Objective: To investigate the mechanism of DNA damage and repair in MLL -rearranged acute myeloid leukemia（ MLL-r AML）cells by the combination of Chidamide and the BRD4 inhibitor(+)-JQ-1.
Methods: MLL-r AML cell lines Molm-13, MV4-11 and non- MLL-r AML cell line Kasumi were divided into control group（contr）, Chidamide group（chida）, (+)-JQ-1 group and Combination group（combi）, respectively. Cell viability of Molm-13 was measured by CCK-8 to determine optimal the concentrations of Chidamide and(+)-JQ-1. The cell cycle was detected by flow cytometry, and apoptosis-related factors Bcl-2, Bax and caspase-3 were detected by Western blot. DNA damage marker γH2AX was detected by immunofluorescence. The protein expressions of DNA damage factor γH2AX, DNA damage checkpoint kinases p-ATR, p-CHK1, p-ATM, p-CHK2 and DNA damage repair factors Rad51 and 53BP1 were detected by Western blot. The expression of DNA damage repair factors Rad51 and 53BP1 mRNA was detected by qRT-PCR.
Results: Under the treatment of Chidamide (300 nmol/L) and (+)-JQ-1 (400 nmol/L), the proportion of G1 phase cells in MLL-r AML cell lines Molm-13 and MV4-11 was increased in combination group compared with control group. In non- MLL-r AML cell line Kasumi, compared with control group, the proportion of G1 phase cells in combination group was increased (P < 0.05). In Molm-13 and MV4-11 cell lines, compared with control group, the expression level of DNA damage marker γH2AX in combination group was increased (P < 0.05). The expression levels of DNA damage checkpoint and damage repair factors p-ATR, p-CHK1, p-ATM, p-CHK2, Rad51, 53BP1 were decreased (P < 0.05). In Kasumi cell line, compared with control group, there was no significant change in the expression of some of the above factors in combination group (P >0.05), but the expression trend of some factors was opposite. In MLL-r AML cell lines Molm-13 and MV4-11, compared with control group, the expression levels of Bax and caspase-3 protein were increased in combination group, while the expression levels of Bcl-2 protein were decreased (P < 0.05). In non- MLL-r AML cell line Kasumi, there was no significant change in apoptotic factor protein expression in combination group compared with control group (P >0.05).
Conclusion: Chidamide combined with (+)-JQ-1 can inhibit the proliferation of MLL-r AML cells, inhibit the initiation of protective self-repair of these leukemia cells by inhibiting the DNA damage response pathway, and ultimately increase the apoptosis of these cells, but non- MLL-r AML cells have no similar results.
题目: 西达本胺联合(+)-JQ-1通过破坏DNA损伤反应途径杀伤 MLL重排急性髓系白血病细胞.
目的: 探讨西达本胺与BRD4抑制剂(+)-JQ-1联合对混合谱系白血病基因重排急性髓系白血病（ MLL-r AML）细胞的DNA损伤及修复机制的影响。.
方法: 分别将 MLL-r AML细胞系Molm-13、MV4-11细胞和非 MLL-r AML细胞系Kasumi细胞分成对照组（contr）、西达本胺组（chida）、(+)-JQ-1组、联合组（combi）4组。采用CCK-8检测Molm-13细胞活力，以确定西达本胺和(+)-JQ-1的给药浓度。采用流式细胞术检测细胞周期变化，Western blot检测凋亡相关因子Bcl-2、Bax、caspase-3的表达。利用免疫荧光检测DNA损伤标志物γH2AX；Western blot检测DNA损伤因子γH2AX，DNA损伤检查点激酶p-ATR、p-CHK1、p-ATM、p-CHK2和DNA损伤修复因子Rad51、53BP1蛋白的表达情况；qRT-PCR检测DNA损伤修复因子Rad51、53BP1 mRNA的表达情况。.
结果: 在药物西达本胺（300 nmol/L）和(+)-JQ-1（400 nmol/L）的联合作用下, MLL-r AML细胞系Molm-13、MV4-11中，与contr组相比，combi组G1期细胞比例升高；非 MLL-r AML细胞系Kasumi中，与contr组相比，combi组G1期细胞比例升高（P < 0.05）。在Molm-13、MV4-11细胞系中，与contr组相比，combi组DNA损伤标志物γH2AX表达水平升高（P < 0.05），DNA损伤检查点和损伤修复因子p-ATR、p-CHK1、p-ATM、p-CHK2、Rad51、53BP1表达水平降低（P < 0.05）；而在Kasumi细胞系中，与contr组相比，combi组上述因子部分表达无明显变化（P >0.05），部分因子的表达趋势与 MLL-r AML细胞系相反。在 MLL-r AML细胞系Molm-13、MV4-11中，与contr组相比，combi组Bax和caspase-3蛋白表达水平升高，而Bcl-2蛋白表达水平下降（P < 0.05）；非 MLL-r AML细胞系Kasumi细胞中，与contr组相比，combi组凋亡因子的表达无明显变化（P >0.05）。.
结论: 西达本胺联合(+)-JQ-1可抑制 MLL-r AML细胞的增殖，并通过抑制DNA损伤反应途径抑制此类白血病细胞保护性自我修复的启动，最终增加细胞的凋亡，而非 MLL-r AML细胞却无类似的结果。.
Keywords: mixed lineage leukemia genes； BRD4； chidamide； DNA damage response； acute myeloid leukemia.