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 (-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 ⌬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 -estradiol: (1) cells that expressed the ⌬MEK1:ER oncoprotein but remained cytokine-dependent and (2) MEK1-responsive cells that grew in response to ⌬MEK1:ER activation. Cytokinedependent 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 ⌬MEK1:ER oncoprotein in relieving cytokine dependence, the cytokine-dependent ⌬MEK1:ERexpressing 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. ⌬MEK1:ER+BCL2 cells remained viable for at least 3 days after estradiol deprivation, whereas viability was readily lost upon withdrawal of -estradiol in the MEK1-responsive cells which lacked BCL2 overexpression. The MAP kinases, ERK1 and ERK2 were activated in response to ⌬MEK1:ER stimulation in both ⌬MEK1:ER and ⌬MEK1:ER+BCL2 cells. As compared to the cytokine-dependent ⌬MEK1:ER and BCL2 infected cells, MEK1-responsive BCL2 infected cells expressed higher levels of BCL2. While both MEK1-responsive ⌬MEK1:ER and ⌬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 ⌬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. Leukemia (2000) 14, 1080-1096.
Previously we documented the transposition of an intracistergration include: promoter insertion, 4,5 enhancer insertion, 6,7 nal A particle (IAP) provirus to the interleukin 3 (IL-3) locus inactivation of a gene, 8,9 and disruption of a mRNA stability ian virus 40 core enhancer sequence (Enh1), a transcriptional
Although insulin-like growth factor (IGF-1) stimulated 3H-thymidine incorporation upon addition to the interleukin-3 (IL-3)-dependent cell line FDC-P1, IGF-1 did not relieve IL-3 dependency for growth. To further examine the effects of IGF-1 on hematopoietic cells, FDC-P1 cells were infected with a retroviral construct (LISN) containing the human IGF-1 receptor (hIGF-1R) and neo genes. IL-3-independent cells were readily isolated after LISN infection when either IGF-1 or supraphysiologic concentrations of insulin were included in the culture medium. These cells were transformed to IL-3 independence by a ligand- dependent mechanism because their growth was dependent on the presence of either IGF-1 or insulin and growth factors capable of supporting autocrine growth were not detected. Furthermore, a monoclonal antibody (MoAb) directed against the human IGF-1R (alpha IR-3) inhibited IGF-1 but not IL-3-induced proliferation and these cells contained 20- to 200- fold more IGF-1 receptors than uninfected FDC-P1 cells. In contrast, when LISN-infected cells were plated in medium without exogenously supplied IGF-1 or insulin, factor-independent cells were rarely isolated. Growth of these cells was also inhibited by the alpha IR-3 MoAb and they expressed 100- to 400-fold more IGF-1 receptors than uninfected FDC-P1 cells. The endogenous IGF-1 and/or insulin present in the calf serum may have enabled their growth because these cells, unlike the parental cells, would proliferate in serum-free defined media and their growth was again inhibited by the alpha IR-3 MoAb. These results demonstrate that IGF-1 can replace IL-3 for growth when FDC-P1 cells overexpress the IGF-1R. Given the fairly ubiquitous expression of the IGF-1 receptor, these and additional experiments might help to determine whether increased expression of endogenous receptors by cells can lead to leukemogenesis and tumorigenesis. Moreover, hIGF-1R-infected cells will be useful in investigating the mechanisms of IGF1-mediated signal transduction because they are now known to proliferate in response to IGF-1.
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