OBJECTIVEThis paper aims to investigate the apoptotic effect of inactivated Sendai virus (hemagglutinating virus of Japan-enveloped, HVJ-E) on murine melanoma cells (B16F10) and the possible mechanisms involved in the putative apoptotic reactions.

METHODSB16F10 cells were treated with HVJ-E at various multiplicities of infection (MOI), and the reactive oxygen species (ROS), cell viability, and apoptosis were measured. Next, the roles of ROS in the regulation of Bcl-2/Bax and the activation of mitogen-activated protein kinase (MAPK) pathways in HVJ-E-treated B16F10 cells were analyzed. To further evaluate the cytotoxic effect of HVJ-E-generated ROS on B16F10 cells, HVJ-E was intratumorally injected, both with and without N-acetyl-L-cysteine (NAC), into melanoma tumors on BALB/c mice. Tumor volume was then monitored for 3 weeks, and the tumor proteins were separated for immunoblot assay.

RESULTSTreatment of B16F10 cells with HVJ-E resulted in a dose-dependent inhibition of cell-viability and an induction of apoptosis. The latter effect was associated with the generation of ROS. Inhibition of ROS generation by NAC resulted in a significant reduction of HVJ-E-induced Erk1/2, JNK, and p38 MAPK activation. Additionally, ROS inhibition caused a decrease in the Bcl-2/Bax ratio as well as promoting activation of apoptosis both in vitro and in vivo.

CONCLUSIONThese results suggest that HVJ-E possesses potential anticancer activity in B16F10 cells through ROS-mediated mitochondrial dysfunction involving the MAPK pathway.

Animals ; Apoptosis ; Cell Line, Tumor ; Mice ; Mitogen-Activated Protein Kinase 1 ; genetics ; metabolism ; Reactive Oxygen Species ; metabolism ; Respirovirus Infections ; virology ; Sendai virus ; physiology ; Virus Inactivation

Recent studies have suggested a probable association between Epstein-Barr virus (EBV) and nasal/nasopharyngeal T cell lymphomas but the role of oncogenes or tumor suppressor genes is poorly understood. We have studied the frequency of p53 expression and its relation to the EBV infection in 33 Korean patients with head and neck (H&N) lymphomas. All cases (23 B cell & 10 T cell) were immunostained for p53 protein using the mAb D07 (Novocastra) and the avidin biotin peroxidase method. EBER in situ hybridization was performed using a fluorescein conjugated EBV oligonucleotide probe (Dako). Among 33 lymphomas, 16 cases stained positively for p53 protein. P53 expression was frequent both in higher grade lymphomas and in advanced stage. Nine cases were EBER positive, EBER was more commonly found in T cell lymphomas than in B cell lymphomas (70% vs 8.7%). EBER positive lymphomas showed a higher frequency of p53 positivity than EBER negative lymphomas (78% vs 38%), although the difference was not statistically significant (p = 0.095). These findings indicate altered expression of p53 protein occurs in H&N lymphomas, especially in late event lymphoma progression and appears to play a role in the development of EBER positive T cell lymphomas.
Follow-Up Studies ; Gene Expression ; Genes, p53 ; Head and Neck Neoplasms/genetics/*metabolism/*virology ; Herpesviridae Infections/genetics/*metabolism ; *Herpesvirus 4, Human ; Human ; Lymphoma, T-Cell/genetics/*metabolism/*virology ; Prognosis ; Protein p53/*biosynthesis ; Support, Non-U.S. Gov't ; Tumor Virus Infections/genetics/*metabolism

OBJECTIVETo investigate whether simian virus 40 (SV40) was related to patients of malignant mesothelioma in China.

METHODSParaffin-embeded samples of 17 patients with malignant mesothelioma were collected. After isolation of DNA from paraffin blocks, polymerase chain reaction (PCR) analyses were performed using three different sets of primer for detection of SV40 large T antigen gene. These samples were also immunohistochemically evaluated for expression of SV40 TAg protein with two different anti-SV40 Tag (Pab101 and Ab-2).

RESULTSOnly one of the three primer pairs successfully amplified SV40 genome in three malignant mesothelioma samples. No immunopositive staining for SV40 TAg was found in any of the samples.

CONCLUSIONSThe study shows that malignant mesothelioma in China may be independent of SV40 infection.

Adult ; Aged ; Antigens, Viral, Tumor ; genetics ; metabolism ; China ; Female ; Host-Pathogen Interactions ; Humans ; Immunohistochemistry ; Male ; Mesothelioma ; pathology ; virology ; Middle Aged ; Polymerase Chain Reaction ; Polyomavirus Infections ; pathology ; virology ; Simian virus 40 ; genetics ; immunology ; physiology ; Tumor Virus Infections ; pathology ; virology ; Young Adult

OBJECTIVETo generate a human papillomavirus (HPV16) prophylactic and therapeutic vaccine candidate for cervical cancer.

METHODSHPV16 major capsid protein L1 gene/minor capsid protein L2 gene and HPV16 early E6/E7 genes were inserted into a vaccinia virus expression vector. A strain of non-recombinant vaccinia virus containing the sequences was obtained through a homologous recombination and identified.

RESULTSDNA hybridization confirmed that the HPV16L1/L2/E6/E7 genes were integrated into vaccinia virus DNA. Western Blot result showed that full-length L1/L2/E6/E7 proteins were co-expressed in CEF cells infected with the recombinant virus.

CONCLUSIONNTVJE6E7CKL1L2 could be taken as a candidate of prophylactic and therapeutic vaccine for HPV-associated tumors and their precancerous transformations.

Animals ; Blotting, Western ; Capsid Proteins ; genetics ; metabolism ; Cells, Cultured ; Chick Embryo ; Cloning, Molecular ; Female ; Gene Expression ; Genetic Vectors ; genetics ; Humans ; Oncogene Proteins, Viral ; genetics ; metabolism ; Papillomaviridae ; genetics ; immunology ; Papillomavirus E7 Proteins ; Papillomavirus Infections ; immunology ; prevention & control ; virology ; Papillomavirus Vaccines ; genetics ; immunology ; therapeutic use ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; Repressor Proteins ; genetics ; metabolism ; Transfection ; Tumor Virus Infections ; immunology ; prevention & control ; virology ; Uterine Cervical Neoplasms ; immunology ; prevention & control ; virology ; Vaccinia virus ; genetics ; Virus Replication

BACKGROUNDTo study the difference in gene expression between the EBV associated gastric carcinoma (EBVaGC) tissues. To explore the mechanism of gastric carcinoma pathogenesis initiated by EBV.

METHODSIn situ hybridization was used to study the frequencies of EBV small RNA expression in 155 cases of gastric carcinoma tissues. The expression levels of P53 protein and P21WAF1 protein were detected by immunohistochemistry in all gastric carcinoma tissues.

RESULTSThe expression of EBV small RNA was positive in 10 out of 155 cases (6.45%). The expression of P53 protein was weakly positive in 4 of the 10 cases. The expression level of P53 protein in EBVaGC was much lower than that in EBVnGC and was weakly positive in 30 of 145 cases with EBVnGC). P21WAF1 expression was detected in 7 of 10 cases with EBVaGC, but in 55 out of 145 cases with EBVaGC, P21WAF1 expression in EBVaGC was much higher than that in EBVnGC.

CONCLUSIONThere seems existing a special mechanism of pathogenesis in EBVaGC. In which P53 gene mutation may not play an important role.

Epstein-Barr Virus Infections ; metabolism ; pathology ; virology ; Herpesvirus 4, Human ; genetics ; physiology ; Host-Pathogen Interactions ; Humans ; Immunohistochemistry ; In Situ Hybridization ; Proto-Oncogene Proteins c-met ; metabolism ; RNA, Viral ; genetics ; Stomach Neoplasms ; metabolism ; pathology ; virology ; Tumor Suppressor Protein p53 ; metabolism

Enterovirus 71 (EV71) is the main causative agent of hand, foot, and mouth disease (HFMD). This article presented the inhibitory activity of pentapeptides on the EV71 infection in rhabdomyosarcoma (RD) and suckling mice. The EV71 VP1 capsid protein expression levels and mRNA levels were analyzed by Western blotting and real-time PCR. The antiviral activity of pentapeptides in vivo was evaluated by weight changes and EV71 VP1 protein expression levels in intestines of suckling mice. Results revealed that the pentapeptide P010157 was able to inhibit EV71 replication in RD cells. After being incubated with the P010157 at a concentration of 100 microg x mL(-1) for 48 h, the level of EV71 vp1 mRNA in RD cells decreased by (92.0 +/- 6.3)%. The estimated EC50 was 2.2 microg x mL(-1). P010157 was able to inhibit EV 71-induced cytopathic effect (CPE) in RD cells. The cytotoxic activity of the compound was evaluated against RD cells by MTS assay. The results showed that P010157 had no obvious toxicity. In addition, the treated mice with P010157 did not exhibit weight loss, as was observed in untreated mice. EV71 replication reduced significantly as revealed by Western blotting. These findings suggest that P010157 could prevent EV71 proliferation in vitro and in vivo. P010157 is a novel compound for antiviral therapies against EV71, which merited further investigation.
Animals ; Antiviral Agents ; pharmacology ; Capsid Proteins ; genetics ; metabolism ; Enterovirus A, Human ; physiology ; Enterovirus Infections ; metabolism ; Humans ; Mice ; Oligopeptides ; pharmacology ; RNA, Messenger ; metabolism ; Rhabdomyosarcoma ; metabolism ; pathology ; virology ; Tumor Cells, Cultured ; Virus Replication ; drug effects

BACKGROUNDExtranodal natural killer/T-cell (NK/T cell) lymphoma, nasal-type, is a rare lymphoma. Skin is the second most common site of involvement after the nasal cavity/nasalpharynx. The aim of this study was to investigate the clinicopathologic features, immunophenotype, T cell receptor (TCR) gene rearrangement, the association with Epstein-Barr virus (EBV) infection and p53 gene mutations of the lymphoma.

METHODSThe clinicopathologic analysis, immunohistochemistry, in situ hybridization for EBER1/2, TCR gene rearrangement by polymerase chain reaction (PCR), mutations of p53 gene analyzed by PCR and sequence analysis were employed in this study.

RESULTSIn the 19 cases, the tumor primarily involved the dermis and subcutaneous layer. Immunohistochemical staining showed that most of the cases expressed CD45RO, CD56, CD3ε, TIA-1 and GrB. Three cases were positive for CD3 and two cases were positive for CD30. Monoclonal TCRγ gene rearrangement was found in 7 of 18 cases. The positive rate of EBER1/2 was 100%. No p53 gene mutation was detected on the exon 4 - 9 in the 18 cases. Fifteen cases showed Pro (proline)/Arg (arginine) single nucleotide polymorphisms (SNPs) on the exon 4 at codon 72. The expression of p53 protein was 72% (13/18) immunohistochemically.

CONCLUSIONSCutaneous NK/T-cell lymphoma is a rare but highly aggressive lymphoma with poor prognosis. No p53 gene mutation was detected on the exon 4 - 9, and Pro/Arg SNPs on p53 codon 72 were detected in the cutaneous NK/T-cell lymphoma. The overexpression of p53 protein may not be the result of p53 gene mutation.

Adolescent ; Adult ; Aged ; Child ; Epstein-Barr Virus Infections ; diagnosis ; genetics ; metabolism ; Female ; Humans ; Immunohistochemistry ; Immunophenotyping ; In Situ Hybridization ; Lymphoma, T-Cell ; diagnosis ; genetics ; metabolism ; Male ; Middle Aged ; Mutation ; Receptors, Antigen, T-Cell ; genetics ; metabolism ; Skin Neoplasms ; diagnosis ; genetics ; metabolism ; Tumor Suppressor Protein p53 ; genetics ; Young Adult

OBJECTIVETo investigate the expression level of COX-2, p16(INK4A) and p53 in patients with classic Hodgkin's lymphoma (cHL), and to evaluate their correlation with prognosis.

METHODSThe clinical data and samples of 52 cHL cases were collected. Immunohistochemical staining was performed to analyze the proteins level mentioned above and in situ hybridization of EBV encoded RNA (EBER) to clarify the tumor EBV infection state. Correlation between the protein expression and prognosis of patients was analyzed.

RESULTSOf 52 cases, the male and female ratio was 1.6∶1, the age was from 22 to 68 years old. All lesions located primarily in lymph nodes. All samples from 52 cases were stained with COX-2, p16(INK4A) and p53, and the positive expression of COX-2 was found in 28 cases (53.8%）, that of p16(INK4A) in 25 cases (48.1%)and p53 in 42 cases (80.8%）. All patients were divided into two groups according to differences in age (<40 years/ ≥ 40 years), gender (male/female), EBV infection (yes/no), B symptoms (yes/no), and the Ann Arbor staging (Ⅰ-Ⅱ/Ⅲ-Ⅳ), the correlation with COX-2, p16(INK4A) and p53 expression were analyzed, and only p53 expression was correlated with Ann Arbor staging (P=0.027). The statistical analysis of correlations between COX- 2, p16(INK4A) and p53 showed that the expression of COX-2 was strongly correlated with p53 (P=0.008), and p16 (INK4A) was not related to either COX-2 or p53 (P=0.246 and 0.958). Kaplan- Meier univariate OS analysis using SPSS17.0 software showed that only COX-2 expression was an adverse prognostic factor for patients'event free survival (EFS) (P=0.003). Meanwhile COX-2 expression was a unique independent prognostic factor analyzed by COX proportional hazards regression model (HR=0.091, 95% CI 0.017-0.505, P=0.006).

CONCLUSIONThe expression rate of COX-2, p16 (INK4A) and p53 in the cHL were relatively high; and they were not statistically correlated with tumor EBV infection status; the COX-2 positive group had poor prognosis, but only event free survival time becomes statistically significant shorter. COX proportional hazard regression model was used to analyze the COX-2 expression as a independent adverse prognostic factors for EFS.

Adult ; Aged ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; metabolism ; Cyclooxygenase 2 ; genetics ; metabolism ; Disease-Free Survival ; Epstein-Barr Virus Infections ; Female ; Hodgkin Disease ; diagnosis ; genetics ; metabolism ; Humans ; Immunohistochemistry ; Kaplan-Meier Estimate ; Male ; Middle Aged ; Prognosis ; Proportional Hazards Models ; Tumor Suppressor Protein p53 ; genetics ; metabolism ; Young Adult

This study aims to construct a eukaryotic expression system for envelope gene of Jaagsiekte sheep retrovirus, observes its localization in 293T cells, and investigates the potential in inducing malignant transformation of NIH3T3 cells. By RT-PCR, the full-length cDNA of envelope gene of Jaagsiekte sheep retrovirus (exJSRV-env) was amplified from the extract of naturally infected sheep lung. The clone of target gene was sub-cloned into eukaryotic expression system pEGFP-C1, and validated by PCR, restriction endonuclease, and sequencing. Bioinformatic analysis concerning biological function and cellular localiza tion of exJSRV-env was also performed. The recombinant clone of exJSRV-env was transfected into 293T cells and NIH3T3 cells by Lipofectamine LTX. The expression and celluar localization in 293T cells were validated by confocal microscopy. Soft agar colony formation assay was employed to test the anchorage-independent growth of NIH3T3. DNA sequencing and restriction enzyme digestion with Kpn I and Hind III indicated the correct construction of the recombinant plasmid, which was named pEGFP-C1/exJSRV-env. Amino acid sequence alignment of exJSRV-env with reference sequences found 85%-100% homogeneity. A YRNM motif was discovered at the cytoplasmic tail of envelope gene, which is exclusively found in exogenous viruses. Phylogenetic tree analysis showed that our clone of exJSRV-env clustered closely with pathogenic exogenous Jaagsiekte sheep retroviruses. Fluorescence microscopy indicated typical membrane localization of exJSRV-env protein. NIH3T3 cells transfected with exJSRV-env lost contact inhibition, and acquired colony forming ability in soft agar. This study indicated that envelope protein of Jaagsiekte sheep retrovirus can induce malignant transformation of mouse fibroblast cell NIH3T3. Discoveries of this study provide a basis for further structural and functional research on Jaagsiekte sheep retrovirus envelope protein.
Amino Acid Sequence ; Animals ; Betaretrovirus ; chemistry ; classification ; genetics ; physiology ; Cell Transformation, Viral ; Green Fluorescent Proteins ; genetics ; metabolism ; Mice ; Molecular Sequence Data ; NIH 3T3 Cells ; Phylogeny ; Retroviridae Infections ; veterinary ; virology ; Sequence Alignment ; Sheep ; Sheep Diseases ; virology ; Transformation, Genetic ; Tumor Virus Infections ; veterinary ; virology ; Viral Envelope Proteins ; chemistry ; genetics ; metabolism

The infiltration of monocytes into the CNS represents one of the early steps to inflammatory events in AIDS-related encephalitis and dementia. Increased activity of selected matrix metalloproteinases (MMPs) such as MMP-9 impairs the integrity of blood-brain barrier leading to enhanced monocyte infiltration into the CNS. In this study, we examined the effect of HIV-1 Tat on the expression of MMP-9 in CRT-MG human astroglioma cells. Treatment of CRT-MG cells with HIV-1 Tat protein significantly increased protein levels of MMP-9, as measured by Western blot analysis, zymography and an ELISA. Treatment of CRT-MG cells with HIV-1 Tat protein markedly increased mRNA levels of MMP-9, as analyzed by RT-PCR. Pretreatment of CRT-MG cells with NF-kappaB inhibitors led to decrease in Tat-induced protein and mRNA expression of MMP-9. Pretreatment of CRT-MG cells with MAPK inhibitors suppressed Tat-induced MMP-9 expression. Furthermore, HIV-1 Tat-induced expression of MMP-9 was significantly inhibited by neutralization of TNF-alpha, but not IL-1beta and IL-6. Taken together, our results indicate that HIV-1 Tat can up-regulate expression of MMP-9 via MAPK-NF-kappaB-dependent mechanisms as well as Tat-induced TNF-alpha production in astrocytes.
AIDS Dementia Complex/*metabolism ; Astrocytes/*drug effects/enzymology ; HIV Infections/*complications ; *HIV-1 ; Humans ; Matrix Metalloproteinase 9/*genetics/immunology ; Mitogen-Activated Protein Kinase Kinases/*metabolism ; NF-kappa B/*metabolism ; Tumor Cells, Cultured ; Tumor Necrosis Factor-alpha/immunology/metabolism ; Up-Regulation/drug effects ; tat Gene Products, Human Immunodeficiency Virus/*metabolism