Conditional cell transformation by doxycycline-controlled expression of the MC29 v-myc allele

To investigate the molecular basis of oncogenesis induced by the v-myc onco gene of avian myelocytomatosis virus MC29, we developed a conditional cell transformation system in which expression of the MC29 v-myc allele is depen dent on a doxycycline-sensitive transactivator (tTA). Clonal lines of quail embryo fibroblasts transformed by doxycycline-controlled v-myc revert to t he normal phenotype and lose their ability to grow in soft agar after the a ddition of doxycycline. Repression of v-myc causes the cells to withdraw fr om 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 t hese cells occurs within 14 h after the addition of doxycycline, the first morphological alterations are observed after 24 h, and after 3 days, the mo rphology changed entirely from small rounded cells showing a typical myc-tr ansformed phenotype to large flat cells resembling normal fibroblasts. Cell s exposed to doxycycline for 3 days reexpressed v-myc within 24 h after wit hdrawal of the drug from the culture medium, partial retransformation occur red after 2 days, and complete morphological transformation was reestablish ed 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 (rt TA) that is activated by doxycycline. The striking differential expression of known transformation-sensitive genes and of new candidate v-myc target g enes revealed the tightness of the doxycycline-controlled v-myc expression system. The data also indicate that expression of v-myc in these cells is i ndispensable for enhanced proliferation, transformation, and immortalizatio n. (C) 1999 Academic Press.