OBJECTIVEThe pEgr-TNFalpha plasmid was constructed to investigate the effect of gene-radiotherapy on melanoma and host immune system.

METHODSpEgr-TNFalpha plasmids were constructed and injected into tumor tissue, 36 hours later, the tumors were given 20 Gy X-ray irradiation. Tumor growth at different timepoints was record and immunologic parameters were detected 15 days later.

RESULTSFrom 3 to 15 d after pEgr-TNFalpha gene-radiotherapy the tumor growth was significantly slower than irradiation or genetherapy alone. NK activity, IL-2, TNFalpha and IL-1beta secretion activities of pEgr-TNFalpha gene-radiotherapy group and pEgr-TNFalpha gene group were higher than those of irradiation alone group significantly.

CONCLUSIONThe anti-tumor effect of pEgr-TNFalpha gene-radiotherapy is better than that of either one applied solely, and it can alleviate the lesion caused by radiation therapy.

Animals ; Combined Modality Therapy ; DNA-Binding Proteins ; genetics ; Early Growth Response Protein 1 ; Female ; Genetic Therapy ; Immediate-Early Proteins ; genetics ; Interleukin-2 ; secretion ; Killer Cells, Natural ; immunology ; Melanoma, Experimental ; immunology ; therapy ; Mice ; Plasmids ; Transcription Factors ; genetics ; Tumor Necrosis Factor-alpha ; genetics

OBJECTIVE12-0-tetradecanoylphorbol-13 acetate (TPA) plays an important role in precipitating cell differentiation for various tumor cells, especially leukemic cells. Changes of many genes may be involved in this process. The purpose of this study was to observe the relationship between the EGR1mRNA expression and cell differentiation during TPA-induced K562 cell differentiation.

METHODSIncubation of human K562 cells in vitro was applied to cultivate K562 cells. The cells were treated in two different ways. K562 cells of experiment group were treated with TPA and those of control group were treated without TPA. Using morphology (Wright's staining and NSE staining) and flow cytometry (FCM), the investigators observed the differentiation characteristics of K562 cells, cell-cycle and the differentiation antigen expressions of CD33 and CD14 on cell membranes. RT-PCR was carried out to assay EGR1 mRNA expression.

RESULTSAfter treated with TPA for 7 d, the morphology of K562 cells obviously tended to mature differentiation, like monocytes. The differentiation rate of induced K562 cells was up to 95% in experiment group and 4.5% in control group, respectively. Using SPSS software, the above result showed statistical significance (P < 0.01). Using NSE staining, K562 cells showed positive reaction. Some of them were densely stained. The positive rate was up to 86%. More than half of the positive cells could be inhibited by NaF. The inhibiting rate of NaF was up to 58.72%, showing statistical difference when compared with that of control group. FCM analysis showed that most of K562 cells stimulated by TPA underwent G1/S phase cell-cycle arrest. The composing rate of cell-cycle in TPA-treated group showed that (53.7 +/- 1.25)% of cells were at G0 + G1 phase and (44.3 +/- 1.32)% were at S phase (P < 0.05). The level of CD33 expression on cell membranes was mildly decreased from 0.997% to 0.893% (P > 0.05). However, the level of CD14 expression was significantly increased from 0.049% to 0.387% (P < 0.05).

CONCLUSIONK562 cells could express EGR1mRNA during TPA-induced differentiation, which suggested that EGR1mRNA might participate in the process of K562 cells differentiating into monocyte/macrophages, and might play an important role in precipitating and maintaining cell differentiation for leukemic cells.

Antigens, CD ; metabolism ; Antigens, Differentiation, Myelomonocytic ; metabolism ; Carcinogens ; pharmacology ; Cell Cycle ; drug effects ; genetics ; Cell Differentiation ; drug effects ; genetics ; Cell Division ; drug effects ; genetics ; Cell Membrane ; chemistry ; drug effects ; DNA-Binding Proteins ; genetics ; Early Growth Response Protein 1 ; Flow Cytometry ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Immediate-Early Proteins ; genetics ; K562 Cells ; Lipopolysaccharide Receptors ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Sialic Acid Binding Ig-like Lectin 3 ; Tetradecanoylphorbol Acetate ; pharmacology ; Transcription Factors ; genetics

OBJECTIVETo study the expression and location of early growth response gene-1 (Egr-1), transforming growth factor-beta(1) (TGF-beta(1)), fibronectin (FN) in silicotic rat and to discuss the role of Egr-1 in the development of silicosis.

METHODSSilicotic animal model of rat was established, and the expressions of Egr-1, TGF-beta(1), FN in various lung cells of silicotic rat were analysed by using immunohistochemical technique (SP) and the image analysis.

RESULTSThe expressions of Egr-1 in bronchial epithelial cell, pulmonary macrophage, alveolar epithelium cell and interstitial cell in lung silicotic tissue (gray values: 118.58 +/- 5.65 - 168.52 +/- 5.67) were higher than those of controls (gray values: 166.23 +/- 5.23 - 188.12 +/- 8.35) during 1 - 28 days, and the expression was mainly in nucleus; the expressions of TGF-beta(1) in these cells (gray values: 123.49 +/- 5.65 - 170.24 +/- 3.56) were also higher than those of controls (166.53 +/- 6.25 - 198.56 +/- 4.53), and the expression was mainly in cytoplasm. The expressions of FN in bronchial epithelial cell, pulmonary macrophage and alveolar epithelial cell (gray values: 150.32 +/- 6.54 - 201.54 +/- 7.38) were lower, while those in interstitial cell (gray values: 121.43 +/- 5.65 - 167.55 +/- 6.35) were higher than those of controls. The changes of TGF-beta(1) and Egr-1 expression level in bronchial epithelial cell, pulmonary macrophage, alveolar epithelium cell and interstitial cell were synchronous during the experiment (1 - 28 days). Both of them were correlated with each other (r = 0.61, P < 0.01), while the expression of FN was not correlated with Egr-1, but correlated to TGF-beta(1) in interstitial cell (r = 0.46, P < 0.01).

CONCLUSIONSilicon dioxide could up-regulate the expression of nuclear transcription factor Egr-1 in several kinds of cell in lung. The activated Egr-1 may coordinate the expression of TGF-beta(1) and FN to regulate the development of silicosis.

Animals ; DNA-Binding Proteins ; analysis ; physiology ; Disease Models, Animal ; Early Growth Response Protein 1 ; Fibronectins ; analysis ; physiology ; Immediate-Early Proteins ; analysis ; physiology ; Immunohistochemistry ; Lung ; chemistry ; physiopathology ; Rats ; Silicosis ; etiology ; metabolism ; Transcription Factors ; analysis ; physiology ; Transforming Growth Factor beta ; analysis ; physiology

OBJECTIVETo study the correlation between the expression of Egr-1 and NF-kappaB and the up-regulation of TNF-alpha and TGF-beta1 in macrophages after stimulation by silica in-vitro.

METHODSMacrophages were treated with antibodies against Egr-1 and NF-kappaB and antisense oligonucleotides. The level of TNF-alpha protein in the cell supernatant was then measured using enzyme-linked immunoadsorbent assay (ELISA). The expression of TGF-beta1 protein was detected by immunocytochemistry. The expression of TNF-alpha and TGF-beta1 mRNAs was also monitored by reverse transcriptase-polymerase chain reaction (RT-PCR).

RESULTSCompared with silica-stimulated macrophages untreated with antibodies, the cells treated with 10 micro g/ml of Egr-1 or NF-kappaB antibodies were associated with reduced expression of TNF-alpha and TGF-beta1 proteins and mRNAs (P < 0.05). Compared with silica-stimulated untransfected group, the antisense group was associated with obvious reduction in the expression of TNF-alpha and TGF-beta1 proteins and mRNAs (P < 0.05).

CONCLUSIONThe expression of TNF-alpha and TGF-beta1 mRNAs and proteins are associated with activation of Egr-1 and NF-kappaB in macrophages, after stimulation by silica. It is possible that the corresponding antibodies and antisense oligonucleotides may become a potential therapeutic tool in the management of silicosis in the future.

Animals ; Antibodies ; immunology ; Cells, Cultured ; DNA-Binding Proteins ; genetics ; immunology ; Early Growth Response Protein 1 ; Immediate-Early Proteins ; genetics ; immunology ; Macrophages ; cytology ; metabolism ; Mice ; NF-kappa B ; genetics ; immunology ; Oligonucleotides, Antisense ; pharmacology ; RNA, Messenger ; biosynthesis ; genetics ; Silicon Dioxide ; pharmacology ; Silicosis ; etiology ; Transcription Factors ; genetics ; immunology ; Transforming Growth Factor beta ; biosynthesis ; genetics ; Transforming Growth Factor beta1 ; Tumor Necrosis Factor-alpha ; biosynthesis ; genetics

BACKGROUNDIn previous work, we suggested that some 2-aminosteroids inhibited proliferation and induced differentiation of both human and murine leukemia cells. Here, we reported the actions of another new 2-aminosteroid designated as H89712 on human leukemia cells.

METHODSCell colony counting and MTT assay were used to determine proliferation. Cell morphology, histochemical staining, UV detection and cytometry were used to determine differentiation. RT-PCR was used to detect gene expression. Standard statistical method was used to analyze data.

RESULTSH89712 inhibited proliferation of HL-60 leukemia cells and the inhibition percentage in MTT assay was 18% at the dose of 10(-8) mol/L and 65% at the dose of 10(-5) mol/L, respectively. The inhibition for HL-60 in colony assay was 23% at the dose of 10(-8) mol/L and 96% at the dose of 10(-5) mol/L, respectively. H89712 also induced HL-60 cells toward macrophage-like differentiation. It was verified by flow cytometry that the percentage of positive CD14 expression in differentiated HL-60 cells was about 9 times higher than that of the control at the dose of 10(-8) mol/L and 20 times higher than that of the control at the dose of 10(-5) mol/L respectively, and this action involved upregulation of MafB gene in HL-60 leukemia cells. On the other hand, H89712 inhibited proliferation of K562 leukemia cells and the inhibition of K562 leukemia cells in MTT assay was shown by 34% at the dose of 10(-8) mol/L and 88% at the dose of 10(-5) mol/L respectively. The inhibition of K562 leukemia cells in colony assay was 53% at the dose of 10(-8) mol/L and 100% at the dose of 10(-5) mol/L respectively. H89712 also induced K562 cells toward erythroid-like differentiation and it was verified by flow cytometry that the percentage of positive CD71 expression in differentiated K562 cells was about 9 times higher than that of the control at the dose of 10(-8) mol/L and 16 times higher than that of the control at the dose of 10(-5) mol/L respectively. This action was related to upregulation of Egr-1 gene in K562 leukemia cells.

CONCLUSIONSOur results showed the important roles played by MafB in macrophage differentiation and Egr-1 in erythroid differentiation of human myeloid leukemia cells.

Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; DNA-Binding Proteins ; genetics ; Early Growth Response Protein 1 ; Gene Expression Regulation, Leukemic ; drug effects ; HL-60 Cells ; Humans ; Immediate-Early Proteins ; genetics ; K562 Cells ; MafB Transcription Factor ; Oncogene Proteins ; genetics ; Steroids ; pharmacology ; Transcription Factors ; genetics ; Up-Regulation

BACKGROUNDGene-radiotherapy, the combination of gene therapy and radiation therapy, is a new paradigm for cancer treatment. To enhance anti-tumor effect of gene-radiotherapy, in this study we construct a radiation-inducible dual-gene co-expression vector pEgr-interferon (IFN)-gamma-endostatin and studied the anti-tumor effect of pEgr-IFN-gamma-endostatin gene-radiotherapy in mice bearing Lewis lung carcinoma and its mechanism.

METHODSGene recombinant technique was used to construct dual-gene co-expression plasmid pEgr-IFN-gamma-endostatin, and single-gene expression plasmid pEgr-IFN-gamma and pEgr-endostatin. The plasmids packed by liposome were injected locally into the tumors of the mice, and the tumors were irradiated with 5 Gy X-ray 36 hours later. The tumor growth rate at different time and mean survival period of the mice were observed. Cytotoxic activity of splenic cytotoxic T-lymphocyte (CTL), natural killer (NK) cell and tumor necrosis factor (TNF)-alpha secretion activity of peritoneal macrophages of the mice in various groups were evaluated 15 days after irradiation. The intratumor micro-vessel density was evaluated by immunohistochemical staining 10 days after irradiation.

RESULTSThe tumor growth rate of the mice in dual-gene-radiotherapy group was significantly lower than those in control group, 5 Gy group and single-gene-radiotherapy group at different time after gene-radiotherapy, and the mean survival period of which was longer. Cytotoxic activity of splenic CTL, NK and TNF-alpha secretion activity of peritoneal macrophages of the mice in dual-gene-radiotherapy group were significantly higher than those in control group, 5 Gy X-ray irradiation group and pEgr-endostatin gene-radiotherapy group 15 days after irradiation. The intratumor micro-vessel density of the mice in dual-gene-radiotherapy group was significantly lower than those in control group, 5 Gy X-ray irradiation group and pEgr-IFN-gammagene-radiotherapy group.

CONCLUSIONThe anti-tumor effect of dual-gene-radiotherapy was significantly better than that of single-gene-radiotherapy by combining the enhancement of anti-tumor immunologic function induced by IFN-gamma with the anti-angiogenesis function of endostatin.

Animals ; Carcinoma, Lewis Lung ; blood supply ; immunology ; therapy ; Cell Line, Tumor ; Combined Modality Therapy ; DNA-Binding Proteins ; genetics ; Early Growth Response Protein 1 ; Endostatins ; genetics ; Female ; Genetic Therapy ; Immediate-Early Proteins ; genetics ; Interferon-gamma ; genetics ; Mice ; Mice, Inbred C57BL ; Neovascularization, Pathologic ; therapy ; Plasmids ; Transcription Factors ; genetics ; X-Ray Therapy

OBJECTIVETo study the expression and localization of early growth response gene-1 (Egr-1) in macrophages after stimulation by silicon dioxide in vivo and in vitro and to discuss the role of Egr-1 in the development of silicosis.

METHODSThe expression of Egr-1 in animal model of silicosis was analyzed by using immunohistochemistry. Western-blot, immunofluorescence and RT-PCR analysis were used to detect the expression and localization of Egr-1 protein and the dynamic changes of Egr-1 mRNA in cultured macrophages RAW264.7, after stimulation by silicon dioxide.

RESULTSIn animal model with induced silicosis, there was an increased expression of Egr-1 in pulmonary macrophages. The expression levels peaked at the 14th day. In vitro, the transcription of Egr-1 increased in RAW264.7 macrophages during 15 to 240 minutes after the administration of silicon dioxide. The response peaked at 15 minutes and diminished to a minimal level at 480 minutes. Nuclear translocation was most apparent at 60 minutes, lasted till 120 minutes and diminished gradually. During the period from 60 to 120 minutes, the expression of Egr-1 protein also reached a peak.

CONCLUSIONSSilicon dioxide can activate the nuclear transcription factor Egr-1 in vivo and in vitro in macrophages. Egr-1 may thus play an important pathogenetic role in the development of silicosis.

Animals ; Blotting, Western ; Cell Line ; DNA-Binding Proteins ; genetics ; metabolism ; Early Growth Response Protein 1 ; Gene Expression Regulation ; drug effects ; Immediate-Early Proteins ; Immunohistochemistry ; Lung ; drug effects ; metabolism ; pathology ; Macrophages ; drug effects ; metabolism ; Macrophages, Alveolar ; drug effects ; metabolism ; pathology ; Male ; RNA, Messenger ; drug effects ; genetics ; metabolism ; Rats ; Rats, Wistar ; Reverse Transcriptase Polymerase Chain Reaction ; Silicon Dioxide ; pharmacology ; Transcription Factors ; genetics ; metabolism

Sex-determining region Y box 18 (Sox18/SOX18) gene is an important regulator of vascular development playing a role in endothelial cell specification or differentiation, angiogenesis and atherogenesis. The aim of this study was to perform comprehensive functional characterization of the human SOX18 promoter, including determination of transcription start point (tsp) and identification of control elements involved in the regulation of SOX18 gene expression, with an emphasis on angiogenesis-related transcription factors. Analyses were performed in HeLa cells, representing a tumor cell line, and in EA.hy926 cells used as an endothelial model system. We have determined unique tsp of SOX18 gene, located 172 nucleotides upstream from ATG codon. Further, we have shown that SOX18 promoter region, -726 to -89 bp relative to tsp, contains positive cis-regulatory element(s) that stimulates SOX18 promoter activity, while region -89 to + 166 represents the minimal promoter. Within this region we have recognized the presence of essential element(s), positioned from -89 to +29, which harbors cluster of three putative early growth response 1 (EGR1) binding sites. By in vitro binding assays and functional analyses we have shown that these three putative binding sites are functionally relevant and sufficient for EGR1-induced SOX18 transcription. Mutations of these binding sites significantly impaired activity of the SOX18 promoter, particularly in EA.hy926 cells, indicating the importance of these regulatory elements for SOX18 promoter activity in endothelial setting. By data presented in this study, we have established SOX18 as a novel target gene regulated by EGR1 transcription factor, thus providing the first functional link between two transcription factors previously shown to be involved in the control of angiogenesis.
Early Growth Response Protein 1/genetics/*metabolism ; Electrophoretic Mobility Shift Assay ; Endothelium/*metabolism/pathology ; Gene Expression Regulation ; Hela Cells ; Humans ; Mutagenesis, Site-Directed ; Neovascularization, Physiologic/genetics ; Promoter Regions, Genetic ; Protein Binding/genetics ; SOXF Transcription Factors/genetics/*metabolism ; Transcription Initiation Site ; Transcriptional Activation

This study was aimed to investigate the influence of TIEG1 on apoptosis of HL-60 cells and the expression of Bcl-2/Bax. Different concentration of TIEG1 were used to treat HL-60 cells, the cell growth inhibition rate was detected by MTT method. After treating HL-60 cells with 12.03 ng/ml TIEG1, cell apoptosis was detected with flow cytometry. Bcl-2 and Bax was detected with RT-PCR. The results showed that TIEG1 had inhibitory effect on HL-60 cell proliferation, and in time-and dose-dependent manners. The more obvious inhibitory effect was observed in HL-60 cells treated with TIEG1 of 12.03 ng/ml. During the course of cell apoptosis, Bax expression increased, but Bcl-2 expression decreased (P < 0.05). It is concluded that TIEG1 inhibits HL-60 cell proliferation and induces apoptosis in time and dose-dependent manners. During the course of HL-60 cells apoptosis induced by TIEG1, Bcl-2/Bax are associated with HL-60 cell apoptosis induced by TIEG1.
Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Early Growth Response Transcription Factors ; pharmacology ; Gene Expression Regulation, Leukemic ; HL-60 Cells ; Humans ; Kruppel-Like Transcription Factors ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; bcl-2-Associated X Protein ; metabolism

We recently reported the use of a gene-trapping approach to isolate cell clones in which a reporter gene had integrated into genes modulated by T-cell activation. We have now tested a panel of clones from that report and identified the one that responds to a variety of G-protein coupled receptors (GPCR). The beta-lactamase tagged EGR-3 Jurkat cell was used to dissect specific GPCR signaling in vivo. Three GPCRs were studied, including the chemokine receptor CXCR4 (Gi-coupled) that was endogenously expressed, the platelet activation factor (PAF) receptor (Gq-coupled), and beta2 adrenergic receptor (Gs-coupled) that was both stably transfected. Agonists for each receptor activated transcription of the beta-lactamase tagged EGR-3 gene. Induction of EGR-3 through CXCR4 was blocked by pertussis toxin and PD58059, a specific inhibitor of MEK (MAPK/ERK kinase). Neither of these inhibitors blocked isoproterenol or PAF-mediated activation of EGR-3. Conversely, beta2- and PAF-mediated EGR-3 activation was blocked by the p38, specific inhibitor SB580. In addition, both beta2- and PAF-mediated EGR-3 activation could be synergistically activated by CXCR4 activation. This combined result indicates that EGR-3 can be activated through distinct signal transduction pathways by different GPCRs and that signals can be integrated and amplified to efficiently tune the level of activation.
Culture Media ; DNA-Binding Proteins ; metabolism ; Early Growth Response Protein 3 ; Gene Expression Regulation ; Genes, Reporter ; Humans ; Jurkat Cells ; MAP Kinase Signaling System ; Receptors, CXCR4 ; metabolism ; Receptors, G-Protein-Coupled ; metabolism ; Signal Transduction ; Transcription Factors ; metabolism ; beta-Lactamases ; metabolism