Levi B Z, Kasik J W, Ozato K
Laboratory of Developmental and Molecular Immunity, National Institute of Child Health and Human Development, Bethesda, MD 20892.
Cell Differ Dev. 1988 Nov;25 Suppl:95-101. doi: 10.1016/0922-3371(88)90105-0.
To study the function of proto-oncogene c-fos, we prepared an antisense plasmid that expresses in mammalian cells c-fos antisense RNA which is complementary to the endogenous c-fos mRNA. Upon transfection into undifferentiated F9 EC cells, the antisense plasmid directed constitutive expression of a large amount of c-fos antisense RNA. These cells were very low in the basal level of c-fos message and were unable to induce c-fos message when stimulated with interferon or phorbol ester. The failure to induce c-fos message led to the blockade of c-fos protein expression in these cells. Thus, these cells represented a c-fos defective phenotype. The blockade of c-fos gene expression seen in antisense-cells could be caused by rapid degradation of the c-fos message, since c-fos mRNA expression was rescued in these cells when treated with protein synthesis inhibitor, cycloheximide. We found that expression of c-myc gene was down-regulated in c-fos antisense-cells: Although control undifferentiated F9 cells constitutively expressed a high level of c-myc message, the antisense cells had a much lower amount of c-myc mRNA. Since p53 and heat shock gene 70 were expressed at comparable levels in control and antisense cells, c-myc gene expression appears to be regulated by c-fos gene in F9 EC cells. Lastly, these antisense cells grew as rapidly as control F9 cells and underwent differentiation after retinoic acid treatment, indicating that c-fos expression is not a prerequisite for differentiation of F9 cells.
为了研究原癌基因c-fos的功能，我们制备了一种反义质粒，该质粒在哺乳动物细胞中表达与内源性c-fos mRNA互补的c-fos反义RNA。转染到未分化的F9胚胎癌细胞后，反义质粒指导大量c-fos反义RNA的组成性表达。这些细胞的c-fos信使基础水平非常低，在用干扰素或佛波酯刺激时无法诱导c-fos信使。无法诱导c-fos信使导致这些细胞中c-fos蛋白表达受阻。因此，这些细胞表现出c-fos缺陷表型。反义细胞中观察到的c-fos基因表达受阻可能是由于c-fos信使的快速降解，因为在用蛋白质合成抑制剂环己酰亚胺处理这些细胞时，c-fos mRNA表达得以恢复。我们发现c-myc基因的表达在c-fos反义细胞中下调：虽然对照未分化的F9细胞组成性地表达高水平的c-myc信使，但反义细胞中的c-myc mRNA量要低得多。由于p53和热休克基因70在对照细胞和反义细胞中的表达水平相当，c-myc基因的表达似乎在F9胚胎癌细胞中受c-fos基因调控。最后，这些反义细胞的生长速度与对照F9细胞一样快，并且在视黄酸处理后发生分化，这表明c-fos表达不是F9细胞分化的先决条件。
