Characterization of proteins binding the 3′ regulatory region of the IL-3 gene in IL-3-dependent and autocrine-transformed hematopoietic cells

Abstract

Previously we documented the prolongation of the IL-3 mRNA half-life in an autocrine-transformed cell line. This cell line has an intracisternal type A particle transposition in the IL-3 mRNA 3′ untranslated region which displaced four out of six AUUUA motifs involved in IL-3 mRNA destabilization. In this study, the proteins binding to the IL-3 mRNA AU-rich elements (ARE) were examined. Specific protein binding was detected to the wildtype IL-3 ARE region which contained 6 AUUUA motifs (AU 6 ). In contrast, no binding was detected to the mutated IL-3 ARE region which contained only two AUUUA motifs (AU 2 ). Proteins with apparent molecular weights of 36, 40, 43, 46, 55, 57, 68 and 95 kDa were bound to AU 6 motif. The hnRNP C and AUF-1 (hnRNP D) proteins were determined to be two of the IL-3 ARE binding proteins. Incubation of protein extracts with antibodies to hnRNP C and AUF-1 significantly decreased the protein binding to the IL-3 ARE. Treatment of IL-3 dependent cells with calcium ionophores eliminated the proteins binding to the ARE in wild-type IL-3-dependent FL5.12 cells and also resulted in the accumulation of IL-3 mRNA transcripts with a long half-life. These results indicated that there was a specific complex which bound the IL-3 mRNA 3′ ARE. Mutations which truncate the IL-3 ARE eliminate the ability of proteins to bind this regulatory region and can result in autocrine transformation due to the presence of IL-3 mRNA transcripts with a long half-life.

Differential abilities of the Raf family of protein kinases to abrogate cytokine dependency and prevent apoptosis in murine hematopoietic cells by a MEK1-dependent mechanism

“…This is most likely due to phorbol esters prolonging the stability of IL-3 and GM-CSF mRNA. 57 Expression of IL-3 and GM-CSF was not detected in 3 and 4). The secretion of GM-CSF was also detected and confirmed by performing 3 H-thymidine incorporation assays in the presence and absence of antibodies specific to GM-CSF and IL-3 (data not presented).…”

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

“…Raf-and MEK1-responsive cells were cultured in DMEM+5% FBS+500 nM 4HT (Sigma, St Louis, MO, USA) to activate the DRaf:ER and DMEK1:ER constructs as described. [23][24][25][26][27][28][29][30][31][40][41][42] Determination of ERK1 and ERK2 activation Activated forms of ERK (p42 and p44 MAP kinase) were detected using a phospho-specific antibody to the activated forms of …”

“…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.…”

“…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).…”