Long E, Lazaris-Karatzas A, Karatzas C, Zhao X
Department of Animal Science, Macdonald Campus of McGill University, 21,111 Lakeshore Rd, Quebec, H9X 3V9, Ste-Anne-de-Bellevue, Canada.
Int J Biochem Cell Biol. 2001 Feb;33(2):133-41. doi: 10.1016/s1357-2725(00)00089-3.
The eukaryotic translation initiation factor 4E regulates the proliferation of many cell types. In the present study, the effect of its overexpression on the growth of an immortalized bovine mammary epithelial cell line, MAC-T, has been investigated. Since involvement of cyclin D1 in growth regulation of other cell types has been suggested previously, the differences in cyclin D1 expression among the 4E-overexpressing and parental cells were also investigated.
The cDNA of mouse eukaryotic translation initiation factor 4E coding region (either wild-type or mutant, where Trp-56 was mutated to Ala) was transfected into MAC-T cells, and its protein expression was detected by Western blot analysis. Growth rates and saturation densities were calculated based on the cell counting data at desired time points.
The cells overexpressing wild-type 4E displayed higher growth rates and saturation densities compared to the parental cells (P<0.05), whereas cells expressing mutant 4E showed lower growth rates and saturation densities than the parental controls (P<0.05). The amounts of cyclin D1 mRNA and protein were higher in the wild-type transfectants than in the parental controls, whereas the mutant transfectants contained lower amounts of cyclin D1 mRNA and protein compared to the parental cells.
Our results suggest that overexpression of eukaryotic translation initiation factor 4E leads to increased cyclin D1 expression at the transcriptional level, which consequently stimulates the proliferation of MAC-T cells.
真核生物翻译起始因子4E调控多种细胞类型的增殖。在本研究中，已对其过表达对永生化牛乳腺上皮细胞系MAC-T生长的影响进行了研究。由于之前已有人提出细胞周期蛋白D1参与其他细胞类型的生长调控，因此还研究了4E过表达细胞和亲本细胞中细胞周期蛋白D1表达的差异。
将小鼠真核生物翻译起始因子4E编码区的cDNA（野生型或突变型，其中色氨酸-56突变为丙氨酸）转染到MAC-T细胞中，并通过蛋白质印迹分析检测其蛋白表达。根据所需时间点的细胞计数数据计算生长速率和饱和密度。
与亲本细胞相比，过表达野生型4E的细胞显示出更高的生长速率和饱和密度（P<0.05），而表达突变型4E的细胞显示出比亲本对照更低的生长速率和饱和密度（P<0.05）。野生型转染子中细胞周期蛋白D1的mRNA和蛋白量高于亲本对照，而突变型转染子中细胞周期蛋白D1的mRNA和蛋白量比亲本细胞少。
我们的结果表明，真核生物翻译起始因子4E的过表达导致细胞周期蛋白D1在转录水平上的表达增加，从而刺激MAC-T细胞的增殖。
