P27(Kip1), cyclin E and endogenous TGF-beta1 changes in apoptosis of NB4 cells induced by As(2)O(3) and/or TGF-beta1 and their significance / 中国实验血液学杂志

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).

Role of P27(Kip1) and TGF-beta1 in APL cell apoptosis induced by As(2)O(3) / 中国实验血液学杂志

The aim of study was to investigate the effects of arsenic trioxide (As(2)O(3)) on cell cycle and apoptosis of APL cells, as well as changes of P27(Kip1), endogenous TGF-beta1, cyclin E and bcl-2, and to explore the relationship between expression of P27(Kip1) and apoptosis induced by As(2)O(3). The apoptosis and cell cycle changes of APL cells treated with As(2)O(3) were detected by morphology and flow cytometry respectively, the protein and mRNA expressions of P27(Kip1), TGF-beta1, cyclin E and BCL-2 were measured by immunohistochemistry and RT-PCR. The results indicated that As(2)O(3) induced APL cell apoptosis in vitro, and cell cycle was arrested at G(1) phase. Apoptotic cells induced by As(2)O(3) 1, 5 and 10 micromol/L for 24 hours were 1.42%, 4.57% and 10.67% respectively; the proportion of apoptotic cells induced by As(2)O(3) of same concentrations for 48 hours increased to 8.92%, 16.07% and 18.90% respectively; the cells induced by As(2)O(3) for 72 hours were mainly in debris. Protein and mRNA expressions of P27(Kip1) and TGF-beta1 of APL cells after treatment with As(2)O(3) increased, accompanying with decrease of cyclin E, bcl-2 protein and mRNA expressions. Apoptotic cells were related to the expressions of P27(Kip1) (r(mRNA) = 0.55, p < 0.05) and TGF-beta1 (r(mRNA) = 0.51, p < 0.05). There was positive correlation between the expression of TGF-beta1 and of P27(Kip1) (r(mRNA) = 0.31, p < 0.05). It is concluded that the apoptosis of APL cells is induced by As(2)O(3), and the cell cycle is arrested at G(1) phase. The expression of P27(Kip1) is closely related to the extent of apoptosis induced by As(2)O(3). Apoptosis of APL cells induced by As(2)O(3) may be caused by up-regulating TGF-beta1 and P27(Kip1), which is antagonistic to cyclin E and BLC-2, leading to arrest of cell cycle at G(1) phase.