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(PARP)-1 N-Terminal Fragment Down Regulates Endogenous PARP-1 Expression and Activity and Sensitises Cells to Oxidative Stress

Ida Rachel Rajiah

Poly (ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme that plays a vital role in DNA repair. This makes it an attractive anticancer therapeutic target. It is activated by DNA breaks and catalyses the synthesis of homopolymers of ADP-ribose from NAD+. Competitive inhibitors as well as the non-catalytic, DNA-binding domain of PARP-1 abolish its polymer synthesising function. Such inhibitors of PARP-1 have been used in clinical trials and are known to cause nausea, fatigue and haematological events and with chronic use the risk of drug-induced DNA damage and tumorigenesis. In this study, I have investigated the effect of PARP-1-N-terminal fragment on endogenous PARP-1 activity and expression in mammalian cells. To elicit DNA damage response, different concentrations of H2O2 were used. For visualisation of the effects by live imaging, 750 bp PARP-1-N-terminal fragment was tagged to EGFPN1 vector. My data shows an inverse correlation between the expression of this fragment and poly (ADP-ribose) synthesis at lower concentrations of H2O2 and induction of apoptosis at higher concentrations. My experimental evidence also supports regulation of endogenous PARP-1 expression by the fragment in the absence of DNA damage. This construct has allowed for the visualisation of its functional ability to sensitise cells to oxidative damage and induce apoptosis as observed by the formation of apoptotic precursors and caspase cleavage products in live cells. The data also provides direct evidence for its therapeutic potential in chemotherapy or radiotherapy to avert necrosis induced inflammatory response.