Long-term genome stability and minimal genotypic and phenotypic alterations in HPV16 E7-, but not E6-, immortalized human uroepithelial cells.

Abstract

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.

20q gain associates with immortalization: 20q13.2 amplification correlates with genome instability in human papillomavirus 16 E7 transformed human uroepithelial cells

“…We observed that two independent E7-transformed HUCs had a clonal marker containing extra 20q material. We also observed that the clonal marker chromosome containing the extra chromosome 20 material remained stable in culture, as did the whole karyotype (Rezniko et al, 1994). Immortalization of E6-transformed HUCs was also associated with chromosome alterations.…”

“…In four E7-HUCs, dE7-1, eE7-1, gE7-1, and yE7-1, cytogenetic analysis indicated that the clonal marker contained 20q material (Table 1). The clonal marker in the two lines previously analysed (i.e., aE7-1 and aE7-2) also contained 20q material by FISH analysis (Rezniko et al, 1994). One of the new lines, yE7-1, contained four clones by cytogenetic analysis, and each of the four clones had an extra copy of chromosome 20 or 20q.…”

“…As noted above, gain of 20q has been observed in clinical bladder cancers. We previously reported that immortalization of HUC after transformation with Human Papillomavirus 16 (HPV16) E7 or E6 rarely occurs (51610 75 ) (Belair et al, 1996) and, when it does occur, is always associated with chromosome changes (Rezniko et al, 1994). We observed that two independent E7-transformed HUCs had a clonal marker containing extra 20q material.…”

“…Normal HUC transformed with HPV16 E7 reproducibly show an extended lifespan, but rarely form immortal cell lines (Rezniko et al, 1994;Belair et al, 1996). When such cells do immortalize, this usually occurs after an extended crisis period of undetectable cell proliferation.…”

A putative serine/threonine kinase encoding gene BTAK on chromosome 20q13 is amplified and overexpressed in human breast cancer cell lines

“…Ampli®cation of chromosome 20q DNA has been detected in colon and bladder cancer cells by CGH technique . 20q ampli®cation has also been reported to accompany human papilloma virus 16/E7 mediated transformation of human uroepithelial cells which undergo immortalization and polyploidization at early passages (Rezniko et al, 1994). In addition to these molecular cytogenetic data, elevated copy number of chromosome 20 has been implicated in several cancer cell types based on their karyotypic analyses.…”