The nucleoside analog ganciclovir (GCV) elicits cytotoxicity in tumor cells via a novel mechanism in which drug incorporation into DNA produces minimal disruption of replication, but numerous DNA double strand breaks occur during the second S-phase after drug exposure. We propose that homologous recombination (HR), a major repair pathway for DNA double strand breaks, can prevent GCV-induced DNA damage, and that inhibition of HR will enhance cytotoxicity with GCV. Survival after GCV treatment in cells expressing a herpes simplex virus thymidine kinase was strongly dependent on HR (>14-fold decrease in IC50 in HR-deficient vs. HR-proficient CHO cells). In a homologous recombination reporter assay, the histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA; vorinostat), decreased HR repair events up to 85%. SAHA plus GCV produced synergistic cytotoxicity in U251tk human glioblastoma cells. Elucidation of the synergistic mechanism demonstrated that SAHA produced a concentration-dependent decrease in the HR proteins Rad51 and CtIP. GCV alone produced numerous Rad51 foci, demonstrating activation of HR. However, the addition of SAHA blocked GCV-induced Rad51 foci formation completely and increased γH2AX, a marker of DNA double strand breaks. SAHA plus GCV also produced synergistic cytotoxicity in HR-proficient CHO cells, but the combination was antagonistic or additive in HR-deficient CHO cells. Collectively, these data demonstrate that HR promotes survival with GCV and compromise of HR by SAHA results in synergistic cytotoxicity, revealing a new mechanism for enhancing anticancer activity with GCV.
Keywords: Anticancer drug; DNA damage; DNA double strand break; DNA repair; DR-GFP; DSB; GCV; GCVMP; GCVTP; HDAC; HR; HSV-TK; Histone deacetylase inhibitor; Homologous recombination; LacZ; MMR; SAHA; ganciclovir; ganciclovir 5′-monophosphate; ganciclovir 5′-triphosphate; green fluorescent protein recombination substrate; herpes simplex virus thymidine kinase; histone deacetylase; homologous recombination; mismatch repair; suberoylanilide hydroxamic acid; β-galactosidase.
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