Flavopiridol Mediated Apoptosis During S Phase

描述(申请人提供)：细胞周期蛋白依赖性激酶(CDK)抑制剂黄烷醇在许多实体肿瘤细胞系中诱导细胞周期停滞。然而，细胞在被招募到S时相后，通过同步或通过化疗药物治疗而使S时相延迟，从而使细胞对黄烷醇增敏。吉西他滨与黄烷醇的组合产生顺序依赖的细胞毒性协同作用。在S期抑制黄烷醇介导的cdk有望阻止适时中和E2F-1活性，导致细胞凋亡反应。在第一个特定目标中，我们将检测黄烷醇对吉西他滨处理的细胞的影响，以确定在S时相穿越过程中E2F-1表达的不适当持续。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，将证实在黄烷醇存在的情况下E2F-1的磷酸化减少。还将测定E2F-1在黄烷醇存在下的半衰期和转录活性。此外，针对E2F-1的siRNA将被引入肿瘤细胞中，以证实吉西他滨诱导S期细胞凋亡是依赖于E2F-1的。E2F-1的一个显性负性突变体将被诱导表达，该突变体保留了DNA结合活性，但缺乏转录反式激活活性，以确定在S期是否需要后者参与黄吡啶诱导的细胞凋亡。此外，还将研究E2F-1与核因子-kappaB途径的组成部分之间的相互作用。在第二个特定目标中，将用吉西他滨和黄烷醇治疗异种移植瘤小鼠，以确认联合用药的顺序依赖性，并确定两种药物之间的最佳间隔。该组合的活性也将在p27Kip1缺失的Barrett相关食道癌动物模型中进行测试。将对异种移植瘤和小鼠肿瘤进行适当的细胞周期蛋白及其磷酸化形式的免疫组织化学检查，以确认黄烷醇对CDK的抑制作用。在第三个具体目标中，吉西他滨和黄烷醇的I期试验将在晚期实体肿瘤受试者中进行。将采用一种新的黄烷醇时间表，旨在达到抑制CDK所必需的持续微摩尔浓度。除了药代动力学分析外，该试验还将纳入肿瘤组织、皮肤和血浆中黄烷醇活性的药效学终点。

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.