David J. Argyle

Equine telomeres and telomerase in cellular immortalisation and ageing

To determine the role of telomeres in cellular ageing in equids, we analysed telomere lengths in peripheral blood derived DNA samples from a panel of donkeys (Equus asinus) ranging from 2 to 30 years of age. The average telomere lengths ranged from 7 to 21 kbp and a statistically significant inverse correlation between telomere lengths and donor age was demonstrated. Similarly, telomere lengths in primary fibroblasts isolated from a horse (Equus equus) demonstrated telomeric loss with in vitro ageing when cultured to senescence. We extended this study to evaluate activity of the enzyme telomerase in various equine cell cultures, normal equine tissues and equine benign tumour samples. Initially a panel of equine immortalised and primary cell cultures were evaluated for telomerase activity using a standard telomere repeat amplification protocol (TRAP) assay. High levels of telomerase activity were detected in equine immortalised cells with no activity evident in primary cell cultures. Similarly, no telomerase activity could be detected in normal equine tissues or equine benign tumour samples of the sarcoid or papilloma type. We conclude that telomere attrition may contribute to ageing in equids. However, it would appear that telomerase does not play a major role in the development of the most common benign tumours of the horse.

Analysis of p53 mutational events and MDM2 amplification in canine soft-tissue sarcomas

Canine cancer is of major significance in terms of animal health and welfare and soft tissue sarcomas are an important group of tumours accounting for approximately 15% of all canine tumours presented. Abnormal p53 protein expression and gene mutations have been identified in a number of different canine tumour types. However, mdm2 gene amplification has only been investigated in a limited number of canine osteosarcomas. In this present study a series of canine soft-tissue sarcomas (STS) were examined for p53 mutations and/or mdm2 amplification. For p53 mutational studies polymerase chain reaction and direct DNA sequencing was used. Gene mutations were identified in 6 of 30 (20%) primary tumour cases including MPNST (n=3) leiomysarcoma (n=1), heamangiosarcoma (n=1) and sarcoma (n=1). mdm2 gene amplification was assessed by Southern Blot. Although there was no evidence for major gene rearrangements, gene amplification was detected in 4 of 35 (11.4 %) primary tumours including MPNST (n=2), rhabdomyosarcoma (n=2). A total of 33 cases were examined for both p53 mutations and mdm2 amplification. Seven of the tumours were positive for p53 mutations, while five were positive for mdm2 amplification. With the exception of one case, a reciprocal relationship between the presence of a p53 mutation and mdm2 gene amplification was demonstrated.

Immunocytochemical analysis of the tumour suppressor protein (p53) in feline neoplasia

Detectable p53 immunostaining in neoplasia generally correlates with the presence of a mutation in the coding region of the p53 gene and may provide insights into the pathogenic mechanisms underlying tumourigenesis. p53 immunoreactivity was examined in 77 feline tumours, selected as a representative sample of 486 specimens submitted for diagnosis and analyzed to estimate the relative frequencies of feline neoplasias. Immunocytochemical staining demonstrated nuclear immunopositivity in 46% of the squamous cell carcinomas (SCCs), 50% of the osteosarcomas, 33% of the mammary carcinomas, 16% of the adenocarcinomas and 14% of the haemangiosarcomas. In contrast, none of the malignant lymphomas or fibrosarcomas examined showed p53 immunoreactivity. These data support a role for p53 aberrations in the pathogenesis of certain feline tumours.

Nucleotide and predicted peptide sequence of feline interferon-gamma (IFN-gamma)

Interferon gamma is a pleiotropic cytokine which is now recognised as an important modulator of the immune response. We now report the cloning and sequencing of feline interferon gamma cDNA which was generated by the polymerase chain reaction. At the nucleotide level feline ifn-gamma shares 78% and 63% homology with human and murine cDNA equivalents. At the amino acid level the feline IFN-gamma shares 63% and 43% homology with human and murine homologs respectively.

Molecular cloning and characterization of canine metalloproteinase-9 gene promoter

This paper describes the cloning and characterization of the canine matrix metalloproteinase-9 (MMP-9) gene promoter. The 5 untranslated region was obtained by genome walking upstream of the canine MMP-9 translation start site using canine genomic DNA as template. A DNA fragment of 1894 bp was isolated and on analysis demonstrated regions of sequence homology with the MMP-9 promoter sequences already determined for other species. In general, conserved regions correlated with DNA binding motifs such as a TATA-like box, AP-1 sites, GC boxes and a nuclear factor-kappaB binding domain. The DNA promoter fragment was sufficient to drive basal expression of a luciferase reporter gene in Madin Darby canine kidney (MDCK) cells and to a lesser extent in feline embryonic fibroblast (FEA) cells. Activity of the promoter was enhanced by the treatment of transfected MDCK cells with phorbol 12-myristate 13-acetate but no effect was observed in the FEA cells. Promoter deletion studies revealed that regions of promoter were necessary for induction of reporter gene expression.

Nucleotide Sequence of a Highly Conserved Region of the Canine p53 Tumour Suppressor Gene

An evolutionary conserved region of the canine tumour suppressor gene, p53, was PCR amplified and its DNA sequence determined. The 1003 bp fragment consisted of exons 5 to 8 and the intervening introns. A high level of sequence homology was demonstrated with human sequences, with the evolutionary conserved domains II, III, IV and V being identical.

Expression of feline recombinant interferon-gamma in baculovirus and demonstration of biological activity.

We have previously reported the cloning of the coding sequence for feline-specific interferon-gamma. Here, we describe the expression of this sequence in a baculovirus system and demonstrate the biological activity of the recombinant protein. The coding sequence for feline interferon was directionally cloned into the baculovirus transfer vector pAcCL29-1. Transfer vector and linearized wild-type AcMNPV (BacPAK6) were used to co-transfect Sf9 cells by calcium phosphate coprecipitation. Subsequently, wild-type and recombinant viruses were separated by plaque assay. Recombinant plaques were expanded and a master stock of virus is produced. Production of biologically active interferon-gamma from infected Sf9 cells was demonstrated using a standard cytopathic effect reduction assay, utilising vesicular stomatitis virus (VSV), and an MHC class II induction assay.

Feline epitheliotrophic T‐cell lymphoma with paraneoplastic eosinophilia – immunochemotherapy with vinblastine and human recombinant interferon α2b

A cat with epitheliotrophic T-cell lymphoma with paraneoplastic eosinophilia is described. Initial attempts to control the disease with conventional therapies failed. The addition of recombinant human interferon alpha(2b) (rhINFalpha(2b)) resulted in a clinical, haematogenous and sonographic improvement for 49 days. The overall survival time from initial diagnosis was 100 days. Relapse was correlated with the development of serum antibodies directed against rhINFalpha(2b). To our knowledge, this is the first report describing the clinical use of IFNalpha in the treatment of neoplasia in the cat.

The cloning and functional analysis of canine matrix metalloproteinase-13 gene promoter

A fragment of the 5 untranslated region corresponding to the canine matrix metalloproteinase-13 (MMP-13), collagenase-3 gene promoter has been isolated and characterized in rat cardiocytes to investigate the role of MMP-13 in cardiac disease. The promoter fragment (1.5 kb) demonstrated regions of sequence homology with the collagenase gene promoter sequences already determined for other species. Conserved regions were identified and shown to correlate with DNA binding motifs including AP-1 sites, a nuclear factor (NF) B-like binding domain, GATA and Nkx2.5 sites. A consensus TATA box was identified and shown to direct transcription initiation approximately 27 bp upstream of the translation start site. The canine MMP-13 promoter fragment was sufficient to drive basal expression of a luciferase reporter gene in both Madin Darby canine kidney cells (MDCK) and primary rat cardiocytes. The activity of the promoter fragment could be significantly increased by the treatment of transfected primary rat cardiocytes with interleukin-1 (IL-1) and basic fibroblastic growth factor (bFGF), with some induction also observed with tumour necrosis factor (TNF). The canine MMP-13 promoter activity has also been compared to the basal and induced activity of the canine MMP-9, gelatinase B promoter in these cell types.

Idiopathic hepatic veno-occlusive disease causing Budd-Chiari-like syndrome in a cat

Budd‐Chiari‐like syndrome (BCLS) is a rare clinical entity characterised by portal hypertension and ascites. This report describes a case of BCLS in a cat due to obstruction at the level of the hepatic veins. The diagnosis was based on the clinical findings and a histopathological assessment of the liver demonstrating perivenular fibrosis around the central and sublobular veins. Although these lesions are similar to those observed in man with BCLS, the aetiology in this case remains unknown.