BIOCHEMICAL PATHWAYS OF APOPTOSIS - NICOTINAMIDE ADENINE DINUCLEOTIDE-DEFICIENT CELLS ARE RESISTANT TO TUMOR-NECROSIS-FACTOR OR ULTRAVIOLET-LIGHT ACTIVATION OF THE 24-KD APOPTOTIC PROTEASE AND DNA FRAGMENTATION

The function of nicotinamide adenine dinucleotide (NAD) and adenosine diphosphate (ADP) ribosylation reactions in the mechanism of apoptotic cell death is controversial, although one theory postulates an essent ial role for NAD depletion by poly-ADP-ribose polymerase. The present study examined the role of intracellular NAD in tumor necrosis factor (TNF) and ultraviolet (UV) light-induced activation of the 24-kD apopt otic protease (AP24) leading to internucleosomal DNA fragmentation and death. Our results demonstrate that nutritional depletion of NAD to u ndetectable levels in two leukemia lines (U937 and HL-60) renders them completely resistant to apoptosis. This was attributed to a block in the activation of AP24 and subsequent DNA cleavage. Normal cells show an elevation of ADP-ribosyl transferase (ADPRT) in both the cytosol an d nucleus after exposure to TNF, but before DNA fragmentation. ADPRT a ctivity as well as cell death was suppressed by an inhibitor specific for mono-ADPRT. Nuclei from NAD-depleted cells were still sensitive to DNA fragmentation induced by exogenous AP24, indicating a selective f unction for NAD upstream of AP24 activation in the apoptotic pathway. We confirmed a requirement for intracellular NAD, activation of ADPRT, and subsequent NAD depletion during apoptosis in KG1a, YAC-1, and BW1 547 leukemia cell lines. However, this mechanism is not universal, sin ce BJAB and Jurkat leukemia cells underwent apoptosis normally, even i n the absence of detectable intracellular NAD. We conclude that TNF or UV Light-induced apoptotic cell death is not due to NAD depletion in some leukemia cell Lines. Rather, NAD-dependent reactions which may in volve mono-ADPRT, function in signal transduction leading to activatio n of AP24, with subsequent DNA fragmentation and cell death.