Abstract
Activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) is an early response of cells exposed to DNA-damaging compounds such as nitric oxide (NO) or reactive oxygen intermediates (ROI). Excessive poly-(ADP-ribose) formation by PARP has been assumed to deplete cellular NAD+ pools and to induce the death of several cell types, including the loss of insulin-producing islet cells in type I diabetes. In the present study we used cells from mice with a disrupted and thus inactivated PARP gene to provide direct evidence for a causal relationship between PARP activation, NAD+ depletion, and cell death. We found that mutant islet cells do not show NAD+ depletion after exposure to DNA-damaging radicals and are more resistant to the toxicity of both NO and ROI. These findings directly prove that PARP activation is responsible for most of the loss of NAD+ following such treatment. The ADP-ribosylation inhibitor 3-aminobenzamide partially protected islet cells with intact PARP gene but not mutant cells from lysis following either NO or ROI treatment. Hence the protective action of 3-aminobenzamide must be due to inhibition of PARP and does not result from its other pharmacological properties such as oxygen radical scavenging. Finally, the use of mutant cells an alternative pathway of cell death was discovered which does not require PARP activation and NAD+ depletion. In conclusion, the data prove the causal relationship of PARP activation and subsequent islet cell death and demonstrate the existence of an alternative pathway of cell death independent of PARP activation and NAD+ depletion.
Highlights
Inactivation of the Poly(ADP-ribose) Polymerase Gene Affects Oxygen Radical and Nitric Oxide Toxicity in Islet Cells*
DNA damage, poly(ADP-ribose) polymerase (PARP) activation, and NAD+ depletion are seen in islets exposed to reactive oxygen intermediates (ROI) [16], and the same pathway has been proposed previously to account for toxic effects of the alkylating agent streptozotocin [17]
Formation of Poly(ADP-ribose) Polymer after DNA Damage-To analyze the relationship between cellular NAD + levels and PARP activity, islet cells from PARP+I + mice were exposed to ROI for different time intervals
Summary
Diabetes Research Institute, Auf'm Hennekamp 65, D-40225 Dusseldorf, Germany. Tel.: 49-211-3382643; Fax: 49-211-3382606. Recent evidence for NO-induced DNA damage in islet cells has led to the proposal that excessive poly(ADP-ribose) polymerase (PARP) activation and the consequent depletion of its substrate, NAD+, might be responsible for cell death [9,10,11]. Excessive poly(ADP-ribose) formation has been assumed to be the reason for the loss of cellular NAD+ in cells exposed to high doses of DNA damaging compounds [17, 18] This led to the hypothesis elaborated by Berger and Okamoto that excessive PARP activation may lead to cell death because of NAD+ depletion [17, 18]. An alternative pathway of cell death could be identified which does not involve PARP activation and NAD+ depletion
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