M Pearce

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

In this study, the abilities of constitutive and conditional forms of the three Raf kinases to abrogate the cytokine dependency of FDC-P1 cells were examined. The constitutively active forms (Δ) of all three Raf kinases were fused to the hormone-binding domain of the estrogen receptor (ER), rendering their activities conditionally dependent upon exogenous β-estradiol. The vast majority of ΔRaf:ER-infected FDC-P1 cells remained cytokine-dependent; however, cells were obtained at low frequency in which expression of ΔRaf:ER abrogated cytokine dependency. Isoform specific differences between the Raf kinases were observed as cytokine-independent cells were obtained more frequently from ΔA-Raf:ER than either ΔRaf-1:ER or ΔB-Raf:ER infected cells. To determine whether the regulatory phosphorylation sites in the Raf proteins were necessary for abrogation of cytokine dependency, they were changed by site-directed mutagenesis. Substitution with phenylalanine eliminated the transforming ability of the ΔB-Raf:ER and ΔRaf-1:ER kinases. However, a similar substitution in A-Raf did not extinguish its transforming activity. The activated Raf proteins induced essential downstream MEK1 activity as treatment with the MEK1 inhibitor, PD98059, suppressed Raf-mediated growth. Activated MAP kinases (ERK1 and ERK2) were detected in ΔRaf:ER-transformed cells, and their presence was dependent upon a functional MEK1 protein. The cytokine-independent phenotype required the continued activity of the ΔRaf:ER proteins as removal of β-estradiol caused the cells to stop growing and undergo apoptosis. The Raf-responsive cells were found to express autocrine growth factors, which promoted their growth. Constitutive activation of the Raf-1 oncogene resulted in malignant transformation as cytokine-independent FDC-P1 cells infected with a retrovirus encoding an activated Raf-1 protein formed tumors upon injection of immunocompromised mice. In summary, Raf kinases can abrogate cytokine dependency, prevent apoptosis and induce the tumorigenicity of a certain subpopulation of FDC-P1 cells by a MEK1-dependent mechanism.

A conditionally-active form of MEK1 results in autocrine transformation of human and mouse hematopoietic cells

The Raf/MEK/MAP kinase cascade plays a critical role in transducing growth signals from activated cell surface receptors. Using ΔMEK1:ER, a conditionally-active form of MEK1, we demonstrate the ability of this dual specificity protein kinase to abrogate the cytokine-dependency of the human and murine hematopoietic cells lines TF-1, FDC-P1 and FL5.12. Cytokine-independent cells were obtained from TF-1, FDC-P1 and FL5.12 cells at frequencies of 2.5×10−3, 5×10−5 and 10−7 respectively, indicating that not all cells expressing ΔMEK1:ER were factor-independent. In general, cells that were converted to a cytokine-independent phenotype displayed a higher level of MAP kinase activity in response to ΔMEK1:ER activation than those that remained cytokine-dependent. ΔMEK1:ER-responsive cells could be maintained long-term in the presence of β-estradiol as well as the estrogen-receptor antagonist 4-Hydroxy-Tamoxifen and the anti-estrogen ICI 164 383. Removal of hormone led to the rapid cessation of cell growth in a manner similar to that observed when cytokine is withdrawn from the parental cells. Treatment of ΔMEK1:ER-responsive cells with a specific and selective inhibitor, PD98059, prevented growth in response to β-estradiol. GM-CSF mRNA transcripts were detected in the MEK1-responsive cells indicating that the activated ΔMEK1:ER may induce a pathway leading to autocrine proliferation. Treatment of MEK1-responsive cells with an anti-GM-CSF antibody, but not a control antibody, suppressed cell growth. The cell lines described here will be useful for elaborating the ability of the MAP kinase pathway to regulate cell proliferation in hematopoietic cells.

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 pathways role in leukemic transformation, a conditionally active (β-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 ΔMEK1: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 β-estradiol: (1) cells that expressed the ΔMEK1:ER oncoprotein but remained cytokine-dependent and (2) MEK1-responsive cells that grew in response to ΔMEK1:ER activation. Cytokine-dependent cells were recovered 102 to 104 times more frequently than MEK1-responsive cells depending upon the particular cell line. To determine whether BCL2 overexpression could synergize with the ΔMEK1:ER oncoprotein in relieving cytokine dependence, the cytokine-dependent ΔMEK1: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. ΔMEK1:ER+BCL2 cells remained viable for at least 3 days after estradiol deprivation, whereas viability was readily lost upon withdrawal of β-estradiol in the MEK1-responsive cells which lacked BCL2 overexpression. The MAP kinases, ERK1 and ERK2 were activated in response to ΔMEK1:ER stimulation in both ΔMEK1:ER and ΔMEK1:ER+BCL2 cells. As compared to the cytokine-dependent ΔMEK1:ER and BCL2 infected cells, MEK1-responsive BCL2 infected cells expressed higher levels of BCL2. While both MEK1-responsive ΔMEK1:ER and ΔMEK1:ER+BCL2 infected cells expressed cDNAs encoding the autocrine cytokine GM-CSF, more GM-CSF cDNAs and bioactivity were detected in the MEK1-responsive ΔMEK1: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.

Effects of inducible MEK1 activation on the cytokine dependency of lymphoid cells.

The Raf/MEK/MAP kinase cascade plays a critical role in transducing growth signals from activated cell surface receptors. Using ΔMEK1:ER, a conditionally active form of MEK1, we demonstrate the ability of this dual specificity protein kinase to abrogate the cytokine dependency of the murine lymphoid hematopoietic cell line FL5.12. Cytokine-independent cells were obtained from FL5.12 cells at a frequency of 1 × 10−7, indicating that a low frequency of cells expressing ΔMEK1:ER were factor-independent. In general, cells that were converted to a cytokine-independent phenotype displayed a higher level of MAP kinase activity in response to ΔMEK1:ER activation than those that remained cytokine-dependent. ΔMEK1:ER-responsive cells could be maintained long-term in the presence of β-estradiol, as well as the estrogen-receptor antagonist 4-hydroxy-tamoxifen. Removal of hormone led to the rapid cessation of cell growth in a manner similar to that observed when cytokine is withdrawn from the parental cells. GM-CSF mRNA transcripts were detected in the MEK1-responsive cells indicating that activated ΔMEK1:ER may induce a pathway leading to autocrine proliferation. Cytokine-dependent ΔMEK1:ER cells were found to increase the expression of GM-CSF receptor α (GM-CSFRα) in response to β-estradiol. In contrast, MEK1-responsive cells were found to express constitutively lower levels of GM-CSFRα and β common (βc) chains indicating that constitutive GM-CSF expression resulted in a decrease in GM-CSFR expression. Treatment of parental cells with supernatant from MEK1-responsive FL5.12 cells was sufficient to promote [3H]-thymidine incorporation. GM-CSF was found to enhance the viability of FL5.12 cells. The cell lines described here will be useful for elaborating the ability of the MAP kinase pathway to regulate cell proliferation in hematopoietic cells.

Synergy between Raf and BCL2 in abrogating the cytokine dependency of hematopoietic cells.

The Raf oncoprotein plays critical roles in the transmission of mitogenic signals from cytokine receptors to the nucleus. There are three Raf family members: A-Raf, B-Raf and Raf-1. Conditionally active forms of the Raf proteins were created by ligating N-terminal truncated activated forms to the estrogen-receptor (ER) hormone-binding domain resulting in β-estradiol-inducible constructs. We introduced these chimeric ΔRaf:ER oncoproteins into the murine FDC-P1 hematopoietic cell line. Two different types of cells were recovered after drug selection in medium containing either cytokine or β-estradiol: (1) cytokine-dependent cells that expressed the ΔRaf:ER oncoproteins; and (2) Raf-responsive cells that grew in response to the ΔRaf:ER oncoprotein. Depending upon the particular ΔRaf:ER oncoprotein, cytokine-dependent cells were recovered 103 to 105 times more frequently than Raf-responsive cells. To determine whether BCL2 could synergize with the ΔRaf:ER oncoproteins and increase the frequency of cytokine-independent cells, cytokine-dependent ΔRaf:ER-expressing cells were infected with either a BCL2 containing retrovirus or an empty retroviral vector. BCL2 overexpression, by itself, did not relieve cytokine dependency of the parental cell line. However, BCL2 overexpression increased the frequency of Raf-responsive cells approximately five- to 100-fold. Cytokine-dependent ΔRaf:ER-infected cells entered the G1 phase of the cell cycle after cytokine withdrawal and entered S phase only after cytokine addition. Raf-responsive ΔRaf:ER cells entered the G1phase of the cell cycle after estrogen deprivation and re-entered the cell cycle after addition of either IL-3 or the estrogen receptor antagonist tamoxifen which activates the ΔRaf:ER constructs. Expression of the BCL2 oncoprotein often delayed the exit from the S and G2/M phases demonstrating the protective effects BCL2 provided to these Raf and BCL2 infected cells. The ΔRaf:ER cells expressed the ΔRaf:ER proteins and downstream MEK and ERK activities after β-estradiol treatment. Raf-responsive cells that were also infected with BCL2 expressed higher levels of BCL2 than the cells that were not infected with BCL2. Thus BCL2 can synergize with the activated Raf in the abrogation of cytokine dependency of certain hematopoietic cells. These cells will be useful in furthering our understanding of the roles of the Raf and BCL2 oncoproteins in hematopoietic cell growth, cell cycle progression and prevention of apoptosis.

Enhanced ability of daniplestim and myelopoietin-1 to suppress apoptosis in human hematopoietic cells.

Modified and chimeric cytokines have been developed to aid in the recovery of hematopoietic precursor cells after myeloablative chemotherapy. The interleukin-3 (IL-3) receptor agonist, daniplestim, binds to the IL-3 receptor-α subunit with 60-fold greater affinity and induces cell proliferation and colony-forming unit formation 10- to 22-fold better than native IL-3. A chimeric cytokine, myelopoietin-1, composed of daniplestim and a G-CSF receptor agonist binds both the IL-3 and G-CSF receptors. While the in vivo effects of daniplestim and myelopoietin-1 are well described, the mechanisms by which they stimulate growth are not well understood. We have investigated the effects of daniplestim and myelopoietin-1 on the prevention of apoptosis in two human hematopoietic cell lines, OCI-AML.5 and AML 193. Daniplestim and myelopoietin-1 prevented apoptosis to a greater degree than native recombinant IL-3 or G-CSF as determined by annexin V/propidium iodide binding and TUNEL assays. Daniplestim and myelopoietin-1 promoted the maintenance of the mitochondrial membrane potential better than native IL-3 or G-CSF. These cytokines promoted a lower redox potential as higher levels of free radicals were detected after cytokine treatment than in cytokine-deprived cells implying increased respiration. These results indicate that daniplestim and myelopoietin-1 are able to prevent apoptosis in hematopoietic cells more effectively than native IL-3 and G-CSF. These effects of daniplestim and myelopoietin-1 may contribute to their effective ability to repopulate hematopoietic precursor cells after chemotherapy.

Enhanced ability of myelopoietins, dual receptor agonists for human IL-3 and g-csf receptors and the IL-3 receptor agonist, daniplestim, to suppress apoptosis and stimulate cytokine-inducible gene expression

Abstract The chimeric receptor agonists, myelopoietins (MPOs), bind and activate the human IL-3 and G-CSF receptors while daniplestim binds the human IL-3 receptor with 20-fold higher affinity than native IL-3. In CFU assays using human bone marrow-derived CD34 + cells, MPOs demonstrated a greater increase in the numbers of CFUs compared to the coaddition of native IL-3 & G-CSF. We have investigated how MPOs and daniplestim stimulate growth and prevent apoptosis using Annexin V/propidium iodide binding, alteration in mitochondrial membrane potential, TUNEL assays, and DNA laddering in the OCI-AML and AML-193 cell lines. MPOs prevented apoptosis more effectively than IL-3 or G-CSF or the co-addition of IL-3 & G-CSF. The expression of apoptosis- and cytokine-regulated genes was examined by RT-PCR. Bcl-X L and Oncostatin M were detected at 2-4 fold higher levels in OCI-AML cells treated with MPOs than cells treated with the co-addition of IL-3 & G-CSF. These cDNAs were detected in 10-fold lower levels in cytokine-deprived cells. Furthermore, IL-3 and daniplestim differed in their abilities to induce LIF expression: native IL-3 induced LIF expression, while daniplestim did not. These results suggest that MPOs inhibit apoptosis more efficiently than the co-addition of IL-3 and G-CSF and induced expression of Bcl-X L and Oncostatin M in cytokine-responsive cell lines.