engleski

HSV-1 thymidine kinase overexpressing chinese hamster ovary cells are hypersensitive to antiherpes virostatic drugs undergoing apoptosis

Stable transfectants overexpressing the thymidine kinase gene (tk) of Herpes simplex virus type 1 were constructed by cotransfection of pHSV-TK (CMV promoter) and pSVneo plasmids into CHO-9 cells. Clones were selected for G418 resistance and checked for expression of the corresponding HSV-TK mRNA and protein by Northern and immunoblotting, respectively. The constructed HSV-TK+ transfectants were used as a sensitive model for studying the action mechanism of antiherpes virostatics, i.e. ganciclovir (GCV) and penciclovir (PCV), notably the end points reproductive cell death and apopptosis. Here we show that 3HGCV becomse more efficiently incorporated than 3HPCV into the DNA of HSV-TK+ cells. Both drugs are very efficient inducers of apoptosis. Thus, 1 mM GCV reduced cell survival to less than 1% and induced a high percentage of apoptotic cells, as measured 72 h after treatment. The corresponding equitoxic concentration of PCV was 5 mM, which induced a significant percentage of apoptosis. The frequency of necrotic cells, as measured by annexin-V/propidium iodide staining and flow cytometry, was not enhanced to the same extent indicating apoptosis to be the major route of cell death by GCV and PCV. Both agents also induced with high frequency chromosomal changes within the same dose range that was efficient in inducing apoptosis. In forthcoming work we are tempted to elucidate apoptosis-inducing signaling upon exposure of HSV-TK+ transfectants with virostatic drugs.

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