Effect of dasatinib in a xenograft mouse model of canine histiocytic sarcoma and in vitro expression status of its potential target EPHA2.

Canine histiocytic sarcoma (HS) is an aggressive and highly metastatic tumor. Previously, the kinase inhibitor dasatinib was shown to have potent growth inhibitory activity against HS cells in vitro, possibly via targeting the EPHA2 receptor. Here, the in vivo effect of dasatinib in HS cells was investigated using a xenograft mouse model. Moreover, the expression status of EPHA2 was examined in six HS cell lines, ranging from insensitive to highly sensitive to dasatinib. In the HS xenograft mouse model, dasatinib significantly suppressed tumor growth, as illustrated by a decrease in mitotic and Ki67 indices and an increase in apoptotic index in tumor tissues. On Western blot analysis, EPHA2 was only weakly detected in all HS cell lines, regardless of sensitivity to dasatinib. Dasatinib likely results in the inhibition of xenograft tumor growth via a mechanism other than targeting EPHA2. The findings of this study suggest that dasatinib is a targeted therapy drug worthy of further exploration for the treatment of canine HS.

Selective growth inhibition by suppression of F1Fo ATPase in canine malignant melanoma cell lines.

Canine malignant melanoma (CMM) is a highly aggressive and fatal neoplasm. To identify potential therapeutic compounds and/or targets, 320 compounds were screened for their growth inhibitory activity in a CMM line (CMM-1) using a chemical library known to target specific signaling pathways/cell growth-related molecules. Among the compounds screened, the F1Fo ATPase inhibitor oligomycin showed potent growth inhibitory effects in CMM-1 cells, while exhibiting less toxic effects in a non-neoplastic control cell line (MDCK cells). The growth inhibitory effect of oligomycin A was then examined using six CMM lines and MDCK cells. Three CMM lines were highly sensitive to oligomycin A, with around 3000-20 000 times lower IC50 compared with oligomycin A-resistant CMM lines and MDCK cells. Oligomycin A-sensitive CMM-1 cells exhibited much greater oligomycin A-induced decreases in cellular ATP compared to oligomycin A-resistant cell lines. Although the oligomycins are clinically unsuitable because of its in vivo toxicity, these findings implicate the potential of F1Fo ATPase as a therapeutic target in a subset of CMM.

Effect of tyrosine kinase inhibition by imatinib mesylate on mast cell tumors in dogs.

BACKGROUND: Imatinib mesylate is a small molecule targeted at dysregulated protein-tyrosine kinase. Mutation of c-kit exon 11, which induces constitutive phosphorylation of KIT, is one of the mechanisms for the development or progression of mast cell tumor (MCT) in dogs. The purpose of this study was to examine the therapeutic potential of imatinib mesylate in canine MCT. HYPOTHESIS: Imatinib mesylate has activity against MCT in dogs, and response to treatment can be correlated to presence of mutation within exon 11 of c-kit. ANIMALS: Twenty-one dogs with MCT with gross tumor burden and median tumor size of 7.2 cm (range, 1.0-25.3 cm) before treatment. METHODS: Tumors were analyzed for mutation of c-kit exon 11. Imatinib mesylate was administered PO to the dogs at a dose of 10 mg/kg daily for 1-9 weeks. RESULTS: Ten of 21 dogs (48%) had some beneficial response to imatinib mesylate treatment within 14 days of treatment initiation. All 5 dogs with a demonstrable c-kit mutation in exon 11 responded to the drug (1 complete remission, 4 partial remission). CONCLUSIONS AND CLINICAL IMPORTANCE: Imatinib mesylate has clinical activity against MCT in dogs. Response could not be predicted based on presence of absence of a mutation in exon 11 of c-kit.

Canine squamous cell carcinoma cell lines with high expression of survivin are sensitive to survivin inhibitor YM155.

Treatment of unresectable canine squamous cell carcinoma (SCC) remains challenging and new therapeutic strategies are needed. Survivin is a member of the inhibitor of apoptosis protein family and its inhibitor, YM155, is a potential anti-tumour agent. In the present study, 10 canine tumour cell lines (representing eight different tumour types) were screened for sensitivity to YM155; the drug potently inhibited the growth of the HAPPY SCC cell line. The growth inhibitory properties of YM155 were then examined in more detail using a panel of seven SCC cell lines. YM155 inhibited the growth of the cell lines HAPPY and SQ4; in contrast to the other lines in the panel, these two cell lines had high levels of expression of survivin. In HAPPY cells, YM155 inhibited expression of the survivin gene at the transcriptional level. In contrast, YM155 down-regulated survivin at the post-transcriptional level in SQ4 cells. YM155 suppressed cell growth in HAPPY cells, mostly via induction of apoptosis, but this was not the case in SQ4 cells. Two canine SCC cell lines with high cellular expression of survivin were sensitive to YM155. The possible underlying mechanisms of the cytotoxic effect of YM155 in these cell lines were different. One cell line had down-regulation of survivin mRNA and protein expression, associated with induction of apoptotic cell death. The other cell line had post-transcriptional down-regulation of survivin expression and subsequent induction of non-apoptotic cell death. Targeting survivin with YM155 is a potential approach for the treatment of canine SCCs with high expression of survivin.

The canine prostate cancer cell line CHP-1 shows over-expression of the co-chaperone small glutamine-rich tetratricopeptide repeat-containing protein α.

Although androgen therapy resistance and poor clinical outcomes are seen in most canine prostate cancer cases, there are only a few tools for analysing canine prostate cancer by using a cell biological approach. Therefore, to evaluate androgen-independent neoplastic cell growth, a new canine prostate cancer cell line (CHP-1) was established in this study. CHP-1 over-expressed the co-chaperone small glutamine-rich tetratricopeptide repeat-containing protein α (SGTA), which is over-expressed in human androgen-independent prostate cancer. The CHP-1 xenograft also showed SGTA over-expression. Although CHP-1 shows poor androgen receptor (AR) signalling upon dihydrotestosterone stimulation, forced expression of AR enabled evaluation of AR signalling. Taken together, these results suggest that CHP-1 will be a useful model for investigating the pathogenesis of androgen-dependent and androgen-independent canine prostate cancer.

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.

Development of the polymerase chain reaction assay based on the canine genome database for detection of monoclonality in B cell lymphoma.

From the canine genome database and its bioinformatic analysis, we identified conserved sequences within the vast majority of 61 variable segments and 1 joining segment of the immunoglobulin heavy chain (IgH) gene, and designed optimal primers for polymerase chain reaction (PCR) amplification directed at these conserved sequences to evaluate the monoclonality of IgH in canine B cell lymphoma. Using the primers, a PCR-based assay was performed on fine-needle aspiration samples of normal, hyperplasia, and malignant lymph nodes and lymphoma cell lines. All fine-needle aspiration samples of five B cell lymphoma cases and the B cell lymphoma line GL-1 exhibited clonal amplification, whereas no amplification was observed in the samples from normal and hyperplasia lymph nodes, cases of T cell lymphoma, and the T cell lymphoma line CL-1. The primers we designed clearly distinguished malignant B lymphocytes from normal, reactive, and malignant T lymphocytes, indicating a potential utility of the primers for PCR-based routine clinical examination for canine B cell lymphoma.

Identification and gene expression of bovine C-type lectin dectin-2.

The C-type lectin receptor has been shown to recognize carbohydrate moieties of self and non-self antigens, thus serving as an innate immune receptor. Using bioinformatics and molecular cloning techniques, we isolated a bovine gene that encodes a polypeptide of 206 amino acids with structural features shared by mouse and human dectin-2, including a high homology with mouse dectin-2 (66%), a type II configuration, a short cytoplasmic domain without tyrosine-based signal motifs, a carbohydrate recognition domain, a putative N-glycosylation site, and an EPN motif involved in the Ca(2+)-dependent binding of hexose carbohydrates. These results reveal this bovine gene to be a counterpart of mouse dectin-2. Moreover, the bovine dectin-2 gene showed heterogeneity in mRNA (the generation of alternatively spliced transcript) and segmentation into six exons, which are also observed in mouse dectin-2. Inconsistent with mouse dectin-2 mRNA, the bovine counterpart is abundantly expressed by Langerhans cells compared to macrophages; however, lymph nodes showed the highest expression level of bovine dectin-2, while spleen and lung showed the highest expression levels of mouse and human dectin-2. In cattle, dectin-2 expressed by dendritic cells may be clinically involved in the recognition of invading antigens in lymph nodes.

Identification and cornification-related gene expression of canine keratinocyte differentiation-associated protein, Kdap.

The outermost layer of skin, the epidermis, is cornified epithelial tissue composed of keratinocytes. To maintain the structure and function of the epidermis, the regulation of proliferation, differentiation, and cornification of keratinocytes is crucial, and various soluble factors secreted by keratinocytes are involved in these regulations. Previously, work has shown that keratinocytes secreted the protein Kdap (keratinocyte differentiation-associated protein) associated with the formation of cornified cell envelopes, a specialized protective barrier structure on the periphery of terminally differentiating keratinocytes. In the present report, the canine counterpart of human Kdap is identified and an attempt has been made to define its physiological role in canine keratinization. Canine Kdap (cKdap) showed structural features commonly observed in other counterparts and is secreted from transfected cells. The expression profile of cKdap mRNA, which was restrictively expressed in cornified epithelial tissues besides skin has also been determined. These findings indicate that there is a strong association between cKdap expression and cornification, which supports previous observations that Kdap is involved in the synthesis and/or degradation of cornified cell envelopes in humans and mice.

Characterization of cDNA and the genomic sequence encoding canine neural-cell adhesion molecule, CD56/N-CAM.

The neural-cell adhesion molecule, CD56/N-CAM is a member of the immunoglobulin superfamily expressed by various tissues and cells, including natural killer (NK) cells. Despite the importance of CD56 as a marker for identifying NK cells in circulating blood, canine CD56 has not been identified. In the present study, we identified the canine counterparts of the 140-kDa (CD56-140) and 120-kDa (CD56-120) isoforms of human DC56. Both of amino acid sequences encoded by the canine CD56-140 and -120 cDNA showed high homology with those of human (both 96% homology), having well-conserved domains (five immunoglobulin, two fibronectin type III, and transmembrane and intracellular or glycosylphosphatidylinositol-linked domain) among various species (human, mouse, and feline). We revealed that the transcripts of canine CD56-140 and -120 arise from alternative mRNA splicing from a single gene located on canine chromosome 5. Moreover, the mRNA encoding canine CD56-140 was expressed at high levels constitutively by nervous system and endocrine tissues as has shown in other animals.

Comparison of expression of glucokinase gene and activities of enzymes related to glucose metabolism in livers between dog and cat.

Plasma glucose and immunoreactive insulin (IRI) concentrations and activities of enzymes related to glucose metabolism in livers were measured in dogs and cats. Nucleotide sequences of the conserved region of glucokinase (GK) cDNA that contained ATP- and glucose-binding domains were determined in canine liver and feline pancreas for design of the species-specific oligonucleotide primers for reverse transcription-polymerase chain reaction (RT-PCR) analysis. There were no significant differences in plasma glucose and IRI concentrations between dogs and cats. In feline liver, although GK activities were not detected, activities of hexokinase, fructokinase, pyruvate kinase, glucose-6-phosphate dehydrogenase, fructose-1,6-bisphosphatase and glucose-6-phosphatase were significantly higher than those in canine liver. The partial sequences of canine liver GK and feline pancreas GK cDNA were respectively 88% and 89% identical with the rat liver GK cDNA. Expression of GK gene was observed in canine liver and pancreas and feline pancreas with RT-PCR using species specific primers based on the cDNA sequences.

Canine epidermal langerhans cells express alpha and gamma but not beta chains of high-affinity IgE receptor.

Epidermal Langerhans cells (LC) express a high-affinity receptor for IgE (FcepsilonRI), consisting of two chains (alpha and gamma chains) in humans that allows LC to perform Fc receptor-mediated uptake of allergens. We found that canine LC express alpha and gamma chains but not beta chain of FcepsilonRI, identical to human but not to mouse LC, which do not express functional FcepsilonRI (only gamma chain is expressed). This finding indicates that canine LC have FcepsilonRI-mediated function similar to or identical to human LC, raising the possibility that canine species provides a better model than mouse to understand the pathogenesis of human atopic dermatitis and investigate the therapeutic effect of drugs.

Mitochondrial function in Babesia microti and Babesia rodhaini.

The role of mitochondria in the energy metabolism of Babesia microti and Babesia rodhaini was investigated. A variety of mitochondrial inhibitors showed greater sensitivity to B. microti than to B. rodhaini. Additionally, alpha-glycerophosphate- and succinate-cytochrome c reductase activities in the crude mitochondrial fraction from B. microti were substantially higher than those from B. rodhaini. Our results suggest that the mitochondria of these parasites possess a series of "classical" apparati for energy production and their relative functional role may be quantitatively greater in B. microti when compared with B. rodhaini.

Activation of HIV in human skin by ultraviolet B radiation and its inhibition by NFkappaB blocking agents.

To determine whether ultraviolet B (UVB) irradiation leads to activation of HIV in human skin, we conducted prospective and controlled studies in two academic medical centers in Texas from July 1995 to April 1999. HIV-positive patients with UV-treatable skin diseases were enrolled at each center, 18 subjects at one and 16 at the other. In one center, specimens from lesional and nonlesional skin biopsies were taken before and after sham- or UVB-irradiation administered in vivo or in vitro. In the other center, UVB phototherapy was administered three times weekly and specimens from skin biopsies were taken before and after 2 weeks (six treatments). Cutaneous HIV load was assessed using reverse transcriptase-polymerase chain reaction and reverse transcriptase-polymerase chain reaction in situ hybridization. UVB irradiation led to a 6-10-fold increase in the number of HIV in skin. To ascertain a role for nuclear factor kappa B (NFkappaB) in UVB-inducible HIV activation, two types of blockers, NFkappaB oligonucleotide decoy and sodium salicylate, were tested; each inhibited UVB-inducible HIV activation in skin partially. We conclude that UVB irradiation leads to increased numbers of HIV in human skin via processes that include release of cytoplasmic NFkappaB.

Proline, leucine, and alanine transport in placental microvillous membrane vesicles prepared from late gestational rats.

To characterize the active transport of amino acids across the placenta, uptakes of proline, leucine, and alanine were kinetically examined in placental microvillous membrane vesicles (PMV) prepared from rats in the late gestational period. Uptake rates of these amino acids in PMV showed saturable hyperbolic curves that obeyed Michaelis-Menten kinetics. Proline, leucine, and alanine transport were demonstrated to be carrier mediated systems with sodium-dependent, -independent, and both manner, respectively. In addition, sodium-dependent L-alanine transport showed two different systems, and new sodium-independent alanine transport system (K(m) of 1.12 mM) was observed in rat placenta. From these results, rat placenta has carrier mediated amino acid transport systems, and possesses at least three different transport systems for alanine.

Effects of epidermal growth factor on maternal and fetal serum amino acid levels in rats.

Pregnant rats were subcutaneously administered with mouse epidermal growth factor (EGF) at the concentration of 0, 100, or 200 micrograms/kg body weight/day from day 18 to 21 of gestation. The amino acid analysis by high-performance liquid chromatography demonstrated that the umbilical venous/maternal and fetal/maternal ratio of serum proline concentration increased in EGF dose-dependent manner accompanied by the increase in the ratios of total fetal weight and placental weight to maternal body weight gain. These results suggested that EGF regulates fetal growth by, as one of its possible mechanism, promoting placental proline supply from mother to fetus.

Hepatic oxygen supply, energy charge, and histological findings in dogs with portal vein arterialization.

Hepatic oxygen supply, energy charge (EC), and histology were examined comparatively in dogs with portal vein anastomosis (PVA group), and PA in addition to PVA (PA group). The PVA group showed a lower level of hepatic oxygen supply than those of the PA group throughout the experimental period, and also showed decreases of adenosine triphosphate (ATP) and EC level after blood perfusion. In contrast, the oxygen supply and consumption were stable in the PA group. A temporary fall of ATP level was followed by recovery to the preperfusion level in the PA group. Histological examination indicated the collapse of hepatic cords with granular and vacuolar degeneration in only the PVA group. These findings suggested that PA, when supplemented to PVA, is an available technique for preventing hepatic failure caused by ischemic conditions.

Changes of hepatic tissue phospholipid peroxidation, malondialdehydes, and antioxidative enzyme activities in dogs with halothane inhalation.

To elucidate the pathogenesis of halothane-induced hepatopathy, the changes of hepatic tissue phospholipid peroxidation, malondialdehydes (MDAs), and antioxidative enzyme activities were examined in the portal vein arterialized dogs with halothane inhalation. In group A, which was given halothane inhalation under the hepatic blood flow volume less than 10% of pre-operation volume designated as a hypoxic condition, peroxidized phosphatidylcholine (PC), and free and protein-bound MDA levels significantly increased after inhalation. Although the level of protein bound MDA in group C, given hypoxic condition alone, also increased during the experimental period, the response of this was smaller than that in group A, suggesting that the halothane inhalation enhanced free radical generation under the hypoxic condition. In contrast, no significant changes of these levels were observed in groups B and D, both of which were supplied with sufficient hepatic oxygen as the normoxic condition. In addition, the significant negative correlations between hepatic oxygen supply and total or protein-bound MDA were observed in only halothane inhaled group. These findings suggested that the cause of halothane-induced hepatopathy is closely related to free radicals mainly generated from halothane anaerobic metabolism under the hypoxic condition.

Lipid peroxidation, antioxidative enzyme activities, and cytosolic free calcium levels in rat hippocampus-derived cells exposed to free radicals.

To elucidate mechanisms of free radical-induced neuronal cell death, lipid peroxidation measured as thiobarbituric acid-reactive substances (TBARS), three antioxidative enzyme activities (superoxide dismutase, glutathione peroxidase, and catalse), and cytosolic free Ca2+ (Ca2+i) were examined in rat hippocampus-derived cells (HV16-4) exposed to free radicals generated by a hypoxanthine-xanthine oxidase system. The viability of cells decreased with an increase in numbers of free radical positive cells in a dose-dependent manner of xanthine oxidase. The protein-bound TBARS did not change, whereas free TBARS increased at 135% of initial value. No remarkable change was observed in three antioxidative enzyme activities. On the other hand, Ca2+i increased after exposure followed by cell death. Furthermore, the addition of Co2+, a nonspecific Ca2+ channel blocker, delayed the increase of Ca2+i and subsequent cell death. These findings suggested that the influx of Ca2+ played a crucial role for HV16-4 cell death induced by free radicals.

Utilization of intestinal triglyceride-rich lipoproteins in mammary gland of cows.

Elution profiles of total lipoproteins, apolipoprotein B (apoB) concentrations in lipoproteins, and plasma triglyceride (TG) levels were examined in early-, late-, and non-lactating cows. Additionally, arteriovenous (A-V) differences were also measured to elucidate the uptake of TG and apoB-containing lipoproteins in mammary gland. Non-lactating cows showed three major peaks corresponding to triglyceride-rich lipoprotein (TRL), low density lipoprotein (LDL), and high density lipoprotein (HDL) fraction, whereas both early- and late-lactating cows revealed two peaks corresponding to TRL and HDL. The peak area of TRL in early- and late-lactating cows were significantly (p < 0.05) smaller than that in non-lactating cows. The plasma TG levels and apoB-48 concentrations of TRL in early- and late-lactating cows were also significantly (p < 0.01) lower. Furthermore, early lactating cows showed significantly (p < 0.05) larger A-V differences in both plasma TG and apoB-48 concentration of TRL than those in late- and non-lactating cows. These results suggested that TG in exogenous (intestinal) TRL was utilized for milk fat synthesis in lactating mammary gland of cows by the receptor-mediated uptake.