APOPTOSIS IN NORMAL AND MALIGNANT LYMPHOCYTES

说明：（申请人的摘要）提高细胞内cAMP的刺激 水平已知在特定的、易感的淋巴细胞中诱导凋亡， 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，慢性淋巴细胞性 白血病（CLL）细胞暴露于 非特异性PDE抑制剂茶碱。申请人最近确认 小鼠PDE 4 B作为胸腺细胞诱导过程中上调的酶 通过体内施用抗CD-β 1诱导细胞凋亡，并解决为 确定在CLL中抑制PDE 4是否诱导cAMP介导的细胞凋亡。 CLL细胞含有PDE 4 B转录物、蛋白质和酶活性。 咯利普兰抑制PDE 4可提高CLL中cAMP水平并诱导细胞凋亡 细胞以剂量依赖性方式，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 咯利普兰诱导的细胞凋亡。有人建议对以下假设进行检验： 淋巴对PDE受体诱导的细胞凋亡的敏感性可能部分地 由PDE家族表达的生理和发育调节决定 或恶性转化。第一，PDE 1B、3B、4A、4 B、4D的表达 将在CLL、ALL、ATL和Sezary白血病细胞中检查7种酶， 以及B细胞和未成熟和成熟T淋巴细胞使用Western印迹 分析、PCR、RNA酶保护、Northerns和酶测定。还将 确定是否刺激激活正常和恶性B和T细胞 改变PDE家族表达，然后将这些研究与 家族特异性PDE抑制剂诱导这些静息细胞凋亡的能力 或激活正常和恶性细胞。将决定家庭是否 特异性PDE抑制剂诱导其他PDE家族代偿性增加 以及抑制几个PDE家族是否更有效， 诱导细胞凋亡。“研究其他机制， 淋巴细胞群对cAMP诱导的细胞凋亡的敏感性不同， 1）GTP酶RAP 1在cAMP介导的细胞凋亡中的作用 淋巴细胞中的凋亡信号传导; 2）cAKI和cAKII在淋巴细胞中的作用， cAMP介导的细胞凋亡; 3）PKA与BAD S112激酶是否相关 淋巴样细胞中线粒体组分的活性预测了对 PDE介导的细胞凋亡。本项目收集的信息将 有助于家族特异性PDE抑制剂的合理应用， 治疗人淋巴恶性肿瘤。

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.