PARP-1 hyperactivation and reciprocal elevations in intracellular Ca²⁺ during ROS-induced nonapoptotic cell death

The generation of reactive oxygen species (ROS) has been implicated in the pathogenesis of renal ischemia/reperfusion injury, and many other pathological conditions. DNA strand breaks caused by ROS lead to the activation of poly(ADP-ribose)polymerase- 1 (PARP-1), the excessive activation of which can result in cell death. We have utilized a model in which 2,3,5-tris(glutathion-Syl) hydroquinone (TGHQ), a nephrotoxic and nephrocarcinogenic metabolite of hydroquinone, causes ROS-dependent cell death in human renal proximal tubule epithelial cells (HK-2), to further elucidate the role of PARP-1 in ROS-dependent cell death. TGHQ-induced ROS generation, DNA strand breaks, hyperactivation of PARP-1, rapid depletion of nicotinamide adenine dinucleotide (NAD), elevations in intracellular Ca²⁺ concentrations, and subsequent nonapoptotic cell death in both a PARP- and Ca²⁺-dependent manner. Thus, inhibition of PARP-1 with PJ34 completely blocked TGHQ-mediated accumulation of poly(ADPribose) polymers and NAD consumption, and delayed HK-2 cell death. In contrast, chelation of intracellular Ca²⁺ with BAPTA completely abrogated TGHQ-induced cell death. Ca²⁺ chelation also attenuated PARP-1 hyperactivation. Conversely, inhibition of PARP-1 modulated TGHQ-mediated changes in Ca²⁺ homeostasis. Interestingly, PARP-1 hyperactivation was not accompanied by the translocation of apoptosis-inducing factor (AIF) from mitochondria to the nucleus, a process usually associated with PARPdependent cell death. Thus, pathways coupling PARP-1 hyperactivation to cell death are likely to be context-dependent, and therapeutic strategies designed to target PARP-1 need to recognize such variability. Our studies provide new insights into PARP-1- mediated nonapoptotic cell death, during which PARP-1 hyperactivation and elevations in intracellular Ca²⁺ are reciprocally coupled to amplify ROS-induced nonapoptotic cell death.