MULTIPLE PATHWAYS OF DNA DISINTEGRATION DURING NEURONAL APOPTOSIS

Apoptotic cell death has been shown to contribute to neurodegeneration. Two biochemical processes are thought to be obligatory in apoptosis, namely, the activation of the caspases and the fragmentation of nuclear DNA. The purpose of this study was to investigate the mechanisms of DNA disintegration during neuronal apoptosis and to clarify whether HMWand internucleosomal DNA cleavage represent distinct and separate types of apoptotic DNA disintegration. It has been shown that the treatment of NB 2A neuroblastoma cells with genotoxic insults (etoposide, Ara-C) led to the formation of HMW-DNA fragments of about 50-100 kb and 300 kb without the formation of an oligonucleosomal DNA ladder. Apoptosis induced by nongenotoxic insults (serum deprivation, OKA) was associated with disintegration of nuclear DNA into 50-100 kb DNA fragments and oligonucleosomal DNA ladder. ATA, Zn-ions, cycloheximide and suramin effectively prevented internucleosomal and HMW-DNA cleavage in serum-deprived and OKA treated cells, but had no inhibitory effect on DNA disintegration in etoposide or Ara-C treated cells. The cell free system experiments allowed further to separate nuclease activities, responsible for these two types of DNA fragmentation. Our results suggest that distinct pathways underlay DNA disintegration during apoptosis induced by genotoxic and nongenotoxic stimuli, and formation of HMW-DNA fragments may proceed independently of the internucleosomal DNA cleavage. We further showed that treatment of CGCs with OKA or L-glutamate both resulted in cell death. The cytotoxicity induced by OKA was associated with activation of caspase-3-like proteases and formation of oligonucleosomal and HMW-DNA fragments. In contrast to OKA, neither activation of caspase-3like proteases, nor formation of oligonucleosomal DNA ladder was observed during L-glutamate induced excitotoxicity. In OKA-treated cells, both internucleosomaland HMWDNA fragmentation were effectively inhibited by caspase inhibitors. However, in L-glutamate treated cells, these inhibitors had no any apparent inhibitory effect on the formation of HMW-DNA fragments. Our further experiments showed that addition of staurosporine to a serum-deficient medium induced in NB 2A cells apoptosis without formation of oligonucleosomal DNA cleavage. Caspase inhibitors effectively prevented activation of caspase-3-like proteases in staurosporine treated neuroblastoma cells without inhibition of HMW-DNA fragmentation. These results suggest that caspase activation is not essential for HMW-DNA fragmentation in cells induced to undergo apoptosis by staurosporine in serum-deficient medium. In summary, this study demonstrates that HMWand internucleosomal DNA cleavage represent independent programmes of apoptotic DNA disintegration. Different mechanisms are involved in the formation of HMW-DNA cleavage that can be induced in both caspase-dependent and -independent manners. Suramin was demonstrated to protect neuronal cells against apoptotic and excitotoxic cell death. National Library of Medicine Classification: WL 102 Medical Subject Headings: neuronal apoptosis, excitotoxicity, cell death, neurodegeneration, NB 2A neuroblastoma cells, cerebellar granule cells, serum deprivation, etoposide, L-glutamate, staurosporine, okadaic acid, caspases, nuclear apoptotic morphology, HMWand internucleosomal DNA fragmentation, nuclease activities, caspase inhibitors, suramin.