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- Author or Editor: Rosa Rabanal x
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Abstract
Objective—To analyze the expression of versican and hyaluronan in melanocytomas and malignant melanomas of dogs, to correlate their expression with expression of the hyaluronan receptor CD44, and to identify enzymes responsible for the synthesis and degradation of hyaluronan in canine dermal fibroblasts and canine melanoma cell lines.
Sample Population—35 biopsy specimens from melanocytic tumors of dogs, canine primary dermal fibroblasts, and 3 canine melanoma cell lines.
Procedures—Versican, hyaluronan, and CD44 were detected in tumor samples by use of histochemical or immunohistochemical methods. Expression of hyaluronan-metabolizing enzymes was analyzed with a reverse transcriptase–PCR assay.
Results—Versican was found only in some hair follicles and around some blood vessels in normal canine skin, whereas hyaluronan was primarily found within the dermis. Hyaluronan was found in connective tissue of the oral mucosa. Versican and, to a lesser extent, hyaluronan were significantly overexpressed in malignant melanomas, compared with expression in melanocytomas. No significant difference was found between malignant tumors from oral or cutaneous origin. The expression of both molecules was correlated, but hyaluronan had a more extensive distribution than versican. Versican and hyaluronan were mainly associated with tumor stroma. Canine fibroblasts and melanoma cell lines expressed hyaluronan synthase 2 and 3 (but not 1) and hyaluronidase 1 and 2.
Conclusions and Clinical Relevance—Versican may be useful as a diagnostic marker for melanocytic tumors in dogs. Knowledge of the enzymes involved in hyaluronan metabolism could reveal new potential therapeutic targets.
Abstract
Objective—To evaluate molecular abnormalities in the c-kit gene of canine mast cell tumors (MCT) with different grades of cellular differentiation.
Sample Population—31 normal tissue specimens from dogs and 45 canine MCT classified according to grade of cell differentiation.
Procedure—Genomic DNA extractions were made from canine MCT and normal tissues. Parts of exon 11, intron 11, and exon 12 of the c-kit gene were amplified by use of polymerase chain reaction. These regions were cloned, sequenced, and compared with GenBank sequences of the National Center for Biotechnology International. A statistical analysis was used to compare sequences from canine MCT and normal tissues.
Results—A significantly higher percentage of homozygous intron 11 deletion was found in canine MCT (49%) than in normal tissues (13%). This percentage was also higher in moderately and poorly differentiated MCT, compared with well-differentiated MCT. Although no mutations were detected in any of the specimens, a polymorphism at amino acid position 606 of the canine c-kit sequence was found in all the studied sequences.
Conclusion and Clinical Relevance—Results indicated a relationship between intron 11 deletion and MCT, and the grade of MCT differentiation. We suggest that intron 11 deletion may be implicated in the pathogenesis of MCT and could be used as a marker for diagnosis and prognosis of canine MCT. (Am J Vet Res 2002;63:1257–1261)
Abstract
Objective—To evaluate expression of matrix metalloproteinase (MMP)-2 and -9 and membrane-type 1 MMP (MT1-MMP) in melanocytomas and malignant melanomas of dogs, analyze in vitro production of MMPs by canine melanoma cell lines and primary dermal fibroblasts, and investigate mutual communication between tumor cells and fibroblasts and the influence of collagen on MMP regulation.
Sample—35 biopsy specimens from melanocytic tumors and primary dermal fibroblasts of dogs and 3 canine melanoma cell lines (CML-1, CML-10c2, and CML-6M).
Procedures—MMP-2, MMP-9, and MT1-MMP were detected in tumor samples by use of immunohistochemical analysis. In vitro production was analyzed via reverse transcriptase-PCR assay, immunocytochemical analysis, zymography, and immunoblotting.
Results—MMP-9 was overexpressed in malignant melanomas, compared with expression in melanocytomas, whereas no significant differences in MMP-2 and MT1-MMP immunostaining were detected. Stromal cells also often had positive staining results. In vitro, all 3 melanoma cell lines and dermal fibroblasts had evidence of MMP-2 and MT1-MMP, but only melanoma cells had evidence of MMP-9. Coculture of CML-1 or CML-10c2 cells and dermal fibroblasts induced an increase in expression of the active form of MMP-2. Culture of melanoma cells on type I collagen increased the activation state of MT1-MMP.
Conclusions and Clinical Relevance—MMP-9 expression was increased in malignant melanomas of dogs. Stromal cells were a source for MMPs. Stromal cells, in combination with matrix components such as type I collagen, can interact with tumor cells to regulate MMP production. Information about MMP production and regulation could help in the development of new treatments.
Summary
Enzyme histochemical and immunohistochemical techniques were used to examine palatine tonsils and aggregated lymphoid follicles (Peyer's patches) of the ileum in 6- to 9-day-old and in 6-month-old pigs. Histochemical techniques were used to detect α-naphthyl-acetate esterase (anae), α-naphthyl-butyrate esterase (anbe), β-glucuronidase, adenosine triphosphatase (ATPase), and acid phosphatase (AcP). Nonspecific esterases (anae, anbe) were detected in macrophages, T-cell area lymphocytes, eosinophils, fibroblastic reticular cells (frc), follicular dendritic cells (fdc), and interdigitating cells (idc). β-Glucuronidase reactivity was strong in macrophages, eosinophils, fdc, and idc, and weaker in frc. Adenosine triphosphatase reactivity was detected in B-cell area lymphocytes, fdc, frc, and idc. Cell types with acid phosphatase reactivity were macrophages, fdc, frc, and idc. Nonepithelial cells of tonsils and aggregated lymphoid follicles of the ileum had similar enzymatic reactions. In Peyer's patches, however, epithelial cells were positive for all enzymes studied; in tonsils, only nonspecific esterases were detected. Immunoperoxidase techniques were used to detect S-100 protein and cytoplasmic immunoglobulins (IgG, IgM, and IgA). The S-100 protein was detected in lymphocytes, fdc, and frc of tonsils and Peyer's patches; in tonsillar epithelial and endothelial cells; and in idc of Peyer's patches. Cytoplasmic immunoglobulins were detected in lymphoblastoid and plasmacytoid cells of follicles and diffuse lymphoid tissue in both organs and in ileal epithelial cells. Compared with those of 6-month-old pigs, cells of 6- to 9-day-old pigs stained less intensely by all enzyme histochemical techniques, and fewer cells were reactive for immunoglobulins.