Molecular and cellular analysis of cell death and drug resistance by controlled expression of genes that influence drug actions or cell death

We have studied the regulation of drug induced apoptosis and the effect of apoptosis regulation on gene amplification. We have also studied the (1) whether the rate of transcription of the mouse DHFR gene affects the rate of amplification of this gene; and (2) how cells achieve resistance to trimetrexate (TMTX). We employed a regulated gene expression system in both studies. This system allowed us to express bcl-2 protooncogene and dihydrofolate reductase gene in Hela S3 cell and Chinese hamster ovary cell, respectively, in a precisely controlled manner. We conclude that (1) Bcl-2 delays the appearance of apoptotic morphology induced by many different drugs; (2) Hela S3 cells are committed to apoptosis upstream of Bcl-2 activity with aphidicolin treatment, but downstream of Bcl-2 activity with TMTX treatment; (3) Bcl-2 increases the number of TMTX selected colonies with amplified DHFR genes by prolonging cell survival during the TMTX induced perturbation; (4) Protein tyrosine phosphorylation and dephosphorylation may be a down-stream event of Bcl-2 activity; (5) TMTX resistance was achieved by amplification or mutations of the DHFR gene or by a novel, yet uncharacterized mechanism, but not by mutations of the MDR gene as speculated; (6) The transfected mouse DHFR gene amplified or otherwise mutated faster than the endogenous hamster DHFR gene; and (7) Because either the amplification of hamster DHFR or the novel mechanism of TMTX resistance could take the TMTX selection pressure away from a repressed mouse DHFR gene, this regulated expression strategy can not be used to accurately assay the rate of amplification of the transfected mouse DHFR at transcriptionally repressed condition. Therefore, no absolute conclusion can be made as to the effect of gene transcription on gene amplification.