The Role of DNA Damage and Repair in Ganciclovir-mediated Cytotoxicity.

HSV-TK/Ganciclovir;Pharmacy and Pharmacology;Health Sciences;Pharmacology

O'Konek, Jessica JaneWilson, Thomas E. ;

University of Michigan

关键词: HSV-TK/Ganciclovir;    Pharmacy and Pharmacology;    Health Sciences;    Pharmacology;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/58441/jjoz_1.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Suicide gene therapy aims to improve selectivity of cancer treatment through expression of herpes simplex virus thymidine kinase (HSV-TK) in tumor cells, permitting phosphorylation of HSV-TK substrates with subsequent cytotoxic incorporation into DNA exclusively within the tumor.This approach results in significantly greater cytotoxicity to cancer cells in a novel delayed manner when applied with the antiviral drug GCV as compared to other HSV-TK substrates.To elucidate the mechanism for the unique cytotoxicity, DNA damage and the repair pathways involved in responding to this damage induced by GCV compared to related analogs were evaluated. Using phosphorylated histone H2AX (-H2AX) as a marker of DNA damage, GCV induced >7-fold more damage than a different HSV-TK substrate, 1--D-arabinofuranosylthymine (araT), at equitoxic concentrations.Although the number of -H2AX foci decreased after removal of either drug, suggesting repair of these early lesions, only GCV produced a late and persistent increase in DNA damage indicating the induction of irreparable DNA damage.Rad51 foci formed primarily following the late increase in -H2AX foci, exposure, implicating homologous recombination repair (HRR) in responding to GCV-induced lesions. A yeast-based screen of DNA damage response mutants was utilized to detect pathways nvolved in cytotoxicity with GCV.Yeast deficient in HRR, cell cycle checkpoint, and mismatch repair (MMR) proteins all exhibited increased sensitivity toGCV.Survival studies in human cells confirmed greater GCV sensitivity in MMR deficient cells, primarily at high concentrations, validating the yeast assay.Thus, MMR and HRR may prevent cytotoxicity with ganciclovir. Previous reports suggested that GCV is genotoxic.DNA mutations induced by GCV were characterized and compared to those resulting from treatment with two other structurally similar HSV-TK substrates, D-carbocyclic 2;;-deoxyguanosine (CdG) and penciclovir (PCV). GCV induced a dose-dependent increase in mutation frequency, while highly cytotoxic concentrations of CdG and PCV failed to increase mutations.GCV predominantly induced GCTA transversions which were significantly less frequent in control cells or those treated with PCV or CdG.Analysis of cell cycle progression revealed different mechanisms of cell cycle arrest for each of these drugs.Thus, alteration of the deoxyribose structure produced profound differences in DNA replication and its fidelity