The isolation of an immortalized and tumorigenic cell line from p21WAF1 null mouse bladders.

Given a role for the deregulation of p21WAF1 in the progression of bladder tumors, we examined the growth of cultured urothelial cells from wild-type and p21WAF1 null bladders. Bladders were excised, minced from euthanized p21WAF1 and wild-type mice, treated overnight with dispase, and then placed into flasks coated with collagen type I in Dulbecco modified Eagle medium with 10% fetal calf serum. After an overnight incubation, the media was replaced with a serum-free media and a portion of explants were treated with 12-O-tetrade-canoylphorbol-13-acetate (TPA) on day 7 and continued for either 4 or 9 wk. The urothelial origin of any surviving epithelial cells was determined by reverse transcription-polymerase chain reaction (RT-PCR) using uroplakin II-specific primers, and the expression of the cell cycle-related proteins, p16INK4 and p19ARF, was examined by semiquantitative RT-PCR and Western blotting. Isolated wild-type and serially passaged p21WAF1 null epithelial-like cells were then injected subcutaneously into nude mice. We found that phorbol ester treatment at two different concentrations significantly enhanced uroepithelial colony formation from isolated wild-type mouse bladder tissue. On the other hand, significantly fewer urothelial colonies were derived from p21WAF1 null bladder cells treated with phorbol ester. Although there was apparent senescence and cell death of epithelial foci and stromal cells in phorbol ester-treated and -untreated p21WAF1 null cultures, after 3 mo there was an apparent subpopulation of epitheloid cells that overgrew each flask. There was a significant decrease in the number of these serially passaged cells in the G1 phase of the cell cycle when compared with initial explant wild-type or p21WAF1 null cells. This subpopulation of epitheloid cells expressed the mouse uroplakin II gene, indicating a urothelial phenotype, but did not express either the p16INKa or p19ARF proteins, whereas p21WAF1 null bladders express both proteins. There was also a high level of expression of the p53 protein and a significant decrease in the expression of the p19ARF transcript in both p21WAF1 null bladder and p21WAF1 null cells. These p21WAF1 null cells could be easily passaged and when injected subcutaneously into nude mice, large tumors developed. Therefore, it appears that a subpopulation of urothelial cells from the p21WAF1 null bladder can develop a tumorigenic phenotype in vitro.