Stat3-dependent induction of interleukin-3 receptor expression in leukemia inhibitory factor-stimulated M1 mouse leukemia cells

M1 mouse leukemia cells differentiate to macrophages/monocytes by the stimulation of interleukin-6 (IL-6)/leukemia inhibitory factor (LIF). To identify new LIF-induced genes, we have performed representational difference analysis using M1 cells and cloned mouse interleukin-3 (IL-3) receptor beta subunit gene. The mRNA expression of both IL-3 receptor (IL-3R) alpha and beta subunits is upregulated after 1 h stimulation of LIF and remains to be elevated along the differentiation of M1 cells. This induction is almost completely suppressed in M1 cells expressing a dominant negative form of Stat3. Furthermore, we show that IL-3-induced Stat5 phosphorylation increases in LIF-stimulated M1 cells. These results suggest that Stat3 may play a role in the differentiation of myeloid cells by regulating IL-3R expression.     

Deregulated c-myb disrupts interleukin-6- or leukemia inhibitory factor-induced myeloid differentiation prior to c-myc: role in leukemogenesis.

The c-myb proto-oncogene is abundantly expressed in tissues of hematopoietic origin, and changes in endogenous c-myb genes have been implicated in both human and murine hematopoietic tumors. c-myb encodes a DNA-binding protein capable of trans-activating the c-myc promoter. Suppression of both of these proto-oncogenes was shown to occur upon induction of terminal differentiation but not upon induction of growth inhibition in myeloid leukemia cells. Myeloblastic leukemia M1 cells that can be induced for terminal differentiation with the physiological hematopoietic inducers interleukin-6 and leukemia inhibitory factor were genetically manipulated to constitutively express a c-myb transgene. By using immediate-early to late genetic and morphological markers, it was shown that continuous expression of c-myb disrupts the genetic program of myeloid differentiation at a very early stage, which precedes the block previously shown to be exerted by deregulated c-myc, thereby indicating that the c-myb block is not mediated via deregulation of c-myc. Enforced c-myb expression also prevents the loss in leukemogenicity of M1 cells normally induced by interleukin-6 or leukemia inhibitory factor. Any changes which have taken place, including induction of myeloid differentiation primary response genes, eventually are reversed. Also, it was shown that suppression of c-myb, essential for terminal differentiation, is not intrinsic to growth inhibition. Taken together, these findings show that c-myb plays a key regulatory role in myeloid differentiation and substantiate the notion that deregulated expression of c-myb can play an important role in leukemogenicity.     

Rapid gene repression triggered by interleukin-6 at the onset of monocyte differentiation

To date, the majority of characterized extracellular ligand-induced rapid changes in gene expression involve upregulation. Hence, rapid gene repression is either less common or less well studied. To study rapid gene repression during cytokine-initiated differentiation programs, we used the mRNA subtractive hybridization technique of representational difference analysis to isolate repressed genes. Cultures of the myeloid leukemia cell line M1 were induced to terminally differentiate by treatment with interleukin-6 (IL-6). The repressed genes identified in our subtraction products include the genes encoding the growth factor receptor Flt3/Flk2/STK-1 (CD135) and the costimulatory protein CD24 [heat-stable antigen] and the c-myb oncogene. Following 4 h of IL-6 treatment, mRNA levels of these genes are decreased by 45-65% relative to controls and after 8 h by 65-80%. Lipopolysaccharide also triggers the repression of these genes. Protein synthesis inhibitors do not block the IL-6-stimulated repression of c-myb, or c-myc, mRNA, yet they do block the repression of flt3 and CD24 mRNA, demonstrating the existence of both protein synthesis-independent and -dependent mechanisms of cytokine-triggered rapid gene repression during differentiation.     

Down-regulation of interleukin 6 receptors of mouse myelomonocytic leukemic cells by leukemia inhibitory factor.

We examined the effect of leukemia inhibitory factor (LIF) on the expression of interleukin 6 receptors (IL-6R) on mouse myelomonocytic leukemic M1 cells. Binding studies using 125I-labeled human and murine IL-6 revealed that LIF caused a decrease in IL-6 binding to M1 cells. The decrease became evident within 1 h, and the maximum decrease was observed at 3-6 h. Scatchard plot analysis revealed that M1 cells had a single class of high affinity receptors for IL-6 and that LIF-induced decrease in IL-6 binding was due to a decrease in the number of IL-6R on the cell surface and not to changes in their affinity. The affinity of IL-6R on M1 cells to human IL-6 (Kd = 2.25 nM) was about 10-fold lower than that to murine IL-6 (Kd = 200 pM). The amount of IL-6 secreted into culture media by M1 cells that were treated with LIF for up to 12 h was not enough to cause receptor down-regulation. Northern blot analysis demonstrated that IL-6R mRNA was down-regulated by LIF treatment, and similar regulation was also observed when the cells were treated with IL-6. The time course of the IL-6R mRNA level was similar to that of IL-6R expression on the cell surface, suggesting that the main mechanism responsible for the loss of high affinity IL-6R was the regulation of IL-6R mRNA. Although the half-life of IL-6R on the cell surface was about 30 min, the addition of LIF reduced it to 16 min, suggesting the existence of an additional mechanism responsible for the loss of high affinity IL-6R on the cell surface.     

Elevated expression of proto-oncogenes during interleukin-5-induced growth and differentiation of murine B lineage cells

Interleukin 5 (IL-5), a lymphokine produced by helper T cells, is involved in the regulation of growth and differentiation of B cells and other hematopoietic cells. To elucidate IL-5-mediated intracellular mechanisms, we have established IL-5-dependent and -independent murine early B cell lines, J6 and MJ88-1, respectively, and examined the effect of IL-5 on the expression of proto-oncogenes during proliferation. Two- to 3.5-fold increases in the levels of c-myb, c-myc, c-fos, and c-fms mRNA were observed in J6 cells, compared with those in MJ88-1 cells. Further, a role of IL-5 in the proto-oncogene expression during differentiation was examined by using thymidine-treated murine B-cell chronic leukemia BCL1-B20 cells with growth arrest. After 4-day culture, the amount of IgM secreted from BCL1-B20 cells was augmented 4-6 fold in the presence of IL-5. Although expression of c-myb, c-fos, and c-fms mRNA did not change, only c-myc mRNA expression was elevated within 30 min of stimulation with IL-5 and reached a maximal level by 1 hr. Addition of phorbol 12-myristate 13-acetate (PMA) or IL-4 to the culture of BCL1-B20 cells inhibited both the IL-5-mediated augmentation of IgM secretion and the elevated expression of c-myc mRNA. These findings suggest that the IL-5 signal may be associated with the up-regulation of c-myc expression.     

Alteration of nuclear proto-oncogene expression by erythropoietin (Epo) in Epo-responsive murine cell lines

The signal transduction system of erythropoietin (Epo) and the accompanying molecular control mechanism of proliferation and differentiation of erythroid progenitors remains largely unknown. In this study, the effect of Epo on the expression of nuclear oncogenes was investigated in two murine cell lines which respond to the hormone in different ways: ELM-I-1 cells proliferate independently of Epo, but differentiate in response to the hormone, while the growth of DA-1ER cells is absolutely dependent on Epo or interleukin (IL) 3. The cell lines were stimulated with Epo or IL-3, and total RNA was extracted. Then expression of nuclear proto-oncogenes (c-myc, c-fos and c-myb) was analyzed by northern blotting. The change in c-fos expression observed during the first two h following stimulation with either stimulant were common to both cell lines; a rapid and temporary increment. Before stimulation, c-myc and c-myb were strongly expressed in both lines. No apparent change in c-myc expression was observed during the first two h of stimulation, while c-myb expression in ELM-I-1 cells was slightly reduced 1 h after stimulation with Epo but not with IL-3. Three days after stimulation with Epo, but not with IL-3, only ELM-I-1 produced hemoglobin and expressed a lower amount of c-myb mRNA. These data suggest the importance of c-fos in the early signaling system of Epo, and the involvement of c-myb in erythroid differentiation but not in proliferation.     

Inhibition by anti-HLA class II monoclonal antibodies of monoclonal antibody OKT3-induced T cell proliferation. Studies at the mRNA level

mAb to monomorphic determinants of HLA class II Ag have been shown to inhibit monocyte-dependent OKT3-induced T cell proliferation, indicating that MHC class II molecules play a regulatory role also in Ag nonrestricted, CD3-induced T cell proliferation. This effect involves several steps in the process of T cell activation and proliferation, including IL-1 beta, IL-6, and IL-2 secretion and IL-2R alpha expression. In the present study, we analyzed the effect of an anti-HLA class II mAb (Q5/6) on the mRNA expression of genes related to monocyte and T cell activation. mRNA levels for early (early c-myc, c-fos) and late (late c-myc, N-ras, c-myb) genes involved in T cell activation were determined as well as mRNA levels for IL-1 beta, IL-6, IFN-gamma, IL-2, and IL-2R alpha. The kinetics of mRNA induction for ICAM-1 was also investigated. The results show that in T lymphocytes the expression of c-fos and early c-myc mRNA was unaffected by mAb Q5/6, whereas the c-myb and N-ras mRNA levels were strongly diminished as well as those of IL-2, IL-2R alpha, and IFN-gamma mRNA. An early increase of ICAM-1 mRNA was partially inhibited. In monocytes, a marked reduction of IL-1 beta and IL-6 mRNA was found. It is concluded that the HLA class II determinant involved in the inhibition mechanism can be engaged in the control of IL-1 beta and IL-6 mRNA levels and constitute an accessory signal up-regulating IL-2 and IL-2R alpha gene activation, through a pathway not affecting c-myc and c-fos expression.     

Contrasting patterns of c-myc and N-myc expression in proliferating, quiescent, and differentiating cells of the embryonic chicken lens.

The present study uses the polymerase chain reaction and in situ hybridization to examine c-myc and N-myc mRNA in the embryonic chicken lens at 6, 10, 14 and 19 days of development and compares the pattern of expression obtained with the developmental pattern of cell proliferation and differentiation. In the central epithelium, c-myc mRNA levels were proportional to the percentage of proliferating cells throughout development. N-myc mRNA expression in this region was relatively low and showed no correlation with cell proliferation. The ratio of N-myc to c-myc mRNA increased markedly with the onset of epithelial cell elongation and terminal fiber cell differentiation, although both c-myc and N-myc mRNAs continued to be expressed in postmitotic, elongating cells of the equatorial epithelium and in terminally differentiating lens fiber cells. Thus, increased expression of N-myc, a gene whose protein product may compete with c-myc protein for dimerization partners, accompanies the dissociation of c-myc expression and cell proliferation during terminal differentiation of lens fiber cells.     

Relationships between the cell cycle and the expression of c-myc and transferrin receptor genes during induced myeloid differentiation

We examined the relationship of cellular oncogene c-myc and transferrin receptor (TfR) gene expression to cell proliferation and cell cycle progression during myeloid differentiation in the HL-60 myeloid leukemia cell line. In order to determine levels of mRNA for these genes in HL-60 cells induced to differentiate along the myeloid pathway, RNA was isolated from HL-60 cells incubated with retinoic acid for 24 h and Northern blots were probed with labeled cDNAs for c-myc and TfR. c-myc mRNA decreased within 3 h of retinoic acid addition, and TfR mRNA decreased after 9 h; both mRNAs continued to decrease over 24 h. RNA was also isolated from HL-60 cells separated by centrifugal elutriation into cell cycle phases. TfR and c-myc cDNA probes hybridized equally to RNA from uninduced cells in all phases of the cell cycle. However, after 24 h incubation with the differentiation inducer retinoic acid, TfR mRNA was expressed substantially less in the G1 stage, whereas c-myc mRNA was still expressed equally in all cell cycle phases. These data indicate that, although TfR and c-myc expression are both associated with cell proliferation in the HL-60 line, TfR is down-regulated specifically in G1 upon induction of terminal differentiation whereas c-myc expression is disassociated from cell cycle control in these cells.     

IL-6 is a differentiation factor for M1 and WEHI-3B myeloid leukemic cells

IL-6 has multiple biologic activities in different cell systems including both the ability to support cell proliferation and to induce differentiation. We reported previously the isolation and functional expression of a mouse IL-6 (mIL-6) cDNA clone derived from bone marrow stromal cells. In this paper, we show that mIL-6 is a potent inducer of terminal macrophage differentiation for a mouse myeloid leukemic cell line, M1. Addition of mIL-6 to cultures of M1 cells rapidly inhibits their proliferation and induces phagocytic activity and morphologic changes characteristic of mature macrophages. These phenotypic changes are accompanied at the molecular level by a decrease in proto-oncogene c-myc mRNA accumulation and increases in Fc gamma R, proto-oncogenes c-fos and c-fms (CSF-1R) mRNA expression. Furthermore, IL-6 enhances the expression of Fc gamma R and c-fms in differentiation-responsive D+, but not unresponsive D- sublines of mouse myelomonocytic leukemic WEHI-3B cells. Together with our previous observation that IL-6 stimulates colony formation by normal myeloid progenitors, these results strongly suggest an important regulatory role for IL-6 in myeloid cell growth and differentiation.     

The novel primary response gene MyD118 and the proto-oncogenes myb, myc, and bcl-2 modulate transforming growth factor beta 1-induced apoptosis of myeloid leukemia cells.

Cell numbers are regulated by a balance among proliferation, growth arrest, and programmed cell death. A profound example of cell homeostasis, controlled throughout life, is the complex process of blood cell development, yet little is understood about the intracellular mechanisms that regulate blood cell growth arrest and programmed cell death. In this work, using transforming growth factor beta 1 (TGF beta 1)-treated M1 myeloid leukemia cells and genetically engineered M1 cell variants, the regulation of growth arrest and apoptosis was dissected. Blocking of early expression of MyD118, a novel differentiation primary response gene also shown to be a primary response gene induced by TGF beta 1, delayed TGF beta 1-induced apoptosis, demonstrating that MyD118 is a positive modulator of TGF beta 1-mediated cell death. Elevated expression of bcl-2 blocked the TGF beta 1-induced apoptotic pathway but not growth arrest induced by TGF beta 1. Deregulated expression of either c-myc or c-myb inhibited growth arrest and accelerated apoptosis, demonstrating for the first time that c-myb plays a role in regulating apoptosis. In all cases, the apoptotic response was correlated with the level of MyD118 expression. Taken together, these findings demonstrate that the primary response gene MyD118 and the c-myc, c-myb, and bcl-2 proto-oncogenes interact to modulate growth arrest and apoptosis of myeloid cells.