Recovery of deficient homologous recombination in Brca2-depleted mouse cells by wild-type Rad51 expression

Lee, Shauna A.; Roques, Céline; Magwood, Alissa C.; Masson, Jean‐Yves; Baker, Mark D.

doi:10.1016/j.dnarep.2008.10.002

Cited by 32 publications

(26 citation statements)

References 59 publications

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“…Rad51 is a crucial downstream protein involved in HR repair, which is relocalized within the nucleus in response to DNA damage to form distinct foci that can be visualized by immunofluorescent microscopy and are thought to represent assemblies of proteins at these sites of HR repair. Therefore, quantification of Rad51 could serve as a marker of HR function to distinguish between HR-proficient and HR-deficient cell lines (19).…”

Section: Methodsmentioning

confidence: 99%

Development of a Functional Assay for Homologous Recombination Status in Primary Cultures of Epithelial Ovarian Tumor and Correlation with Sensitivity to Poly(ADP-Ribose) Polymerase Inhibitors

Mukhopadhyay

Elattar

Cerbinskaite

et al. 2010

Clinical Cancer Research

244

230

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Purpose: Poly(ADP-ribose) polymerase (PARP) inhibitors selectively target homologous recombination (HR)-defective cells and show good clinical activity in hereditary breast and ovarian cancer associated with BRCA1 or BRCA2 mutations. A high proportion (up to 50%) of sporadic epithelial ovarian cancers (EOC) could be deficient in HR due to genetic or epigenetic inactivation of BRCA1/BRCA2 or other HR genes. Therefore, there is a potential for extending the use of PARP inhibitors to these patients if HR status can be identified. We developed a functional assay of HR status in primary cultures of EOCs based on Rad51 focus formation that correlates well with sensitivity to the potent PARP inhibitor AG014699.Experimental Design: Primary cultures were derived from ascitic fluid from patients with EOCs. HR status was investigated by γH2AX and Rad51 focus formation by immunofluorescence. Cytotoxicity to PARP inhibitors was tested by sulforhodamine B and survival assay.Results: Twenty-five cultures were evaluated for HR status and cytotoxicity to PARP inhibitor. Following exposure to AG014699, there was an increase in Rad51 foci (HR competent) in 9 of 24 (36%) but no increase (HR deficient) in 16 of 24 (64%) cultures. Cytotoxicity was observed in 15 of 16 (93%) HRdeficient samples but not in 9 of 9 HR-competent samples following 24-hour exposure to 10 μmol/L AG014699.Conclusion: HR status can be determined in primary cancer samples by Rad51 focus formation, and this correlates with in vitro response to PARP inhibition. Use of this assay as a biomarker now needs testing in the setting of a clinical trial.

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Section: Methodsmentioning

confidence: 99%

Development of a Functional Assay for Homologous Recombination Status in Primary Cultures of Epithelial Ovarian Tumor and Correlation with Sensitivity to Poly(ADP-Ribose) Polymerase Inhibitors

Mukhopadhyay

Elattar

Cerbinskaite

et al. 2010

Clinical Cancer Research

244

230

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“…To determine the requirement for Brca2 in 39 extension, we utilized mouse hybridoma cell line 12-21 that is 75% siRNA depleted for mouse Brca2 (Lee et al 2009) along with 12-21 derivatives that express either ectopic wild-type human BRCA2 or a human BRCA2 mutant bearing an in-frame deletion of BRC repeats 1-8 (Magwood et al 2012(Magwood et al , 2013. As shown in Figure 7, at 6 hr, the frequency of the 39 extension/vector backbone is significantly reduced in the mouse Brca2 knockdown cell line 12-21 compared to control igm482 cells.…”

Section: Brca2 Is Required For 39 Extensionmentioning

confidence: 99%

Nascent DNA Synthesis During Homologous Recombination Is Synergistically Promoted by the Rad51 Recombinase and DNA Homology

et al. 2014

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In this study, we exploited a plasmid-based assay that detects the new DNA synthesis (39 extension) that accompanies Rad51-mediated homology searching and strand invasion steps of homologous recombination to investigate the interplay between Rad51 concentration and homology length. Mouse hybridoma cells that express endogenous levels of Rad51 display an approximate linear increase in the frequency of 39 extension for homology lengths of 500 bp to 2 kb. At values below 500 bp, the frequency of 39 extension declines markedly, suggesting that this might represent the minimal efficient processing segment for 39 extension. Overexpression of wild-type Rad51 stimulated the frequency of 39 extension by 3-fold for homology lengths ,900 bp, but when homology was .2 kb, 39 extension frequency increased by as much as 10-fold. Excess wild-type Rad51 did not increase the average 39 extension tract length. Analysis of cell lines expressing N-terminally FLAG-tagged Rad51 polymerization mutants F86E, A89E, or F86E/A89E established that the 39 extension process requires Rad51 polymerization activity. Mouse hybridoma cells that have reduced Brca2 (Breast cancer susceptibility 2) due to stable expression of small interfering RNA show a significant reduction in 39 extension efficiency; expression of wild-type human BRCA2, but not a BRCA2 variant devoid of BRC repeats 1-8, rescues the 39 extension defect in these cells. Our results suggest that increased Rad51 concentration and homology length interact synergistically to promote 39 extension, presumably as a result of enhanced Brca2-mediated Rad51 polymerization. Homologous recombination is a complex, multi-step process (reviewed in Brugmans et al. 2007). In eukaryotes, the central step in the repair of a DNA DSB involves the binding of multiple monomers of the RAD51 protein (a functional homolog of the Escherichia coli RecA recombinase) to 39-ending single-stranded DNA overhangs created by nucleolytic resection (Van Den Bosch et al. 2003). The resulting RAD51 nucleoprotein filament promotes formation of a joint molecule between the processed broken DNA and the homologous repair template by the orchestrated steps of homology searching and DNA strand invasion and exchange. Joint molecule formation is followed by new DNA synthesis, which replaces nucleotides lost through DSB formation and 39-end resection (Pâques and Haber 1999;Symington 2002;Li and Heyer 2008). Depending on the subpathway of HR, subsequent steps may involve unwinding (dissolution) of the DNA strand containing the newly synthesized DNA and ligation to the processed second end or the formation of a stably joined Holliday junction intermediate that can be processed further by structure-specific endonucleases yielding recombinant products (Li and Heyer 2008).At the heart of the HR process is the Rad51 (or RecA) nucleoprotein filament, whose formation requires the initial association of four to five monomers with 39-ending singlestranded DNA in the step known as nucleation (Galletto

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“…Although mutations in the RAD51 open-reading frame are rare in cancer, overexpression of Rad51 has been reported in a wide variety of cancers, especially those harboring p53 mutations (Klein, 2008). Rad51 overexpression can lead to resistance to both drug-and radiation-induced DNA damage and has been shown to compensate for the homologous recombination defects caused by BRCA1 or BRCA2 deficiency (Martin et al, 2007;Brown and Holt, 2009;Lee et al, 2009;Yang et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

Dynamic Regulation of Rad51 by E2F1 and p53 in Prostate Cancer Cells upon Drug-Induced DNA Damage under Hypoxia

Wang

McGregor

et al. 2014

Mol Pharmacol

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Intratumoral hypoxia has been proposed to create a "mutator" phenotype through downregulation of DNA repair, leading to increased genomic instability and drug resistance. Such downregulation of DNA repair has been proposed to sensitize hypoxic cancer cells to DNA-damaging agents and inhibitors of DNA repair. Here, we showed that prostate cancer cells with mutant p53 were resistant to the poly(ADP-ribose) polymerase inhibitor, veliparib (2-[(2R)-2-methylpyrrolidin-2-yl]-1H-benzimidazole-4-carboxamide, dihydrochloride; Abbott Laboratories, Abbott Park, IL), and the DNA-damaging topoisomerase I inhibitor camptothecin-11 (CPT-11) or SN38 (7-ethyl-10-hydroxycamptothecin) under hypoxia. Upregulation of Rad51 by E2F1 upon DNA damage under hypoxia contributed to such resistance, which was reversed by either inhibiting RAD51 transcription with small interfering RNA or by expressing wild-type p53 in the p53 null prostate cancer line. Accumulation of endogenous p53 but not E2F1 and suppressed RAD51 transcription was observed in prostate cancer line with wild-type p53 after DNA damage under hypoxia. Combining veliparib with CPT-11 significantly enhanced DNA damage and apoptosis under both hypoxic and normoxic culture conditions. Such enhanced DNA damage and antitumor activities were seen in the presence of Rad51 upregulation and confirmed in vivo with PC3 mouse xenografts. These data illustrate a dynamic regulation of Rad51 by E2F1 and p53 in prostate cancer cells' response to hypoxia and DNA damage. The veliparib and CPT-11 combination can be further explored as a treatment of metastatic castrationresistant prostate cancers that have frequent p53 mutations and enriched genomic instability.

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Recovery of deficient homologous recombination in Brca2-depleted mouse cells by wild-type Rad51 expression

Cited by 32 publications

References 59 publications

Development of a Functional Assay for Homologous Recombination Status in Primary Cultures of Epithelial Ovarian Tumor and Correlation with Sensitivity to Poly(ADP-Ribose) Polymerase Inhibitors

Development of a Functional Assay for Homologous Recombination Status in Primary Cultures of Epithelial Ovarian Tumor and Correlation with Sensitivity to Poly(ADP-Ribose) Polymerase Inhibitors

Nascent DNA Synthesis During Homologous Recombination Is Synergistically Promoted by the Rad51 Recombinase and DNA Homology

Dynamic Regulation of Rad51 by E2F1 and p53 in Prostate Cancer Cells upon Drug-Induced DNA Damage under Hypoxia

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