CELL CYCLE CHECKPOINTS AND CHEMOSENSITIVITY OF HUMAN CANCER CELLS

本研究的重点是细胞周期检查点及其作用。 这些系统在确定化疗敏感性方面发挥了作用。我们的目标还包括 利用我们对这些检查点系统的新知识来设计新的 癌症治疗的治疗策略。我们正在澄清 对DNA损伤做出反应以阻止G1和G2中细胞的检查点 阶段。我们还定义了癌细胞中这些系统的缺陷。我们 目的是从DNA损伤或未复制的角度追踪这些系统 DNA被细胞感知到与细胞相互作用的反应元件 细胞周期蛋白依赖性激酶阻止细胞周期进程或在某些情况下 诱导细胞凋亡。我们的结果表明，淋巴瘤细胞中存在P53突变 品系降低了对DNA损伤剂的敏感性。但不是 抗有丝分裂药物。敏感度降低与逃避 P53介导的细胞凋亡，尽管其他因素也可以促进 结果。我们一直在探索p53调控基因中的区域。 与细胞周期蛋白E/CDK2和增殖细胞核抗原相互作用的产物Waf1/Cip1。我们的 结果表明，Waf1/Cip1的氨基末端有两个区域 参与细胞周期蛋白E/CDK2相互作用和/或抑制该激酶 而Waf1/Cip1的羧基末端区域参与了增殖细胞核抗原 互动。G2检查点似乎保护细胞免受DNA损伤 通过延长DNA修复的时间。我们发现， 己酮可可碱优先取消细胞中的G2检查点 被破坏的p53和这样的细胞可以优先被组合杀死 一种DNA损伤剂和己酮可可碱。我们目前正在探索 这些活动背后的机制，特别侧重于 G2细胞周期蛋白依赖性激酶的形成和激活。我们也是 调查几个可能在临床上使用的G2检查点删除者 在优先杀死突变型p53肿瘤的联合方案中很有用。 结合其他几个实验室，我们发现 NCI细胞筛选中的大多数细胞系都扰乱了P53的功能。 我们还发现，这些p53基因突变的细胞往往对 目前使用的化疗药物种类繁多。一个值得注意的问题 这项分析中的例外是微管制剂，它的活性 似乎与P53状态无关。搜索45,000个复合数据 BASE揭示了一些可能优先活跃在 P53基因被破坏的细胞。这些代理人将接受详细的分析 P53功能被干扰的同源细胞系 HPV-E6基因或显性阴性突变体P53的转染法 吉恩。

This research project focuses on cell cycle checkpoints and the roles these systems play in determining chemosensitivity. We also aim to utilize our emerging knowledge of these checkpoint systems to design new therapeutic stratagems for cancer treatment. We are elucidating the checkpoints that respond to DNA damage to arrest cells in G1 and G2 phases. We are also defining defects in these systems in cancer cells. We aim to trace these systems from the point where DNA damage or unreplicated DNA is sensed by the cell to the response elements that interact with the cyclin-dependent kinases to arrest cell cycle progression or in some cases induce apoptosis. Our results suggest that p53 mutations i lymphoma cell lines confer decreased sensitivity to DNA damaging agents. but not antimitotic agents. Decreased sensitivity is related to an evasion of p53-mediated apoptosis, although other factors can also contribute to the outcome. We have been exploring domains in the p53 regulated gene product, Waf1/Cip1, that interact with cyclin E/Cdk2 and PCNA. Our results suggest two regions in the amino terminus of Waf1/Cip1 are involved in cyclin E/Cdk2 interaction and/or inhibition of this kinase while the carboxy-terminal region of Waf1/Cip1 is involved in PCNA interaction. The G2 checkpoint appears to protect cells from DNA damage by extending the time available for DNA repair. We have found that pentoxifylline preferentially abrogates the G2 checkpoint in cells with disrupted p53 and such cells can be preferentially killed by a combination of a DNA damaging agent and pentoxifylline. We are presently exploring the mechanisms underlying these activities with a special focus on the formation and activation of the G2 cyclin-dependent kinases. We are also investigating several G2 checkpoint abrogators that might be clinically useful in combination protocols to preferentially kill mutant p53 tumors. In conjunction with several other laboratories we have found that the majority of cell lines in the NCI cell screen have disrupted p53 function. We have also found these p53 disrupted cells tend to be less sensitive to a wide variety of currently used chemotherapeutic agents. A notable exception in this analysis was the microtubule agents, whose activity appeared independent of p53 status. Searching the 45,000 compound data base revealed a number of agents which might be preferentially active in cells with disrupted p53. These agents will undergo detailed analysis in isogeneic cell lines in which p53 function has been disrupted by transfection with either the HPV-E6 gene or a dominant negative mutant p53 gene.