Molecular cloning of canine Wilms' tumor 1 for immunohistochemical analysis in canine tissues.
Ontology highlight
ABSTRACT:
SUBMITTER: Sakai O
PROVIDER: S-EPMC5559376 | biostudies-literature | 2017 Jul
REPOSITORIES: biostudies-literature
Publications
Molecular cloning of canine Wilms' tumor 1 for immunohistochemical analysis in canine tissues.
Sakai Osamu O Sakurai Masashi M Sakai Hiroki H Kubo Masahito M Hiraoka Hiroko H Baba Kenji K Okuda Masaru M Mizuno Takuya T
The Journal of veterinary medical science 20170712 7
Wilms' tumor 1 (WT1) expression has been investigated in various human cancers as a target molecule for cancer immunotherapy. However, few studies have focused on WT1 expression in dogs. Firstly, cDNA of canine WT1 (cWT1) was molecularly cloned from normal canine kidney. The cross-reactivity of the anti-human WT1 monoclonal antibody (6F-H2) with cWT1 was confirmed via Western blotting using cells overexpressing cWT1. Immunohistochemical staining revealed that cWT1 expression was detected in all ...[more]
Similar Datasets
Project description:Fragile X mental retardation-related protein 1 (FXR1) is a cytoplasmic RNA-binding protein highly conserved among vertebrates. It has been studied for its role in muscle development, inflammation, and tumorigenesis, being related, for example, to metastasizing behavior in human and canine uveal melanoma. Anti-FXR1 antibodies have never been validated in the canine species. To investigate FXR1 expression in canine melanocytic tumors, the present study tested two commercially available polyclonal anti-human FXR1 antibodies, raised in goat and rabbit, respectively. The cross-reactivity of the anti-FXR1 antibodies was assessed by Western blot analysis, and the protein was localized by IHC in a set of normal canine tissues and in canine melanocytic tumors (10 uveal and 10 oral). Western blot results demonstrated that the antibody raised in rabbit specifically recognized the canine FXR1, while the antibody raised in goat did not cross-react with this canine protein. FXR1 protein was immunodetected using rabbit anti-FXR1 antibody, in canine normal tissues with different levels of intensity and distribution. It was also detected in 10/10 uveal and 9/10 oral melanocytic tumors. The present study validated for the first time the use of anti-FXR1 antibody in dogs and highlighted different FXR1 protein expression in canine melanocytic tumors, the significance of which is undergoing further investigations.
| S-EPMC6071178 | biostudies-literature
Project description:Macrophages have been confirmed to play a significant role in the behavior of human lymphomas, albeit no consistent data are so far available in canine lymphomas. The present study characterizes the macrophages present in cases of canine nodal lymphoma and their relationship with the histological grade and the immunophenotype. Samples from the lymph nodes of 25 dogs diagnosed with lymphoma were selected. Immunohistochemistry was used to determine the tumor immunophenotype (CD3 and CD20 antibodies) and macrophage characterization (Iba1, MAC387, CD204, CD163 and iNOS antibodies). Macrophage counting was performed in 10 randomly selected, high-power fields per sample. Generalized linear models with Poisson distribution were used for statistical analysis. A significantly greater number of macrophages (Iba1+) were detected in high-grade and B-cell lymphomas. The highest amount of both M1 (iNOS+) and M2 (CD204+ and CD163+) subtypes were observed in B-cell lymphomas. High-grade lymphomas showed a greater number of CD204+ and CD163+ cells and recently recruited MAC387+ macrophages. The latter were most abundant in T than in B-cell lymphomas. In conclusion, a significant population of macrophages is present in canine lymphomas, which constitute a heterogeneous population that shows variations in the amount and immunohistochemical profile according to the histological grade and immunophenotype.
| S-EPMC8388421 | biostudies-literature
Project description:Oral melanoma is a common canine tumor whose prognosis is considered ominous, but poorly predicted by histology alone. In the present study the gene and protein expression of Leukotriene A4 hydrolase (LTA4H) and Fragile-X-mental retardation-related protein1 (FXR1), both reported as related to metastatic potential in different tumors, were investigated in canine oral melanoma. The main aim of the study was to confirm and quantify the presence of LTA4H and FXR1 genes and protein in oral melanomas. A secondary aim was to investigate their association with histologic prognostic criteria (mitotic count, Ki-67 index). Formalin-fixed-paraffin-embedded canine oral melanomas (36) were collected and histopathological evaluation carried out. Immunolabelling for LTA4H and FXR1 and Ki-67 were performed. RT-PCR evaluated LTA4H and FXR1 gene expressions. Histologically, most tumors were epithelioid cell melanomas (19/36) and were amelanotic, mildly or moderately pigmented (5, 12 and 13/36 respectively), only 6 were highly pigmented. Mitotic count ranged 1-106, Ki-67 index ranged 4.5-52.3. Thirty-two (32/32) melanomas immunolabelled for LTA4H and 33/34 for FXR1. RT-PCR values ranged 0.76-5.11 ΔCt for LTA4H and 0.22-6.24 ΔCt for FXR1. Molecular and immunohistochemical expression of both LTA4H and FXR1 did not statically correlate with mitotic count or Ki-67 index. The present study demonstrates LTA4H and FXR1 gene and protein in canine oral melanoma, however their expression is apparently unrelated to histopathologic prognostic criteria. Although LTA4H and FXR1 seem unrelated to tumor behavior, their extensive expression in the present cohort of cases suggest that they may play a role in canine oral melanoma oncogenesis.
| S-EPMC8710725 | biostudies-literature
Project description:Somatic defects at five loci, WT1, CTNNB1, WTX, TP53 and the imprinted 11p15 region, are implicated in Wilms tumor, the commonest childhood kidney cancer. In this study we analysed all five loci in 120 Wilms tumors. We identified epigenetic 11p15 abnormalities in 69% of tumors, 37% were H19 epimutations and 32% were paternal uniparental disomy (pUPD). We identified mutations of WTX in 32%, CTNNB1 in 15%, WT1 in 12% and TP53 in 5% of tumors. We identified several significant associations: between 11p15 and WTX (P=0.007), between WT1 and CTNNB1 (P less than 0.001), between WT1 and pUPD 11p15 (P=0.01), and a strong negative association between WT1 and H19 epimutation (P less than 0.001). We next used these data to stratify Wilms tumor into three molecular Groups, based on the status at 11p15 and WT1. Group 1 tumors (63%) were defined as 11p15-mutant and WT1-normal; a third also had WTX mutations. Group 2 tumors (13%) were WT1-mutant. They either had 11p15 pUPD or were 11p15-normal. Almost all had CTNNB1 mutations but none had H19 epimutation. Group 3 tumors (25%) were defined as 11p15-normal and WT1-normal and were typically normal at all five loci (P less than 0.001). We also identified a novel clinical association between H19 epimutation and bilateral disease (P less than 0.001). These data provide new insights into the pattern, order, interactions and clinical associations of molecular events in Wilms tumor.
| S-EPMC3359888 | biostudies-other
Project description:Apoptosis inhibitor of macrophage (AIM) plays roles in survival of macrophages. In this study, we cloned canine AIM cDNA and observed its transcriptional expression levels in various tissues. The coding sequence of canine AIM was 1,023 bp encoding 340 amino acid residues, which had around 65% homology with those of the human, mouse and rat. Transcriptional expression of AIM was observed in the spleen, lung, liver and lymph node, which confirmed the expression of canine AIM in tissue macrophages. Moreover, AIM was highly expressed in one of the canine histiocytic sarcoma cell lines. CD36, the receptor of AIM, was also expressed in various tissues and these cell lines. These findings are useful to reveal the actual functions of canine AIM.
| S-EPMC4300382 | biostudies-literature
Project description:Mammalian beta-defensins are small cationic peptides possessing broad antimicrobial and physiological activities. Because dogs are particularly resilient to sexually transmitted diseases, it has been proposed that their antimicrobial peptide repertoire might provide insight into novel antimicrobial therapeutics and treatment regimens. To investigate this proposal, we cloned the full-length cDNA of three canine beta-defensin isoforms (cBD-1, -2, and -3) from canine testicular tissues. Their predicted peptides share identical N-terminal 65-amino-acid residues, including the beta-defensin consensus six-cysteine motif. The two longer isoforms, cBD-2 and -3, possess 4 and 34 additional amino acids, respectively, at the C terminus. To evaluate the antimicrobial activity of cBD, a 34-amino-acid peptide derived from the shared mature peptide region was synthesized. Canine beta-defensin displayed broad antimicrobial activity against gram-positive bacteria (Listeria monocytogenes and Staphylococcus aureus; MICs of 6 and 100 mug/ml, respectively), gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, and Neisseria gonorrhoeae; MICs of 20 to 50, 20, and 50 mug/ml, respectively), and yeast (Candida albicans; MIC of 5 to 50 mug/ml) and lower activity against Ureaplasma urealyticum and U. canigenitalium (MIC of 200 mug/ml). Antimicrobial potency was significantly reduced at salt concentrations higher than 140 mM. All three canine beta-defensins were highly expressed in testis. In situ hybridization indicated that cBD-1 was expressed primarily in Sertoli cells within the seminiferous tubules. In contrast, cBD-2 was located primarily within Leydig cells. The longest isoform, cBD-3, was detected in Sertoli cells and to a lesser extent in the interstitium. The tissue-specific expression and broad antimicrobial activity suggest that canine beta-defensins play an important role in host defense and other physiological functions of the male reproductive system.
| S-EPMC1087356 | biostudies-literature
Project description:P-glycoprotein (P-gp) is one of the best-known ATP-dependent efflux transporters, contributing to differences in pharmacokinetics and drug-drug interactions. Until now, studies on pig P-gp have been scarce. In our studies, the full-length porcine P-gp cDNA was cloned and expressed in a Madin-Darby Canine Kidney (MDCK) cell line. P-gp expression was then determined in tissues and its role in the pharmacokinetics of oral enrofloxacin in pigs was studied. The coding region of pig Abcb1 gene was 3,861?bp, encoding 1,286 amino acid residues (Mw?=?141,966). Phylogenetic analysis indicated a close evolutionary relationship between porcine P-gp and those of cow and sheep. Pig P-gp was successfully stably overexpressed in MDCK cells and had efflux activity for rhodamine 123, a substrate of P-gp. Tissue distribution analysis indicated that P-gp was highly expressed in brain capillaries, small intestine, and liver. In MDCK-pAbcb1 cells, enrofloxacin was transported by P-gp with net efflux ratio of 2.48 and the efflux function was blocked by P-gp inhibitor verapamil. High expression of P-gp in the small intestine could modify the pharmacokinetics of orally administrated enrofloxacin by increasing the Cmax, AUC and Ka, which was demonstrated using verapamil, an inhibitor of P-gp.
| S-EPMC5004175 | biostudies-literature
Project description:Phosphatase and tensin homolog (PTEN) is a critical tumor suppressor gene with a vital role in regulating cell proliferation, migration, and survival. The loss of PTEN function, either by genetic alterations or decreased protein expression, is frequent in human gliomas and has been correlated with tumor progression, grade, therapeutic resistance, and decreased overall survival in patients with glioma. While different genetic mutations in PTEN gene have been occasionally reported in canine gliomas, no alterations in protein expression have been reported. This study investigates the immunohistochemical expression of PTEN in canine gliomas to evaluate possible alterations, as those reported in human gliomas. Immunohistochemical PTEN expression and pattern distribution were analyzed in 37 spontaneous canine gliomas. Among gliomas, 52.6% cases showed high PTEN expression and 48.6% displayed reduced (13.5%) or highly reduced (35.1%) immunopositivity. Most oligodendrogliomas showed high expression (73.7%), while the majority of astrocytomas (69.2%) showed a reduced or highly reduced expression. A reduced PTEN expression was mostly associated with a heterogeneous loss of PTEN immunopositivity. These observations are in line with those reported in human gliomas and provide a rationale for future studies regarding abnormalities in PTEN expression and PI3K/Akt/mTor pathway in canine gliomas, to evaluate its prognostic and therapeutic implications.
| S-EPMC11273977 | biostudies-literature
Project description:1 The full-length, canine cholecystokinin 1 (CCK1) receptor was cloned from gallbladder tissue using RT-PCR with a combination of primers designed to interact with conserved regions of the human and rat CCK1 receptor, which also shared homology with the canine genomic sequence. 2 Analysis of the sequence of the canine CCK1 receptor revealed a 1287 base pair product, which encoded a 429 amino-acid protein. This protein was 89% identical to the human and 85% identical to the rat CCK1 receptor. 3 The canine CCK1 receptor was expressed in CHO-K cells for pharmacological characterization. In competition studies, using [(125)I]BH-CCK-8S as radioligand, the affinity values estimated for CCK receptor-selective compounds were not significantly different between the canine and human CCK1 receptors (pK(I)+/-s.e.m. at canine CCK1 receptor; L-364,718=8.82+/-0.08, L-365,260=6.61+/-0.05, YF476=7.91+/-0.15, YM022=8.28+/-0.06 and dexloxiglumide=7.53+/-0.11). Furthermore, the selectivity of these compounds between canine CCK1 and CCK2 receptors was consistent with the selectivity between the human CCK1 and CCK2 receptors. 4 Two additional forms of the canine CCK1 receptor were identified during the cloning procedure. These had three (variant #1) and six (variant #2) amino-acid differences from the wild-type canine CCK1 receptor. Variant #1 bound [(125)I]BH-CCK-8S and displayed an identical pharmacological profile to the wild-type receptor using the ligands described above. No significant binding was measured with variant #2. 5 In conclusion, we have cloned and pharmacologically characterized the canine CCK1 receptor. The data obtained will facilitate the interpretation of numerous pharmacological experiments that have been performed using canine tissue to elucidate the actions of CCK and gastrin.
| S-EPMC1576148 | biostudies-literature
Project description:In spite of major advances over the past several decades in diagnosis and treatment, breast cancer remains a global cause of morbidity and premature death for both human and veterinary patients. Due to multiple shared clinicopathological features, dogs provide an excellent model of human breast cancer, thus, a comparative oncology approach may advance our understanding of breast cancer biology and improve patient outcomes. Despite an increasing awareness of the critical role of fibrillar collagens in breast cancer biology, tumor-permissive collagen features are still ill-defined. Here, we characterize the molecular and morphological phenotypes of type I collagen in canine mammary gland tumors. Canine mammary carcinoma samples contained longer collagen fibers as well as a greater population of wider fibers compared to non-neoplastic and adenoma samples. Furthermore, the total number of collagen cross-links enriched in the stable hydroxylysine-aldehyde derived cross-links was significantly increased in neoplastic mammary gland samples compared to non-neoplastic mammary gland tissue. The mass spectrometric analyses of type I collagen revealed that in malignant mammary tumor samples, lysine residues, in particular those in the telopeptides, were markedly over-hydroxylated in comparison to non-neoplastic mammary tissue. The extent of glycosylation of hydroxylysine residues was comparable among the groups. Consistent with these data, expression levels of genes encoding lysyl hydroxylase 2 (LH2) and its molecular chaperone FK506-binding protein 65 were both significantly increased in neoplastic samples. These alterations likely lead to an increase in the LH2-mediated stable collagen cross-links in mammary carcinoma that may promote tumor cell metastasis in these patients.
| S-EPMC8060395 | biostudies-literature