Combined effects of aberrant MEK1 activity and BCL2 overexpression on relieving the cytokine dependency of human and murine hematopoietic cells

The MEK1 oncoprotein plays a critical role in Ras/Raf/ MEK/MAPK-mediated transmission of mitogenic signals from cell surface receptors to the nucleus. In order to examine this pathway's role in leukemic transformation, a conditionally active (p-estradiol-inducible) form of the MEK1 protein was created by ligating a cDNA encoding an N-terminal truncated form of MEK1 to the hormone-binding domain of the estrogen receptor (ER), We introduced this chimeric Delta MEK1:ER oncoprotein into cytokine-dependent human TF-1 and murine FDC-P1 hematopoietic cell lines. Two different types of cells were recovered after drug selection in medium containing either cytokine or beta-estradiol: (1) cells that expressed the Delta MEK1:ER oncoprotein but remained cytokine-dependent and (2) MEK1-responsive cells that grew in response to Delta MEK1:ER activation. Cytokine-dependent cells were recovered 10(2) to 10(4) times more frequently than MEK1-responsive cells depending upon the particular cell line. To determine whether BCL2 overexpression could synergize with the Delta MEK1:ER oncoprotein in relieving cytokine dependence, the cytokine-dependent Delta MEK1:ER-expressing cells were infected with a BCL2-containing retrovirus, and the frequency of MEK1-responsive cells determined. BCL2 overexpression, by itself, did not relieve cytokine dependency of the parental cells, however, it did increase the frequency at which MEK1-responsive cells were recovered approximately 10-fold. Delta MEK1:ER+BCL2 cells remained viable for at least 3 days after estradiol deprivation, whereas viability was readily last upon withdrawal of p-estradiol in the MEK1-responsive cells which lacked BCL2 overexpression. The MAP kinases, ERK1 and ERK2 were activated in response to Delta MEK1:ER stimulation in both Delta MEK1:ER and Delta MEK1:ER+BCL2 cells. As compared to the cytokine-dependent Delta MEK1:ER and BCL2 infected cells, MEK1-responsive BCL2 infected cells expressed higher levels of BCL2, While both MEK1-responsive Delta MEK1:ER and Delta MEK1:ER+BCL2 infected cells expressed cDNAs encoding the autocrine cytokine GM-CSF, more GM-CSF cDNAs and bioactivity were detected in the MEK1-responsive Delta MEK1:ER+BCL2 cells than in the MEK1-responsive cells lacking BCL2 or cytokine-dependent cells. These conditionally transformed cells will be useful in furthering our understanding of the roles MEK1 and BCL2 play in the prevention of apoptosis in hematopoietic cells.