Resistance of primary cultured mouse hepatic tumor cells to cellular senescence despite expression of p16(Ink4a), p19(Arf), p53, and p21(Waf1/Cip1).

Primary cultured mouse hepatic cells become senescent within a short period, although rare cells form colonies from which continuously proliferating cell lines can be established. In contrast, hepatic tumor (HT) cells show little senescence and higher colony-forming capacity. To assess this difference, we investigated p16(Ink4a)/p19(Arf)/p53/p21(Waf1/Cip1) expression in primary normal and HT cells, together with cell lines established from both. In primary normal cells, p16(Ink4a)/p19(Arf) were expressed only in association with senescence and disappeared at later stages of colony formation. In contrast, primary HT cells showed sustained p16(Ink4a)/p19(Arf) expression from the beginning. No p16(Ink4a)/p19(Arf) alterations, such as deletion, mutations, or hypermethylation, were detected in the primary HT cells, although most cell lines derived from either normal or HT cell colonies lost p16(Ink4a) or p19(Arf) expression owing to hypermethylation or homozygous deletion of p16(Ink4a)/p19(Arf). On the other hand, primary normal and HT cells and most cell lines showed constitutively elevated expression of p53/p21(Waf1/Cip1), with a further increment after ultraviolet ir-radiation, indicating a functionally normal p53 pathway. These results indicate that primary HT cells are resistant to senescence despite retaining p16(Ink4a)/p19(Arf)/p53/p21(Waf1/Cip1) expression and that loss of p16(Ink4a)/p19(Arf) function is associated only with establishment of the cell lines.