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Differential expression of keratin 5 gene in non-tumorigenic and tumorigenic rat bladder cell lines
CANCER LETTERS Cancer Letters 75 (1993) 87-93
Differential
expression of keratin 5 gene in non-tumorigenic and tumorigenic rat bladder cell lines
Li Nan”, Hitoshi ‘Department
of Pathology.
Kawamataa, Xiaodi Tanb, Shuji Kameyama”, Ryoichi Oyasu* a Nmthweslem Cllicugo.
hDepartment
of Pathology.
University
Medical
School,
303 E. Chicago
.4 wnue,
IL 606 I I-3OON. US24
Children’s Chicogo.
Menmriul
Hospitul.
IL 606/4.
USA
Northwrstem
University.
(Received 16 August 1993; accepted 21 September 1993)
Abstract
In this study, we attempted to find a gene or genes which were differentially expressed between a non-tumorigenic rat bladder cell line and a highly tumorigenicimetastatic bladder carcinoma cell line that was derived from the former after treatment in vitro with N-methyl-N-nitrosourea. We cloned a rat keratin 5 cDNA by a differential hybridization technique and found that all of the non-tumorigenic cells (717) and normal bladder tissue expressed keratin 5, but most of the tumorigenic cells (g/IO) did not express keratin 5. Furthermore, in a spontaneously transformed cell line, keratin 5 expression was lost during the transformation process. These results suggest that loss of keratin 5 expression is closely associated with acquisition of a tumorigenic phenotype by rat bladder non-tumorigenic cells. Kql:
words;
Bladder carcinoma:
Cytokeratin:
Differential
expression
1. Introduction Urinary bladder cancer is the fifth most common cancer in men in western society; its incidence in the United States is more than 45 000 cases per year [l]. Bladder cancers can be divided into two types: low-grade, low-stage and high-grade, highstage. The majority of bladder cancers are of the first type and are characterized by a high rate of recurrence after successful surgical excision. The
* Corresponding 0304-3835/931$06.00 SSDI
author.
0
0304-3835(93)03192-8
1993 Elsevier
Scientific
Publishers
Ireland
second type of tumor is deeply invasive and is associated with a poor prognosis [I]. Very little is known about the molecular mechanisms of urinary bladder carcinogenesis and tumor-associated markers. Although a mutated c-Ha-ras 1 gene was first isolated from a human bladder cancer cell line [3], analysis of human bladder tumors showed that only lo%, had mutant Ha-ras 1 [4-61. It has been reported that mutation of tumor suppressor gene ~53 is associated with high-nuclear-grade invasive human bladder carcinomas [7]. We have tested the mRNA expression of the c-rnyc-. c-jun. Ltd. All righta reserved
88
L Nun ct ul. / Cuncer Letr. 75 (1993)
c-fos, c-erbB1,
nm23, Ha-ras, TGF-CY and TGF-PI genes in more than 15 rat bladder carcinoma cell lines of various degrees of biologic aggressiveness. We could not find any significant alterations in them except in TGF-/31 [8]. Therefore, we have undertaken to find a gene or genes that are differentially expressed in a nontumorigenic rat bladder cell line and in a highly tumorigenicimetastatic bladder cell line derived from the former after treatment in vitro with Nmethyl-N-nitrosourea (MNU). We isolated a cDNA clone which was found only in a cDNA library constructed from non tumorigenic cells by a differential hybridization technique. Analysis of the nucleotide sequence for the cDNA clone revealed that the isolated clone encoded the keratin 5 protein. Keratin 5 was found to be expressed in all of the non-tumorigenic cells (717) and in normal bladder tissue but not in most tumorigenic cells (8110). These results suggested
Table I Characteristics
Tumorigenicity nude mice”
In vitro MNU-converted Parental D44c
2. Material and methods 2. I. Cell lines and cell culture D44c cells were isolated from a small nodule developed in a heterotopically transplanted rat urinary bladder (HTB) [9] treated with MNU. The D44c cells were treated further with MNU in vitro and a number of subclones were isolated [lo]. We selected ten representative subclones on the basis of their biological characteristics and, in addition, we used six rat bladder cell lines which were recently established in our laboratory from tumors developed in HTBs [11] (Table 1). Cells were grown in Ham’s F12 medium supplemented with 5% fetal calf serum (FCS), non-essential amino
in
+ + + + + + + + +
Cells established MYU2 MYU4 MYU6s MYKUIL MYP3 MYU3L
Metastases lungsh
Histopathology turnoF
to
of
clones
Cl-3 Cl-16 Cl-17 Cl-2 Cl-5 Cl-8 Cl-4 Cl-6 Cl-l
Abbreviations: differentiated
that, at least in our rat bladder cell system, loss of keratin 5 expression was associated with acquisition of a tumorigenic phenotype by rat bladder cells.
of cell lines
Cell line
GZ-100 GI-200 Gl-200 G4-100 G4-50 G3-50 G3-50 G4-50 G2-100 LMCl9
87-93
_ _ _ + + + + +
w.d.Sq. w.d.Sq. p.d.Sq. w.d.Sq. ud.Ca. p.d.Ad. w.d.Sq. p.dAd. p.d.Ad.
_
u.d.Ca
from tumors _ _ _ _ +
w.d.Sq.. well-differentiated adenocarcinoma; u.d.Ca..
“Refer to reference hRefer to reference CRefer to reference
number number number
8. IO. I I.
squamous carcinoma; p.d.Sq.. undifferentiated carcinoma.
poorly differentiated
squamous
carcinoma:
p.d.Ad..
poorly
L. Nan er al. /Cancer Lert. 75 (1993) 87-93
acids, 100 &ml streptomycin, and 100 U/ml penicillin in a humidified atmosphere of 95% air and 5% CO1 at 37°C. All of the cell lines used were undergone fewer than 25 passages. Passage-42 cells of G2-100 Cl-3 were also used. 2.2. Construction of cDNA libraries Total RNA was isolated from D44c non-tumorigenic cells and from G2-100 Cl-l tumorigenicl metastatic cells by lysis with guanidinium isothiocyanate and centrifugation over a 5.7 M CsCl cushion. Poly(A)+ RNA was purified by oligo(dT) cellulose chromatography and cDNA libraries were constructed in hgtl 1 by standard protocols [ 121. 2.3. Screening of cDNA libraries by differential h_ybridization 32P-labeled cDNA, generated by reverse transcription of 5 pg mRNA of D44c or G2- 100 Cl-l cells, was used as a probe for screening of cDNA libraries constructed from both types of cells [ 121. Approximately 1.0 x lo6 plaques were screened for each library.
89
tionated by electrophoresis in 1.0 % agarose/2.2 M formaldehyde slab gels, and blotted to nylon filters (ICN, Costa Mesa, CA). The filters were prehybridized for 4 h at 42°C in 50% formamidel5 x Denhardt’s solution/5 x SSPE/O. 1% SDS containing sonicated salmon sperm DNA at 100 &ml. Hybridization with 32P-labeled probes was carried out for 18-20 h at 42°C. Filters were washed for 30 min in 0.1 x SSC/O.S% SDS at room temperature, followed by two changes of the same solution at 50°C and then exposure to Kodak XARS X-ray film. 2.6. Southern blot analysis High-molecular-weight DNA was isolated from the cells by the proteinase K-phenol-chloroform extraction method [ 121. DNA (10 pg) was digested with restriction endonuclease EcoRI before being fractionated on a 1.0% agarose gel, transferred to a nylon filter (ICN) and hybridized with 3’Plabeled cDNA probes.
2.4. Nucleotide sequencing The cDNA insert in hgt 11 was amplified by the polymerase chain reaction with the forward and reverse Xgt 11 primers (3 ‘-GCCCAGAGGTCCTCAGCAGCGGTGG-5 ’ and 5 ‘-TTGACACCAGACCAACTGGTAATG-3 ‘) (Promega, Madison, WI) [ 131 and cloned into pUC19 plasmid at the EcoRI cloning site. Nucleotide sequencing of the cDNA clone was performed by the dideoxynucleotide termination method with a DNA sequencing kit (United States Biochemical, Cleveland, OH). The nucleotide sequence of the cDNA clone was compared with the known genes in the GenBank.
2.7. Western blotting Cells were sonicated in 20 mM Tris-HCl (pH 7.4)/150 mM NaCl/O.S% deoxycholate/l% Nonidet-40/l mM EDTA/2 mM phenylmethylsulfonyl fluoride/200 KIU/ml aprotinin and the homogenates were centrifuged at 14 000 rev./min at 4°C for 20 min. The supernatants were used for Western blot analysis. The samples (20 pg) were electrophoresed on SDS-polyacrylamide gel by the method of Laemmli [16]. Proteins from gels were transferred to nitrocellulose membrane (BIORAD, Richmond, CA) [17] and keratin 5 was detected on nitrocellulose by the ABC method [ 181 with monoclonal anti-cytokeratin 8.13 (Sigma, St. Louis, MO) and Vectastain ABC kit (Vector Lab., Burlingame, CA).
2.5. Northern blot analysis Cells grown in monolayers were harvested at early confluence. RNA was prepared by lysing of cells in hypotonic buffer containing Nonidet P-40, followed by removal of nuclei as described previously [ 141. Fresh tumor tissue (0.1 g) was used for RNA extraction by the acid guanidium thiocyanate-phenol-chloroform (AGPC) method [ 151. Total RNA (10 or 20 pg) was denatured, frac-
2.8. Tumorigenicity in nude mice After trypsinization, cells were washed twice with Ham’s F12 medium without serum and resuspended in 0.1 ml of the same medium. Three male nude mice (Harlan Bioproducts for Science, Inc., Indianapolis, IN) were injected subcutaneously in the dorsal flank with 2 x lo6 cells. The mice were monitored once a week for the appearance of tumors. They were killed 3.5 months after
90
inoculation of cells. Tumors at the incubation site were removed and examined microscopically. One tumor was used for explant culture. The lungs, liver, kidneys, brain, intestine and axillary and supraclavicular lymph nodes were examined microscopically for evidence of metastases. 2.9. Explant culture Tumor tissue was minced to 0.5 mm7 in size and seeded on 100 mm’ Petri dishes coated with rat collagen (Collaborative Biomedical Products; Becton Dickinson Labware, Bedford, MA) containing 2 ml of Ham’s F-12 supplemented with 10% FCS. Epithelial cells were isolated and cloned. The cloned cells were cultured on uncoated plastic Petri dishes.
L. Nun et 01. /Cancer
Lrrt.
75 i 1993) 87-93
28s -
‘keratin 5
18s1
p-actin
23456
Fig. I. Northern blot analysis of RNA from cell lines. Total RNA (10 &lane) was fractionated on I .O”%denaturing agarose gel. transferred to a nylon filter and hybridized to “P-labeled keratin 5 and human p-actin probes. Lane 1. D44c; lane 2. G2-100 Cl-3; lane 3. Gl-200 Cl-17; lane 4. G3-50 Cl-8; lane 5. G2-100 Cl-l and lane 6. LMCIY.
3. Results 3.1. Isolation of a cDNA clone differentially e.upressed between non-tumorigenic cells and tumorigenic/ metastatic cells Approximately 1.0 x lo6 recombinant phage plaques for each library (D44c non-tumorigenic or G2- 100 C 1- 1 tumorigenic/metastatic cells) were screened by the differential hybridization method. Thirty-two recombinant phages (20 from the D44c cDNA library and 12 from the G2-100 Cl-l library) were subjected to a second screening. After the second screening, we found that one clone existed only in D44c cDNA library and not in G2100 Cl-l library. The cDNA insert (1.7 kb) of this clone was amplified and cloned into pUC19 plasmid. Analysis for nucleotide sequence revealed that this cDNA insert encoded the 213 C-terminus of keratin 5. but did not contain the initial codon for translation. 3.2. Keratin 5 expression in non-tumorigenic und tumorigenic cells To determine the level of expression of keratin 5 mRNA, we isolated total RNA from 17 cell lines of various degrees of biologic potential. By Northern blot analysis, high levels of expression of keratin 5 mRNA were observed in all of the nontumorigenic cell lines (D44c, G2-100 Cl-3, MYU2, MYU6s, MYU4, MYP3 and MYKUlL) and a normal rat bladder, whereas expression was com-
pletely absent in eight of the tumorigenic cell lines (Gl-200 Cl-17, G4-100 Cl-2, G4-50 Cl-5, G3-50 Cl-8, G3-50 Cl-4, G4-50 Cl-6, G2-100 Cl-l and LMC19) (Fig. 1). Two tumorigenic cell lines (Gl200 Cl-16 and MYU3L) expressed keratin 5. We tested keratin 5 protein (58 KD) expression in 17 clones by western blot analysis. The protein was detected in all of the non-tumorigenic cells and two of the tumorigenic cells (Gl-200 Cl-16 and MYU3L). In Myu3L, however, the keratin 5 expression was very faint indicating a trace level of
&Da) i-3
.I
s
r
97.4 66.2
-
45.0
-
31.0
1234567 Fig. 2. Western blot analysis of keratin 5 (58 kDa) of the cell lines. The cells lysate (20 &lane) was fractionated by 12.5% SDS-polyacrylamide gel. transferred to nitrocellulose membrane and keratin 5 was detected on nitrocellulose by the ABC method with monoclonal anti-cytokeratin 8.13 and a Vectastain ABC kit. Lane 1. G4-50 Cl-6; lane 2. G3-50 Cl-4; lane 3, G4-50 Cl-5; lane 4, GI-200 Cl-16; lane 5, G4-100 Cl-2; lane 6. LMC19: lane 7, D44c.
L. Nan et al. /Cancer
Table 2 Keratin 5 expression
Lert. 75 (1993)
91
87-93
of cell lines
Cell line
Tumorigenicity nude mice
D44c GZ-100 Cl-3 Gl-200 Cl-16 GI-200 Cl-17 G4-100 Cl-2 G4-50 Cl-5 G3-50 Cl-8 G3-50 Cl-4 G4-50 Cl-6 G2-100 Cl-l LMCl9 MYU2 MYU4 MYU6s MYKUIL MYP3 MYU3L
in
+ + + •t+ + + + + _ _ +
expression. This finding was in accord with the result of Northern blot analysis (Fig. 2; Table 2). DNA was extracted from six representative clones, D44c, G2-100 Cl-3, Gl-200 Cl-17, G3-50 Cl-g, G2-100 Cl-l and LMC19, digested with EcoRI and subjected to Southern blot hybridization analysis with the keratin 5 probe. The same
28s -
-
keratin 5
-
p-actin
18s -
Fig. 3. Northern blot analysis of RNA (the same conditions as for Fig. I, except 20 pg RNA/lane). Lane I, passage 42 G2- IO0 Cl-3 cells; lane 2. a tumor formed by passage 42 G?-100 Cl-3 cells; lane 3. cells established from the tumor (lane 2).
K5 mRNA expression
KS protein expression
+ f + _
+ + +
_
_
_ _ _ _
_ _ _
+ + + + + +
+ + + + + f
banding patterns and densities of DNA were evident in all cell lines, indicating that the differences in expression of keratin 5 mRNA were not due to gross gene rearrangements, deletion, or amplitication (data not shown). 3.3. Disappearance
of keratin 5 expression taneously transformed clone
in spon-
One of the non-tumorigenic clones, G2-100 Cl3, transformed spontaneously when passaged more than 40 times. We tested the keratin 5 expression in the transformed cells. We injected the passage 42 G2-100 Cl-3 cells subcutaneously into three nude mice. All of the mice grew tumors at the injection site and developed pulmonary metastases in 3.5 months. Total RNA was extracted from one of the subcutaneous tumors and from cells established from the tumor and it was used for Northern blot analysis. The result showed that passage 42 G2- 100 Cl-3 cells still expressed keratin 5 mRNA at a high level. However, its expression in the nude mouse tumor and in cells derived from this tumor was entirely absent (Fig. 3). These results can be interpreted as showing that most of the passage 42 G2-100 Cl-3 cells still expressed keratin 5 mRNA, but that some cells transformed spontaneously and lost the expression
92
of keratin 5. Because only the transformed cells could grow in nude mice, the tumors, or cells established from these tumors, did not express keratin 5 mRNA. 4. Discussion We have cloned a rat keratin 5 cDNA by a differential hybridization technique and we tested the expression of keratin 5 in several rat bladder cell lines by Northern and Western blot analysis. Keratin 5 mRNA expression was observed in all of the non-tumorigenic cells (7/7), but was absent in most of the tumorigenic cells (8/10). Furthermore, in spontaneously transformed cells. the expression of the keratin 5 gene was lost. These observations suggested that at least in our rat bladder cell system, loss of keratin 5 gene expression was closely associated with acquisition in vitro of a tumorigenie phenotype. Our results are consistent with those of other investigators in other organ systems. For example, Trask et al. [19] demonstrated that keratin 5 mRNA and protein were expressed in normal mammary epithelial cells in culture, but were absent from tumor-derived cell lines. Paine et al. [20] found that, in their isologous rat neoplastic epithelial cell lines, the keratin 5 mRNA level of T952iF7 (a clonal anaplastic cell line) was reduced lOOO-fold when compared with that of parental ASP/B10 (a clonal benign epithelial line). From these observations, it appears that keratin 5 was expressed only in the well-differentiated cells which did not show a tumorigenic phenotype and was not expressed in undifferentiated tumorigenic cells. In our study when non-tumorigenic D44c cells became tumorigenic by carcinogen (MNU) treatment or spontaneously, some genes which protect cells from a transformed phenotype and thus induce differentiation may have been suppressed. or some gene or genes that can block differentiation may have been activated. The former genes are considered to be tumor suppressor genes, and the latter are considered to be oncogenes. As a result of these gene alterations, cells become undifferentiated and tumorigenic and this is associated with loss of keratin 5 expression. To test the direct effect of keratin 5 on the
L. Nun ct al. / Cunwr
Lrrt.
75 (1993) 87-93
tumorigenic phenotype, we attempted to suppress keratin 5 cDNA expression in D44c cells. We cotransfected the cells with an antisense keratin 5 expression vector and pSV2-neo. Although we isolated several G418-resistant clones, we could not obtain transfectants which did not express the keratin 5, eventhough we performed several transfection experiments. We confirmed that the expression vector works in rodent cells by successful transfection of 3T3 cells with the antisense keratin 5 expression vector. The reason for our inability to obtain the transfectant may be that keratin 5 expression is essential for supporting the cell structure of well-differentiated D44c cells. The pattern of cytokeratins expressed in normal urothelium and bladder cancers has been reported. Schaafsma et al. [21] and Moll et al. [22] demonstrated immunohistochemically that these was a decrease in cytokeratin 13 and an increase in cytokeratin 14 in human bladder tumors. Schaafsma et al. [23] also reported changes in the epitope configuration of cytokeratin 8 and 18 in human invasive transitional cell carcinomas. Reedy et al. [24] noted the loss of cytokeratin 13 and 19 expression in MNU induced invasive rat bladder carcinomas. The percentage of aneuploid cell population in tumors was negatively correlated to the expression of cytokeratin 19 by immunohistochemical staining. We do not regard loss of one of the intermediate filaments as a diagnostic marker for malignant conversion. However, as shown in this experiment, it is clear that the expression of the keratin 5 gene was limited to the tumorigenic rat bladder cell lines. Thus, loss or marked decrease of keratin 5 expression was well correlated with acquisition of a tumorigenic phenotype of bladder cancer. 5. References Henderson. B.E.. Ross. R.K. and Pike. M.C. ( 1991) Toward the primary prevention of cancer. Science. 254. I131-1138. Greene. LF.. Hanash. K.A. and Farrow. G.M. ( 1973) Benign papilloma or papillary carcinoma of the bladder’? J. Urol., I IO. 205-207. Reddy. E.P.. Reynolds. R.K. Santos, E. and Barbacid, M. (1982) A point mutation is responsible for the acquisition of transforming properties by the T24 human bladder carcinoma oncogene. Nature. 300. 149- 152.
L. Nan et al. /Cancer
Lett. 75 (1993)
93
87-93
Feinberg. A.P.. Vogelstein. B.. Droller, M.J.. Baylin. S.B. and Nelkin. B.D. (1983) Mutation affecting the 12th amino acid of the c-Ha-m oncogene product occurs infrequently in human cancer. Science. 220, 1175-l 177. 5 Fujita, J., Yoshida, 0.. Yuasa. Y., Rhim, J.S.. Hatanaka, M. and Aaronson, S.A. (1984) Ha-ras oncogenes are activated by somatic alterations in human urinary tract tumors. Nature, 309. 464-466. 6 Knowles, M.A. and Williamson, M. (1993) Mutation of H-ras is infrequent in bladder cancer: confirmation by single-strand conformation polymorphism analysis. designed restrictions fragment length polymorphism and direct sequencing. Cancer Res., 53. 133-139. I Fujimoto. K.. Yamday, Y., Okajima, E.. Kakizoe. T., Sasaki, H., Sugimura. T. and Terada. M. (1992) Frequent association of ~53 gene mutations in invasive bladder cancer. Cancer Res., 52, 1393-1398. 8 Kawamata, H.. Azuma, M., Kameyama. S.. Nan, L. and Oyasu. R. (1992) Effect of epidermal growth factoritransforming growth factor o and transforming growth factor /3l on growth in vitro of rat urinary bladder carcinoma cells. Cell Growth Differ.. 3. 819-825. 9 Samma, S., Homma, Y. and Oyasu, R. (1987) Rat urinary bladder denuded of urothelium. An in vivo model for the epithelial stromal interactions of carcinogenesis. Am. J. Pathol.. 128. 328-337. IO Azuma, M.. Momose. H. and Oyasu, R (1990) In vitro malignant conversion of low-grade rat urinary bladder carcinoma cells by exposure to N-methyl-N-nitrosourea. Cancer Res., 50, 7062-7067. II Kawamata. H., Kameyama, S.. Nan. L.. Yamamoto, M. and Oyasu, R. (1993) Response to epidermal growth factor (EGF) and transforming growth factor /3l (TGF-PI) of newly established rat bladder carcinoma cell lines, Proc. Am. Assoc. Cancer Res., 34. 146. I2 Maniatis. T.. Fritsch. R. E. and Sambrook. J. (1982) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 13 Goueli, S.A. and Ahmed. K. (1991) A procedure for rapid isolation of cloned DNA inserts using the polymerase chain reaction technique. BioTechniques, IO, 306-309. I4 Berger. S.L. and mrkenmeter. C.S. (1979) Inhibition of 4
I5
intractable nucleases with ribonucleotide-vanadyl complexes: isolation of messenger ribonucleic acid from resting lymphocytes. Biochemistry, 18, 5143-5149. Chirgwin. J.M.. Prrybyla. A.E.. MacDonald, R.J. and
16
17
I8
19
20
21
22
23
24
Rutter. W.J. (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry, 18. 5294-5299. Laemmli, U.K.. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227. 680-685. Towbin, H.. Staehelin. T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gel to nitrocellulose sheets: procedure and some applications. Proc. Nat]. Acad. Sci. USA, 76, 4350-4354. Hsu. SM.. Raine, L. and Fanger. H. (1981) Use of avidinbiotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J. Histochem. Cytochem.. 29. 577-580. Trask, D.K.. Band. V., Zajchowski, D.A., Yaswen. P.. Suh, T. and Sager, R. (1990) Keratins as markers that distinguish normal and tumor-derived mammary epithelial cells. Proc. Nat]. Acad. Sci. USA, 87. 2319-2323. Paine, M.L. Gibbins. J.R.. Chew, K.E., Demetriou. A. and Kefford. R.F. (1992) Loss of keratin expression in anaplastic carcinoma cells due to post-transcriptional down-regulation acting in trans. Cancer Res.. 52. 6603-661 I. Schaafsma, H.E.. Ramaekers, F.C.S.. van Muijen, G.N.P., Lane. E.B.. Leigh, I.M., Robben. H.. Huijsmans, A.. Ooms. E.C.M. and Ruiter. D.J. (1990) Distribution of cytokeratin polypeptides in human transitional cell carcinomas. with special emphasis on changing expression patterns during tumor progression. Am. J. Pathol.. 136. 329-343. Mall. R.. Achtstatter. T.. Becht. E., Balcarova-Stander. J., Ittensohn. M. and Franke, W.W. (1988) Cytokeratins in normal and malignant transitional epithelium: maintenance of expression of urothelial features in transitional cell carcinomas and bladder carcinoma cell culture lines. Am. J. Pathol.. 132. 123-144. Schaafsma. H.E., Ramaekers. F.C.S.. van Muijen. G.N.P., Robben. H.. Lane. E.B.. Leigh. I.M. , Ooms, E.C.M.. Schalken, J.A., van Moorselaar. R.J.A. and Ruiter. D.J. (1991) Cytokeratin expression patterns in metastatic transitional cell carcinoma of the urinary tract. Am. J. Pathol., 139, 1389-1400. Reedy. E.A.. Cottrell. J.R. and Resau, J.H. (1991) Correlation of DNA ploidy levels with altered cytokerdtin patterns in rat bladder tumors. Pathology, 59. 62-6X.
Differential
expression of keratin 5 gene in non-tumorigenic and tumorigenic rat bladder cell lines
Li Nan”, Hitoshi ‘Department
of Pathology.
Kawamataa, Xiaodi Tanb, Shuji Kameyama”, Ryoichi Oyasu* a Nmthweslem Cllicugo.
hDepartment
of Pathology.
University
Medical
School,
303 E. Chicago
.4 wnue,
IL 606 I I-3OON. US24
Children’s Chicogo.
Menmriul
Hospitul.
IL 606/4.
USA
Northwrstem
University.
(Received 16 August 1993; accepted 21 September 1993)
Abstract
In this study, we attempted to find a gene or genes which were differentially expressed between a non-tumorigenic rat bladder cell line and a highly tumorigenicimetastatic bladder carcinoma cell line that was derived from the former after treatment in vitro with N-methyl-N-nitrosourea. We cloned a rat keratin 5 cDNA by a differential hybridization technique and found that all of the non-tumorigenic cells (717) and normal bladder tissue expressed keratin 5, but most of the tumorigenic cells (g/IO) did not express keratin 5. Furthermore, in a spontaneously transformed cell line, keratin 5 expression was lost during the transformation process. These results suggest that loss of keratin 5 expression is closely associated with acquisition of a tumorigenic phenotype by rat bladder non-tumorigenic cells. Kql:
words;
Bladder carcinoma:
Cytokeratin:
Differential
expression
1. Introduction Urinary bladder cancer is the fifth most common cancer in men in western society; its incidence in the United States is more than 45 000 cases per year [l]. Bladder cancers can be divided into two types: low-grade, low-stage and high-grade, highstage. The majority of bladder cancers are of the first type and are characterized by a high rate of recurrence after successful surgical excision. The
* Corresponding 0304-3835/931$06.00 SSDI
author.
0
0304-3835(93)03192-8
1993 Elsevier
Scientific
Publishers
Ireland
second type of tumor is deeply invasive and is associated with a poor prognosis [I]. Very little is known about the molecular mechanisms of urinary bladder carcinogenesis and tumor-associated markers. Although a mutated c-Ha-ras 1 gene was first isolated from a human bladder cancer cell line [3], analysis of human bladder tumors showed that only lo%, had mutant Ha-ras 1 [4-61. It has been reported that mutation of tumor suppressor gene ~53 is associated with high-nuclear-grade invasive human bladder carcinomas [7]. We have tested the mRNA expression of the c-rnyc-. c-jun. Ltd. All righta reserved
88
L Nun ct ul. / Cuncer Letr. 75 (1993)
c-fos, c-erbB1,
nm23, Ha-ras, TGF-CY and TGF-PI genes in more than 15 rat bladder carcinoma cell lines of various degrees of biologic aggressiveness. We could not find any significant alterations in them except in TGF-/31 [8]. Therefore, we have undertaken to find a gene or genes that are differentially expressed in a nontumorigenic rat bladder cell line and in a highly tumorigenicimetastatic bladder cell line derived from the former after treatment in vitro with Nmethyl-N-nitrosourea (MNU). We isolated a cDNA clone which was found only in a cDNA library constructed from non tumorigenic cells by a differential hybridization technique. Analysis of the nucleotide sequence for the cDNA clone revealed that the isolated clone encoded the keratin 5 protein. Keratin 5 was found to be expressed in all of the non-tumorigenic cells (717) and in normal bladder tissue but not in most tumorigenic cells (8110). These results suggested
Table I Characteristics
Tumorigenicity nude mice”
In vitro MNU-converted Parental D44c
2. Material and methods 2. I. Cell lines and cell culture D44c cells were isolated from a small nodule developed in a heterotopically transplanted rat urinary bladder (HTB) [9] treated with MNU. The D44c cells were treated further with MNU in vitro and a number of subclones were isolated [lo]. We selected ten representative subclones on the basis of their biological characteristics and, in addition, we used six rat bladder cell lines which were recently established in our laboratory from tumors developed in HTBs [11] (Table 1). Cells were grown in Ham’s F12 medium supplemented with 5% fetal calf serum (FCS), non-essential amino
in
+ + + + + + + + +
Cells established MYU2 MYU4 MYU6s MYKUIL MYP3 MYU3L
Metastases lungsh
Histopathology turnoF
to
of
clones
Cl-3 Cl-16 Cl-17 Cl-2 Cl-5 Cl-8 Cl-4 Cl-6 Cl-l
Abbreviations: differentiated
that, at least in our rat bladder cell system, loss of keratin 5 expression was associated with acquisition of a tumorigenic phenotype by rat bladder cells.
of cell lines
Cell line
GZ-100 GI-200 Gl-200 G4-100 G4-50 G3-50 G3-50 G4-50 G2-100 LMCl9
87-93
_ _ _ + + + + +
w.d.Sq. w.d.Sq. p.d.Sq. w.d.Sq. ud.Ca. p.d.Ad. w.d.Sq. p.dAd. p.d.Ad.
_
u.d.Ca
from tumors _ _ _ _ +
w.d.Sq.. well-differentiated adenocarcinoma; u.d.Ca..
“Refer to reference hRefer to reference CRefer to reference
number number number
8. IO. I I.
squamous carcinoma; p.d.Sq.. undifferentiated carcinoma.
poorly differentiated
squamous
carcinoma:
p.d.Ad..
poorly
L. Nan er al. /Cancer Lert. 75 (1993) 87-93
acids, 100 &ml streptomycin, and 100 U/ml penicillin in a humidified atmosphere of 95% air and 5% CO1 at 37°C. All of the cell lines used were undergone fewer than 25 passages. Passage-42 cells of G2-100 Cl-3 were also used. 2.2. Construction of cDNA libraries Total RNA was isolated from D44c non-tumorigenic cells and from G2-100 Cl-l tumorigenicl metastatic cells by lysis with guanidinium isothiocyanate and centrifugation over a 5.7 M CsCl cushion. Poly(A)+ RNA was purified by oligo(dT) cellulose chromatography and cDNA libraries were constructed in hgtl 1 by standard protocols [ 121. 2.3. Screening of cDNA libraries by differential h_ybridization 32P-labeled cDNA, generated by reverse transcription of 5 pg mRNA of D44c or G2- 100 Cl-l cells, was used as a probe for screening of cDNA libraries constructed from both types of cells [ 121. Approximately 1.0 x lo6 plaques were screened for each library.
89
tionated by electrophoresis in 1.0 % agarose/2.2 M formaldehyde slab gels, and blotted to nylon filters (ICN, Costa Mesa, CA). The filters were prehybridized for 4 h at 42°C in 50% formamidel5 x Denhardt’s solution/5 x SSPE/O. 1% SDS containing sonicated salmon sperm DNA at 100 &ml. Hybridization with 32P-labeled probes was carried out for 18-20 h at 42°C. Filters were washed for 30 min in 0.1 x SSC/O.S% SDS at room temperature, followed by two changes of the same solution at 50°C and then exposure to Kodak XARS X-ray film. 2.6. Southern blot analysis High-molecular-weight DNA was isolated from the cells by the proteinase K-phenol-chloroform extraction method [ 121. DNA (10 pg) was digested with restriction endonuclease EcoRI before being fractionated on a 1.0% agarose gel, transferred to a nylon filter (ICN) and hybridized with 3’Plabeled cDNA probes.
2.4. Nucleotide sequencing The cDNA insert in hgt 11 was amplified by the polymerase chain reaction with the forward and reverse Xgt 11 primers (3 ‘-GCCCAGAGGTCCTCAGCAGCGGTGG-5 ’ and 5 ‘-TTGACACCAGACCAACTGGTAATG-3 ‘) (Promega, Madison, WI) [ 131 and cloned into pUC19 plasmid at the EcoRI cloning site. Nucleotide sequencing of the cDNA clone was performed by the dideoxynucleotide termination method with a DNA sequencing kit (United States Biochemical, Cleveland, OH). The nucleotide sequence of the cDNA clone was compared with the known genes in the GenBank.
2.7. Western blotting Cells were sonicated in 20 mM Tris-HCl (pH 7.4)/150 mM NaCl/O.S% deoxycholate/l% Nonidet-40/l mM EDTA/2 mM phenylmethylsulfonyl fluoride/200 KIU/ml aprotinin and the homogenates were centrifuged at 14 000 rev./min at 4°C for 20 min. The supernatants were used for Western blot analysis. The samples (20 pg) were electrophoresed on SDS-polyacrylamide gel by the method of Laemmli [16]. Proteins from gels were transferred to nitrocellulose membrane (BIORAD, Richmond, CA) [17] and keratin 5 was detected on nitrocellulose by the ABC method [ 181 with monoclonal anti-cytokeratin 8.13 (Sigma, St. Louis, MO) and Vectastain ABC kit (Vector Lab., Burlingame, CA).
2.5. Northern blot analysis Cells grown in monolayers were harvested at early confluence. RNA was prepared by lysing of cells in hypotonic buffer containing Nonidet P-40, followed by removal of nuclei as described previously [ 141. Fresh tumor tissue (0.1 g) was used for RNA extraction by the acid guanidium thiocyanate-phenol-chloroform (AGPC) method [ 151. Total RNA (10 or 20 pg) was denatured, frac-
2.8. Tumorigenicity in nude mice After trypsinization, cells were washed twice with Ham’s F12 medium without serum and resuspended in 0.1 ml of the same medium. Three male nude mice (Harlan Bioproducts for Science, Inc., Indianapolis, IN) were injected subcutaneously in the dorsal flank with 2 x lo6 cells. The mice were monitored once a week for the appearance of tumors. They were killed 3.5 months after
90
inoculation of cells. Tumors at the incubation site were removed and examined microscopically. One tumor was used for explant culture. The lungs, liver, kidneys, brain, intestine and axillary and supraclavicular lymph nodes were examined microscopically for evidence of metastases. 2.9. Explant culture Tumor tissue was minced to 0.5 mm7 in size and seeded on 100 mm’ Petri dishes coated with rat collagen (Collaborative Biomedical Products; Becton Dickinson Labware, Bedford, MA) containing 2 ml of Ham’s F-12 supplemented with 10% FCS. Epithelial cells were isolated and cloned. The cloned cells were cultured on uncoated plastic Petri dishes.
L. Nun et 01. /Cancer
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75 i 1993) 87-93
28s -
‘keratin 5
18s1
p-actin
23456
Fig. I. Northern blot analysis of RNA from cell lines. Total RNA (10 &lane) was fractionated on I .O”%denaturing agarose gel. transferred to a nylon filter and hybridized to “P-labeled keratin 5 and human p-actin probes. Lane 1. D44c; lane 2. G2-100 Cl-3; lane 3. Gl-200 Cl-17; lane 4. G3-50 Cl-8; lane 5. G2-100 Cl-l and lane 6. LMCIY.
3. Results 3.1. Isolation of a cDNA clone differentially e.upressed between non-tumorigenic cells and tumorigenic/ metastatic cells Approximately 1.0 x lo6 recombinant phage plaques for each library (D44c non-tumorigenic or G2- 100 C 1- 1 tumorigenic/metastatic cells) were screened by the differential hybridization method. Thirty-two recombinant phages (20 from the D44c cDNA library and 12 from the G2-100 Cl-l library) were subjected to a second screening. After the second screening, we found that one clone existed only in D44c cDNA library and not in G2100 Cl-l library. The cDNA insert (1.7 kb) of this clone was amplified and cloned into pUC19 plasmid. Analysis for nucleotide sequence revealed that this cDNA insert encoded the 213 C-terminus of keratin 5. but did not contain the initial codon for translation. 3.2. Keratin 5 expression in non-tumorigenic und tumorigenic cells To determine the level of expression of keratin 5 mRNA, we isolated total RNA from 17 cell lines of various degrees of biologic potential. By Northern blot analysis, high levels of expression of keratin 5 mRNA were observed in all of the nontumorigenic cell lines (D44c, G2-100 Cl-3, MYU2, MYU6s, MYU4, MYP3 and MYKUlL) and a normal rat bladder, whereas expression was com-
pletely absent in eight of the tumorigenic cell lines (Gl-200 Cl-17, G4-100 Cl-2, G4-50 Cl-5, G3-50 Cl-8, G3-50 Cl-4, G4-50 Cl-6, G2-100 Cl-l and LMC19) (Fig. 1). Two tumorigenic cell lines (Gl200 Cl-16 and MYU3L) expressed keratin 5. We tested keratin 5 protein (58 KD) expression in 17 clones by western blot analysis. The protein was detected in all of the non-tumorigenic cells and two of the tumorigenic cells (Gl-200 Cl-16 and MYU3L). In Myu3L, however, the keratin 5 expression was very faint indicating a trace level of
&Da) i-3
.I
s
r
97.4 66.2
-
45.0
-
31.0
1234567 Fig. 2. Western blot analysis of keratin 5 (58 kDa) of the cell lines. The cells lysate (20 &lane) was fractionated by 12.5% SDS-polyacrylamide gel. transferred to nitrocellulose membrane and keratin 5 was detected on nitrocellulose by the ABC method with monoclonal anti-cytokeratin 8.13 and a Vectastain ABC kit. Lane 1. G4-50 Cl-6; lane 2. G3-50 Cl-4; lane 3, G4-50 Cl-5; lane 4, GI-200 Cl-16; lane 5, G4-100 Cl-2; lane 6. LMC19: lane 7, D44c.
L. Nan et al. /Cancer
Table 2 Keratin 5 expression
Lert. 75 (1993)
91
87-93
of cell lines
Cell line
Tumorigenicity nude mice
D44c GZ-100 Cl-3 Gl-200 Cl-16 GI-200 Cl-17 G4-100 Cl-2 G4-50 Cl-5 G3-50 Cl-8 G3-50 Cl-4 G4-50 Cl-6 G2-100 Cl-l LMCl9 MYU2 MYU4 MYU6s MYKUIL MYP3 MYU3L
in
+ + + •t+ + + + + _ _ +
expression. This finding was in accord with the result of Northern blot analysis (Fig. 2; Table 2). DNA was extracted from six representative clones, D44c, G2-100 Cl-3, Gl-200 Cl-17, G3-50 Cl-g, G2-100 Cl-l and LMC19, digested with EcoRI and subjected to Southern blot hybridization analysis with the keratin 5 probe. The same
28s -
-
keratin 5
-
p-actin
18s -
Fig. 3. Northern blot analysis of RNA (the same conditions as for Fig. I, except 20 pg RNA/lane). Lane I, passage 42 G2- IO0 Cl-3 cells; lane 2. a tumor formed by passage 42 G?-100 Cl-3 cells; lane 3. cells established from the tumor (lane 2).
K5 mRNA expression
KS protein expression
+ f + _
+ + +
_
_
_ _ _ _
_ _ _
+ + + + + +
+ + + + + f
banding patterns and densities of DNA were evident in all cell lines, indicating that the differences in expression of keratin 5 mRNA were not due to gross gene rearrangements, deletion, or amplitication (data not shown). 3.3. Disappearance
of keratin 5 expression taneously transformed clone
in spon-
One of the non-tumorigenic clones, G2-100 Cl3, transformed spontaneously when passaged more than 40 times. We tested the keratin 5 expression in the transformed cells. We injected the passage 42 G2-100 Cl-3 cells subcutaneously into three nude mice. All of the mice grew tumors at the injection site and developed pulmonary metastases in 3.5 months. Total RNA was extracted from one of the subcutaneous tumors and from cells established from the tumor and it was used for Northern blot analysis. The result showed that passage 42 G2- 100 Cl-3 cells still expressed keratin 5 mRNA at a high level. However, its expression in the nude mouse tumor and in cells derived from this tumor was entirely absent (Fig. 3). These results can be interpreted as showing that most of the passage 42 G2-100 Cl-3 cells still expressed keratin 5 mRNA, but that some cells transformed spontaneously and lost the expression
92
of keratin 5. Because only the transformed cells could grow in nude mice, the tumors, or cells established from these tumors, did not express keratin 5 mRNA. 4. Discussion We have cloned a rat keratin 5 cDNA by a differential hybridization technique and we tested the expression of keratin 5 in several rat bladder cell lines by Northern and Western blot analysis. Keratin 5 mRNA expression was observed in all of the non-tumorigenic cells (7/7), but was absent in most of the tumorigenic cells (8/10). Furthermore, in spontaneously transformed cells. the expression of the keratin 5 gene was lost. These observations suggested that at least in our rat bladder cell system, loss of keratin 5 gene expression was closely associated with acquisition in vitro of a tumorigenie phenotype. Our results are consistent with those of other investigators in other organ systems. For example, Trask et al. [19] demonstrated that keratin 5 mRNA and protein were expressed in normal mammary epithelial cells in culture, but were absent from tumor-derived cell lines. Paine et al. [20] found that, in their isologous rat neoplastic epithelial cell lines, the keratin 5 mRNA level of T952iF7 (a clonal anaplastic cell line) was reduced lOOO-fold when compared with that of parental ASP/B10 (a clonal benign epithelial line). From these observations, it appears that keratin 5 was expressed only in the well-differentiated cells which did not show a tumorigenic phenotype and was not expressed in undifferentiated tumorigenic cells. In our study when non-tumorigenic D44c cells became tumorigenic by carcinogen (MNU) treatment or spontaneously, some genes which protect cells from a transformed phenotype and thus induce differentiation may have been suppressed. or some gene or genes that can block differentiation may have been activated. The former genes are considered to be tumor suppressor genes, and the latter are considered to be oncogenes. As a result of these gene alterations, cells become undifferentiated and tumorigenic and this is associated with loss of keratin 5 expression. To test the direct effect of keratin 5 on the
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tumorigenic phenotype, we attempted to suppress keratin 5 cDNA expression in D44c cells. We cotransfected the cells with an antisense keratin 5 expression vector and pSV2-neo. Although we isolated several G418-resistant clones, we could not obtain transfectants which did not express the keratin 5, eventhough we performed several transfection experiments. We confirmed that the expression vector works in rodent cells by successful transfection of 3T3 cells with the antisense keratin 5 expression vector. The reason for our inability to obtain the transfectant may be that keratin 5 expression is essential for supporting the cell structure of well-differentiated D44c cells. The pattern of cytokeratins expressed in normal urothelium and bladder cancers has been reported. Schaafsma et al. [21] and Moll et al. [22] demonstrated immunohistochemically that these was a decrease in cytokeratin 13 and an increase in cytokeratin 14 in human bladder tumors. Schaafsma et al. [23] also reported changes in the epitope configuration of cytokeratin 8 and 18 in human invasive transitional cell carcinomas. Reedy et al. [24] noted the loss of cytokeratin 13 and 19 expression in MNU induced invasive rat bladder carcinomas. The percentage of aneuploid cell population in tumors was negatively correlated to the expression of cytokeratin 19 by immunohistochemical staining. We do not regard loss of one of the intermediate filaments as a diagnostic marker for malignant conversion. However, as shown in this experiment, it is clear that the expression of the keratin 5 gene was limited to the tumorigenic rat bladder cell lines. Thus, loss or marked decrease of keratin 5 expression was well correlated with acquisition of a tumorigenic phenotype of bladder cancer. 5. References Henderson. B.E.. Ross. R.K. and Pike. M.C. ( 1991) Toward the primary prevention of cancer. Science. 254. I131-1138. Greene. LF.. Hanash. K.A. and Farrow. G.M. ( 1973) Benign papilloma or papillary carcinoma of the bladder’? J. Urol., I IO. 205-207. Reddy. E.P.. Reynolds. R.K. Santos, E. and Barbacid, M. (1982) A point mutation is responsible for the acquisition of transforming properties by the T24 human bladder carcinoma oncogene. Nature. 300. 149- 152.
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Feinberg. A.P.. Vogelstein. B.. Droller, M.J.. Baylin. S.B. and Nelkin. B.D. (1983) Mutation affecting the 12th amino acid of the c-Ha-m oncogene product occurs infrequently in human cancer. Science. 220, 1175-l 177. 5 Fujita, J., Yoshida, 0.. Yuasa. Y., Rhim, J.S.. Hatanaka, M. and Aaronson, S.A. (1984) Ha-ras oncogenes are activated by somatic alterations in human urinary tract tumors. Nature, 309. 464-466. 6 Knowles, M.A. and Williamson, M. (1993) Mutation of H-ras is infrequent in bladder cancer: confirmation by single-strand conformation polymorphism analysis. designed restrictions fragment length polymorphism and direct sequencing. Cancer Res., 53. 133-139. I Fujimoto. K.. Yamday, Y., Okajima, E.. Kakizoe. T., Sasaki, H., Sugimura. T. and Terada. M. (1992) Frequent association of ~53 gene mutations in invasive bladder cancer. Cancer Res., 52, 1393-1398. 8 Kawamata, H.. Azuma, M., Kameyama. S.. Nan, L. and Oyasu. R. (1992) Effect of epidermal growth factoritransforming growth factor o and transforming growth factor /3l on growth in vitro of rat urinary bladder carcinoma cells. Cell Growth Differ.. 3. 819-825. 9 Samma, S., Homma, Y. and Oyasu, R. (1987) Rat urinary bladder denuded of urothelium. An in vivo model for the epithelial stromal interactions of carcinogenesis. Am. J. Pathol.. 128. 328-337. IO Azuma, M.. Momose. H. and Oyasu, R (1990) In vitro malignant conversion of low-grade rat urinary bladder carcinoma cells by exposure to N-methyl-N-nitrosourea. Cancer Res., 50, 7062-7067. II Kawamata. H., Kameyama, S.. Nan. L.. Yamamoto, M. and Oyasu, R. (1993) Response to epidermal growth factor (EGF) and transforming growth factor /3l (TGF-PI) of newly established rat bladder carcinoma cell lines, Proc. Am. Assoc. Cancer Res., 34. 146. I2 Maniatis. T.. Fritsch. R. E. and Sambrook. J. (1982) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 13 Goueli, S.A. and Ahmed. K. (1991) A procedure for rapid isolation of cloned DNA inserts using the polymerase chain reaction technique. BioTechniques, IO, 306-309. I4 Berger. S.L. and mrkenmeter. C.S. (1979) Inhibition of 4
I5
intractable nucleases with ribonucleotide-vanadyl complexes: isolation of messenger ribonucleic acid from resting lymphocytes. Biochemistry, 18, 5143-5149. Chirgwin. J.M.. Prrybyla. A.E.. MacDonald, R.J. and
16
17
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19
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24
Rutter. W.J. (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry, 18. 5294-5299. Laemmli, U.K.. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227. 680-685. Towbin, H.. Staehelin. T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gel to nitrocellulose sheets: procedure and some applications. Proc. Nat]. Acad. Sci. USA, 76, 4350-4354. Hsu. SM.. Raine, L. and Fanger. H. (1981) Use of avidinbiotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J. Histochem. Cytochem.. 29. 577-580. Trask, D.K.. Band. V., Zajchowski, D.A., Yaswen. P.. Suh, T. and Sager, R. (1990) Keratins as markers that distinguish normal and tumor-derived mammary epithelial cells. Proc. Nat]. Acad. Sci. USA, 87. 2319-2323. Paine, M.L. Gibbins. J.R.. Chew, K.E., Demetriou. A. and Kefford. R.F. (1992) Loss of keratin expression in anaplastic carcinoma cells due to post-transcriptional down-regulation acting in trans. Cancer Res.. 52. 6603-661 I. Schaafsma, H.E.. Ramaekers, F.C.S.. van Muijen, G.N.P., Lane. E.B.. Leigh, I.M., Robben. H.. Huijsmans, A.. Ooms. E.C.M. and Ruiter. D.J. (1990) Distribution of cytokeratin polypeptides in human transitional cell carcinomas. with special emphasis on changing expression patterns during tumor progression. Am. J. Pathol.. 136. 329-343. Mall. R.. Achtstatter. T.. Becht. E., Balcarova-Stander. J., Ittensohn. M. and Franke, W.W. (1988) Cytokeratins in normal and malignant transitional epithelium: maintenance of expression of urothelial features in transitional cell carcinomas and bladder carcinoma cell culture lines. Am. J. Pathol.. 132. 123-144. Schaafsma. H.E., Ramaekers. F.C.S.. van Muijen. G.N.P., Robben. H.. Lane. E.B.. Leigh. I.M. , Ooms, E.C.M.. Schalken, J.A., van Moorselaar. R.J.A. and Ruiter. D.J. (1991) Cytokeratin expression patterns in metastatic transitional cell carcinoma of the urinary tract. Am. J. Pathol., 139, 1389-1400. Reedy. E.A.. Cottrell. J.R. and Resau, J.H. (1991) Correlation of DNA ploidy levels with altered cytokerdtin patterns in rat bladder tumors. Pathology, 59. 62-6X.