Liang Ying, Li Yan, Wang Yue, Li Xia, Wang Ping-Ping, Wang Bai-Xun
Department of Hematology, The First Affiliated Hospital, China Medical University, Shenyang 110001, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2009 Feb;17(1):74-9.
This study was aimed to investigate the effects of arsenic trioxide (As(2)O(3)) and/or transforming growth factor-beta1 (TGF-beta1)on cell apoptosis and the changes of P27(Kip1), cyclin E and endogenous TGF-beta1 mRNA levels in NB4 cells. As(2)O(3) cytotoxicity to NB4 cells and the IC(50) were assayed with MTT, the apoptotic morphological changes were observed by Wright-Giemsa staining; the cell cycle and apoptosis were detected with flow cytometry. Semiquantitative RT-PCR was used to examine P27(Kip1), cyclin E and endogenous TGF-beta1 mRNA levels. The results showed that the As(2)O(3) and TGF-beta1 significantly suppressed the growth of NB4 cells, and promoted the apoptosis of these cells. The growth inhibition and apoptosis of NB4 cells treated with As(2)O(3) were in dose-and time-dependent manners. IC(50) were about 12 micromol/L for 24 hours, about 5 micromol/L for 48 hours, and about 3 micromol/L for 72 hours respectively. Cell cycle arrest in NB4 cells was induced by As(2)O(3) and/or TGF-beta1. The arrest of NB4 cells treated by 5 micromol/L As(2)O(3) was in G(2)/M phase, and 5 ng/ml TGF-beta1 in G(1) phase. However, the arrest of NB4 cells caused by combination of As(2)O(3) and TGF-beta1 was in S phase. After treating with As(2)O(3), P27(Kip1) and endogenous TGF-beta1 mRNA expressions of NB4 cells were up-regulated, and cyclin E mRNA expression was down-regulated. When NB4 cells were treated with TGF-beta1 alone, P27(Kip1) and cyclin E mRNA expressions were the same as that treated by As(2)O(3). Exogenous TGF-beta1 enhanced the above effects of As(2)O(3) in combination group. It is concluded that As(2)O(3) and TGF-beta1 are able to induce apoptosis and cell cycle abnormal distribution in NB4 cells. As(2)O(3) and exogenous TGF-beta1 may up-regulate endogenous TGF-beta1, which induce apoptosis of NB4 cells through consequently high expression of P27(Kip1). TGF-beta1 may lead to cell cycle arrest by inhibiting the expression of cyclin E directly, or by the activity of cyclin E through the increased expression of P27(Kip1).
本研究旨在探讨三氧化二砷（As₂O₃）和/或转化生长因子-β1（TGF-β1）对NB4细胞凋亡以及P27^（Kip1）、细胞周期蛋白E和内源性TGF-β1 mRNA水平变化的影响。采用MTT法检测As₂O₃对NB4细胞的细胞毒性及半数抑制浓度（IC₅₀），用瑞氏-吉姆萨染色观察凋亡形态学变化；采用流式细胞术检测细胞周期和凋亡情况。运用半定量逆转录聚合酶链反应（RT-PCR）检测P27^（Kip1）、细胞周期蛋白E和内源性TGF-β1 mRNA水平。结果显示，As₂O₃和TGF-β1均能显著抑制NB4细胞生长，并促进其凋亡。As₂O₃对NB4细胞的生长抑制和凋亡作用呈剂量和时间依赖性。作用24小时的IC₅₀约为12 μmol/L，48小时约为5 μmol/L，72小时约为3 μmol/L。As₂O₃和/或TGF-β1可诱导NB4细胞发生细胞周期阻滞。5 μmol/L As₂O₃作用后NB4细胞阻滞于G₂/M期，5 ng/ml TGF-β1作用后阻滞于G₁期。然而，As₂O₃与TGF-β1联合作用导致NB４细胞阻滞于S期。As₂O₃作用后，NB4细胞的P27^（Kip1）和内源性TGF-β1 mRNA表达上调，细胞周期蛋白E mRNA表达下调。单独用TGF-β1处理NB4细胞时，P27^（Kip1）和细胞周期蛋白E mRNA表达变化与As₂O₃处理时相同。联合组中外源性TGF-β1增强了As₂O₃的上述作用。结论：As₂O₃和TGF-β1能够诱导NB4细胞凋亡及细胞周期异常分布。As₂O₃和外源性TGF-β1可能上调内源性TGF-β1，进而通过高表达P27^（Kip1）诱导NB4细胞凋亡。TGF-β1可能通过直接抑制细胞周期蛋白E的表达，或通过增加P27^（Kip1）的表达影响细胞周期蛋白E的活性，从而导致细胞周期阻滞。
