腺苷A2A受体与HIF-1α交互作用对CLL细胞凋亡的调控及机制

Background: Hypoxia-CD39/CD73-A2A/A2B system plays important roles in tumor growth and therapy. Overexpression of hypoxia inducible factor-1α (HIF-1α), an important mediator controlling the expression of a wide variety of apoptotic genes, has been observed in bone marrow leukemic cells from chronic lymphocytic leukemia (CLL) patients. ADO has been shown to protect CLL cells from apoptosis and affect HIF-1α expression in our previous study. However, the reciprocal action of adenosine A2A receptor and HIF-1α in CLL remains elusive...Objective: We aim to explore the molecular mechanisms underlying the relationship between A2AR activation and HIF-1α in CLL...Methods: To study the impacts of A2AR activation on CLL cells, CLL cells will be treated with ADO or A2AR agonist CGS21680, followed by determination of cell apoptosis, HIF-1α expression, and intracellular A2AR downstream signals i.e. cAMP levels and PI3K/AKT and MAPK pathways. To delineate the pathways modulating HIF-1α expression and cells apoptosis, specific pharmacological inhibitors will be employed in parallel. HIF-1α gene knockdown cells will be used to determine specific role of HIF-1α in A2AR mediated blockade of cell apoptosis. ADO level can be measured quantitatively by HPLC. CD39 and CD73 activity will be detected by TLC analysis. In order to detect the effects of HIF-1α on CD39/CD73-A2AR/A2BR system, CLL cells are pre-treated with DMOG, a prolyl-hydroxylase inhibitor that stabilizes HIF under normoxic conditions or 17-DMAG, a potent HIF-1 inhibitor. And then, cells are exposured to hypoxia for 48 hours. CD39, CD73, ADO and A2AR expressions in CLL cells were detected in the following experiments. CLL cells apoptosis are also analyzed by FACS. HIF-1α, CD39, CD73, ADO and A2AR genes knock down cells were used to determine the mediator in HIF-1α related CLL cells apoptosis. Expression of A2AR and HIF-1αin CLL patient specimens will be analyzed by qPCR and Western blot and their utilities in CLL prognosis will be evaluated...Expected results: 1. Demonstrate the effects of A2AR activation on HIF-1α expression and CLL cell apoptosis. Explore involved mechanism of HIF-1α expression regulated by A2AR activation. 2. Explore the effects of HIF-1α on CD39/CD73-A2AR/A2BR system in CLL. 3. Discuss the importance of the reciprocal action of HIF-1α and adenosine A2A in anti-tumor apoptosis in CLL. 4. Determine the levels of ADO and expression of HIF-1α, CD39, CD73 and ADO receptors in CLL patients, analyze their correlations with clinical characteristics and parameters, explore their clinical potential as biomarkers for diagnosis, therapy response and prognosis. Provide novel molecular insights for CLL therapeutics.

低氧-CD39/CD73-腺苷（ADO）-A2A/A2B系统在肿瘤细胞凋亡过程中发挥重要作用。慢性淋巴细胞白血病（CLL）患者骨髓白血病细胞高表达的低氧诱导因子-1α（HIF-1α）通过调节其靶基因的表达影响CLL细胞凋亡，但是ADO与HIF-1α交互作用对CLL凋亡的调控及机制目前尚未阐明。我们的前期研究发现，ADO不仅影响CLL细胞凋亡，同时影响HIF-1α表达。为明确HIF-1α与腺苷A2AR受体交互作用对CLL的影响及机制，我们通过细胞、分子、基因工程等技术，以CLL细胞株、转基因小鼠及CLL临床标本为研究对象，从体内、体外水平探讨①A2AR与HIF-1α的交互作用机制，②A2AR与HIF-1α交互作用对CLL细胞凋亡的调控机制，③A2AR与HIF-1α共同作为CLL治疗靶点的可能性。

低氧-CD39/CD73-腺苷（ADO）-A2A/A2B系统在肿瘤细胞凋亡过程中发挥重要作用。慢性淋巴细胞白血病（CLL）患者骨髓白血病细胞高表达的低氧诱导因子-1α（HIF-1α）在CLL细胞的凋亡过程中也发挥重要作用。ADO与HIF-1α交互作用对CLL凋亡的调控及机制目前尚未阐明。我们的研究发现，CLL患者骨髓中及外周血中CD39、CD73、A2AR和HIF-1α蛋白的表达显著升高，且CD73及HIF-1α的表达水平与CLL患者的分期相关，A2AR的活化与CLL细胞增殖及HIF-1α的表达相关。应用HIF-1α慢病毒shRNA 对CLL细胞进行转染，检测转染细胞的增殖及凋亡水平。结果提示HIF-1α+CLL细胞增殖率升高，凋亡减低，与预处理组（选择性阻断A2AR后转染细胞）相比，细胞增殖水平较高，而凋亡率无明显差异。应用Western-blotting明确HIF-1α+CLL细胞CD39、CD73、A2AR表达水平，结果提示CD39、CD73、A2AR表达随HIF-1α表达升高而升高。利用TCL-1转基因CLL小鼠模型，抽取其外周血及骨髓，我们的结果显示，与正常小鼠相比，CLL小鼠外周血及骨髓ADO水平均增高，CD39、CD73、A2A受体、HIF-1α的mRNA及蛋白水平也均高于正常小鼠。小鼠血清中CD39及CD73均具有酶活性，可以水解ADP生成ADO。在小鼠CLL动物模型中，通过A2AR拮抗剂阻断ADO对CLL白血病细胞的直接影响，我们发现，A2AR拮抗剂在体内可以显著降低CLL小鼠外周血及骨髓的淋巴细胞计数，瘤负荷减轻。在小鼠CLL动物模型中，应用17-DMAG阻断HIF-1α对CLL细胞的作用，CD39、CD73、ADO受体的表达均减低，CLL小鼠外周血及骨髓的淋巴细胞计数也低于未治疗组。我们的结果首次深入探讨了A2AR与HIF-1α的交互作用机制及对CLL细胞凋亡的调控机制，为CLL治疗提供新的靶点。