Differential sensitivity of p53(-) and p53(+) cells to caffeine-induced radiosensitization and override of G2 delay.
Differential sensitivity of p53(-) and p53(+) cells to caffeine-induced radiosensitization and override of G2 delay.
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DOI：
10.1016/0360-3016(95)97825-l
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发表时间：
1995-04
期刊：
影响因子：
11.2
通讯作者：
S. Powell;J. DeFrank;Paul Connell;M. Eogan;FredericI. Preffer;D. Dombkowski;Wei Tang;S. Friend
中科院分区：
文献类型：
--
作者：
S. Powell;J. DeFrank;Paul Connell;M. Eogan;FredericI. Preffer;D. Dombkowski;Wei Tang;S. Friend
目的大多数药物发现工作都集中在寻找新的DNA损伤剂以优先杀死肿瘤细胞。另一种方法是通过改变肿瘤对损伤的特异性反应来寻找增加肿瘤特异性杀伤力的方法。我们探讨了缺乏对X射线反应的G1/S停滞的细胞在用超过G2/M停滞的药物处理时是否对X射线损伤更敏感。材料和方法用咖啡因和不加咖啡因(一种已知的检查点抑制物)照射基因上匹配的(+/+)或(-/-)p53基因的小鼠胚胎成纤维细胞和通过转基因获得(+)或(-)野生型P53功能的大鼠胚胎成纤维细胞(REF)。咖啡因治疗从照射前1小时开始持续24小时。用克隆形成法检测电离辐射后细胞存活率。在细胞周期分析中，细胞在照射时处于指数级的非同步生长。记录照射前、照射后即刻、照射后3、6、9、12、24、48h细胞周期中G1期、S期和G2/M期细胞的比例。结果咖啡因在低剂量(0.5 mM)时可引起(-/-)细胞的放射敏感性，但对(+/+)细胞无明显影响。致敏增敏比(SER)分别为1.45和1.56(0.1和0.01)。在这种咖啡因剂量下，由于对(+/+)细胞没有可检测到的影响，这种SER反映了治疗效果。在咖啡因浓度为1 mM时，(-/-)细胞的敏感度为1.77，但(+/+)细胞也表现出敏化(SER=1.25)。在(-/-)细胞中，咖啡因浓度为0.1 mM时，SER为1.5，存活率为0.01。在0.5 mM和2 mM咖啡因作用下，转基因的REF细胞也表现出对咖啡因的放射增敏作用，2 mM的SER=1.45，存活率为0.1。在相同剂量的咖啡因下，REF细胞未表现出明显的致敏作用。携带野生型P53基因的REF细胞单独转染pCMVneo后，放射敏感性无明显变化，细胞周期中G1/S期阻滞无明显变化。在不含咖啡因的情况下，以及在含有0.5和2 mM咖啡因的情况下，检测4或8GyX射线对细胞周期检查点的影响。在(+/+)细胞中，G_1/S和G_2/M期被阻滞，咖啡因对这两个检查点均无明显影响。相反，(-/-)细胞在0.5 mM咖啡因作用下，G2/M期细胞明显减少(50%)，而在2 mM咖啡因作用下则完全被抑制。这些数据表明，缺乏P53的细胞被低剂量的咖啡因敏化，其G2/M检查点发生改变，这从咖啡因对G2/M超载的不同影响可以看出。也对有和没有功能性P53的肿瘤细胞进行了评估。初步数据显示，可以得出类似的结论。低剂量咖啡因仅对携带E6基因的MCF7细胞有增敏作用。结论咖啡因对P53(-)细胞具有较强的放射增敏作用，提示已被证明控制G1/S检查点的P53也可能影响G2/M期阻滞。这些数据表明，在缺乏功能性p53的肿瘤中，通过将DNA损伤剂与扰乱G2/M期停滞的化合物相结合，有机会获得治疗收益。P53在G2/M期转化中的作用尚不明确。
PurposeMost drug discovery efforts have focused on finding new DNA damaging agents to kill tumor cells preferentially. An alternative approach is to find ways to increase tumor specific killing by modifying tumor specific responses to that damage. We asked whether cells lacking the G1/S arrest in response to X-rays are more sensitive to X-ray damage when treated with agents that override G2/M arrest.Materials and MethodsMouse embryonic fibroblasts genetically matched to be (+/+) or (-/-) p53 and rat embryonic fibroblasts (REF) made (+) or (-) for wild-type p53 function by transfection were irradiated with and without caffeine, a known checkpoint inhibitor. Caffeine treatment was maintained for 24 hours from 1 hour prior to irradiation. Cell survival following ionizing radiation was measured by clonogenic assay. For cell-cycle analysis, cells were in exponential asynchronous growth at the time of irradiation. The proportion of cells in G1, S and G2/M phases of the cell cycle were recorded immediately before and following irradiation and subsequently at 3, 6, 9, 12, 24 and 48 hours following irradiation.ResultsCaffeine was found to cause radiosensitzation at low dose (0.5 mM) in (-/-) cells but not in (+/+) cells. The sensitization enhancement ratio (SER) was 1.45 at 0.1 survival and 1.56 at 0.01 survival. At this dose of caffeine, this SER reflected therapeutic gain as there was no detectable effect on (+/+) cells. At 1mM caffeine, sensitization of (-/-) cells was 1.77, but (+/+) cells now also showed sensitization (SER= 1.25). In (-/-) cells at 0.1 mM caffeine the SER was 1.5 at 0.01 survival. The transfected REF cells (functionally for p53) also exhibited caffeine-induced radiosensitization at both 0.5 and 2mM caffeine with a SER= 1.45 for 2mM at 0.1 survival. No significant sensitization could be demonstrated for REF cells at the same doses of caffeine. The REF cells, with wild-type p53, transfected with pCMVneo alone showed no change in radiosensitivity or G1/S arrest. Cell cycle checkpoint arrest in response to 4 or 8 Gy X-rays was measured without caffeine and with 0.5 and 2mM caffeine. In (+/+) cells, G1/S and G2/M arrest was seen and there was no demonstrable impact of caffeine at either dose on either checkpoint. By contrast (-/-) cells showed a clear reduction (50%) of the size of G2/M arrest at 0.5 mM caffeine and complete override at 2mM caffeine. These data imply that cells which lack p53, are sensitized by low-dose caffeine and their G2/M checkpoint is altered, seen by the different effects of caffeine upon G2/M override. Tumor cells which do and do not have functional p53 have also been evaluated. Preliminary data suggest a similar conclusion can be drawn. MCF-7 cells transfected with control plasmid or plasmids containing the gene for HPV-E6 protein also show sensitization with low dose caffeine only in cells which have E6.ConclusionsThe greater caffeine-induced radiosensitization in p53 (-) cells suggests that p53, already shown to control the G1/S checkpoint, may also influence aspects of G2/M arrest. These data indicate an opportunity for therapeutic gain by combining DNA damaging agents with compounds that disrupt G2/M arrest in tumors lacking functional p53. The role of p53 in G2/M transition remains to be defined.