OBJECTIVE: To screen for aberrantly expressed genes in osteosarcoma cells and investigate the role of RHPN2 in regulating the proliferation, apoptosis, migration and tumorigenic abilities of osteosarcoma cells. METHODS: We used GEO2R to analyze the differential gene expression profile between osteosarcoma cells and normal cells in the GSE70414 dataset. RTqPCR and Western blotting were performed to detect RHPN2 expression in osteosarcoma cell lines MG-63, 143B and SAOS2. Two RHPN2-shRNA and a control NC-shRNA were designed to silence the expression of RHPN2 in 143B cells, and CCK8 assay, colony-forming assay, annexin V-FITC/PI staining and scratch assays were carried out to examine the changes in proliferation, apoptosis and migration of the cells. We also established nude mouse models bearing osteosarcoma xenografts derived 143B cells and RHPN2-shRNA-transfected 143B cells, and assessed the effect of RHPN2 silencing on osteosarcoma cell tumorigenesis using HE staining. Kaplan-Meier survival curves were used to analyze the correlation between RHPN2 expression and survival outcomes of patients with osteosarcoma. RESULTS: RHPN2 expression was significantly upregulated in osteosarcoma cell lines MG-63, 143B and SAOS2 (P < 0.01). Silencing of RHPN2 significantly inhibited the proliferation and migration of 143B cells in vitro, promoted cell apoptosis (P < 0.01), and suppressed tumorigenic capacity of the cells in nude mice. A high expression of RHPN2 was significantly correlated with a poor prognosis of patients with osteosarcoma (P < 0.05). CONCLUSION RHPN2 is highly expressed in osteosarcoma cells to promote cell proliferation and migration and inhibits cell apoptosis. A high expression of RHPN2 is associated with a poorer prognosis of the patients with osteosarcoma.
Adaptor Proteins, Signal Transducing/metabolism* ; Animals ; Apoptosis ; Bone Neoplasms/metabolism* ; Carcinogenesis ; Cell Line, Tumor ; Cell Movement/physiology* ; Cell Proliferation/physiology* ; Humans ; Immediate-Early Proteins ; Mice ; Mice, Nude ; Osteosarcoma/metabolism* ; RNA, Small Interfering/genetics*

OBJECTIVEThe aim of this study was to construct a new oncolytic virus oHSV2hGM-CSF and evaluate its oncolytic activity in vitro and in vivo in parallel with oHSV1hGM-CSF.

METHODSoHSV2hGM-CSF was a replication-competent, attenuated HSV2 based on the HG52 virus (an HSV2 strain). It was engineered to be specific for cancer by deletion of the viral genes ICP34.5 and ICP47 and insertion of the gene encoding hGM-CSF. To measure the in vitro killing effect of the virus, 15 human tumor cell lines (HeLa, Eca-109, PG, HepG2, SK/FU, CNE-2Z, PC-3, SK-OV3, A-549, 786-0, MCF-7, Hep-2, HT-29, SK-Mel-28, U87-MG) and mouse melanoma (B16R) cell line were seeded into 24-well plates and infected with viruses at MOI = 1 (multiplicity of infection, MOI), or left uninfected. The cells were harvested 24 and 48 hours post infection, and observed under the microscope. For animal studies, the oncolytic viruses were administered intratumorally (at 3-day interval) at a dose of 2.3 x 10(6) PFU (plaque forming unit, PFU) for three times when the tumor volume reached 7-8 mm3. The tumor volume was measured at 3-day intervals and animal survival was recorded.

RESULTSBoth oHSV2hCM-CSFand oHSV1hGM-CSF induced widespread cytopathic effects at 24 h after infection. OHSV2hGM-CSF, by contrast, produced more plaques with a syncytial phenotype than oHSV1hGM-CSF. In the in vitro killing experiments for the cell lines HeLa, HepG2, SK-Mel-28, B16R and U87-MG, oHSV2hGM-CSF eradicated significantly more cells than oHSV1hGM-CSF under the same conditions. For the mouse experiments, it was observed that oHSV2hGM-CSF significantly inhibited the tumor growth. At 15 days after B16R tumor cells inoculation, the tumor volumes of the PBS, oHSV1hGCM-CSF and oHSV2hGM-CSF groups were (374.7 +/- 128.24) mm3, (128.23 +/- 45.32) mm3 (P < 0.05, vs. PBS group) or (10.06 +/- 5.1) mm3 (P < 0.01, vs. PBS group), respectively (mean +/- error). The long term therapeutic effect of oHSV2hGM-CSF on the B16R animal model was evaluated by recording animal survival over 110 days after tumor cells inoculation whereas all the mice in the PBS group died by day 22 (P < 0.01). The anti-tumor mechanism of the newly constructed oHSV2hGM-CSF against B16R cell tumor appeared to include the directly oncolytic activity and the induction of anti-tumor immunity to some degree.

CONCLUSIONThe findings of our study demonstrate that the newly constructed oHSV2hGM-CSF has potent anti-tumor activity in vitro to many tumor cell lines and in vive to the transplanted B16R tumor models.

Animals ; Cell Line, Tumor ; Female ; Gene Deletion ; Genetic Engineering ; Granulocyte-Macrophage Colony-Stimulating Factor ; genetics ; Herpesvirus 2, Human ; genetics ; immunology ; Humans ; Immediate-Early Proteins ; genetics ; metabolism ; Melanoma, Experimental ; pathology ; therapy ; virology ; Mice ; Mice, Inbred C57BL ; Oncolytic Virotherapy ; methods ; Oncolytic Viruses ; genetics ; physiology ; Random Allocation ; Tumor Burden ; Viral Proteins ; genetics ; metabolism ; Xenograft Model Antitumor Assays

Herpes simplex virus type 1 (HSV-1) is a common human pathogen causing cold sores and even more serious diseases. It can establish a latent stage in sensory ganglia after primary epithelial infections, and reactivate in response to stress or sunlight. Previous studies have demonstrated that viral immediate-early protein ICP0 plays a key role in regulating the balance between lytic and latent infection. Recently, It has been determined that promyelocytic leukemia (PML) nuclear bodies (NBs), small nuclear sub-structures, contribute to the repression of HSV-1 infection in the absence of functional ICP0. In this review, we discuss the fundamentals of the interaction between ICP0 and PML NBs, suggesting a potential link between PML NBs and ICP0 in regulating lytic and latent infection of HSV-1.
Herpes Simplex ; virology ; Herpesvirus 1, Human ; genetics ; physiology ; Humans ; Immediate-Early Proteins ; metabolism ; Intranuclear Inclusion Bodies ; metabolism ; virology ; Leukemia, Promyelocytic, Acute ; metabolism ; Ubiquitin-Protein Ligases ; metabolism ; Virus Latency ; physiology

HSV-1, a neurotropic virus, always leads to severe nervous symptoms. It is hard to completely eradicate the latent viruses after conventional therapy so that recurrence is inevitable. ICP is a key regulator for HSV replication and transcription that determines the cytolytic infection or latent state. In search of new anti-virus strategy targeting HSV-1ICP4, two pairs of siRNA were designed, and a recombinant eukaryotic lentiviral expression plasmid pLKO-puro(r)-hU6-siRNA was constructed. Vero cells were transfected with the designed siRNAs by Lipofectamine 2000 and four stable monoclonal cell lines were established by puromycin screening method. The ICP4 expression at mRNA level was detected with real-time PCR, and the HSV-1 replication was measured with TCID50 assay. SiRNA was shown as an effective way to inhibit the expression of ICP4 in monoclonal cell lines. Meanwhile, HSV-1 replication was significantly inhibited when ICP4 was shut down by siRNA. We conclude that siRNA targeting ICP4 attenuates HSV-1 replication. Further more, multi-site siRNAs show stronger inhibitory effect on viral replication, which may be an effective and feasible approach for biological anti-viral drugs.
Animals ; Base Sequence ; Cercopithecus aethiops ; Genetic Therapy ; Genetic Vectors ; genetics ; Herpesvirus 1, Human ; physiology ; Humans ; Immediate-Early Proteins ; deficiency ; genetics ; RNA Interference ; RNA, Messenger ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; Vero Cells ; Virus Replication ; genetics

The role of serum and glucocorticoid-induced kinase 1 (SGK1) pathway in the connective tissue growth factor (CTGF) expression was investigated in cultured human mesangial cells (HMCs) under high glucose. By using RT-PCR and Western blot, the effect of SGK1 on the CTGF expression in HMCs under high glucose was examined. Overexpression of active SGK1 in HMCs transfected with pIRES2-EGFP-S422D hSGK1 (SD) could increase the expression of phosphorylated SGK1 and CTGF as compared with HMCs groups transfected with pIRES2-EGFP (FP) under high glucose or normal glucose. Overexpression of inactive SGK1 in HMCs transfected with pIRES2-EGFP-K127N hSGK1 (KN) could decrease phosphorylated SGK1 and CTGF expression as compared with HMCs groups transfected with FP under high glucose. In conclusion, these results suggest that high glucose-induced CTGF expression is mediated through the active SGK1 in HMCs.
Cells, Cultured ; Connective Tissue Growth Factor/genetics ; Connective Tissue Growth Factor/*metabolism ; Glucose/*pharmacology ; Immediate-Early Proteins/metabolism ; Immediate-Early Proteins/*physiology ; Mesangial Cells/cytology ; Mesangial Cells/*metabolism ; Protein-Serine-Threonine Kinases/metabolism ; Protein-Serine-Threonine Kinases/*physiology ; Signal Transduction/drug effects

OBJECTIVE: To characterize the antisense c-fos oligonucleotides that control the expression of immediate-early gene c-fos in retina in order to better understand the mechanism by which antisense c-fos oligonucleotides induced myopia. In this study the signal transduction in the pathway linking visual experience and the regulation of the eye's growth was investigated. METHODS: Thirty-one 3-week guinea pigs were assigned into 3 groups: antisense and sense c-fos oligonucleotides were intravitreally injected every 3 days to the eyes of the experimental guinea pigs at different concentrations; and saline vehicle to control guinea pigs in the same way. The refraction and axial length of the eyes were measured before and after the treatment, and the immediate-early gene c-fos expression in the retina was quantified by immunohistochemistry and RT-PCR. RESULTS: The moderate myopia was induced in high (1 nmol) and low (0.1 nmol) level of antisense c-fos oligonucleotide intravitreous injection (-5.425 D and -5.575 D, respectively) compared with the control ateral eyes. The refraction and axial length of the treated eyes increased, and the expression of immediate-early gene c-fos decreased significantly in the antisense c-fos oligonucleotides intravitreously injected eyes compared with the sense c-fos oligonucleotide intravitreously and saline vehicle injected eyes (P<0.01). The refraction and axial length were of no statistically significant differences among the sense c-fos oligonucleotides-treated eyes and saline-treated eyes and non-treated eyes (P>0.05). CONCLUSION The obvious myopia can be induced by antisense c-fos oligonucleotides in guinea pigs; antisense c-fos oligonucleotides inhibit c-fos expression in the retina. Immediate-early gene c-fos may be a potential factor in the prevention of myopia and plays an important role in the signal transduction of the retina.
Animals ; Genes, Immediate-Early ; genetics ; Guinea Pigs ; Immunohistochemistry ; Microinjections ; Myopia ; chemically induced ; genetics ; physiopathology ; Oligonucleotides, Antisense ; administration & dosage ; genetics ; toxicity ; Proto-Oncogene Proteins c-fos ; biosynthesis ; genetics ; RNA, Messenger ; genetics ; metabolism ; Random Allocation ; Retina ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; physiology

OBJECTIVETo investigate the mechanisms for the cytopathic effect (CPE) of human cytomegalovirus (HCMV) in ECV304 endothelial-like cells.

METHODSPCR and indirect immunofluorescence were used to detect HCMV infection by examining immediate-early (IE) gene and protein expression of the virus in ECV304 cells. Phase-contrast and electron microscopies were performed to observe the morphological changes of the infected and uninfected cells, and DNA ladder analysis and flow cytometry were carried out to study HCMV-induced cell apoptosis.

RESULTSIn HCMV-infected ECV304 cells, cytopathic effects were first observed at approximately 72 h post-infection. The cells with CPE changes exhibited detachment from the monolayer, cell rounding and shrinkage. The expression of the IE gene was detected. Chromatin condensation and nuclear fragmentation along with dramatic changes of the mitochondria were observed by electron microscopy at 96 h post-infection. Cellular DNA fragmentation was observed in the infected cells, which had cells apoptotic rates of 4.1% and 45.7% at 96 h and 144 h post-infection, respectively.

CONCLUSIONHCMV can induce apoptosis of ECV304 endothelial-like cells.

Antigens, Viral ; genetics ; metabolism ; Apoptosis ; physiology ; Cell Line ; Cytomegalovirus ; genetics ; growth & development ; metabolism ; DNA, Viral ; analysis ; Endothelial Cells ; cytology ; ultrastructure ; virology ; Fluorescent Antibody Technique, Indirect ; Humans ; Immediate-Early Proteins ; genetics ; metabolism ; Microscopy, Electron ; Microscopy, Phase-Contrast ; Polymerase Chain Reaction ; Umbilical Veins ; cytology ; virology

Angiotensin II (Ang II), which is an important mediator of both vascular responsiveness and growth, has been shown to induce vascular smooth muscle cell (VSMC) hypertrophy via the activation of a complex series of intracellular signaling events. Heat shock protein 70 (Hsp70) has recently been shown to protect against Ang II-induced hypertension. In this study, we tested the hypothesis that Hsp70 can protect VSMC from Ang II-induced hypertrophy. We treated VSMCs with Ang II to induce hypertrophy and to activate MAPK signaling pathway. We observed that the augmentation of Hsp70 expression inhibited Ang II-stimulated VSMC hypertrophy. This inhibitory effect of Hsp70 appears to be partly due to extracellular signal-regulated kinase (ERK1/2) inactivation, which in turn, may possibly result from the accumulation of MAP kinase phosphatase-1 (MKP-1).
Rats, Sprague-Dawley ; Rats ; RNA, Small Interfering/pharmacology ; Protein-Tyrosine-Phosphatase/metabolism/physiology ; Phosphoprotein Phosphatase/metabolism/physiology ; Muscle, Smooth, Vascular/*cytology/*drug effects ; Mitogen-Activated Protein Kinase 3/antagonists & inhibitors ; Mitogen-Activated Protein Kinase 1/antagonists & inhibitors ; Male ; MAP Kinase Kinase 2/metabolism ; MAP Kinase Kinase 1/metabolism ; Immediate-Early Proteins/metabolism/physiology ; Hypertrophy ; HSP70 Heat-Shock Proteins/antagonists & inhibitors/*pharmacology ; Flavonoids/pharmacology ; Enzyme Stability/drug effects ; Cells, Cultured ; Cell Cycle Proteins/metabolism/physiology ; Aorta/drug effects/pathology ; Animals ; Angiotensin II/*pharmacology

In order to investigate the mechanism of mechanical stress-mediated arterial remodeling, we studied the pressure-induced expression of immediate-early response gene product c-Jun in common carotid arteries in rats. The common carotid arteries were perfused with both high pressure (160 mmHg) and normal pressure (80 mmHg) for 0.5, 1, 3 and 6 hours. Expression of immediate-early response gene product c-Jun in the arteries was examined by immunohistochemistry and computer image processing. c-Jun was weakly expressed at 1 h, then increased at 3 h and 6 h after exposure of the arteries to normal pressure. Positive immunohistochemical product of c-Jun appeared in the arteries at 0.5 h after the onset of high pressure, then it increased markedly till 6 h. There was significant difference between the two groups. These results indicate that expression of c-Jun of the arteries can be induced by pressure, which may play an important role in mechanical stress-mediated arterial remodeling.
Animals ; Biomechanical Phenomena ; Carotid Artery, Common ; cytology ; metabolism ; physiology ; Genes, Immediate-Early ; Male ; Muscle, Smooth, Vascular ; cytology ; metabolism ; Pressure ; Proto-Oncogene Proteins c-jun ; biosynthesis ; genetics ; Rats ; Rats, Sprague-Dawley ; Stress, Mechanical

OBJECTIVETo investigate the effect of allitridin injection on the expression of human cytomegalovirus (HCMV) immediate-early antigens (IEAs including IE72 and IE86) in human embryonic lung cells.

METHODHCMV AD 169 Virus strain infected cell model (MOI = 2.5 and 0.25, respectively) were established, and then treated with ICm5 and MTC doses of allitridin. Western blot was used to analyze the of IE72 and IE86 expression after the treatment, ganciclovir(GCV, IC50 and 2.3 x IC50) treatment as control.

RESULTNo matter what kind of MOI was used, both IE86 and IE72 antigens' expression was effectively suppressed by allitridin treatment, and the inhibitory rate of IE86 was almost twice of IE72's. Compared with GCV, allitridin had stronger inhibitory effect on IE86 expressing, although its efficacy on IE72 was weaker than GCV.

CONCLUSIONAllitridin could suppress the expression of IE72 and IE86, especially for IE86 expressing, maybe it is ore of key role in the mechanism of allitridin against HCMV.

Allyl Compounds ; administration & dosage ; isolation & purification ; pharmacology ; Antiviral Agents ; pharmacology ; Cells, Cultured ; Cytomegalovirus ; genetics ; physiology ; Fibroblasts ; cytology ; metabolism ; Garlic ; chemistry ; Gene Expression Regulation, Viral ; drug effects ; Humans ; Immediate-Early Proteins ; metabolism ; Injections ; Lung ; cytology ; Sulfides ; administration & dosage ; isolation & purification ; pharmacology ; Trans-Activators ; metabolism ; Viral Proteins ; metabolism