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 cyto sine 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 nong enotoxic agents was associated with the disintegration of nuclear DNA into high molecular weight (HMW) and oligonucleosomal-DNA fragments, while the f ormation of HMW-DNA fragments, but not oligonucleosomal-DNA ladder accompan ied apoptosis induced by genotoxic agents. Combination of genotoxic and non genotoxic insults, i.e, incubation of etoposide-treated cells in the serum- free medium, resulted in an additive effect on the profile of DNA disintegr ation, which involved both HMW fragmentation pattern as in etoposide alone treated cells and the oligonucleosomal-DNA ladder observed with serum-depri ved cells. On the other hand, incubation of serum-deprived cells in the pre sence of Zn2+-ions led to the abrogation of internucleosomal DNA fragmentat ion but accumulation of HMW-DNA fragments. Differences in the pattern of DN A fragmentation were reproducible in a cell free apoptotic system after tre atment of isolated normal nuclei with cytosolic extracts prepared from the cells treated with genotoxic or nogenotoxic apoptotic inducers. Cell free e xperiments also revealed that activities responsible for the formation of H MW- and oligonucleosomal-BNA fragments are separable in cytosolic extract p repared from the serum-deprived cells. Finally, DNA fragmentation induced b y nongenotoxic apoptotic inducers was effectively prevented by cycloheximid e and suramin, while both cycloheximide and suramin had only a slight inhib itory 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 t hat the formation of HMW- and oligonucleosomal-DNA fragments proceeds via s eparate mechanisms in NB-2a neuroblastoma cells. (C) 1999 Elsevier Science Ltd. All rights reserved.