Zhang Qing, Li Feng-Mei, Wang Wei, Zhang Zhi-Hua, Zhang Rong-Juan, Ma Ming-Shuai, Wang Li-Hong
Department of Hematology, The Affiliated Hospital of Chengde Medical University, Chengde 067000, Hebei Province, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2024 Oct;32(5):1323-1333. doi: 10.19746/j.cnki.issn.1009-2137.2024.05.004.
To investigate the mechanism of DNA damage and repair in -rearranged acute myeloid leukemia（ -r AML）cells by the combination of Chidamide and the BRD4 inhibitor(+)-JQ-1.
-r AML cell lines Molm-13, MV4-11 and non- -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 and mRNA was detected by qRT-PCR.
Under the treatment of Chidamide (300 nmol/L) and (+)-JQ-1 (400 nmol/L), the proportion of G phase cells in -r AML cell lines Molm-13 and MV4-11 was increased in combination group compared with control group. In non- -r AML cell line Kasumi, compared with control group, the proportion of G phase cells in combination group was increased ( < 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 ( < 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 ( < 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 ( >0.05), but the expression trend of some factors was opposite. In -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 ( < 0.05). In non- -r AML cell line Kasumi, there was no significant change in apoptotic factor protein expression in combination group compared with control group ( >0.05).
Chidamide combined with (+)-JQ-1 can inhibit the proliferation of -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- -r AML cells have no similar results.
探讨西达本胺与BRD4抑制剂（+）-JQ-1联合作用于重排急性髓系白血病（-r AML）细胞时DNA损伤与修复的机制。
将-r AML细胞系Molm-13、MV4-11及非-r AML细胞系Kasumi分别分为对照组（contr）、西达本胺组（chida）、（+）-JQ-1组和联合组（combi）。采用CCK-8法检测Molm-13细胞活力，以确定西达本胺和（+）-JQ-1的最佳浓度。采用流式细胞术检测细胞周期，采用蛋白质免疫印迹法检测凋亡相关因子Bcl-2、Bax和caspase-3。采用免疫荧光法检测DNA损伤标志物γH2AX。采用蛋白质免疫印迹法检测DNA损伤因子γH2AX、DNA损伤检查点激酶p-ATR、p-CHK1、p-ATM、p-CHK2以及DNA损伤修复因子Rad51和53BP1的蛋白表达。采用qRT-PCR法检测DNA损伤修复因子及mRNA的表达。
在西达本胺（300 nmol/L）和（+）-JQ-1（400 nmol/L）处理下，联合组中-r AML细胞系Molm-13和MV4-11的G期细胞比例相较于对照组增加。在非-r AML细胞系Kasumi中，联合组的G期细胞比例相较于对照组增加（P<0.05）。在Molm-13和MV4-11细胞系中，联合组的DNA损伤标志物γH2AX表达水平相较于对照组增加（P<0.05）。DNA损伤检查点及损伤修复因子p-ATR、p-CHK1、p-ATM、p-CHK2、Rad51、53BP1的表达水平降低（P<0.05）。在Kasumi细胞系中，联合组上述部分因子的表达与对照组相比无显著变化（P>0.05），但部分因子的表达趋势相反。在-r AML细胞系Molm-13和MV4-11中，联合组的Bax和caspase-3蛋白表达水平相较于对照组增加，而Bcl-2蛋白表达水平降低（P<0.05）。在非-r AML细胞系Kasumi中，联合组凋亡因子蛋白表达与对照组相比无显著变化（P>0.05）。
西达本胺与（+）-JQ-1联合可抑制-r AML细胞增殖，通过抑制DNA损伤反应途径抑制这些白血病细胞保护性自我修复的启动，最终增加这些细胞的凋亡，但非-r AML细胞无类似结果。
