Flavopiridol Mediated Apoptosis During S Phase in NSCLC

说明：（申请人的摘要）Flavopiridol是第一个有效的抑制剂 细胞周期蛋白依赖性激酶（CDKs）进入临床试验。临床前数据 表明对非小细胞肺癌（NSCLC）有希望活性， 这种疾病迫切需要新的药物。在大多数NSCLC细胞 细胞毒性仅在高浓度下长时间暴露后发生。 这些药物浓度在体内难以达到。然而，在这方面， Flavopiridol处理细胞在早期S期，从一个 羟基脲诱导的阻滞在G1/S边界，导致更早，显着 增强细胞死亡。类似地，在S 由化疗剂如顺铂或吉西他滨施加的相位延迟 导致细胞凋亡增强，因此存在序列依赖性协同作用。 在第一个具体目标中，将分析BrdU标记细胞的命运， 细胞凋亡测定以确定S期细胞是否是最敏感的 到flavopiridol。此外，将检查适当配对的细胞系 为了确定在S期Flavopiridol介导的凋亡是否 对转化细胞的选择性以及它是否需要p53。在第二 针对S相敏感性的机理进行了研究。数据 将产生支持一个模型，其中flavopiridol介导的细胞周期蛋白 在S期期间A-cdk 2抑制阻止E2 F-1的磷酸化及其在细胞中的表达。 伴侣DP-1，导致不适当的持续E2 F-1活性， 凋亡细胞周期蛋白A激酶活性以及E2 F-1磷酸化及其 反式激活和DNA结合活性将在 在募集至S期后，用flavopiridol处理的细胞。E2 F-1活性 将被野生型和突变物种的异位表达改变， 确定在S期对flavopiridol的凋亡反应是否是 影响。此外，flavopiridol介导的细胞凋亡将与cdk 2 通过产生耐夫拉吡醇细胞系和通过 保留活性但具有改变的ATP结合的cdk 2突变体的表达 网站.在第三个具体目标，毒性和潜在的抗肿瘤 吉西他滨，一种S期特异性抗肿瘤药物， 药物，然后是flavopiridol，将在I期临床试验中进行评估。 将进行体外研究，以确定flavopiridol是否影响 吉西他滨的代谢。两者的药代动力学相互作用 还将探索患者的药物。

DESCRIPTION: (Applicant's Abstract) Flavopiridol is the first potent inhibitor of cyclin-dependent kinases (cdks) to enter clinical trial. Preclinical data suggest promising activity against non-small cell lung cancer (NSCLC), a disease for which there is a pressing need for new agents. In most NSCLC cell lines, cytotoxicity only occurs after prolonged exposures at high concentrations of drug that are difficult to achieve in vivo. However, flavopiridol treatment of cells during early S phase, following release from a hydroxyurea-induced block at the G1/S boundary, results in earlier, markedly enhanced cell death. Similarly, treatment with flavopiridol following an S phase delay imposed by chemotherapeutic agents such as cisplatin or gemcitabine results in enhanced apoptosis, so that there is sequence-dependent synergism. In the first specific aim, the fate of BrdU-labeled cells will be analyzed in apoptosis assays to determine whether cells in S phase are the most sensitive to flavopiridol. In addition, appropriately paired cell lines will be examined to determine whether flavopiridol-mediated apoptosis during S phase is selective for transformed cells and whether it requires p53. In the second specific aim, the mechanism of S phase sensitivity will be investigated. Data will be generated to support a model in which flavopiridol-mediated cyclin A-cdk2 inhibition during S phase prevents the phosphorylation of E2F-l and its partner DP-1, resulting in inappropriately persistent E2F-1 activity and apoptosis. Cyclin A-kinase activity, as well as E2F-1 phosphorylation and its transactivation and DNA binding activities will be assessed in flavopiridol-treated cells following recruitment to S phase. E2F-1 activity will be altered by ectopic expression of wild-type and mutant species to determine whether the apoptotic response to flavopiridol during S phase is affected. Furthermore, flavopiridol-mediated apoptosis will be linked to cdk2 inhibition by the generation of flavopiridol-resistant cell lines and by expression of cdk2 mutants that retain activity but harbor altered ATP binding sites. In the third specific aim, the toxicity and potential anti-tumor activity of the sequential combination of gemcitabine, an S phase-specific agent, followed by flavopiridol, will be assessed in a phase I clinical trial. In vitro studies will be performed to determine whether flavopiridol affects the metabolism of gemcitabine. The pharmacokinetic interactions of the two drugs in patients will also be explored.