RESUMO
The 92 kDa type VI collagenase (matrix metalloproteinase-9 (MMP-9)) activities on zymography assay were found to be 1-6 times higher in benign tumor breast tissues of 12 canines and 4-26 times higher in adenocarcinoma breast tissues of nine canines than that of control tissues, respectively. A full-length canine MMP-9 cDNA was cloned from the adenocarcinoma tissue by reverse transcription-PCR and 5'- and 3'-RACE. The isolated cDNA contained an open reading frame coding for a polypeptide of 704 amino acids. The predicted protein sequence displayed extensive similarity to that of known MMP-9s and contained a putative signal sequence, a propeptide, an active site with three zinc-binding histidine residues, a calcium-binding domain, a hemopexin region, and three key cysteine residues. Western blotting using MMP-9-specific antibodies prepared against the peptide corresponding to Arg(642)-Asp(704) of canine MMP-9 and Northern blotting using a MMP-9-specific cDNA fragment as a probe confirmed that MMP-9 (the 92 kDa protein band) was highly expressed in canine mammary adenocarcinoma tissues. Higher levels of MMP-9 activity were found in the sera of canines with mammary adenocarcinoma. The results indicated that MMP-9 plays an important role in the progression of a canine mammary tumor and that assay of serum MMP-9 is helpful for early diagnosis as progress of adenocarcinoma.
Assuntos
Adenocarcinoma/enzimologia , Doenças do Cão/enzimologia , Neoplasias Mamárias Animais/enzimologia , Metaloproteinase 9 da Matriz/biossíntese , Adenocarcinoma/sangue , Adenocarcinoma/veterinária , Sequência de Aminoácidos , Animais , Sequência de Bases , Biomarcadores Tumorais/sangue , Clonagem Molecular , DNA Complementar/biossíntese , DNA Complementar/química , Doenças do Cão/sangue , Cães , Neoplasias Mamárias Animais/sangue , Metaloproteinase 9 da Matriz/sangue , Metaloproteinase 9 da Matriz/química , Metaloproteinase 9 da Matriz/genética , Dados de Sequência Molecular , PrognósticoRESUMO
Expression of matrix metalloproteinases (MMPs) was examined immunohistochemically (MMP-9 and -2) and by gelatin zymography (MMP-9) in 12 benign and 12 malignant canine mammary tumours. In nine of 12 benign tumours, weak expression of MMP-9 was demonstrated immunohistochemically in the cytoplasm of neoplastic cells. In the remaining three benign tumours (mixed or complex) MMP-9 expression was observed in the neoplastic luminal epithelial cells and myoepithelial cells in some areas. The neoplastic luminal epithelial cells in 12 malignant tumours reacted strongly for MMP-9. MMP-2 immunolabelling was observed in both benign and malignant tumour tissues, but was stronger in the latter. Zymography and densitometry showed that expression of MMP-9 was higher in the benign mammary tumours than in normal canine mammary tissues, but highest in the malignant mammary tumours. In benign and malignant canine mammary tumours, the zymography and densitometry results for MMP-9 accorded well with the immunohistochemical results obtained with anti-human MMP-9 monoclonal antibody.