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.     

Role of c-myc and other genes in interleukin 2 regulated CT6 T lymphocytes and their malignant variants

The cloned murine cytotoxic T cell line CT6 solely requires interleukin 2 (IL 2) for viability and cell cycle progression. Treatment of G arrested cultures of CT6 cells with recombinant IL 2 induces the rapid sequential expression of the nuclear proto-oncogenes c-fos, c-myc, and c-myb but does not affect the expression of several cytosolic or membrane-associated proto-oncogenes. A comparison of early genes induced by growth factor treatment of quiescent NIH/3T3 fibroblasts and CT6 cells demonstrated that only c-fos and c-myc induction is shared in the two different lineages. Factor-independent lines derived from CT6 cells show no mitogenic response to IL 2, yet binding of IL 2 with its receptor in the cells was capable of inducing the expression of c-fos and c-myc. In factor-independent cell lines, c-myc was uniformly expressed at high constitutive levels, suggesting that c-myc abrogates growth factor requirements of these cells. The levels of c-myc expression in the factor-independent lines was not due to an autocrine production of IL 2 but may be a consequence of constitutively activated IL 2 receptors.     

Opposite effects of inhibitors of mitogen-activated protein kinase pathways on the egr-1 and beta-globin expression in erythropoietin-responsive murine erythroleukemia cells

The effect of erythropoietin (Epo) on the expression of mitogen-activated protein kinase (MAPK) target genes egr-1 and c-fos was investigated in Epo-responsive murine erythroblastic cell line ELM-I-1. Epo induced a transient rise in egr-1 mRNA without a similar effect on c-fos expression. The induction of egr-1 correlated with a rapid ERK1/2 phosphorylation and was prevented with MEK1/2 inhibitors PD 98059 and UO126. The p38 inhibitor SB 203580 enhanced ERK1/2 phosphorylation and egr-1 mRNA levels. Longer incubations of ELM-I-1 cells with Epo revealed a second later phase of increase in egr-1 expression which was also prevented by MEK1/2 inhibitors, whereas SB 203580 had a stimulatory effect. In contrast, the beta-globin mRNA production was enhanced in the presence of PD 98059 and UO126 and reduced by SB 203580. The results suggest a regulatory role of egr-1 expression in Epo signal transduction and provide pharmacological evidence for the negative modulation of differentiation-specific gene expression by the ERK1/2 pathway in murine erythroleukemia cells.     

Interleukin-6- and leukemia inhibitory factor-induced terminal differentiation of myeloid leukemia cells is blocked at an intermediate stage by constitutive c-myc.

Interleukin-6 (IL-6) and leukemia inhibitory factor (LIF), two multifunctional cytokines, recently have been identified as physiological inducers of hematopoietic cell differentiation which also induce terminal differentiation and growth arrest of the myeloblastic leukemic M1 cell line. In this work, it is shown that c-myc exhibited a unique pattern of expression upon induction of M1 terminal differentiation by LIF or IL-6, with an early transient increase followed by a decrease to control levels by 12 h and no detectable c-myc mRNA by 1 day; in contrast, c-myb expression was rapidly suppressed, with no detectable c-myb mRNA by 12 h. Vectors containing the c-myc gene under control of the beta-actin gene promoter were transfected into M1 cells to obtain M1myc cell lines which constitutively synthesized c-myc. Deregulated and continued expression of c-myc blocked terminal differentiation induced by IL-6 or LIF at an intermediate stage in the progression from immature blasts to mature macrophages, precisely at the point in time when c-myc is normally suppressed, leading to intermediate-stage myeloid cells which continued to proliferate in the absence of c-myb expression.     

Heparin prevents vascular smooth muscle cell progression through the G1 phase of the cell cycle

To gain insight into the mechanism of the antiproliferative effects of heparin on vascular smooth muscle cells (SMC), the influence of this glycosaminoglycan on cell cycle progression and the expression of the c-fos, c-myc, and c-myb proto-oncogenes and two other growth-regulated genes was examined. SMC, synchronized by a serum-deprivation protocol, enter S phase 12-16 h after serum stimulation. Pretreatment with heparin for 48 h blocked the induction of histone H3 RNA, an S phase-expressed product, and prevented cell replication. Thus, heparin prevents entry of cells into S phase. Conversely, heparin had essentially no effect on changes in expression of the c-fos and c-myc proto-oncogenes during the G0 to G1 transition. Normal increases in c-fos and c-myc RNA were observed 30 min and 2 h following serum addition, respectively. However, the increase in expression of the mRNA of the c-myb proto-oncogene and the mitochondrial ATP/ADP carrier protein, 2F1, which begins to occur 8 h following serum addition to SMC, was completely inhibited by heparin. Two-dimensional polyacrylamide gel electrophoresis of the products of a rabbit reticulocyte cell-free translation of RNA isolated at various times confirmed this temporal assessment of the effects of heparin. These results suggest that heparin does not inhibit cell proliferation by blocking the G0 to G1 transition. Rather, heparin may affect a critical event in the mid-G1 phase of the cell cycle which is necessary for subsequent DNA synthesis.     

Interleukin-2 (IL-2) induces erythroid differentiation and tyrosine phosphorylation in ELM-I-1 cells transfected with a human IL-2 receptor beta chain cDNA.

The molecular mechanism of erythroid differentiation has been still ill-defined. In this study, we introduced a human interleukin-2 receptor (IL-2R) beta chain cDNA into ELM-I-1 cells which differentiated into hemoglobin-positive cells in the presence of erythropoietin (Epo), and established the transformant which expressed IL-2R beta chain. In this transformant, we revealed that IL-2 induced erythroid differentiation and the same pattern of tyrosine phosphorylation as Epo. These data suggest that tyrosine phosphorylation is involved in signal transduction pathway of erythroid differentiation. It is also implicated that the Epo and IL-2 receptor system share a common signal transduction pathway.     

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.     

Molecular analysis of cell cycle-related gene expression in anti-HLA class I monoclonal antibody (01.65) treated PHA-activated human T-lymphocytes.

HLA class I antigens seem to be involved in the proliferative response of PHA-activated human T-lymphocytes. We have previously reported that the treatment of PHA-activated peripheral blood mononuclear cells (PBMC) with an anti-HLA class I monoclonal antibody, 01.65, (i) inhibits the tritiated thymidine incorporation, (ii) inactivates cytosolic protein kinase C (PKC) and (iii) causes an increase in the duration of the cell cycle. Northern Blot kinetic analysis of c-fos, c-myc, cdc2, IL-2R, c-myb, ODC, TK and H3, from 10 minutes to 120 hours, was performed in MAb 01.65 treated cultures. We found that the expression of four genes (c-myc, IL-2R, cdc2 and TK) was depressed 24 hours after PHA stimulation.     

IL-2 modulation of murine T-cell oncogene expression

C-myb, a cellular oncogene associated with normal thymic development, was found to be highly expressed in four interleukin 2 (IL-2)-independent T-cell lines, but not in two of three IL-2-dependent T-cell lines. The IL-2-dependent lines, HT2 and CTLL-2, were found to have low levels of c-myb mRNA in the presence of IL-2. However, short-term IL-2 depletion resulted in at least fivefold increases in c-myb message. Add-back of IL-2 after 30 hr IL-2 depletion of CTLL-2 cells resulted in return to baseline low-level c-myb mRNA. Expression of the oncogenes myc, bas, raf, and abl as well as the T-cell genes Thy-1 and CT beta did not parallel that of c-myb. These studies indicate that removal of a growth factor can result in increased levels of a specific cellular oncogene and that two nuclear protooncogenes (c-myb and c-myc) are expressed differentially during cell growth. These results may help to explain aspects of intrathymic T-cell differentiation where there is very high c-myb expression in the face of limiting amounts of growth factors such as IL-2.