Subject(s)
Genes, Transgenic, Suicide/genetics , Genetic Therapy/adverse effects , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/therapeutic use , Antineoplastic Agents/toxicity , Genetic Vectors , Humans , Mutation , Prodrugs/pharmacokinetics , Prodrugs/toxicity , Safety , Virus Replication , Virus SheddingABSTRACT
BACKGROUND: Ganciclovir exhibits broad-spectrum activity against DNA viruses such as cytomegaloviruses, herpes simplex viruses, varicella-zoster virus, Epstein-Barr virus and human herpes virus-6. Ganciclovir is widely applied for anti-herpetic treatment, cytomegalovirus prophylaxis after organ transplantation, and, more recently, in experimental gene therapy to eradicate cycling cells that express the herpes simplex virus thymidine kinase gene. Although ganciclovir supposedly acts as a chain terminator, there is compelling evidence demonstrating the presence of ganciclovir, but not of acyclovir, incorporated internally into DNA, leaving the precise mechanism by which ganciclovir inhibits DNA synthesis enigmatic. METHODS: To study the potential involvement of mitochondria in the ganciclovir nucleotide cytotoxicity, we used adenovirus-mediated gene transfer to express herpes simplex virus thymidine kinase in rat liver and administered ganciclovir 2 days post-infection. The integrity and function of mitochondria in the rat liver cells were evaluated by several techniques. In addition, we analyzed the nucleotide pools in cellular extracts and in isolated mitochondria. RESULTS: We show that ganciclovir nucleotides are abundantly present in the mitochondria of rat livers that express the HSVtk gene. Already 48 h after administration, 10-30% of the total mitochondrial nucleotide pool consists of ganciclovir nucleotides. Their presence is correlated with a lower amount of mitochondrial DNA, a reduced mitochondrial-membrane potential, morphological abnormalities, and liver dysfunction. CONCLUSIONS: These data provide evidence for the involvement of mitochondria in the hepatotoxicity of the HStk/ganciclovir combination. This may explain the toxicity of the HSVtk/gancilovir combination in some metabolically active but non-proliferating cells, such as liver cells. This toxicity limits the applicability of this enzyme/prodrug combination.
Subject(s)
Antiviral Agents/adverse effects , Antiviral Agents/pharmacology , DNA, Mitochondrial/biosynthesis , Ganciclovir/adverse effects , Ganciclovir/pharmacology , Mitochondria/chemistry , Prodrugs , Simplexvirus/enzymology , Simplexvirus/genetics , Thymidine Kinase/genetics , Adenoviridae , Animals , Antiviral Agents/metabolism , Ganciclovir/metabolism , Gene Transfer Techniques , Genetic Therapy/adverse effects , Genetic Therapy/methods , Genetic Vectors , Liver/drug effects , Liver/pathology , Liver/ultrastructure , Male , Mitochondria/physiology , Phosphorylation , RatsABSTRACT
The chicken anemia virus-derived Apoptin protein shows remarkable specificity; namely, it induces apoptosis in tumor cells, but not in normal diploid cells. We have exploited the Apoptin gene for use in cancer gene therapy. Here we demonstrate that adenovirus-mediated intratumoral transfer and expression of the Apoptin gene results in regression or complete remission of human hepatomas grown as xenografts in immune-deficient mice, and significantly increases their survival long term. Early after intratumoral injection, Apoptin could be detected in significant quantities by Western blot analyses and immunohistochemistry. Furthermore, cell death and disruption of the tumor integrity were apparent in the transduced regions. This experimental gene therapeutic strategy constitutes a unique example of specific antitumor activity using a virus-derived gene with broad-spectrum applicability.
