Distinct mechanisms underlay DNA disintegration during apoptosis induced by genotoxic and nongenotoxic agents in neuroblastoma cells.

Exposure of mouse NB-2a neuroblastoma cells to genotoxic (etoposide or cytosine arabinoside) or nongenotoxic challenges (serum deprivation or okadaic acid) resulted in progressive cell death with biochemical and morphological characteristics typical of apoptosis. Apoptotic cell death induced by nongenotoxic agents was associated with the disintegration of nuclear DNA into high molecular weight (HMW) and oligonucleosomal-DNA fragments, while the formation of HMW-DNA fragments, but not oligonucleosomal-DNA ladder accompanied apoptosis induced by genotoxic agents. Combination of genotoxic and nongenotoxic insults, i.e. incubation of etoposide-treated cells in the serum-free medium, resulted in an additive effect on the profile of DNA disintegration, which involved both HMW fragmentation pattern as in etoposide alone treated cells and the oligonucleosomal-DNA ladder observed with serum-deprived cells. On the other hand, incubation of serum-deprived cells in the presence of Zn2+-ions led to the abrogation of internucleosomal DNA fragmentation but accumulation of HMW-DNA fragments. Differences in the pattern of DNA fragmentation were reproducible in a cell free apoptotic system after treatment of isolated normal nuclei with cytosolic extracts prepared from the cells treated with genotoxic or nogenotoxic apoptotic inducers. Cell free experiments also revealed that activities responsible for the formation of HMW- and oligonucleosomal-DNA fragments are separable in cytosolic extract prepared from the serum-deprived cells. Finally, DNA fragmentation induced by nongenotoxic apoptotic inducers was effectively prevented by cycloheximide and suramin, while both cycloheximide and suramin had only a slight inhibitory effect on DNA fragmentation induced by genotoxic agents. The results presented suggest that distinct pathways underlay disintegration of nuclear DNA during apoptosis induced by genotoxic and nongenotoxic inducers, and that the formation of HMW- and oligonucleosomal-DNA fragments proceeds via separate mechanisms in NB-2a neuroblastoma cells.