Interleukin-3 (IL-3) is required for the proliferation and survival of growth factor-dependent myeloid cell lines. In the absence of IL-3, cells rapidly accumulate in the G1 phase of the cell cycle and subsequently undergo programmed cell death or apoptosis. A key regulator of proliferation appears to be the Raf-1 serine/threonine kinase. In this report we demonstrate that Raf-1 activity is required for the growth of the IL-3-dependent murine myeloid 32D cell line and it is regulated at least at two levels through regulation of its kinase activity and through ligand-dependent regulation of steady state Raf-1 protein levels. Steady state levels of Raf-1 protein, but not mRNA, were tightly regulated by IL- 3, suggesting that translation of c-raf mRNA is ligand-dependent. Introduction of retroviral v-raf vectors, which constitutively expressed gag-v-Raf protein, had beneficial consequences on both the proliferation and survival of 32D cells, whereas c-raf viral vectors had no benefit and were susceptible to ligand-dependent regulation of Raf-1 protein levels. v-raf clones proliferated at an increased rate with a shortened G1 phase, had decreased requirements for IL-3 for growth, and when deprived of IL-3 had delayed onset of apoptosis. When introduced in combination with v-myc into myeloid cells, v-raf promoted IL-3-dependent growth, whereas c-raf did not. Autonomous growth of these cultures was dependent upon Raf activity since vectors expressing raf null mutants compromised their ability to grow in the absence of IL-3. These studies therefore suggest that synergistic transformation by Raf and Myc involves combination of signals that promote ligand-independent myeloid cell cycle progression (Myc) and survival (Raf).
