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

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

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 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

OBJECTIVETo study influence of lanthanum chloride (LaCl(3)) on the expression of immediate early genes (IEGs) including c-jun, early growth response gene 1 (Egr1) and activity-regulated cytoskeletal gene (Arc) in the hippocampus of rats, and discuss the mechanism of LaCl(3) undermining learning and memory capability.

METHODSForty female Wistar adult rats were divided into control group, low LaCl(3)-contaminated group (0.25%), medium LaCl(3)-contaminated group (0.50%), and high LaCl(3)-contaminated group (1.00%) by randomized design. Each group had ten female rats along with five male rats and mated by the ratio of 2:1. The amounts of pups in the above four groups were 80, 83, 78 and 75 separately. The pups in respective group were La-dyed by lactation, and then the pups in LaCl(3)-contaminated groups drank 0.25%, 0.50% and 1.00% LaCl(3) separately for one month. Learning and memory capability of pups were measured in jumping stairs experiment. Hippocampal lanthanum content was determined by inductively coupled plasma mass spectrometry (ICP-MS). Hippocampal c-jun, Egr1 and Arc mRNA expression was detected by RT-PCR, and corresponding protein expression was measured by Western blotting method.

RESULTSIn the jumping stairs experiment, pups in 0.25%, 0.50% and 1.00% LaCl(3)-contaminated groups respectively made (1.75 ± 0.71), (2.38 ± 0.92) and (3.00 ± 0.76) mistakes; significantly higher than control group (1.25 ± 0.46) (q values were 4.386, 6.793, P < 0.05). However, the incubation period of 0.25%, 0.50% and 1.00% LaCl(3)-contaminated groups were (174.13 ± 33.72), (139.25 ± 45.83) and (75.50 ± 18.56) respectively, which were all significantly lower than that of control group (206.75 ± 20.47) (q values were 2.958, 6.121, 11.902, P < 0.05). Hippocampal c-jun mRNA expression were (0.89 ± 0.08), (0.77 ± 0.12), (0.58 ± 0.14) and (0.29 ± 0.10); while the c-jun protein expression were (0.72 ± 0.13), (0.64 ± 0.11), (0.43 ± 0.11) and (0.31 ± 0.14), and the Egr1 mRNA expression were (0.78 ± 0.09), (0.61 ± 0.13), (0.53 ± 0.10) and (0.22 ± 0.08), Egr1 protein expression were (0.65 ± 0.18), (0.40 ± 0.15), (0.32 ± 0.13) and (0.14 ± 0.09) in 0.25%, 0.50% and 1.00% LaCl(3)-contaminated groups; and all of which presented a dose-effect relationship that the correlation coefficients of these parameters with dose were -0.900 (t = 11.309, P = 0.000), -0.969 (t = 7.058, P = 0.000), -0.898 (t = 11.179, P = 0.000) and -0.962 (t = 6.739, P = 0.000).

CONCLUSIONLaCl(3) undermines the learning and memory capability of rats, which is possibly related to lower expression of c-jun and Egr1 gene and protein induced by lanthanum in hippocampus.

Animals ; Early Growth Response Protein 1 ; metabolism ; Female ; Gene Expression ; Genes, Immediate-Early ; drug effects ; genetics ; Hippocampus ; drug effects ; metabolism ; Lanthanum ; pharmacology ; Learning ; drug effects ; Male ; Memory ; drug effects ; Proto-Oncogene Proteins c-jun ; metabolism ; Rats ; Rats, Wistar

OBJECTIVETo develop a new kind of vector system called gene-viral vector, which combines the advantages of gene and virus therapies.

METHODSUsing recombinant technology, an anti-tumor gene was inserted into the genome of replicative virus specific for tumor cells. The cell killing effect, reporter gene expression of the green fluorescence protein, anti-tumor gene expression of mouse interleukin-12 (mIL-12) and replication of virus were observed by the methods of cell pathology, fluorescence microscopy, ELISA and electron microscopy, respectively.

RESULTSA new kind of gene-viral vector system of adenovirus, in which the E1b-55 kD gene was deleted but the E1a gene was preserved, was constructed. The vector system, like the replicative virus ONYX-015, replicated and proliferated in tumor cells but not in normal ones. Our vector had an advantage over ONYX-015 in that it carried different kinds of anti-tumor genes to enhance its therapeutic effect. The reporter gene expression of the green fluorescence protein in tumor cells was much better than the adenovirus vector employed in conventional gene the rapy, and the expression in our vector system was as low as or even less than that in the conventional adenovirus gene therapy system. Similar results were observed in experiments with this vector system carrying the anti-tumor gene mIL-12. Replication and proliferation of the virus carrying the mIL-12 gene in tumor cells were confirmed by electron microscopy.

CONCLUSIONSGene-viral vectors are new vectors with an anti-tumor gene inserted into the genome of replicative virus specific for tumor cells. Because of the specific replication and proliferation of the virus in tumor cells, expression of the anti-tumor gene is increased hundreds to thousands of times. This approach takes full advantages of gene therapy and virus therapy to enhance the effect on the tumor. It overcomes the disadvantages of conventional gene therapy, such as low transfer rate, low gene expression, lack of target tropism, and low anti-tumor activity. We believe that this is a promising means for future tumor treatment.

Adenoviridae ; genetics ; Adenovirus E1A Proteins ; genetics ; Adenovirus E1B Proteins ; genetics ; Genetic Therapy ; methods ; Genetic Vectors ; genetics ; Humans ; Interleukin-12 ; genetics ; Neoplasms ; therapy ; Recombination, Genetic ; Tumor Cells, Cultured ; Virus Replication

BACKGROUNDOur previous studies have demonstrated potent oncolysis efficacy of the E1A, E1B double-restricted replication-competent oncolytic adenovirus AxdAdB-3 for treatment of bladder cancer. Here, we reported the feasibility and efficacy of AxdAdB-3 alone, or in combination with gemcitabine for treating renal cell carcinoma.

METHODSCytopathic effects of AxdAdB-3 were evaluated in human renal cell carcinoma cell lines TOS-1, TOS-2, TOS-3, TOS-3LN, SMKT-R3, SMKT-R4 and ACHN, and in normal human renal proximal tubule epithelial cells (RPTEC). AxdAdB-3 induced down-regulation of the cell cycle was determined by flow cytometry. Combination therapies of AxdAdB-3 with gemcitabine were evaluated in vitro and in vivo on subcutaneous TOS-3LN tumors in a severe combined immunodeficiency disease (SCID) mouse model.

RESULTSAxdAdB-3 was potently cytopathic against the tested most renal cell carcinoma cell lines including TOS-2, TOS-3, TOS-3LN, SMKT-R3 and SMKT-R4, while normal human RPTEC were not destroyed. AxdAdB-3 effectively induced cell cycle S-phase entry. Combined therapy of AxdAdB-3 with gemcitabine demonstrated stronger antitumor effects in vitro and in vivo compared with either AxdAdB-3 or gemcitabine alone.

CONCLUSIONAxdAdB-3 alone, or in combination with gemcitabine may be a promising strategy against renal cell carcinoma.

Adenoviridae ; genetics ; metabolism ; physiology ; Adenovirus E1A Proteins ; genetics ; Adenovirus E1B Proteins ; genetics ; Animals ; Antimetabolites, Antineoplastic ; pharmacology ; therapeutic use ; Carcinoma, Renal Cell ; drug therapy ; therapy ; Cell Cycle ; drug effects ; genetics ; Cell Line ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Coxsackie and Adenovirus Receptor-Like Membrane Protein ; Deoxycytidine ; analogs & derivatives ; pharmacology ; therapeutic use ; Flow Cytometry ; Humans ; Immunohistochemistry ; Male ; Mice ; Mice, Inbred BALB C ; Mice, SCID ; Oncolytic Virotherapy ; Receptors, Virus ; genetics ; metabolism ; Xenograft Model Antitumor Assays