Comparison of the genotoxic and apoptosis-inducing properties of ganciclovir and penciclovir in Chinese hamster ovary cells transfected with the thymidine kinase gene of herpes simplex virus-1: Implications for gene therapeutic approaches

Abstract

We studied the genotoxic and apoptosis-inducing properties of ganciclovir (GCV) and penciclovir (PCV) using Chinese hamster ovary cells stably transfected with the thymidine kinase (tk) gene of herpes simplex virus-1 (HSV-1). Cells expressing HSVtk were 300 and 100 times more sensitive than their isogenic HSVtk Ϫ counterparts to the cytotoxic effects of GCV and PCV, respectively.Using radiolabeled drugs, GCV was found to be incorporated into the genomic DNA much more effectively than PCV. GCV was highly potent in inducing chromosomal aberrations compared with PCV, which provoked less sister chromatid exchanges and chromosomal changes using equimolar or equitoxic doses. For both agents, apoptosis was shown to be the major route of cell killing. Time course experiments revealed that neither genotoxicity nor apoptosis were induced within the cell cycle exposed to the drug; they are late events provoked in the following cell cycle(s). This indicates that the incorporation/exposure step of GCV or PCV into DNA is not decisive for triggering genotoxicity and apoptosis, but that events occurring subsequently, presumably during replication of a DNA containing the nucleotide analogs, are of major importance. Because PCV, unlike GCV, induced highly effectively apoptosis without exerting much genotoxicity, the use of PCV as a relatively safe alternative drug for suicide gene therapy of malignant diseases is recommended.

Ganciclovir-induced apoptosis in HSV-1 thymidine kinase expressing cells: critical role of DNA breaks, Bcl-2 decline and caspase-9 activation

“…The membranes were stained with Poencaue R to con®rm equal loading GCV is incorporated into the virus DNA thus blocking its replication. GCV becomes also incorporated into the genomic DNA of mammalian cells during replication (Rubsam et al, 1998;Thust et al, 2000a;Tomicic and Kaina, unpublished data). Unlike acyclovir (ACV), which is an absolute chain terminator, GCV only poorly inhibits replication of cellular DNA in the exposure cell cycle (Thust et al, 2000a).…”

“…GCV becomes also incorporated into the genomic DNA of mammalian cells during replication (Rubsam et al, 1998;Thust et al, 2000a;Tomicic and Kaina, unpublished data). Unlike acyclovir (ACV), which is an absolute chain terminator, GCV only poorly inhibits replication of cellular DNA in the exposure cell cycle (Thust et al, 2000a). GCV incorporation into DNA does not lead to genotoxic alterations per se.…”

“…The incorporated GCV however was not genotoxic by its own, but appeared to generate critical DNA lesions in the post-exposure cell cycle triggering genotoxicity (such as SCEs and aberrations) and apoptosis (Thust et al, 2000a). These secondary DNA lesions were hypothesized to be DNA double-strand breaks (DSBs) that were previously shown to trigger genotoxicity (Obe et al, 1985) and apoptosis (Lips and Kaina, 2001).…”

“…The wide application of the drug raises the question as to its genotoxic potential. GCV was found to be a potent recombinogenic and chromosomal breakage-inducing agent in metabolically competent cells expressing HSVtk (Thust et al, 2000a). Regarding the biochemical and molecular mechanism involved in GCV-induced genotoxicity, evidence has been provided that GCV is incorporated into the cellular DNA, thus causing sister-chromatid exchanges (SCEs) and chromosomal aberrations, reproductive cell death and apoptosis (Rubsam et al, 1998;Wei et al, 1998;Thust et al, 2000a,b).…”

Characterization of HIV-1 TAT peptide as an enhancer of HSV-TK/GCV cancer gene therapy

“…The cytotoxic mechanism of HSV-TK/GCV therapy is shown to be mediated mainly via apoptosis, and necrotic cell death has been shown to play a minor role. 30,31 However, during apoptosis, some of the intracellular proteins are degraded by proteases. This may raise a question whether also TAT-TK-GFP fusion proteins are degraded before their release into the extracellular milieu and uptake by the neighboring cells.…”