Flavopiridol Mediated Apoptosis During S Phase

描述（由申请人提供）：细胞周期蛋白依赖性激酶（cdk）抑制剂flavopiridol在许多实体瘤细胞系中诱导细胞周期停滞。然而，细胞在通过同步化或通过用施加S期延迟的化疗剂处理募集到S期后对flavopiridol敏感。吉西他滨与夫拉吡醇的组合产生序列依赖性细胞毒性协同作用。预期在S期期间Flavopiridol介导的cdk抑制可防止E2 F-1活性的适当定时中和，从而导致凋亡反应。在第一个具体目标中，将检查夫拉普利醇在吉西他滨处理的细胞中的作用，以确定在S期穿越期间E2 F-1表达的不适当持续性。在flavopiridol存在下E2 F-1的磷酸化降低将使用正磷酸盐标记的细胞以及针对cdk 2-和cdk 7-磷酸化位点的磷酸特异性抗体来证实。还将测定在夫拉吡醇存在下E2 F-1的半衰期和转录活性。此外，靶向E2 F-1的siRNA将被引入肿瘤细胞中，以证实吉西他滨募集至S期后flavopiridol诱导的细胞凋亡是E2 F-1依赖性的。保留DNA结合活性但缺乏转录反式激活活性的E2 F-1的显性负突变体将被诱导表达，以确定后者是否是S期期间flavopiridol介导的细胞凋亡所需的。此外，还将检查E2 F-1与NF-κ B途径组分的相互作用。在第二个具体目标中，将用吉西他滨和夫拉匹醇治疗异种移植荷瘤小鼠，以确认组合的序列依赖性并确定两种药物之间的最佳间隔。还将在巴雷特相关食管癌的p27 Kip 1缺陷动物模型中测试组合的活性。将对异种移植物和鼠肿瘤进行免疫组织化学，以确定适当的细胞周期蛋白及其磷酸化形式，以确认flavopiridol对cdk的抑制作用。在第三个具体目标中，将在晚期实体瘤受试者中进行吉西他滨随后flavopiridol的I期试验。将采用一种新的flavopiridol方案，旨在达到持续的微摩尔浓度，这是cdk抑制所必需的。除了药代动力学分析外，该试验还将纳入肿瘤组织、皮肤和血浆中flavopiridol活性的药效学终点。

DESCRIPTION (provided by applicant): The cyclin-dependent kinase (cdk) inhibitor flavopiridol induces cell cycle arrest in many solid tumor cell lines. However, cells are sensitized to flavopiridol following recruitment to S phase by synchronization or by treatment with chemotherapy agents that impose S phase delay. The combination of gemcitabine, followed by flavopiridol, produces sequence-dependent cytotoxic synergy. Flavopiridol-mediated cdk inhibition during S phase is expected to prevent the appropriately timed neutralization of E2F-1 activity, resulting in an apoptotic response. In the first specific aim, the effects of flavopiridol in gemcitabine-treated cells will be examined to establish the inappropriate persistence of E2F-1 expression during S phase traversal. Reduced phosphorylation of E2F-1 in the presence of flavopiridol will be confirmed using cells labeled with orthophosphate as well as with phospho-specific antibodies directed at cdk2- and cdk7-phosphorylation sites. The half-life and transcriptional activity of E2F-1 in the presence of flavopiridol will also be determined. Furthermore, siRNAs targeting E2F-1 will be introduced into tumor cells to confirm that flavopiridol-induced apoptosis following recruitment to S phase by gemcitabine is E2F-1-dependent. A dominant negative mutant of E2F-1, retaining DNA binding activity, but lacking transcriptional transactivation activity, will be inducibly expressed to determine if the latter is required for flavopiridol-mediated apoptosis during S phase. In addition, the interaction of E2F-1 with components of the NF-kappaB pathway will be examined. In the second specific aim, xenograft-bearing mice will be treated with gemcitabine and flavopiridol to confirm the sequence dependence of the combination and to determine the optimal interval between the two drugs. The activity of the combination will also be tested in a p27Kip1-deficient animal model of Barrett's-associated esophageal carcinoma. Xenografts and murine tumors will be subjected to immunohistochemistry for appropriate cell cycle proteins and their phosphorylated forms to confirm cdk inhibition by flavopiridol. In the third specific aim, a phase I trial of gemcitabine followed by flavopiridol will be performed in subjects with advanced solid tumors. A novel flavopiridol schedule will be employed, designed to achieve sustained micromolar concentrations that are necessary for cdk inhibition. In addition to pharmacokinetic analyses, the trial will incorporate pharmacodynamic endpoints for flavopiridol activity in tumor tissue, skin and plasma.