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. © 2001 Wiley-Liss, Inc.