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 tr ansmission of mitogenic signals from cell surface receptors to the nucleus. In order to examine this pathway's role in leukemic transformation, a cond itionally active (p-estradiol-inducible) form of the MEK1 protein was creat ed 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 ch imeric Delta MEK1:ER oncoprotein into cytokine-dependent human TF-1 and mur ine FDC-P1 hematopoietic cell lines. Two different types of cells were reco vered after drug selection in medium containing either cytokine or beta-est radiol: (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 p articular cell line. To determine whether BCL2 overexpression could synergi ze with the Delta MEK1:ER oncoprotein in relieving cytokine dependence, the cytokine-dependent Delta MEK1:ER-expressing cells were infected with a BCL 2-containing retrovirus, and the frequency of MEK1-responsive cells determi ned. BCL2 overexpression, by itself, did not relieve cytokine dependency of the parental cells, however, it did increase the frequency at which MEK1-r esponsive cells were recovered approximately 10-fold. Delta MEK1:ER+BCL2 ce lls remained viable for at least 3 days after estradiol deprivation, wherea s viability was readily last upon withdrawal of p-estradiol in the MEK1-res ponsive cells which lacked BCL2 overexpression. The MAP kinases, ERK1 and E RK2 were activated in response to Delta MEK1:ER stimulation in both Delta M EK1: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 a nd Delta MEK1:ER+BCL2 infected cells expressed cDNAs encoding the autocrine cytokine GM-CSF, more GM-CSF cDNAs and bioactivity were detected in the ME K1-responsive Delta MEK1:ER+BCL2 cells than in the MEK1-responsive cells la cking BCL2 or cytokine-dependent cells. These conditionally transformed cel ls will be useful in furthering our understanding of the roles MEK1 and BCL 2 play in the prevention of apoptosis in hematopoietic cells.