Liang Yu-Long, Lei Ting-Wen, Wu Heng, Su Jian-Min, Wang Li-Ying, Lei Qun-Ying, Zha Xi-Liang
Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, Shanghai 200032, China.
World J Gastroenterol. 2003 Aug;9(8):1689-96. doi: 10.3748/wjg.v9.i8.1689.
To clarify the mechanisms of integrin overexpression in negatively regulating the cell cycle control of hepatocellular carcinoma cells SMMC-7721.
The cell cycle pattern was determined by flow cytometry. The mRNA and protein expression levels were assayed by RT-PCR and Western blot, respectively. Stable transfection was performed by Lipofectamine 2000 reagent, and cells were screened by G418.
Overexpression of alpha5beta1 or beta1 integrin induced S-phase delay in SMMC-7721 cells, and this delay was possibly due to the accumulation of cyclin-dependent kinase inhibitors (CKIs) p21(cip1) and p27(kip1). The decrease of protein kinase B (PKB) phosphorylation was present in this signaling pathway, but focal adhesion kinase (FAK) was not involved. When phosphorylation of PKB was solely blocked by wortmannin, p27(kip1) protein level was increased. Moreover, S-phase delay was recurred when attachment of the parental SMMC-7721 cells was inhibited by the preparation of poly-HEME, and this cell cycle pattern was similar to that of beta1-7721 or alpha5beta1-7721 cells.
S-phase delay induced by overexpression of integrin beta1 subunit is attributed to the decrease of PKB phosphorylation and subsequent increases of p21(cip1) and p27(kip1) proteins, and may be involved in the unoccupied alpha5beta1 because of lack of its ligands.
阐明整合素过表达对肝癌细胞SMMC - 7721细胞周期调控产生负向作用的机制。
采用流式细胞术检测细胞周期模式。分别通过逆转录聚合酶链反应（RT - PCR）和蛋白质免疫印迹法（Western blot）检测mRNA和蛋白质表达水平。使用脂质体2000试剂进行稳定转染，并用G418筛选细胞。
α5β1或β1整合素的过表达诱导SMMC - 7721细胞出现S期延迟，这种延迟可能是由于细胞周期蛋白依赖性激酶抑制剂（CKIs）p21（cip1）和p27（kip1）的积累所致。该信号通路中存在蛋白激酶B（PKB）磷酸化水平降低的情况，但粘着斑激酶（FAK）未参与其中。当渥曼青霉素单独阻断PKB的磷酸化时，p27（kip1）蛋白水平升高。此外，当聚血红素制剂抑制亲代SMMC - 7721细胞的附着时，再次出现S期延迟，且这种细胞周期模式与β1 - 7721或α5β1 - 7721细胞相似。
整合素β1亚基过表达诱导的S期延迟归因于PKB磷酸化水平降低以及随后p21（cip1）和p27（kip1）蛋白水平升高，并且可能由于缺乏配体而涉及未占据的α5β1。
