Intrinsic and acquired resistance to cisplatin remains a primary hurdle to treatment of high-grade serous ovarian cancer (HGSOC). Cisplatin selectively kills tumor cells by inducing DNA crosslinks that block replicative DNA polymerases. Single-stranded DNA (ssDNA) generated at resulting stalled replication forks (RF) is bound and protected by heterotrimeric replication protein A (RPA), which then serves as a platform for recruitment and activation of replication stress response factors. Cells deficient in this response are characterized by extensive ssDNA formation and excessive RPA recruitment that exhausts the available pool of RPA, which (i) inhibits RPA-dependent processes such as nucleotide excision repair (NER) and (ii) causes catastrophic failure of blocked RF. Here, we investigated the influence of RPA availability on chemosensitivity using a panel of human HGSOC cell lines. Our data revealed a striking correlation among these cell lines between cisplatin sensitivity and the inability to efficiently repair DNA via NER, specifically during S phase. Such defects in NER were attributable to RPA exhaustion arising from aberrant activation of DNA replication origins during replication stress. Reduced RPA availability promoted Mre11-dependent degradation of nascent DNA at stalled RF in cell lines exhibiting elevated sensitivity to cisplatin. Strikingly, defective S-phase NER, RF instability, and cisplatin sensitivity could all be rescued by ectopic overexpression of RPA. Taken together, our findings indicate that RPA exhaustion represents a major determinant of cisplatin sensitivity in HGSOC cell lines. The influence of replication protein A exhaustion on cisplatin sensitivity harbors important implications toward improving therapy of various cancers that initially respond to platinum-based agents but later relapse due to intrinsic or acquired drug resistance. .

Nucleotide excision repair (NER) is a highly conserved pathway that removes helix-distorting DNA lesions induced by a plethora of mutagens, including UV light. Our laboratory previously demonstrated that human cells deficient in either ATM and Rad3-related (ATR) kinase or translesion DNA polymerase (i.e. key proteins that promote the completion of DNA replication in response to UV-induced replicative stress) are characterized by profound inhibition of NER exclusively during S phase. Toward elucidating the mechanistic basis of this phenomenon, we developed a novel assay to quantify NER kinetics as a function of cell cycle in the model organism Saccharomyces cerevisiae. Using this assay, we demonstrate that in yeast, deficiency of the ATR homologue Mec1 or of any among several other proteins involved in the cellular response to replicative stress significantly abrogates NER uniquely during S phase. Moreover, initiation of DNA replication is required for manifestation of this defect, and S phase NER proficiency is correlated with the capacity of individual mutants to respond to replicative stress. Importantly, we demonstrate that partial depletion of Rfa1 recapitulates defective S phase-specific NER in wild type yeast; moreover, ectopic RPA1-3 overexpression rescues such deficiency in either ATR-or polymerase -deficient human cells. Our results strongly suggest that reduction of NER capacity during periods of enhanced replicative stress, ostensibly caused by inordinate sequestration of RPA at stalled DNA replication forks, represents a conserved feature of the multifaceted eukaryotic DNA damage response.

The aim of the study was to characterize if the development of cardiac hypertrophy ( CH ) caused by severe left ventricle ( LV ) volume overload ( VO ) from chronic aortic valve regurgitation ( AR ) in male rats was influenced by androgens. We studied Wistar rats with/without orchiectomy (Ocx) either sham‐operated (S) or with severe AR for 26 weeks. Loss of testosterone induced by Ocx decreased general body growth. Cardiac hypertrophy resulting from AR was relatively more important in intact (non‐Ocx) animals than in Ocx ones compared to their respective S group (60% vs. 40%; P = 0.019). The intact AR group had more LV dilation, end‐diastolic LV diameter being increased by 37% over S group and by 17% in ARO cx rats ( P < 0.0001). Fractional shortening (an index of systolic function) decreased only by 15% in ARO cx compared to 26% for intact AR animals ( P = 0.029). Changes in LV gene expression resulting from CH were more marked in intact rats than in ARO cx animals, especially for genes linked to extracellular matrix remodeling and energy metabolism. The ratio of hydroxyacyl‐Coenzyme A dehydrogenase activity over hexokinase activity, an index of the shift of myocardial substrate use toward glucose from the preferred fatty acids, was significantly decreased in the AR group but not in ARO cx. Finally, pJ nk2 LV protein content was more abundant in AR than in ARO cx rats, indicating decreased activation of this stress pathway in the absence of androgens. In summary, testosterone deficiency in rats with severe LV VO resulted in less CH and a normalization of the LV gene expression profile.