Inhibition of G₁-Phase Arrest Induced by Ionizing Radiation in Hematopoietic Cells by Overexpression of Genes Involved in the G₁S-Phase Transition

D-type cyclins and cyclin-dependent kinase (cdk-4) are likely involved in regulating passage of cells through the G₁ phase of the cell cycle. A decrease in the proportion of cells in G₁, a relatively radiation-sensitive phase of the cell cycle, should result in increased resistance to ionizing radiation; however, the effect of such overexpression on X-ray-induced G₁-phase arrest is not known. Radiation survival curves were obtained at a dose rate of either 8 cGy/min or 1 Gy/min for subclones of the IL-3-dependent hematopoietic progenitor cell line 32D cl 3 expressing transgenes for either cyclin-D1, D2 or D3 or cdk-4. We compared the results to those with overexpression of the transgene for Bcl-2, whose expression enhances radiation survival and delays apoptosis. Cells overexpressing transgenes for each D-type cyclin or Bcl-2 had an increased number of cells in S phase compared to parent line 32D cl 3; however, overexpression of cdk-4 had no effect on cell cycle distribution. Cell death resulting from withdrawal of IL-3 was not affected by overexpression of cyclins D1 and D3 but was delayed by overexpression of D2, cdk-4 or Bcl-2. Flow cytometry 24 h after 5 Gy irradiation demonstrated that overexpression of each G₁-phase regulatory transgene decreased the proportion of cells at the G₁/ S-phase border. Western analysis revealed induction of cyclin-D protein levels by irradiation, but no change in the levels of cdk-4, p53 or p21. There was no significant change in the D₀, but a significant increase in the n̄ for cyclin-D or cdk-4 transgene-overexpressing clones at 1 Gy/min (P < 0.017). At a lower dose rate of 8 cGy/min, the n̄ for cyclin or cdk-4-overexpressing clones was also increased (P < 0.07). Thus overexpression of cyclin-D or cdk-4 in hematopoietic cells induces detectable effects on hematopoietic cell radiation biology including a broadening of the shoulder on the radiation survival curve and a decrease in radiation-induced G₁/ S-phase arrest.