Effects of inducible MEK1 activation on the cytokine dependency of lymphoid cells

W L Blalock, M Pearce, F Chang, J T Lee, S C Pohnert, C Burrows, L S Steelman, R A Franklin, M McMahon, J A McCubrey
Leukemia 2001, 15 (5): 794-807
The Raf/MEK/MAP kinase cascade plays a critical role in transducing growth signals from activated cell surface receptors. Using deltaMEK1:ER, a conditionally active form of MEK1, we demonstrate the ability of this dual specificity protein kinase to abrogate the cytokine dependency of the murine lymphoid hematopoietic cell line FL5.12. Cytokine-independent cells were obtained from FL5.12 cells at a frequency of 1 x 10(-7), indicating that a low frequency of cells expressing deltaMEK1:ER were factor-independent. In general, cells that were converted to a cytokine-independent phenotype displayed a higher level of MAP kinase activity in response to deltaMEK1:ER activation than those that remained cytokine-dependent. deltaMEK1:ER-responsive cells could be maintained long-term in the presence of beta-estradiol, as well as the estrogen-receptor antagonist 4-hydroxy-tamoxifen. Removal of hormone led to the rapid cessation of cell growth in a manner similar to that observed when cytokine is withdrawn from the parental cells. GM-CSF mRNA transcripts were detected in the MEK1-responsive cells indicating that activated deltaMEK1:ER may induce a pathway leading to autocrine proliferation. Cytokine-dependent deltaMEK1:ER cells were found to increase the expression of GM-CSF receptor alpha (GM-CSFRalpha) in response to beta-estradiol. In contrast, MEK1-responsive cells were found to express constitutively lower levels of GM-CSFRalpha and beta common (betac) chains indicating that constitutive GM-CSF expression resulted in a decrease in GM-CSFR expression. Treatment of parental cells with supernatant from MEK1-responsive FL5.12 cells was sufficient to promote [3H]-thymidine incorporation. GM-CSF was found to enhance the viability of FL5.12 cells. The cell lines described here will be useful for elaborating the ability of the MAP kinase pathway to regulate cell proliferation in hematopoietic cells.

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