TSLC1 tumour-suppressor gene expression in canine mast cell tumours

Tumour suppressor in lung cancer-1 (TSLC1) is a tumour-suppressor gene coding for an adhesion molecule that is expressed by mast cells. Reduced TSLC1 expression is associated with a poor prognosis in several human tumours, and this study sought to investigate if TSLC1 expression could be used to predict outcome in dogs with mast cell tumours (MCTs). Sections of MCTs of different tumour grades from 45 dogs (Group 1) were immunohistochemically assessed for TSLC1 and Ki67 expression. In addition, 35 intermediate-grade MCTs (Group 2) from dogs with known clinical follow-up were immunohistochemically stained for TSLC1 and Ki67. The TSLC1 staining intensity was found to strongly inversely correlate with tumour grade for Group 1 (P = 0.002857). For Group 2 there was a trend towards dogs with lower TSLC1 scores being more likely to die from MCT-related disease (P = 0.058). The intensity of TSLC1 staining inversely correlated with Ki67 expression for both groups.     

Immunohistochemical evaluation of p53 expression with different antibodies in malignant canine tumours with or without p53 gene mutation

Six monoclonal antibodies and a polyclonal antibody (CM1) were used to investigate the overexpression of p53 protein by immunohistochemistry (IHC) in six sarcomas and 21 mammary carcinomas from 27 dogs. IHC was compared with p53 gene mutation analysis performed on the same samples. Only the monoclonal PAb240, PAb421 and the CM1 antibodies were able to detect expression of canine p53 protein. CM1 was found to give the highest concordance (8/11) between positive expression of the p53 protein by IHC and the presence of a gene mutation. In the samples that were negative for p53 expression by IHC, but contained a p53 gene mutation according to DNA analysis, the mutation often affected the epitopes that could have been recognized by these antibodies. Only one out of 16 tumours without a p53 gene mutation had a weakly positive IHC result. These findings indicate that in these two types of canine tumours, IHC – particularly with CM1 – can detect many alterations in p53 expression owing to a gene mutation. False‐positive results were very infrequent.     

Differential expression of cell cycle regulators p21, p27 and p53 in metastasizing canine mammary adenocarcinomas versus normal mammary glands

The cyclin dependent kinase inhibitors p21 and p27 are important regulators of cell cycle progression. To analyze their role in the malignant progression of canine mammary tumors expression levels of p27 and p21 and its major regulator p53 were compared in simple adenomas, adenocarcinomas of the mammary gland and lymph node metastases with normal mammary gland. Laser microdissection of tissue samples and real-time PCR were used for quantification of mRNA expression levels. p21 was overexpressed in adenocarcinomas, whereas adenomas and metastases expressed p21 more heterogeneously. Comparison of p21 expression in adenocarcinomas and their metastases revealed a significant decrease in expression in metastases. In contrast, p27 expression was reduced in the adenocarcinomas but heterogeneously expressed in adenomas and metastases. Taken together the results suggest that loss of p21 overexpression is associated with tumor metastasis while reduced cell cycle inhibition by p27 is associated with malignant progression.     

Relationship between retinoic acid receptor alpha gene expression and growth-inhibitory effect of all-trans retinoic acid on canine tumor cells

Retinoids show antitumor effects on human acute promyelocytic leukemia and other tumors via retinoid receptors. In dogs, the role of retinoid receptors in inhibiting tumor development remains unclear. To evaluate the correlation between the degree of expression of retinoic acid receptor alpha (RARalpha) mRNA and the antiproliferative effects of all-trans retinoic acid (ATRA) treatments, expression analysis of RARalpha mRNA and cell growth inhibition assay were performed on 17 established canine tumor cell lines, including 6 mammary gland tumor (MGT) cell lines, 3 osteosarcoma cell lines, 5 melanoma cell lines, and 3 mast cell tumor (MCT) cell lines. Among the cell lines investigated, all 3 MCT cell lines showed high expression of RARalpha, and the most effective cell growth inhibition was observed in ATRA-treated MCT cell lines. However, remarkable antiproliferative effects of ATRA treatments were not observed on other tumor cell lines with moderate or low RARalpha mRNA expression. As a result of the relationship between RARalpha mRNA expression and ATRA treatment with regression analysis, statistically significant correlation was suggested. Furthermore, real-time quantitative polymerase chain reaction analysis of RARalpha was performed on MCT tissue samples of dogs with spontaneous disease, and 5 of 9 tissues showed high expression. These results suggest that ATRA may be an effective antitumor agent for MCT in dogs, and that prior measurement of expression of RARalpha mRNA may be a good indicator of the effectiveness of ATRA treatment.     

Loss of p27 expression in canine mammary tumors and their metastases

The p27 gene is a member of the cyclin-dependent kinase inhibitors, which arrest G1- to S-phase transition of the cell cycle. We have previously shown a significant reduction of p27 mRNA expression level in laser-microdissected mammary carcinomas and their lymph node metastases when compared to non-neoplastic mammary gland of the same dog. Here, p27 expression was analyzed on the protein level in non-neoplastic mammary gland, primary mammary carcinomas, their lymph node metastases and intravascular tumor cells of 49 dogs, adenomas of 49 dogs and non-neoplastic mammary gland of 98 dogs by immunohistochemistry. A significantly (p ? 0.05) decreased percentage of p27 positive tissue samples was found when normal gland was compared with adenomas, carcinomas and lymph node metastases. Specifically, 91% of normal gland epithelium displayed nuclear p27 expression. In contrast, only 22% of the adenomas, 20% of carcinomas, 12% of lymph node metastases and 32% of intravascular tumor cells had p27 reactivity. Cell cycle control by p27 is therefore lost in the majority of canine mammary tumors. The lack of significant differences between benign and malignant mammary tumors indicates that decreased p27 expression is an early step in carcinogenesis of canine mammary tumors and hinders the use of p27 as a marker of malignancy for this tumor type.     

p62/SQSTM1 expression in canine mammary tumours: Evolutionary notes

Recent studies highlighted the role of autophagy as a cardinal regulatory system for homeostasis and cancer‐related signalling pathways. In this context, the deregulated expression of p62 – Sequestosome1 (p62/SQSTM1) – a protein acting both as an autophagy receptor and signalling hub, has been associated with tumour development and chronic inflammation. Multiple clinical studies test drugs targeting autophagy, and even more research is on the way to clinical trials. However, no comparative investigations have been carried out to identify adequate preclinical models to assess p62‐based medicine. In veterinary oncology the role of p62 in cancer‐related pathways has been largely ignored. We compared p62 sequences in multiple organisms and found that canine p62 significantly diverges from the humans and from other animals sequences. Then, we chart by immunohistochemistry the expression levels of p62 in canine mammary tumours. A total of 66 tumours and 10 non‐neoplastic mammary samples were examined. The expression of p62 was higher in normal tissue and adenomas than carcinomas, with lowest levels of p62 protein detected in high grade carcinomas. In all cases examined the tumour stroma appeared to be p62‐negative. Taken together our results would suggest that in dogs the association between p62 expression and cancer cells overturns that reported in human breast carcinoma, where p62 accumulates in malignant cells as compared to normal epithelium. Thus, at least in canine mammary tumours, p62 should be not considered a tumour‐rejection antigen for an anti‐cancer immunotherapy.     

Occurrence and expression of p53 suppressor gene and c‐Myc oncogene in dog eyelid tumors

Purpose To detect the occurrence and expression of the suppressor gene p53 and of the oncogene c‐Myc in eyelid tumors of dogs using the PCR, RT‐PCR, PCR‐ELISA and RT‐PCR‐ELISA techniques. These genes have not been described in dog eyelid tumors before. Methods Nine samples of eyelid or third eyelid epithelial tumors were obtained from the archives of the Department of Veterinary Pathology. Tumor diagnosis was confirmed by evaluation of hematoxylin‐eosin stained sections, and immunohistochemistry for cytokeratin AE1/AE3 and vimentin V9. A canine mammary tumor was used for positive control. Agarose gel electrophoresis, PCR‐ELISA and RT‐PCR‐ELISA were used to detect p53 and c‐Myc genes. Results The occurrence of p53 was detected in most of the eyelid tumors and third eyelid tumors studied (88.8%, n = 8) and was expressed in 75% of the positive samples, as indicated by ELISA. The c‐Myc gene was found in 77.7% (n = 7) of the samples and was expressed in eight samples. Conclusions Eyelid and third eyelid tumors of dogs express both the p53 and the c‐Myc genes as shown by PCR and RT‐PCR. However, PCR ELISA and RT‐PCR ELISA were more efficient in assessing occurrence and expression of these genes because they identified amplified products that were not detected by agarose gel electrophoresis.     

A novel NF-κB inhibitor improves glucocorticoid sensitivity of canine neoplastic lymphoid cells by up-regulating expression of glucocorticoid receptors

Lymphoid neoplasms including lymphoma and leukemia are one of the most life-threatening disorders in dogs. Many lymphoid malignancies are well-treated with glucocorticoid (GC); however, GC resistance sometimes develops and its mechanism remains uncertain. Since constitutive activation of nuclear factor-κB (NF-κB) has been reported to play roles in lymphoid malignancies, we examined whether inhibition of NF-κB activity with a synthetic inhibitor IMD-0354 affected GC sensitivity of canine neoplastic lymphoid cells, CL-1 and GL-1. Dexamethasone failed to inhibit proliferation of these cells, in which low expression of glucocorticoid receptors (GR) was identified. In the presence of IMD-0354, GR expressions in CL-1 and GL-1 were increased, consequently dexamethasone inhibited their proliferation. These results indicated that GR expression might be down-regulated by spontaneous activation of NF-κB, resulting in GC resistance. Taken together, interference of NF-κB activity may have the synergistic effect in combination chemotherapy with GC for treatment against lymphoid malignancies.     

Properties of the feline tumour suppressor reduced expression in immortalized cells (REIC/Dkk‐3)

Reduced expression in immortalized cells (REIC/Dkk‐3), a member of the human Dickkopf (Dkk) family, is a growth suppressor in human and canine mammary tumours. Mammary gland tumours are common neoplasms with high malignancy in female cats. The purpose of this study was to clone the feline REIC/Dkk‐3 homolog, investigate its expression in cell lines established from feline mammary gland tumours, and test its tumour suppressor function. Western blot analysis revealed that expression of the REIC/Dkk‐3 protein was reduced in feline mammary carcinoma cell lines. Forced expression of REIC/Dkk‐3 induced apoptosis in feline mammary tumour cell lines. These results demonstrate that REIC/Dkk‐3 expression, which is downregulated in feline mammary tumour cell lines, results in the induction of apoptosis in these cells. Our findings suggest that feline REIC/Dkk‐3 represents a potential molecular target for the development of therapies against feline mammary cancers.     

Detection of canine oral papillomavirus-DNA in canine oral squamous cell carcinomas and p53 overexpressing skin papillomas of the dog using the polymerase chain reaction and non-radioactive in situ hybridization

Nineteen cutaneous and mucocutaneous papillomas, as well as 29 oral and 25 non-oral squamous cell carcinomas of dogs were analyzed immunohistologically for the presence of papillomavirus (PV)-antigens. Canine oral papillomavirus (COPV)-DNA was detected in formalin-fixed, paraffin-embedded tissues by polymerase chain reaction (PCR) and non-radioactive in situ hybridization (ISH). Furthermore, the expression of the tumor suppressor protein p53 was investigated. PV-antigens were detectable in more than 50% of the oral and cutaneous papillomas, while no PV-antigens could be demonstrated in venereal papillomas. One squamous cell carcinoma was PV-antigen positive. Only two cutaneous papillomas of the head showed a strong p53-specific immunostaining, while overexpressed p53 was detectable in approximately 35% of all squamous cell carcinomas. It was possible to amplify fragments of the E6, E7 and L1 gene by polymerase chain reaction (PCR) from five of eight oral and from five of eight cutaneous papillomas as well as from three oral squamous cell carcinomas. Nine of 10 papillomas showed a strong nucleus-associated hybridization signal typical for COPV-DNA. In three squamous cell carcinomas COPV-DNA was located in nests of the epithelial tumor cells surrounding ‘horn pearls' or disseminated in the carcinoma tissue. These observations support the view that COPV may also induce non-oral papillomas in the dog and confirm the opinion that a progression of viral papillomas into carcinomas in dogs may occur.     

牛瑟氏泰勒虫p23-IL-18融合基因的原核表达

为了研发牛瑟氏泰勒虫病基因工程亚单位疫苗,本实验将p23-IL-18融合基因克隆到pET-28a原核表达载体,构建pET-28a-p23-IL-18重组原核表达质粒,经BamHⅠ和EcoRⅠ双酶切鉴定后在37 ℃用IPTG诱导表达,对表达产物进行SDS-PAGE和Western blotting分析。结果表明,成功构建牛瑟氏泰勒虫pET-28a-p23-IL-18原核表达质粒,目的基因在大肠杆菌中成功获得表达,融合蛋白的分子量约为48ku,并被牛瑟氏泰勒虫阳性血清所识别,具有良好的反应原性。本研究结果为牛瑟氏泰勒虫病基因工程亚单位疫苗的制备奠定了基础。     

Radiosensitivity of canine osteosarcoma cells transfected with wild‐type p53 in vitro*

The p53 gene is one of the important tumour suppressor genes that are involved with the cell survival signal pathway. One of the major functions of the p53 protein is to organize cell cycle regulation and induction of apoptosis for cellular genetic stability. It has been documented that more than 50% of all human cancers include a p53 mutation. We evaluated the difference in radiosensitivity between upregulating the expression of canine wild‐type p53 (cp53) in cultured osteosarcoma (D17) cells and naive D17 cells in vitro. We found that upregulating transfected cp53 D17 cells increased their radiation sensitivity in vitro, and there was a significant decrease (P < 0.009) in survival between cp53‐transfected D17 cells and naive D17 cells. In this experiment, a p53 enhancement ratio (p53ER) reached approximately 3.0 at high doses. The transfected cp53 D17 cells were significantly more radiosensitive at all doses evaluated than naive D17 cells, except at 1 Gy where too few data points were available. The p53ER increased rapidly at doses less than 4 Gy, achieving a maximum of about 3.0 for doses of 4 Gy and above. This study shows the enhanced radiosensitivity of the transfected p53 at clinically relevant doses.     

Characterization of HOX gene expression in canine mammary tumour cell lines from spontaneous tumours

Spatial/temporal controls of development are regulated by the homeotic (HOX) gene complex and require integration with oncogenes and tumour suppressors regulating cell cycle exit. Spontaneously derived neoplastic canine mammary carcinoma cell models were investigated to determine if HOX expression profiles were associated with neoplasia as HOX genes promote neoplastic potential in human cancers. Comparative assessment of human and canine breast cancer expression profiles revealed remarkable similarity for all four paralogous HOX gene clusters and several unlinked HOX genes. Five canine HOX genes were overexpressed with expression profiles consistent with oncogene‐like character (HOXA1, HOXA13, HOXD4, HOXD9 and SIX1) and three HOX genes with underexpressed profiles (HOXA11, HOXC8 and HOXC9) were also identified as was an apparent nonsense mutation in HOXC6. This data, as well as a comparative analysis of similar data from human breast cancers suggested expression of selected HOX genes in canine mammary carcinoma could be contributing to the neoplastic phenotype.     

Novel p53 tumour suppressor mutations in cases of spindle cell sarcoma,pleomorphic sarcoma and fibrosarcoma in cats

Twenty feline neoplasms were sequenced in the region from exons 5 to 8 for the presence of tumour suppressor gene p53 mutations. In a spindle cell sarcoma of the bladder, a missense mutation (codon 164 AAGGAG, lysineglutamic acid) in exon 5 was detected. In a pleomorphic sarcoma, a 23 bp deletion involving the splicing junction between intron 5 and exon 6 was observed. In a fibrosarcoma, a 6 bp deletion of p53 covering 2 bp of exon 7 and 4 bp of intron 7, including the splicing junction, was found. The study demonstrates three new p53 mutations in different types of sarcomas in cats.     

犬细小病毒结构蛋白VP2基因的克隆和在毕赤酵母中表达

表达犬细小病毒VP2蛋白（CPV—VP2）,用于重组蛋白免疫小鼠制备单克隆抗体,并为犬细小病毒病的诊断奠定基础。采用PCR方法对CPVVP2基因进行扩增,将CPVVP2基因克隆到毕赤酵母分泌表达载体pPICZAA中,构建真核重组表达载体pPICZAA-VP2,将该重组质粒线性化后,转化巴斯德毕赤酵母（Pichiapastoris）X-33中,甲醇诱导表达CPVVP2,SDs_PAGE和Westernblotting鉴定表达蛋白。结果,成功扩增了CPV—VP2基因,构建了真核重组表达载体pPICZAA—VP2,在毕赤酵母菌中表达出约68000蛋白。Western—blotting鉴定表明,表达蛋白为目的蛋白VP2。表达菌株扩大表达体系于培养基中培养,上清液用70％过硫酸铵4℃沉淀浓缩,采用His选择镍-亲和层析柱分离纯化获得重组的酵母表达的带组氨酸标签的VP2蛋白,表达量约3mg／L。结果表明,在毕赤酵母中成功地表达了CPV—VP2蛋白,且能被犬细小病毒VP2单克隆抗体特异识别。