转录因子Pax5可不依赖与启动子结合促进B细胞淋巴瘤生长

Transcription Factor Pax5 Promotes B-lymphomagenesis in a Promoter-Independent Manner

目的:研究B淋巴细胞转录因子Pax5是否可以经过不依赖与启动子直接结合方式控制B细胞淋巴瘤的生长。方法:将可以产生各种突变型pax5并带GFP荧光的逆转录病毒感染小鼠淋巴瘤细胞系myc3和38B9,48 h后应用流式细胞术检测GFP+淋巴瘤细胞的比例,然后将GFP+和GFP-淋巴瘤细胞一起继续培养或皮下移植至小鼠体内生长,体外每隔3 d或体内成瘤后重新检测GFP+细胞比例并确定其细胞周期参数和凋亡细胞的数量。结果:与正常对照组(migR-GFP,空载体)相比,突变型Pax5 mt 1-357及缺乏DNA结合功能的突变型Pax5 mt 304-358和野生型Pax5一样均能明显增加GFP+细胞比例(P<0.01),且S及G2/M期GFP+肿瘤细胞明显增多(P<0.01),但只有DNA结合功能的Pax5 mt 1-143与空载体对照一样无法使GFP+细胞比例增加(P>0.05),细胞周期也无明显改变(P>0.05)。结论:Pax5可以经过不依赖与起动子直接结合方式促进B细胞淋巴瘤在体内体外的生长,其主要功能是加速肿瘤细胞分裂而不是降低细胞凋亡。本研究为以Pax5蛋白特定区域作为治疗B细胞淋巴瘤新靶点提供了理论依据。

Objective： To determine whether B lymphocyte-specific transcription factor Pax5 regulates B-lymphomagenesis without direct binding to promoter. Methods： Mouse B-lymphoma cell line myc3 and 38 B9 were infected with GFP-tagged retrovirus that encodes wide type or various mutant pax5 genes. After viral infection for 48 hours,the percentage of GFP positive lymphoma cells was determined by flow cytomety. The percentage of GFP positive tumor cells was further monitored every 3 days in vitro or once the tumor was formed in vivo. Both cell cycle and apoptic cell number of GFP positive lymphoma cells were analyzed using flow cytometry. Results： Similar to the infection with wild type Pax5 retrovirus,infection with Pax5 mt 1-357 and Pax5 mt 304-358 that lacks of DNA binding motif can strongly increase the percentage of GFP＋B-lymphoma cells both in vitro and in vivo（ P〈0. 01）,while infection with empty viral vector migRGFP and Pax5 mt 1-143 containing only DNA binding motif failed to increase the percentage of GFP positive tumor cells（ P〈0. 05）. Moreover,the analysis of flow cytometry demonstrated that more B-lymphoma cells infected with wild type Pax5,Pax5 mt 1-357 and Pax5 mt 304-358 retroviruses entered S and G2/M phases in comparison with those infected with empty viral vector migR-GFP and Pax5 mt 1-143. Apoptotic rates among different groups were not significantly changed. Conclusion： Pax5 can promote B-lymphoma cell growth both in vitro and in vivo in a promoter-independent manner. This is mainly due to the accelerating of cell cycle rather than decreasing apoptosis. Our studies provide potential theory for restraing B-lymphomagenesis by targeting the specific Pax5 domains.