Serine/threonine protein phosphatase type 1γ1 is required for the completion of cytokinesis in human A549 lung carcinoma cells
Serine/threonine protein phosphatase type 1γ1 is required for the completion of cytokinesis in human A549 lung carcinoma cells
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DOI：
10.1074/jbc.275.3.1846
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发表时间：
2000-01-21
影响因子：
4.8
通讯作者：
Honkanen, RE
中科院分区：
文献类型：
--
作者：
Cheng, AY;Dean, NM;Honkanen, RE
在低等真核生物中，丝氨酸/苏氨酸蛋白磷酸酶1型（PP 1）的缺失导致有丝分裂开始后的生长停滞。在人类中，已经鉴定了PP 1的四种高度同源的同种型（PP 1 α、PP 1 δ、PP 1 γ 1和PP 1 γ 2）。然而，由于缺乏亚型特异性抑制剂，确定这些磷酸酶的作用已被证明是困难的。在这项研究中，我们开发了嵌合反义2 '-O-（2-甲氧基）乙基硫代磷酸寡核苷酸靶向人PP 1 γ 1，特异性抑制PP 1 γ 1基因的表达。然后使用两种有效的反义寡核苷酸（ISIS 14435和14439; IC 50类似于50 nM）来阐明PP 1 γ 1在细胞周期进程中的细胞功能。在A549细胞中，PP 1 γ 1表达的抑制导致细胞增殖的剂量依赖性抑制，当PP 1 γ 1 mRNA表达被抑制> 85%时，生长停滞发生在类似于36-48 h后。增殖激活细胞分选仪分析显示，ISIS 14435/14439诱导的生长停滞与含有4 N DNA的细胞数量增加相关。免疫染色处理的细胞显示，PP 1 γ 1表达的抑制有丝分裂纺锤体的形成没有明显的影响。然而，表达下降与细胞分裂后期的细胞分裂失败和双核体的形成有关。
In lower eukaryotic organisms, the loss of serine/threonine protein phosphatase type 1 (PP1) results in growth arrest after the onset of mitosis. In humans, four highly homologous isoforms of PP1 (PP1 alpha, PP1 delta, PP1 gamma 1, and PP1 gamma 2) have been identified. Determining the roles of these phosphatases, however, has proven difficult due to the lack of subtype-specific inhibitors. In this study, we developed chimeric antisense 2'-O-(2-methoxy)ethylphosphothioate oligonucleotides targeting human PP1 gamma 1 that specifically inhibit PP1 gamma 1 gene expression. Two potent antisense oligonucleotides (ISIS 14435 and 14439; IC50 similar to 50 nM) were then employed to elucidate the cellular functions of PP1 gamma 1 during cell cycle progression. In A549 cells, the inhibition of PP1 gamma 1 expression resulted in a dose-dependent inhibition of cellular proliferation, with growth arrest occurring after similar to 36-48 h, when PP1 gamma 1 mRNA expression was inhibited by >85%. Fluorescence-activated cell sorter analysis revealed that ISIS 14435/14439-induced growth arrest was associated with an increase in the number of cells containing 4N DNA. Immunostaining of treated cells revealed that the inhibition of PP1 gamma 1 expression had no apparent effect on the formation of mitotic spindles. However, decreased expression was associated with the failure of cell division in a late stage of cytokinesis and the formation of dikaryons.