In parallel to delayed reproductive death and delayed micronuclei in surviving V79 cells that have been reported, the frequency of apoptosis and dicentrics was investigated in the same cell line, 1-2 weeks after X-ray doses between 1 and 12 Gy. Apoptosis was assessed by morphological criteria after staining with Giemsa and using the Apoptag method in parallel. The frequency of apoptosis peaked 48 h after irradiation at about 35% as a function of dose and then decreased to about 10% on day 3. It remained 10 times higher than in unirradiated cells until day 14. The frequency of dicentric chromosomes 24 h after irradiation increased according to a linear-quadratic function, up to about 110% after 12 Gy, decreased 3 days later to about 10% and remained about 5-fold higher than in control cells. The dose-response relationship was similar for all the various end-points of expression of radiation induced genomic instability, showing a steep increase of the frequency with dose up to 3-4 Gy and with no further increase at higher radiation doses.

The inhibitor of apoptosis wild-type survivin is a multifunctional protein that suppresses apoptosis and regulates cell cycle progression. An association between wild-type survivin expression and radiosensitivity has been described in different tumor cells. The effects of siRNA-induced knockdown of wild-type survivin and survivin-splice variants survivin-2B and survivin-D3 were investigated under normoxic and hypoxic conditions in the human sarcoma cell line US 8-93 (mutant p53). Inhibition of the survivin isoforms by siRNA resulted in a decrease of target mRNA down to 14-70% compared to cells treated with control siRNA independent of the oxygen level. The mRNA expression of survivin isoforms was decreased by the factor of 1-12 when the cells were cultivated under hypoxic conditions. Moreover, the knockdown of wild-type survivin reduced colony formation independent of oxygen concentration down to 70% and induced formation of polyploid cells. Less reduction of plating efficiency was observed after specific knockdown of survivin-2B and survivin-D3 under hypoxic or normoxic conditions. A knockdown of wild-type survivin, survivin-D3 and survivin-2B isoforms in combination with irradiation caused no radiosensitization in cell line US 8-93, neither under hypoxic nor under normoxic conditions tested in the colony-forming assay. However, knockdown of wild-type survivin caused radiosensitization in the megacolony assay.

V79 Chinese hamster cells were irradiated with doses of 1-12 Gy 300 kV X-rays. Their colony-forming ability and the frequency of micronuclei in binucleate cells after treatment with cytochalasin B was determined at various times after irradiation. The frequency of micronuclei determined within the first 24 h after irradiation increased with doses up to 4 Gy and decreased as the dose increased further. Up to 4 Gy there was a close correlation between surviving fraction and the fraction of cells without micronuclei although the surviving fraction was 2-3 times lower than the fraction of cells without micronuclei. Six, 10 or 13 days after irradiation with either 9 or 12 Gy the plating efficiency and the frequency of micronuclei after cytokinesis block with cytochalasin B was determined in the irradiated, but surviving, cells. The delayed plating efficiency of irradiated cells was significantly decreased. The proportion of binucleated cells was in the normal range at 6-13 days after irradiation, indicating that the proliferative activity of irradiated but surviving cells was not depressed at that time. The micronucleus frequency, however, was significantly increased at all times after irradiation. There was little heterogeneity of plating efficiency and micronucleus frequency among 12 clones which had been isolated for irradiated cultures, 3 weeks after 12 Gy.

No abstract