APOPTOSIS IN NORMAL AND MALIGNANT LYMPHOCYTES

描述：(申请者摘要)提高细胞内cAMP的刺激 已知水平可诱导特定的、易受影响的淋巴组织中的细胞凋亡 人口。CAMP代谢由至少10个不同的家族控制 环核苷酸磷酸二酯酶(PDE)。最近，慢性淋巴细胞 白血病(CLL)细胞被显示在暴露于 非特异性PDE抑制剂茶碱。申请人最近确认了 小鼠PDE4B作为一种在胸腺细胞诱导中上调的酶 通过体内给予抗CD-epsilon诱导细胞凋亡，并解决了 确定抑制PDE4是否可诱导CLL中cAMP介导的细胞凋亡。 CLL细胞具有PDE4B转录本、蛋白和酶活性。 罗利普兰抑制PDE4升高cAMP水平并诱导CLL细胞凋亡 以剂量依赖的方式，需要48小时才能达到最大 效果。WMC和抗-Ig刺激的原代人B细胞对 罗利普兰诱导细胞凋亡。建议对以下假设进行检验： 淋巴系统对PDE抑制剂诱导的细胞凋亡的敏感性可能部分是 PDE家族表达的生理和发育调节决定 或者通过恶变。第一，PDE1B、3B、4A、4B、4D的表达 将在CLL、ALL、ATL和Sezary白血病细胞中检测7种酶 以及B细胞和未成熟和成熟T淋巴细胞的蛋白质印迹 分析、聚合酶链式反应、核糖核酸酶保护、Northerns和酶分析。它也将是 确定激活正常和恶性B和T细胞的刺激是否 改变PDE家族的表达，随后这些研究与 家族特异性PDE抑制剂诱导静息状态下细胞凋亡的能力 或激活正常和恶性细胞。将确定家人是否 特定的PDE抑制剂诱导其他PDE家族的代偿性增加 以及对几个PDE家族的抑制是否在 比“单一疗法”更能诱导细胞凋亡。调查其他机制，以 淋巴组织对cAMP诱导的细胞凋亡的敏感性不同， 研究内容如下：1)GTP酶RAP1在cAMP介导中的作用 淋巴细胞中的凋亡信号；2)Caki和cAKII在 CAMP介导的细胞凋亡；3)PKA和Bad S112激酶是否存在关联 淋巴样细胞中线粒体组分的活性可预测对 PDE抑制剂诱导细胞凋亡。在这个项目中收集的信息将 协助家族特异性PDE抑制剂的合理应用 人类淋巴系恶性肿瘤的治疗。

DESCRIPTION: (Applicant's Abstract) Stimuli which raise intracellular cAMP levels are know to induce apoptosis in specific, susceptible lymphoid populations. cAMP metabolism is controlled by at least ten distinct families of cyclic nucleotide phosphodiesterases (PDEs). Recently, chronic lymphocytic leukemia (CLL) cells were shown to undergo apoptosis following exposure to the nonspecific PDE inhibitor theophylline. The applicant recently identified murine PDE4B as an enzyme upregulated during the induction of thymocyte apoptosis by in vivo administration of anti-CD-epsilon, and resolved to determine whether inhibition of PDE4 induced cAMP-mediated apoptosis in CLL. CLL cells contained PDE4B transcript, protein and enzymatic activity. Inhibition of PDE4 by rolipram raised cAMP levels and induced apoptosis in CLL cells in a dose dependent fashion that required 48 hours to achieve maximal effect. WMC and anti-Ig stimulated primary human B cells were resistant to rolipram-induced apoptosis. It is proposed to examine the hypothesis that lymphoid sensitivity to PDE inhibitor-induced apoptosis may in part be determined by physiologic and developmental regulation of PDE family expression or by malignant transformation. First, the expression of PDE1B, 3B, 4A, 4B, 4D and 7 enzymes will be examined in CLL, ALL, ATL and Sezary leukemic cells as well as B cells and immature and mature T lymphocytes using Western blot analysis, PCR, RNase protection, Northerns and enzyme assays. It will also be determined whether stimuli which activate normal and malignant B and T cells alter PDE family expression, followed by correlation of these studies with the ability of family-specific PDE inhibitors to induce apoptosis in these resting or activated normal and malignant cells. It will be determined whether family specific PDE inhibitors induce compensatory increases in other families of PDEs and whether inhibition of several families of PDEs is more effective in inducing apoptosis than "monotherapy." To investigate other mechanisms by which lymphoid populations differ in their sensitivity to cAMP-induced apoptosis, the following will be examined: 1) The role of the GTPase RAP1 in cAMP-mediated apoptotic signaling in lymphoid cells; 2) The role of cAKI and cAKII in cAMP-mediated apoptosis; 3) Whether the association of PKA and BAD S112 kinase activity with mitochondrial fractions in lymphoid cells predicts sensitivity to PDE inhibitor-induced apoptosis. The information gathered in this project will assist in the rational application of family-specific PDE inhibitors to the treatment of human lymphoid malignancies.