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 (beta-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 deltaMEK1: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 deltaMEK1:ER oncoprotein but remained cytokine-dependent and (2) MEK1-responsive cells that grew in response to deltaMEK1: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 deltaMEK1:ER oncoprotein in relieving cytokine dependence, the cytokine-dependent deltaMEK1: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. DeltaMEK1:ER+BCL2 cells remained viable for at least 3 days after estradiol deprivation, whereas viability was readily lost upon withdrawal of beta-estradiol in the MEK1-responsive cells which lacked BCL2 overexpression. The MAP kinases, ERK1 and ERK2 were activated in response to deltaMEK1:ER stimulation in both deltaMEK1:ER and deltaMEK1:ER+BCL2 cells. As compared to the cytokine-dependent deltaMEK1:ER and BCL2 infected cells, MEK1-responsive BCL2 infected cells expressed higher levels of BCL2. While both MEK1-responsive deltaMEK1:ER and deltaMEK1:ER+BCL2 infected cells expressed cDNAs encoding the autocrine cytokine GM-CSF, more GM-CSF cDNAs and bioactivity were detected in the MEK1-responsive deltaMEK1: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.
Blalock
W L
WL
Department of Microbiology and Immunology, East Carolina University School of Medicine, Greenville, NC 27858, USA.
Moye
P W
PW
Chang
F
F
Pearce
M
M
Steelman
L S
LS
McMahon
M
M
McCubrey
J A
JA
D000818:Animals; D017209:Apoptosis / Q000502:physiology; D001483:Base Sequence; D001854:Bone Marrow Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D002453:Cell Cycle / Q000187:drug effects; D002455:Cell Division / Q000502:physiology; D002460:Cell Line; D017931:DNA Primers; D018076:DNA, Complementary; D004958:Estradiol / Q000494:pharmacology; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000502:physiology*; D006801:Humans; D007377:Interleukin-3 / Q000502:physiology*; D048369:MAP Kinase Kinase 1; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D013629:Tamoxifen / Q000031:analogs & derivatives / Q000494:pharmacology; D013936:Thymidine / Q000378:metabolism
D017931:DNA Primers; D018076:DNA, Complementary; D007377:Interleukin-3; D019253:Proto-Oncogene Proteins c-bcl-2; D013629:Tamoxifen; C016601:afimoxifene; D004958:Estradiol; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D017346:Protein Serine-Threonine Kinases; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; C482127:Map2k1 protein, mouse; D020929:Mitogen-Activated Protein Kinase Kinases; D013936:Thymidine