Adenoviral vectors for modulation of poly(ADP-ribose) polymerase-1 (PARP1) – dependent DNA repair as a predictive tool for chemotherapy

Abstract

Loss of the DNA homologous recombination repair pathway (DHR) sensitizes cells to the effects of clastogenic chemotherapy agents when the alternative base excision DNA repair pathway (BER) is blocked by inhibitors of poly(ADP- ribose) polymerases (PARP). The mutation of BRCA is an example of defective DHR, where several PARP inhibitors are now in advanced stages of clinical trials due to their ability to form synthetic lethality with chemotherapy drugs in breast and ovarian cancer. Because the inability of cells to perform DHR is the determining factor for successful PARP inhibitor administration in a chemotherapy protocol, testing of patient cells for sensitization by PARP inhibitors would be advantageous for the prediction of therapy outcome. This proof-of-principle study set out to design recombinant adenoviruses as a simple tool for the functional block of PARP1-mediated DNA strand break repair important for the execution of BER that could potentially be used to test cultured cells from patients for the sensitizing effects of PARP inhibitors in a chemotherapy setting. Recombinant adenoviruses for the expression of the PARP1 DNA binding domain (DBD) as a dominant-negative PARP1 mutant, for full- length PARP1 expression and other control viruses were generated and used to infect normal human fibroblasts. Transgene expression and the modulation of poly(ADP-ribose) (PAR) were studied using immunofluorescence and immunoblotting techniques. Overexpression of PARP1 increased, but expression of the PARP1 DBD blocked PAR formation in response to methylnitronitrosoguanidine (MNNG) treatment of virally transduced cells. Ectopic DBD expression caused the formation of caspase 3 cleavage products of PARP1 protein, which are a marker of apoptotic cell death. Cells infected with control virus without PARP DBD did not induce caspase 3-mediated cleavage of PARP1. In conclusion, the set of adenoviral vectors presented in this study can be used to modulate PARP activity in normal human cells and may therefore be used as a tool for testing cellular sensitization by blocking DNA BER by administration of PARP inhibitors and to gain better understanding of potential PARP inhibitor resistance.