Khanna K K, Beamish H, Yan J, Hobson K, Williams R, Dunn I, Lavin M F
Queensland Cancer Fund Research Unit, Queensland Institute of Medical Research, Bancroft Centre, Brisbane, Australia.
Oncogene. 1995 Aug 17;11(4):609-18.
We have previously demonstrated that cells from patients with ataxia-telangiectasia (A-T) fail to show initial delay at several cell cycle checkpoints post-irradiation. In addition a defect in the induction of p53 by ionizing radiation was evident. We demonstrate here that the radiation signal transduction pathway operating through p53, its target gene WAF1, cyclin-dependent kinases and the retinoblastoma (Rb) protein is defective in A-T cells. The defective p53 induction after ionizing radiation, observed previously in A-T cells, was also reflected at the functional level using p53-DNA binding activity, transactivation and transfection with wild type p53. Correction of the defect at the G1/S checkpoint was observed when wild type p53 was constitutively expressed in A-T cells. Exposure of control cells to radiation gave rise to p53 induction and as a consequence increased expression of WAF1 mRNA and protein, but A-T cells were defective in this response. As expected the WAF1 response in irradiated control cells resulted in an inhibition of cyclin-dependent kinase activity including cyclin E-cdk2, which plays an important role in the transition from G1 to S phase. No inhibition of cyclin-dependent kinase activity was observed in A-T cells correlating with the delayed WAF1 response. On the contrary an enhancement of cyclin-dependent kinase activity was seen in A-T cells post-irradiation. An accumulation of the hypophosphorylated form of Rb protein occurred in irradiated control cells compatible with the G1/S phase delay observed in these cells after exposure to radiation. In unirradiated A-T cells the amount of Rb protein was much higher compared to controls and it was mainly in the hyperphosphorylated (functionally inactive) form. In addition, accumulation of the hypophosphorylated form of Rb in A-T cells post-irradiation was defective, consistent with the lack of cell cycle arrest. Thus the failure of the G1/S checkpoint in A-T cells after exposure to ionizing radiation is consistent with a defective radiation signal transduction pathway operating through p53.
我们之前已经证明，共济失调毛细血管扩张症（A-T）患者的细胞在辐射后未能在几个细胞周期检查点表现出初始延迟。此外，电离辐射诱导p53明显存在缺陷。我们在此证明，通过p53、其靶基因WAF1、细胞周期蛋白依赖性激酶和视网膜母细胞瘤（Rb）蛋白起作用的辐射信号转导途径在A-T细胞中存在缺陷。先前在A-T细胞中观察到的电离辐射后p53诱导缺陷，在功能水平上也通过p53-DNA结合活性、反式激活以及用野生型p53转染得以体现。当野生型p53在A-T细胞中组成性表达时，观察到G1/S检查点的缺陷得到纠正。对照细胞暴露于辐射会导致p53诱导，结果WAF1 mRNA和蛋白表达增加，但A-T细胞在这种反应中存在缺陷。正如预期的那样，受辐射的对照细胞中的WAF1反应导致细胞周期蛋白依赖性激酶活性受到抑制，包括在从G1期到S期转变中起重要作用的细胞周期蛋白E-cdk2。在A-T细胞中未观察到细胞周期蛋白依赖性激酶活性受到抑制，这与WAF1反应延迟相关。相反，在辐射后的A-T细胞中观察到细胞周期蛋白依赖性激酶活性增强。在受辐射的对照细胞中出现了低磷酸化形式的Rb蛋白积累，这与这些细胞在暴露于辐射后观察到的G1/S期延迟相符。在未受辐射的A-T细胞中，Rb蛋白的量比对照细胞高得多且主要处于高磷酸化（功能无活性）形式。此外，辐射后A-T细胞中低磷酸化形式的Rb积累存在缺陷，这与缺乏细胞周期停滞一致。因此，A-T细胞在暴露于电离辐射后G1/S检查点的失效与通过p53起作用的缺陷辐射信号转导途径一致。
