Paiameteis of genome instability and morphological alterations associated with cell transformation were studied in an isogeneic set of clonal human uroepithelial cell (HUG) lines immortalized by the human papilloma virus 16 (HPV16) E6 and/or E7 gene(s). HPV16 E6 binds p53, leading to rapid degradation of p53, whereas E7 binds and alters pRb and other proteins. We report that two independent E7-immortalized HUG lines showed minimal phenotypic or genotypic alterations, except that both lines contained amplification of 20q DNA sequences and a greater polyploidization at an early passage. The E7-immortalized HUG line resembled normal HUG lines, except that they failed to senesce. In contrast, the E6-immortalized HUG lines were morphologically altered, contained numerous random chromosome aberrations, and showed unstable evolving karyotypes with passage in culture. No amplified DNA sequences were detected in E6-immortalized HUG lines. Instead, clonal losses of chromosome regions (i.e., -3p, -6q, -9p], putatively containing tumor suppressor or senescence genes, accompanied the E6-HUG immortalization event. E6-immortalized HUG lines showed transformed phenotypes similar to E6/E7-HUG lines. The difference in genome stability between E6-and £7-immortalized HUG was highly significant statistically (p-value <10~^). Thus, the HPV16 E7 gene led to HUG immortalization by a pathway that blocked cellular senescence, but did not disrupt genome stability. These results implicate p53 loss, but not pRb alteration, in genome destabilization.

Breast, bladder, colon, and ovarian carcinomas show frequent low level 20q gain and less frequently high level 20q13.2 ampli®cation, but the signi®cance of these 20q ampli®cations in transformation has not been de®ned. Using karyotypic and comparative genomic hybridization (CGH) analyses, chromosome losses and gains were analysed in six newly immortalized human uroepithelial cell (HUC) lines transformed by Human Papillomavirus 16 (HPV16) E7. Results showed clonal chromosomes with 20q11-4qter gain in all six lines. CGH revealed a peak of 20q13.2 ampli®cation in two cell lines. FISH with whole chromosome 20 paint showed expanded chromosome regions (ECRs) and double minute chromosomes (DMs) that contained chromosome 20 material in cell lines with 20q13.2 ampli®cation. FISH with probes from the center of the 20q13.2 human breast cancer amplicon showed as many as 24 signals in cells with 20q13.2 ampli®cation. The acquisition of genome instability in these E7-HUCs did not correlate with TP53 mutation, as all E7-HUCs contained only wildtype TP53. These results suggest that low level 20q gain is associated with overcoming cellular senescence in E7 transformed cells (P-value=2610 77 ), but does not confer genome instability, while high level 20q13.2 ampli®cation is associated with chromosome instability. Loss of 10p (P-value=3610 75 ) was also important in immortalization of E7-transformed HUCs. Thus, these results have profound implications for interpreting the signi®cance of high versus low level 20q gains in human cancers.

Telomerase activity is readily detected in most cancer biopsies, but not in premalignant lesions or in normal tissue samples with a few exceptions that include germ cells and hemopoietic stem cells. Telomerase activity may, therefore, be a useful biomarker for diagnosis of malignancies and a target for inactivation in chemotherapy or gene therapy. These observations have led to the hypothesis that activation of telomerase may be an important step in tumorigenesis. To test this hypothesis, we studied telomerase activity in isogeneic samples of uncultured and cultured specimens of normal human uroepithelial cells (HUCs) and in uncultured and cultured biopsies of superficial and myoinvasive transitional cell carcinoma (TCC) of the bladder. Our results demonstrated that four of four TCC biopsies, representing both superficial and myoinvasive TCCs, were positive for telomerase activity, but all samples of uncultured HUC were telomerase negative. However, when the same normal HUC samples were established as proliferating cultures in vitro, telomerase activity was readily detected but usually at lower levels than in TCCs. Consistent with the above observation of the telomerase activity in HUCs, telomeres did not shorten during the HUC in vitro lifespan. Demonstration of telomerase in proliferating human epithelial cells in vitro was not restricted to HUCs, because it was also present in prostate and mammary cell cultures. Notably, telomerase activity was relatively low or undetectable in nonproliferating HUC cultures. These data do not support a model in which telomerase is inactive in normal cells and activated during tumorigenic transformation. Rather, these data support a model in which the detection of telomerase in TCC biopsies, but not uncultured HUC samples, ref lects differences in proliferation between tumor and normal cells in vivo.

Telomerase activity is readily detected in most cancer biopsies, but not in premalignant lesions or in normal tissue samples with a few exceptions that include germ cells and hemopoietic stem cells. Telomerase activity may, therefore, be a useful biomarker for diagnosis of malignancies and a target for inactivation in chemotherapy or gene therapy. These observations have led to the hypothesis that activation of telomerase may be an important step in tumorigenesis. To test this hypothesis, we studied telomerase activity in isogeneic samples of uncultured and cultured specimens of normal human uroepithelial cells (HUCs) and in uncultured and cultured biopsies of superficial and myoinvasive transitional cell carcinoma (TCC) of the bladder. Our results demonstrated that four of four TCC biopsies, representing both superficial and myoinvasive TCCs, were positive for telomerase activity, but all samples of uncultured HUC were telomerase negative. However, when the same normal HUC samples were established as proliferating cultures in vitro, telomerase activity was readily detected but usually at lower levels than in TCCs. Consistent with the above observation of the telomerase activity in HUCs, telomeres did not shorten during the HUC in vitro lifespan. Demonstration of telomerase in proliferating human epithelial cells in vitro was not restricted to HUCs, because it was also present in prostate and mammary cell cultures. Notably, telomerase activity was relatively low or undetectable in nonproliferating HUC cultures. These data do not support a model in which telomerase is inactive in normal cells and activated during tumorigenic transformation. Rather, these data support a model in which the detection of telomerase in TCC biopsies, but not uncultured HUC samples, ref lects differences in proliferation between tumor and normal cells in vivo.