Epperly M, Berry L, Halloran A, Greenberger J S
Department of Radiation Oncology, University of Pittsburgh Medical Center, Pennsylvania 15213, USA.
Radiat Res. 1995 Sep;143(3):245-54.
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.
D型细胞周期蛋白和细胞周期蛋白依赖性激酶（cdk - 4）可能参与调控细胞通过细胞周期的G1期。G1期是细胞周期中相对对辐射敏感的阶段，G1期细胞比例的降低应导致对电离辐射的抗性增加；然而，这种过表达对X射线诱导的G1期阻滞的影响尚不清楚。对于表达细胞周期蛋白D1、D2或D3或cdk - 4转基因的IL - 3依赖性造血祖细胞系32D cl 3的亚克隆，以8 cGy/分钟或1 Gy/分钟的剂量率获得了辐射存活曲线。我们将结果与过表达Bcl - 2转基因的结果进行了比较，Bcl - 2的表达可提高辐射存活率并延迟细胞凋亡。与亲本细胞系32D cl 3相比，过表达每种D型细胞周期蛋白或Bcl - 2转基因的细胞S期细胞数量增加；然而，cdk - 4的过表达对细胞周期分布没有影响。因撤除IL - 3导致的细胞死亡不受细胞周期蛋白D1和D3过表达的影响，但受D2、cdk - 4或Bcl - 2过表达的延迟。5 Gy照射24小时后的流式细胞术显示，每种G1期调节转基因的过表达降低了处于G1/S期边界的细胞比例。蛋白质免疫印迹分析显示照射可诱导细胞周期蛋白D蛋白水平升高，但cdk - 4、p53或p21的水平没有变化。D0没有显著变化，但在1 Gy/分钟时，细胞周期蛋白D或cdk - 4转基因过表达克隆的n显著增加（P < 0.017）。在较低剂量率8 cGy/分钟时，细胞周期蛋白或cdk - 4过表达克隆的n也增加了（P < 0.07）。因此，造血细胞中细胞周期蛋白D或cdk - 4的过表达对造血细胞辐射生物学产生了可检测的影响，包括辐射存活曲线上肩部变宽以及辐射诱导的G1/S期阻滞减少。
