(CANCER RESEARCH 52, 1457-1462, March 15, 1992] Elevated Expression of pp60c~srcin Low Grade Human Bladder Carcinomas1 Paul Fanning, Kathleen Bulovas, Kulvinder S. Saini, John A. Libertino, Adrian D. Joyce, and Ian C. Summerhayes Laboratory of Cancer Biology, Department of Surgery, Neve England Deaconess Hospital, Boston, Massachusetts 02115 [P. F., K. B., K. S. S., /. C. SJ; Division of Surgery, Lahey Clinic Medical Center, Burlington, Massachusetts 01803 [J. A. L.J; and Department of Urology, Kings College Hospital, London SE59RS, United Kingdom [A. D. JJ ABSTRACT The results presented in this report demonstrate that the tyrosine- specific protein kinase activity of pp60' M'is elevated over that recorded in normal bladder mucosa in a subset of human bladder carcinomas. Increased kinase activity was observed mainly in low grade bladder lesions and was associated, at least in part, with elevated levels of pp60c""cexpression. Extension of this analysis to a panel of human blad der carcinoma cell lines confirmed previous observations of low pp60c""c kinase activity in three lines established from high grade (GUI) bladder tumors and revealed increased kinase activity in three alternative bladder lines derived from Gìor GII tumors. Use of an anti-phosphotyrosine antibody in Western blot analysis revealed the presence of novel phos- photyrosyl cellular substrates in human bladder cell lines and tumors displaying elevated pp6()' "' kinase activity. These observations suggest an association for the ire protooncogene in urothelial cell differentiation events. INTRODUCTION The molecular events underlying neoplastic progression in the bladder represent multiple genetic alterations contributing to the malignant transformation of normal bladder mucosa. Such events implicate both the activation (1-3) and inactivation (4, 5) of oncogenes and antioncogenes, respectively, suggesting a role for both in the regulation of cell growth. In this respect, the protein products encoded by cellular protooncogenes are believed to play a role in normal growth and differentiation pathways. Consequently, the events underlying differentiation and neoplastic progression are closely intertwined and it is often difficult to separate, or relate, one from or to another. This is exemplified by studies demonstrating the differential action of a single oncogene in the induction or blockage of differentiation events in alternative systems (6-11). The protooncogene c-src has been implicated in both differ entiation and neoplastic pathways in human tumors, where increased protein kinase activity was found associated with the differentiation status in tumors of neuroectodermal origin (12, 13) and in neoplastic progression in colon carcinomas (14, 15). The involvement of the src protooncogene in differentiation events of the nervous system emanates from the observation of elevated expression of pp60°srein the developing vertebrate nervous system (16-18). Differentiated neurons from embry onic rat brain express high levels of two forms of pp60c srcwhich display elevated specific activity (19). In addition, primary neurons induced to differentiate in culture, display increased expression of pp60c'src with elevated kinase activity (20). A correlation of the level of pp60c srcexpression with differentia tion events in this tissue is further strengthened by the obser vation of similar biochemical events in human tumors of neu ronal and neuroendocrine origin (12, 13, 22). In contrast, human tumor cells of neuroectodermal origin, which do not express neural characteristics, were found to have moderate to Received 9/16/91; accepted 1/8/92. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by NIH Grants CA42944 and CA44704. low levels of pp60csrc kinase activity (13). More recently, acti vation of pp60c~srcprotein kinase has been reported in preneo- plastic colonie adenomatous lesions (15) and in 70% of colon carcinomas (14, 21) suggestive of a role for src activation in neoplastic progression in this organ. A more extensive analysis of human tumor cell lines, derived from tissues other than those of neuroectodermal origin, has revealed low src kinase activity in most cases, with the exception of rhabdomyosarcomas, os- teogenic sarcoma, Ewing's sarcoma, and colon carcinomas (13). This screening study included three human bladder carcinoma cell lines (13) and one normal/tumor bladder tissue pairing (12), all of which displayed pp60csrc kinase activity comparable to that observed in normal bladder epithelium. In this study we report that the tyrosine-specific protein kinase activity of pp60c srcis elevated over that recorded in normal bladder mu cosa in a subset of human bladder carcinomas. From the use of a panel of bladder cell lines we demonstrate increased ppoO0"51* expression and kinase activity associated with the differentia tion status of cells with identification of novel phosphotyrosyl proteins in this system. MATERIALS AND METHODS Preparation of Tumor Material. Human bladder tumors were snap frozen in liquid nitrogen and stored at -70°C prior to use. Tissue segments were embedded in OCT compound and a frozen tissue section was stained to assess the tumor content of material. Blocks were trimmed to remove stremai elements and only tissue with >80% tumor content was included in this study. On the average 50 frozen sections were required for each assay and in all cases sections 1, SI, 101, and 1S1 were stained to continuously monitor the tumor profile of the material. Preparation of tumor in this manner was considered critical for histológica! evaluation of tissue and subsequent interpretation of results. Immunoprecipitation of ppoOâ„¢". Subconfluent dishes of cell lines or frozen tissue sections from tumors were lysed in PBSTDS2 buffer. Lysis was carried out on ice for 20 min followed by clarification in a microfuge at 4°C. Supernatants were removed and a \Q-ui aliquot was taken for protein estimation using the bovine serum albumin protein assay system (Pierce Chemical Co., Rockford, IL). For immunoprecipitation, protein concentrations were standardized between samples, using 200 up, pro tein from each preparation. Lysates were incubated overnight at 4"( ' with Mab 327 (Oncogene Science, Manhasset, NY), followed by the addition of protein A-Sepharose beads and a further 90-min incubation. Immune complexes were washed three times in PBSTDS and three times in 0.1 x phosphate-buffered saline. pp60c"*"Western Blot Analysis. Precipitations for Western blot analy sis were run in 7.5% polyacrylamide gels followed by overnight transfer to nitrocellulose using standard procedures. Nitrocellulose blots were blocked in TBS buffer containing 3% gelatin followed by incubation with Mab 327 for 90 min at room temperature. Blots were washed in three changes of TBS-Tween 20 (0.5% v/v) and incubated with phos- phatase-labeled goat anti-mouse (KJrkegaard and Perry Labs, Gaithers- burg, MD) followed by extensive washing in TBST. Blots were devel- 2The abbreviations used are: PBSTDS, phosphate-buffered saline, pH 7.4-1 % Triton X-100-0.5% sodium deoxycholate-0.1 % sodium dodecyl sulfate-2 HIM phenyl-methylsulfonyl fluoride-100 units/ml aprotinin; Mab, monoclonal anti body; TBS, triethanolamine-buffered saline (10 mM Tris-150 HIMNaCI, pH 7.5); TBST, TBS-Tween 20. 1457 Research. on October 4, 2017. © 1992 American Association for Cancer cancerres.aacrjournals.org Downloaded from