Oikawa T, Yamada T, Kubota Y, Kondoh N, Hitomi Y, Uchiumi F, Yamamoto T
Department of Cell Genetics, Sasaki Institute, University of Tokyo, Japan.
Cytogenet Cell Genet. 1996;72(1):12-9. doi: 10.1159/000134152.
Somatic cell hybrids were obtained by cell fusions between Lck-positive EL4 mouse T lymphoma cells and Lck-negative B82 mouse fibroblasts of S194 mouse plasmacytoma cells to examine negative control of lck gene expression in the resulting hybrids. Western blot analysis using a monoclonal antibody against the Lck protein showed a marked decrease in p56lck expression in B82 x EL4 (BEL) hybrids. In contrast to BEL hybrids, the level of p56lck was not changed significantly in S194 x EL4 (SEL) hybrids and was approximately one-half of that seen in EL4 cells. Diminished expression of the Lck protein in BEL hybrids paralleled downregulation of lck mRNA, which was exclusively transcribed from the distal promoter in EL4 cells. It is unlikely that the suppression was simply a consequence of chromosome segregation critical for lck gene expression, since BEL hybrids retained the EL4-derived lck gene and most of the chromosomes from both parental cells. The results from treatment of BEL hybrids with actinomycin D or cycloheximide suggested that suppression of lck gene expression in the hybrids might not be due to posttranscriptional control. DNA methylation status in the lck distal promoter and the coding regions did not appear to correlate with the expression of the gene. Our results suggest that negative control of lck gene expression differs between fibroblasts and B cells, in that lck gene expression in T cells can be shut down by transfer of a putative repressor factor or factors in fibroblasts but not in B cells.
通过Lck阳性的EL4小鼠T淋巴瘤细胞与S194小鼠浆细胞瘤细胞的Lck阴性B82小鼠成纤维细胞进行细胞融合，获得了体细胞杂种，以检查所得杂种中lck基因表达的负调控。使用抗Lck蛋白的单克隆抗体进行的蛋白质印迹分析显示，B82×EL4（BEL）杂种中p56lck的表达明显降低。与BEL杂种相反，S194×EL4（SEL）杂种中p56lck的水平没有明显变化，约为EL4细胞中水平的一半。BEL杂种中Lck蛋白表达的减少与lck mRNA的下调平行，lck mRNA仅从EL4细胞中的远端启动子转录。这种抑制不太可能仅仅是lck基因表达关键的染色体分离的结果，因为BEL杂种保留了EL4来源的lck基因以及来自两个亲本细胞的大部分染色体。用放线菌素D或环己酰亚胺处理BEL杂种的结果表明，杂种中lck基因表达的抑制可能不是由于转录后控制。lck远端启动子和编码区的DNA甲基化状态似乎与该基因的表达无关。我们的结果表明，成纤维细胞和B细胞中lck基因表达的负调控有所不同，因为T细胞中的lck基因表达可以通过成纤维细胞中一种或多种假定的抑制因子的转移而关闭，但B细胞中则不然。
