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
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)
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.
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.