(Fujita et al., 1985;Visvanathan et al., 1988;Czerniak et al., 1990;Knowles and Williamson, 1993;Levesque et al., 1993), p53 (Sidransky et al., 1991; Fujimoto et al., 1992), retinoblastoma (Rb;Horowitz et al., 1990;Cairns et al., 1991; CordonCardo et al., 1992;Logothetis et al., 1992), c-erbB-2 (Wright et al., 1990(Wright et al., , 1991Coombs et al., 1991; Moriyama et al., 1991;Wood et al., 1991;Sauter et al., 1993), epidermal growth factor receptor (EGFR;Neal et al., 1985Neal et al., , 1990, c-src (Fanning et al., 1992), MDM2 (Habuchi et al., 1994;Lianes et al., 1994) and MTS1 (Cairns et al., 1994; Kamb et al., 1994;Spruck et al., 1994).It has become clear that a series of diverse genetic changes are involved in generating the different phenotypes observed in bladder cancer. However, how these different genetic elements interact with other cellular proteins and complement each other in neoplastic progression is little understood. To address these issues, investigators have developed both in vitro and in vivo models of progression, including the use of human carcinoma derived cell lines. Such cell lines have been used as a resource to identify the involvement of change at a particular locus in specific tissues (Der et al., 1982;Parada et al., 1982;Horowitz et al., 1990;Fanning et al., 1992) and in studies directed at identifying the function of a gene following restoration of expression (Takahashi et al., 1991;Goodrich et al., 1992). It is argued that changes found in cell lines are not always a reflection of events in vivo; however, molecular events associated with H-ras, Rb and c-src (Der et al., 1982;Horowitz et al., 1990; Fanning et al., 1992, respec-
Materials and methdsCell lines 5637, CUBIII, EJ, HT1376, HU456, J82, KK47, PSI, RT4, RT112, TCCSUP, UM-UC-3, HT1197, SCaBER (ATCC) and BC16 (kindly provided by Dr C Remikoff, University of Wisconsin) cell lines were maintained in Dulbecco's modified medium supplemented with 7.5% fetal bovine serum and penicillin/streptomycin. Immunoprecipitation MDM2 Subconfluent cells were washed in phosphatebuffered saline (PBS), lysed in ice-cold PBSTDS lysis buffer (PBS pH 7.4, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate, 100 U ml-aprotinin) for 20 min and clarified by centrifuging at 14 000 g for 20 min at 4'C. Protein for the immunoprecipitations was standardised using the BCA method (Pierce, Rockford, IL, USA) and incubated overnight at 4°C with 15 jil of anti-MDM2 (Ab-1; Oncogene Science, Manhasset, NY, USA) followed by the addition of protein A-Sepharose beads for an additional 90 min incubation at 4°C. Immunocomplexes were washed three times in PBSTDS and once in 0.1% PBS. Samples were run on a 7.5% polyacrylamide gel and proteins were transferred to nitrocellulose. Blots were blocked overnight at 4°C in 10% non-fat dried milk in triethanolamine-buffered saline (TBS) with 0.05% followed by incubation with MDM2 (Ab-1; Oncogene Science) for 2 h at room temperature. The blots were washed three times with TBST followed by incubation with...
