MECHANISTIC STUDIES OF PMA AND FLAVOPIRIDOL INTERACTIONS

该项目的目标是确定一种结合分化诱导剂(例如PMA、Bryostatin、丁酸钠等)的新治疗策略的机制基础。在白血病治疗中使用药物周期素依赖的激酶抑制剂(如黄吡哆醇；FP)。最近的证据表明，联合应用PMA和FP可显著诱导白血病细胞的凋亡，显著降低克隆形成能力，阻断PMA相关的成熟，并抑制CDKI p21(WAF1/CIP1)的表达。我们推测，这些作用源于FP介导的PMA诱导分化的中断和/或相关的G_1抑制程序。具体地说，我们认为FP通过以下方式干扰PMA相关的成熟：1)阻断p21(WAF1/CIP1)的表达；2)促进pRb去磷酸化，从而扰乱正常的G期、停滞事件；3)破坏E2F-1/pRb结合，从而促进触发细胞凋亡的E2F1相关基因的不适当表达。这些假设将在各种U937转染者和亲本白血病细胞系中进行测试。还将寻找FP和其他分化诱导剂(例如丁酸钠和Bryostatin)之间协同作用的证据，并确定这些组合的共同特征。最后，我们将使用连续的基因芯片分析来确定一系列与细胞凋亡、分化和细胞周期相关的基因，这些基因的表达可能会受到PMA/FP共同处理的干扰。我们的最终目标是提供一个合理的基础，将分化诱导剂和CDK抑制剂结合起来，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.