Conditional Cell Transformation by Doxycycline-Controlled Expression of the MC29 v-mycAllele

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To investigate the molecular basis of oncogenesis induced by the v-myconcogene of avian myelocytomatosis virus MC29, we developed a conditional cell transformation system in which expression of the MC29 v-mycallele is dependent on a doxycycline-sensitive transactivator (tTA). Clonal lines of quail embryo fibroblasts transformed by doxycycline-controlled v-mycrevert to the normal phenotype and lose their ability to grow in soft agar after the addition of doxycycline. Repression of v-myccauses the cells to withdraw from the cell cycle, and long-term survival in culture requires reexpression of v-myc. Although complete repression of v-mycmRNA 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 typicalmyc-transformed phenotype to large flat cells resembling normal fibroblasts. Cells exposed to doxycycline for 3 days reexpressed v-mycwithin 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-mycallele 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-myctarget genes revealed the tightness of the doxycycline-controlled v-mycexpression system. The data also indicate that expression of v-mycin these cells is indispensable for enhanced proliferation, transformation, and immortalization.