Jou Y S, Matesic D, Dupont E, Lu S C, Rupp H L, Madhukar B V, Oh S Y, Trosko J E, Chang C C
Department of Pediatrics and Human Development, College of Human Medicine, Michigan State University, East Lansing 48824-1317.
Mol Carcinog. 1993;8(4):234-44. doi: 10.1002/mc.2940080406.
To study the biochemical basis of gap-junctional intercellular communication (GJIC) and its role in tumorigenesis, a mammalian cell expression vector carrying both a rat connexin 43 (Cx43) cDNA and an amplifiable dihydrofolate reductase (DHFR) gene was transfected into the GJIC-deficient rat liver mutant cell line aB1. Two stable transfectants were selected for further amplification of the transfected Cx43 gene by increasing stepwise the concentration of methotrexate (MTX) in the culture medium. The results indicate that GJIC was restored in these two Cx43 cDNA transfectants after they became highly resistant to MTX but not in the control-vector transfectants, in which the DHFR gene was similarly amplified. The amount of Cx43 DNA revealed by Southern blot analysis and the expression of Cx43 gene revealed by northern and western blot analyses were concomitantly increased in the Cx43 cDNA transfectants resistant to high concentrations of MTX. Western blot analysis, using an antipeptide antibody that specifically recognizes Cx43 protein, further revealed that an approximately 46-kDa phosphorylated Cx43 protein that was prominent in the parental GJIC-competent cells was absent in the aB1 cells. This Cx43 protein, however, reappeared in the two Cx43 cDNA transfectants after amplification. After treatment of the membrane proteins with alkaline phosphatase in vitro, the approximately 46- and 44-kDa proteins disappeared, whereas the approximately 42-kDa proteins remained with increasing intensity, indicating that the higher molecular-weight proteins were the phosphorylated Cx43. These results indicate that a defect in posttranslational phosphorylation of Cx43 protein associated with low expression of the Cx43 gene might be responsible for the GJIC deficiency in aB1 cells and that increased expression of Cx43 by gene amplification might restore this phosphorylated Cx43 protein and so reestablish GJIC.
为研究间隙连接细胞间通讯（GJIC）的生化基础及其在肿瘤发生中的作用，将携带大鼠连接蛋白43（Cx43）cDNA和可扩增二氢叶酸还原酶（DHFR）基因的哺乳动物细胞表达载体转染至缺乏GJIC的大鼠肝突变细胞系aB1中。通过逐步增加培养基中甲氨蝶呤（MTX）的浓度，筛选出两个稳定转染子以进一步扩增转染的Cx43基因。结果表明，这两个Cx43 cDNA转染子在对MTX产生高度抗性后恢复了GJIC，但对照载体转染子未恢复，在对照载体转染子中DHFR基因同样被扩增。在对高浓度MTX具有抗性的Cx43 cDNA转染子中，Southern印迹分析显示的Cx43 DNA量以及Northern和Western印迹分析显示的Cx43基因表达均随之增加。使用特异性识别Cx43蛋白的抗肽抗体进行的Western印迹分析进一步显示，在亲本具有GJIC能力的细胞中突出的约46 kDa磷酸化Cx43蛋白在aB1细胞中不存在。然而，该Cx43蛋白在扩增后的两个Cx43 cDNA转染子中重新出现。用碱性磷酸酶体外处理膜蛋白后，约46 kDa和44 kDa的蛋白消失，而约42 kDa的蛋白强度增加，表明较高分子量的蛋白是磷酸化的Cx43。这些结果表明，与Cx43基因低表达相关的Cx43蛋白翻译后磷酸化缺陷可能是aB1细胞中GJIC缺陷的原因，并且通过基因扩增增加Cx43的表达可能会恢复这种磷酸化的Cx43蛋白，从而重新建立GJIC。
