Expression of receptor tyrosine kinases VEGFR-1 (FLT-1), VEGFR-2 (KDR), EGFR-1, PDGFRa and c-Met in canine primary brain tumours

Inhibition of tumour growth and angiogenesis by targeting key growth factor receptors is a promising therapeutic strategy for central nervous system tumours. Characterization of these growth factor receptors in canine primary brain tumours has not been done. Using quantitative real‐time TaqMan polymerase chain reaction (PCR), we evaluated the expression of messenger RNA (mRNA) for five tyrosine kinase growth factor receptors (vascular endothelial growth factor receptor [VEGFR]‐1, VEGFR‐2, endothelial growth factor receptor [EGFR]‐1, platelet‐derived growth factor receptor a [PDGFRa], and c‐Met) relative to normal cerebral cortex in 66 spontaneous canine primary brain tumours. Increased expression of VEGFR‐1 and VEGFR‐2 mRNA was greatest in grade IV astrocytomas (glioblastoma multiforme) and grade III (anaplastic) oligodendrogliomas. EGFR‐1 mRNA expression was more consistently increased than the other receptors in all tumour types, while increased PDGFRa mRNA expression was mostly restricted to oligodendrogliomas. The similarities in increased expression of these tyrosine kinase growth factor receptors in these canine tumours, as compared to data from their human counterparts, suggest that common molecular mechanisms may be present.     

Cloning of canine Toll-like receptor 7 gene and its expression in dog tissues

Toll-like receptor 7 (TLR7) is activated by single strand RNA and imidazoquinoline compounds, and induces interferon production. In this study, canine TLR7 cDNA was cloned and sequenced. The full-length cDNA of canine TLR7 gene was 3419bp, encoding 1032 amino acids. The similarities of canine TLR7 with human and mouse TLR7 were 84 and 80% at the nucleotide sequence level, and 86 and 79% at amino acid sequence level, respectively. Further, the expression of TLR7 mRNA was investigated in canine normal tissues by semiquantitative RT-PCR analysis. The common expression level of TLR7 mRNA in tissues from three dogs examined was in large intestine, lung, pancreas, small intestine and skin, though the expression level in each tissue was varied among these healthy dogs. In other tissues (kidney, liver, lymph node, spleen, adrenal gland, and PBMCs), the level of TLR7 mRNA expression was different in individuals.     

Molecular cloning of canine thymus and activation-regulated chemokine (TARC) gene and its expression in various tissues

Thymus and activation-regulated chemokine (TARC) is known as a functional ligand for CC chemokine receptor 4 (CCR4), which is selectively expressed on Th2 lymphocytes and induces selective migration of the cells to allergic lesions. In this study, we cloned canine TARC cDNA from canine thymus by RT-PCR with rapid amplification of cDNA ends (RACE) method. The canine TARC clone contained a full-length open reading frame encoding 99 amino acids and included four cysteine residues characteristic to CC chemokine family. The canine TARC cDNA showed 77.5%, 67.4%, and 68.5% amino acid sequence similarity with human, mouse and rat homologues, respectively. Expression of TARC mRNA was detected not only in thymus but also in spleen, lymph node, lung and heart of the various normal dog tissues examined. TARC cDNA clone obtained in this study will be useful for further investigation on allergic diseases in dogs.     

Expression of c- yes oncogene product in various animal tissues and spontaneous canine tumours

An immunohistochemical study of various visceral organs of normal adult dogs, cats, pigs, horses, cows, and chickens (five of each species) and of 185 spontaneous canine tumours was carried out using paraffin wax sections and a commercially available antibody to the human c- yes oncogene product. Among the adult normal tissues of six animal species, epithelial cells of the proximal and distal renal tubules, the myocardium, hepatocytes, cerebellar Purkinje cells and adrenal cortical cells were positive for c- yes product. Among the foetal tissues of dogs and chickens, a positive reaction was observed on canine chorionic villi cells and chick yolk sac surface epithelium, and on epithelial cells of the renal tubules, hepatocytes and the myocardium. These findings suggest that the c- yes proto-oncogene may play a physiological role in the cell growth and metabolism of these adult and foetal tissues. Of the 185 tumours tested, 59 (31.9 per cent) expressed the c- yes oncogene product. The c- yes -positive tumours accounted for 44.4 per cent (12/27) of the skin tumours, 5.5 per cent (1/18) of the round cell tumours, 35. 7 per cent (10/28) of the soft tissue tumours, 21.4 per cent (3/14) of the testicular tumours, 29.1 per cent (23/79) of the mammary tumours, and 52.6 per cent (10/19) of the other tumours types. Expression of the c- yes oncogene appeared to be common in spontaneously arising canine tumours, and the degree of expression varied considerably by tumour type.     

Molecular cloning and expression analysis of the canine chemokine receptor CCR9

The chemokine receptor CCR9, which interacts with the thymus-expressed chemokine TECK/CCL25, contributes to the localization of lymphocytes to the small intestine, and is implicated in the development of human inflammatory bowel disease (IBD); however, their role in canine IBD is unknown. The objective of this study was to isolate cDNA encoding CCR9 and to investigate CCR9 expression in normal canine tissues and lymphoid cell lines. The complete open reading frame contained 1104 bp, encoding 367 amino acids, with 85% and 81% identity to human and mouse homologs, respectively. CCR9 mRNA was detected in all tissues investigated with the highest expression level in the small intestine. CCR9 mRNA was also expressed in GL-1, a canine B cell leukemia cell line, but not in CLBL-1, a canine B cell lymphoma cell line. Immunoblot and flow cytometry analyses of these cell lines using an anti-human CCR9 monoclonal antibody revealed that CCR9 protein expression was detected only in GL-1, indicating the cross-reactivity of the antibody. Using the antibody, flow cytometry showed that the proportions of CCR9(+) cells were small (mean, 4.88%; SD, 2.15%) in the normal canine PBMCs. This study will be useful in understanding canine intestinal immunity and the immunopathogenesis of canine IBD.     

Molecular cloning of the canine c-Met/HGF receptor and its expression in normal and regenerated liver

The c-Met proto-oncogene is the receptor for hepatocyte growth factor (HGF), which is a member of the tyrosine kinase family. Activation of the HGF/c-Met signal pathway leads to cell proliferation, motility, regeneration, and morphogenesis. In this study, the complete nucleotide sequence of complementary DNA (cDNA) of canine c-Met was cloned, and its distribution was determined in tissues. The canine c-Met cDNA clone had an open reading frame of 4419 bp that encoded a putative polypeptide of 1383 amino acids. The c-Met mRNA was expressed in a variety of canine tissues including peripheral blood mononuclear cells (PBMC), bone marrow, liver, kidney, lung, stomach, uterus, testis, thymus, lymph node, small intestine, colon, adrenal gland, thyroid gland, heart, muscle, skin, pancreas, ovary, prostate, spleen, fat, cerebrum, and cerebellum. In addition, the c-Met mRNA expression in normal and regenerated liver was examined. The levels of the mRNA increased 2-fold in regenerated liver compared to that found in normal liver, indicating that c-Met is involved in various functions including remodeling of canine hepatocytes.     

Differences in expression of retinal pigment epithelium mRNA between normal canines

A reference database of differences in mRNA expression in normal healthy canine retinal pigment epithelium (RPE) has been established. This database identifies non-informative differences in mRNA expression that can be used in screening canine RPE for mutations associated with clinical effects on vision. Complementary DNA (cDNA) pools were prepared from mRNA harvested from RPE, amplified by PCR, and used in a subtractive hybridization protocol (representational differential analysis) to identify differences in RPE mRNA expression between canines. The effect of relatedness of the test canines on the frequency of occurrence of differences was evaluated by using 2 unrelated canines for comparison with 2 female sibling canines of blue heeler/bull terrier lineage. Differentially expressed cDNA species were cloned, sequenced, and identified by comparison to public database entries. The most frequently observed differentially expressed sequence from the unrelated canine comparison was cDNA with 21 base pairs (bp) identical to the human epithelial membrane protein 1 gene (present in 8 of 20 clones). Different clones from the same-sex sibling RPE contained repetitions of several short sequence motifs including the human epithelial membrane protein 1 (4 of 25 clones). Other prevalent differences between sibling RPE included sequences similar to a chicken genetic marker sequence motif (5 of 25), and 6 clones with homology to porcine major histocompatibility loci. In addition to identifying several repetitively occurring, noninformative, differentially expressed RPE mRNA species, the findings confirm that fewer differences occurred between siblings, highlighting the importance of using closely related subjects in representational difference analysis studies.     

Decorin concentrations in canine normal and neoplastic mammary gland tissues

In the present study, the concentration of decorin in canine normal and neoplastic mammary gland tissues was examined to understand the potential role of decorin in development and progression of canine mammary tumours. The homogenates of 48 mammary gland tumours (10 benign and 38 malignant) and 10 samples of normal canine mammary gland tissue were used in the study. The presence and quantification of decorin was examined in the homogenates using Western blot and specific canine ELISA. Western blotting confirmed the presence of decorin both in the normal mammary gland tissues and in the mammary gland tumours. The concentration of decorin was significantly higher (p < .05) in the benign tumours and non-metastatic malignant tumours than in the normal mammary gland. The concentration of decorin was significantly lower (p < .05) in the malignant tumours with metastasis to regional lymph nodes compared with benign tumours and non-metastatic malignant tumours. No significant differences were found in the level of decorin between the benign and the non-metastatic malignant tumours. Both the histological type of malignant tumours and the histological grade did not significantly affect the concentration of decorin. These findings suggest that neoplastic transformation in the canine mammary gland leads to increase in the decorin protein synthesis. The reducing decorin concentration in canine malignant mammary tumours appears to facilitate the metastatic spread of these tumours.     

Expression and significance of PTEN and VEGF in canine mammary gland tumours

To investigate the relationship between the expression of the PTEN (phosphatase and tensin homolog deleted on chromosometen) and VEGF (vascular endothelial growth factor) and the clinicopathological features in canine mammary gland tumours, the expression levels of PTEN and VEGF protein were assessed in 50 cases of canine mammary gland tumours tissues and 4 cases of normal mammary gland tissues with using immunohistochemical method. The over-expression rate of PTEN protein was 100% in normal and well-differentiated mammary gland tissues and 67% in breast cancer cases respectively with a significant difference between the two groups (P<0.01). Expression of PTEN was not related to age and tumour size, but closely correlated to lymph node metastasis (P<0.01). The over-expression rate of VEGF protein was 33.3% in normal mammary gland tissues, and 78% in canine mammary gland tumours with a significant difference between the two groups (P<0.01).Expression of VEGF was not related to age or tumour size, but closely correlated with lymph node metastasis and clinical stage (P<0.05).Therefore the combination detection of PTEN and VEGF could serve as an important index to estimate the biological behavior and prognosis of canine mammary gland tumours. Reduced expression of PTEN might be involved in carcinogenesis and progression of canine breast cancer by up-regulating the VEGF expression to enhance angiogenesis.     

Expression patterns of the slit subfamily mRNA in canine malignant mammary tumors

Slit, a secreted protein, functions as a chemorepellent factor in axon guidance and neuronal migration and as an inhibitor in leukocyte chemotaxis. In humans, slit2 protein attracts endothelial cells and promotes tube formation in the tumor angiogenic mechanism. In this study, we cloned a part of the canine slit subfamily and examined the expression of slit subfamily mRNAs in 3 normal canine mammary glands and 11 mammary tumor samples by RT-PCR. The cloned part of the slit gene sequences showed high similarity to those of the human, mouse, and rat. The mRNAs were expressed at low levels in the normal mammary gland. The expression levels of slit1 mRNA were low in both the normal and tumor tissues. In contrast, the expression of slit2 mRNA increased in most of the malignant mammary tumors, and an increase in slit3 mRNA expression was observed in 2 of the malignant mixed tumors. These results suggest that the expression of slit2 plays an important role in tumor angiogenesis in canine mammary gland tumors and that slit2 can be a putative marker for malignancy diagnosis of these tumors.     

Messenger ribonucleic acid expressions of hepatocyte growth factor, angiopoietins and their receptors during follicular development in gilts

Angiogenic factors are associated with angiogenesis during follicular development in the mammalian ovary. The aim of the present study was to determine the relationships between the vascular network and mRNA expressions of angiopoietins (Ang)-1, Ang-2 and hepatocyte growth factor (HGF), and their receptors in follicles at different developmental stages during follicular development. Ovaries in gilts were collected 72 h after equine chorionic gonadotropin (eCG, 1250 IU) treatment for histological observation of the capillary network. Granulosa cells and thecal tissues in small (<4 mm), medium (4-5 mm) or large (>5 mm) individual follicles were collected for detection of mRNA expression of HGF, Ang-1 and Ang-2 in granulosa cells, and HGF receptor (HGF-R) and Tie-2 in the theca cells by semi-quantitative RT-PCR. The number of capillaries in the thecal cell layer increased significantly in healthy follicles at all developmental stages in the eCG group compared with those in controls. The expression of Ang-1 mRNA declined in granulosa cells of medium and large follicles and the level of Ang-2 mRNA increased in granulosa cells of small follicles after eCG treatment. The ratio of Ang-2/Ang-1 increased in small, medium and large follicles from ovaries after eCG treatment, but Tie-2 mRNA expression in the theca cells did not change. The level of HGF mRNA increased in granulosa cells of small follicles after eCG treatment but HGF-R in theca cells was not increased by eCG. These data suggested that the angiopoietins might be associated with thecal angiogenesis during follicular development in eCG-treated gilts.     

Cloning and sequence analysis of the complementary DNA for feline preproparathyroid hormone

OBJECTIVES: To clone and sequence the cDNA for feline preproparathyroid hormone (preproPTH) and to compare that sequence with other known parathyroid hormone (PTH) sequences. SAMPLE POPULATION: Parathyroid glands from 1 healthy cat. PROCEDURES: A cDNA library was constructed in lambda phage from feline parathyroid gland mRNA and screened with a radiolabeled canine PTH probe. Positive clones were sequenced, and nucleic acid and deduced amino acid sequences were analyzed and compared with known preproPTH and PTH sequences. RESULTS: Screening of approximately 2 X 10(5) recombinant plaques revealed 3 that hybridized with the canine PTH probe; 2 clones comprised the complete sequence for feline preproPTH. Feline preproPTH cDNA consisted of a 63-base pair (bp) 5'-untranslated region (UTR), a 348-bp coding region, and a 326-bp 3'-UTR. The coding region encoded a 115-amino acid peptide. Mature feline PTH consisted of 84 amino acids. Amino acid sequence analysis revealed that feline PTH was > 83% identical to canine, bovine, swine, equine, human, and macaque PTH and 69, 71, and 44% identical to mouse, rat, and chicken PTH, respectively. Within the region responsible for hormonal activity (amino acids 1 to 34), feline PTH was > 79% identical to other mammalian PTH sequences and 64% identical to the chicken sequence. CONCLUSIONS AND CLINICAL RELEVANCE: The amino acid sequence of PTH is conserved among mammalian species. Knowledge of the cDNA sequence for feline PTH may be useful to investigate disturbances of calcium metabolism and alterations in PTH expression in cats.     

Molecular cloning of canine monoamine oxidase subtypes A (MAOA) and B (MAOB) cDNAs and their expression in the brain

The role of monoamine oxidase has been shown to be related to some behavioral changes including aggression and cognitive dysfunction. In order to demonstrate the basic expression patterns of monoamine oxidase in the canine brain, we determined the full-length nucleotide sequences of cDNA for canine monoamine oxidase type A (MAOA) and type B (MAOB) genes that were isolated from the canine brain cDNA library. Oligonucleotide primers for PCR were constructed based on the conserved sequences reported thus far for other mammalian species. The nucleotide sequences had open reading frames of 1584 and 1563 bp for MAOA and MAOB, respectively. Both of these genes showed relatively high homology with other species in both nucleotide (> 81%) and deduced amino acid (> 85%) sequences. In Northern blot analyses MAOA mRNA was expressed broadly in various parts of the canine brain, whereas MAOB mRNA was found only in specific brain regions, such as the hypothalamus, hippocampus, brain stem and olfactory bulb. These results suggest that MAOA and MAOB mRNAs have subtype-specific expression patterns in the canine brain.     

Immunohistochemical expression of calretinin in canine testicular tumours and normal canine testicular tissue

Calretinin is a calcium-binding protein expressed abundantly in the central and peripheral neural tissues. It has been demonstrated to be a valuable marker in human testicular neoplasia. The immunohistochemical expression of calretinin has been studied in 102 samples of normal (n=25) and three different neoplastic canine testicular tumours (n=77). In normal canine testis, calretinin expression was restricted to Leydig and Sertoli cells of the testis. In tumour tissues, calretinin expression was detected in all tumours investigated (interstitial cell tumours, seminoma, and Sertoli cell tumours), with a cytoplasmic and nuclear pattern of cellular distribution. The present work reports, for the first time, calretinin immunohistochemical expression in normal and neoplastic canine testis.     

Insulin receptor is expressed in normal canine mammary gland and benign adenomas but decreased in metastatic canine mammary carcinomas similar to human breast cancer

Insulin receptor (INSR) or insulin-like growth factor (IGF) signalling is speculated to be involved in mammary tumour development. Expression levels of members of the insulin receptor family (INSR, IGF1R, IGF2R, GHR) and their ligands IGF1and IGF2 were quantified in macro- and microdissected tissue samples of normal canine mammary gland, adenomas, carcinomas and their lymph node metastases to evaluate their potential impact on the carcinogenesis of canine mammary tumours. Normal mammary gland and adenomas had strong INSR expression, while carcinomas and metastases had significantly decreased expression. No differences were observed for IGF1R expression. IGF1, IGF2 and GHR mRNA expressions were strongly decreased in adenomas, carcinomas and metastases. INSR and IGF1R are therefore expressed in normal gland and adenomas and an increased stimulus by their ligands may be a proliferative stimulus in those tissues. However, decreased INSR expression carcinomas and their metastases render questionable its impact at late stages of carcinogenesis.