DNA damage cell checkpoint activities are altered in monocrotaline pyrrole-induced cell cycle arrest in human pulmonary artery endothelial cells
DNA damage cell checkpoint activities are altered in monocrotaline pyrrole-induced cell cycle arrest in human pulmonary artery endothelial cells
复制标题
DOI：
10.1006/taap.2000.8966
复制
发表时间：
2000-07-15
影响因子：
3.8
通讯作者：
Segall, HJ
中科院分区：
文献类型：
--
作者：
Wilson, DW;Lamé, MW;Segall, HJ
单罗塔碱吡咯（MCTP）引起培养的牛肺动脉内皮细胞在细胞周期的G2期细胞和核增大和持续的细胞周期阻滞。为了更好地表征这一过程的细胞周期调控机制，并确定这一过程是否会发生在人源性细胞中，我们用MCTP处理人肺动脉内皮细胞（HPAEC）培养物，并通过行细胞术测定细胞周期蛋白B1和p53的表达与DNA含量的关系。我们还通过Western blots验证了cdc2在失活磷酸化状态下的持久性，之前在牛细胞培养中描述过，发生在HPAEC中，p53、细胞周期蛋白A、细胞周期蛋白B1和cdc25c表达的改变也在处理过的HPAEC提取物中进行了Western blots检测，HPAEC对MCTP的反应与阿霉素和诺可唑的反应进行了比较，这两种药物已知会导致细胞周期改变。这些实验结果表明，MCTP处理的HPAEC在G2中形成了细胞群，细胞周期蛋白B1的表达增加。这些细胞表达cdc2的量增加，但cdc25c的量没有增加。与对照培养物相比，非活性三磷酸化cdc2与活性单磷酸化形式的比例适度增加，而在诺可达唑处理的培养物中，活性形式占主导地位。此外，第二组表达cyclin B1的细胞继续掺入BrdU， DNA含量与8n染色体一致。在诺可达唑处理的细胞中，明显存在类似的8n细胞群，但这些细胞同时具有细胞周期蛋白B1阳性和阴性成分。与已知的p53诱导剂阿霉素相比，MCTP处理的HPAEC仅在高浓度下表达p53，并且p53的表达与G2阻滞或多倍体不协调。我们得出结论，低浓度MCTP处理的HPAEC发生G2阻滞与持续的cyclin B1表达有关，不能完全激活cdc2，并且通过与p53表达改变无关的途径继续DNA合成。(C) 2000年学术出版社。
Monocrotaline pyrrole (MCTP) causes cyto- and karyomegaly and persistent cell cycle arrest in the G2 stage of the cell cycle in cultured bovine pulmonary artery endothelial cells. To better characterize the cell cycle regulatory mechanisms of this process as well as determine whether this process would occur in cells of human origin, we treated human pulmonary artery endothelial cell (HPAEC) cultures with MCTP and determined, by Row cytometry, the expression of cyclin B1 and p53 in conjunction with DNA content. We also validated by Western blots that the persistence of cdc2 in its inactivated phosphorylated state, previously described in bovine cell cultures, occurred in HPAEC, Alterations in p53, cyclin A, cyclin B1, and cdc25c expression were also examined in Western blots of treated HPAEC extracts, The response of HPAEC to MCTP was compared with that of adriamycin and nocodazole, agents known to cause cell cycle alterations. Results of these experiments demonstrate that HPAEC treated with MCTP develop a population of cells in G2 that has increased cyclin B1 expression. These cells express increased amounts of cdc2 but not cdc25c, The ratio of inactive triphosphorylated cdc2 to the active monophosphorylated form increased moderately from control cultures in contrast to predominance of the active form in nocodazole-treated cultures. In addition, a second population of cells expressing cyclin B1 had continued incorporation of BrdU and DNA content consistent with 8 N chromosomes. A similar 8 N cell population was evident in nocodazole-treated cells but these cells had both cyclin B1 positive and negative components. Compared with adriamycin, a known inducer of p53, MCTP-treatcd HPAEC expressed p53 only at high concentrations and p53 expression was not coordinated with G2 arrest or polyploidy, We conclude that HPAEC treated with low concentrations of MCTP develop G2 arrest in association with persistent cyclin B1 expression, failure to completely activate cdc2, and continued DNA synthesis through a pathway that is unrelated to altered expression of p53. (C) 2000 Academic Press.