Kang K S, Yun J W, Yoon B, Lim Y K, Lee Y S
Department of Veterinary Public Health, College of Veterinary Medicine and School of Agricultural Biotechnology, Seoul National University, 103 Seodun-dong, Kwonsun-ku, 441-744, Suwon, South Korea.
Cancer Lett. 2001 May 26;166(2):147-53. doi: 10.1016/s0304-3835(01)00446-3.
Gap junctional intercellular communication (GJIC) is thought to be essential for maintaining cellular homeostasis and growth control. In order to detect any protective agent against tumor formation, we examined the anticarcinogenic effect of a germanium dioxide (GeO(2)) using a model system of GJIC in F344 rat liver epithelial cells, named WB cells. 12-O-tetradecanoylphorbol-13-acetate (TPA), known as tumor promoters, inhibited GJIC in the epithelial cells as determined by the scrape loading/dye transfer (SL/DT) assay. And GeO(2) recovered this inhibition of GJIC. Immunostaining of connexin 43 (Cx43) protein in WB cells indicated that TPA caused a loss of Cx43 protein from the cell membranes. However, GeO(2) treatment showed re-appearance of Cx43 protein on the membrane. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blots were analyzed to determine whether the test compounds might have altered the steady-state levels of gap junction mRNA and/or connexin protein levels or phosphorylation. The inhibition of GJIC by TPA in WB cells was correlated with the hyperphosphorylation of Cx43 as measured by mobility shifts of the western blot bands of Cx43. TPA induced hyperphosphorylation of Cx43 protein, while GeO(2) appeared to partially block this hyperphosphorylation. Here, we showed that pre- and co-incubation with GeO(2) in TPA-treated WB-cells abolished down-regulation of GJIC by TPA. These data suggest that GeO(2) may inhibit tumor promotion by enhancing GJIC.
间隙连接细胞间通讯（GJIC）被认为对于维持细胞内环境稳定和生长控制至关重要。为了检测任何抗肿瘤形成的保护剂，我们使用F344大鼠肝上皮细胞（称为WB细胞）中的GJIC模型系统，研究了二氧化锗（GeO₂）的抗癌作用。12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯（TPA），即众所周知的肿瘤促进剂，通过刮擦加载/染料转移（SL/DT）测定法确定，它抑制上皮细胞中的GJIC。而GeO₂恢复了这种对GJIC的抑制作用。WB细胞中连接蛋白43（Cx43）蛋白的免疫染色表明，TPA导致细胞膜上Cx43蛋白缺失。然而，GeO₂处理显示膜上Cx43蛋白重新出现。通过逆转录 - 聚合酶链反应（RT - PCR）和蛋白质免疫印迹分析来确定测试化合物是否可能改变了间隙连接mRNA的稳态水平和/或连接蛋白的蛋白质水平或磷酸化。WB细胞中TPA对GJIC的抑制与通过Cx43蛋白质免疫印迹条带迁移率变化测量的Cx43过度磷酸化相关。TPA诱导Cx43蛋白过度磷酸化，而GeO₂似乎部分阻断了这种过度磷酸化。在这里，我们表明在TPA处理的WB细胞中预先和共同孵育GeO₂消除了TPA对GJIC的下调作用。这些数据表明，GeO₂可能通过增强GJIC来抑制肿瘤促进作用。
