International Mammalian Genome Society

The 14th International Mouse Genome Conference (2000)

B20. Indistinct Replication Senescence of Primary Cultured Mouse Hepatocellular Carcinoma Cells Despite the Expression of Normal p16Ink4a, p19Arf and p53

Katsuhiro Ogawa, Emi Imamura, Masahiko Obata, Kan Kishibe and Junichi Goto.
Department of Pathology, Asahikawa Medical College, Asahikawa, Japan

The primary cells isolated from the normal mouse liver undergo extensive replicative senescence in vitro within a short period, although rare cells can pass through the crisis, forming the colonies that can be established as continuously growing cell lines. In contrast, the primary hepatocellular carcinoma (HCC) cells show less prominent senescence, less decline in cell number and higher colony forming capacity. To assess the basis of this difference, we investigated the status and expression of the genes involved to replicative senescence in the primary cells and cell lines derived from the normal liver and HCC. In the primary normal cells, although p16Ink4a and p19Arf were not expressed during early phase, they were up-regulated in association with senescence, but disappeared in the later stage of colony formation. In contrast, the primary HCC cells expressed both p16Ink4a/p19Arf from the beginning, sustaining the expression during cultivation. No alterations of p16Ink4a/p19Arf such as allelic loss at the Ink4a/Arf locus and deletion, mutations and hypermethylation of p16Ink4a/p19Arf were detected in the primary cells from the normal livers and HCC. On the other hand, majority of the normal and HCC cell lines lost the p16Ink4a and/or p19Arf expression, associating with p16Ink4a promoter hypermethylation or homozygous deletion at the Ink4a/Arf locus. When the primary cells and cell lines were exposed to UV light, they were arrested at the G1 phase of cell cycle with up-regulation of p21Waf1/Cip1 and MDM2, indicating that the p53-p21Waf1/Cip1-Rb pathway is functionally normal. These results indicate that the primary HCC cells can compromise replicative senescence despite retaining the normal p16Ink4a/p19Arf/p53 functions, although both normal and HCC cell lines frequently loose the p16Ink4a/p19Arf function.

Research supported by the grant from the Japanese Ministry of Education, Science, Sports and Culture.

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