Abstract
Abstract The use of DNA damaging agents remains one of the most common approaches to cancer therapy. The type of damage caused by the agent will determine which of the five main DNA damage repair (DDR) processes will respond. Initially we are developing multiplex quantitative immunoflourescence assays (qIFAs) for biomarkers of active Homologous Recombination (HR) and Nucleotide Excision Repair (NER) pathway and plan to develop mutiplex assays for the other repair pathways in the near future. The first multiplex panel to quantify DDR response has been developed to include Nbs1 phosphorylated at Serine343 and γH2AX, both early markers of DNA double strand breaks as well as ERCC1, which plays a key role in NER. Quantitation of repair pathway activation after drug treatment builds upon our validated qIFA for γH2AX, which scores individual nuclei as positive or negative for the biomarker and quantifies the γH2AX response as the fraction of nuclear area that is occupied by γH2AX foci. In contrast, the multiplex qIFA was designed to quantify the global DNA repair protein response to a variety of cytotoxic, genotoxic and targeted agents administered singly and in combination. Initial evaluations were performed in vitro on a panel of cell lines, characterized for repair pathway defects: the BRCA1-null human ovarian cancer line UWB1.289 (ATCC# CRL-2945), its isogenic line with restored BRCA1 named UWB1.289+BRCA1 (ATCC# CRL-2946), and SKOV3 (BRCA1 WT). Both gemcitabine and cisplatin increased pNbs1 both in UWB1.289 and UWB1.289+BRCA1, but not in SKOV3. Co-expression of pNbs1 with γH2AX was observed but was not 100% concordant. Combination treatment with a topoisomerase 1 inhibitor and PARP1/2 inhibitor (the indenoisoquinoline NSC 724998 with ABT-888) increased nuclear ERCC1 only in UWB1.289, but not in the isogenic UWB1.289+BRCA1 line, or in the ovarian lines SKOV3 and IGROV1, or the human fibroblast line GM00637. Assessment of drug-induced DDR response in a panel of cell lines with known repair defects is planned with the expectation that this will provide a scientific foundation for interpreting qIFA results when the assay is used to analyze clinical biopsies, and will assist in the evaluation of combination therapy strategies, such as synthetic lethality, designed to take advantage of DNA repair defects. Funded by NCI Contract No. HHSN261200800001E. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3620. doi:1538-7445.AM2012-3620
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