INDUCTION OF CELL-DIFFERENTIATION POTENTIATES APOPTOSIS TRIGGERED BY PRIOR EXPOSURE TO DNA-DAMAGING DRUGS

At the end of their life span, differentiated cells die by apoptosis. Subsets of cells also die, in some cell systems, shortly after exposur e to differentiating agents. This suggests that early during different iation the cells may undergo ''priming,'' during which synthesis and/o r activation and accumulation of effecters of apoptosis occurs. The ob jective of the present study was to test the hypothesis that the signa l for apoptosis provided by DNA-damaging drugs given prior to inductio n of differentiation will be more effective in triggering apoptosis th an when given following induction of differentiation. Human promyelocy tic HL-60 cells were treated with the topoisomerase I inhibitor campto thecin, the alkylating agent nitrogen mustard, or 5'-azacytidine, an a ntimetabolite affecting predominantly RNA metabolism. Following drug r emoval, the cells were postincubated with n-butyrate, which induces di fferentiation of HL-60 cells along the monocytic pathway, or with all- trans-retinoic acid, which triggers myelocytic differentiation. Multip arameter flow cytometry using two different methods of analysis of apo ptosis-associated DNA breakage in site, as well as evaluation of cell morphology and DNA gel electrophoresis, were used to ascertain the mod e of cell death. Increases of 100-200% in the percentage of apoptotic cells were seen when cells were first treated with camptothecin or nit rogen mustard, followed by n-butyrate or retinoic acid, compared to th e combined percentage of apoptotic cells when these agents were used i ndividually. In contrast to the DNA-damaging agents camptothecin and n itrogen mustard, no enhancement of apoptosis was observed when n-butyr ate or retinoic acid was added after cell preexposure to 5'-azacytidin e. The data suggests that although the sensitivity of the DNA damage d etection and/or the apoptosis trigger mechanism may be higher in proli ferating cells, the execution of apoptosis is potentiated in cells und ergoing either monocytic or myelocytic differentiation. The data also support the antitumor strategy of using differentiating agents subsequ ent to DNA-damaging drugs or radiation.