Gamma-irradiation is a usual method to inactivate whole-cellular anticancer vaccines consisting viable tumor cells. To evaluate the effect of gamma-irradiation to transgene expression in tumor cells we constructed several stably transfected clones of human and mouse cell lines expressing transgenic GM-CSF or GFP under control of IE-CMV promoter. Irradiation of those cells with different doses (ranged from 20 to 100 Gr) of gamma-radiation caused loss of proliferation capacity with survival of the cells population clearly depended on irradiation dose. Cell-cycle staining reveals accumulation of the cells with G2/M DNA content and almost loss of cells in S-phase. Substantial proportion of irradiated cells shows beta-galactosidase activity and morphological changes associated with cell senescence. An irradiated cell shows no changes in the level of mitochondrial dehydrogenase activity regardless irradiation dose exposed. Irradiated cells retain their ability to express transgene. Moreover, amount of the secreted GM-CSF as well as MFI in GFP-expressing cells significantly increases after gamma-irradiation up to 10 fold for cells exposed with 100 Gr. Enhancing of the transgene expression in both human and mouse cells positively correlates with total dose of gamma-irradiation gained by the cells and demonstrates gradual nature. Overall, our results supports using of 100 Gr of gamma-irradiation as the optimal dose for whole-cell anticancer vaccine inactivation.