0022-534 7/90/1431-0167$02.00/0 Vol. 143, January Printed in U.S.A.
THE JOURNAL OF UROLOGY
Copyright© 1990 by AMERICAN UROLOGICAL ASSOCIATION, INC.
DIFFERENTIAL CYTOKERATIN EXPRESSION IN NORMAL, HYPERPLASTIC AND MALIGNANT EPITHELIAL CELLS FROM HUMAN PROSTATE EDWARD R. SHERWOOD,* LORI A. BERG, NANCY J. MITCHELL, JOHN E. McNEAL, JAMES M. KOZLOWSKI and CHUNG LEE From the Genitourinary Oncology Program, Department of Urology, Northwestern University Medical School, Chicago, Illinois and the Division of Urology, Stanford University Medical Center, Stanford, California
ABSTRACT
Studies were undertaken to define the expression of cytokeratins in normal, hyperplastic and malignant epithelial cells from human prostate. Cytokeratin (CK) polypeptides, separated by twodimensional electrophoresis, were identified by immunoblotting with CK-specific monoclonal antibodies. CK polypeptides 5, 7,8,15,18 and 19 were identified in fresh normal and hyperplastic prostate. Expression of CK 15 has not been previously reported in human prostate. Analysis of central and peripheral zone tissues from human prostate did not reveal qualitative differences in CK expression between these areas. Epithelial cells harvested from fresh BPH tissue by percoll gradient centrifugation and propagated in vitro using selective culture techniques showed alterations in CK expression compared to intact human prostate. Specifically, CKs 6,14,16 and 17 were noted in cultured BPH epithelial cells but not fresh normal prostate or BPH tissue. Immunoblot analysis of the established prostate cancer cell lines PC3, DU145 and LNCAP showed expression of CKs 8 and 18 but not CKs 5, 7 and 15 which were observed in benign prostate. These studies further characterize CK expression in benign and malignant human prostate and provide insights which may be useful in differentiating normal, hyperplastic and malignant epithelial cells in the human prostate gland. (J. Ural., 143: 167-171, 1990) The prostate gland is the major accessory organ of the male reproductive tract and the most common site of benign and malignant neoplastic disease in men. Benign prostatic hyperplasia (BPH) arises from fibroadenomatous growth of the prostate resulting in expansion of well differentiated acinar structures and stromal elements. 1 Carcinoma of the prostate has recently been identified as the most frequently encountered malignant neoplasm in men. 3 The morphology of lesions in prostatic carcinoma range from well differentiated to highly anaplastic tumors. 3 - 4 Although the incidence of prostatic neoplasia is high, little is known about the cell biology of normal and transformed prostatic epithelium. The cytokeratins are a class of cytoskeletal intermediate filaments found in epithelial and mesothelial cells. 5 - 6 Each epithelial type has a distinct profile of cytokeratin polypeptides which can be utilized to distinguish cells of simple and complex epithelia. 5 In addition, the pattern of cytokeratin expression within a specific tissue can serve as a marker of epithelial differentiation. 5 For example, the luminal and basal populations of prostatic epithelium can be distinguished based on the expression of specific cytokeratins. 7 Furthermore, alterations in cytokeratin polypeptide expression have been observed in benign and malignant neoplasia when compared to normal epithelium in several tissue types. 5 In the present study, the cytokeratin profiles of normal, hyperplastic and malignant epithelial cells from human prostate were assessed by immunoblotting of cytokeratin polypeptides separated by two-dimensional electrophoresis. These studies were undertaken to fully characterize the cytokeratin content of prostatic epithelial cells and determine the utility of Accepted for publication August 7, 1989. * Requests for reprints: Dept. of Urology, Northwestern University School of Medicine, 303 E. Chicago Ave., Chicago IL 60611. Supported by the Edwin and Lucy Kretschmer Fund of the Northwestern University Medical School, NIH Grants DK-08204 and DK39250, and the William 0. Jeffery, III fellowship.
specific cytokeratin antibodies in differentiating normal, hyperplastic and malignant epithelium in human prostate. MATERIALS AND METHODS
Cell lines and tissues. The established prostate cancer cell lines PC3 and DU145 were obtained from the tumor depository at the National Cancer Institute (Frederick, MD). LNCAP was purchased from the American Type Culture Collection (Rockville, MD). Epithelial cells were harvested from fresh prostate specimens by percoll gradient centrifugation and selective culture techniques as previously outlined. 8- 9 PC3, DU145 and LNCAP were cultured in RPMI-1640 with 10% fetal bovine serum and penicillin(lOO U/ml.)/streptomycin(lOO µ,g./ml.). Fresh prostatic epithelial cells were cultured in WAJC 404 medium containing ITS (insulin at five µ,g./ml, transferrin at five µ,g./ml., selenous acid at five ng./ml., Collaborative Research, Bedford, MA), prolactin (three ng./ml., Sigma Chemical, St. Louis, MO), epidermal growth factor (10 ng./ml., Sigma), bovine pituitary extract (30 µ,g./ml., Collaborative Research), polyvinyl pyrrolidone (two mg./ml., Sigma), cholera toxin (10 ng./ml.) and penicillin/streptomycin. Normal prostatic tissue was obtained from organ donors of less than 30 years of age. H&E stained slides were prepared from portions of formalin-fixed normal tissue to ascertain the absence of benign or malignant neoplasia. Hyperplastic human prostate was obtained from men undergoing suprapubic or retropubic prostatectomy for benign prostatic hyperplasia (BPH). Central and peripheral zone tissues were dissected as previously outlined10 from tissues obtained by radical prostatectomy. Sections of benign prostate were obtained from the respective zones and the absence of carcinoma was confirmed by histological analysis. Fresh PC3 tumors were obtained by injecting PC3 cells (1 X 106/mouse) subcutaneously into nude mice. Tumors were allowed to grow for four weeks and harvested from the subcutaneous sites with special care being taken to remove any skin or attached connective tissue. All fresh
167
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tissues were flash frozen in liquid nitrogen at the time of procurement and stored at -70C until utilized. Sample preparation. PC3, DU145 and LNCAP cells were harvested from ongoing cultures by quick trysinization. The cells were washed (3X) with Hanks' Balanced Salt Solution (HBSS) and dissolved in urea mix (9M urea, LKB ampholytes pH 9 to 11, 2% mercaptoethanol, 4% NP-40). Fresh prostate epithelial cells were detached from culture vessels using type I collagenase (675 U/ml.) in HBSS. The cells were washed (3x) with HBSS and dissolved in urea mix. Fresh prostate epithelial cells were also harvested from the second and third bands of discontinuous percoll gradients as previously described. 8 - 9 The cells were washed (3X) with HBSS and dissolved in urea mix. One million cells were loaded onto each 2D gel. Fresh tissues were homogenized by grinding tissue in a glass tissue homogenizer containing phosphate buffered saline (three µ,l./mg. of tissue). Tissue homogenates was mixed with urea mix at a 1:3 ratio and periodically vortexed for two hours. Debris was pelleted by centrifugation (2000 g for one minute) and the supernatant was harvested for analysis by two-dimensional electrophoresis. Seventy µg. of protein was loaded onto each 2D gel. Two-dimensional electrophoresis. The ISO-DALT system for 2D electrophoresis was performed according to the method of Anderson and Anderson. 11- 12 Briefly, the first dimension (the ISO system), which separates proteins according to their isoelectric points, was carried out for 14,000 volt hours in 5% polyacrylamide containing eight M urea and 2% total ampholytes with a pH range of three to 10. At the end of isoelectric focussing, each gel was extracted from the elongated glass tube (17.8 x 0.15 cm.) and equilibrated. The tube gels were layered over slab gels consisting of a linear polyacrylamide gradient (nine to 18%) containing 1 % SDS. Electrophoresis was carried out at a constant voltage of 100 to 150 volts for 16 hours. Protein in gels was detected by the silver stain method of Guevera and colleagues. 13 Immunoprotein blotting. Cytokeratin specific monoclonal antibodies 4.62, 8.12 and 8.13 14- 16 were purchased from Sigma Chemical Co. The region of specific staining for each antibody is demonstrated in a silver stained gel of cultured prostate epithelial cells (figure 1). Anti-CK 4.62 has previously demonstrated reactivity with CK polypeptide 19 in cultured human breast and colon carcinoma cells as well as bovine tissues. 17 CKs 13,15 and 16 are immunoreactive with anti-CK 8.12. 15 Anti-CK 8.13 is immunoreactive with CK polypeptides 1,5,6, 7,8 and 18 in human cervical tissues. 16 Cytokeratins on 2D gels were detected by the immunoblotting procedure of Towbin and colleagues 18 using the cytokeratin specific monoclonal antibodies. Briefly, proteins resolved by 2D electrophoresis were transferred to nitrocellulose paper (BioRad, 0.45 µ,m.) at 120 volts for two hours using a transfer buffer consisting of 25 mM Tris, 192 mM glycine and 20% methanol (pH 8.3). Blots were washed with distilled water, incubated (25C) with blocking buffer (Carnation non-fat dry milk) for two hours and with primary anticytokeratins at a 1:500 dilution overnight (16 to 18 hrs). The blots were washed with blocking solution and incubated (25C) with horseradish peroxidase-conjugated rabbit anti-mouse IgG for two hours (1:1000). The color reaction was induced using 4chloro-1-naphthol substrate.
FIG. 1. Silver stained 2D gel of cultured epithelial cells from hyperplastic human prostate. Areas of immunoblot reactivity with monoclonal anti-cytokeratins 4.62, 8.12 and 8.13 are designated. Molecular weight and pH markers are indicated. IEF = isoelectric focussing, first dimension; SDS-PAGE = polyacrylamide gel electrophoresis, second dimension.
RESULTS
Immunoreactivity of benign and malignant prostate epithelial cells with anti-cytokeratin 4.62. The cytokeratin (CK) content of cultured benign and malignant prostate epithelial cells as well as fresh prostatic tissues was assessed by immunoblot analysis of prostatic proteins separated by 2D electrophoresis. Epithelial cells from fresh normal prostate, representing a mixture of the three anatomical zones, exhibited expression of CKs 15 and 19, according to the convention of Moll and colleagues,5 following immunoblot analysis using anti-CK 4.62 (figure 2A). Similarly, CKs 15 and 19 were noted following
FIG. 2. Immunoblot analysis of prostate epithelial cell proteins separated by 2D electrophoresis using monoclonal anti-CK 4.62. A) normal prostate, B) whole BPH tissue, C) central zone tissue, D) peripheral zone tissue, E) BPH epithelial cells from discontinuous percoll gradient, F) cultured BPH epithelial cells, G) cultured PC3 cells, H) solid PC3 tumor from athymic nude mice.
169
CYTOKERATINS IN BENIGN AND MALIGNANT PROSTATE
immunoblot analysis of fresh BPH tissue as well as tissue from the central or peripheral zones of radical prostatectomy specimens (figures 2B-2D). BPH epithelial cells harvested directly from percoll gradients exhibited expression of CKs 15 and 19 while CKs 13,14,15,16,17 and 19 were noted in BPH epithelial cells obtained from primary cultures (figure 2E and 2F). The prostate cancer cell line PC3 showed expression of CK19 both in vitro and in vivo (figure 2G and 2H). The prostate cancer cell lines DU145 and LNCAP did not exhibit immunoreactivity with anti-CK 4.62 (data not shown). Immunoblot analysis of cytokeratin expression in prostatic cell lines and tissues using anti-cytokeratin 8.12. Analysis of cytokeratin expression in normal prostate tissue and fresh BPH as well as central and peripheral zone tissues using anti-CK 8.12 showed immunoreactivity with CK 15 (figure 3A-3D). In addition, a protein with the same electrophoretic mobility as CK 13 was noted in some central and peripheral zone tissues (figure 3C-3D) as well as whole BPH tissue (data not shown). The presence of CK13 in fresh prostate tissues was variable and inconsistent. BPH epithelial cells harvested from percoll gradients showed expression of CKs 13 and 15 while CKs 13,14,15,16,17 and 19 were noted in BPH epithelial cells obtained from primary cultures (figure 3E-3F). The prostate cancer cell lines PC3, DU145 and LNCAP did not exhibit immunoreactivity with anti-CK 8.12 (data not shown). Immunoreactivity of benign and malignant prostate epithelial cells with monoclonal anti-cytokeratin 8.13. Immunoblotting with anti-CK 8.13 showed the presence of cytokeratin polypeptides 5,7,8 and 18 in fresh normal prostate as well as whole BPH tissue and tissue dissected from the central and peripheral zones of the prostate (figure 4A-4D). Likewise, CKs 5,7,8 and 18 were noted in BPH epithelial cells obtained from discontinuous percoll gradients (figure 4E). Cultured BPH epithelial cells showed expression of cytokeratin polypeptides 5,6, 7,8 and 18 while exhibiting a diffuse immunostaining pattern that was not observed in freshly derived benign prostatic tissue and cells (figure 4F). The diffusely immunoreactive proteins were actively synthesized by cultured epithelial cells since the same proteins were observed on fluorographs of 35 S-methionine labelled proteins (data not shown). The prostate cancer cell line PC3 showed immunoreactive cytokeratins 8 and 18 following in vitro propagation but only CK 18 was noted in fresh PC3
FIG. 4. Immunoreactivity of prostate epithelial cell proteins with anti-CK 8.13. A) normal prostate, B) whole BPH tissue, C) central zone tissue, D) peripheral zone tissue, E) BPH epithelial cells from discontinuous percoll gradient, F) cultured BPH epithelial cells, G) cultured PC3 cells, H) PC# solid tumor from athymic nude mice, I) cultured DU145 cells, J) cultured LNCAP cells.
TABLE 1.
Cytokeratin expression in epithelial cells from normal, hyperplastic and malignant prostate Cytokeratin Expression
Specimen Molecular wt. (kD) Isoelectric point (pH) Normal prostate WholeBPH Central zone Peripheral
5
6
7
8
58
56
54
52
7.4
7.8
6.0
6.1
13 54 5.1
14 50 5.3
15 50 4.9
16 48 5.1
17 46 5.1
18 45 5.7
19 40 5.2
++
++ ++
++
++ ++
++ ++ ++
++ ++ +/++ ++ +/++ ++ +/-
++ ++ ++
++ ++ ++ ++ ++ ++
++
++ ++
++
++ ++
++ ++ ++ ++ ++
++ ++
zone
BPH epithelia Percoll BPH epithelia cultured PC3 in situ PC3 cultured DU145cultured LNCAPcultured
++ ++ ++ ++
++
++ ++
++ ++ ++ ++ ++
++
++
++, major expression, +/-, minor or variable expression.
FIG. 3. lmmunoblot analysis of prostate epithelial cells with antiCK 8.12. A) normal prostate, B) whole BPH tissue, C) central zone tissue, D) peripheral zone tissue, E) BPH epithelial cells from discontinuous percoll gradient, F) cultured BPH epithelial cells.
tumors harvested from nude mice (figures 4G-4H). Immunostaining of CKs 8 and 18 was noted in the cultured prostate cancer cell lines DU145 and LNCAP (figure 4G-4J). A composite presentation of cytokeratin expression in benign and malignant prostate epithelial cells and tissues as determined by immunoblot analysis with anti-cytokeratin monoclonals 4.62, 8.12 and 8.13 is provided as Table 1. The molecular weight and isoelectric points of the respective cytokeratin polypeptides are also indicated (table 1).
170
SHERWOOD AND ASSOCIATES DISCUSSION
The current studies were undertaken to further define the cytokeratin composition of benign and malignant epithelial cells from human prostate. Our results indicate that: 1) cytokeratin polypeptide 15, which had not been previously identified in human prostate, is expressed in normal prostate and BPH; 2) comparison of central and peripheral zone tissues from human prostate did not reveal qualitative differences in CK expression; 3) cultured BPH epithelial cells exhibit altered cytokeratin expression compared to BPH epithelia in situ and 4) the cytokeratin content of cultured and in vivo propagated prostate cancer cells is different from fresh normal prostate andBPH. Cytokeratin polypeptides 5,7,8,15,18 and 19 were identified in normal and hyperplastic human prostate by western immunoblot analysis. The expression of CKs 5,7,8,18 and 19 in human prostate has been reported7• 19 but CK 15 has not been previously observed in normal or hyperplastic prostate. Huszar and colleagues 15 reported negative immunoreactivity following immunohistochemical analysis of a single prostate specimen using anti-CK 8.12 which stains for CK 15. However, CK 15 was repeatedly demonstrated in benign prostatic tissue by immunoblot analysis with anti-CKs 4.62 and 8.12 in the present study. In addition, CKs 5 and 15 are considered to be a closely associated pair of cytokeratin polypeptides. 20 The presence of CK 5 in benign human prostate has been demonstrated by several investigators. Therefore, the presence of CK 15 in human prostate is not an unexpected finding. These observations indicate that cytokeratins characteristic of both simple (CKs 7 8 18 19) and stratified (CKs 5,15) epithelium are present in nor~ltl a~d hyperplastic human prostate. 5 The similarity in cytokeratin expression between normal prostate and BPH is not suprising because the expanded acinar structures in BPH maintain well differentiated morphology with intact basal and luminal epithelia characteristic of normal prosta,te. Differences in the cytokeratin pattern of central and peripheral zones from human prostate were not observed. Morphologic studies indicate that BPH arises primarily in the transition zone or periurethral region of the prostate. 21 The present study indicates that cytokeratin expression may not serve as a useful marker for distinguishing central and peripheral zone tissues in the human prostate. Recent advances in tissue culture techniques have allowed for the successful isolation and cultivation of epithelial cells from fresh human prostate.a-9 , 22- 23 These procedures have facilitated the study of prostate cell biology. The cellular characteristics of cultured prostate epithelial cells was partially defined in the present study by comparing the CK profile of fresh BPH tissue with epithelial cells isolated from the human prostate by percoll gradient centrifugation and selective culture techniques. Epithelial cells obtained directly from percoll gradients prior to introduction into tissue culture did not exhibit a demonstrable difference in cytokeratin expression compared to whole prostate. However, prostate epithelial cells harvested from primary cultures grown in plastic tissue culture flasks showed alterations in cytokeratin expression. Specifically, CKs 6,14,16 and 17 were observed in cultured epithelial cells but not in fresh prostatic tissue. In addition, cultured epithelial cells exhibited a widely variant cytokeratin profile compared to fresh tissue following analysis with specific cytokeratin antibodies. For example, proteins with the same 2D electrophoretic mobility as CKs 13,14,15,16,17 and 19 were noted in cultured epithelial cells following immunoblotting with anti-CKs 4.62 and 8.12. However, anti-CK 4.62 reacted with CKs 15 and 19 in fresh prostate while CK 15 was noted in fresh prostate following immunoblotting with anti-CK 8.12. Our previous studies also show that cultured prostate epithelial cells express the intermediate filament polypeptide vimentin. 9 Vimentin is generally present in mesenchymal cells but not epithelial cells in situ.
These findings indicate that alterations in cytokeratin and vimentin gene expression occur following in vitro propagation of freshly isolated prostate epithelial cells on plastic resulting in an atypical cytokeratin and vimentin profile. Altered intermediate filament expression in cultured cells has been previously reported for a variety of tissues. 5 Immunoblot analysis of CK expression in the established prostate cancer cell lines PC3, LNCAP and DU145 revealed the presence of CKs 8 and 18. Additionally, PC3 cells maintained in vitro and as subcutaneous solid tumors in nude mice exhibited expression of CK19. Compared to benign prostatic tissues, the established tumor lines lacked expression of CKs 5, 7 and 15. Previous reports indicate that CK5 is selectively expressed in the basal cell population of the human prostate. 24 Because CK5 is not expressed in prostatic carcinoma, several investigators have postulated that transformed prostate epithelium arises from the luminal epithelium of the prostate. 24- 25 The present study supports this hypothesis by showing the presence of CK5 in benign prostatic tissue but not in the established carcinoma lines. The expression of CK 15 was also noted in benign prostate but not in the prostate cancer lines following immunoblot analysis with anti-CK 8.12. As previously indicated, CKs 5 and 15 are a closely associated pair of cytokeratin polypeptides. The loss of both CK 5 and 15 expression in cultured prostatic carcinoma cells further supports the interrelationship of the two proteins. These findings also indicate that differential expression of CK 15 in benign and malignant prostate may provide an additional marker for the identification of prostatic carcinoma. Immunohistochemical studies on fresh benign and malignant prostate are currently underway in our laboratory to determine the utility of anti-CK 8.12 in differentiating benign prostatic epithelium from well differentiated and poorly differentiated carcinoma of the prostate. In summary, we have utilized cytokeratin specific monoclonal antibodies to further define cytokeratin expression in epithelial cells from benign and malignant prostate. Using anti-CK 8.12, CK polypeptide 15 was identified in benign human prostate which was previously unreported. In addition, alterations in CK expression were noted in cultured benign prostate epithelial cells and established carcinoma cell lines when compared to fresh benign prostatic tissue. Based on these observations, CK specific monoclonals may provide useful tools for the study of benign and malignant prostate epithelium. REFERENCES 1. Grayhack, J. T. and Kozlowski, J.M.: Benign prostatic hyperplasia.
2. 3. 4. 5. 6.
7. 8.
9.
In: Adult and Pediatric Urology. Edited by J. Y. Gillenwater, J. T. Grayhack, S. S. Howards and J. W. Duckett. Chicago:Year Book Publishers, pp. 1062-1125, 1988. Cancer Statistics. Ca-A Cancer Journal for Clinicians, American Cancer Society, p. 12, 1989. Kern, W. H.: Well differentiated carcinoma of the prostate. Cancer, 41: 2046, 1978. Whitmore, W. F.: The natural history of prostatic carcinoma. Cancer, 32: 1104, 1973. Moll, R., Franke, W. W. and Schiller, D. L.: The catalog of human cytokeratins:patterns of expression in normal epithelia, tumors and cultured cells. Cell, 31: 11, 1982. Wu, Y., Parker, L. M., Binder, N. E., Beckert, M. A., Senard, J. H., Griffiths, C. T. and Rheinwald, J. G.: The mesothelial keratins:A new family of cytoskeletal proteins identified in cultured mesothelial cells and non-keratinizing epithelia. Cell, 31: 693, 1982. Barwick, K. and Mardi, A.: An immunohistochemical study of the myoepithelial cells in prostate hyperplasia and neoplasia. Lab. Invest., 48: 7A, 1983. Kozlowski, J.M., McEwan, R. N., Keer, H., Sensibar, J., Sherwood, E., Lee, C., Albini, A. and Martin, G.: Prostate cancer and the invasive phenotype: Application of new in vitro and in vivo approaches. In: Tumor Progression and Metastasis. Edited by I. J. Fidler and J. Nicholson. New York:Alan R. Liss, pp. 189-231, 1988. Sherwood, E., Berg, L. A., McEwan, R. N., Pasciak, R. M., Ko-
CYTOKERA'TINS KN BENIGN AND MALIGNANT PROSTATE
10. 11.
12.
13.
14.
15.
16.
17.
zlowski, J. M. and Lee, C.: Two-dimensional protein profiles of cultured epithelial and stromal cells from hyperplastic human prostate. J. Cell. Biochem., 40: 201, 1989. McNeal, J. E.: Normal histology of the prostate. Am. J. Surg. Pathol., 12: 619, 1988. Anderson, N. G. and Anderson, N. L.: Analytical techniques for cell fractions. XXL Two-dimensional analysis of serum and tissue proteins: Multiple isoelectric focussing. Anal. Biochem., 85: 331, 1978. Anderson, N. L. and Anderson, N. G.: Analytical techniques for cell fractions. XXL. Two-dimensional analysis of serum and tissue proteins: multiple gradient slab electrophoresis. Anal. Biochem., 85: 341, 1978. Guevera, J., Johnston, D. A., Ranagoli, L. S., Martin, B. A., Capatillo, S. and Rodriguez, L. V.: Qualitative aspects of silver deposition in proteins resolved by complex polyacrylamide gels. Electrophoresis, 3: 197, 1982. Gigi, 0., Geiger, B., Eshhar, Z., Moll, R., Schmid, E., Winter, S., Schiller, D. L. and Franke, W. W.: Detection of a cytokeratin determinant common to diverse epithelial cells by a broadly cross-reacting monoclonal antibody. EMBO J., 1: 1429, 1982. Huszar, M., Gigi-Leitner, 0., Moll, R., Franke, W.W. and Geiger, B.: Monoclonal antibodies to various acidic (type I) cytokeratins of stratified epithelia. Differentiation, 31: 141, 1986. Gigi-Leitner, 0., Geiger, B., Levy, R. and Czernobilsky, B.: Cytokeratin expression in squamous metaplasia of the uterine cervix. Differentiation, 31: 191, 1986. Gigi-Leitner, 0. and Geiger, B.: Antigenic interrelationship be-
18. 19. 20.
21.
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23. 24.
25.
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tween the 40-kilodalton cytokeratin polypeptide and desmoplakins. Cell Motil. and the Cytoskeleton, 6: 628, 1986. Towbin, H., Staehelin, J., Gordon, J.: Electrophoretic transfer of proteins from acrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci., 46: 4350, 1979. Achtstater, T., Moll, R., Moore, B. and Franke, W.W.: Cytokeratin polypeptide patterns of different epithelia of the human male urogenital tract. J. Histochem. Cytochem., 33: 415, 1985. Sun, T-T., Tseng, S. C. G., Huang, A. J-W., Cooper, D., Schermer, A., Lynch, M. H., Weiss, R. and Eichner, R.: Monoclonal antibody studies of mammalian epithelial keratins: a review. Ann. N.Y. Acad. Sci., 455: 307, 1986. McNeal, J. E.: Anatomy of the prostate and morphogenesis of BPH. In: New Approaches to the Study of Benign Prostatic Hyperplasia. Edited by F. A. Kimball, A. E. Buhl and D. B. Carter. New York:Alan R. Liss, pp. 27-53, 1984. Chaproniere, D. M. and McKeehan, W. L.: Serial culture of single adult human prostatic epithelial cells in serum-free medium containing low calcium and a new growth factor from bovine brain. Cancer Res., 46: 819, 1986. Peehl, D. M. and Stamey, T. A.: Serum-free growth of human prostatic epithelial cells. In Vitro Cell. Dev. Biol., 22: 82, 1986. Nagle, R. B., Ahmann, F. R., McDaniel, K. M., Paquin, M. L., Clark, V. A. and Celmiker, A.: Cytokeratin characterization of human prostate carcinoma and its derived cell lines. Cancer Res., 47: 281, 1987. Brawer, M. K., Peehl, D. M., Stamey, T. A. and Bostwick, D. G.: Keratin immunoreactivity in the benign and neoplastic human prostate. Cancer Res., 45: 3663, 1985.
THE JOURNAL OF UROLOGY
Copyright© 1990 by AMERICAN UROLOGICAL ASSOCIATION, INC.
DIFFERENTIAL CYTOKERATIN EXPRESSION IN NORMAL, HYPERPLASTIC AND MALIGNANT EPITHELIAL CELLS FROM HUMAN PROSTATE EDWARD R. SHERWOOD,* LORI A. BERG, NANCY J. MITCHELL, JOHN E. McNEAL, JAMES M. KOZLOWSKI and CHUNG LEE From the Genitourinary Oncology Program, Department of Urology, Northwestern University Medical School, Chicago, Illinois and the Division of Urology, Stanford University Medical Center, Stanford, California
ABSTRACT
Studies were undertaken to define the expression of cytokeratins in normal, hyperplastic and malignant epithelial cells from human prostate. Cytokeratin (CK) polypeptides, separated by twodimensional electrophoresis, were identified by immunoblotting with CK-specific monoclonal antibodies. CK polypeptides 5, 7,8,15,18 and 19 were identified in fresh normal and hyperplastic prostate. Expression of CK 15 has not been previously reported in human prostate. Analysis of central and peripheral zone tissues from human prostate did not reveal qualitative differences in CK expression between these areas. Epithelial cells harvested from fresh BPH tissue by percoll gradient centrifugation and propagated in vitro using selective culture techniques showed alterations in CK expression compared to intact human prostate. Specifically, CKs 6,14,16 and 17 were noted in cultured BPH epithelial cells but not fresh normal prostate or BPH tissue. Immunoblot analysis of the established prostate cancer cell lines PC3, DU145 and LNCAP showed expression of CKs 8 and 18 but not CKs 5, 7 and 15 which were observed in benign prostate. These studies further characterize CK expression in benign and malignant human prostate and provide insights which may be useful in differentiating normal, hyperplastic and malignant epithelial cells in the human prostate gland. (J. Ural., 143: 167-171, 1990) The prostate gland is the major accessory organ of the male reproductive tract and the most common site of benign and malignant neoplastic disease in men. Benign prostatic hyperplasia (BPH) arises from fibroadenomatous growth of the prostate resulting in expansion of well differentiated acinar structures and stromal elements. 1 Carcinoma of the prostate has recently been identified as the most frequently encountered malignant neoplasm in men. 3 The morphology of lesions in prostatic carcinoma range from well differentiated to highly anaplastic tumors. 3 - 4 Although the incidence of prostatic neoplasia is high, little is known about the cell biology of normal and transformed prostatic epithelium. The cytokeratins are a class of cytoskeletal intermediate filaments found in epithelial and mesothelial cells. 5 - 6 Each epithelial type has a distinct profile of cytokeratin polypeptides which can be utilized to distinguish cells of simple and complex epithelia. 5 In addition, the pattern of cytokeratin expression within a specific tissue can serve as a marker of epithelial differentiation. 5 For example, the luminal and basal populations of prostatic epithelium can be distinguished based on the expression of specific cytokeratins. 7 Furthermore, alterations in cytokeratin polypeptide expression have been observed in benign and malignant neoplasia when compared to normal epithelium in several tissue types. 5 In the present study, the cytokeratin profiles of normal, hyperplastic and malignant epithelial cells from human prostate were assessed by immunoblotting of cytokeratin polypeptides separated by two-dimensional electrophoresis. These studies were undertaken to fully characterize the cytokeratin content of prostatic epithelial cells and determine the utility of Accepted for publication August 7, 1989. * Requests for reprints: Dept. of Urology, Northwestern University School of Medicine, 303 E. Chicago Ave., Chicago IL 60611. Supported by the Edwin and Lucy Kretschmer Fund of the Northwestern University Medical School, NIH Grants DK-08204 and DK39250, and the William 0. Jeffery, III fellowship.
specific cytokeratin antibodies in differentiating normal, hyperplastic and malignant epithelium in human prostate. MATERIALS AND METHODS
Cell lines and tissues. The established prostate cancer cell lines PC3 and DU145 were obtained from the tumor depository at the National Cancer Institute (Frederick, MD). LNCAP was purchased from the American Type Culture Collection (Rockville, MD). Epithelial cells were harvested from fresh prostate specimens by percoll gradient centrifugation and selective culture techniques as previously outlined. 8- 9 PC3, DU145 and LNCAP were cultured in RPMI-1640 with 10% fetal bovine serum and penicillin(lOO U/ml.)/streptomycin(lOO µ,g./ml.). Fresh prostatic epithelial cells were cultured in WAJC 404 medium containing ITS (insulin at five µ,g./ml, transferrin at five µ,g./ml., selenous acid at five ng./ml., Collaborative Research, Bedford, MA), prolactin (three ng./ml., Sigma Chemical, St. Louis, MO), epidermal growth factor (10 ng./ml., Sigma), bovine pituitary extract (30 µ,g./ml., Collaborative Research), polyvinyl pyrrolidone (two mg./ml., Sigma), cholera toxin (10 ng./ml.) and penicillin/streptomycin. Normal prostatic tissue was obtained from organ donors of less than 30 years of age. H&E stained slides were prepared from portions of formalin-fixed normal tissue to ascertain the absence of benign or malignant neoplasia. Hyperplastic human prostate was obtained from men undergoing suprapubic or retropubic prostatectomy for benign prostatic hyperplasia (BPH). Central and peripheral zone tissues were dissected as previously outlined10 from tissues obtained by radical prostatectomy. Sections of benign prostate were obtained from the respective zones and the absence of carcinoma was confirmed by histological analysis. Fresh PC3 tumors were obtained by injecting PC3 cells (1 X 106/mouse) subcutaneously into nude mice. Tumors were allowed to grow for four weeks and harvested from the subcutaneous sites with special care being taken to remove any skin or attached connective tissue. All fresh
167
168
SHERWOOD AND ASSOCIATES
tissues were flash frozen in liquid nitrogen at the time of procurement and stored at -70C until utilized. Sample preparation. PC3, DU145 and LNCAP cells were harvested from ongoing cultures by quick trysinization. The cells were washed (3X) with Hanks' Balanced Salt Solution (HBSS) and dissolved in urea mix (9M urea, LKB ampholytes pH 9 to 11, 2% mercaptoethanol, 4% NP-40). Fresh prostate epithelial cells were detached from culture vessels using type I collagenase (675 U/ml.) in HBSS. The cells were washed (3x) with HBSS and dissolved in urea mix. Fresh prostate epithelial cells were also harvested from the second and third bands of discontinuous percoll gradients as previously described. 8 - 9 The cells were washed (3X) with HBSS and dissolved in urea mix. One million cells were loaded onto each 2D gel. Fresh tissues were homogenized by grinding tissue in a glass tissue homogenizer containing phosphate buffered saline (three µ,l./mg. of tissue). Tissue homogenates was mixed with urea mix at a 1:3 ratio and periodically vortexed for two hours. Debris was pelleted by centrifugation (2000 g for one minute) and the supernatant was harvested for analysis by two-dimensional electrophoresis. Seventy µg. of protein was loaded onto each 2D gel. Two-dimensional electrophoresis. The ISO-DALT system for 2D electrophoresis was performed according to the method of Anderson and Anderson. 11- 12 Briefly, the first dimension (the ISO system), which separates proteins according to their isoelectric points, was carried out for 14,000 volt hours in 5% polyacrylamide containing eight M urea and 2% total ampholytes with a pH range of three to 10. At the end of isoelectric focussing, each gel was extracted from the elongated glass tube (17.8 x 0.15 cm.) and equilibrated. The tube gels were layered over slab gels consisting of a linear polyacrylamide gradient (nine to 18%) containing 1 % SDS. Electrophoresis was carried out at a constant voltage of 100 to 150 volts for 16 hours. Protein in gels was detected by the silver stain method of Guevera and colleagues. 13 Immunoprotein blotting. Cytokeratin specific monoclonal antibodies 4.62, 8.12 and 8.13 14- 16 were purchased from Sigma Chemical Co. The region of specific staining for each antibody is demonstrated in a silver stained gel of cultured prostate epithelial cells (figure 1). Anti-CK 4.62 has previously demonstrated reactivity with CK polypeptide 19 in cultured human breast and colon carcinoma cells as well as bovine tissues. 17 CKs 13,15 and 16 are immunoreactive with anti-CK 8.12. 15 Anti-CK 8.13 is immunoreactive with CK polypeptides 1,5,6, 7,8 and 18 in human cervical tissues. 16 Cytokeratins on 2D gels were detected by the immunoblotting procedure of Towbin and colleagues 18 using the cytokeratin specific monoclonal antibodies. Briefly, proteins resolved by 2D electrophoresis were transferred to nitrocellulose paper (BioRad, 0.45 µ,m.) at 120 volts for two hours using a transfer buffer consisting of 25 mM Tris, 192 mM glycine and 20% methanol (pH 8.3). Blots were washed with distilled water, incubated (25C) with blocking buffer (Carnation non-fat dry milk) for two hours and with primary anticytokeratins at a 1:500 dilution overnight (16 to 18 hrs). The blots were washed with blocking solution and incubated (25C) with horseradish peroxidase-conjugated rabbit anti-mouse IgG for two hours (1:1000). The color reaction was induced using 4chloro-1-naphthol substrate.
FIG. 1. Silver stained 2D gel of cultured epithelial cells from hyperplastic human prostate. Areas of immunoblot reactivity with monoclonal anti-cytokeratins 4.62, 8.12 and 8.13 are designated. Molecular weight and pH markers are indicated. IEF = isoelectric focussing, first dimension; SDS-PAGE = polyacrylamide gel electrophoresis, second dimension.
RESULTS
Immunoreactivity of benign and malignant prostate epithelial cells with anti-cytokeratin 4.62. The cytokeratin (CK) content of cultured benign and malignant prostate epithelial cells as well as fresh prostatic tissues was assessed by immunoblot analysis of prostatic proteins separated by 2D electrophoresis. Epithelial cells from fresh normal prostate, representing a mixture of the three anatomical zones, exhibited expression of CKs 15 and 19, according to the convention of Moll and colleagues,5 following immunoblot analysis using anti-CK 4.62 (figure 2A). Similarly, CKs 15 and 19 were noted following
FIG. 2. Immunoblot analysis of prostate epithelial cell proteins separated by 2D electrophoresis using monoclonal anti-CK 4.62. A) normal prostate, B) whole BPH tissue, C) central zone tissue, D) peripheral zone tissue, E) BPH epithelial cells from discontinuous percoll gradient, F) cultured BPH epithelial cells, G) cultured PC3 cells, H) solid PC3 tumor from athymic nude mice.
169
CYTOKERATINS IN BENIGN AND MALIGNANT PROSTATE
immunoblot analysis of fresh BPH tissue as well as tissue from the central or peripheral zones of radical prostatectomy specimens (figures 2B-2D). BPH epithelial cells harvested directly from percoll gradients exhibited expression of CKs 15 and 19 while CKs 13,14,15,16,17 and 19 were noted in BPH epithelial cells obtained from primary cultures (figure 2E and 2F). The prostate cancer cell line PC3 showed expression of CK19 both in vitro and in vivo (figure 2G and 2H). The prostate cancer cell lines DU145 and LNCAP did not exhibit immunoreactivity with anti-CK 4.62 (data not shown). Immunoblot analysis of cytokeratin expression in prostatic cell lines and tissues using anti-cytokeratin 8.12. Analysis of cytokeratin expression in normal prostate tissue and fresh BPH as well as central and peripheral zone tissues using anti-CK 8.12 showed immunoreactivity with CK 15 (figure 3A-3D). In addition, a protein with the same electrophoretic mobility as CK 13 was noted in some central and peripheral zone tissues (figure 3C-3D) as well as whole BPH tissue (data not shown). The presence of CK13 in fresh prostate tissues was variable and inconsistent. BPH epithelial cells harvested from percoll gradients showed expression of CKs 13 and 15 while CKs 13,14,15,16,17 and 19 were noted in BPH epithelial cells obtained from primary cultures (figure 3E-3F). The prostate cancer cell lines PC3, DU145 and LNCAP did not exhibit immunoreactivity with anti-CK 8.12 (data not shown). Immunoreactivity of benign and malignant prostate epithelial cells with monoclonal anti-cytokeratin 8.13. Immunoblotting with anti-CK 8.13 showed the presence of cytokeratin polypeptides 5,7,8 and 18 in fresh normal prostate as well as whole BPH tissue and tissue dissected from the central and peripheral zones of the prostate (figure 4A-4D). Likewise, CKs 5,7,8 and 18 were noted in BPH epithelial cells obtained from discontinuous percoll gradients (figure 4E). Cultured BPH epithelial cells showed expression of cytokeratin polypeptides 5,6, 7,8 and 18 while exhibiting a diffuse immunostaining pattern that was not observed in freshly derived benign prostatic tissue and cells (figure 4F). The diffusely immunoreactive proteins were actively synthesized by cultured epithelial cells since the same proteins were observed on fluorographs of 35 S-methionine labelled proteins (data not shown). The prostate cancer cell line PC3 showed immunoreactive cytokeratins 8 and 18 following in vitro propagation but only CK 18 was noted in fresh PC3
FIG. 4. Immunoreactivity of prostate epithelial cell proteins with anti-CK 8.13. A) normal prostate, B) whole BPH tissue, C) central zone tissue, D) peripheral zone tissue, E) BPH epithelial cells from discontinuous percoll gradient, F) cultured BPH epithelial cells, G) cultured PC3 cells, H) PC# solid tumor from athymic nude mice, I) cultured DU145 cells, J) cultured LNCAP cells.
TABLE 1.
Cytokeratin expression in epithelial cells from normal, hyperplastic and malignant prostate Cytokeratin Expression
Specimen Molecular wt. (kD) Isoelectric point (pH) Normal prostate WholeBPH Central zone Peripheral
5
6
7
8
58
56
54
52
7.4
7.8
6.0
6.1
13 54 5.1
14 50 5.3
15 50 4.9
16 48 5.1
17 46 5.1
18 45 5.7
19 40 5.2
++
++ ++
++
++ ++
++ ++ ++
++ ++ +/++ ++ +/++ ++ +/-
++ ++ ++
++ ++ ++ ++ ++ ++
++
++ ++
++
++ ++
++ ++ ++ ++ ++
++ ++
zone
BPH epithelia Percoll BPH epithelia cultured PC3 in situ PC3 cultured DU145cultured LNCAPcultured
++ ++ ++ ++
++
++ ++
++ ++ ++ ++ ++
++
++
++, major expression, +/-, minor or variable expression.
FIG. 3. lmmunoblot analysis of prostate epithelial cells with antiCK 8.12. A) normal prostate, B) whole BPH tissue, C) central zone tissue, D) peripheral zone tissue, E) BPH epithelial cells from discontinuous percoll gradient, F) cultured BPH epithelial cells.
tumors harvested from nude mice (figures 4G-4H). Immunostaining of CKs 8 and 18 was noted in the cultured prostate cancer cell lines DU145 and LNCAP (figure 4G-4J). A composite presentation of cytokeratin expression in benign and malignant prostate epithelial cells and tissues as determined by immunoblot analysis with anti-cytokeratin monoclonals 4.62, 8.12 and 8.13 is provided as Table 1. The molecular weight and isoelectric points of the respective cytokeratin polypeptides are also indicated (table 1).
170
SHERWOOD AND ASSOCIATES DISCUSSION
The current studies were undertaken to further define the cytokeratin composition of benign and malignant epithelial cells from human prostate. Our results indicate that: 1) cytokeratin polypeptide 15, which had not been previously identified in human prostate, is expressed in normal prostate and BPH; 2) comparison of central and peripheral zone tissues from human prostate did not reveal qualitative differences in CK expression; 3) cultured BPH epithelial cells exhibit altered cytokeratin expression compared to BPH epithelia in situ and 4) the cytokeratin content of cultured and in vivo propagated prostate cancer cells is different from fresh normal prostate andBPH. Cytokeratin polypeptides 5,7,8,15,18 and 19 were identified in normal and hyperplastic human prostate by western immunoblot analysis. The expression of CKs 5,7,8,18 and 19 in human prostate has been reported7• 19 but CK 15 has not been previously observed in normal or hyperplastic prostate. Huszar and colleagues 15 reported negative immunoreactivity following immunohistochemical analysis of a single prostate specimen using anti-CK 8.12 which stains for CK 15. However, CK 15 was repeatedly demonstrated in benign prostatic tissue by immunoblot analysis with anti-CKs 4.62 and 8.12 in the present study. In addition, CKs 5 and 15 are considered to be a closely associated pair of cytokeratin polypeptides. 20 The presence of CK 5 in benign human prostate has been demonstrated by several investigators. Therefore, the presence of CK 15 in human prostate is not an unexpected finding. These observations indicate that cytokeratins characteristic of both simple (CKs 7 8 18 19) and stratified (CKs 5,15) epithelium are present in nor~ltl a~d hyperplastic human prostate. 5 The similarity in cytokeratin expression between normal prostate and BPH is not suprising because the expanded acinar structures in BPH maintain well differentiated morphology with intact basal and luminal epithelia characteristic of normal prosta,te. Differences in the cytokeratin pattern of central and peripheral zones from human prostate were not observed. Morphologic studies indicate that BPH arises primarily in the transition zone or periurethral region of the prostate. 21 The present study indicates that cytokeratin expression may not serve as a useful marker for distinguishing central and peripheral zone tissues in the human prostate. Recent advances in tissue culture techniques have allowed for the successful isolation and cultivation of epithelial cells from fresh human prostate.a-9 , 22- 23 These procedures have facilitated the study of prostate cell biology. The cellular characteristics of cultured prostate epithelial cells was partially defined in the present study by comparing the CK profile of fresh BPH tissue with epithelial cells isolated from the human prostate by percoll gradient centrifugation and selective culture techniques. Epithelial cells obtained directly from percoll gradients prior to introduction into tissue culture did not exhibit a demonstrable difference in cytokeratin expression compared to whole prostate. However, prostate epithelial cells harvested from primary cultures grown in plastic tissue culture flasks showed alterations in cytokeratin expression. Specifically, CKs 6,14,16 and 17 were observed in cultured epithelial cells but not in fresh prostatic tissue. In addition, cultured epithelial cells exhibited a widely variant cytokeratin profile compared to fresh tissue following analysis with specific cytokeratin antibodies. For example, proteins with the same 2D electrophoretic mobility as CKs 13,14,15,16,17 and 19 were noted in cultured epithelial cells following immunoblotting with anti-CKs 4.62 and 8.12. However, anti-CK 4.62 reacted with CKs 15 and 19 in fresh prostate while CK 15 was noted in fresh prostate following immunoblotting with anti-CK 8.12. Our previous studies also show that cultured prostate epithelial cells express the intermediate filament polypeptide vimentin. 9 Vimentin is generally present in mesenchymal cells but not epithelial cells in situ.
These findings indicate that alterations in cytokeratin and vimentin gene expression occur following in vitro propagation of freshly isolated prostate epithelial cells on plastic resulting in an atypical cytokeratin and vimentin profile. Altered intermediate filament expression in cultured cells has been previously reported for a variety of tissues. 5 Immunoblot analysis of CK expression in the established prostate cancer cell lines PC3, LNCAP and DU145 revealed the presence of CKs 8 and 18. Additionally, PC3 cells maintained in vitro and as subcutaneous solid tumors in nude mice exhibited expression of CK19. Compared to benign prostatic tissues, the established tumor lines lacked expression of CKs 5, 7 and 15. Previous reports indicate that CK5 is selectively expressed in the basal cell population of the human prostate. 24 Because CK5 is not expressed in prostatic carcinoma, several investigators have postulated that transformed prostate epithelium arises from the luminal epithelium of the prostate. 24- 25 The present study supports this hypothesis by showing the presence of CK5 in benign prostatic tissue but not in the established carcinoma lines. The expression of CK 15 was also noted in benign prostate but not in the prostate cancer lines following immunoblot analysis with anti-CK 8.12. As previously indicated, CKs 5 and 15 are a closely associated pair of cytokeratin polypeptides. The loss of both CK 5 and 15 expression in cultured prostatic carcinoma cells further supports the interrelationship of the two proteins. These findings also indicate that differential expression of CK 15 in benign and malignant prostate may provide an additional marker for the identification of prostatic carcinoma. Immunohistochemical studies on fresh benign and malignant prostate are currently underway in our laboratory to determine the utility of anti-CK 8.12 in differentiating benign prostatic epithelium from well differentiated and poorly differentiated carcinoma of the prostate. In summary, we have utilized cytokeratin specific monoclonal antibodies to further define cytokeratin expression in epithelial cells from benign and malignant prostate. Using anti-CK 8.12, CK polypeptide 15 was identified in benign human prostate which was previously unreported. In addition, alterations in CK expression were noted in cultured benign prostate epithelial cells and established carcinoma cell lines when compared to fresh benign prostatic tissue. Based on these observations, CK specific monoclonals may provide useful tools for the study of benign and malignant prostate epithelium. REFERENCES 1. Grayhack, J. T. and Kozlowski, J.M.: Benign prostatic hyperplasia.
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