RESUMO
Stromelysin-3 (ST3) has two highly conserved domains in the pro-domain. In particular, an unusual 10-amino acid residue sandwiched between the pro-domain and the catalytic domain of ST3 exists in ST3 but not in other matrix metalloproteinases (MMPs). To specifically detect ST3 expression in human tumors, we have made two kinds of ST3-specific polyclonal antibodies. One was raised against the synthetic 10-amino acid residue (88GLSARNRQKR97) specific to ST3, and the other against recombinant ST3 pro-domain (62APATQEAPRPASSLRPPRCGVPDPSDGLSARNRQKR97) containing the decapeptide and PRCGVPD sequence obtained by expression in Escherichia coli. Two protein species, 59 kDa and 45 kDa which were consistent with those expected for pro-ST3 and the mature form of ST3, were specifically detected in 100-fold concentrated conditioned media of fetal lung fibroblast by Western blot analysis. Immunohistochemical staining indicated that in infiltrating ductal breast carcinoma and squamous cell carcinoma of the uterine cervix, reactivity of those antibodies was found not only in fibroblastic cells surrounding cancer cells but also in neoplastic cells. However, reactivity of two ST3 antibodies was inhibited by excess of the synthetic peptide (10-amino acid residue) not only in fibroblastic cells but also in neoplastic cells. These findings suggest that antibodies against the ST3 specific region may cross react with the recently known membrane type-metalloproteinase (MT-MMP), which have RXKR sequences between the pro- and catalytic domain.
Assuntos
Anticorpos , Especificidade de Anticorpos , Metaloproteinase 11 da Matriz/metabolismo , Oligopeptídeos/metabolismo , Anticorpos/imunologia , Western Blotting , Carcinoma Ductal de Mama/metabolismo , Carcinoma de Células Escamosas/metabolismo , Reações Cruzadas , Feminino , Fibroblastos/metabolismo , Humanos , Imuno-Histoquímica , Metaloproteinase 11 da Matriz/química , Metaloproteinase 11 da Matriz/imunologia , Oligopeptídeos/imunologia , Estrutura Terciária de Proteína , Neoplasias Uterinas/metabolismoRESUMO
The expression of urokinase-type plasminogen activator (u-PA), its receptor (u-PAR) and metalloproteases activity were analyzed in 4 human gastric-cancer cell lines (AGS, Hs746T, SNU-1, and SNU-5), in an attempt to relate these activities to their invasive potential and tumorigenicity on the modified chorioallantoic membranes (CAM) of chick embryos. Only 1 of the 4 cell lines tested, Hs746T, expressed both u-PA and u-PAR as well as MMP-2, but not MMP-9. This cell line was both tumorigenic and highly invasive (51.3 +/- 13.1%) on a modified CAM. Its invasive capacity was comparable with that of a highly malignant human epidermoid-carcinoma cell line (HEp3), which usually showed 40 to 50% invasiveness. The 3 other cell lines all produced MMP-2 and MMP-9, but only AGS showed moderate invasiveness (24.2 +/- 8.8%). While antibodies to u-PA were significantly effective in reducing CAM invasiveness of Hs746T cells by approximately 40%, the invasiveness of the t-PA-expressing AGS cell line was not affected by anti-t-PA antibodies. These results suggest that when one of the components of the u-PA/u-PAR system (the enzyme and/or the receptor) is not produced and u-PA/u-PAR-dependent cell-surface proteolytic activity is thereby diminished, the malignant phenotype that can be determined by tumorigenicity and invasion of connective tissue on a CAM is compromised. Production of both type-IV collagenases (MMP-2 and MMP-9) cannot offset this deficiency.
Assuntos
Expressão Gênica , Invasividade Neoplásica , Receptores de Superfície Celular/genética , Neoplasias Gástricas/enzimologia , Neoplasias Gástricas/patologia , Ativador de Plasminogênio Tipo Uroquinase/genética , Animais , Northern Blotting , Embrião de Galinha , Colagenases/genética , Gelatinases/genética , Humanos , Metaloproteinase 2 da Matriz , Metaloproteinase 9 da Matriz , Metaloendopeptidases/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Ativador de Plasminogênio Tecidual/antagonistas & inibidores , Ativador de Plasminogênio Tecidual/fisiologia , Células Tumorais CultivadasRESUMO
Salmonella penetrates the basement membrane of intestinal epithelial cells into deeper tissues, in which process extracellular matrix proteases should be required. Hypothesizing that the proteases might be provided by host cells, we investigated the changes of expression of urokinase type plasminogen activator(u-PA), plasminogen activator inhibitor-1(PAI-1), and collagenases in epithelial cells(Caco-2) infected with Salmonella typhimurium. The change of mRNA levels, amount of the enzyme secretion and functional activity were analyzed by Northern blot, ELISA, and Zymography. The mRNA level of u-PA was elevated by Salmonella infection itself without any exogenous transcription regulators. u-PA was actively secreted into the medium and was enzymatically active. The synthesis and secretion of PAI-1 was increased over time from 2 hrs post infection(pi) to 8 hrs pi. Zymographic assay revealed that the secretion of collagenases (type IV, type V and interstitial collagenase) were also increased. Taken together, S. typhimurium infection might induce accumulation of pericellular proteolytic activity and consequently degrade the extracellular matrix surrounding the infected cells. These in turn might enable Salmonella to invade into deeper tissues.