Inhibition of homologous recombination with vorinostat synergistically enhances ganciclovir cytotoxicity.
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.
Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
Brendon
B
Ladd
Jeffrey J
JJ
Ackroyd
J Kevin
JK
Hicks
Christine E
CE
Canman
Sheryl A
SA
Flanagan
Donna S
DS
Shewach
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D016466:CHO Cells; D002352:Carrier Proteins / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D003412:Cricetulus; D004706:Endodeoxyribonucleases; D015774:Ganciclovir / Q000494:pharmacology*; D006367:HeLa Cells; D059765:Homologous Recombination / Q000187:drug effects*; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology*; D009687:Nuclear Proteins / Q000378:metabolism; D051135:Rad51 Recombinase / Q000378:metabolism; D000077337:Vorinostat
D000970:Antineoplastic Agents; D002352:Carrier Proteins; D006877:Hydroxamic Acids; D009687:Nuclear Proteins; D000077337:Vorinostat; C495153:RAD51 protein, human; D051135:Rad51 Recombinase; D004706:Endodeoxyribonucleases; C410741:RBBP8 protein, human; D015774:Ganciclovir
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