Inhibition of G1-phase arrest induced by ionizing radiation in hematopoietic cells by overexpression of genes involved in the G1/S-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 n 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 n 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.
Epperly
M
M
Department of Radiation Oncology, University of Pittsburgh Medical Center, Pennsylvania 15213, USA.
Berry
L
L
Halloran
A
A
Greenberger
J S
JS
D000818:Animals; D002460:Cell Line; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases; D016213:Cyclins / Q000235:genetics*; D016193:G1 Phase / Q000528:radiation effects*; D005786:Gene Expression Regulation; D006412:Hematopoietic Stem Cells / Q000528:radiation effects*; D007377:Interleukin-3 / Q000502:physiology; D051379:Mice; D008809:Mice, Inbred C3H; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D011518:Proto-Oncogene Proteins
D016213:Cyclins; D007377:Interleukin-3; D011518:Proto-Oncogene Proteins; D017346:Protein Serine-Threonine Kinases; C495902:Cdk4 protein, mouse; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases