Oberst C, Hartl M, Weiskirchen R, Bister K
Institute of Biochemistry, University of Innsbruck, Peter-Mayr-Str. 1a, Innsbruck, A-6020, Austria.
Virology. 1999 Jan 20;253(2):193-207. doi: 10.1006/viro.1998.9499.
To investigate the molecular basis of oncogenesis induced by the v-myc oncogene of avian myelocytomatosis virus MC29, we developed a conditional cell transformation system in which expression of the MC29 v-myc allele is dependent on a doxycycline-sensitive transactivator (tTA). Clonal lines of quail embryo fibroblasts transformed by doxycycline-controlled v-myc revert to the normal phenotype and lose their ability to grow in soft agar after the addition of doxycycline. Repression of v-myc causes the cells to withdraw from the cell cycle, and long-term survival in culture requires reexpression of v-myc. Although complete repression of v-myc mRNA and v-Myc protein in these cells occurs within 14 h after the addition of doxycycline, the first morphological alterations are observed after 24 h, and after 3 days, the morphology changed entirely from small rounded cells showing a typical myc-transformed phenotype to large flat cells resembling normal fibroblasts. Cells exposed to doxycycline for 3 days reexpressed v-myc within 24 h after withdrawal of the drug from the culture medium, partial retransformation occurred after 2 days, and complete morphological transformation was reestablished after 6 days. Analogous results were obtained with a cell line in which expression of the v-myc allele is dependent on a reverse transactivator (rtTA) that is activated by doxycycline. The striking differential expression of known transformation-sensitive genes and of new candidate v-myc target genes revealed the tightness of the doxycycline-controlled v-myc expression system. The data also indicate that expression of v-myc in these cells is indispensable for enhanced proliferation, transformation, and immortalization.
为了研究禽成髓细胞瘤病毒MC29的v-myc癌基因诱导肿瘤发生的分子基础，我们开发了一种条件性细胞转化系统，其中MC29 v-myc等位基因的表达依赖于强力霉素敏感的反式激活因子（tTA）。由强力霉素控制的v-myc转化的鹌鹑胚胎成纤维细胞克隆系在添加强力霉素后恢复到正常表型，并失去在软琼脂中生长的能力。v-myc的抑制导致细胞退出细胞周期，并且在培养中的长期存活需要v-myc的重新表达。尽管在添加强力霉素后14小时内这些细胞中的v-myc mRNA和v-Myc蛋白完全被抑制，但在24小时后观察到首次形态学改变，并且在3天后，形态完全从显示典型myc转化表型的小圆形细胞变为类似于正常成纤维细胞的大扁平细胞。暴露于强力霉素3天的细胞在从培养基中撤出药物后24小时内重新表达v-myc，2天后发生部分重新转化，6天后重新建立完全的形态转化。用v-myc等位基因的表达依赖于由强力霉素激活 的反向反式激活因子（rtTA）的细胞系获得了类似的结果。已知的转化敏感基因和新的候选v-myc靶基因的显著差异表达揭示了强力霉素控制 的v-myc表达系统的严密性。数据还表明，v-myc在这些细胞中的表达对于增强增殖、转化和永生化是必不可少的。
