Bcl-2 overexpression does not prevent but retards adriamycin toxicity in CC531 colon carcinoma cells.

BACKGROUND: The Bcl-2 protein is a critical regulator of susceptibility towards cell death induced by antineoplastic drugs. Reduced growth activity and increased glutathione (GSH) levels protect against adriamycin toxicity. We recently demonstrated statistically significantly reduced growth activity and elevated cellular GSH levels in exponentially growing rat CC531 colon carcinoma cells overexpressing the full-length human Bcl-2 protein (CCbcl2#A3). METHODS: To assess the importance of reduced growth activity or increased GSH levels, we determined the mitochondrial function, 24 h after adriamycin treatment, in CCbcl2#A3 cells, parental CC531 cells and cells overexpressing the Bcl-2 protein lacking the N-terminal BH4 domain (CC Delta BH4): these latter cells contained elevated cellular GSH levels but were not reduced in growth activity. RESULTS: CCbcl2#A3, but not CC Delta BH4, cells were 3-fold less susceptible than parental cells suggestive of a protective role for reduced growth but not for increased GSH levels in BCL-2 transfectants. This was confirmed in several growth-inhibited CC531 transfectants and in slowly proliferating (ca. 100% confluent) cell populations compared to exponentially growing (ca. 50% confluent) cell populations. Reduced growth activity might delay the onset of cell death. Therefore, we tested the effect of adriamycin five days after treatment. In this long-term assay we found no differences between the various cells. CONCLUSION: Reduction of growth activity, for instance by an overexpression of the Bcl-2 protein, only transiently reduced the susceptibility towards adriamycin treatment.

Ganciclovir nucleotides accumulate in mitochondria of rat liver cells expressing the herpes simplex virus thymidine kinase gene.

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.