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Many studies showed that the p300/CBP coactivator is limiting. Here we review three studies that showed howtranscription complexes formed on viral cis-regulator elements compete with cellular transcription complexesby sequestrating the p300/CBP coactivator. According to the microcompetition model, this sequestering cancause disease. We use the microcompetition model to explain how a specific type of sequestering, caused by alatent virus that has an active cis-regulatory element in its promoter/enhancer that binds the transcriptioncomplex p300/CBPGABP can cause diseases such as cancer, atherosclerosis, diabetes, and certain autoimmune diseases.
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Objective To construct the promoter enhanced green fluorescent protein (pEGFP1) reporter gene vector of different truncated fragments of human cellular repressor of E1A-stimulated genes (hCREG), and compare the transcriptional activity of each promoter to determine the hCREG core promoter region. Methods The promoter fragment with length of 2003 (–1925/+78) bp was obtained by querying the hCREG sequence from US National Center of Biotechnology Information (NCBI) database and combining with the characteristics of the promoter. Five promoter fragments were truncated by PCR and double enzyme digestion and cloned into pEGFP1 to construct pEGFP1_hCREG_2003, pEGFP1_hCREG_945, pEGF P1_hCREG_586, pEGFP1_ hCREG_478 and pEG FP1_hCREG_358 reporter gene vector plasmid. The 293T cells were transiently co-transfected with the internal reference plasmid pGL4.73 [hRluc/SV40] for 48 hours. The green fluorescence expression of pEGFP1_hCREG promoter reporter gene was observed under fluorescence microscope, and the mRNA expression of each promoter was detected by real-time quantitative PCR, and the core promoter region was determined. Bioinformatics was used to predict the transcription factors that might bind to the core promoter region. Results Five hCREG promoter reporter gene vectors were successfully constructed by double enzyme digestion and gene sequencing. The results showed that the transcription activity of pEGFP1_ hCREG_586 was the highest (P0.05), implying that –867/ –509 bp is a negative regulatory region, and there existed enhancer sequences in –400/–281 bp and –508/–401 bp, so the core promoter region of hCREG gene is located in the upstream sequence of –508/–281 bp. Bioinformatics predicted that the possibly bound transcription factors in key promoter region –508/–281 bp were Pax5/P53, C/EBPβ, GR-β, GATA-1, GR-α, c-Jun, PRB/ PRA, YY1, RXR-α, AP-2, FOXP3, GR, TFIID, STAT4 and c-Ets-1. Conclusion The recombinant plasmid of hCREG gene promoter has been successfully constructed, the core promoter of which is located in –508/–281 bp, where several transcription factors might be bound.
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Objective To investigate the effect of E1A gene on the radiosensitivity of human nasopharyngeal carcinoma cells and its possible mechanism. Methods The E1A gene was transfected into nasopharyngeal carcinoma CNE-2R cells by adenovirus vector. The expression of E1A gene was detected by RT-PCR. Untransfected CNE-2R cells(PBS group)and CNE-2R cells transfected with empty vector Ad-β-gal(Ad-β-gal group)and E1A(Ad-E1A group)were given 0 Gy,2 Gy,4 Gy,6 Gy,8 Gy 6 MV X-ray irradiation. The changes in radiosensitivity of CNE-2R cells were determined by colony-forming assay. Flow cytometry was used to analyze cell apoptosis in each group. The expression of NF-κB, CK2α, Bcl-2, and cleaved caspase-3 was measured by Western blot. Results RT-PCR confirmed that the E1A gene was transfected into CNE-2R cells and stably expressed. The Ad-E1A group had a significantly lower plating efficiency than the PBS group and the Ad-β-gal group(P<0.05). The Ad-E1A group had significantly lower cell survival rate at 2 Gy irradiation than the PBS group and the Ad-β-gal group(0.217 vs. 0.602, P<0.05;0.217 vs. 0.585, P<0.05). The Ad-E1A group had a significantly higher α/β value than the PBS group and the Ad-β-gal group(24.680 vs. 5.268, P<0.05;24.680 vs. 5.132, P<0.05). Flow cytometry results showed that irradiation alone could promote the apoptosis of CNE-2R cells,when combined with E1A gene,the apoptosis rate was significantly increased(P<0.05). Western blot results showed that E1A gene down-regulated the expression of NF-κB/p65,CK2α,and Bcl-2 and up-regulated the expression of cleaved caspase-3. Conclusions E1A gene can enhance the radiosensitivity of nasopharyngeal carcinoma cells by inhibiting the expression of CK2 to block the NF-κB signaling pathway and promoting cell apoptosis.
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Objective To investigate the effect of cellular repressor of E1A stimulated genes (CREG1) on cardiac function in mouse with myocardial fibrosis. Methods CREG1 knockout mice (CREG1+/-) and CREG1 wild-type mice (CREG1+/+) were used to reproduce the model of myocardial fibrosis by subcutaneous pump burying of angiotensin Ⅱ (AngⅡ). After being stimulated with AngⅡ for 14 days, myocardial fibrosis was verified by HE staining and Masson trichrome staining. Western blotting and immunohistochemistry were used to detect the expression of CREG1 in myocardium before stimulation and 3, 7, 14 days after the AngⅡ stimulation. The cardiac function was evaluated by echocardiography after AngⅡ stimulation for 14 days. The CREG+/+ mice were given AngⅡ for 14 days, and at the same time recombinant CREG1 protein [respectively 15, 30, 60 and 300μg/(kg.d), intraperitoneal (IP) injections] (treatment group) and NaCl (control group) were administered for treatment, and then cardiac function and myocardiac apoptosis were examined. Results Western blotting and immunohistochemistry showed that the expression of CREG1 in heart tissue was significantly lower in CREG+/-mice than in CREG+/+ mice (P<0.05). After AngⅡ stimulation for 3, 7 and 14 days, the expression of CREG1 in heart tissue declined significantly in both CREG+/-and CREG+/+ mice (P<0.05), especially in CREG+/-mice (P<0.01). With HE and Masson staining, it was also found that CREG1 deficiency aggravated myocardial fibrosis and cardiac function deterioration in response to AngⅡ stimulation (P<0.05). Conversely, exogenous infusion of recombinant CREG1 protein significantly inhibited the occurrence of myocardial apoptosis (P<0.05), thus ameliorated cardiac function (P<0.05). Conclusions CREG1 deficiency may aggravate the deterioration of cardiac function in mouse with myocardial fibrosis induced by AngⅡ stimulation. The deterioration of cardiac function can be improved by administration of exogenous recombinant CREG1 protein.
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Objective To explore possible role of cellular repressor of E1A-stimulated genes(CREG) in the process of phenotypic switching of adventitial fibroblasts(AFs). Methods Immunofluorescent staining was performed with tissue sections from mouse carotid arteries to evaluate the relationship between the expression of CREG and smooth muscle actin-α(α-SMA) in injured arteries, especially in the adventitia. Tissue block pasted culture method was used to isolate and culture AFs. RT-PCR and Western-blot were used to detect the change of CREG andα-SMA mRNA and protein expression in AFs in the presence of different concentrations of AngⅡfor 12 h/24 h or in the presence of 100 nmol/L Ang Ⅱ for different times. Results Normal mouse carotid arteries had little α-SMA expression throughout the tunica adventitia. Arteries at day 1 and day 3 post-injury exhibited significantly higher immunofluorescence of α-SMA compared with non-injured arteries. Alpha-SMA expression began to decrease on day 7 and progressively declined on day 14. In contrast, immunofluorescent staining revealed that CREG was expressed in the adventitia of normal arteries. Expression of CREG in the adventitia of injured arteries was decreased on the 1st day, reached its lowest value on the 3rd day, and increased gradually from the 7th day, and was higher compared with that in non-injured arteries on the 14th day after injury. Similarly, the expression of CREG in AFs was very high, and AngⅡremarkably decreased mRNA and protein expression levels of CREG in a dose-dependent and time-dependent manner. Conclusions The changes in CREG expression correlate with AF phenotypic modulation, and CREG down-regulation may facilitate AF phenotypic switching into myofibroblasts (MFs).
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Objective To construct a new conditionally replicative adenovirus (CRAds) targeting carcinoembryonic antigen (CEA) positive colorectal cancer cells. Methods The DNA fragment of the CEA gene promoter was amplified through PCR and cloned into the vector carrying fusion reporter gene EGFP-Luc to construct expression plasmid pCEA-EGFPLuc. The constructed plasmid pCEA-EGFPLuc was transfected into CEA positive and negative cells by liposome. The activity of CEA gene promoter was evaluated by detecting the expression of EGFP and luciferase activity. The conditionally replicative adenovirus Ad.CEA-E1A/CMV-TK carrying suicide gene HSVtk was constructed, in which the E1A gene was controlled under CEA promoter. CEA positive(Lovo and SW620)and negative tumor cells(HeLa) were infected with Ad.CEA-E1A/ CMV-TK. The selective cytotoxicity of Ad.CEA-E1A/CMV-TK and the synergistic effect of the virus with GCV in CEA positive tumor cells were evaluated by the expression of E1A, cytopathic effect and cell survival rate. Results CEA promoter possesses a good specificity as well as high activity. The expression of E1A only presented in CEA positive tumor cells. After infection with Ad. CEA-E1A/CMV-TK, the cell survival rates of Lovo and SW620 were (36.72±2.49)% and (39.82±4.76)%, respectively, significantly lower than that of Hela[(87.44±2.76)%1 (P<0.01). When combined with GCV, Ad.CEA-E1A/CMV-TK had better oncolytic effect on Lovo and SW620 cells, with cell survival rates of (17.26±3.65) % and (23.93±5.40) %, respectively, significantly lower than those without GCV[(36.72±2.49) % and (39.82±4.76) %, respectively] (P<0.01). Conclusion Ad. CEA-E1A/CMV-TK under the control of CEA promoter has selective cytotoxic effect on CEA positive colorectal cancer cells, and the effect can be enhanced when combined with GCV.
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Objective To study the effect of E1A gene on the radiosensitivity of nasopharyngeal carcinoma (NPC) cells and its mechanism. Methods Ad-E1A gene was transfected into human NPC cells (CNE2), then the positive clones (CNE2-Ad-E1A) were identified by RT-PCR. CNE2 cells, CNE2 cells transfected with Ad-β-gal (CNE2-Ad-β-gal) and CNE2-Ad-E1A cells were irradiated with 0 Gy,2 Gy,4 Gy,6 Gy and 8 Gy respectively using 6 MV X-ray. Clone forming assays were carried out, cell survival curves were drawn and the sensitivity enhancing ratio (SER) was calculated. The redistributions of cell cy-cle were analyzed by flow cytometry. RT-PCR was used to detect the expression of wtp53. Results RT-PCR confirmed that E1A gene had been integTated into positively transfected cells and stably expressed. Cell survival curves showed that the SER of D0,Dq and SF_2 value was 1.37, 1.95 and 1.46 in CNE2-Ad-E1A cells. The D_0,D_q and SF_2 value was 1.57 Gy,1.82 Gy, 0.89 in CNE-2 cells and 1.53 Gy,1.78 Gy,0.82 in CNE2-Ad-β-gal cells, respectively. The G_2/M arrest was shown in CNE2-Ad-E1A cells. Moreover, the expression of wtp53 gene was markedly enhanced in Ad-E1A-CNE2 cells. Conclusions E1A gene can ef-fectively enhance the radiosensitivity of human NPC cells, which may be associated the enhancement of wt-p53 expression and G_2/M arrest.
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Objective:To study the inhibitory effect of E1A gene on the growth of tumors in nude mice implanted with nasopharygeal carcinoma CNE2 cells and its promotion effect on the racliosensitivity of CNE2-implanted tumors, and to investigate the related mechanism. Methods: E1A gene was transfected into CNE2 cells using adenovirus system, and sta-ble E1A positive clones were established. The inhibitory effect of E1A on tumor formation-ability of CNE2 cells was ob-served in nude mice. The efficacy of E1A gene therapy with or without radiotherapy against CNE2 cell-implanted tumors was evaluated. The effect of E1A gene therapy on the expression of P53 was detected by RT-PCR. Results: CNE2 cells stably transfected with E1A gene (CNE2-Ad-E1A) were successfully established. The tumor formation time was later and tumor size was smaller in CNE2-Ad-E1A cell-implanted mice compared with those in CNE2 cell- and CNE2-Ad-β-gal cell-implanted mice (CNE2 cells stably transfected with Ad-β-gal). Radiotherapy, E1A gene therapy and E1A gene + radio-therapy all suppressed the growth of implanted tumors, with the tumor suppression rates being (60.32±5.34) %, (70.53±6.12) %, and (97.15±4.87) % , respectively. E1A gene therapy significantly increased the expression of P53 gene in tumor tissues. Conclusion: E1A can inhibit the growth of tumors in mice implanted with nasopharygeal carcinoma cells, and enhance its sensitivity to radiotherapy, which may be related to the increased expression of P53 gene in tumor tissues.
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Oncolytic adenovirus-mediated E1A gene therapy may be a useful therapeutic approach for cancer treatment.In addition to direct lysis,viruses also mediate exogenous tumor suppressor genes inducing antitumor efficacy.Vectors need further investigation to improve the targeting and effectiveness in vivo.
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Objective To analyze the influence of adenovirus latent infection on gamma-glutamylcysteine systhetase(γ-GCS) in rat alveolar epithelial cells. Methods The rat alveolar epithelial cells were stably transfected with the plasmid pE1Aneo and control plasmid. Glutathione(GSH) contents, the activity of γ-GCS were detected in oxidant stress. Then the leuel of protein expression, mRNA expression, and promoter transcriptional activity of glutamate-cysteine ligase catalytic subunit(GCLC) were further detected. Results GSH contents decreased because of adenovirus E1A expression in oxidant stress. E1A repressed the expression and activity of γ-GCS, messenger RNA expression, and promoter transcriptional activity of GCLC. Conclusion Adenovirus E1A decreased the activity of γ-GCS probably by repressed promoter transcriptional activity of GCLC. As a result, GSH contents were downregulated in oxidant stress. Thus Adenovirus latent infection amplified the oxidant/antioxidant imbalance in rat alveolar epithelial cells in oxidants stress, which may be an important mechanism of COPD.
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Objective:To study the inhibitory effect of E1A gene on the growth of tumors in nude mice implanted with nasopharygeal carcinoma CNE2 cells and its promotion effect on the radiosensitivity of CNE2-implanted tumors,and to investigate the related mechanism.Methods: E1A gene was transfected into CNE2 cells using adenovirus system,and stable E1A positive clones were established.The inhibitory effect of E1A on tumor formation-ability of CNE2 cells was observed in nude mice.The efficacy of E1A gene therapy with or without radiotherapy against CNE2 cell-implanted tumors was evaluated.The effect of E1A gene therapy on the expression of P53 was detected by RT-PCR.Results: CNE2 cells stably transfected with E1A gene(CNE2-Ad-E1A)were successfully established.The tumor formation time was later and tumor size was smaller in CNE2-Ad-E1A cell-implanted mice compared with those in CNE2 cell-and CNE2-Ad-?-gal cell-implanted mice(CNE2 cells stably transfected with Ad-?-gal).Radiotherapy,E1A gene therapy and E1A gene+radiotherapy all suppressed the growth of implanted tumors,with the tumor suppression rates being(60.32?5.34)%,(70.53?6.12)%,and(97.15?4.87)%,respectively.E1A gene therapy significantly increased the expression of P53 gene in tumor tissues.Conclusion: E1A can inhibit the growth of tumors in mice implanted with nasopharygeal carcinoma cells,and enhance its sensitivity to radiotherapy,which may be related to the increased expression of P53 gene in tumor tissues.
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Objectives The cellular repressor of E1A-activated genes (CREG), a novel gene, was recently found to play a role in inhibiting cell growth and promoting cell differentiation. The purpose of this study was to obtain antibody against CREG protein and to study the expression of CREG protein in human internal thoracic artery cells (HITASY) which express different patterns of differentiation markers after serum withdrawal. Methods The open reading frame of CREG gene sequence was amplified by PCR and cloned into the pGEX-4T-1 vector. Glutathione-S-transferase (GST)-CREG fusion protein was expressed in E. Coli BL21 and purified from inclusion bodies by Sephacryl S-200 chromatography. Rabbits were immunized with the purified GST-CREG protein. Western blot examined with immunohistochemistry staining and the protein expression level was analyzed by Western blot in HITASY cells after serum removal. Results It was confirmed by using endonuclease digesting and DNA sequencing that the PCR product of CREG was correctly inserted into the vector. The GST-CREG protein was purified with gel filtration chromatography. Polyclonal antibody against GST-CREG was obtained from rabbits. CREG protein immunohistochemistry staining displayed a perinuclear distribution in the cytoplasm of HITASY cells. Results from Western blot suggested that comparing with the untreated cells upregulation of CREG polyclonal antibody against CREG was comfirmed. Using this antibody, the changes of CREG protein expression was observed in the process of phenotypic modulation of HITASY cells. These results provide basic understanding on the relationship of CREG gene with the cell phenotypic conversion.
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Objective:To explore the expression of intercellular adhesion molecule-1(ICAM-1 )induced by cigarette smoking extract(CSE) as well as the influence of the adenovirus E1A gene in pulmonary epithelial cells.Methods:A549 cells were transfected with a plasmid carrying the adenovirus E1A gene and stable trasfectants expressing E1A protein were selected. The cells were tested for the expression of ICAM-1 after stimulation with different concentrations of CSE.Results:ICAM-1expression was increased in A549 markedly with CSE stimulation,and the higher CSE concentration were,the more expression of ICMA-1 was observed. As compared with parental cells or cells transfected with control plasmid, ICAM-1 expression was markedly increased with stimulation of different concentrations of CSE in E1A-positive A549 cells.Conclusion:CSE exposure induces the surface expression of ICAM-1 and adenovirus E1A gene can markedly increase ICAM-1 expression induced by CSE in pulmonary epithelial cells, which suggest that latent adenovirus infection may amplify the inflammation process present in airways of smokers.
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Objective To analyze the human cellular repressor of E1A-stimulated genes (hCREG)using bioinformatics tools and predict the promoter position and transciption factor binding sites.Methods Complete coding hCREG sequences were obtained from Genbank.hCREG was analyzed with First EF software in order to obtain the promoter sequence of hCREG.Moreover,transcription factor binding sites of hCREG was convinced by Motif software.Subsequently,the transcription factor of hCREG by bioinformatics tools was related between hCREG and smooth muscle cells differentiation observed by both Western blot and immunoflourescence.Results The promoter of hCREG was located in -109~-359 bp of up-stream of transcriptional site,which was predicted with 945bp length.Transciption factor binding sites of hCREG was predicted by Motif software which was found with 80 transciption factors.The expression of hCREG and smooth muscle ?-actin(SM ?-actin)increased in HITASY after 72 hours with serum deprivation detected by both Western blot and immunoflourescence.Meanwhile,the results showed that the expression of wtp53 increased significantly.These results suggested that transcription factor wtp53 might regulate the expression of hCREG and promoted dedifferentiated phenotype of VSMCs.Conclusion The transcriptional information of the proximal promoter of hCREG obtained.As up-stream regulational factor of hCREG,wtp53 can up-regulate the expression of hCREG and may play a vital role in the process of VSMCs differentiation and phenotype modulation.
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AIM:To observe the influence of adenovirus latent infection on the oxidant/antioxidant balance in rat alveolar epithelial cells.METHODS:The rat alveolar epithelial cells were stably transfected with the plasmid pE1Aneo and control plasmid pneo.GSH and MDA contents,the activities of major anti-oxidative enzymes including SOD,CAT,GPx,GST and ?-GCS were detected in oxidant stress.RESULTS:Adenovirus E1A expression repressed the activity of ?-GCS,and decreased GSH contents in oxidant stress.As a result,the activity of GPx and GST was decreased.The contents of MDA maintained high in oxidant stress.CONCLUSION:Adenovirus latent infection amplifies the oxidant/antioxidant imbalance in rat alveolar epithelial cells in oxidants stress,and adenovirus E1A protein decreases the activity of ?-GCS,which plays an important role in this process.