Fusion protein and hemagglutinin of canine distemper virus co-induce apoptosis in canine mammary tumor cells.

BACKGROUND: Canine distemper virus (CDV) has been shown to have oncolytic activity against primary canine tumors. Previous studies from this laboratory had confirmed that CDV induces apoptosis in canine mammary tumor (CMT) cells, although the molecular mechanism remains unknown. METHODS: The CDV N, P, M, F, H, L, C, and V genes were identified in CDV-L and cloned separately. Mutants with deletions in the 5' region (pCMV-F L△60, pCMV-FL△107, and pCMV-FL△114) or with site-directed mutagenesis in the 3' region (pCMV-FLA602-610) of the F gene were generated. Late-stage apoptotic cells were detected by Hoechst 33342. Early-stage apoptotic cells were detected by AnnexinV-FITC/PI. Quantitative real-time PCR was performed to detect the mRNA levels of target genes of apoptotic and NF-κB pathway. Western blot analysis was performed to detect the expression or phosphorylation levels of target proteins of apoptotic or NF-κB pathway. Immunofluorescence assay was performed to detect the nuclear translocation of p65 protein. Recombinant viruses (rCDV-FL△60 and rCDV-FLA602-610) were rescued by a BHK-T7-based system. 5-week-old female BALB/c nude mice were used to detect the oncolytic activity of recombinant viruses. RESULTS: In this study, it was first confirmed that none of the structural or non-structural proteins of CDV-L, a vaccine strain, was individually able to induce apoptosis in canine mammary tubular adenocarcinoma cells (CIPp) or intraductal papillary carcinoma cells (CMT-7364). However, when CIPp or CMT-7364 cells were co-transfected with glycoprotein fusion (F) and hemagglutinin (H) proteins of CDV-L, nuclear fragmentation was observed and a high proportion of early apoptotic cells were detected, as well as cleaved caspase-3, caspase-8 and poly (ATP ribose) polymerase (PARP). Cleaved caspase-3 and PARP were down-regulated by apoptosis broad-spectrum inhibitor Z-VAD-FMK and caspase-8 pathway inhibitor Z-IETD-FMK, confirming that the F and H proteins coinduced apoptosis in CMT cells via the caspase-8 and caspase-3 pathways. F and H proteins co-induced phosphorylation of p65 and IκBα and nuclear translocation of p65, confirming activation of the NF-κB pathway, inhibition of which down-regulated cleaved caspase-3 and cleaved PARP. Recombinant F protein with enhanced fusion activity and H protein co-induced more cleaved caspase-3 and PARP than parental F protein, while the corresponding recombinant virus exhibited the same properties both in CIPp cells and in a subcutaneous xenograft mouse model. CONCLUSIONS: F and H proteins of CDV-L co-induce apoptosis in CMT cells, while the NF-κB pathway and fusion activity of F protein paly essential roles in the process.

Oncolytic activity of canine distemper virus in canine mammary tubular adenocarcinoma cells.

Canine distemper virus (CDV), bearing a close resemblance to measles virus, represents a promising candidate for oncolytic therapy; however, its application and underlying oncolytic mechanisms in canine mammary carcinoma cells remain to be explored. Here, we found that an attenuated canine distemper vaccine strain, CDV-L, efficiently infected and inhibited the growth of canine mammary tubular adenocarcinoma CIPp cells but not MDCK cells in vitro. Transcriptomic analysis of CDV-L-infected CIPp cells revealed substantially differentially expressed genes in apoptotic and NF-κB signalling pathways. Subsequent validations confirmed that CDV-L-induced apoptosis of CIPp cells through the caspase-8 and caspase-3 pathway. Identification of phosphorylated-IκBα, phosphorylated-p65 and the nuclear translocation of p65 confirmed the activation of the NF-κB signalling pathway. Inhibition of the NF-κB pathway abrogated CDV-L-induced cleaved-caspase-3 and cleaved-PARP. In a CIPp subcutaneous xenograft mouse model, intratumoural injections of CDV-L significantly restricted tumour growth without apparent pathology, and virus remained localized within the tumour. Taken altogether, these findings indicate that CDV-L exerts an antitumour effect in CIPp cells, and that apoptosis and the NF-κB pathway play essential roles in this process.

Naturally-occurring right terminal hairpin mutations in three genotypes of canine parvovirus (CPV-2a, CPV-2b and CPV-2c) have no effect on their growth characteristics.

We have isolated 4 naturally-occurring strains of CPV in mainland China and have identified them as CPV-2, 2a, 2b and 2c genotypes according to their VP2 sequences which also revealed substitutions within their right terminal regions. To determine if these substitutions affected the growth characteristics of the 4 strains, we constructed plasmids based on their genomic sequences minus their right terminal sequences, with the latter replaced by a single right terminal region. Analysis of rescued recombinants showed that the substitutions within their natural right termini had no significant effect on their growth characteristics.

An enhanced anti-tumor effect of apoptin-cecropin B on human hepatoma cells by using bacterial magnetic particle gene delivery system.

The gene therapy of cancer, due to the limit of its efficiency and safety, has not been widely used in clinical. Recently, bacterial magnetic particles (BMPs), which are membrane-bound nanocrystals found in magnetotactic bacteria, have been exploited as a new gene delivery system. However, its application on gene therapy remains to be explored. In our previous study, we found that a combination of cecropin B (ABPs) and apoptin (VP3) could serve as an effective gene therapeutic agent. Thus, in this study, we used BMPs to deliver the co-expression plasmid of these two gene, namely pVAX1-VA, and evaluated its therapeutic effect on human hepatocellular carcinoma (HepG2). Our results showed that BMPs significantly improved the efficiency of gene transfection (almost 3-fold than Lipofectamine 2000 at 48 h, P < .001), which led to stronger apoptosis (in a peak almost 2-fold than Lipofectamine 2000-pVAX1-VA, P < .01) and growth inhibition of HepG2 cells. More importantly, compared with Lipofectamine 2000-pVAX1-VA group, BMP-pVAX1-VA strikingly inhibited tumor growth (0.60 ±â€¯0.09 g vs. 0.88 ±â€¯0.11 g, P < .05) in nude mouse tumor models and increased the tumor-infiltrating lymphocytes considerably without apparent cytotoxicity. These findings suggest that BMPs could be an attractive gene delivery system for gene therapy and provide a potential available treatment for human hepatocellular carcinoma and maybe some other kinds of tumors.

The phosphorylation of Ser221 in VP2 of mink enteritis virus and its roles in virus amplification.

Recent reports have indicated that phosphorylation of capsid proteins plays an important role in virion assemblage. Autonomous parvoviruses are among the smallest known viruses with an ssDNA genome enclosed within an icosahedral capsid. Here, we demonstrate that a structural protein (VP2) of one member, mink enteritis virus (MEV), is phosphorylated at serine-221 (Ser221) in vivo. Mutant viruses containing an S221A non-phosphorylatable alanine substitution, or an S221E glutamic acid substitution to mimic serine phosphorylation, were able to express VP2 but had either limited ability or were unable to propagate in feline F81 cells. We propose a new mechanism whereby VP2 phosphorylation plays an essential role in amplification during MEV infection.

A recombinant canine distemper virus expressing a modified rabies virus glycoprotein induces immune responses in mice.

Canine distemper virus (CDV) and rabies virus (RV) are two important pathogens of the dog. CDV, a member of the morbillivirus genus, has shown promise as an expression vector. The glycoprotein from RV is a main contributor to protective immunity and capable of eliciting the production of virus-neutralizing antibodies. In this study, we recovered an attenuated strain of canine distemper virus and constructed a recombinant virus, rCDV-RV-G, expressing a modified (R333Q) rabies virus glycoprotein (RV-G) of RV Flury strain LEP. RV-G expression by the recombinant viruses was confirmed. Furthermore, G was proved to be incorporated into the surface of CDV particles. While replication of the recombinant virus was slightly reduced compared with the parental CDV, it stably expressed the RV-G over ten serial passages. Inoculation of mice induced specific neutralizing antibodies against both RV-G and CDV. Therefore, the rCDV-RV-G has the potential as a vaccine that may be used to control rabies virus infection in dogs and other animals.

Overexpression of Banna mini-pig inbred line fatty acid binding protein 3 promotes adipogenesis in 3T3-L1 preadipocytes.

Fatty acid binding protein 3 (H-FABP, FABP3) has been significantly associated with intramuscular fat (IMF) content in pigs, which is positively correlated with palatability of pork. However, its underlying function is not fully elucidated. We have investigated the effects of overexpression of the FABP3 gene on differentiation and adipogenesis of 3T3-L1 preadipocytes in the fat Banna mini-pig inbred line (fBMIL). Eukaryotic vectors that expressed the FABP3 protein were constructed, and stably established in the 3T3-L1 preadipocytes cell line. Cells were induced in a standard differentiation cocktail. Morphological changes and the degree of adipogenesis were measured by Oil Red O staining assay and triacylglycerol content measurement, respectively. mRNA expression levels of triacylglycerol metabolism-related genes were measured by qPCR. FABP3 significantly promoted differentiation of 3T3-L1 cells and enhanced triacylglycerol levels (P < 0.05). mRNA of the peroxisome proliferator-activated receptor γ (PPARγ), adipocyte fatty acid binding protein (422/aP2) and glycerol-3-phosphate dehydrogenase (GPDH) gene increased markedly (P < 0.05). In conclusion, expression of the FABP3 gene enhances adipogenesis in 3T3-L1 preadipocytes primarily by upregulating lipogenic PPARγ, 422/aP2 and GPDH genes.

Histopathological and gene expression analysis of mice exposed to diethylstilbestrol.

The estrogenic compound diethylstilbestrol (DES) has been widely studied to understand its potential involvement in endocrine function and carcinogenesis. This study examined the influence of DES on adult mice by histopathological analysis and studied the gene expression changes using mRNA differential display. Pathological changes in the mice following DES exposure included testicular atrophy, ovarian and hepatic fibrosis, and reduced numbers of mature oocytes and spermatogenic cells. Other pathological changes, such as cirrhosis of the liver, were also found. To elucidate the molecular mechanism underlying these effects, we used mRNA differential display to analyze changes in gene expression following DES exposure. In total, 20 genes were differentially expressed in liver, kidney, ovary, uterus, and testis. All putative target genes were validated by QRT-PCR. The study provides evidence that DES has an acute effect on gene expression. The results may facilitate the discovery of the genotoxic mechanism of DES and allow one to discover new DES-responsive genes.

In vitro induction of apoptosis in tumor cells by inactivated NDV and IAV.

We examined how Newcastle disease virus (NDV) and influenza A virus (IAV) inactivated by 5% formaldehyde, used either alone or in combination, can induce apoptosis in both HeLa and SP2/0 cells. Inactive NDV and IAV demonstrated enhanced rates of lysis in apoptotic tumor cells and greater antitumor effects when combined. Our study supports the argument that viral replication does not cause virally induced apoptosis.