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

Abstract

The effects of deregulated Raf activation on the growth and differentiation of hematopoietic cells were investigated. The cytokine-dependent murine myeloid FDC-P1 and human erythroleukemic TF-1 cell lines were transformed to grow in response to deregulated Raf expression in the absence of exogenous cytokines. The conditionally active Raf proteins were regulated by ␤-estradiol as cDNAs containing the Raf catalytic, but lacking negative-regulatory domains, were ligated to the hormone binding domain of the estrogen receptor (⌬Raf:ER). Continuous ⌬Raf expression prevented apoptosis in the absence of exogenous cytokines and altered the morphology of the FD/⌬Raf:ER cells as they grew in large aggregated masses (Ͼ100 cells) whereas the parental cytokinedependent FDC-P1 cells grew in smaller grape-like clusters (Ͻ10 cells). FD/⌬Raf-1:ER cells growing in response to Raf activation displayed decreased levels of the Mac-2 and Mac-3 molecules on their cell surface. In contrast, when these cells were cultured in IL-3, higher levels of these adhesion molecules were detected. Expression of activated Raf oncoproteins also abrogated cytokine dependency and prevented apoptosis of TF-1 cells. Moreover, the differentiation status of these Raf-responsive cells was more immature upon Raf activation as culture with the differentiation-inducing agent phorbol 12 myristate 13-acetate (PMA) and ␤-estradiol resulted in decreased levels of the CD11b and CD18 integrin molecules on the cell surface. In contrast when the Raf-responsive cells were induced to differentiate with PMA and GM-CSF, in the absence of ⌬Raf:ER activation, increased levels of the CD11b and CD18 molecules were detected. Retinoic acid (RA) inhibited 3 H-thymidine incorporation in response to GM-CSF. Interestingly, Raf activation counterbalanced the inhibition of DNA synthesis caused by RA but not PMA. Thus deregulated Raf expression can alter cytokine dependency, integrin expression and the stage of differentiation. These Raf-responsive cell lines will be useful in elucidating the roles of the MAP kinase cascade on hematopoietic cell differentiation and malignant transformation. Leukemia

Sex differences and estrogen modulation of the cellular immune response after injury

“…P-selectin, CINC-2β, and MCP-1 stayed lowered in the estrogen treated rats 24 hours after injury. The decrease in adhesion molecules and chemokines is modulated by estrogen and could be a result of a reduction of cell-surface integrin expression [57], or reductions of oxidative stress [58][59][60].…”

Kinases: positive and negative regulators of apoptosis

“…[259][260][261] However, when TF-1 cells are stimulated with PMA they are induced to differentiate. [262][263][264] Growth inhibition mediated by PMA may be mediated in part by p21 WAF1 induction 265 and this occurs in the absence of apoptosis. Thus the direct effects of PMA on apoptosis are hard to interpret.…”

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.…”