Development of multidrug resistance in a canine lymphoma cell line

New multidrug resistant cell lines developed from the canine B cell lymphoma cell line (GL-1) were characterized in terms of chemosensitivity to some antineoplastics and P-glycoprotein (Pgp) expression. GL-1 was continuously exposed to a culture medium containing gradually increasing levels of doxorubicin and the cells that could grow in the presence of doxorubicin were obtained. Chemosensitivity of these cells to various antineoplastics were investigated with or without verapamil, which reversed Pgp-mediated drug resistance. The expression of Pgp on the cells was also examined by Western blot analysis. As a result, three kinds of resistant cell lines, designated as GL-DOX60, 300, and 4000 were obtained. These cell lines showed stable proliferation in the medium containing 60, 300, and 4000 ng/ml, respectively. These cells were much more resistant to vincristine than doxorubicin. This resistance was strongly reversed by the presence of verapamil. On the other hand, cisplatin was effective enough in killing these derived cells. In the Western Blot analysis, some bands that reacted to the anti-human Pgp monoclonal antibodies were observed in GL-DOX4000. The cells derived from GL-1 have multidrug resistance potential mediated by canine Pgp. The cells produced in this experimental trial are considered to be useful models for various investigations on canine multidrug resistance.     

Effects of enrofloxacin and ciprofloxacin hydrochloride on canine and equine chondrocytes in culture

OBJECTIVE: To study chondrotoxic effects of enrofloxacin (ENR) and ciprofloxacin hydrochloride (CFX) on canine and equine articular chondrocytes in culture and to compare the effects with that of cultivation in Mg2+-free medium. SAMPLE POPULATION: Chondrocytes from articular cartilage of 4- and 6 -month old dogs and 2- to 4- year-old horses. PROCEDURE: Chondrocytes were cultivated with 10, 40, 80, and 160 microg of CFX/ml, 10, 50, 100, and 150 microg of ENR/ml, or in Mg2+-free medium. A live-to-dead test was performed to test cytotoxic effects. Morphologic changes were evaluated by electron microscopy. An attachment assay was used to test the ability of chondrocytes to adhere to collagen type-II coated-chamber slides in the presence of CFX and with Mg2+-free medium. RESULTS: Chondrocytes cultivated in quinolone-supplemented medium or Mg2+-free medium had a decreased ability to adhere to culture dishes. Cell shape and the actin and vimentin cytoskeleton changed in a concentration-dependent manner. These effects were not species-specific and developed with both quinolones. On day 1 of culture, adhesion of chondrocytes to collagen type II was reduced to 70 and 45% of control values in the CFX treatment and Mg2+-free treatment groups, respectively. On day 5 of culture, adhesion of chondrocytes was reduced to 45 and 40% of control values in the CFX treatment and Mg2+-free treatment groups, respectively. CONCLUSION AND CLINICAL RELEVANCE: In vitro, chondrotoxic effects of quinolones appear to be the result of irregular integrin signaling and subsequent cellular changes. Drug concentrations leading to morphologic changes in vitro may be achieved in articular cartilage in vivo.     

Comparison of growth and differentiation of normal and neoplastic canine keratinocyte cultures

Neoplastic canine keratinocytes derived from a spontaneous oral squamous cell carcinoma were maintained in culture for more than 45 passages. The presence of desmosomes and keratin filaments was demonstrated by electron microscopy and immunohistochemistry. The keratinocytes were grown in two different culture conditions to induce variations in the stage of differentiation, i.e., in submerged cultures and at the air-liquid interface. For comparison, normal canine keratinocytes were grown under the same conditions. Anisocytosis was present in neoplastic cultures grown submerged in medium. Grown at the air-liquid interface, neoplastic keratinocytes differentiated into a well-organized, multilayered stratified squamous epithelium analogous to normal keratinocytes. Rare areas of irregular growth and formation of whorls were detected. Expression of lectin binding sites and specific cell surface antigens of neoplastic and normal keratinocytes demonstrated marked similarities between the two cell lines. Neoplastic cells lacked certain surface antigens that are present on normal cells. Squamous cell carcinoma cells grew faster than normal canine keratinocytes as demonstrated by growth curve evaluation. Neoplastic keratinocytes responded to growth stimulation by epidermal growth factor and cholera toxin as do normal keratinocytes. Neoplastic cells grown in medium lacking these factors proliferated faster than growth factor stimulated normal keratinocytes.     

Novel diabetes mellitus treatment: mature canine insulin production by canine striated muscle through gene therapy

Muscle-targeted gene therapy using insulin genes has the potential to provide an inexpensive, low maintenance alternative or adjunctive treatment method for canine diabetes mellitus. A canine skeletal muscle cell line was established through primary culture, as well as through transdifferentiation of canine fibroblasts after infection with a myo-differentiation gene containing adenovirus vector. A novel mutant furin-cleavable canine preproinsulin gene insert (cppI4) was designed and created through de novo gene synthesis. Various cell lines, including the generated canine muscle cell line, were transfected with nonviral plasmids containing cppI4. Insulin and desmin immunostaining were used to prove insulin production by muscle cells and specific canine insulin ELISA to prove mature insulin secretion into the medium. The canine myoblast cultures proved positive on desmin immunostaining. All cells tolerated transfection with cppI4-containing plasmid, and double immunostaining for insulin and desmin proved present in the canine cells. Canine insulin ELISA assessment of medium of cppI4-transfected murine myoblasts and canine myoblast and fibroblast mixture proved presence of mature fully processed canine insulin, 24 and 48 h after transfection. The present study provides proof of principle that canine muscle cells can be induced to produce and secrete canine insulin on transfection with nonviral plasmid DNA containing a novel mutant canine preproinsulin gene that produces furin-cleavable canine preproinsulin. This technology could be developed to provide an alternative canine diabetes mellitus treatment option or to provide a constant source for background insulin, as well as C-peptide, alongside current treatment options.     

Evaluation of cell-surface IgE receptors on the canine mastocytoma cell line C2 maintained in continuous culture

OBJECTIVE: To assess expression and function of cell-surface IgE receptors on the canine mastocytoma cell line C2 maintained in continuous culture. SAMPLE POPULATION: C2 cells maintained in medium lacking IgE for up to 10 passages before being stored at -80 C. PROCEDURE: Cells were thawed, cultured in medium without IgE for 1 to 3 passages, sensitized for 7 days with IgE-rich serum from dogs naturally sensitized to Ascaris suum, and stimulated with antigen Asc S1 from A suum, goat polyclonal anti-canine IgE, or calcium ionophore and phorbol myristate acetate (PMA). Percentage of intracellular beta-hexosaminidase released and concentration of tumor necrosis factor-alpha (TNF-alpha) synthesized after stimulation were determined. Expression of cell-surface IgE receptors was assessed by use of a flow cytometry. RESULTS: Immunologic stimulation (antigen or anti-IgE) failed to induce release or synthesis of detectable amounts of beta-hexosaminidase or TNF-alpha. In contrast, nonimmunologic stimulation (calcium ionophore and PMA) led to release of beta-hexosaminidase (mean +/- SEM maximum release, 23.95+/-1.96%) and synthesis of TNF-alpha (maximum concentration, 34.34+/-2.34 pg/10(6) cells). As revealed by use of flow cytometry, C2 cells expressed surface IgE receptors that bound canine IgE in vitro. CONCLUSIONS: Continuous culture of the canine mastocytoma cell line C2 in medium without exogenous IgE or cytokines and other growth factors resulted in cell-surface expression of nonfunctional IgE receptors. However, C2 cells maintained in continuous culture may still be a useful tool for the evaluation of mast cell responses to nonimmunologic stimulation and IgE receptor differentiation and maturity.     

Cellular characterization of multidrug resistance P-glycoprotein, alpha fetoprotein, and neovascular endothelium-associated antigens in canine hepatocellular carcinoma and cirrhotic liver

P-glycoprotein (P-gp), which is encoded by the multidrug resistance gene (MDR-1); alpha fetoprotein (AFP); and vascular endothelium-associated antigens are well-known markers for human and canine hepatic diseases. We obtained liver tissues from 5 dogs with hepatocellular carcinoma (HCC) and 12 dogs with cirrhosis, and we performed histopathologic and immunohistochemical evaluations using anti-P-gp, anti-AFP, anti-CD31, and anti-CD34 antibodies. P-gp was expressed at higher levels in HCC than in cirrhotic livers ( P < .01), and was most commonly localized in biliary canaliculi and small ductuli. AFP was localized mainly in the cytoplasm in HCC ( P < .01) and in a few cases of cirrhosis. In both HCC and cirrhosis, the AFP-positive cells were morphologically similar to normal hepatocytes and showed an even cytoplasmic distribution of AFP. The endothelial markers CD31 and CD34 were used to investigate vascular distribution. CD31 was expressed strongly in the portal area and parenchyma in HCC, but it was rarely observed in the parenchyma in cirrhosis. CD34 expression could not be detected in both HCC and cirrhosis. This study constitutes the first comprehensive study of P-gp, AFP, and endothelial markers in canine HCC and cirrhosis. The importance of these markers in HCC and cirrhosis in dogs was demonstrated and provides a more accurate basis for a definitive diagnosis of HCC and cirrhosis in dogs.     

Alpha-fetoprotein in serum and tumor tissues in dogs with hepatocellular carcinoma.

Serum alpha-fetoprotein (AFP) concentrations were measured before and after surgical removal of tumor masses in four dogs with hepatocellular carcinoma (HCC). Localization of AFP was also examined immunohistochemically in tumor tissues. In three cases, the serum AFP concentration was 10 to 20 times higher than that of normal dogs. One to two months after surgery, the serum AFP concentration had decreased to normal range. AFP was localized in the tumor tissues in these three cases. One case, which had a low serum AFP, did not show AFP localization in tumor tissue.     

Detection of serum alpha-fetoprotein in dogs with hepatic tumors.

Serum alpha-fetoprotein (AFP) concentration was detected by use of 2 commercially available kits containing antibodies to human AFP--a radioimmunoassay and an enzymetric test. Using neonatal canine serum (a source high in AFP), it was determined that reagents from both kits were able to bind to canine AFP, but a significant difference was detected in AFP concentration. The enzymetric test was superior in detecting canine AFP. Sera from dogs were classified into 6 groups: from dogs with primary hepatic tumors only (group 1); from dogs with primary hepatic tumors and other tumors (group 2); from dogs with normal liver but with other types of neoplasia (group 3); from dogs with nonneoplastic hepatic disease and tumors originating in other organs (group 4); from dogs with nonneoplastic hepatic disease only (group 5); and from clinically normal dogs (group 6). Serum biochemical determinations (alkaline phosphatase, alanine transaminase, albumin, total protein, total bilirubin, and serum bile acids) and values from the 2 AFP assays were obtained for all dogs. Serum AFP concentration detected by the enzymetric test was significantly higher in dogs with hepatocellular carcinoma and cholangiocarcinoma. Values greater than 250 ng/ml were detected in 5 of 9 dogs with cholangiocarcinoma and in 3 of 4 dogs with hepatocellular carcinoma. High serum AFP concentration also was indicative of liver involvement in 2 of 3 dogs with primary hepatic lymphosarcoma; 2 dogs had values greater than 225 ng/ml. Serum AFP concentration in dogs with other types of hepatic tumors was less than 250 ng/ml, and serum AFP concentration could not be correlated with such tumors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prolonging hypothermic storage (4 C) of bovine embryos with fish antifreeze protein

Embryos obtained via superovulation are necessary for mammalian artificial reproduction, and viability is a key determinant of success. Nonfreezing storage at 4 C is possible, but currently used storage solutions can maintain embryo viability for only 24–48 h. Here we found that 10 mg/ml antifreeze protein (AFP) dissolved in culture medium 199 with 20% (v/v) fetal bovine serum and 25 mM HEPES could keep bovine embryos alive for 10 days at 4 C. We used a recombinant AFP isolated from the notched-fin eelpout (Zoarces elongatus Kner). Photomicroscopy indicated that the AFP–embryo interaction was enhanced at 37 C. Embryos pre-warmed with the AFP solution at 37 C for 60 min maintained high viability, whereas those that were not pre-warmed could live no longer than 7 days. Thus, short-term storage of bovine embryos was achieved by a combination of AFP-containing medium and controlled pre-warming.     

High level activity of 2', 5'-oligoadenylate synthetase in dog serum

Most animal cells that are exposed to interferon (IFN) experience an increase in the activity of 2', 5'-oligoadenylate synthetase (OAS), which is an important effector of IFN's antiviral action. OAS activity has been widely used in clinical chemistry as an indicator of IFN activity. In this study, we found that OAS activity in canine serum is 46.0 +/- 40.4 nmol/dl/hr, which is 10- to 100-fold higher than in other animals such as the cat (1.9 +/- 2.1), rabbit (4.0 +/- 1.1), and guinea pig (0.3 +/- 0.6). The canine OAS protein was detected by Western blotting using a 68M-10 monoclonal anti-murine OAS antibody, and was found to be composed of at least three distinct molecular species of p40 class OAS. Among these, the 40 and 42 kDa components were determined to be the major species in serum and fibroblast cell lines, respectively.     

德国牧羊犬成纤维细胞培养及其生长特性研究

试验旨在探讨犬耳部和腹部皮肤成纤维细胞体外培养和纯化方法,为下一步犬体细胞核移植提供基础。结果表明,在组织块培养后的4 d,耳组织周围开始有细胞出现,8~10 d呈优势生长,2周后可长满细胞瓶。腹部皮肤组织则要6~7 d方可见有细胞沿组织块周围爬出,10 d左右可以在组织块周围形成成片细胞,20 d左右可以长满整个细胞瓶。腹部皮肤成纤维细胞建系初期生长速度低于耳成纤维细胞,但传代至第4代时,两种细胞的生长曲线基本相同。     

2例犬死亡原因的实验室诊断

对昆明某犬场2例死亡犬的死亡原因进行实验室鉴定。采集犬肠内容物分别做细菌和病毒检测。细菌检测包括:犬肠内容物划线培养,纯化,分离,革兰氏染色,镜检,生化试验,血清试验,药敏试验。病毒检测包括:提取病料的总DNA,用犬细小病毒的特异引物进行PCR扩增,扩增产物经胶回收后克隆到pMD18-T载体,转化受体菌DH5α,挑取阳性克隆质粒进行序列测定,并与已知参考毒株序列进行比对,分析核苷酸的同源性。分离到一株有部分耐药性的侵袭性大肠埃希菌;检测到一株犬细小病毒,由引物P1/P2扩增的基因与参考毒株CPV-b同源性为98.58%,由引物P3/P4扩增的基因与参考毒株CPV-b同源性为92.1%。结果表明,2例死亡犬的死亡原因分别为侵袭性大肠埃希菌感染和犬细小病毒感染。     

Establishment and characterization of the growth and pulmonary metastasis of a highly lung metastasizing cell line from canine osteosarcoma in nude mice.

Highly lung metastasizing model of canine osteosarcoma in nude mice was established from five subcutaneous implantation cycles of lung tumor deposits. The selection of cells with increased metastatic properties from the parent POS canine osteosarcoma cell line recovered medium sized and polygonal Highly Metastasizing POS cells (HMPOS). The doubling time of HMPOS and POS in culture averaged 30 +/- 1.2 hr and 32 +/- 1.3 hr respectively, and their cell growth patterns in vitro were comparable to their in vivo growth patterns. HMPOS cells produced more tumor deposits (> 20 nodules, > 1 -mm in diameter) of various sizes with replacement of lung tissues at 12 weeks after implantation. POS cells produced fewer and smaller lung deposits (< 10 nodules, 1-mm in diameter). Tumor size and number of metastatic tumor deposits showed a regular association. HMPOS cells developed an osteoblastic type of cellular differentiation subcutaneously and in the lungs. HMPOS micrometastasis along the alveolar walls and blood vessels at 4 weeks averaged 6-7 small tumor locus. Each micrometastatic locus contained an average of 5-7 tumor cells, and developed a pleomorphic osteoblastic type of cellular differentiation. An average of 4 macrometastatic nodules could be seen at 6 weeks, composed of an average of 23 tumor cells, 10 nodules at 8 weeks, 12 nodules at 10 weeks and 20 nodules at 12 weeks. These model provides an opportunity for the evaluation of new treatments against canine lung metastatic osteosarcoma in a nude mice model.     

Comparison of induced corpora lutea from prepuberal gilts and spontaneous corpora lutea from mature gilts: in vitro progesterone production

Prepuberal (P) gilts were induced to ovulate with pregnant mare serum gonadotropin followed 72 h later by human chorionic gonadotropin (hCG). Three P gilts and three mature (M) gilts each were ovariectomized on d 10, 14, 18, 22 and 26 (d 0 = day of hCG for P gilts and onset of estrus for M gilts). Gilts ovariectomized on d 14, 18, 22 and 26 were hysterectomized on d 6 to ensure maintenance of the corpora lutea (CL). Two to five grams of minced luteal tissue were dispersed using collagenase and hyaluronidase in HEPES buffered salt solution supplemented with glucose and bovine serum albumin. Dispersed cells were rinsed in Dulbecco's Modified Eagle Medium (DMEM), counted (ratio of large to total number of luteal cells determined) and then incubated for 1 h in DMEM. With aliquots standardized to 2.5 X 10(4) viable, large cells (greater than 25 micron diameter) were incubated in 1 ml DMEM for 2 h in the presence of either 10, 50, 100 or 1,000 ng luteinizing hormone (LH); .1, 1, 10 or 100 ng hCG; 10, 100 or 1,000 ng norepinephrine (NE) or either .75, or 1.5 mM dibutyrl cyclic adenosine monophosphate (dbcAMP). Progesterone (P4) in the medium was quantified by radioimmunoassay. Basal P4 production (no P4 stimulator added to the medium) on d 10, 14, 18, 22 and 26 for P gilts was 246 +/- 9, 66 +/- 4, 64 +/- 6, 41 +/- 3 and 69 +/- 6 ng/ml medium, respectively, and for M gilts was 281 +/- 12, 128 +/- 8, 53 +/- 4, 82 +/- 6, 101 +/- 5 ng/ml medium, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Therapeutic efficacy of ABT-737, a Bcl-2 inhibitor, in a canine melanoma cell line

The small molecule inhibitor, ABT-737, inhibits Bcl-2 that is overexpressed in many tumor cell lines and, in combination with an anticancer drug, can strongly enhance proapoptotic activity. In the present study, we evaluated the inhibitory activity of ABT-737 on the survival of a canine melanoma cell line (MCM-N1). MCM-N1 cell viability was decreased following 24- and 48-hr culture with ABT-737, depending on ABT-737 concentration, while cell viability was unchanged in controls. ABT-737 synergized with carboplatin to promote cell death. Notably, approximately 50% of MCM-N1 cells survived following culture with 2-4 μg/ml of carboplatin; whereas, less than 20% of MCM-N1 cells survived following culture with ABT-737 (1 mM) plus carboplatin (2-10 μg/ml).     

Biological and molecular characterization of a canine hemangiosarcoma-derived cell line

Canine hemangiosarcoma (HSA) is a devastating disease. Investigation of novel therapies has been limited by the limited availability of canine HSA-derived cell lines. We report the development of a canine HSA-derived cell line, DEN-HSA, which recapitulates features of angiogenic endothelium. DEN-HSA cells were derived from a spontaneous HSA arising in the kidney of a dog. DEN-HSA displayed surface molecules distinctive of endothelial histogenesis, including factor VIII-related antigen, ICAM-1 and alpha(v)beta3 integrin. In vitro, DEN-HSA formed microvascular tube-like structures on Matrigel, and proliferated in response to a variety of angiogenic growth factors. The cells expressed mRNA and protein specific for bFGF and its receptors, and VEGF and its receptors, among others. DEN-HSA conditioned medium evoked a marked angiogenic response in a murine corneal pocket assay, indicating potent proangiogenic activity of substances secreted by this cell line. This research confirms the DEN-HSA cell line as endothelial in origin, suggests the presence of angiogenic growth factor autocrine loops, and offers the potential to utilize DEN-HSA cells for the study of novel therapies that modulate endothelial proliferation.     

猪繁殖与呼吸道综合征病毒(PRRSV)单克隆抗体的研制

利用猪繁殖与呼吸道综合征病毒（ＰＲＲＳＶ）国内分离株Ｊ１,采用反复差速离心法制备免疫抗原,长程免疫法免疫ＢＡＬＢ／ｃ小鼠,用间接ＥＬＩＳＡ方法检测抗体,通过细胞融合技术,并经３次亚克隆获得了１０株能稳定分泌抗ＰＲＲＳＶ单抗的杂交瘤细胞单克隆株（Ａ１Ｄ７Ｈ１０,Ａ１Ｄ７Ｈ１１,Ａ１Ｅ７Ｈ９,Ａ１Ｅ７Ｄ９,Ａ２Ｄ８Ｅ７,Ａ２Ｄ８Ｂ１１,Ｂ３Ｄ１１Ｄ６,Ｂ２Ｇ９Ａ９,Ｂ２Ｇ９Ｆ２）。这些细胞经体外连续传     

小鼠胚胎干细胞培养、克隆、分离、传代研究

对影响小鼠胚胎干细胞（Embryonic stem cells,ES细胞）培养、克隆、分离、传代效果的因素进行了探索研究。应用223枚昆明白小鼠胚胎和20枚129品系小鼠胚胎的研究结果表明，129品系小鼠胚胎比昆明白小鼠胚胎更适合作为ES细胞建系的材料，两者FS出现率差异显著（P＜0．05）；以DMEM＋10％NBS＋10％FCS为基础培养液，分别加入LIF、胰岛素、LIF＋SCF，极显著提高昆明白小鼠胚胎贴壁率，ICM生长率及F1、F2出现率（P＜0．01），而在DMEM＋10％NBS＋10％FCS＋LIF＋SCF为培养液，得到昆明白小鼠胚胎最高贴壁率、ICM生长率及传代率；4dpc胚胎传代情况显著好于3．5dpc胚胎（P＜0．05）。