Matesic D F, Rupp H L, Bonney W J, Ruch R J, Trosko J E
Department of Pediatrics/Human Development, Michigan State University, East Lansing 48824.
Mol Carcinog. 1994 Aug;10(4):226-36. doi: 10.1002/mc.2940100407.
The effects of three tumor promoters on gap-junction permeability; connexin 43 and 26 mRNA levels, protein levels, and phosphorylation; and the numbers of gap-junctional membrane plaques were studied in the rat liver epithelial cell line WB-F344 to determine whether changes in these parameters correlated with the inhibition of gap-junction function. 12-O-tetradecanoylphorbol-13-acetate (TPA; 10 ng/mL), dieldrin (10 micrograms/mL), and heptachlor epoxide (10 micrograms/mL) inhibited gap-junctional intercellular communication (GJIC) assayed by fluorescent dye transfer by 80-90% after a 5-min exposure and by more than 90% within 1 h. Decreases in steady-state connexin 43 mRNA levels were detected by northern blot analysis within 1 h and paralleled changes in steady-state beta-actin mRNA, but these changes did not occur rapidly enough to account for the rapid loss of gap-junction function. A substantial loss in the number of connexin 43 immunostained gap-junctional membrane plaques was detected after a 15-min exposure to all three promoters, but little change had occurred at 5 min. Western blot analyses using connexin 43-specific antibodies showed changes in the degree of connexin 43 phosphorylation for all three tumor promoters. TPA induced the appearance of a fourth connexin 43-immunoreactive band (P3) and a concomitant decrease in the relative intensity of the unphosphorylated (P0) band within 5 min of treatment. P3, in addition to bands P1 and P2, disappeared after treatment with alkaline phosphatase. In contrast, dieldrin and heptachlor expoxide induced loss of P2 with a concomitant increase in the relative staining intensity of P0 within 1 h of exposure, but no changes were seen after 5 min. Connexin 43 phosphorylation levels recovered in parallel with the recovery of GJIC for all three tumor promoters. Connexin 26 mRNA levels showed little change after a 1-h exposure to three promoters, but reductions in connexin 26 immunofluorescent staining were observed. These results suggest that (i) TPA-induced hyperphosphorylation of connexin 43 occurred fast enough to account for inhibition of GJIC, (ii) dieldrin and heptachlor expoxide modulated connexin phosphorylation in a manner different from TPA by promoting hypophosphorylation of connexin 43, (iii) redistribution of plasma membrane gap-junctional plaques after treatment with phorbol ester and non-phorbol-ester tumor promoters occurred subsequent to changes in gap-junction permeability, and (iv) changes in connexin mRNA levels could not account for the losses in fluorescent dye coupling induced by these promoters.
在大鼠肝上皮细胞系WB-F344中研究了三种肿瘤启动子对缝隙连接通透性、连接蛋白43和26的mRNA水平、蛋白水平及磷酸化作用以及缝隙连接膜斑数量的影响，以确定这些参数的变化是否与缝隙连接功能的抑制相关。12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯（TPA；10 ng/mL）、狄氏剂（10微克/mL）和环氧七氯（10微克/mL）在暴露5分钟后通过荧光染料转移检测发现，缝隙连接细胞间通讯（GJIC）被抑制了80 - 90%，1小时内抑制率超过90%。通过Northern印迹分析在1小时内检测到连接蛋白43的稳态mRNA水平下降，且与稳态β - 肌动蛋白mRNA的变化平行，但这些变化发生得不够迅速，无法解释缝隙连接功能的快速丧失。暴露于所有三种启动子15分钟后，检测到连接蛋白43免疫染色的缝隙连接膜斑数量大幅减少，但在5分钟时几乎没有变化。使用连接蛋白43特异性抗体的Western印迹分析显示，所有三种肿瘤启动子均使连接蛋白43的磷酸化程度发生了变化。TPA在处理5分钟内诱导出现第四条连接蛋白43免疫反应带（P3），同时未磷酸化带（P0）的相对强度随之降低。用碱性磷酸酶处理后，除了P1和P2带外，P3带消失。相反，狄氏剂和环氧七氯在暴露1小时内诱导P2带消失，同时P0带的相对染色强度增加，但在5分钟后未见变化。所有三种肿瘤启动子的连接蛋白43磷酸化水平均与GJIC的恢复平行恢复。暴露于三种启动子1小时后，连接蛋白26的mRNA水平变化不大，但观察到连接蛋白26免疫荧光染色减少。这些结果表明：（i）TPA诱导的连接蛋白43过度磷酸化发生得足够快，足以解释GJIC的抑制；（ii）狄氏剂和环氧七氯通过促进连接蛋白43的低磷酸化，以与TPA不同的方式调节连接蛋白磷酸化；（iii）用佛波酯和非佛波酯肿瘤启动子处理后，质膜缝隙连接斑的重新分布发生在缝隙连接通透性变化之后；（iv）连接蛋白mRNA水平的变化无法解释这些启动子诱导的荧光染料偶联的丧失。
