Identification and quantification of DNA repair protein poly(ADP ribose) polymerase 1 (PARP1) in human tissues and cultured cells by liquid chromatography/isotope-dilution tandem mass spectrometry

Abstract

Poly(ADP ribose) polymerase 1 (PARP1) is a multifunctional DNA repair protein of the base excision repair pathway and plays a major role in the repair of DNA strand breaks and in replication and transcriptional regulation among other functions. Mounting evidence points to the predictive and prognostic value of PARP1 expression in human cancers. Thus, PARP1 has become an important target in cancer therapy, leading to the development of inhibitors as anticancer drugs. In the past, PARP1 expression levels in tissue samples have generally been estimated by indirect and semi-quantitative immunohistochemical methods. Accurate measurement of PARP1 in normal tissues and malignant tumors of patients will be essential for evaluating PARP1 as a predictive and prognostic biomarker in cancer and other diseases, and for the development and use of its inhibitors in cancer therapy. In this work, we present an approach involving liquid chromatography–isotope-dilution tandem mass spectrometry to positively identify and accurately quantify PARP1 in human tissues and cultured cells. We identified and quantified PARP1 in human normal ovarian tissues and malignant ovarian tumors, and in three pairs of human cell lines, each pair consisting of a normal cell line and its cancerous counterpart. Significantly greater expression of PARP1 was observed in malignant ovarian tissues than in normal ovarian tissues. In the case of one pair of cell lines, the cancerous cell line also exhibited greater expression of PARP1 than in normal cell line. We also show the simultaneous measurement of PARP1 and apurinic/apyrimidinic endonuclease 1 (APE1) in a given protein extract. The approach presented in this work is expected to contribute to the accurate quantitative assessment of PARP1 levels in basic research and clinical studies.