Evaluation of a side population of canine lymphoma cells using Hoechst 33342 dye

Cancer stem cell (CSC) research has increased exponentially to gain further insight into the mechanisms underlying both carcinogenesis and chemotherapy resistance. The present study was performed to explore the potential value of a side population (SP) assay for identifying and characterizing putative CSCs among canine lymphoma cells. Canine lymphoma cells from cell lines and clinical samples were subjected to the SP assay consisting of Hoechst 33342 staining and subsequent flow cytometric analysis. The SP assay revealed various amounts of a SP fraction among the canine lymphoma cells. The percentages of SP were not affected by inhibitors of membrane transporters, verapamil hydrochloride, or fumitremorgin C. Most of the canine lymphoma cells expressed high levels of Bmi-1 and membrane transporter proteins such as ABCG2 and phosphorylated (p)-glycoprotein. This investigation lays the groundwork for further studies of the biological behaviors and molecular characteristics of CSCs in cases of canine lymphoma.     

Isolation and characterisation of cancer stem cells from canine osteosarcoma

There is increasing evidence that cancer is a stem cell disease. This study sought to isolate and characterise cancer stem cells from canine osteosarcoma. One human and three canine cell lines were cultured in non-adherent culture conditions using serum-starved, semi-solid media. Primitive sarcosphere colonies from all cell lines were identified under these conditions and were characterised using molecular and cytochemical techniques for embryonic stem cell markers. Expression of the embryonic stem cell-associated genes Nanog, Oct4 and STAT3 indicated a primitive phenotype. Sarcospheres could be reproduced consistently when passaged multiple times and produced adherent cell cultures when returned to normal growth conditions. Similarities between human and canine osteosarcoma cell lines add credence to the potential of the dog as a model for human disease.     

Expression of leukemia inhibitory factor and leukemia inhibitory factor receptor in the canine pituitary gland and corticotrope adenomas

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine of the IL-6 family that activates the hypothalamic-pituitary-adrenal axis and promotes corticotrope cell differentiation during development. The aim of this study was to investigate the expression of LIF and its receptor (LIFR) in the canine pituitary gland and in corticotrope adenomas, and to perform a mutation analysis of LIFR. Using immunohistochemistry, immunofluorescence, and quantitative expression analysis, LIF and LIFR expression were studied in pituitary glands of control dogs and in specimens of corticotrope adenoma tissue collected through hypophysectomy in dogs with pituitary-dependent hypercortisolism (PDH, Cushing's disease). Using sequence analysis, cDNA was screened for mutations in the LIFR. In the control pituitary tissues and corticotrope adenomas, there was a low magnitude of LIF expression. The LIFR, however, was highly expressed and co-localized with ACTH_1-24 expression. Cytoplasmatic immunoreactivity of LIFR was preserved in corticotrope adenomas and adjacent nontumorous cells of pars intermedia. No mutation was found on mutation analysis of the complete LIFR cDNA. Surprisingly, nuclear to perinuclear immunoreactivity for LIFR was present in nontumorous pituitary cells of the pars distalis in 10 of 12 tissue specimens from PDH dogs. These data show that LIFR is highly co-expressed with adrenocorticotropic hormone (ACTH) and α-melanocyte-stimulating hormone (α-MSH) in the canine pituitary gland and in corticotrope adenomas. Nuclear immunoreactivity for LIFR in nontumorous cells of the pars distalis may indicate the presence of a corticotrope adenoma.     

Intranuclear cisternal and vesicular structures in canine circumanal gland carcinoma cells

Intranuclear cisternal and vesicular structures, bound by a single membrane, were observed for the first time in some glandular "bird's eye" cells of canine circummanal gland carcinomas. The role of these intranuclear structures is not clear. It is suggested that they may be involved in cellular adaptation to an environment of nutrient deficiency, as a result of cell overcrowding.     

FSH分泌细胞在皖西白鹅脑垂体中的分布与定位

采用免疫组化SABC法并结合DAB显色技术时皖西白鹩脑垂体结构和促卵泡素(follicular stimulating hormone,FSH)分泌细胞进行了研究和定位.结果发现,皖西白鹅的垂体前叶由远侧部和结节部构成,没有中间部.垂体FSH免疫阳性细胞分布广泛,但前叶远侧部分布较多,从而表明皖西白鹅整个垂体都有FSH分泌,主要分泌部位在垂体前叶的远侧部.     

Identification of novel tumour‐associated antigens in canine mammary gland tumour

Canine mammary gland tumour (MGT) is the most common neoplasm in female dogs and has similar biological characteristics to human MGT. Spontaneous canine MGT is a more attractive clinical model in oncological research than that of the murine experimental model. Tumour‐associated antigens (TAAs), which are produced in tumour cells, are applied as tumour markers, tumour vaccine antigens and molecular targets of therapeutic drugs. In this study, we have primarily identified 13 different TAAs of canine MGT by serological immunoscreening of cDNA expression library. The results of serological mini‐arrays of identified antigens showed that CCDC41 antigen specially reacted with 35% of sera from MGT‐dogs and did not react with control sera. We also found that HSPH1 mRNA expression levels increased significantly in MGT tissues. These findings will contribute to the development of diagnostic technologies and translational target therapies for dogs. Clinical relevance : HSPH1, which is strongly expressed in the tumour tissue, will be a possible vaccine antigen of canine MGT.     

Regulation of adrenocorticotropin secretion from cultured canine anterior pituitary cells.

Pituitary cells, collected from five healthy dogs, were cultured and treated with various doses of ovine corticotropin-releasing hormone (CRH), arginine vasopressin (AVP), oxytocin (OT), or angiotensin II (AII) to determine which of these hypothalamic peptides affected adrenocorticotropin (ACTH) secretion. Of the 4 peptides, only CRH significantly increased ACTH secretion from cultured canine anterior pituitary cells. The lowest dose of CRH tested, 0.01 nM, significantly stimulated ACTH release. Co-addition of AVP, OT, or AII with CRH did not increase ACTH secretion beyond that caused by addition of CRH alone. Similarly, neither co-addition of AVP with OT, AVP with AII, or OT with AII significantly stimulated ACTH secretion. These results support a role for CRH in the physiologic regulation of ACTH secretion from the canine anterior pituitary, but do not support regulatory roles for AVP, OT, or AII.     

Unstable side population phenotype of mouse spermatogonial stem cells in vitro

Stem cells of the side population (SP) phenotype are found in many self-renewing tissues and can be identified by their unique ability to effectively exclude the dye Hoechst 33342. We previously established a method for expanding spermatogonial stem cells (SSCs) in vitro, but the frequency of SSCs is only about 1 to 2%, limiting detailed SSC analyses. In this study, we sought to isolate SSCs from in vitro cultures by exploiting their ability to exclude Hoechst 33342. In contrast to the findings of previous in vivo studies, we found that SP cells developed in a stochastic manner in vitro. Moreover, SP cells in culture were not enriched in SSCs, but they were interconvertible with non-SP cells. Although SP cells were consistently found in testes after transplantation of cultured cells, they were not enriched in SSCs. These results show that SSCs have an unstable SP phenotype and provide evidence that SSCs change their phenotype characteristics in response to their microenvironment.     

Ghrelin-stimulation test in the diagnosis of canine pituitary dwarfism

This study investigated whether ghrelin, a potent releaser of growth hormone (GH) secretion, is a valuable tool in the diagnosis of canine pituitary dwarfism. The effect of intravenous administration of ghrelin on the release of GH and other adenohypophyseal hormones was investigated in German shepherd dogs with congenital combined pituitary hormone deficiency and in healthy Beagles. Analysis of the maximal increment (i.e. difference between pre- and maximal post-ghrelin plasma hormone concentration) indicated that the GH response was significantly lower in the dwarf dogs compared with the healthy dogs. In none of the pituitary dwarfs, the ghrelin-induced plasma GH concentration exceeded 5 microg/l at any time. However, this was also true for 3 healthy dogs. In all dogs, ghrelin administration did not affect the plasma concentrations of ACTH, cortisol, TSH, LH and PRL . Thus, while a ghrelin-induced plasma GH concentration above 5 microg/l excludes GH deficiency, false-negative results may occur.     

Secretory carcinoma of the canine mammary gland

Secretory carcinoma is an uncommon variant of breast cancer, characterized by the presence of intracellular and extracellular eosinophilic secretion. Here, we report the cytologic, histologic, and immunohistochemical findings of a secretory carcinoma diagnosed in the left inguinal mammary gland of a 3-year-old female German Shepherd Dog. The fine-needle aspiration cytology showed numerous large branching sheets of neoplastic cells and isolated cells with cytoplasmic vacuoles. Histologically, the tumor was composed of cells with clear cytoplasm and prominent vacuoles that pushed the nuclei to the periphery, resembling signet ring cells. These cells were arranged in solid or tubular structures with lumenal spaces filled with eosinophilic secretion. Immunohistochemical reactions to cytokeratin (CAM 5.2) and alpha-lactalbumin were strongly positive in all neoplastic cells, and staining for vimentin and S100 protein was negative. The cytomorphologic and immunohistochemical features of this tumor are similar to those seen in tumors in women, hence enabling the diagnosis of a rare case of primary secretory carcinoma of the canine mammary gland.     

Lipomatous infiltration of the canine salivary gland

Benign connective tumours of the canine salivary glands are rare. This report describes lipomatous infiltration of parotid or submandibular salivary glands in seven dogs in which the glands were enlarged as a result of infiltration by fat cells; they appeared to have been successfully treated by local excision. The precise cause of the lipomatous infiltration in the dogs is unclear but different causes of similar lesions in humans are discussed.     

Mast cells in canine mammary gland tumour: number, distribution and EPOR positivity

Erythropoietin (EPO)-mediated mitogenic and anti-apoptotic effects involve all the cells expressing functional receptors for EPO (EPOR), as demonstrated by in vitro and in vivo studies. EPO shows pleiotropic effects and acts as an endogenous mediator of adaptive tissue response to metabolic stress protecting tissues from different injuries. Recently, the EPO/EPOR complex has been identified in several neoplastic cell lines and solid tumours. In this study, the authors investigated the mast cells (MCs) number, distribution and their immunoreactivity for EPOR in normal, dysplastic and neoplastic canine mammary gland. The results showed that MCs were more numerous in displastic glands compared with normal and neoplastic glands. As far as the EPOR immunoreactivity is concerned, we did not observe MCs reaction on cancer, in contrast with previously published data where epithelium of neoplastic gland showed an increase in EPOR expression along with the neoplastic progression. Overall, our results might be suggestive for MCs role in oncogenesis and offer new insight regarding to the expression of EPOR in mammary gland cancer in dog.     

Immunohistochemical characterisation of the lesions of canine idiopathic pericarditis

Pericardial tissue was obtained from 14 dogs with idiopathic pericarditis, and from three dogs with pericardial effusion associated with neoplastic disease, for histopathological assessment and characterisation of infiltrating leucocytes by immunohistochemistry. The major pathological change was extensive pericardial fibrosis which was generally accompanied by a mixed inflammatory response that was of greatest intensity at the cardiac surface of the tissue. Perivascular lymphoplasmacytic aggregates were present at the pleural surface and within the fibrosed pericardium. There were no features that clearly distinguished the samples from dogs with neoplastic disease from dogs with idiopathic pericarditis. The pericardial infiltrates were dominated by MAC 387+ monocyte-macrophages and plasma cells expressing immunoglobulin (Ig)A or IgG. CD3+ T lymphocytes and major histocompatibility complex (MHC) class II+ macrophages were less common, although the perivascular aggregates were mixtures of T and B lymphocytes and a proportion of fibroblasts expressed MHC class II. There was no vascular pathology or deposition of immunoglobulin or complement within vessel walls. These findings are consistent with an immune response dominated by humoral effector mechanisms (Th2 immunity) but do not clearly support a primary immune-mediated pathogenesis for idiopathic pericarditis.     

Identification of tumor-initiating cells in a canine hepatocellular carcinoma cell line

Tumor-initiating cells (TICs) or cancer stem cells (CSCs), a small subset of tumor cells, are involved in tumor initiation, progression, recurrence and metastasis. In human hepatocellular carcinoma (HCC), TICs are enriched with cell surface markers and have the ability to self-renew and differentiate tumors at a high frequency. We established a canine HCC cell line, HCC930599, and analyzed it for stem and progenitor cell marker expression using flow cytometry. HCC930599 showed high CD44 and CD29, moderate CD90, and low CD133, CD34, CD24, CD117, and CD13 expression. CD90⁺CD44⁺ and CD90⁻CD44⁺ cells were characterized using the in vitro sphere assay and an in vivo transplant model. CD90⁺CD44⁺ cells acquired enhanced self-renewal capacity, proliferative activity and tumourigenicity compared with CD90⁻CD44⁺ cells, suggesting that TICs exist in the HCC930599 cell line and that CD90 is a marker for enriched TICs. Understanding TIC characteristics may help elucidate hepatic carcinogenesis and HCC therapy development.