PARP-1 participates in regulation of cell cycle signaling in the hydroquinone-induced TK6 malignant transformation

Abstract

Objective To investigate the expression of polyadenosine diphospho-ribose polymerase 1 (PARP-1) and p16/retinoblastoma (Rb) protein in hydroquinone (HQ)-induced TK6 cells and their regulatory mechanisms.

Methods According to the 2×2 factorial design model, TK6 cells were divided into PBS-TK6 group and HQ-TK6 group based on HQ exposure, and then sub-divided into non-DOX intervention subgroup and DOX intervention subgroup based on DOX intervention, a total of four groups. The PBS-TK6 group was treated with phosphate buffer saline, and the HQ-TK6 group was treated with HQ at a final concentration of 20.0 μmol/L. The non-DOX intervention subgroup was added with 0.05% dimethyl sulfoxide; and the DOX intervention subgroup was added with PARP-1 agonist DOX at a final concentration of 0.5 μmol/L. The distribution of cell cycle was detected by flow cytometry. The protein expression of p16/Rb, cyclin D1 (cyclinD1), multifunctional protein E2 transcription factor 1 (E2F1), Rb, and p-Rb were detected by Western blot, and the level of p16 ribosylation was detected by immunofluorescence and immunoprecipitation.

Results Compared with the PBS-TK6 group, the cell cycle distribution percentage in G0/G1 phase and the relative expression levels of p16 proteins were decreased in the cells of the HQ-TK6 group (all P<0.05). The cell cycle distribution percentage in S phase and the relative expression levels of cyclinD 1 and p-Rb proteins were up-regulated (all P<0.05). Compared with the non-DOX intervention group, the cell cycle distribution percentage in G0/G1 and G2/M phases and the relative expression level of p16 protein increased in the DOX intervention group (all P<0.05). The percentage of cells in S phase and the relative expression levels of cyclinD 1 and p-Rb proteins were down-regulated (all P< 05). The results of interaction effect analysis showed that compared with the non-DOX PBS-TK6 cells, the relative expression levels of Rb and E2F1 protein in the DOX PBS-TK6 cells intervention group were down-regulated (all P<0.05). The relative expression level of Rb protein in non-DOX HQ-TK6 cell group was down-regulated ( P<0.05), and the relative expression of E2F1 protein was up-regulated ( P<0.05). Compared with DOX PBS-TK6 cell group, the relative expression level of Rb protein in DOX HQ-TK6 cell group was down-regulated and that of E2F1 protein was up-regulated (all P<0.05). Compared with the non-DOX HQ-TK6 cell group, the relative expression level of Rb protein in the DOX HQ-TK6 cell group was up-regulated and that of E2F1 protein was down-regulated (all P<0.05).

Conclusion PARP-1 participates in cell cycle regulation by regulating the p16/Rb signaling pathway in TK6 cells.

Abstract

摘要： 目的 探讨聚腺苷二磷酸-核糖聚合酶 (PARP-1) 和 p16/视网膜母细胞瘤 (Rb) 信号通路在氢醌 (HQ) 诱导的TK6 细胞中的表达及其相关调控机制。 方法 按照 2×2 析因设计模型, 将 TK6 细胞按 HQ 染毒水平分为磷酸缓冲盐溶液 (PBS)-TK6 组和 HQ-TK6 组, 再按照多柔比星 (DOX) 干预水平分为无 DOX 干预组和 DOX 干预组, 共 4 组。PBS-TK6 组细 胞以 PBS 处理, HQ-TK6 组细胞予终浓度为 20.0 μmol/L 的 HQ 染毒处理;无 DOX 干预组细胞加人体积分数为 0.05% 的二 甲基亚砜, DOX干预组细胞加人终浓度为 0.5 μmol/L 的 DOX。采用流式细胞术检测细胞周期分布情况, 蛋白免疫印迹法 检测 p16/Rb 信号通路 p16、细胞周期蛋白 D1(cyclinD1)、多功能蛋白 E2 转录因子 1 (E2F1)、Rb、p-Rb 蛋白表达水平, 免疫 荧光和免疫共沉淀法检测p16核糖化水平。 结果 主效应分析结果显示, 与 PBS-TK6 组比较, HQ-TK6 组细胞 G0/G1 期 细胞周期分布百分比和 p16 蛋白相对表达水平均下调 ( P 值均<0.05), S 期细胞周期分布百分比和 cydinD1、p-Rb 蛋白相 对表达水平均上升 ( P 值均<0.05); 与无 DOX 干预组比较, DOX 干预组细胞在 G0/G1 期、G2/M 期的细胞周期分布百分比 和 p16 蛋白相对表达水平均上升 ( P 值均<0.05), 在 S 期细胞百分比和 cydinD1、p-Rb 蛋白相对表达水平均下调 ( P 值均<0.05)。交互效应分析结果显示, 与 PBS-TK6 细胞无 DOX 干预组比较, PBS-TK6 细胞 DOX 干预组中 Rb 和 E2F1 蛋白相对 表达水平均下调 ( P 值均<0.05), HQ-TK6 细胞无 DOX 干预亚组的 Rb 蛋白相对表达水平均下调 ( P<0.05), E2F1 蛋白相对 表达水平上调 ( P<0.05); 与 PBS-TK6 细胞 DOX 干预组比较, HQ-TK6 细胞DOX干预组的Rb蛋白相对表达水平下调 ( P<0.05), E2F1 蛋白相对表达水平上调 ( P<0.05); 与 HQ-TK6 细胞无DOX干预组比较, HQ-TK6 细胞 DOX 干预组的 Rb 蛋白 相对表达水平上调 ( P<0.05), E2F1 蛋白相对表达水平下调 ( P<0.05)。 结论 PARP-1 可能通过调控 p16/Rb 信号通路参 与 TK6 细胞的周期调控。