Elizabeth A. Gault

Telomere loss in relation to age and early environment in long-lived birds

Shortening of telomeres, specific nucleotide repeats that cap eukaryotic chromosomes, is thought to play an important role in cellular and organismal senescence. We examined telomere dynamics in two long–lived seabirds, the European shag and the wandering albatross. Telomere length in blood cells declines between the chick stage and adulthood in both species. However, among adults, telomere length is not related to age. This is consistent with reports of most telomere loss occurring early in life in other vertebrates. Thus, caution must be used in estimating annual rates of telomere loss, as these are probably not constant with age. We also measured changes within individuals in the wild, using repeat samples taken from individual shags as chicks and adults. We found high inter–individual variation in the magnitude of telomere loss, much of which was explained by circumstances during growth. Individuals laying down high tissue mass for their size showed greater telomere shortening. Independently of this, individuals born late in the season showed more telomere loss. Early conditions, possibly through their effects on oxidative stress, appear to play an important role in telomere attrition and thus potentially in the longevity of individuals.

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.

Establishment and characterization of equine fibroblast cell lines transformed in vivo and in vitro by BPV-1: Model systems for equine sarcoids

It is now widely recognized that BPV-1 and less commonly BPV-2 are the causative agents of equine sarcoids. Here we present the generation of equine cell lines harboring BPV-1 genomes and expressing viral genes. These lines have been either explanted from sarcoid biopsies or generated in vitro by transfection of primary fibroblasts with BPV-1 DNA. Previously detected BPV-1 genome variations in equine sarcoids are also found in sarcoid cell lines, and only variant BPV-1 genomes can transform equine cells. These equine cell lines are morphologically transformed, proliferate faster than parental cells, have an extended life span and can grow independently of substrate. These characteristics are more marked the higher the level of viral E5, E6 and E7 gene expression. These findings confirm that the virus has an active role in the induction of sarcoids and the lines will be invaluable for further studies on the role of BPV-1 in sarcoid pathology.

Bovine papillomavirus type 1 oncoprotein E5 inhibits equine MHC class I and interacts with equine MHC I heavy chain.

Bovine papillomavirus type 1 is one of the aetiological agents of equine sarcoids. The viral major oncoprotein E5 is expressed in virtually all sarcoids, sarcoid cell lines and in vitro-transformed equine fibroblasts. To ascertain whether E5 behaves in equine cells as it does in bovine cells, we introduced the E5 open reading frame into fetal equine fibroblasts (EqPalF). As observed in primary bovine fibroblasts (BoPalF), E5 by itself could not immortalize EqPalF and an immortalizing gene, such as human telomerase (hTERT/hT), was required for the cells to survive selection. The EqPalF-hT-1E5 cells were morphologically transformed, elongated with many pseudopodia and capable of forming foci. Equine major histocompatibility complex class I (MHC I) was inhibited in these cells at least at two levels: transcription of MHC I heavy chain was inhibited and the MHC I complex was retained in the Golgi apparatus and prevented from reaching the cell surface. We conclude that, as in bovine cells and tumours, E5 is a player in the transformation of equine cells and the induction of sarcoids, and a potential major cause of MHC I downregulation and hence poor immune clearance of tumour cells.

Transcriptional Changes Induced by Bovine Papillomavirus Type 1 in Equine Fibroblasts

ABSTRACT Bovine papillomavirus type 1 (BPV-1) and, less commonly, BPV-2 are associated with the pathogenesis of common equine skin tumors termed sarcoids. In an attempt to understand the mechanisms by which BPV-1 induces sarcoids, we used gene expression profiling as a screening tool to identify candidate genes implicated in disease pathogenesis. Gene expression profiles of equine fibroblasts transformed by BPV-1 experimentally or from explanted tumors were compared with those of control equine fibroblasts to identify genes associated with expression of BPV-1. Analysis of the microarray data identified 81 probe sets that were significantly (P < 0.01) differentially expressed between the BPV-1-transformed and control cell lines. Expression of several deregulated genes, including MMP-1, CXCL5, FRA-1, NKG7, TLR4, and the gene encoding the major histocompatibility complex class I (MHC-I) protein, was confirmed using other BPV-1-transformed cell lines. Furthermore, expression of these genes was examined using a panel of 10 sarcoids. Increased expression of MMP-1, CXCL5, FRA-1, and NKG7 was detected in a subset of tumors, and TLR4 and MHC I showed robust down-regulation in all tumors. Deregulated expression was confirmed at the protein level for MMP-1 and MHC-I. The present report identifies genes modulated by BPV-1 transformation and will help identify the molecular mechanisms involved in disease pathogenesis.

Different contribution of bovine papillomavirus type 1 oncoproteins to the transformation of equine fibroblasts.

Equine sarcoids represent the most common skin tumours in equids worldwide, characterized by localized invasion, rare regression and high recurrence following surgical intervention. Bovine papillomavirus type 1 (BPV-1) and less commonly BPV-2 are now widely recognized as the causative agents of the disease. Fibroblasts isolated from sarcoids are highly invasive. Invasion is associated with a high level of viral gene expression and matrix metalloproteinase upregulation. However, it remains unclear to what extent BPV-1 proteins are involved in the transformation of equine cells. To address this question, the individual viral genes E5, E6 and E7 were overexpressed in normal equine fibroblasts (EqPalF cells) and in the immortal but not fully transformed sarcoid-derived EqS02a cell line. The proliferation and invasiveness of these cell lines were assessed. E5 and E6 were found to be responsible for the enhanced cell proliferation and induction of increased invasion in EqS02a cells, whilst E7 appeared to enhance cell anchorage independence. Knockdown of BPV-1 oncogene expression by small interfering RNA reversed the transformed phenotype of sarcoid fibroblasts. Together, these observations strongly suggest that BPV-1 proteins play indispensable roles in the transformation of equine fibroblasts. These data also suggest that BPV-1 proteins are potential drug targets for equine sarcoid therapy.

Telomerase activity and telomerase reverse transcriptase catalytic subunit expression in canine lymphoma: correlation with Ki67 immunoreactivity

Increased telomerase activity (TA) has been found in human and canine solid tumours, stem cells and somatic tissues with enhanced proliferative potential. The relationship between TA in normal and malignant lymphoid tissues remains unclear. The TA and the expression of canine telomerase reverse transcriptase catalytic subunit (dogTERT) messenger RNA (mRNA) were analyzed in malignant lymph nodes from 30 dogs with lymphoma, from two dogs with non-neoplastic illness and from two clinically normal dogs, demonstrating a statistically significant difference between TA in lymphoma lymph nodes (n = 30) and normal nodes (n = 4) but no significant difference in dogTERT mRNA expression. In addition, the expression of telomerase reverse transcriptase catalytic subunit (TERT) protein and Ki67 was analyzed in malignant lymph nodes from 10 dogs with lymphoma and from two clinically normal dogs by immunohistochemistry. TERT expression was associated with Ki67 in all lymphoma nodes (n = 10), and differences were illustrated between TERT and Ki67 expression between lymphoma (n = 10) and non-lymphoma (n = 2) nodes. This data support further investigation of telomerase in canine haematopoietic neoplasia through large-scale prospective studies.

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.

p38 mitogen-activated protein kinase is crucial for bovine papillomavirus type-1 transformation of equine fibroblasts

Equine sarcoids represent the most common skin tumours in equids worldwide, characterized by extensive invasion and infiltration of lymphatics, rare regression and high recurrence after surgical intervention. Bovine papillomavirus type-1 (BPV-1) and less commonly BPV-2 are the causative agents of the diseases. It has been demonstrated that BPV-1 viral gene expression is necessary for maintaining the transformation phenotype. However, the underlying mechanism for BPV-1 transformation remains largely unknown, and the cellular factors involved in transformation are not fully understood. Previously mitogen-activated protein kinase (MAPK) signalling pathway has been shown to be important for cellular transformation. This study investigated the role of p38 MAPK (p38) in the transformation of equine fibroblasts by BPV-1. Elevated expression of phosphorylated p38 was observed in BPV-1 expressing fibroblasts due to the expression of BPV-1 E5 and E6. The phosphorylation of the MK2 kinase, a substrate of p38, was also enhanced. Inhibition of p38 activity by its selective inhibitor SB203580 changed cell morphology, reduced the proliferation of sarcoid fibroblasts and inhibited cellular invasiveness, indicating the indispensable role of p38 in BPV-1 transformation of equine fibroblasts. These findings provide new insights into the pathogenesis of equine sarcoids and suggest that p38 could be a potential target for equine sarcoid therapy.

Small interfering RNA targeting bovine papillomavirus type 1 E2 induces apoptosis in equine sarcoid transformed fibroblasts.

Equine sarcoids are skin tumours of horses caused by infection with BPV-1 or 2. Maintenance and replication of the viral genome depend upon the viral proteins E1 and E2. We examined the effects of an E2 specific siRNA on E2 and E1 viral gene expression, viral load and cell growth in BPV-1 transformed sarcoid-derived cells. Transfection with E2-siRNA caused a reduction in E2 and E1 mRNA expression as well as viral load, growth inhibition and decreased anchorage-independent growth. siRNA treated cells showed significantly higher apoptosis rates than control cells. Thus sequence specific targeting of E2 provides a powerful strategy to eliminate BPV-1 genomes and induce cell death in BPV-1 transformed cells.