Genomic characterization and distribution of bovine foamy virus in Japan.

Bovine foamy virus (BFV) is distributed through worldwide cattle herds. Although the biological features of BFV are not well understood, appearance of clinical manifestation by superinfection with other microorganisms is inferred. In Japan, reports of genomic characterizations and epidemiology of this virus are limited. In this study, we performed whole genomic sequencing of BFV strains Ibaraki and No.43, which were isolated in this country. Additionally, we investigated BFV in geographically distant four daily farms in Japan, to estimate the distribution of BFV and its correlation to bovine leukemia virus (BLV). BFV was distributed throughout Japan; the average positive rate was 12.7%. The nucleotide sequence identities of the isolates were 99.6% when compared with BFV strain isolated in the USA. The phylogenetic tree using env gene sequence showed strains Ibaraki, No.43 and Kagoshima were sorted in the same cluster including the USA and Chinese strains, while Hokkaido strain was in the other cluster including European strains. Although no clear correlation between BFV and BLV could be found, BFV and BLV infections were likely to increase with ages. Our data on epidemiology and characteristics of BFV will provide important information to reveal biological features of BFV.

Survey of bovine foamy virus infection among cattle in Japan and comparison with bovine leukemia virus infection.

The prevalence of bovine foamy virus (BFV) infections in cattle on farms in the Kanto region of Japan was determined using agar gel immunodiffusion (AGID) test and polymerase chain reaction (PCR). Six out of 20 farms contained BFV-positive cattle. Furthermore, 16.7% (91/545) of all cattle tested positive for BFV. This suggested that BFV-infected cattle are widely prevalent in Japan. Positive results for BFV infection were consistent between AGID and PCR tests. Additionally, we tested for bovine leukemia virus (BLV) infections at nine farms, primarily those containing BFV-infected cows. At each farm, the infection rate of BFV was lower than that of BLV. Further, cattle that were PCR-positive but antibody-negative, indicating immune tolerance to BFV, were not detected.

Transcriptional activation of long terminal repeat of bovine leukemia virus by bovine heat shock factor 1.

Bovine leukemia virus (BLV) causes enzootic bovine leukosis (EBL). The BLV genome encodes Tax protein, a transcriptional activator of viral gene expression that binds to the BLV long terminal repeat (LTR). Heat shock factor 1 (HSF1) is a known regulator of the heat shock response proteins, including heat shock proteins. In the present study, the BLV LTR was investigated for interaction of heat shock element (HSE) with HSF1 and the viral Tax protein. It could be confirmed that a functional HSE is well conserved in different BLV strains. The LTR transcriptional activity, as measured by luciferase reporter assay, was upregulated by bovine HSF1 - without Tax expression - in feline CC81 cells. The HSF1 activated LTR transcription by binding to the HSE. LTR-activation was lost upon HSE removal from the LTR and upon expression of a mutant HSF1 lacking the DNA-binding domain. We conclude that BLV LTR is activated to a basal level by host transcriptional factor HSF1, but without Tax protein involvement.

Enzootic bovine leukosis in a two-month-old calf.

A two-month-old calf was diagnosed with leukosis on the basis of the clinical sign of enlarged, superficial lymph nodes. Serological and genetic tests for bovine leukemia virus (BLV) were performed because the calf was born from a cow infected with BLV. The serum had a weakly positive BLV antibody, and the BLV provirus was detected within neoplastic cells on performing polymerase chain reaction (PCR). Analysis of the BLV provirus integration site using inverse PCR revealed that the BLV integration site location was identical on all chromosomes in all tumor tissues examined. Thus, the tumor cells monoclonally proliferated following BLV infection. The present study shows that enzootic bovine leukosis can occur in a young animal, as in the two-month-old calf in our study.

Overexpression of feline tripartite motif-containing 25 interferes with the late stage of feline leukemia virus replication.

Tripartite motif-containing 25 (TRIM25) regulates various cellular processes through E3 ubiquitin ligase activity. Previous studies have revealed that the expression of TRIM25 is induced by type I interferon and that TRIM25 is involved in the host cellular innate immune response against retroviral infection. Although retroviral infection is prevalent in domestic cats, the roles of feline TRIM25 in the immune response against these viral infections are poorly understood. Because feline TRIM25 is expected to modulate the infection of feline leukemia virus (FeLV), we investigated its effects on early- and late-stage FeLV replication. This study revealed that ectopic expression of feline TRIM25 in HEK293T cells reduced viral protein levels leading to the inhibition of FeLV release. Our findings show that feline TRIM25 has a potent antiviral activity and implicate an antiviral mechanism whereby feline TRIM25 interferes with late-stage FeLV replication.

Efficient propagation of equine viruses in a newly established equine cell line, FHK-13.1 cells.

Equine cells are required for isolation of viruses that infect the horse. However, only a few equine cell lines and cell cultures are available so far. Fetal horse kidney (FHK)-Tcl3.1 cell is a novel cell line established by introducing simian virus 40 (SV40) large T antigen. In the present study, the ability to propagate equine viruses was compared between FHK-Tcl3.1 cells and other equine cells. FHK-Tcl3.1 cells efficiently increased many viruses derived from or having pathogenicity to horses and produced high infective titers in culture fluids. These results indicate that FHK-Tcl3.1 cells would be useful for propagation and serological tests of viruses that affect Equidae.

Activation of epidermal growth factor receptor signaling by the prostaglandin E(2) receptor EP4 pathway during gastric tumorigenesis.

Cyclooxygenase-2 (COX-2) plays an important role in tumorigenesis through prostaglandin E(2) (PGE(2)) biosynthesis. It has been shown by in vitro studies that PGE(2) signaling transactivates epidermal growth factor receptor (EGFR) through an intracellular mechanism. However, the mechanisms underlying PGE(2)-induced EGFR activation in in vivo tumors are still not fully understood. We previously constructed transgenic mice that develop gastric tumors caused by oncogenic activation and PGE(2) pathway induction. Importantly, expression of EGFR ligands, epiregulin, amphiregulin, heparin-binding EGF-like growth factor, and betacellulin, as well as a disintegrin and metalloproteinases (ADAMs), ADAM8, ADAM9, ADAM10, and ADAM17 were significantly increased in the mouse gastric tumors in a PGE(2) pathway-dependent manner. These ADAMs can activate EGFR by ectodomain shedding of EGFR ligands. Notably, the extensive induction of EGFR ligands and ADAMs was suppressed by inhibition of the PGE(2) receptor EP4. Moreover, EP4 signaling induced expression of amphiregulin and epiregulin in activated macrophages, whereas EP4 pathway was required for basal expression of epiregulin in gastric epithelial cells. In contrast, ADAMs were not induced directly by PGE(2) in these cells, suggesting indirect mechanism possibly through PGE(2)-associated inflammatory responses. These results suggest that PGE(2) signaling through EP4 activates EGFR in gastric tumors through global induction of EGFR ligands and ADAMs in several cell types either by direct or indirect mechanism. Importantly, gastric tumorigenesis of the transgenic mice was significantly suppressed by combination treatment with EGFR and COX-2 inhibitors. Therefore, it is possible that inhibition of both COX-2/PGE(2) and EGFR pathways represents an effective strategy for preventing gastric cancer.

Prostaglandin E2 signaling and bacterial infection recruit tumor-promoting macrophages to mouse gastric tumors.

BACKGROUND & AIMS: Helicobacter pylori infection induces an inflammatory response, which can contribute to gastric tumorigenesis. Induction of cyclooxygenase-2 (COX-2) results in production of prostaglandin E(2) (PGE(2)), which mediates inflammation. We investigated the roles of bacterial infection and PGE(2) signaling in gastric tumorigenesis in mice. METHODS: We generated a germfree (GF) colony of K19-Wnt1/C2mE mice (Gan mice); these mice develop gastric cancer. We examined tumor phenotypes, expression of cytokines and chemokines, and recruitment of macrophages. We also investigated PGE(2) signaling through the PGE(2) receptor subtype 4 (EP4) in Gan mice given specific inhibitors. RESULTS: Gan mice raised in a specific pathogen-free facility developed large gastric tumors, whereas gastric tumorigenesis was significantly suppressed in GF-Gan mice; reconstitution of commensal flora or infection with Helicobacter felis induced gastric tumor development in these mice. Macrophage infiltration was significantly suppressed in the stomachs of GF-Gan mice. Gan mice given an EP4 inhibitor had decreased expression of cytokines and chemokines. PGE(2) signaling and bacterial infection or stimulation with lipopolysaccharide induced expression of the chemokine C-C motif ligand 2 (CCL2) (which attracts macrophage) in tumor stromal cells or cultured macrophages, respectively. CCL2 inhibition suppressed macrophage infiltration in tumors, and depletion of macrophages from the tumors of Gan mice led to signs of tumor regression. Wnt signaling was suppressed in the tumors of GF-Gan and Gan mice given injections of tumor necrosis factor-α neutralizing antibody. CONCLUSIONS: Bacterial infection and PGE(2) signaling are required for gastric tumorigenesis in mice; they cooperate to up-regulate CCL2, which recruits macrophage to gastric tumors. Macrophage-derived tumor necrosis factor-α promotes Wnt signaling in epithelial cells, which contributes to gastric tumorigenesis.

Inflammation, tumor necrosis factor and Wnt promotion in gastric cancer development.

Infection-associated chronic inflammation plays an important role in tumorigenesis, and macrophages are a key player in both inflammation and tumorigenesis. Tumor-associated macrophages accelerate tumorigenesis through the enhancement of angiogenesis, remodeling and the suppression of antitumor immunity. Helicobacter pylori infection induces inflammatory responses, which are closely associated with gastric cancer development. Recent studies using mouse models indicate that activated macrophages in the infected and inflamed gastric mucosa express TNF-alpha, which stimulates the surrounding epithelial cells to promote Wnt signaling activity. Such a promotion of Wnt signaling activity beyond the threshold for tumorigenesis may, therefore contribute to gastric cancer development. Accordingly, it is possible that the TNF-alpha-induced promotion of Wnt signaling is a novel protumorigenic mechanism of inflammation in gastric carcinogenesis.

Prostaglandin E2, Wnt, and BMP in gastric tumor mouse models.

The development of gastric cancer is closely associated with Helicobacter pylori (H. pylori) infection. The expression of cylooxigenase-2 (COX-2), a rate-limiting enzyme for prostaglandin biosynthesis, is induced in H. pylori-associated chronic gastritis, which thus results in the induction of proinflammatory prostaglandin, PGE(2). The COX-2/PGE(2) pathway plays a key role in gastric tumorigenesis. On the other hand, several oncogenic pathways have been shown to trigger gastric tumorigenesis. The activation of Wnt/beta-catenin signaling is found in 30-50% of gastric cancers, thus suggesting that Wnt signaling plays a causal role in gastric cancer development. Mutations in the bone morphogenetic protein (BMP) signaling pathway are responsible for the subset of juvenile polyposis syndrome (JPS) that develops hamartomas in the gastrointestinal tract. BMP suppression appears to contribute to gastric cancer development because gastric cancer risk is increased in JPS. Wnt signaling is important for the maintenance of gastrointestinal stem cells, while BMP promotes epithelial cell differentiation. Accordingly, it is possible that both Wnt activation and BMP suppression can cause gastric tumorigenesis through enhancement of the undifferentiated status of epithelial cells. Recent mouse model studies have indicated that induction of the PGE(2) pathway is required for the development of both gastric adenocarcinoma and hamartoma in the Wnt-activated and BMP-suppressed gastric mucosa, respectively. This article reviews the involvement of the PGE(2), Wnt, and BMP pathways in the development of gastric cancer, and gastric phenotypes that are found in transgenic mouse models of PGE(2) induction, Wnt activation, BMP suppression, or a combination of these pathways.

Induction and down-regulation of Sox17 and its possible roles during the course of gastrointestinal tumorigenesis.

BACKGROUND & AIMS: The activation of Wnt/beta-catenin signaling causes the development of gastric and colon cancers. Sox17 represses Wnt/beta-catenin signaling and is down-regulated in colon cancer. This study was designed to elucidate the role of Sox17 during the course of gastrointestinal tumorigenesis. METHODS: Sox17 expression was examined in gastrointestinal tumors of mouse models and humans. The roles of Sox17 in gastric tumorigenesis were examined by cell culture experiments and by construction of Sox17 transgenic mice. RESULTS: Sox17 was induced in K19-Wnt1/C2mE mouse gastric tumors and K19-Wnt1 preneoplastic lesions, where Wnt/beta-catenin signaling was activated. Consistently, Wnt activation induced Sox17 expression in gastric cancer cells. In contrast, Sox17 was rarely detected by immunohistochemistry in gastric and colon cancers, whereas strong nuclear staining of Sox17 was found in >70% of benign gastric and intestinal tumors. Treatment with a demethylating agent induced Sox17 expression in gastric cancer cells, thus indicating the down-regulation of Sox17 by methylation. Moreover, transfection of Sox17 in gastric cancer cells suppressed both the Wnt activity and colony formation efficiency. Finally, transgenic expression of Sox17 suppressed dysplastic tumor development in K19-Wnt1/C2mE mouse stomach. CONCLUSIONS: Sox17 plays a tumor suppressor role through suppression of Wnt signaling. However, Sox17 is induced by Wnt activation in the early stage of gastrointestinal tumorigenesis, and Sox17 is down-regulated by methylation during malignant progression. It is therefore conceivable that Sox17 protects benign tumors from malignant progression at an early stage of tumorigenesis, and down-regulation of Sox17 contributes to malignant progression through promotion of Wnt activity.

Activated macrophages promote Wnt signalling through tumour necrosis factor-alpha in gastric tumour cells.

The activation of Wnt/beta-catenin signalling has an important function in gastrointestinal tumorigenesis. It has been suggested that the promotion of Wnt/beta-catenin activity beyond the threshold is important for carcinogenesis. We herein investigated the role of macrophages in the promotion of Wnt/beta-catenin activity in gastric tumorigenesis. We found beta-catenin nuclear accumulation in macrophage-infiltrated dysplastic mucosa of the K19-Wnt1 mouse stomach. Moreover, macrophage depletion in Apc(Delta716) mice resulted in the suppression of intestinal tumorigenesis. These results suggested the role of macrophages in the activation of Wnt/beta-catenin signalling, which thus leads to tumour development. Importantly, the conditioned medium of activated macrophages promoted Wnt/beta-catenin signalling in gastric cancer cells, which was suppressed by the inhibition of tumour necrosis factor (TNF)-alpha. Furthermore, treatment with TNF-alpha induced glycogen synthase kinase 3beta (GSK3beta) phosphorylation, which resulted in the stabilization of beta-catenin. We also found that Helicobacter infection in the K19-Wnt1 mouse stomach caused mucosal macrophage infiltration and nuclear beta-catenin accumulation. These results suggest that macrophage-derived TNF-alpha promotes Wnt/beta-catenin signalling through inhibition of GSK3beta, which may contribute to tumour development in the gastric mucosa.

The effect of small interfering RNA (siRNA) against the Bcl-2 gene on apoptosis and chemosensitivity in a canine mammary gland tumor cell line.

The aim of this study was to investigate whether downregulation of Bcl-2 expression by small interfering RNA (siRNA) against the canine Bcl-2 gene would enhance the apoptosis and sensitivity of a canine mammary gland tumor cell line (CF33) to doxorubicin. Transfections of CF33 with siRNA were performed using cationic liposomes. Sequence-specific downregulation of Bcl-2 expression was measured by semiquantitative RT-PCR and Western blot analysis. Total viable cells were determined by MTS assay and apoptotic cell rates were determined by the immunohistochemical analysis on ssDNA. Our data showed the siRNA downregulated Bcl-2 expression which increased apotosis and also increased the sensitivity of CF33 to doxorubicin. This study indicated that downregulation of Bcl-2 expression by siRNA would be useful as a new protocol to increase the effect of doxorubicin on treatment of canine mammary gland tumors, requiring a detailed evaluation of siRNA in vivo.

Apoptosis of canine mammary tumor cells induced by small interfering RNA (siRNA) against Bcl-xL gene.

The inhibition of Bcl-xL mRNA expression and the acceleration of apoptotic cell rates in canine mammary tumor cell line (CF33) by the small interfering RNA (siRNA) were analyzed. The level of Bcl-xL transcripts in CF33 was decreased when cultured with siRNA, suggesting that siRNA might inhibit the expression of Bcl-xL mRNA in the CF33. Apoptotic cell rates in CF33 cultured with siRNA in Oligofectamine medium, with double strand RNA in Oligofectamine medium, without siRNA in Oligofectamine medium and in DMEM alone were 60.9%, 30%, 28.7% and 11.6% at 48-hr incubation, respectively, when evaluated by TUNEL assay. From these results, it was suggested that canine Bcl-xL might be an anticancer target of canine tumors.

In vitro effect of recombinant human tumor necrosis factor-alpha on canine neutrophil apoptosis.

Neutrophils (polymorphonuclear leukocytes: PMNs) are essential for the host defense against various infections and are often injurious to the host, causing inflammatory diseases where tumor necrosis factor-alpha (TNF-alpha) is suggested to play an important role. Since an effect of TNF-alpha on canine PMN apoptosis has not been studied, canine PMNs were stimulated with recombinant human (rh)TNF-alpha in the present study to investigate the effect of TNF-alpha on canine PMN apoptosis. PMN apoptosis and function to produce ROS were assessed by flow cytometry. Delayed apoptosis was observed in the PMNs treated with rhTNF-alpha at 100 ng/ml, accompanied by retention of capability to produce ROS. However, PMN apoptosis was accelerated by rhTNF-alpha combined with cycloheximide. Therefore, it is indicated that TNF-alpha is able to activate anti- and pro-apoptotic pathways in PMNs and that the inhibition of PMN apoptosis by TNF-alpha requires protein synthesis in the PMNs.

In vitro effect of recombinant human granulocyte colony-stimulating factor on canine neutrophil apoptosis.

Apoptosis is essential in eliminating neutrophils (polymorphonuclear leukocytes: PMNs) in animals. The suppression of PMN apoptosis is believed to be beneficial in eradicating pathogens and is implicated in the pathogenesis of human inflammatory diseases. In the present study, canine PMNs were stimulated with recombinant human granulocyte colony-stimulating factor (rhG-CSF) to investigate the in vitro effect on the apoptosis of canine PMNs. Apoptotic cell rates were assessed by flow cytometry in relation to the ability of PMNs to produce reactive oxygen species (ROS). Canine PMN apoptosis was markedly suppressed by rhG-CSF treatment, in association with the retention of the PMN ability to produce ROS. The addition of cycloheximide abolished this suppression by rhG-CSF. Moreover, canine PMNs, which were stimulated by rhG-CSF, expressed high levels of anti-apoptotic mcl-1 gene mRNA, as quantified by real-time polymerase chain reaction method. The results suggest that PMNs, stimulated by G-CSF, could work effectively over a longer period to eliminate pathogens, and that the prolongation of the PMN life-span might occasionally aggravate tissue injuries in dogs. In addition, the suppression of PMN apoptosis seems to be mediated by the induction of anti-apoptotic mcl-1 gene expression.

Effect of antineoplastic drugs on the expression of Bcl-2 and Bcl-xL genes in the feline T-cell leukemia cell line.

The Bcl-2 gene is the first member of a rapidly expanding family of genes that regulate apoptosis. Bcl-2 has been shown to repress cell death triggered by a diverse array of stimuli including chemotherapy and gamma-irradiation. Chemotherapy of feline lymphoma is generally carried out with antineoplastic drugs, which are reported to induce apoptosis in tumor cells. However, the precise apoptotic signals, induced by chemotherapeutic drugs against feline tumors have not been fully characterized. Therefore, we have evaluated the expression of Bcl-2 and Bcl-xL in FT-1 upon in vitro treatment with these drugs. In the present study, full length of feline Bcl-xL gene was sequenced, and the expressions of Bcl-2 and Bcl-xL mRNAs in feline lymphoma cell line (FT-1) cultured with doxorubicin, prednisolone or vincristine were investigated. Feline Bcl-xL clone was 1163 base pairs in length and encoded 233 amino acids. The predicted amino acid sequence was 99.1%, 98.7%, 96.1%, 97.4%, 97.0% and 97.9% homologous to predicted Bcl-xL of dog, human, mouse, pig, rat and sheep, respectively. The levels of Bcl-2 transcripts at 24h incubation in FT-1 stimulated with doxorubicin (0.3mug/ml), prednisolone (0.2mug/ml) and vincristine (5ng/ml) were increased to about 41.0-, 62.0- and 11.1-fold to those in non-stimulated FT-1, respectively. On the other hand, the level of Bcl-xL transcripts at 24h incubation in FT-1 stimulated by doxorubicin and prednisolone were significantly increased about 4.2- and 5.8-folds to the controls and inducible level of Bcl-xL by vincristine was decreased about 0.35-folds.

High expression of Bcl-xL in delayed apoptosis of canine neutrophils induced by lipopolysaccharide.

The expression of Bcl-2 family members, Bcl-xL, Bcl-2, Mcl-1 and Bax was investigated in delayed apoptosis of canine neutrophils induced by lipopolysaccharide (LPS). Apoptotic cell rates in neutrophils stimulated by LPS (100 ng/ml) were measured at 24 h incubation by TUNEL assay. The incidence of apoptotic neutrophils stimulated by LPS at 24 h incubation was 17.0+/-2% and that in non-stimulated neutrophils was 29.9+/-3%. By real-time quantitative PCR analysis, it was indicated that Bcl-xL and Bax levels in canine neutrophils were significantly affected by LPS stimulation. The levels of Bcl-xL, Bcl-2, Mcl-1 and Bax transcripts at 9 h incubation in neutrophils stimulated by LPS (100 ng/ml) were increased by about 80.4-, 1.9-, 1.4- and 5.3-folds, in comparison to those in non-stimulated neutrophils, respectively. These results indicated that Bcl-xL was proved have an important role in the inhibition of canine neutrophil apoptosis by LPS.

Molecular cloning of canine Mcl-1 gene and its expression in tumor cell lines.

The canine Mcl-1 gene was cloned and sequenced. Canine Mcl-1 clone was 2694 base pairs in length and encoded 350 amino acids. The predicted amino acid sequence was 87.7%, 77.1% and 75.7% homologous to predicted human, mouse and rat Mcl-1, respectively. RT-PCR analysis revealed that canine Mcl-1 mRNA was expressed in PBMCs (peripheral blood mononuclear cells), bone marrow cells, MDCK (Madin-Darby canine kidney) and GL-1 (canine B cell leukemia) whereas undetectable in CL-1 (canine T cell lymphoma) cell line.

Accelerated neutrophil apoptosis in 2 canine cases of hepatic disorder.

Accelerated neutrophil apoptosis was confirmed by TUNEL assay in two canine cases of hepatic disorder. One dog was diagnosed as having lymphocytic hepatitis and the other lymphocytic cholangitis by histopathology of liver biopsy specimen.