OBJECTIVETo study the effect of binding activities of the NH(2) terminus of E1A to the proteins regulating cell growth on ras-induced cell senescence and explore the mechanism of E1A-mediated escape from ras-induced senescence by E1A in human fibroblast.

METHODSIn primary human fibroblasts, the proteins regulating cell growth in association with E1A NH(2) terminus, including the Rb family proteins, p300/CBP, and p400, were inactivated or interfered. The effect of alterations in the binding activities of these proteins on cell senescence bypass mediated by E1A was evaluated by cell growth curve.

RESULTSThe Inactivation of Rb family proteins alone was not sufficient to rescue ras-induced cell senescence, whereas inactivation of both the Rb proteins and p300/CBP blocked ras-induced senescence of human fibroblasts.

CONCLUSIONRb and p300/CBP binding activities are both required for E1A to bypass ras-induced senescence in human fibroblasts.

Adenovirus E1A Proteins ; pharmacology ; Cellular Senescence ; drug effects ; Fibroblasts ; cytology ; Humans ; Primary Cell Culture ; Retinoblastoma Protein ; metabolism ; Skin ; cytology ; p300-CBP Transcription Factors ; metabolism ; ras Proteins ; antagonists & inhibitors ; pharmacology

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

OBJECTIVEThe relationship between latent adenovirus infection and airway inflammation have not been well documented. The aim of this study is to illustrate the roles of adenovirus E1A protein on the level of glutathione (GSH) in response to oxidative stress and the effect of the oxidant/antioxidant imbalance upon the transactivation of NF-kappaB triggered by E1A protein.

METHODSRat alveolar epithelial cell stably expressing adenoviral E1A or control plasmid were developed. For isolation of nuclear extracts, 5 x 10(5) cells were plated and grown overnight in 60 mm dishes. Experiments were repeated three times. The cell model of stably expressing adenoviral E1A was stimulated by H2O2. The level of GSH were measured. E1A positive clone was stimulated by LPS or TNF-alpha and treated with L-Buthionine-sulfoximine (BSO). The expression of NF-kappaB was measured by Western blot. Differences between groups were assessed for significance by Student' t test; multiple comparisons by the one-way ANOVA.

RESULTSThere is no difference of GSH level without stimulation between E1A-positive clones and E1A-negative clones. For E1A-positive clones, the level of GSH did not increase in response to H2O2 as E1A-negative clones. The quantitation by densitometry of the NF-kappaB expression in E1A-positive clones were (79.3 +/- 4.6), (80.3 +/- 3.8) respectively without treatment and were (81.8 +/- 3.9) - (89.9 +/- 1.6) and (94.1 +/- 1.9) - (99.8 +/- 1.6) respectively under LPS or TNF-alpha stimulation, which were significantly higher than that of the control group (68.3 +/- 3.8), (69.4 +/- 4.3) respectively without stimulation and (70.1 +/- 2.8) - (80.8 +/- 3.6), (73.4 +/- 4.9) - (83.2 +/- 6.7) respectively under stimulation. The quantitation by densitometry of the NF-kappaB expression in E1A-negative clones were (1.25 +/- 0.18) and (1.69 +/- 0.19) respectively under LPS and TNF-alpha-stimulation and (1.22 +/- 0.16) and (1.75 +/- 0.13) respectively upon treatment for LPS and TNF-alpha with BSO preincubation. There did not show difference upon treatment with LPS or TNF-alpha with or without BSO in E1A-negative cell clone. The quantitation by densitometry of the NF-kappaB expression in E1A-positive clone were (1.75 +/- 0.10) and (2.26 +/- 0.21) respectively upon treatment for LPS and TNF-alpha with BSO preincubation which were significantly higher than that of LPS or TNF-alpha-stimulation alone (1.35 +/- 0.12), (1.80 +/- 0.14) respectively.

CONCLUSIONThese results indicate that E1A protein decreased GSH levels in oxidant stress and upregulated NF-kappaB transcription activity. The oxidant/antioxidant imbalance in rat alveolar epithelial cells enhances E1A-modulated transcriptional activation of NF-kappaB. The mechanism underlying transactivation of NF-kappaB involved by E1A may be related to oxidative stress.

Adenovirus E1A Proteins ; genetics ; metabolism ; Animals ; Cell Line ; Epithelial Cells ; metabolism ; NF-kappa B ; genetics ; metabolism ; Oxidative Stress ; Pulmonary Alveoli ; cytology ; Rats ; Transcriptional Activation

We constructed the recombinant adenovirus vector expressing IL-24 and E1A (Ad-IL-24-E1A) and investigated the inhibition of Ad-IL-24-E1A on SMMC-7721 hepatocellular carcinoma in vitro. We amplified IL-24 gene by PCR using pAdTrack-IL-24 as template. The IL-24 gene was cloned into pAdTrack-IRES at the Bgl II and Sal I site to form pAdTrack-IL-24-IRES. E1A digested from pAdTrack-E1A was cloned into the pAdTrack-IL-24-IRES at the Xho I and EcoR V site to form the pAdTrack-IL-24-IRES-E1A. We co-transformed both pAdTrack-IL-24-IRES-E1A and pAdeasy-1 digested by Pme I and packaged to obtain Ad-IL-24-E1A. Ad-IL-24-E1A at 50 MOI infected SMMC-7721 cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay determined cell proliferation. Flow cytometry detected Cell apoptosis. The apoptotic rate of SMMC-7721 cells was 52% 48 h after infection with Ad-IL-24-E1A. The result showed that the growth of SMMC-7721 cells was significantly inhibited by Ad-IL-24-E1A at the MOI of 50.
Adenoviridae ; genetics ; metabolism ; Adenovirus E1A Proteins ; biosynthesis ; genetics ; Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Carcinoma, Hepatocellular ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Genetic Vectors ; genetics ; Humans ; Interleukins ; biosynthesis ; genetics ; Liver Neoplasms ; pathology ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology

OBJECTIVE: To determine the effect of Ad-E1A gene therapy on in vivo radiosensitivity to nasopharyngeal carcinoma. METHODS: CNE-2Z cells (2 x 10(5)) were subcutaneously injected into nude mice to develop tumor (1-3 mm) 6 days later. The tumor-bearing mice were then randomly divided into 6 groups (10 mice per group) for PBS treatment or treatment with radiotherapy, Ad-E1A, or Ad-beta-gal alone or radiotherapy in combination with Ad-E1A or Ad-beta-gal. The mice were treated with Ad-E1A or Ad-beta-gal (5 x 10(9) plaque forming units) by intratumoral injection twice weekly for 2 weeks at beginning of week 2. The mice treated with radiotherapy in combination with Ad-E1A or Ad-beta-gal received 2 Gy radiotherapy daily for 5 days following the first week of treatment with Ad-E1A or Ad-beta-gal. Control mice received PBS therapy or radiotherapy only after tumor cells were injected. When the size of tumor exceeded 2 cm, the mice were killed and the tumors underwent immunohistochemical analysis for VEGF and CD34 expression and TUNEL assay for apoptosis. RESULTS: The growth delay time was longest in the Ad-E1A plus radiotherapy group. Tumors treated with Ad-E1A plus radiotherapy were 4.7-fold smaller than those treated with radiotherapy alone and 5.3-fold smaller than those treated with Ad-E1A alone. The survival rate of tumor-bearing mice treated with Ad-E1A plus radiotherapy was significantly higher than that of other treatment groups. The vessel density and the VEGF expression were significantly lower in tumors treated with Ad-E1A plus radiotherapy than those treated with radiotherapy alone, Ad-E1A alone, Ad-beta-gal alone, or Ad-beta-gal plus radiotherapy (P<0.01). TUNEL staining revealing apoptosis can be detected in the Ad-E1A group, radiotherapy group, Ad-E1A plus radiotherapy group, and more apoptosis can be detected in tumors treated with Ad-E1A plus radiotherapy than those of other treatment groups. CONCLUSION E1A gene therapy can effectively enhance the nasopharyngeal carcinoma sensitivity to the radiotherapy by down-regulating VEGF expression and inducing apoptosis.
Adenovirus E1A Proteins ; biosynthesis ; genetics ; Animals ; Apoptosis ; physiology ; Cell Line, Tumor ; Genetic Therapy ; methods ; Humans ; Mice ; Mice, Nude ; Nasopharyngeal Neoplasms ; blood supply ; pathology ; radiotherapy ; Neoplasm Transplantation ; Radiation Tolerance ; genetics ; Random Allocation ; Vascular Endothelial Growth Factor A ; genetics ; metabolism

OBJECTIVE: To investigate the inhibitive effect of E1A gene carried by PEI-Fe(3)O(4) nanometer particle (NP) on the cell growth of human cervical carcinoma cell in vitro and its mechanism, and to provide the experimental evidence for the feasibility of gene therapy for human cervical carcinoma. METHODS: E1A gene conjugated to PEI-Fe3O4 NP was transfected into human cervical carcinoma cell line Hela. The cell growth curve of Hela was drawn, the doubling time and the number of colony formations on the soft agar were calculated based on the cell count. RT-PCR and Western blot were used to detect the expression of the E1A and HER-2/neu in Hela cells. RESULTS: The cell doubling time of Hela cells transfected with E1A gene (Hela-E1A) was 1.53 times and 1.58 times longer than that of the Hela transfected with blank vector (Hela-vector) and blank Hela control (Hela), respectively. The E1A transfected Hela cells grew slower than those of the control group. The cell colony formation efficiency in the Hela-E1A (6.62%) group was significantly lower than that of Hela (30.48%) and Hela-vector (28.3%) groups (P<0.05). As compared to Hela and Hela-vector, the inhibition rate of Hela-E1A was 78.28% and 76.62% respectively. RT-PCR and Western blot demonstrated that the overexpression of E1A through gene transfection significantly inhibited mRNA and protein expression of HER-2/neu in Hela cells. CONCLUSION E1A gene can suppress the cell growth of human cervical carcinoma cell Hela in vitro. Down-regulated expression of HER-2/neu gene by E1A overexpression in Hela might contribute to the Hela growth inhibitive effect of E1A.
Adenovirus E1A Proteins ; genetics ; Cell Proliferation ; Female ; Gene Expression Regulation, Neoplastic ; Genetic Therapy ; methods ; HeLa Cells ; Humans ; Transfection ; Uterine Cervical Neoplasms ; genetics ; therapy

OBJECTIVETo investigate the role of human adenovirus type 5 (Ad5) early-region 1A (E1A) in premature senescence of human fibroblasts induced by Ras activation.

METHODSHuman fibroblasts were cotransduced with E1A, E1A1-143, or their vector (BP) and Ha-RasV12 or its vector (WH). The growth curves and percentages of the apoptotic cells were determined.

RESULTSExpression of E1A or Ha-RasV12 in human fibroblasts significantly inhibited the cell growth. Transduction of Ha-RasV12 along with E1A or E1A 1-143 into human fibroblast cells resulted in active and rapid cell proliferation.

CONCLUSIONE1A can rescue human fibroblasts from Ras-induced premature senescence, and the senescence bypassing activity of E1A resides in its NH2 terminus.

Adenovirus E1A Proteins ; genetics ; metabolism ; Apoptosis ; genetics ; physiology ; Cell Transformation, Neoplastic ; genetics ; metabolism ; Cells, Cultured ; Cellular Senescence ; genetics ; physiology ; Fibroblasts ; cytology ; metabolism ; Genes, ras ; physiology ; Humans ; Skin ; cytology ; Transfection

OBJECTIVE: To explore the molecular mechanism of proliferation inhibition of human breast cancer cell MCF-7 regulated by E1A gene. METHODS: E1A gene was transfected into MCF-7 cells by liposome reagents. RT-PCR and Western blot were used to detect E1A mRNA and protein expression and HER-2 mRNA in MCF-7. The proliferation and colony formation of MCF-7 were measured by 3-(4,5-dinmethylthiahiazo-z-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and soft agar formation assay. The apoptosis of MCF-7 cells regulated by E1A expression was examined by flow cytometry. RESULTS: E1A was not endogenously expressed in MCF-7. E1A expression in MCF-7 could significantly decrease HER-2 mRNA and protein expression. Flow cytometry indicated that the apoptosis of MCF-7 could be induced by E1A. Meanwhile, E1A gene could significantly inhibit MCF-7 proliferation and colony formation in soft agar. CONCLUSION E1A gene can decrease HER-2 expression and induce the apoptosis of human breast cancer cell MCF-7, and inhibit the proliferation and colony formation of MCF-7.
Adenovirus E1A Proteins ; biosynthesis ; genetics ; Apoptosis ; genetics ; Breast Neoplasms ; genetics ; metabolism ; Cell Proliferation ; Female ; Genes, erbB-2 ; genetics ; Humans ; RNA, Messenger ; biosynthesis ; genetics ; Receptor, ErbB-2 ; biosynthesis ; genetics ; Transfection ; Tumor Cells, Cultured

OBJECTIVETo prepare nanoparticles containing E1A gene and observe the efficiency and feasibility of transfecting E1A gene into human undifferentiated thyroid cancer cell line HTC/3. To examine the sensitivity of transgene cells to X-ray and X-ray-induced apoptosis in those cells.

METHODSNanoparticle-DNA complex was prepared with PLGA coating adenoviral early expression gene E1A, and the package efficiency, release progress in vitro, and size of the complex were determined. The nanoparticle-DNA was transfected into the HTC/3 cells. Lipofectamine was used to transfect E1A gene as a control. RT-PCR was used to examine E1A gene mRNA expression in the transfected cells. The survival ratio of HTC/3-E1A and control cells, and the growth inhibition ratio induced by different doses of X-ray in HTC/3-E1A cells were examined by MTT assay. The apoptosis in HTC/3-E1A cells induced by 2 Gy X-ray iradiation was examined by flow cytometry and DNA electrophoresis.

RESULTSThe package efficiency, release progress in vitro, and size of the nanoparticle-DNA complex were 0.78%, 18 days, and 150-280 nm, respectively when transfected the plasmid at the same level, the nanoparticle group got more positive transgene cell clones than that in lipofectamine group, with a statistically significant difference (P < 0.05). RT-PCR showed that transgenic cells from both nanoparticle-DNA and lipofectamine groups had E1A gene mRNA expression. The HTC/3-E1A cells grew slowly, and their doubling time was prolongated (1.44 times in comparison with that in parental cells). According to IC50, the sensitivity of HTC/3-E1A cells to X-ray was improved 2.9 and 2.8 times, respectively, in comparison with that in HTC/3-Vect and HTC/3 cells. The ratio of subG0/G1 phase of HTC/3-E1A cells was significantly higher than that in HTC/3-Vect and HTC/3 cells (P < 0.01). The ratio of S phase of HTC/3-E1A cells was significantly lower than that in HTC/3-Vect and HTC/3 cells (P < 0.01). A typical DNA ladder pattern of apoptosis in HTC/3-E1A cells was observed by electrophoresis, but not found in HTC/3-Vect and HTC/3 cells.

CONCLUSIONA nanoparticle-DNA complex has been successfully prepared, and it may carry a foreign gene into cells. The sensitivity of HTC/3-E1A cells to X-ray is significantly improved. Moreover, apoptosis is induced by x-ray in the E1A gene-transfected cells.

Adenovirus E1A Proteins ; biosynthesis ; genetics ; physiology ; Apoptosis ; radiation effects ; Cell Cycle ; radiation effects ; Cell Line, Tumor ; Cell Proliferation ; DNA ; genetics ; Humans ; Lactic Acid ; chemistry ; Nanoparticles ; Particle Size ; Plasmids ; Polyglycolic Acid ; chemistry ; RNA, Messenger ; metabolism ; Thyroid Neoplasms ; metabolism ; pathology ; Transfection ; X-Rays

The E1A gene was obtained by PCR with QBI-293A cell genome DNA as template. After enzyme digestion, the E1A gene was ligated to transfer vector pAdTrack-CMV. The positive clone pAdTrack-CMV-E1A were lineared by PmeI and co-transformed with pAdEasy-1 in BJ5183 E. coli. The recombinant adenovirus vector pAdEasy-1-pAdTrack-CMV-E1A were digested by PacI and transfected into QBI-293A cells with liposomes. The oncolytic recombinant adenovirus Ad-E1A was obtained after 7 days. The results showed that this oncolytic adenovirus Ad-E1A can replicate in ECV304 cells and inhibit growth of ECV304 cell. In addition, it also decreased the secretion of VEGF and expression of NF-kappaB of ECV304 cells, indicating that Ad-E1A have potential of inhibition of tumor metastasis.
Adenoviridae ; genetics ; physiology ; Adenovirus E1A Proteins ; biosynthesis ; genetics ; Cell Proliferation ; Cells, Cultured ; Endothelial Cells ; cytology ; metabolism ; Humans ; Oncolytic Virotherapy ; Oncolytic Viruses ; genetics ; physiology ; Promoter Regions, Genetic ; Umbilical Veins ; cytology ; metabolism