Fang M Z, Mar W C, Cho M H
School of Agricultural Biotechnology, Seoul National University, 441-744, Suwon, South Korea.
Toxicology. 2001 Mar 21;161(1-2):117-27. doi: 10.1016/s0300-483x(01)00344-4.
During the multistage carcinogenesis, functions of several key genes involved in the cell cycle control and cell-cell communication can be damaged. Gap junction intercellular communication (GJIC) is known to transfer small, water-soluble molecules through intercellular channels composed of proteins called connexins (Cxs). Therefore, aberrant expression of Cx may be one of the critical factors for the clonal expansion of initiated cells during the two-stage transformation. We already improved the classical in vitro two-stage transformation method using N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) as an initiator and cadmium as a promoter on Balb/3T3 A31 cells, and reconfirmed the promotional effect of cadmium with this method (Fang, M.Z., Cho, M.H., Lee, H.W., 2001. Improvement of in vitro two-stage transformation assay and detection of the promotional effect of cadmium, Toxicol. In Vitro (in press). In this study, precise roles of Cd on Cx expression in normal Balb/3T3 A31 and during the promotion stage of the in vitro two-stage transformation were elucidated. For this purpose, the Cx43, Cx32 and Cx26 protein levels, Cx43 and Cx26 mRNA levels and the cellular distribution location of Cx43 protein were determined. Normal Balb/3T3 cells expressed Cx43 and Cx32, but not Cx26. After a short-term treatment of cadmium on normal cells, phosphorylation of Cx43 protein increased and Cx32 protein level decreased. However, during the promotion stage of the in vitro two-stage transformation, transformed cells treated with cadmium for long periods expressed Cx43 and Cx32 highly, similar to the level of normal Balb/3T3 cells, compared to the nontransformed cells. Moreover, Cx43 of the transformed cells was distributed mostly in the perinuclear region rather than the intercellular membrane. These data suggest that cadmium may inhibit the GJIC by increasing the phosphorylation of Cx43 and decreasing the expression of Cx32 in the normal Balb/3T3 A31 cells. Our results also suggest that these changes are not associated with the cell transformation; transformed cells may reexpress Cx43 and Cx32 similar to the normal cells, though Cx43 protein is distributed aberrantly during the transformation process. Further studies are needed to clarify the relationship between transformation and posttranslational modification of the Cx proteins.
在多阶段致癌过程中，参与细胞周期调控和细胞间通讯的几个关键基因的功能可能会受损。已知间隙连接细胞间通讯（GJIC）通过由连接蛋白（Cxs）组成的细胞间通道转运小的水溶性分子。因此，Cx的异常表达可能是启动细胞在两阶段转化过程中克隆扩增的关键因素之一。我们已经改进了经典的体外两阶段转化方法，该方法使用N-甲基-N'-硝基-N-亚硝基胍（MNNG）作为启动剂，镉作为促进剂作用于Balb/3T3 A31细胞，并通过该方法再次证实了镉的促进作用（Fang, M.Z., Cho, M.H., Lee, H.W., 2001. 体外两阶段转化试验的改进及镉促进作用的检测，《毒理学体外研究》（即将发表））。在本研究中，阐明了镉对正常Balb/3T3 A31细胞中Cx表达以及体外两阶段转化促进阶段Cx表达的精确作用。为此，测定了Cx43、Cx32和Cx26蛋白水平、Cx43和Cx26 mRNA水平以及Cx43蛋白的细胞分布位置。正常Balb/3T3细胞表达Cx43和Cx32，但不表达Cx26。正常细胞经镉短期处理后，Cx43蛋白的磷酸化增加，Cx32蛋白水平降低。然而，在体外两阶段转化的促进阶段，长期用镉处理的转化细胞与未转化细胞相比，高度表达Cx43和Cx32，与正常Balb/3T3细胞水平相似。此外，转化细胞的Cx43主要分布在核周区域而非细胞间膜。这些数据表明，镉可能通过增加正常Balb/3T3 A31细胞中Cx43的磷酸化和降低Cx32的表达来抑制GJIC。我们的结果还表明，这些变化与细胞转化无关；转化细胞可能重新表达与正常细胞相似的Cx43和Cx32，尽管在转化过程中Cx43蛋白的分布异常。需要进一步研究来阐明转化与Cx蛋白翻译后修饰之间的关系。
