PTEN 基因在犬乳腺肿瘤组织中的表达及临床意义

有关酪氨酸磷酸酶基因（Phosphatase and tensin homolog deleted on chromosometen,PTEN）在乳腺肿瘤中的检测在人医早有报道。为了研究PTEN基因在犬乳腺肿瘤组织中的表达情况,笔者运用实时荧光PCR定量检测了38例不同的犬乳腺肿瘤组织（包括15例良性乳腺肿瘤和23例恶性乳腺肿瘤）、4例正常犬乳腺组织。结果发现：PTEN基因在犬恶性乳腺肿瘤组织中表达量明显低于其在良性乳腺肿瘤和正常乳腺组织中的表达量,两者差异极显著（P〈0.001）;PTEN在良性乳腺肿瘤组织中的表达与正常犬乳腺组织相比,差异不显著（P〉0.05）;发生了淋巴结转移的乳腺癌PTEN基因的表达量与未发生转移的乳腺癌组织的表达量间差异亦不显著（P〉0.05）,且PTEN的表达量与肿瘤组织的大小和发病动物年龄无关。结论：PTEN蛋白表达异常可能与乳腺肿瘤发生、发展相关,可考虑作为判断犬乳腺肿瘤生物学行为和预测的指标。     

Alteration of somatostatin receptor 2 expression in canine mammary gland tumor

Somatostatin receptor 2 (SSTR2) is a negative regulator of cell proliferation in human breast cancer. Since there is little information about SSTR2 in canine mammary gland tumor (MGT), we clarified its distribution and expression level in normal mammary gland, benign MGT and malignant MGT. SSTR2 expression determined by immunohistochemical staining was observed in the cytoplasm of luminal epithelial cells. The intensity was negatively correlated with malignancy: normal tissues and some of the benign tumors had the highest levels, while the malignant tumors had little or no SSTR2 expression. As for the Western blotting, SSTR2 protein level in benign tumors was significantly lower than the normal mammary gland. On the other hand, SSTR2 protein levels in two of three malignant tumors were higher than the other groups. These results suggest that SSTR2 expression alters according to the malignancy of canine MGT.     

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.     

Expression patterns of the BRCA1 splicing variants in canine normal tissues and mammary gland tumors

Human BRCA1 is familial breast cancer susceptibility gene. Recently, decreased BRCA1 mRNA and protein expression has been identified in sporadic breast tumors. In the reported human BRCA1 splicing variants, delta11b lacks the majority of exon11 and is suspected to have a distinct function in normal tissues. The splicing variants display a variety of expression pattern in breast cancer samples. Although mammary gland tumor is important disease in dog, there are few reports for BRCA1 in the canine tumors. In this study, we examined the relative amounts of BRCA1 splicing variants mRNA in canine normal and mammary tumor samples by RT-PCR to investigate whether there is the altered expression of variant mRNAs in the canine tumor as reported in human. The exon11b-defecting RT-PCR products were observed in all the normal tissues examined and the nucleotide sequence was quite similar to that of human BRCA1 delta11b. In some tumor samples, we did not detect the products targeted for exon10-13 and exon14-15, while these products were observed in all the normal samples examined. Especially, the relative amounts of the exon11-defecting products were remarkably decreased in most of the tumors (11/16).     

The expression of carbonic anhydrase in canine mammary gland and mammary gland tumor

Carbonic anhydrase (CA), a metallo-enzyme containing zinc, broadly distributes in mammalian tissues and participates in physiological regulation such as respiration, acid-base balance, ion transport, bone resorption, as well as the development of tumor by the reversible hydration of carbon dioxide. However the expression of CA in the tissue of mammary gland tumor was not documented. In this study we examine the histolocalization and gene expression of CA in both normal canine mammary gland tissue and mammary gland tumor by histochemical examination, and RT-PCR. Four mRNA expression of CA isoenzymes, such as CA II, IV, VI and IX were found under RT-PCR analysis and different band patterns were found between normal canine mammary tissue and canine mammary gland tumor tissue. CA II, IV, VI and IX gene mRNA expression were found in the normal mammary gland tissue, indicating CA II, IV, VI and IX are likely to be the essential enzymes to maintain the normal physiological condition of canine mammary gland tissue cells. However the expression of CA IV was not found in the tissue of malignant mammary gland tumor that may become the marker for the prognostic recognition of canine mammary gland tumor.     

NLRP3炎症小体及其下游炎症因子在犬乳腺肿瘤组织中的表达

本研究旨在明确NLRP3炎症小体及下游炎症因子在犬乳腺肿瘤中的表达及临床意义.采用RT-qPCR方法检测45例犬乳腺肿瘤组织(15例良性肿瘤和30例恶性肿瘤)以及16例肿瘤旁正常乳腺组织中的NLRP3、Caspase-1、IL-1β和IL-18 mRNA的表达水平,并分析NLRP3与肿瘤临床病理学特征的关系(肿瘤大小、...     

Immunohistochemical expression of estrogen receptor beta in normal and tumoral canine mammary glands

To date, two isoforms of estrogen receptors (ER) have been identified, cloned, and characterized from several species, estrogen receptor-alpha (ERalpha) and estrogen receptor-beta (ERbeta). Although the presence of ERalpha has been demonstrated in normal and tumoral canine mammary tissues, the issue of ERbeta expression has not been addressed in the dog. In this study, we have analyzed the expression of ERbeta in formalin-fixed, paraffin-embedded tissue samples of nonaltered mammary gland, 30 malignant (six complex carcinoma, 12 simple carcinoma, three carcinosarcoma, and nine carcinoma or sarcoma in benign tumor), and five benign (one fibroadenoma, one complex papilloma, one complex adenoma, and two benign mixed tumors) mammary tumors of the dog by using a polyclonal ERbeta antibody and the avidin-biotin-peroxidase complex immunohistochemical technique. Our results show that high numbers of normal ductal and acinar epithelium and approximately one third of canine mammary tumors express ERbeta. This expression was higher in benign than in malignant tumors. Furthermore, expression was higher in complex and mixed histologic subtypes of malignant tumors when compared with simple subtypes.     

Expression of a tumor-associated antigen, RCAS1, in canine mammary tumors

Receptor-binding cancer antigen expressed on SiSo cells (RCAS1), one of novel cancer cell-surface antigens, is strongly expressed in invasive cancers. RCAS1 inhibits the in vitro growth of lymphocytes such as T cells and natural killer (NK) cells, and induces apoptotic cell death. We investigated the expression of RCAS1 in canine mammary tumor cell lines and tumor cells by immunohistochemistry, and also in situ deoxyribonucleic acid (DNA) fragmentation in tumor-infiltrating lymphocytes (TILs) by the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling (TUNEL) method. All canine mammary tumor cell lines expressed RCAS1 at both the messenger ribonucleic acid (mRNA) and protein level. Immunohistochemically, RCAS1 was negative in 100% of normal mammary glands, but was expressed in 100% of malignant tumors examined. In most malignant mammary tumors, RCAS1 was localized in the cytoplasm with no polarity of expression. In benign mammary tumors, it was detected on the luminal surface of the tumor cell. RCAS1 expression or localization was significantly correlated with malignancy. In situ DNA fragmentation of CD3-positive TILs was observed in RCAS1-expressing tumors. RCAS1-expressing tumors, indicating a possible induction of apoptotic cell death in TILs through RCAS1 expression. These observations suggest that RCAS1 probably plays an important role in tumor progression and escape from immune surveillance in canine mammary tumors.     

Calreticulin expression in neoplastic versus normal dog mammary glands: a cDNA subtraction-based study

This is the first report describing the expression of canine calreticulin (cCRT) in canine mammary gland tumour (MGT). Using cDNA subtraction method, it is found that mRNAs of CRT, cathepsin A, ovostatin, and lactotransferrin were differentially expressed in mammary adenocarcinoma as compared to hyperplasia, both of which were obtained from the dog. Furthermore, the mRNA expression levels of CRT and cathepsin A were significantly higher in canine MGT samples than in nontumour samples. In contrast, immunohistochemical studies have indicated that the expression of cCRT protein found to be detected in most of mammary gland tissues and was not correlated to the types of canine MGTs. Furthermore, cCRT was molecularly cloned, and the amino acid sequence of cCRT was found to be very similar to those of other species. Further studies are required to elucidate additional roles of cCRT in canine MGT.     

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.     

Cloning and expression analysis of prohibitin mRNA in canine mammary tumors

Prohibitin is an antiproliferative protein that is a product of a putative tumor suppressor gene. However, there is little information on prohibitins in companion animals. In this study, we cloned canine prohibitin mRNA using RT-PCR and 3′-RACE (Rapid Amplification of cDNA Ends). The sequence was well conserved compared with those of other mammals, including human. The deduced amino acid sequence translated from the open reading frame completely corresponded to the human sequence. Canine prohibitin mRNA was expressed in all normal mammary and tumor samples examined. These results suggest that this protein plays a vital role in cell growth mechanisms and may be related to the occurrence of canine mammary tumors.     

Computerized morphometry of mean nuclear diameter and nuclear roundness in canine mammary gland tumors on cytologic smears

BACKGROUND: The use of computer-based image analysis systems in veterinary oncology has increased. Computerized morphometry is a part of image analysis that describes geometric figures of cellular structures in any dimension. Most investigators have performed morphometric analysis on histologic specimens. Computer-assisted nuclear cytomorphometry can provide important preoperative information on neoplastic lesions in animals. OBJECTIVES: The aim of this study was to define whether the morphometric parameters of mean nuclear diameter and nuclear roundness could be used to differentiate benign from malignant canine mammary gland tumors on cytologic specimens. METHODS: Mean nuclear diameter and nuclear roundness were determined by computer-assisted morphometry of epithelial cells in Hemacolor-stained cytologic smears from normal canine mammary gland (n = 7) and from canine mammary adenomas (n = 8), tubulopapillary carcinomas (n = 9), and solid carcinomas (n = 6). Data were analyzed by the Mann-Whitney U test. RESULTS: Significant differences (P <.001) were found in mean nuclear diameter and nuclear roundness among all tumor types and in comparison with normal canine mammary gland epithelial cells (except for nuclear roundness between tubulopapillary carcinomas and solid carcinomas). CONCLUSIONS: The morphometric parameters of mean nuclear diameter and nuclear roundness can be used in the preoperative differentiation of benign from malignant canine mammary gland tumors.     

Expression of maspin in mammary gland tumors of the dog

Maspin is a serine protease inhibitor that inhibits tumor invasion and metastasis in human breast cancer and is consistently expressed by mammary myoepithelial cells (MECs). To analyze the value of maspin as a marker of the MEC layer of the normal and tumoral canine mammary gland, the immunohistochemical expression of maspin was studied in formalin-fixed tissues from 55 benign and malignant tumors (40 tumors also contained the surrounding normal mammary gland) using a commercially available monoclonal antibody. Periacinar and periductal MECs of all 40 normal mammary glands were stained by the anti-human maspin monoclonal antibody, and immunoreactivity was observed in the nucleus and cytoplasm of these cells. In addition, maspin was found in 53 (98%) of the tumors studied, reacting with the MECs in 100% of benign tumors and 93% of malignant tumors and to the epithelial cells of 16% of benign and 73% of malignant tumors. In the MEC compartment, immunoreactivity was observed in the cytoplasm of hypertrophic MECs, fusiform MECs, stellate MECs, rounded (myoepithelial) cells, and chondroblasts. In the epithelial cell compartment, immunoreactivity was observed in the cytoplasm of cells with and without squamous differentiation. Stromal myofibroblasts were unreactive. Maspin appears to be a very sensitive marker of the normal and neoplastic myoepithelium that, contrary to smooth muscle differentiation markers, does not stain stromal myofibroblasts. In addition, a subset of neoplastic epithelial cells reacted with the maspin antibody. The relationship between maspin expression in different cellular compartments of canine mammary carcinomas and the biologic aggressiveness of the disease remains to be elucidated.     

Cyclooxygenase-2 expression in canine mammary tumors

Mammary tumors are the most common neoplasms in female dogs. Induction of cyclooxygenase-2 (COX-2) has been implicated in various cancers in humans. However, expression of COX-2 has not been investigated in canine mammary tumors. Normal mammary gland (n = 4), simple or complex adenomas (n = 63), and simple or complex adenocarcinomas (n = 84) were studied by immunohistochemistry. Results showed that COX-2 was not expressed in the normal gland but was detected in 24% of adenomas and in 56% of adenocarcinomas (P < 0.001). The incidence of COX-2 expression and the intensity of the COX-2 signal were higher in adenocarcinomas than in adenomas (P < 0.001). These results demonstrate for the first time that COX-2 is induced in a proportion of canine mammary tumors and that COX-2 expression is more frequent and more intense in malignant than in benign tumors, suggesting a potential role for COX-2 in canine mammary tumorigenesis.     

Fractal dimension of canine mammary gland epithelial tumors on cytologic smears

BACKGROUND: Fractal geometry is a tool that can be used for describing, modeling, analyzing, and processing irregular and complex figures. Past investigations in medicine have revealed that fractal analysis could also be applied in tumor pathology to characterize irregular boundaries of the nuclei of tumor cells. OBJECTIVE: The aim of this study was to define whether the fractal dimension parameter could be used on cytologic specimens to differentiate benign from malignant canine mammary gland epithelial tumors. METHODS: The fractal dimension of nuclear surface was determined by computer-assisted morphometry on Hemacolor-stained cytologic smears obtained by fine needle aspiration of normal canine mammary gland epithelial cells, and cells in mammary adenomas, tubulopapillary carcinomas, solid carcinomas, and anaplastic carcinomas. Data were analyzed by Mann-Whitney U test. RESULTS: Significant differences (P <.001) were observed in mean fractal dimension among all tumor types and in comparison with normal canine mammary gland epithelial cells (except for the fractal dimension between solid carcinomas and anaplastic carcinomas). CONCLUSION: The morphometric parameter, fractal dimension, could help in the diagnostic discrimination between benign and malignant canine mammary gland epithelial tumors on cytologic specimens.     

一例犬乳腺导管乳头状癌的诊断与治疗

犬乳腺导管乳头状癌是犬乳腺肿瘤的一种,为恶性肿瘤,多发于中老年未绝育母犬。临床症状为乳腺出现肿块,大小不等,生长快速,肿瘤可以在一个或多个腺体中发展。在晚期转移的情况下,癌变会扩散到淋巴结,肺,结肠,肾脏,有时还会扩散到骨骼,及早施行手术通常预后良好。本文对一例8岁母犬的乳腺导管内乳头状癌病例的临床诊断与手术治疗情况作详细介绍,以期为犬乳腺肿瘤的临床诊治提供参考。     

The oncolytic effects of reovirus in canine solid tumor cell lines

Oncolytic virotherapy is a new strategy for cancer treatment for humans and dogs. Reovirus has been proven to be a potent oncolytic virus in human medicine. Our laboratory has previously reported that canine mast cell tumor and canine lymphoma were susceptible to reovirus. In this study, canine solid tumor cell lines (mammary gland tumor, osteosarcoma and malignant melanoma) were tested to determine their susceptibility towards reovirus. We demonstrated that reovirus induces more than 50% cell death in three canine mammary gland tumors and one canine malignant melanoma cell line. The reovirus-induced cell death occurred via the activation of caspase 3. Ras activation has been shown to be one of the important mechanisms of reovirus-susceptibility in human cancers. However, Ras activation was not related to the reovirus-susceptibility in canine solid tumor cell lines, which was similar to reports in canine mast cell tumor and canine lymphoma. The results of this study highly suggest that canine mammary gland tumor and canine malignant melanoma are also potential candidates for reovirus therapy in veterinary oncology.     

Alpha basic crystallin expression in canine mammary tumors

The aim of this study was to evaluate prognostic and/or diagnostic factors of canine mammary tumors by immunohistochemically analyzing the expression of alpha basic crystallin (αB-c). For this, formalin-fixed, paraffin-embedded blocks of 51 naturally-occurring canine mammary tumors (11 benign and 40 malignant) were used. Tissue from eight normal canine mammary glands were served as a control. Immunohistochemically, in the control mammary tissues, a few luminal epithelial cells were αB-c positive but myoepithelial cells were negative. In benign or simple type malignant tumors, αB-c expression was observed in luminal epithelial cells while the myoepithelial basal cells were negative. In benign or complex type malign tumors, positive staining was predominantly found in the cytoplasm of epithelial cells. Immunoreactivity of αB-c was also observed in neoplastic myoepithelial cells. Statistically, the number of cells immunolabeled with αB-c was found to be significantly different among tissues from normal canine mammary glands, benign lesions, and malignant tumors (p < 0.05). αB-c immunoreactivity was higher in malignant tumors than the control mammary tissues (p < 0.001). Data obtained in the current study revealed a strong association between high expression levels of αB-c and primary mammary gland tumors in canines.