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Phenobarbital Causes Apoptosis in Conditionally Immortalized Mouse Hepatocytes Depending on Deregulated c-myc Expression: Characterization of an Unexpected Effect¹

Department of Pathology, Asahikawa Medical College, 4-5-3-11 Nishikagura, Asahikawa 078, Japan

The CHST8 mouse hepatocyte cell line, conditionally immortalized with the temperature-sensitive SV40 large T antigen gene, rapidly proliferates at 33°C with active expression of the c-myc proto-oncogene but, due to the heat-labile nature of the mutant T antigens, becomes growth arrested and morphologically senescent at 39°C; this is accompanied by the disappearance of c-myc transcripts. In a previous study, we transfected the CHST8 cells at 33°C with an activated c-H-ras or a c-myc, both of which are frequently involved in mouse hepatocarcinogenesis in vivo. When the temperature was shifted to 39°C, cells with only one of the exogenous oncogenes did not escape from the senescence, but those containing both exhibited an immortal phenotype. In the present study, using this in vitro model of hepatocarcinogenesis, we demonstrated that phenobarbital, a tumor promoter of rodent hepatocarcinogenesis, triggers remarkable apoptosis specifically in the c-myc-transfected CHST8 cells at 39°C, which show abundant c-myc expression despite growth arrest. Dissociation of p53 proteins from degrading T antigens followed by a phenobarbital and c-myc-dependent, 15-fold induction of Bax protein, known to activate the apoptotic pathway downstream of p53, occurred in association with this phenomenon. The effects of phenobarbital and c-myc in increasing Bax on shifting the temperature from 33°C to 39°C were additive, with both having similar degrees of influence on the protein level. Interestingly, subsequent introduction of an activated c-H-ras oncogene into the c-myc-transfected CHST8 cells resulted not only in escape from the growth arrest at 39°C but also in complete inhibition of the phenobarbital-inducible apoptosis along with de novo induction of the Bax antagonist, Bcl-2. These findings strongly suggest that the phenobarbital-inducible apoptosis is mediated by Bax. Although it is a common notion that phenobarbital promotes liver tumor development through suppression of apoptosis, our results, together with the known fact that phenobarbital occasionally inhibits hepatocarcinogenesis in mice, indicate a problematic complexity in its biological activities.

¹ This work was supported in part by grants from the Ministry of Education, Science, Sports and Culture; the Ministry of Health and Welfare; and the Akiyama Memorial Foundation, Japan.

² To whom requests for reprints should be addressed. Fax: 81-0166-65-8669.

Received 1/14/97. Accepted 5/14/97.

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