Synergism between stem cell factor and granulocyte-macrophage colony-stimulating factor on cell proliferation by induction of cyclins

Synergism between stem cell factor (SCF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) has been shown to be essential for hematopoietic cell proliferation. Since HML-2 cells proliferate exponentially in the presence of SCF and GM-CSF together, we analyzed the molecular mechanism of the interaction between these two factors in the cells. An immediate-early gene product, c-myc, was additively upregulated in HML-2 cells by addition of a combination of SCF and GM-CSF. c-myc antisense oligonucleotides effectively suppressed cell proliferation and downregulated the induction of D3, E, A, and B cyclins in HML-2 cells stimulated with the two-factor combination. HML-2 cells arrested at the G0/G1 phase with SCF alone and expressed modest amounts of c-myc and cyclin D3, but not cyclin E. With GM-CSF treatment alone, the cells could not progress to the G2/M phase and expressed c-myc, cyclin D3 and cyclin E but not cyclins A or B. The addition of the counterpart cytokine resulted in cell cycle completion by induction of the deficient cyclins. Taken together, it appears that the induction of c-myc is an indispensable event in the proliferation of HML-2 cells and that the cytokines SCF and GM-CSF interact reciprocally for expression of all cyclins required for cell cycle progression.     

Hydrogen peroxide-induced expression of the proto-oncogenes, c-jun, c-fos and c-myc in rabbit lens epithelial cells

The involvement of H2O2 in cataract development has been established inboth human patients and animal models. At the molecular level H2O2 has beenobserved to cause damage to DNA, protein and lipid. To explore the oxidativestress response of the lens system at the gene expression level, we haveexamined the effects of H2O2 on the mRNA change of the proto-oncogenes,c-jun, c-fos and c-myc in a rabbit lens cell line, N/N1003A. H2O2 treatmentof the rabbit lens epithelial cells for 60 min induces quick up-regulationof both c-jun and c-fos mRNAs. The maximal induction is 38 fold for c-jun at150 µM and 72 fold for c-fos at 250 µM H2O2. Treatment ofN/N1003A cells with 50-250 µM H2O2 for 60 min leads to a 2-5 foldincrease of the c-myc mRNA level. H2O2 also induces an up-regulation intransactivity of the activating protein-1 (AP-1) as shown with a reportergene driven by a prolactin gene promoter with 4 copies of AP-1 binding sitesinserted in the upstream of the promoter. Maximal induction occurs with 150µM H2O2. In the same system, the antioxidants, N-acetyl-cysteine (NAC)and pyrrolidine dithiocarbamate (PDTC) at concentrations shown toup-regulate the mRNAs of both c-jun and c-fos, also enhance thetransactivity of AP-1. NAC and PDTC have different effects in modulating theinduction of AP-1 activity by H2O2 and TPA. These results reveal thatoxidative stress regulates expression of various regulatory genes in lenssystems, which likely affects cell proliferation, differentiation andviability and thus affect normal lens functions.     

Characterization of cis-regulatory elements of the c-myc promoter responding to human GM-CSF or mouse interleukin 3 in mouse proB cell line BA/F3 cells expressing the human GM-CSF receptor.

Interleukin 3 (IL-3) or granulocyte macrophage colony-stimulating factor (GM-CSF) activates c-fos, c-jun, and c-myc genes and proliferation in both hematopoietic and nonhematopoietic cells. Using a series of deletion mutants of the beta subunit of human GM-CSF receptor (hGMR) and inhibitors of tyrosine kinase, two distinct signaling pathways, one for activation of c-fos and c-jun genes, and the other for cell proliferation and activation of c-myc gene have been elucidated. In contrast to wealth of information on the pathway leading to activation of c-fos/c-jun genes, knowledge of the latter is scanty. To clarify the mechanisms of activation of c-myc gene by cytokines, we established a transient transfection assay in mouse proB cell line BA/F3 cells expressing hGMR. Analyses of hGMR beta subunit mutants revealed two cytoplasmic regions involved in activation of the c-myc promoter, one is essential and the other is dispensable but enhances the activity. These regions are located at the membrane proximal and the distal regions covering amino acid positions 455-544 and 544-589, respectively. Characterization of cis-acting regulatory elements of the c-myc gene showed that the region containing the P2 promoter initiation site is sufficient to mediate the response to mIL-3 or hGM-CSF. Electrophoretic mobility shift assay using an oligonucleotide corresponding to the distal putative E2F binding site revealed that p107/E2F complex, the negative regulator of E2F, decreased, and free E2F increased after mIL-3 stimulation. These results support the thesis that mIL-3 or hGM-CSF regulates the c-myc promoter by altering composition of the E2F complexes at E2F binding site.     

Mitogenic growth factors regulate differentially early gene mRNA expression: a study on two clones of 3T3 fibroblasts.

The relationship between cell proliferation and mRNA levels of the immediate early genes c-fos, c-jun, and jun B has been investigated in two clones of 3T3 fibroblasts (D1-3T3 and N2-3T3) upon treatment with basic fibroblast growth factor (bFGF), thrombin, phorbol 12-myristate 13-acetate (PMA) and dibutyryl cyclic AMP (Bt2cAMP). The 3T3-derived clone D1-3T3 almost stops dividing upon serum deprivation, while the N2-3T3 clone does not. The proliferation of the two clones was stimulated by thrombin and PMA and inhibited by Bt2cAMP. Basic FGF stimulated the growth of D1-3T3 but partly inhibited that of N2-3T3 cells. In spite of variable mitogenic response, immediate early genes, c-fos, c-jun, jun B, and c-myc, were induced by the growth factors and by PMA in both cell clones. In our experimental conditions the early gene mRNAs were expressed independently; i.e., the expression of one protooncogene had no bearing on the expression of the other. The cell growth was not directly related to the expression of a particular protooncogene mRNA. Data are presented showing that early gene mRNA expression induced by bFGF or thrombin was not mediated by protein kinase C activation while thrombin-induced mitosis was. Basic FGF induced a part of c-jun mRNA expression, but not mitosis, through a pertussis toxin-sensitive mechanism.     

Progestins both stimulate and inhibit breast cancer cell cycle progression while increasing expression of transforming growth factor alpha, epidermal growth factor receptor, c-fos, and c-myc genes.

This study documents a biphasic change in the rate of cell cycle progression and proliferation of T-47D human breast cancer cells treated with synthetic progestins, consisting of an initial transient acceleration in transit through G1, followed by cell cycle arrest and growth inhibition. Both components of the response were mediated via the progesterone receptor. The data are consistent with a model in which the action of progestins is to accelerate cells already progressing through G1, which are then arrested early in G1 after completing a round of replication, as are cells initially in other phases of the cell cycle. Such acceleration implies that progestins act on genes or gene products which are rate limiting for cell cycle progression. Increased production of epidermal growth factor and transforming growth factor alpha, putative autocrine growth factors in breast cancer cells, does not appear to account for the initial response to progestins, since although the mRNA abundance for these growth factors is rapidly induced by progestins, cells treated with epidermal growth factor or transforming growth factor alpha did not enter S phase until 5 to 6 h later than those stimulated by progestin. The proto-oncogenes c-fos and c-myc were rapidly but transiently induced by progestin treatment, paralleling the well-known response of these genes to mitogenic signals in other cell types. The progestin antagonist RU 486 inhibited progestin regulation of both cell cycle progression and c-myc expression, suggesting that this proto-oncogene may participate in growth modulation by progestins.     

NAB2, a corepressor of EGR-1, inhibits vascular endothelial growth factor-mediated gene induction and angiogenic responses of endothelial cells

In this study we have investigated the role of a specific corepressor of EGR-1, NAB2, to down-regulate vascular endothelial growth factor (VEGF)-induced gene expression in endothelial cells and to inhibit angiogenesis. Firstly, we show a reciprocal regulation of EGR-1 and NAB2 following VEGF treatment. During the initial phase EGR-1 is rapidly induced and NAB2 levels are down-regulated. This is followed by a reduction of EGR-1 and a concomitant increase of NAB2. Secondly, using the tissue factor gene as a readout for VEGF-induced and EGR-1-regulated gene expression we demonstrate that NAB2 can completely block VEGF-induced tissue factor reporter gene activity. Thirdly, by adenovirus-mediated expression we show that NAB2 inhibits up-regulation of tissue factor, VEGF receptor-1, and urokinase plasminogen activator mRNAs even when a combination of VEGF and bFGF is used for induction. In addition, NAB2 overexpression significantly reduced tubule and sprout formation in two different in vitro angiogenesis assays and largely prevented the invasion of cells and formation of vessel-like structures in the murine Matrigel model. These data suggest that NAB2 regulation represents a mechanism to guarantee transient EGR-1 activity following exposure of endothelial cells to VEGF and that NAB2 overexpression could be used to inhibit signals involved in the early phase of angiogenesis.     

Effect of trichloroethylene on DNA methylation and expression of early-intermediate protooncogenes in the liver of B6C3F1 mice.

Trichloroethylene (TCE) is a multimedia environmental pollution that is carcinogenic in mouse liver. The ability of TCE to modulate DNA methylation and the expression of immediate-early protooncogenes was evaluated. Female B6C3F1 mice were administered 1000 mg/kg TCE by gavage 5 days/week and killed after 5, 12, or 33 days of exposure. Methylation of DNA as 5-methylcytosine was decreased by 5 days of treatment with TCE and remained reduced for 33 days. TCE also decreased the methylation of the promoter regions for the protooncogenes, c-jun and c-myc. The expression of the mRNA for the two protooncogenes was increased between 60 and 120 minutes after administering the last dose of TCE and returned to control level by 24 hours. The expression of the mRNA for c-fos remained undetectable after administering TCE. Hence, TCE decreased the methylation both of total DNA and the promoters for the c-jun and c-myc genes and increased the expression of their mRNA. The decreased methylation and increased expression of the two immediate-early protooncogenes might be associated with TCE-induced increase in cell proliferation and promotion of tumors.