Abstract

The effects of deregulated Raf activation on the growth and differentiation of hematopoietic cells were investigated. The cytokine-dependent murine myeloid FDC-P1 and human erythroleukemic TF-1 cell lines were transformed to grow in response to deregulated Raf expression in the absence of exogenous cytokines, The conditionally active Raf proteins were regulated by beta -estradiol as cDNAs containing the Raf catalytic, but lacking negative-regulatory domains, were ligated to the hormone binding domain of the estrogen receptor (Delta Raf:ER). Continuous Delta Raf expression prevented apoptosis in the absence of exogenous cytokines and altered the morphology of the FD/Delta Raf:ER cells as they grew in large aggregated masses (>100 cells) whereas the parental cytokine-dependent FDC-P1 cells grew in smaller grape-like clusters (<10 cells). FD/Raf-1:ER cells growing in response to Raf activation displayed decreased levels of the Mac-2 and Mac-3 molecules on their cell surface. In contrast, when these cells were cultured in IL-3, higher levels of these adhesion molecules were detected. Expression of activated Raf oncoproteins also abrogated cytokine dependency and prevented apoptosis of TF-1 cells. Moreover, the differentiation status of these Raf-responsive cells was more immature upon Raf activation as culture with the differentiation-inducing agent phorbol 12 myristate 13-acetate (PMA) and beta -estradiol resulted in decreased levels of the CD11b and CD18 integrin molecules on the cell surface. In contrast when the Raf-responsive cells were induced to differentiate with PMA and GM-CSF, in the absence of Delta Raf:ER activation, increased levels of the CD11b and CD18 molecules were detected. Retinoic acid (RA) inhibited H-3-thymidine incorporation in response to GM-CSF. Interestingly, Raf activation counterbalanced the inhibition of DNA synthesis caused by RA but not PMA. Thus deregulated Raf expression can alter cytokine dependency, integrin expression and the stage of differentiation. These Raf-responsive cell lines will be useful in elucidating the roles of the MAP kinase cascade on hematopoietic cell differentiation and malignant transformation.