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

Abstract

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.

Ceramide regulates cellular homeostasis via diverse stress signaling pathways

“…27 Important upstream regulators of the ERKs including Raf and MEK-1 have been shown to synergize with Bcl2 in blocking cell killing induced by factor withdrawal in both human and murine hematopoietic cell lines. 106,107 It will be interesting to determine if ceramide affects this synergy. Meanwhile, other important regulatory proteins localized in the membranes of other organelles and in the plasma membrane would undergo similar regulation.…”

Differential effects of kinase cascade inhibitors on neoplastic and cytokine-mediated cell proliferation

“…18 Spontaneous factorindependent cells are rarely recovered from this cell line, which makes it an attractive model system to analyze the effects that various oncogenes have on signal transduction and the transition to cytokine independence and leukemogenesis. 7,18,[19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34] Oncogene-transformed cytokine-independent cells have been obtained after infection/transfection of this cell line. 7 These cytokine-dependent and oncogene-transformed derivative cells represent appropriate tools to screen for novel therapeutic compounds as well as to evaluate their effectiveness in suppressing the growth and differentiation capacity of certain types of leukemias containing various oncogene mutations and chromosomal translocations.…”

“…7,8 N-terminal deleted forms of Raf and MEK1 proteins, which remove the Ras binding domain and the negative regulatory sites in CR1 and CR2 in Raf and the negative regulatory domain of MEK1, result in activated oncoproteins due to the aberrant stimulation of downstream kinases, transcription factors and molecules involved in the prevention of apoptosis. [23][24][25][26][27][28][29][30][31][40][41][42] Conditional Raf and MEK oncogenes were developed by ligation of the 5 0 deleted Raf and MEK cDNAs to the cDNA encoding the hormone binding domain of the estrogen receptor (ER). The activity of the kinases encoded by these cDNAs are dependent upon estrogen (b-estradiol).…”

“…The activity of the kinases encoded by these cDNAs are dependent upon estrogen (b-estradiol). [23][24][25][26][27][28][29][30][31][40][41][42] The ER antagonist, 4-hydroxytamoxifen (4HT) also activates the ER-coupled oncoprotein and does not have the estrogenic effects of b-estradiol that can both promote as well as inhibit growth. Conditional oncoproteins are in some cases better tools to understand the effects of oncogenes on proliferation and apoptosis because once the regulator is taken away, the cells revert to the 'cytokine-dependent' state.…”

Effects of deregulated Raf activation on integrin, cytokine-receptor expression and the induction of apoptosis in hematopoietic cells

“…11 This cell line has a low spontaneous frequency of conversion to factor independence and TF-1 cells do not normally form tumors after injection in immunocompromised mice. [11][12][13][14][15][16][17] Thus, these cell lines represent models for analysis of the effects of aberrant oncogene expression on human hematopoietic cell growth, differentiation and malignant transformation.…”