Guan X, Ruch R J
Department of Pathology, Medical College of Ohio, Toledo 43699, USA.
Carcinogenesis. 1996 Sep;17(9):1791-8. doi: 10.1093/carcin/17.9.1791.
Treatment of WB-F344 rat liver epithelial cells with DDT (1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane) or lindane induces a loss of gap junction plaques and a decrease in the phosphorylated gap junction protein connexin43-P2 (Cx43-P2), which is associated with the plaques. In this study we have considered several mechanisms. The loss of junctional plaques could be due to disaggregation of junctional particles or to endocytosis of the plaques, while the loss of Cx43-P2 could be due to dephosphorylation or degradation. Immunohistochemical analyses of DDT- or lindane-treated cells revealed a reduction in plasma membranous Cx43-positive gap junction plaques coincident with the appearance of Cx43-positive punctate cytoplasmic structures. The cytoplasmic Cx43-positive structures eventually disappeared after 4 h treatment. Diffuse Cx43-positive plasma membranous staining was not seen following DDT or lindane treatment. Western blot analyses of these cells indicated that Cx43-P2 decreased in a time-dependent manner that paralleled the disappearance of gap junction plaques from the plasma membrane. The loss of Cx43-P2 was not due to dephosphorylation, since no increase in non-phosphorylated (Cx43-NP) or other phosphorylated (Cx43-P1) forms of the protein were evident. The decrease in Cx43-P2 and the disappearance of cytoplasmic Cx43-positive structures were prevented by colchicine and chloroquine, which suggests that Cx43-P2-containing plaques were internalized and degraded in lysosomes. In addition, two small (approximately 18 and approximately 22 kDa) bands appeared in Western blots coincident with the loss of Cx43-P2 and may be degradation products of the protein. These immunohistochemical and biochemical data strongly suggest that the loss of gap junction plaques and of Cx43-P2 in WB-F344 cells treated with DDT and lindane were due to endocytosis of the plaques and degradation of Cx43-P2 in lysosomes.
用滴滴涕（1,1-双（对氯苯基）-2,2,2-三氯乙烷）或林丹处理WB-F344大鼠肝上皮细胞，会导致间隙连接斑的丧失以及与这些斑相关的磷酸化间隙连接蛋白连接蛋白43-P2（Cx43-P2）减少。在本研究中，我们考虑了几种机制。连接斑的丧失可能是由于连接颗粒的解聚或斑的内吞作用，而Cx43-P2的丧失可能是由于去磷酸化或降解。对经滴滴涕或林丹处理的细胞进行免疫组织化学分析发现，质膜上Cx43阳性间隙连接斑减少，同时出现Cx43阳性点状细胞质结构。经4小时处理后，细胞质中Cx43阳性结构最终消失。滴滴涕或林丹处理后未观察到弥漫性Cx43阳性质膜染色。对这些细胞进行的蛋白质印迹分析表明，Cx43-P2以时间依赖性方式减少，这与质膜上间隙连接斑的消失平行。Cx43-P2的丧失并非由于去磷酸化，因为未观察到该蛋白的非磷酸化（Cx43-NP）或其他磷酸化（Cx43-P1）形式增加。秋水仙碱和氯喹可阻止Cx43-P2的减少以及细胞质中Cx43阳性结构的消失，这表明含Cx43-P2的斑被内化并在溶酶体中降解。此外，在蛋白质印迹中出现了两条小带（约18 kDa和约22 kDa），与Cx43-P2的丧失同时出现，可能是该蛋白的降解产物。这些免疫组织化学和生化数据有力地表明，用滴滴涕和林丹处理的WB-F344细胞中间隙连接斑和Cx43-P2的丧失是由于斑的内吞作用以及Cx43-P2在溶酶体中的降解。
