosome 11, to the
region corresponding to band 11q23.
[0012] Oligonucleotides primer pairs corresponding to human
uroplakin genes such as UP II can be prepared in accordance with
well known techniques. These uroplakin specific oligonucleotide
primers are useful in a number of methods.
[0013] In one embodiment, oligonucleotide primer pairs
corresponding to a selected uroplakin gene can be used to detect
the presence of mutations in the selected uroplakin gene. For
example, experiments were performed to identify polymorphisms and
potential tumor-specific mutations of human UP II gene. In these
experiments, SSCP assays were performed on genomic DNAs from 57
cases of human transitional cell carcinomas. PCR was performed
using 5 pairs of primers as depicted in Example 1 corresponding to
intron sequences that flanked exons 1 to 5. One major
mobility-shifted band was identified in exon 2 of the UP II gene in
one tumor. This was due to a polymorphism consisting of a G to A
substitution at position 114. Additional polymorphisms and
potential tumor specific mutations can be identified for human UP
II and other uroplakin genes in accordance with this method.
[0014] Oligonucleotide primers to human uroplakin genes are also
useful in distinguishing different forms of bladder cancer and
identifying human bladder cancer cells in blood and tissue. In this
embodiment, total RNA is extracted from the blood or tissue cells.
The extracted total RNA is then reverse transcribed and amplified
by polymerase chain reaction using an oligonucleotide primer
complementary to a uroplakin gene so that the presence of the
uroplakin RNA in the cell can be detected and human bladder cancer
cells identified.
[0015] For example, experiments were performed to determine whether
different forms of bladder cancer could be distinguished based upon
expression of a selected uroplakin genes. In these experiments, two
major forms of bladder cancer were considered, transitional cell
carcinomas and squamous cell carcinomas. Patients in the study
included cases of bilharzial-related bladder cancer (a
parasite-related disease), both transitional cell and squamous
cell, and conventional transitional cell carcinoma cases.
Immunohistochemical staining studies were first performed using a
rabbit antiserum to a synthetic peptide corresponding to amino acid
residues 7-19 located at the N-terminus of mature UP II. This
antibody has previously been shown to be monospecific for UP II by
immunoblotting (Lin, J. H. et al. 1994. J. Biol. Chem.
269:1775-1784). Results of these experiments showed that 40% of the
transitional cell carcinoma specimens were uroplakin
II-positive.
[0016] Based upon this information indicating that UP II gene is
present in the majority of transitional cell carcinomas, an RT-PCR
assay for detection of blood-borne tumor cell dissemination by UP
II detection in patients with transitional cell carcinoma was
developed. Various specific oligonucleotide primers for human
uroplakin II as described in Example 5 were designed on the basis
of the sequence of the human UP II gene. Two primer pairs were
identified which were particularly effective in amplifying UP II
mRNA from the specimens. These primers are located in exons 2 (UP
II outer sense and nested sense; SEQ ID NOs: 14 and 15,
respectively) and exon 4 (UP II outer antisense and nested
antisense; SEQ ID NOs: 21 and 22, respectively). The outer primers
produce a PCR fragment of 330 bp while the nested primers produce a
fragment of 270 bp.
[0017] The PCR products were then cloned into the PCRII plasmid
vector using the TA cloning system. These plasmids were then
transformed into competent E. coli cells using standard methods.
Plasmid DNA was then isolated and screened by restriction analysis
using EcoRI. The TA clones were sequenced by the dideoxy method
using SP6 and T7 primers. These sequences were compared to the
original human UP II DNA sequence (SEQ ID NO: 1).
[0018] Total RNAs were extracted from peripheral blood of patients
with transitional cell carcinoma. The RNAs were then
reverse-transcribed and subjected to PCR amplification using the
identified oligonucleotide primers. As controls, RNAs were also
extracted from normal blood, normal tissue, and bladder cancer
specimens.
[0019] Results showed that UP II mRNAs were only amplified in
normal urothelium and transitional cell carcinoma tissue. The
response was specific in that there was no response in negative
control tissues (human-prostate, skin, ovary, uterus, liver, and
blood). All 10 (2 superficial and 8 invasive) transitional cell
carcinoma tissue specimens were positive for UP II. In the blood
samples from patients with transitional cell carcinoma, the RT-PCR
assay was used to identify metastasized cells. Of the 50
non-metastatic patients, none of the blood samples were positive
for UP II. In contrast, 2 of 10 of the metastatic blood samples
were positive for UP II. Southern blotting and DNA sequencing
established that the PCR products amplified from these two patients
were UP II sequence. Accordingly, the UP II primers of the present
invention are useful for the identification of blood-borne bladder
cancer cells and in the diagnosis of metastatic disease at an early
stage.
[0020] As will be obvious to those of skill in the art upon this
disclosure, similar RT-PCR methods for other uroplakin genes can be
developed in accordance with the methods described herein based
upon the detection of other uroplakin genes. Based upon the
teachings provided herein, one of skill could routinely screen the
human genomic library for other uroplakin genes (i.e., III, Ia, and
Ib) to develop specific oligonucleotide primers for screening for
different types of bladder cancer.
[0021] The following nonlimiting examples are presented to
illustrate the claimed invention.
EXAMPLES
Example 1
Genomic Cloning and Single Strand Conformation Polymorphism
[0022] A human genomic library in lamda Fix-II phage (Stratagene,
La Jolla, Calif.) was screened with a .sup.32P-labeled bovine
uroplakin-II cDNA described by Lin, J. H. et al. 1994. J. Biol.
Chem. 269:1775-84. PCR-SSCP assays were performed on a subset of 57
bladder tumors using a modification of the method described by
Orita, M. et al. 1989. Genomics 5:874-879 and Cordon-Cardom C. et
al. 1994. Int. J. Cancer 56:347-353. The sequences of the five sets
of PCR primers used to amplify exons 1-5 of the human uroplakin II
gene are as follows:
1 Exon 1: 5'-CTGCCAGCACCTATTCCACCTC-3' (SEQ ID NO: 3)
5'-CCATCGGAGCTCCCTCTGC-3' (SEQ ID NO: 4) Exon 2:
5'-CCATCGGAGCTCCCTCTGC-3' (SEQ ID NO: 5) 5'-GGGACTAGAGGGATGCCTTG--
3' (SEQ ID NO: 6) Exon 3: 5'-GAAACTTGACCCAGTCTTCC-3' (SEQ ID NO: 7)
5'-CTTCCCTAGGTGCCTCAGG-3' (SEQ ID NO: 8) Exon 4:
5'-CTCTTCCTGTAAGTCCCAAATAC-3' (SEQ ID NO: 9)
5'-GAATGGTCAGGGAAGCGTTTG-3' (SEQ ID NO: 10) Exon 5:
5'-CCACAGTGGTCTCCCCTCTC-3' (SEQ ID NO: 11)
5'-CTGGAGAAGCTGCTGCTCCG3' (SEQ ID NO: 12)
[0023] Each PCR reaction mixture contained 100 ng of tumor genomic
DNA in 10 .mu.l of 10 mM Tris-HDl (pH 8.3), 50 mM KCl, 2.5 mM
MgCl.sub.2, 250 .mu.M each of cold deoxynucleotide-5'-triphosphate,
1.5 .mu.M of each PCR primer, 0.5 unit of Tag DNA-polymerase, and
01 .mu.l of .sup.32P-dCTP (NEN, Cambridge, Mass.). PCR reactions
were performed using a Thermal Cycler (Perkin-Elmer Cetus, Foster
City, Calif.) by denaturation at 94.degree. C. for 3 minutes,
followed by 40 cycles of amplification (45 seconds at 94.degree.
C., 45 seconds at 65.degree. C. and 45 seconds at 72.degree. C.)
and were then kept at 72.degree. C. for 3 minutes. The PCR products
were denatured and loaded onto a denaturing 8% MDE polyacrylamide
gel (J.T. Baker, Phillipsburg, N.J.) and electrophoresed at room
temperature for 14 to 18 hours. After electrophoresis, the gels
were dried and exposed to X-ray film at 70.degree. C. overnight.
Mobility-shifted DNA bands wee reamplified and directly sequenced
by the dideoxy method described by Sanger, F. et al. 1977. Proc.
Natl Acad. Sci. USA 74:5463-5467.
Example 2
Fluorescence In Situ Hybridization
[0024] A human uroplakin II genomic fragment was labeled with
digoxigenin-dUTP by nick translation (Genome systems, St. Louis,
Mo.). The labeled probes were combined with sheared human DNA and
hybridized to normal metaphase chromosomes derived from
PHA-stimulated peripheral blood lymphocytes from a male donor in a
solution containing 50% formamide, 10% dextran sulfate and 2.times.
SSC. Specific signals for one-color experiments were detected by
incubating the hybridized specimens with fluoresceinated
anti-digoxigenin antibodies followed by counter-staining with DAPI.
Two-color experiments were conducted using the same protocol,
except for the inclusion of Texas Red-labeled avidin as described
by Stokke, T. et al. 1995 Genomics 26:134-7.
[0025] A cohort of 121 patients with primary bladder tumors wee
evaluated. Seventy-eight cases were bilharzial-related bladder
cancer; these samples were obtained from the Pathology Department
at the National Cancer Institute in Cairo, Egypt. Schistosomiases
infection was confirmed in all these 78 cases by the presence of
ova on histological sections. Forty-two of these cases were
transitional cell carcinomas, while the remaining 36 were squamous
cell carcinomas. An additional forty-three cases of conventional
transitional cell carcinoma were obtained from the Pathology
Department at the Memorial-Sloan Kettering cancer center in New
York. Hematoxylin-eosin stained sections of all cases were examined
to evaluate the pathological type, tumor grade and stage. Tumors
were staged as Pis (carcinoma in situ; 1 case) , P1 (17 cases), P2
(4 cases), P3 (92 cases) and P4 (7 cases). Twenty-five tumors were
classified as low grade (grade 1), 71 as intermediate grade (grade
2) and 25 as high grade (grade 3).
Example 3
Immunohistochemical Techniques
[0026] Three rabbit antisera against uroplakin proteins or peptides
were used in this study. First, a rabbit antiserum was generated
against total uroplakins of highly purified bovine AUM; this
antiserum reacts strongly with the 47-kD uroplakin III, moderately
with the 27-kD uroplakin Ib, and weakly with the 15-kD uroplakin II
as disclosed by Wu, X. R. and Sun, T. T. 1993 J. Cell Science
106:31-43 and Wu, X. R. et al. 1994 J. Biol. Chem. 269:13716-24.
Second, an anti-47-kDa antibody was affinity-purified from the
above anti-total uroplakins against the electrophoretically
purified 47-kD uroplakin band as disclosed by Wu, X. R. and Sun, T.
T. 1993 J. Cell Science 106:31-43 and Wu, X. R. et al. 1990. J.
Biol. Chem. 265:19170-9. Third, a rabbit antiserum was raised
against a synthetic peptide corresponding to amino acid residues
7-19 (DSGSGFTVTRLLA (SEQ ID NO: 13)) of the mature bovine uroplakin
as described by Lin, J. H. et al. 1994. J. Biol. Chem. 269:1775-84.
The latter two antibodies were shown to be monospecific for the
47-kD UP III and the 15-kD UP II, respectively, by
immunoblotting.
[0027] Avidin-biotin immunohistochemical staining was performed as
described by Osman, I. et al. 1997 Clin. Can. Res. 3:531-536. In
this method, deparaffinized tissue sections were treated with 1%
hydrogen peroxide in phosphate-buffered saline (PBS) to block the
endogenous peroxidase activity, incubated with normal goat serum
(diluted 1:10 in 2% bovine serum albumin or BSA), followed by
overnight incubation at 4.degree. C. with various antisera to
uroplakins (diluted 1:10,000 in 2% BSA/PBS). Biotinylated goat
anti-rabbit IgG was applied for 1 hour at 25.degree. C., 1:800
(Vector Laboratories, Burlingame, Calif.), followed by
avidin-biotin peroxidase complexes for 30 minutes, 1:25 dilution
(Vector Laboratories). Diaminobenzidine and hematoxylin were used
as the final chromogen and nuclear counter-stain, respectively. The
immunoreactivities were classified as positive if more than 20%
tumor cells showed positive membrane and cytoplasmic staining.
[0028] The associations between the uroplakin expression patterns
and clinicopathological parameters, including tumor stage, grade,
and tumor type, were assessed by Fisher's Exact test as described
by Metha, C. R. and Patel, N. R. 1983. J. Am. Stat. Assoc.
78:427-434; the two tailed p-values were employed to assess the
significance level. For variables with more than two categories,
the dose-response relationship was assessed by the trend-test using
the Mantel-Haenszel method as described by Landis, R. J. et al.
1978 Int. Stat. Rev. 46:237-254. The FREQ procedure in SAS was used
for analyzing the data as described in accordance with the SAS/STAT
Use Guide.
Example 4
Blood Sample Processing and RNA Extraction
[0029] All blood specimens used in the study were from patients
seen at the New York University Medical Center. Specimen
procurement was conducted according to the approval of the
institutional review board. Two heparinized (blue top) tubes per
patient were obtained via venipuncture technique. Samples were
promptly brought to the laboratory on ice for immediate proceeding.
A total of 50 nonmetastatic and 10 metastatic blood from patients
with bladder cancer were obtained. The 6 blood specimens used as
negative controls wee from healthy volunteers. In addition, 10
bladder tumor tissues were obtained after specimens were removed
from either radical cystoprostatectomy or transurethrally.
[0030] RNA extraction was performed using the RNAgents Total RNA
Isolation System by Promega. A total of 5 ml whole anticoagulated
venous blood was mixed with 1.5.times.volume of ice cold
diethylpyrocarbonate-treated water. After 5 minutes of incubation,
the samples were centrifuged for 20 minutes at 10,000 rpm at
4.degree. C. in a 50 ml polystyrene tube. After decanting the
supernatant, the platelet was washed with 1.5.times.volume ice cold
1.times.phosphate buffered solution and centrifuged for 15 minutes
at 10,000 rpm at 4.degree. C. The supernatant was carefully
decanted and the pellet was resuspended with 5 ml of denaturing
buffer, 0.5 ml of 2 M NaAcetate (pH 4.0), and 5 ml of
Phenol:Chloroform:Isoamyl alcohol. This tube was incubated on ice
for 15 minutes and then centrifuged for 20 minutes at 10,000 rpm at
4.degree. C. The supernatant was carefully isolated and was mixed
with an equal volume of isopropanol. this mixture was incubated at
-20.degree. C. overnight.
[0031] The solution was centrifuged for 15 minute at 10,000 rpm at
4.degree. C. The pellet was resuspended in 1 ml of denaturing
buffer and 1 ml of isopropanol. The mixture was incubated at
-20.degree. C. for 2 hours and transferred to two eppendorf tubes.
This solution was centrifuged for 15 minutes at 10,000 rpm at
4.degree. C. The collected pellet was washed with 0.5 ml of 75%
ethanol and then centrifuged for 15 minutes at 10,000 rpm at
4.degree. C. The supernatant was decanted and the pellet was dried
at room temperature for 10 minutes. The RNA pellet was dissolved in
30 .mu.l of RNAse-free water. TNA concentrations and purity were
determined by ultraviolet spectrophotometer. Approximately 15-25
.mu.g of RNAs was extracted from 5 ml of human whole blood. Bladder
tissue specimens were homogenized initially and RNA was extracted
in a similar method.
Example 5
Polymerase Chain Reaction Assay
[0032] Specific oligonucleotide primers for human uroplakin II were
designed on the basis of SEQ ID NO: 1. Ten different primer pairs
were selected to locate in different exons as described in Example
1. Two pair of primers were identified that were most effective in
amplifying UP II mRNA from these specimens. The primers were
located in exons 2 (UP II outer sense and nested sense), exons 4
(UP II outer antisense and nested antisense). The outer primers
produce a PCR fragment of 330 bp and the nested primers a fragment
of 270 bp.
[0033] Upon the recommendation of the manufacturer Promega and
using the Reverse Transcription Kit, 1 .mu.g of the total RNA was
reversely transcribed into complementary DNA. The total volume of
the reaction was 20 .mu.l, consisting of 2 .mu.l of 10.times. RT
buffer, 4 .mu.l of 25 mM MgCl2, 2 .mu.l of 10 mM DNTP mix, 0.5
.mu.l of rRNAsin ribonuclease inhibitor (1 unit/.mu.l), 0.6 .mu.l
of AMV transcriptase (15 units/.mu.l), 0.5 .mu.l of oligo
dTprimers, and RNAse free water. The reaction was incubated at
42.degree. C. for 25 minutes, heated at 99.degree. C. for 5 minutes
and then placed on ice immediately.
[0034] The polymerase chain reaction was performed in a total
volume of 50 .mu.l containing 1.5 .mu.l of the complementary DNA,
1.5 units of Tag polymerase, Promega buffer solution, 100 .mu.M of
each deoxynucleotide triphosphates and 10 ng of each primer. This
mix with the outer sense and outer antisense was transferred to a
Perkin-Elmer model PCR machine. The polymerase chain reaction
profile was (a) 1 cycle of 94.degree. C. for 5 minutes, then
60.degree. C. for 1 minute, then 72.degree. C. for 2 minutes; (b) 5
cycles of 94.degree. C. for 2 minutes, then 60.degree. C. for 1
minute, then 72.degree. C. for 2 minutes; (c) 1 cycle of 94.degree.
C. for 2 minutes, then 60.degree. C. for 1 minute, then 72.degree.
C. for 8 minutes. For reamplification, 2.5 .mu.l of the first PCR
product was further amplified with the nested primers in a 50 .mu.l
reaction volume under the same condition for 35 cycles. Fifteen
microliters of the product were electrophoresed on a 1.5% agarose
gel and visualized by ethidium bromide staining.
[0035] Polymerase chain reaction products were cloned into the
PCRII plasmid vector using the TA cloning system. These plasmids
were transformed into competent Escherichia coli cells using
standard methods; plasmid DNA was isolated and screened by
restriction analysis using EcoRI. The TA clones were then sequenced
by the dideoxy method using SP6 and T7 primers. These sequences
were then compared to the original Human Uroplakin II DNA
sequence.
Example 6
Southern Transfer Analysis
[0036] A 1.2% agarose gel containing the samples was soaked with
0.25 M HCl solution for 30 minutes followed by 1.5 M NaCl mixed
with 0.5 M NaOH for 20 minutes, twice. The gels were then pressure
blotted on nylon membrane overnight. The membranes were then soaked
and rinsed with 2.times.SSC twice, and then crosslinked with the
ultraviolet Stratalinker for 1 minute. The blots were prehybridized
at 60.degree. C. for three hours and eventually hybridized with
.sup.32P-labelled human uroplakin complementary DNA probes
overnight. The membranes were then washed twice with the low
stringent buffer solution of 0.15 M NaP/0.1% sodium dodecyl sulfate
at 60.degree. C. for 20 minutes. The membranes were then air dried
and autoradiographed for 2 hours at 70.degree. C.
[0037] The following is a list of primers used for PCR
amplification. Primers HUPS1 and HUPAS4 were found to be the most
effective in amplifying UP II mRNA from blood of metastatic bladder
cancer patients.
2 Human Uroplakin II Sense Primer HUPS1
5'-AACATCTCAAGCCTCTCTGGTCTG-3' (SEQ ID (outer sense primer) NO: 14)
HUPS2 5'-TGTCACCTCACAGGAGGCAATGCC-3' (SEQ ID (nested sense primer)
NO: 15) HUPS3 5'-ACTGATGGTCCGGAGAGCCAATGA-3' (SEQ ID NO: 16) HUPS4
5'-GTCCCCGGCGCTAACGGAGAGCCTG-3' (SEQ ID NO: 17) Human Uroplakin II
Antisense Primers HUPAS1 5'-AGCCCAGTGCCAGGGCAATGATGAA-3' (SEQ ID
NO: 15) HUPAS2 5'-GCACCAGCAGCAACATGGCGACAGAG-3' (SEQ ID NO: 19)
HUPAS3 5'-CAGCACCGTGATGACCACCATGCCC-3' (SEQ ID NO: 20) HUPAS4
5'-TGTGGACATTGGGATCTCTCTGCTG-3' (SEQ ID (outer antisense primer)
NO: 21) HUPAS5 5'-GCTGTCCCCTTCTTCACTAGGTAGG-3' (SEQ ID (nested
antisense primer) NO: 22) Human Uroplakin II DNA probe
5'-TGCCGTGGGCGCAGGGAACTGGTGAG (SEQ ID TGTGGTGGACAGTGGTGCTGGCTT-
CACAGTCACT-3' NO: 23)
[0038]
Sequence CWU 1
1
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