INHIBITION OF APOPTOSIS BY OVEREXPRESSING BCL-2 ENHANCES GENE AMPLIFICATION BY A MECHANISM INDEPENDENT OF APHIDICOLIN PRETREATMENT

To study the effect of apoptosis on gene amplification, we have constr ucted HeLa S3 cell lines in which the expression of bcl-2 (BCL2) can b e controlled by tetracycline in the growth medium, Induction of Bcl-2 expression caused a temporary delay of apoptosis and resulted in rough ly a 3-fold increase in the frequency of resistant colonies when cells were selected with trimetrexate. This resistance was due to amplifica tion of the dihydrofolate reductase gene, Cells grown out of the poole d resistant colonies retained the same level of resistance to trimetre xate whether Bcl-2 was induced or repressed, consistent with the theor y that Bcl-2 functions by facilitating gene amplification, rather than being the resistance mechanism per se, Pretreating cells with aphidic olin is another method to increase gene amplification frequency, When Bcl-2-expressing cells were pretreated with aphidicolin, the resulting increase in gene amplification frequency was approximately the produc t of the increases caused by aphidicolin pretreatment or Bcl-2 express ion alone, indicating that Bcl-2 increases gene amplification through a mechanism independent of that of aphidicolin pretreatment. These res ults are consistent with the concept that gene amplification occurs at a higher frequency during drug-induced cell cycle perturbation, Bcl-2 evidently increases the number of selected amplified colonies by prol onging cell survival during the perturbation.