MECHANISTIC STUDIES OF PMA AND FLAVOPIRIDOL INTERACTIONS

该项目的目标是确定一种新的治疗策略的机制基础，该策略结合了分化诱导剂（例如，PMA、苔藓抑素、丁酸钠等）与药理学细胞周期蛋白依赖性激酶抑制剂（例如，flavopiridol; FP）在白血病治疗中的应用。最近的证据表明，PMA和FP在白血病细胞中的共同给药显著诱导细胞凋亡，显著降低克隆形成，阻断PMA相关的成熟，并使CDKI p21（WAF 1/CIP 1）的表达失调。我们假设这些行动源于FP介导的破坏PMA诱导的分化和/或相关的G，逮捕程序。具体而言，我们认为FP通过以下方式干扰PMA相关成熟：1）阻断p21（WAF 1/CIP 1）的表达，这是一种凋亡抑制剂; 2）加速pRB去磷酸化，从而失调正常的G1阻滞事件; 3）破坏E2 F-1/pRB结合，从而促进触发凋亡的E2 F1相关基因的不适当表达。这些假设将在各种U937转染子和亲本白血病细胞系中进行测试。FP和其他分化诱导剂（如丁酸钠和苔藓抑素）之间的协同作用的证据也将寻求，并确定这些组合的共同特征。最后，我们将使用一系列的cDNA微阵列分析，以确定一系列的细胞凋亡，分化和细胞周期相关的基因，其表达可能会受到干扰PMA/FP共治疗。我们的最终目标是为联合分化诱导剂和CDK抑制剂提供合理的基础，最终可能转化为AML和其他恶性肿瘤的新治疗方案。

The goal of the project is to define a mechanistic basis for a novel therapeutic strategy combining differentiation-inducing agents (e.g., PMA, bryostatin, sodium butyrate, etc.) with pharmacologic cyclin-- dependent kinase inhibitors (e.g., flavopiridol; FP) in leukemia treatment. Recent evidence indicates that co-administration of PMA and FP in leukemic cells dramatically induces apoptosis, markedly reduces clonogenicity, blocks PMA-related maturation, and dysregulates expression of the CDKI p21(WAF1/CIP1). We hypothesize that these actions stem from FP-mediated disruptions in PMA-induced differentiation and/or the associated G, arrest program. Specfically, we propose that FP interferes with PMA-related maturation by 1) blocking the expression of p21(WAF1/CIP1), an inhibitor of apoptosis; 2) accelerating pRB dephosphorylation, thereby dysregulating normal G, arrest events, and 3) disrupting E2F-1/pRB binding, thereby promoting the inappropriate expression of E2F1-related genes that trigger apoptosis. These hypotheses will be tested in a variety of U937 transfectant and parental leukemia cell lines. Evidence of synergism between FP and other differentiation-inducing agents (e.g. sodium butyrate and bryostatin) will also be sought, and features common to these combinations identified. Finally, we will use serial cDNA microarray analysis to identify a spectrum of apoptosis -, differentiation-, and cell cycle-related genes whose expression may be perturbed by PMA/FP co-- treatment. Our ultimate goal is to provide a rational basis for combining differentiation-inducers and CDK inhibitors that may eventually be translated into novel treatment protocols for AML and other malignancies.