Cyclic AMP-mediated apoptosis in lymphoid malignancies

描述(申请人提供)：4型cAMP磷酸二酯酶(PDE4)的抑制剂，如罗利普兰，可诱导B细胞慢性淋巴细胞白血病患者的细胞凋亡，即使在没有激活腺苷环化酶的外源性药物的情况下，如二萜Forsklin。相反，T细胞或T-CLL对罗利普兰诱导的细胞凋亡具有抵抗力。罗利普兰诱导B-CLL细胞线粒体死亡途径，可能部分是由于磷酸酶PP2A的激活和BAD的去磷酸化。值得注意的是，cAMP还可以通过EPAC1激活的RAP1 GDP交换因子激活B-CLL中的抗凋亡途径。B-CLL细胞在外周血造血细胞中具有独特的EPAC1功能表达，提示EPAC1信号可能在本病的病理生理过程中起一定作用。具体目的1.为了阐明PDE4抑制剂激活B-CLL线粒体凋亡途径的机制，我们将：a)确定PDE4抑制剂诱导的CLL细胞凋亡是否由PKA介导。B)确定罗利普兰诱导的CLL细胞凋亡是否不依赖于EPAC。C)确定罗利普兰诱导CLL PP2A活性增强的机制。D)确定罗利普兰介导的CLL细胞凋亡是否需要PP2A。具体目的II.为了对比EPAC和PKA信号在CLL中的作用，我们将：a)确定PDE4抑制剂激活PKA是否阻断EPAC激活以提高B-CLL趋化因子转录水平的能力。B)确定EPAC诱导的趋化因子和/或Mcl-1转录本的增强是否是循环造血细胞中B-CLL所特有的。C)确定EPAC诱导的B-CLL存活率增加是否源于IL-8自分泌环路的上调。D)确定PKA和EPAC是否对PI3K或其下游靶点进行不同的调控。为了确定功能性EPAC在CLL细胞中的独特表达是否具有病理生理学意义，我们将：a)检测EPAC激活对CLL黏附和迁移的影响。B)比较EPAC和PKA激活对基础和化疗诱导的CLL存活率的影响。C)比较EPAC和PKA对CLL和B细胞增殖的影响。

DESCRIPTION (provided by applicant): Inhibitors of type 4 cAMP phosphodiesterase (PDE4) such as rolipram-induced apoptosis in B cell chronic lymphocytic leukemia, even in the absence of exogenous pharmacologic agents that activate adenylate cyclase such as the diterpene forskolin. T cells or T-CLL, in contrast, are resistant to rolipram-induced apoptosis. Rolipram induces a mitochondrial death pathway in B-CLL cells, perhaps in part due to activation of the phosphatase PP2A and dephosphorylation of BAD. Remarkably, cAMP can also activate an antiapoptotic pathway in B-CLL through EPAC1, a cAMP-activated Rap1 GDP exchange factor. B-CLL cells are unique among circulating hematopoietic cells in their functional expression of EPAC1, suggesting EPAC1 signaling may play a role in the pathophysiology of this disease. Specific Aim I. To elucidate the mechanism by which PDE4 inhibitors activate a mitochondrial pathway of apoptosis in B-CLL, we will: A) Determine whether PDE4 inhibitor-induced apoptosis in CLL is PKA-mediated. B) Determine whether rolipram-induced apoptosis in CLL is EPAC-independent. C) Determine the mechanism for rolipram-induced augmented CLL PP2A activity. D) Determine whether PP2A is required for rolipram-mediated apoptosis in CLL. Specific Aim II. To contrast the roles of EPAC and PKA signaling in CLL, we will: A) Determine whether PKA activation by PDE4 inhibitors blocks the ability of EPAC activation to augment B-CLL chemokine transcript levels. B) Determine if EPAC-induced augmentation of chemokine and/or Mcl-1 transcripts is unique to B-CLL among circulating hematopoietic cells. C) Determine if EPAC-induced augmentation of B-CLL survival results from up-regulation of an IL-8 autocrine loop. D) Determine whether PKA and EPAC differentially regulate PI3K or its downstream targets. Specific Aim III. To determine whether the unique expression of functional EPAC in CLL cells is of pathophysiologic importance, we will: A) Examine the effects of EPAC activation on CLL adhesion and migration. B) Compare the effects of EPAC and PKA activation on both basal and chemotherapy-induced CLL survival. C) Compare the effects of EPAC and PKA on CLL and B cell proliferation.