Flavopiridol Mediated Apoptosis During S Phase

黄吡醇介导 S 期细胞凋亡

• 批准号: 7032446
• 负责人: GEOFFREY I SHAPIRO
• 金额: $ 32.25万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-01 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7032446
• 关键词: antineoplastics; apoptosis; cell cycle proteins; clinical research; clinical trial phase I; combination chemotherapy; cyclin dependent kinase; drug interactions; enzyme activity; enzyme inhibitors; flavopiridol; gemcitabine; gene expression; human subject; human therapy evaluation; immunocytochemistry; laboratory mouse; neoplasm /cancer; neoplasm /cancer chemotherapy; neoplasm /cancer pharmacology; patient oriented research; pharmacokinetics; phosphorylation; protein protein interaction; transcription factor

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.

描述（由申请人提供）：周期蛋白依赖性激酶（cdk）抑制剂黄匹吡醇在许多实体肿瘤细胞系中诱导细胞周期阻滞。然而，通过同步或使用化疗药物延迟S期，细胞在S期募集后对黄吡醇敏感。吉西他滨与黄吡醇联合使用可产生序列依赖性的细胞毒性协同作用。在S期黄烷醇介导的cdk抑制有望阻止适当时间的E2F-1活性中和，导致凋亡反应。在第一个特定目的中，将检测黄匹吡醇对吉西他滨处理的细胞的影响，以确定在S期穿越期间E2F-1表达的不适当持久性。使用正磷酸盐标记的细胞以及针对cdk2-和cdk7磷酸化位点的磷酸化特异性抗体，将证实黄吡醇存在下E2F-1磷酸化的降低。E2F-1在黄吡醇存在下的半衰期和转录活性也将被确定。此外，靶向E2F-1的sirna将被引入肿瘤细胞，以证实黄嘌呤诱导的细胞凋亡在吉西他滨募集到S期后是E2F-1依赖性的。E2F-1的显性负突变体保留DNA结合活性，但缺乏转录反激活活性，将被诱导表达，以确定后者是否需要黄烷醇介导的S期凋亡。此外，E2F-1与NF-kappaB通路组分的相互作用将被研究。在第二个特定目标中，将使用吉西他滨和黄匹吡醇治疗异种移植小鼠，以确认组合的序列依赖性，并确定两种药物的最佳间隔。该组合的活性也将在p27kip1缺失的巴雷特相关食管癌动物模型中进行测试。异种移植物和小鼠肿瘤将通过免疫组化检测合适的细胞周期蛋白及其磷酸化形式，以证实黄匹吡醇对cdk的抑制作用。在第三个具体目标中，吉西他滨和黄匹吡醇的I期临床试验将在晚期实体瘤患者中进行。一种新的黄匹吡醇计划将被采用，旨在达到持续的微摩尔浓度，这是抑制cdk所必需的。除了药代动力学分析外，该试验还将纳入黄吡醇在肿瘤组织、皮肤和血浆中的药效学终点。