An extensive literature documents the role of poly(ADP-ribose)polymerase 1 (PARP1) in DNA repair. PARP1 recognizes and is activated by single strand DNA breaks. Upon activation, it poly(ADP-ribosyl)ates a variety of proteins involved in chromatin organization and DNA repair. Recent studies suggest that the prosurvival effects of PARP1 may, in addition to its role in DNA repair, reflect its contributions to the induction of autophagy, a process whereby cytosol and damaged organelles are encapsulated in vacuoles termed autophagosomes that fuse with lysosomes, leading to degradation of the sequestered cargo. PARP1 presumably mediates this induction via its utilization of NAD for poly(ADP-ribosyl)ation, which results in a metabolic deficiency. Consequently, it activates AMPK and in turn inhibits mTOR activity, thus negating the latter's active suppression of autophagy. In contrast, we have data suggesting that PARP1 may play a role in autophagy unrelated to its establishment of conditions that ultimately inhibit mTOR. Specifically, we found that PARP1 knockout (PARP1-/-) murine embryonic fibroblasts (MEFs), relative to wild type MEFs, exhibit reduced autophagosome formation following induction of autophagy with the mTOR inhibitor rapamycin. Furthermore, immunoprecipitation (IP) studies with human ovarian cancer cell lines actively engaged in autophagy suggested that PARP1 and ATG12-ATG5 exist as a complex. Such complexes have never been reported and are significant since ATG12-ATG5 is a component of the ATG12-ATG5/ATG16 complex that resides on the autophagosome, which plays a key role in the synthesis and elongation of the autophagosome membrane. We hypothesize that a portion of the PARP1 pool exists in a complex with members of the autophagic synthetic machinery and is required for optimal autophagosome synthesis. The overall aims of this application are to determine: 1) if PARP1 is required for the synthesis of autophagosomes following receipt of an autophagy-inducing signal, and 2) if PARP1 exists in a complex with ATG12-ATG5 or ATG12- ATG5/ATG16 on the autophagosomes. An additional component of both aims will be to determine if either property requires the poly(ADP-ribosyl)ating activity of PARP1. To achieve these goals confocal fluorescence microscopy and immunobiochemical assays will be used to monitor rapamycin- and starvation-induced autophagosome synthesis in cultures of human ovarian cancer cell lines transfected with vector or PARP1 shRNA, wild type MEFs and PARP1-/- MEFs. The cells will be used in IP and confocal fluorescence colocalization studies to determine if PARP1 localizes to ATG12-ATG5, ATG12-ATG5/ATG16 complexes and autophagosomes. Engineered cell lines expressing poly(ADP-ribosyl)ation dead PARP1 and pharmacological inhibitors of PARP1 will be used to examine if PARP1 must be active for complex formation and autophagosome synthesis. Significant efforts are being made to use inhibitors of PARP1 and autophagy to potentiate the therapeutic effectiveness of cytotoxic drugs. Our studies investigate a potentially novel function for PARP1 that links it to autophagy.
PARP1 plays a critical role in DNA repair, which contributes to cell survival. One of the survival pathways is mediated by autophagy. This application will study the mechanism by which PARP1 regulates autophagy that is independent of its role in DNA repair. Many anticancer agents cause cellular damage that is counteracted by a parallel induction of autophagy. Since both PARP1 inhibitors and autophagy inhibitors often enhance the cytotoxicities of chemotherapeutic drugs and have advanced to use in clinical trials, the proposed research has relevance to public health.
|Wang, Juan; Wu, Gen Sheng (2014) Role of autophagy in cisplatin resistance in ovarian cancer cells. J Biol Chem 289:17163-73|