OBJECTIVETo assess the protection against cisplatin-induced ototoxicity by adenovirus-mediated overexpression of the bcl-2 gene in cultured spiral ganglion cells (SGC).

METHODSSGC from P3 rats were cultured in vitro and exposed to adenovirus vector carrying green fluorescent protein gene (Ad-GFP), followed by immunocytochemical analysis for expression of the neuron-specific marker Neurofilament 200 (NF200) and detection under laser scanning confocal fluorescence microscope. Then, SGC were transduced by Ad-bcl-2 and the expression of human bcl-2 protein was evaluated by Western Blot. Finally, the cultures of SGC were divided into 4 groups: the group of Ad-bcl-2 transfection followed by cisplatin treatment, the group of Ad-GFP transfection followed by cisplatin treatment, the group of cisplatin treatment only and the untreated group. Cisplatin worked for 48 hours at a concentration of 2 microg/ml. Outcome measures included survival number of SGC and longest neurite length by using ImageJ software.

RESULTSSGC were cultured successfully in vitro and transfected by adenovirus vector safely and efficiently. By Western Blot, human bcl-2 protein was expressed in the group after exposure to Ad-bcl-2, but not in the Ad-GFP transfected SGC. Cisplatin exposure resulted in shrinking of neuritis and pyknosis of cell body, even cell death. Expression of bcl-2 in the SGC provided a significant level of protection against cisplatin-induced SGC degeneration.

CONCLUSIONSOur results suggest that SGC can be transduced by adenovirus vector safely and efficiently in vitro. Adenovirus-mediated delivery of the bcl-2 gene attenuates cisplatin-induced SGC degeneration.

Adenoviridae ; genetics ; Animals ; Apoptosis ; drug effects ; Cisplatin ; pharmacology ; Genes, bcl-2 ; Humans ; Spiral Ganglion ; cytology

AIMTo investigate the protective effects of adenovirus-mediated hepatocyte growth factor (Ad-HGF) on injury of rat cortex neurons induced by in vitro serum-free culture.

METHODSFlow cytometry was used to assay the transfection rate of rat cortex neurons infected by adenovirus-mediated green fluorescent protein(Ad-GFP) at different multiplicity of infection (MOI) to find out the best MOI in experiment. ELISA was used to elucidate the expression patterns of cortex neuron. Neutral red stain and PI-Hoechst 33342 double stain were used to compare the viability of cortex neurons, which were cultured in serum-free medium for 6 h, 12 h, 24 h and 48 h respectively, among the Ad-HGF transfected group, the Ad-GFP transfected group and the control group.

RESULTSIt was found that when MOI was 50 PFU per cell, a transfection rate as high as 99.3% was maintained and Ad-HGF was able to express in cortex neurons effectively and persistently. In addition, the death rate and apoptotic rate of cortex neurons (infected 2 hours after seeding) cultured in serum-free medium for 12 h in Ad-HGF transfected group was significantly lower than that in both the Ad-GFP group and the control group (P < 0.05).

CONCLUSIONAd-HGF plays a protective role against in vitro serum-free culture induced injury on rat cortex neurons infected 2 hours after seeding. Though its effects on rat cortex neurons infected 5 days after seeding are not so remarkable, Ad-HGF also has the potential to protect cortex neurons from serum-free culture induced injury.

Adenoviridae ; genetics ; Animals ; Animals, Newborn ; Cells, Cultured ; Cerebral Cortex ; drug effects ; Culture Media, Serum-Free ; Hepatocyte Growth Factor ; genetics ; pharmacology ; Neurons ; drug effects ; Rats ; Rats, Wistar ; Transfection

To investigate the inhibitory effect and anti-cancer mechanism of adenovirus mediated IL-24 gene expression on the human U251 glioma cell. U251 glioma cells were infected with Ad-IL-24 at various multiplicity of infection (MOIs). Cell proliferation was determined by MTT assay. Cell apoptosis was detected by flow cytometry and Hochest staining. The transcription of apoptosis-related genes was analyzed by reverse transcription-PCR (RT-PCR), and the expression of Cleaved Caspase-3 was analyzed by Western blotting. The result showed that the growth of U251 glioma cells was significantly inhibited by Ad-IL-24 at the MOI of 100. The apoptotic rate of U251 glioma cells was 42% 72 h after infection with Ad-IL-24. Four days after infection, the growth of the U251 glioma cells was inhibited to 50%. RT-PCR showed that Ad-IL-24 not only up-regulated expression of bax/bcl-2, ICE, C-myc, p53 and down-regulated the expression of HIF-1alpha, but also enhanced Caspase-3 activation, eventually resulting apoptosis. Taken together, these results suggest that infection of U251 glioma cells with Ad-IL-24 can inhibit growth and induce apoptosis significantly by the regulation of apoptosis-related genes.
Adenoviridae ; genetics ; metabolism ; Apoptosis ; Brain Neoplasms ; genetics ; pathology ; Cell Proliferation ; drug effects ; Genetic Therapy ; Glioma ; genetics ; pathology ; Humans ; Interleukins ; genetics ; metabolism ; Recombination, Genetic ; Tumor Cells, Cultured

PURPOSE: Antimicrobial peptides have an important role in self-protection of the ocular surface. Human cationic antimicrobial protein (hCAP)-18 is a linear, alpha-helical peptide that consists of a conserved pro-sequence called a cathelin-like domain and a C-terminal peptide named LL-37. We investigated the in vitro anti-adenoviral activity of hCAP-18/LL-37 in several adenovirus types, inducing keratoconjunctivitis. METHODS: A549 cells were used for viral cell culture, and human adenovirus (HAdV) types 3 (HAdV3, species B), 4 (species E), 8, 19a, and 37 (species D) were used. The cytotoxicity of LL-37 was evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay to obtain 50% cytotoxic concentration. After pretreatment of A549 cells with serial dilutions of LL-37 for 24 hours, adenovirus was cultured for seven days, and adenoviral DNA was quantitatively measured by real-time polymerase chain reaction (PCR). RESULTS: The 50% effective concentration of LL-37 obtained by real-time PCR ranged between 118 and 270 microM. LL-37 showed a significant inhibitory effect on adenoviral proliferation in all adenovirus types except HAdV4 in a dose-dependent manner. CONCLUSIONS: LL-37 has significant inhibitory activity against HAdV3, 8, and 19, which induce keratoconjunctivitis. These results indicate that hCAP-18/LL-37 may be a possible candidate for the treatment of HAdV keratoconjunctivitis.
Adenocarcinoma ; Adenoviridae/*drug effects/*genetics ; Adenoviridae Infections/*drug therapy/virology ; Antimicrobial Cationic Peptides/*pharmacology ; Cell Line, Tumor ; DNA, Viral/genetics ; Humans ; Keratoconjunctivitis/*drug therapy/virology ; Lung Neoplasms ; Reverse Transcriptase Polymerase Chain Reaction/methods

BACKGROUNDSteroids inhibit osteogenic differentiation and decrease bone formation while concomitantly inducing adipose deposition in osteocytes. This leads to the fatty degeneration and necrosis of bone cells commonly seen in osteonecrosis of the femoral head. The peroxisome proliferator-activated receptor-γ (PPARγ) is an adipogenic transcription factor linked to the development of this disease and responsible for inducing adipogenesis over osteogenesis in bone marrow mesenchymal stem cells (BMSCs). The aim of this study was to assess whether adipogenic differentiation could be suppressed, and thus osteogenic potential retained, by inhibiting PPARγ expression in BMSCs.

METHODSCells from the bone marrow of New Zealand rabbits were treated with 10(-7) mol/L dexamethasone and infected with one of three small interference RNA (siRNA) adenovirus vectors (S1, S2, and S3) or non-targeting control siRNA (Con) and compared with dexamethasone-treated (model) and untreated (normal) cells. Cells were grown for 21 days and stained with Sudan III for adipocyte formation. At various time points, cells were also assessed for changes in PPARγ, osteocalcin (OC), Runx2, alkaline phosphatase (ALP) activity, and triglyceride (TG) content.

RESULTSDexamethasone-treated model and control groups showed a significant increase in fatty acid-positive staining, which was inhibited in cells treated with PPARγ siRNA-treated, similar to normal untreated cells. All three siRNA groups significantly inhibited PPARγ mRNA and protein, adipocyte number, and TG content compared with the dexamethasone-treated model and control groups, matching that seen in normal cells. OC and Runx2 mRNA and protein, as well as ALP activity, were significantly higher in cells treated with siRNA against PPARγ, similar to that seen in the normal cells. These osteogenic markers were significantly lower in the dexamethasone-treated cell cultures.

CONCLUSIONSThe siRNA adenovirus vector targeting PPARγ can efficiently inhibit steroid-induced adipogenic differentiation in rabbit BMSCs and retain their osteogenic differentiation potential.

Adenoviridae ; genetics ; Adipogenesis ; drug effects ; genetics ; Animals ; Cell Differentiation ; drug effects ; genetics ; Mesenchymal Stromal Cells ; cytology ; drug effects ; metabolism ; PPAR gamma ; genetics ; metabolism ; pharmacology ; RNA, Small Interfering ; Rabbits ; Steroids

OBJECTIVETo explore the influence of two different endolymphatic infusion ways on cochlear morphology and function.

METHODSForty healthy pigment guinea pigs (250 - 350 g) with normal Prey's reflex were divided into A and B group with 20 animals respectively. The right ears served as treated ears and the left ones as control ones. In group A, 5 microl of adenovirus 5-enhanced green fluorescence protein (Ad5-EGFP) suspension was infused into the scala media through an opened cochleostomy on the lateral wall of the scala media (LWS). In group B, the same volume of Ad5-EGFP suspension was infused into the scala media through punctured round window membrane and the basilar membrane (RBM). Cochlear morphology was examined under scan electric microscope and phalloidin staining was used to observe the hair cells in the infused ears after the animals were sacrificed. Auditory brainstem thresholds of the ears of all the animals were measured before and after treatment.

RESULTSAll the animals recovered well after operation. The holes on the lateral wall of the scala media and punctures on the round window membrane were healed completely. EGFP labeling appeared in the organ of Corti and lining wall of the stria vascularis indicated that adenovirus suspension was injected into the scala media using LWS (succeed in 14 animals accounted for 70%) and RBM (in 8 animals accounted for 40%) ways. Viruses were inoculated into the scala media with only locally inflammation reaction. In group A the hearing threshold decreased significantly in the treated ears compared with the control ears after the operation [(33.1+/-10.3) dB, (9.4+/-3.9) dB, F=46.34, P=0.0005]. However, in group B there was no significantly different between the treated ears and the control ears after the operation [(2.5+/-3.8) dB, (2.5+/-3.8) dB, F=0.00, P=1.000]. Phalloidin staining indicated that in group A the extension of hair cells loss was bigger than in group B. In some animals of two groups, EGFP labeling appeared in the extra-lymphatic system indicating that some of the injected suspension leaked out of the scala media.

CONCLUSIONSAd5-EGFP could be infused into the scala media through LWS or RBM and adenovirus could infect the lining cells of scala media and supporting cells in the basal membrane successfully without causing immunoreaction in the whole cochlea. LWS caused more hair cell loss and hearing loss than RBM. However, the cochlear morphology could be recovered completely after surgery. The positive inoculation rate was relatively higher that through LWS than that through RBM.

Adenoviridae ; genetics ; Animals ; Cochlea ; drug effects ; pathology ; physiopathology ; Drug Administration Routes ; Endolymph ; Evoked Potentials, Auditory, Brain Stem ; Green Fluorescent Proteins ; genetics ; metabolism ; Guinea Pigs

OBJECTIVETo study the protective effect of recombinant adenovirus-mediated human cytosolic glutathione peroxidase (hCGPx) gene transfection on vascular endothelial cells ECV304 from oxidative damage.

METHODSpGEM-T Easy Vector containing hCGPx cDNA and recombinant adenovirus shuttle plasmid pACCMV-pLpA were used to construct the shuttle plasmid pACCMV-hCGPx for cotransfection of 293 cells with pJM17, thereby to obtain the recombinant adenovirus AdCMV-hCGPx. Cultured ECV304 cells were transfected with AdCMV-hCGPx for 24, 48 and 72 h, respectively, with the cells transfected with the empty vector serving as control, and hCGPx gene expression was then examined in the transfected cells. The transfected cell viability and apoptotic cell ratio were evaluated after treatment of the cells with H(2)O(2).

RESULTSThe expression ratio of hCGPx gene was significantly higher in the AdCMV-hCGPx-transfected cells than in those with empty vector transfection (P<0.01). The hCGPx gene-transfected cells showed significantly higher viability and significantly lower apoptotic ratio than the control cells following challenge with H(2)O(2)-induced oxidative damage.

CONCLUSIONhCGPx gene transfer mediated by recombinant adenovirus protects the vascular endothelial cells from oxidative damage in vitro, possibly due to the antioxidative and apoptosis-inhibiting effect of hCGPx.

Adenoviridae ; genetics ; Apoptosis ; drug effects ; Cell Line ; Cell Survival ; drug effects ; Cytosol ; enzymology ; Endothelial Cells ; cytology ; drug effects ; metabolism ; Flow Cytometry ; Genetic Vectors ; Glutathione Peroxidase ; biosynthesis ; genetics ; Humans ; Hydrogen Peroxide ; pharmacology ; Oxidative Stress ; Plasmids ; genetics ; Time Factors ; Transfection

OBJECTIVETo explore the proliferation of primary cultured rats hepatocytes after delivery of exogenous hepatocyte growth factor (HGF) gene which was inserted into the genome of replication-deficient recombinant adenovirus vector.

METHODSThe recombinant adenovirus-AdHGF which could express HGF was generated by homologous recombination. After the HGF gene was delivered into the hepatocytes, the expression of both HGF and c-met/HGF receptor mRNA in the cells was detected by RT-PCR and the level of HGF in the culture supernatant was also assayed by ELISA. On the other hand, cell proliferation was compared between before and after delivery of the HGF gene by MTS assay and the percentages of cell cycles were analyzed by flow cytometry. In addition, the expression of proliferating cell nuclear antigen (PCNA) was determined by immunocytofluorescent stain.

RESULTS4 x 10(10) efu/ml titer of AdHGF was obtained after recombination, RT-PCR indicated that the expression of HGF mRNA in hepatocytes increased on the third day after infected by the viruses and c-met/HGF receptor mRNA was also up-regulated. The HGF level in the culture supernatant assayed by ELISA was (5,939.0+/-414.39) pg/ml, which was much higher than that in the control (208.1pg/ml+/-37.20pg/ml, F=13.661, P<0.01). In addition, the proliferation of hepatocytes infected with AdHGF increased significantly according to MTS method (F>or=15.158, P<0.01) and more hepatocytes in G0/G1 stages changed into S stage (chi2=41.616, P<0.01), accordingly, PCNA index increased from 6.42+/- 1.88 to 14.56+/-2.85 (F=42.122, P<0.01).

CONCLUSION

THE RESULTSshow that HGF gene delivered into hepatocytes by AdHGF can be expressed with high efficiency in the cells, which can stimulate hepatocytes proliferation. It may be an effective tool for hepatocyte transplantation by gene modified donor hepatocytes.

Adenoviridae ; genetics ; metabolism ; Animals ; Cell Division ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Genetic Vectors ; Hepatocyte Growth Factor ; biosynthesis ; genetics ; pharmacology ; Hepatocytes ; cytology ; drug effects ; metabolism ; Rats ; Rats, Sprague-Dawley ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology

The present study is aimed to study the effect of sarcoplasmic reticulum Ca(2+)-ATPase 2a (SERCA2a) gene transfer on the contractile function of isolated cardiomyocytes of canines. The cardiomyocytes were isolated with collagenases. The isolated cardiac cells were divided into untransfected group, empty vector group and SERCA2a-transfected group. Recombinant adenovirus vector carrying enhanced green fluorescent protein gene was used for SERCA2a gene delivery. The expression of SERCA2a protein in cardiomyocytes was determined by Western blot. Contractile function of cardiomyocytes was measured with motion edge-detection system of single cell at 48 h after transfection. The results showed, compared with untransfected group, SERCA2a protein level, percentage of peak contraction amplitude under normal condition, percentages of peak contraction amplitude under Ca(2+) or isoproterenol stimulation, time-to-peak contraction (TTP) and time-to-50% relaxation (R50) in SERCA2a-transfected group all increased significantly. While all the above indices in empty vector group did not show any differences with those in untransfected group. These results suggest that the overexpression of SERCA2a by gene transfer may enhance the contraction function of canine myocardial cells.
Adenoviridae ; genetics ; metabolism ; Animals ; Dogs ; Male ; Myocardial Contraction ; drug effects ; physiology ; Myocytes, Cardiac ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; genetics ; metabolism ; Transfection

Cancer stem cells (CSCs) are resistant to chemo- and radio-therapy, and can survive to regenerate new tumors. This is an important reason why various anti-cancer therapies often fail to completely control tumors, although they kill and eliminate the bulk of cancer cells. In this study, we determined whether or not adenine nucleotide translocator-2 (ANT2) suppression could also be effective in inducing cell death of breast cancer stem-like cells. A sub-population (SP; CD44+/CD24-) of breast cancer cells has been reported to have stem/progenitor cell properties. We utilized the adeno-ANT2 shRNA virus to inhibit ANT2 expression and then observed the treatment effect in a SP of breast cancer cell line. In this study, MCF7, MDA-MB-231 cells, and breast epithelial cells (MCF10A) mesenchymally-transdifferentiated through E-cadherin knockdown were used. ANT2 expression was high in both stem-like cells and non-stem-like cells of MCF7 and MDA-MB-231 cells, and was induced and up-regulated by mesenchymal transdifferentiation in MCF10A cells (MCF10AEMT). Knockdown of ANT2 by adeno-shRNA virus efficiently induced apoptotic cell death in the stem-like cells of MCF7 and MDA-MB-231 cells, and MCF10AEMT. Stem-like cells of MCF7 and MDA-MB-231, and MCF10AEMT cells exhibited increased drug (doxorubicin) resistance, and expressed a multi-drug resistant related molecule, ABCG2, at a high level. Adeno-ANT2 shRNA virus markedly sensitized the stem-like cells of MCF7 and MDA-MB-231, and the MCF10AEMT cells to doxorubicin, which was accompanied by down-regulation of ABCG2. Our results suggest that ANT2 suppression by adeno-shRNA virus is an effective strategy to induce cell death and increase the chemosensitivity of stem-like cells in breast cancer.
ATP-Binding Cassette Transporters/*genetics/metabolism ; Adenine Nucleotide Translocator 2/antagonists & inhibitors/genetics ; Adenoviridae/*genetics ; Antineoplastic Agents/pharmacology ; Apoptosis/drug effects/genetics ; Breast Neoplasms ; Cadherins/antagonists & inhibitors/genetics ; Cell Line, Tumor ; Cell Survival/drug effects/genetics ; Cell Transdifferentiation/drug effects ; Doxorubicin/pharmacology ; Drug Resistance, Neoplasm/drug effects/*genetics ; Epithelial-Mesenchymal Transition/drug effects ; Female ; Gene Expression Regulation, Neoplastic/drug effects ; Gene Knockdown Techniques ; Humans ; Neoplasm Proteins/*genetics/metabolism ; Neoplastic Stem Cells/drug effects/*metabolism/pathology ; RNA, Small Interfering/*genetics ; Signal Transduction/drug effects