RESUMO
The degradation of the extracellular matrix is part of many pathological and physiological processes. Of the several proteases involved in extracellular matrix turnover, the plasmin/plasminogen activator system and the family of matrix metalloproteases have received the most attention. Recent investigations in the field of matrix metalloprotease biochemistry have focused on the functions of the various enzyme domains and their interactions with inhibitor domains. Research into physiological activation mechanisms has demonstrated a plasmin/plasminogen activator-metalloprotease cascade, as well as providing an initial characterization of cell surface associated metalloprotease activation.
Assuntos
Matriz Extracelular/enzimologia , Metaloendopeptidases/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Éxons , Glicoproteínas/metabolismo , Humanos , Metaloendopeptidases/antagonistas & inibidores , Metaloendopeptidases/genética , Dados de Sequência Molecular , Inibidores Teciduais de MetaloproteinasesRESUMO
Structural changes in the extracellular matrix are necessary for cell migration during tissue remodeling and tumor invasion. Specific cleavage of laminin-5 (Ln-5) by matrix metalloprotease-2 (MMP2) was shown to induce migration of breast epithelial cells. MMP2 cleaved the Ln-5 gamma2 subunit at residue 587, exposing a putative cryptic promigratory site on Ln-5 that triggers cell motility. This altered form of Ln-5 is found in tumors and in tissues undergoing remodeling, but not in quiescent tissues. Cleavage of Ln-5 by MMP2 and the resulting activation of the Ln-5 cryptic site may provide new targets for modulation of tumor cell invasion and tissue remodeling.
Assuntos
Mama/citologia , Moléculas de Adesão Celular/metabolismo , Movimento Celular , Matriz Extracelular/metabolismo , Gelatinases/metabolismo , Metaloendopeptidases/metabolismo , Animais , Mama/metabolismo , Adesão Celular , Divisão Celular , Linhagem Celular , Tamanho Celular , Colagenases/metabolismo , Células Epiteliais , Epitélio/metabolismo , Feminino , Fibrinolisina/metabolismo , Gelatinases/antagonistas & inibidores , Humanos , Metaloproteinase 2 da Matriz , Metaloproteinase 9 da Matriz , Metaloendopeptidases/antagonistas & inibidores , Camundongos , Fenilalanina/análogos & derivados , Fenilalanina/farmacologia , Inibidores de Proteases/farmacologia , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Tiofenos/farmacologia , CalininaRESUMO
We previously demonstrated that TIMP-2 increases the association of Crk with C3G and via subsequent activation of Rap1 enhances the expression of RECK, a membrane-anchored MMP inhibitor. In the present study, we investigate the mechanism of how the TIMP-2 signal is transduced from the alpha3beta1 integrin receptor to the Crk-C3G-Rap1 molecular complex. TIMP-2 treatment of human microvascular endothelial cells (hMVECs) increased the phosphorylation levels of Src at Tyr-527, the negative regulatory site, through enhanced association of Src with Csk. This results in the reduction of Src kinase activity and dephosphorylation of paxillin at Tyr-31/118, the target sites for Src kinase phosphorylation and also the binding sites for the downstream effector Crk. Such TIMP-2 effects accompany the disassembly of paxillin-Crk-DOCK180 molecular complex and, in turn, Rac1 inactivation. On the contrary, levels of paxillin-Crk-C3G complex formation are not reduced, rather slightly increased, which is consistent with our previous finding. Therefore, TIMP-2-mediated inhibition of Src kinase activity leads to the signaling switch from Rac1 to Rap1, thereby leading to enhanced RECK expression.
Assuntos
Glicoproteínas de Membrana/biossíntese , Paxilina/metabolismo , Inibidor Tecidual de Metaloproteinase-2/fisiologia , Tirosina/metabolismo , Regulação para Cima/fisiologia , Células Cultivadas , Proteínas Ligadas por GPI , Humanos , Glicoproteínas de Membrana/genética , Fosforilação , Transdução de Sinais/fisiologia , Tirosina/antagonistas & inibidores , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rap1 de Ligação ao GTP/fisiologia , Quinases da Família src/antagonistas & inibidoresRESUMO
Cellular pathways for induction of programmed cell death (PCD) have been identified, but little is known about specific extracellular matrix processes that may affect apoptosis along those pathways. In this study, a series of Burkitt's lymphoma (BL) cell lines were assayed for their expression of tissue inhibitor of metalloproteinases (TIMP)-1. Results indicate that TIMP-1-positive BL lines show resistance to cold-shock-induced apoptosis. Furthermore, recombinant TIMP-1, but not TIMP-2 or a synthetic metalloproteinase inhibitor (BB-94), confers resistance to apoptosis induced by both CD95-dependent and -independent (cold shock, serum deprivation, and gamma-radiation) pathways in TIMP-1-negative BL lines. TIMP-1 suppression of PCD is not due to metalloproteinase inhibition, as reduction and alkylation of the TIMP-1 did not abolish this activity. Retroviral induction of TIMP-1 not only resulted in cell survival but also in continued DNA synthesis for up to 5 d in the absence of serum, while controls underwent apoptosis. This resistance to apoptosis is reversed by anti-TIMP-1 antibodies, demonstrating that secreted TIMP-1 is active in blocking apoptosis. Furthermore, TIMP-1 upregulation induced expression of Bcl-XL but not Bcl-2 as well as decreased NF-kappaB activity as compared with controls. These results demonstrate that TIMP-1 suppresses apoptosis in B cells and suggests a novel activity for TIMP-1 in tissue homeostasis.
Assuntos
Apoptose/efeitos dos fármacos , Linfócitos B/efeitos dos fármacos , Células-Tronco Neoplásicas/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-1/farmacologia , Linfócitos B/patologia , Linfoma de Burkitt/patologia , Células Cultivadas , Depressão Química , Inibidores Enzimáticos/farmacologia , Humanos , Hiperplasia , NF-kappa B/metabolismo , Tonsila Palatina/patologia , Fenilalanina/análogos & derivados , Fenilalanina/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Recombinantes/farmacologia , Tiofenos/farmacologia , Inibidor Tecidual de Metaloproteinase-1/fisiologia , Inibidor Tecidual de Metaloproteinase-2/farmacologia , Células Tumorais Cultivadas , Proteína bcl-XRESUMO
We report here that a 92-kD gelatinolytic metalloproteinase is expressed as protein and mRNA in human osteoarthritic cartilage, but not in normal adult articular cartilage. Western immunoblotting demonstrated that the 92-kD gelatinolytic activity corresponded to 92-kD type IV collagenase/gelatinase (gelatinase B); mRNA for gelatinase B was identified by Northern blotting. Chondrocytes from normal cartilage also exhibited mRNA for 72-kD type IV collagenase/gelatinase (gelatinase A), tissue collagenase, and stromelysin-1, and these mRNAs were increased in osteoarthritic cartilage. Regional analysis of osteoarthritic cartilage samples from four individuals revealed that gelatinase B mRNA was expressed in grossly fibrillated areas; two of four nonfibrillated cartilage samples failed to exhibit the mRNA, but did have increased levels of mRNA for other neutral metalloproteinases. IL-1 alpha treatment of normal human cartilage explants or isolated chondrocytes induced increased levels of gelatinase B and increased mRNA for tissue collagenase and stromelysin-1. Under identical conditions, mRNA levels for gelatinase A were not increased indicating that regulation of this enzyme in human articular chondrocytes is distinct from that of other metalloproteinases. Our data showing expression of gelatinase B in fibrillated cartilage suggest that it is a marker of progressive articular cartilage degradation in osteoarthritis.
Assuntos
Cartilagem Articular/enzimologia , Colagenases/metabolismo , Interleucina-1/farmacologia , Metaloendopeptidases/metabolismo , Adulto , Expressão Gênica/efeitos dos fármacos , Humanos , Metaloproteinase 2 da Matriz , Metaloproteinase 9 da Matriz , Pessoa de Meia-Idade , Peso Molecular , Técnicas de Cultura de Órgãos , RNA Mensageiro/genéticaRESUMO
Loss of function in the von Hippel-Lindau (VHL) tumor suppressor gene occurs in familial and most sporadic renal cell carcinomas (RCCs). VHL has been linked to the regulation of cell cycle cessation (G(0)) and to control of expression of various mRNAs such as for vascular endothelial growth factor. RCC cells express the Met receptor tyrosine kinase, and Met mediates invasion and branching morphogenesis in many cell types in response to hepatocyte growth factor/scatter factor (HGF/SF). We examined the HGF/SF responsiveness of RCC cells containing endogenous mutated (mut) forms of the VHL protein (VHL-negative RCC) with that of isogenic cells expressing exogenous wild-type (wt) VHL (VHL-positive RCC). We found that VHL-negative 786-0 and UOK-101 RCC cells were highly invasive through growth factor-reduced (GFR) Matrigel-coated filters and exhibited an extensive branching morphogenesis phenotype in response to HGF/SF in the three-dimensional (3D) GFR Matrigel cultures. In contrast, the phenotypes of A498 VHL-negative RCC cells were weaker, and isogenic RCC cells ectopically expressing wt VHL did not respond at all. We found that all VHL-negative RCC cells expressed reduced levels of tissue inhibitor of metalloproteinase 2 (TIMP-2) relative to the wt VHL-positive cells, implicating VHL in the regulation of this molecule. However, consistent with the more invasive phenotype of the 786-0 and UOK-101 VHL-negative RCC cells, the levels of TIMP-1 and TIMP-2 were reduced and levels of the matrix metalloproteinases 2 and 9 were elevated compared to the noninvasive VHL-positive RCC cells. Moreover, recombinant TIMPs completely blocked HGF/SF-mediated branching morphogenesis, while neutralizing antibodies to the TIMPs stimulated HGF/SF-mediated invasion in vitro. Thus, the loss of the VHL tumor suppressor gene is central to changes that control tissue invasiveness, and a more invasive phenotype requires additional genetic changes seen in some but not all RCC lines. These studies also demonstrate a synergy between the loss of VHL function and Met signaling.
Assuntos
Carcinoma de Células Renais/genética , Genes Supressores de Tumor , Fator de Crescimento de Hepatócito/farmacologia , Neoplasias Renais/genética , Ligases , Proteínas/genética , Proteínas Supressoras de Tumor , Ubiquitina-Proteína Ligases , Doença de von Hippel-Lindau/genética , Carcinoma de Células Renais/patologia , Carcinoma de Células Renais/fisiopatologia , Endopeptidases/metabolismo , Espaço Extracelular/enzimologia , Expressão Gênica , Fator de Crescimento de Hepatócito/antagonistas & inibidores , Fator de Crescimento de Hepatócito/fisiologia , Humanos , Neoplasias Renais/patologia , Neoplasias Renais/fisiopatologia , Invasividade Neoplásica , Fenótipo , Receptores Proteína Tirosina Quinases/metabolismo , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Inibidor Tecidual de Metaloproteinase-2/genética , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Células Tumorais Cultivadas , Proteína Supressora de Tumor Von Hippel-LindauRESUMO
The 72-kd type IV collagenase is a member of the collagenase enzyme family that has been closely linked with the invasive phenotype of cancer cells. Previous studies have shown that both normal cells and highly invasive tumor cells produce the 72-kd type IV procollagenase enzyme in a complexed form consisting of the proenzyme and a novel tissue inhibitor of metalloproteinases, TIMP-2. The balance between activated enzyme and available inhibitor is thought to be a critical determinant of the matrix proteolysis associated with a variety of pathologic processes, including tumor cell invasion. In the present study, we demonstrate that alteration of the metalloproteinase-metalloproteinase-inhibitor balance in favor of excess inhibitor blocks human fibrosarcoma HT-1080 tumor cell invasion of a reconstituted basement membrane. The HT-1080 cell line produces both the 72-kd and the 92-kd type IV collagenases. Alteration of the type IV collagenase-inhibitor balance was achieved by addition of free TIMP-2 or antibodies to 72-kd type IV collagenase. Native, purified TIMP-2 was inhibitory in the range of 1-25 micrograms/mL. Addition of specific antiserum against the 72-kd type IV collagenase, which did not cross-react with the 92-kd type IV collagenase, inhibited HT-1080 cell invasion to the same extent. These results suggest that metalloproteinases, in particular the 72-kd type IV collagenase, are critical for tumor cell invasion of the reconstituted basement membrane. Our findings demonstrate that addition of the endogenous inhibitor TIMP-2 is able to block invasion. Thus, we recommend initiation of in vivo studies of the therapeutic potential of TIMP-2 to block tumor cell invasion and intravasation into the circulation.
Assuntos
Antineoplásicos/farmacologia , Metaloendopeptidases/antagonistas & inibidores , Invasividade Neoplásica , Proteínas de Neoplasias/farmacologia , Animais , Humanos , Soros Imunes/imunologia , Camundongos , Colagenase Microbiana/antagonistas & inibidores , Colagenase Microbiana/imunologia , Colagenase Microbiana/fisiologia , Inibidor Tecidual de Metaloproteinase-2 , Células Tumorais CultivadasRESUMO
BACKGROUND: Most (70%-100%) ovarian carcinomas express high levels of the epidermal growth factor receptor (EGFR). To examine the relationship between EGFR and the invasive phenotype, we assessed integrin expression, adhesion, matrix metalloproteinase (MMP) activity, and migration in ovarian cancer cells in which EGFR expression was modified. METHODS: NIH:OVCAR-8 human ovarian carcinoma cells were transfected with an expression vector containing the human EGFR complementary DNA in an antisense orientation (EGFR-antisense cells) or the vector alone (vector control cells). We compared vector control and EGFR-antisense cells for cell morphology and adhesion by light microscopy, expression of alpha(6)- and alpha(3)-integrin subunits by flow cytometry, MMP and tissue inhibitor of MMP (TIMP) activity by zymography, and migration by a wound migration assay. In some experiments, EGFR kinase activity in parental cells was inhibited by treatment with PD153035. All statistical tests were two-sided. RESULTS: EGFR-antisense cells were morphologically distinct from vector control cells and had a selective decrease in adhesion to laminin-1 that was not observed with vector control cells (P = .008) or on other extracellular matrix substrates. Compared with vector control cells, cell surface alpha(6)-integrin expression decreased by approximately 80% (difference = 78.7%; 95% confidence interval [CI] = 77.8% to 79.6), MMP-9 activity decreased by approximately 50%, and TIMP activity increased by approximately 50% in EGFR-antisense cells. Vector control cells were highly motile (5.51 arbitrary distance unit; 95% CI = 4.98 to 6.04), whereas the EGFR-antisense cells were not (0.99 arbitrary distance units; 95% CI = 0.38 to 1.60). The morphology and integrin profile of NIH:OVCAR-8 parental cells treated with PD153035 were similar to those of the EGFR-antisense cells. CONCLUSIONS: Reduced EGFR expression in ovarian carcinoma cells decreased their adhesion to laminin-1, expression of the alpha(6)-integrin subunit (a well-characterized laminin-1 receptor), and MMP-9 activity. These data support the hypothesis that EGFR overexpression in ovarian cancer cells results in multiple phenotypic changes that enhance the invasive phenotype.
Assuntos
Carcinoma/patologia , Receptores ErbB/fisiologia , Invasividade Neoplásica/genética , Proteínas de Neoplasias/fisiologia , Neoplasias Ovarianas/patologia , Antígenos CD/biossíntese , Antígenos CD/genética , Comunicação Autócrina , Carcinoma/metabolismo , Adesão Celular , Movimento Celular , Tamanho Celular , DNA Antissenso/genética , DNA Complementar/genética , Indução Enzimática , Inibidores Enzimáticos/farmacologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Proteínas da Matriz Extracelular/química , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Integrina alfa3 , Integrina alfa6 , Integrinas/biossíntese , Integrinas/genética , Laminina/química , Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/genética , Proteínas de Neoplasias/efeitos adversos , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Neoplasias Ovarianas/metabolismo , Fenótipo , Quinazolinas/farmacologia , Transdução de Sinais , Inibidor Tecidual de Metaloproteinase-1/biossíntese , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-2/biossíntese , Inibidor Tecidual de Metaloproteinase-2/genética , Transfecção , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismoRESUMO
A basal promoter for the Mr 72,000 type IV collagenase gene was specifically defined by chloramphenicol acetyltransferase assays of a nested set of 5' upstream fragments containing the promoter region. This core promoter is TACATCT and is a noncanonical TATA box that fits the TATA consensus sequence. This sequence begins 26 base pairs in the upstream direction from the start site of transcription for the type IV collagenase gene. This basal promoter is active in the highly metastatic A2058 melanoma cell line. A putative enhancer was found between nucleotides -223 to -422 that produces a 7-fold increase in transcriptional activity in the A2058 melanoma cell line. The region immediately 5' of the basal promoter, upstream to position -422, contains a silencer and represses transcriptional activity in the nonmetastatic HT144 melanoma cell line. The results of this study are consistent with previous data that found high expression of Mr 72,000 type IV collagenase mRNA and enzymatic activity in the A2058 cell line, whereas low mRNA expression and type IV collagenase activity were found in the HT144 cell line.
Assuntos
Melanoma/genética , Colagenase Microbiana/genética , Regiões Promotoras Genéticas , Sequência de Bases , Elementos Facilitadores Genéticos , Humanos , Melanoma/enzimologia , Dados de Sequência Molecular , Peso Molecular , Metástase Neoplásica , Transcrição Gênica , Células Tumorais CultivadasRESUMO
A group of coordinated cellular processes, not just one gene product, is responsible for invasion and metastasis, the most life-threatening aspect of cancer. It is now recognized that negative factors may be just as important as positive elements. Genetic changes causing an imbalance of growth regulation lead to uncontrolled proliferation necessary for both primary tumor and metastasis expansion. However, unrestrained growth does not, by itself, cause invasion and metastasis. This phenotype may require additional genetic changes. Thus, tumorigenicity and metastatic potential have both overlapping and separate features. Invasion and metastasis can be facilitated by proteins which stimulate tumor cell attachment to host cellular or extracellular matrix determinants, tumor cell proteolysis of host barriers, such as the basement membrane, tumor cell locomotion, and tumor cell colony formation in the target organ for metastasis. Facilitory proteins may act at many levels both intracellularly or extracellularly but are counterbalanced by factors which can block their production, regulation, or action. A common theme has emerged. In addition to loss of growth control, an imbalanced regulation of motility and proteolysis appears to be required for invasion and metastasis.
Assuntos
Invasividade Neoplásica/genética , Metástase Neoplásica/genética , Animais , Humanos , Invasividade Neoplásica/patologia , Metástase Neoplásica/patologia , Inibidores de Proteases/farmacologiaRESUMO
The metastasis associated 72-kDa type IV collagenase is secreted as a latent proenzyme which is converted to an active 62-kDa form by autoproteolytic removal of an amino terminal profragment. The region immediately upstream from the cleavage site contains a highly conserved peptide sequence, MRKPRCGNPDV, which is present in all known members of the matrix metalloproteinase family. Evidence implicates the cysteine residue of this sequence as critical for maintenance of the latent form through coordination with the catalytic zinc atom of the active site. A synthetic peptide, TMRKPRCGNPDVAN (peptide 74), encompassing this conserved sequence, has been shown to inhibit the activated form of the 72-kDa type IV collagenase in vitro. In the present study we examine the ability of this peptide inhibitor to modulate tumor cell invasiveness. Peptide 74 and the control peptide 78, which contains a single substitution of serine for the "critical" cysteine residue, were added at 30 microM concentrations to the upper compartment of the Boyden chamber in the chemoinvasion assay using HT1080 and A2058 human tumor cells. In this assay a layer of reconstituted basement membrane, Matrigel, is coated onto chemotaxis filters and acts as a barrier to the migration of cells in the Boyden chambers. Only cells with invasive capacity can cross the Matrigel barrier. Peptide 74 containing the cysteine residue inhibited the invasion of both the HT1080 and A2058 cells through the Matrigel barrier; control peptide 78 was not inhibitory. Both peptides were shown to be without cytotoxic action and did not inhibit chemotaxis or affect cell number. This study demonstrates that addition of an excess peptide containing the matrix metalloproteinase prosegment inhibitory sequence can inhibit invasive activity at the cellular level and suggests that this may be a useful strategy to modulate tumor cell invasiveness in vivo.
Assuntos
Colagenase Microbiana/antagonistas & inibidores , Invasividade Neoplásica , Peptídeos/farmacologia , Sequência de Bases , Fibrossarcoma/enzimologia , Fibrossarcoma/patologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Metaloproteinase 9 da Matriz , Melanoma/enzimologia , Melanoma/patologia , Dados de Sequência Molecular , Peptídeos/química , Células Tumorais CultivadasRESUMO
The regulation of Mr 72,000 type IV collagenase and interstitial collagenase expression was studied in vitro. Three tumorigenic human cell lines were used, together with human fetal lung fibroblasts as a nontumorigenic control. Mr 72,000 type IV collagenase was expressed constitutively by all four cell lines, whereas only A2058 melanoma cells exhibited constitutive expression of interstitial collagenase. Treatment of cells with transforming growth factor beta 1 (TGF-beta 1) and 12-O-tetradecanoylphorbol-13-acetate (TPA) revealed an opposite pattern of regulation of these two metalloproteinases. Specifically, TPA increased interstitial collagenase mRNA levels in each cell line and decreased type IV collagenase mRNA levels in control fibroblasts and the tumorigenic cell lines, HT-1080 and A2058. TGF-beta 1 treatment increased type IV collagenase mRNA levels in each cell line and decreased interstitial collagenase mRNA levels in A2058 melanoma cells. Interstitial collagenase mRNA induction was accompanied in all cell lines by elevated interstitial procollagenase in the conditioned medium, as detected by zymography. Changes in Mr 72,000 type IV collagenase expression revealed a more complex pattern of regulation. TPA and TGF-beta 1 treatment of HT-1080 cells resulted in the appearance of two bands of gelatinolytic activity with a molecular weight of approximately 62,000 and 59,000. The Mr 62,000 species was also induced by TGF-beta 1 treatment of A2058 cells. Addition of affinity-purified radiolabeled Mr 72,000 type IV procollagenase to TPA-treated HT-1080 cells demonstrated that both species were products of the Mr 72,000 proenzyme and that exogenous proenzyme could be processed by these cells. Western blot analysis with specific antipeptide antibodies revealed that both the Mr 62,000 and 59,000 species were derived from the Mr 72,000 proenzyme by amino-terminal cleavage. There was no evidence for cellular processing of either interstitial procollagenase or the Mr 92,000 type IV procollagenase. These results demonstrate that the Mr 72,000 type IV collagenase is under the control of different regulatory elements from interstitial collagenase, at the level of both mRNA expression and cellular processing, and that this processing appears to be the result of a phorbol ester and TGF-beta 1-inducible cellular activation mechanism. The ratio of active enzyme species to latent Mr 72,000 proenzyme may provide a better correlation with invasive potential than overall levels of this widely expressed metalloproteinase.
Assuntos
Espaço Extracelular/enzimologia , Fibrossarcoma/enzimologia , Melanoma/enzimologia , Colagenase Microbiana/metabolismo , RNA Mensageiro/metabolismo , RNA Neoplásico/metabolismo , Western Blotting , Meios de Cultura , Precursores Enzimáticos/metabolismo , Humanos , Colagenase Microbiana/genética , Peso Molecular , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais Cultivadas/enzimologiaRESUMO
We have recently reported that concomitant with an increase in invasiveness, there is an increase in the expression and secretion of the matrix-degrading 72 kDa gelatinase A/type IV collagenase (MMP-2) in a moderately invasive human melanoma cell line (A375M) upon perturbation of the alpha v beta 3 classic vitronectin receptor. In the present study, we have extended these observations to include a highly invasive and metastatic melanoma cell line (C8161) which expresses a comparable amount of the alpha 5 beta 1 integrin (classic fibronectin receptor), but very little alpha v beta 3 integrin on its surface. When perturbed with an anti-alpha 5 beta 1 antibody, C8161 cells are 89% more invasive in vitro, and express and secrete increased levels of the gelatinase A. These changes were not elicited using antibodies to the alpha v beta 3 integrin. In addition, a 73% increase in invasion of C8161 cells through a fibronectin-enhanced matrix occurred, which could be abrogated by neutralizing antibodies to gelatinase A. Furthermore, we attempted to transiently mimic the invasive phenotype of the C8161 cells by diminishing the alpha v beta 3 integrin from the A375M cell surface through fluorescence-activated cell sorting selection or deoxynojirimycin treatment, and found these cells to be 30-50% more invasive than the parental population. These data suggest that alternative modulation and signaling events could be involved in melanoma tumor cell invasion as a result of the differential expression of integrins, and strictly cataloging the presence of these integrins is but an initial step in the analysis of their functional activity.
Assuntos
Colagenases/metabolismo , Integrinas/metabolismo , Melanoma/metabolismo , Invasividade Neoplásica , Receptores de Citoadesina/metabolismo , Anticorpos/administração & dosagem , Humanos , Integrinas/análise , Metaloproteinase 9 da Matriz , Melanoma/patologia , Receptores de Citoadesina/análise , Receptores de Fibronectina , Receptores de VitronectinaRESUMO
Local invasive growth is one of the key features of primary malignant brain tumors accompanied by remodeling of the vasculature and destruction of normal brain tissue. Tissue invasiveness is an essential biological function used by a tumor to overcome the various barriers to its progression. The expression of metalloproteases has been shown to play a critical role in the invasive process in a number of tumors; however, their expression in human brain tumors has not been previously reported. In this study we showed metalloprotease activities at M(r) 240,000, 123,000, 92,000, 72,000, and 67,000 in brain tumor extracts. These enzyme activities were inhibited by EDTA, an inhibitor of metalloproteases. Significant increases in levels of protease bands at M(r) 92,000, 123,000, and 240,000 were observed in glioblastoma and metastatic lung tumors. Enzymatic inhibition and Western blotting with M(r) 92,000 type IV collagenase antibody confirmed the presence of M(r) 92,000 type IV collagenase in all samples. Quantitative analysis by densitometry showed 8-10-fold and 6-8-fold increases in M(r) 92,000 type IV collagenase activity in glioblastoma and metastatic lung carcinoma samples, respectively, when compared with normal brain, meningioma, astrocytoma, metastatic colon, and breast carcinoma samples. These findings provide evidence for elevated levels of metalloproteases in glioblastomas and suggest a therapeutic target for minimizing the invasive propensity of gliomas using protease inhibitors.
Assuntos
Neoplasias Encefálicas/enzimologia , Colagenases/metabolismo , Glioma/enzimologia , Anticorpos/farmacologia , Western Blotting , Neoplasias Encefálicas/patologia , Ácido Edético/farmacologia , Eletroforese em Gel de Poliacrilamida , Glioma/patologia , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/secundário , Metaloproteinase 9 da Matriz , Inibidores de Metaloproteinases de Matriz , Peso MolecularRESUMO
A full-length complementary DNA (cDNA) for interstitial collagenase was isolated from an A2058 melanoma cDNA library using the pCD-X Okayama-Berg vector. The tumor interstitial collagenase cDNA was sequenced and compared to the published sequences for human fibroblast collagenase. The sequence for the tumor collagenase has two DNA base pairs which differ from the sequence of normal fibroblast collagenase. Restriction enzyme digestion of a specific DNA fragment produced by polymerase chain reaction amplification of genomic DNA from human placenta resolves a discrepancy in the previously reported DNA and amino acid sequences for the fibroblast collagenase. A high level of expression of interstitial collagenase message was found in human A2058 melanoma cells by Northern blot analysis, and this level was slightly increased by phorbol ester (phorbol myristate acetate) stimulation. Interstitial collagenase mRNA expression was significantly decreased by treatment with either transforming growth factor-beta 1 or retinoic acid in A2058 melanoma cells. A high level of the collagenase protein secreted into conditioned media was identified by Western blotting. As shown by gelatin zymogram analysis interstitial collagenase was one of at least two metalloproteinases secreted by this same cell line. Thus, human melanoma cells can directly produce interstitial collagenase without a requirement for host cell interaction.
Assuntos
Isoenzimas/genética , Melanoma/enzimologia , Colagenase Microbiana/genética , Células Tumorais Cultivadas/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Western Blotting , Clonagem Molecular/métodos , DNA de Neoplasias/genética , DNA de Neoplasias/isolamento & purificação , Fibroblastos/enzimologia , Humanos , Melanoma/genética , Colagenase Microbiana/análise , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Reação em Cadeia da Polimerase , Mapeamento por RestriçãoRESUMO
Degradation (turnover) of collagenous matrix occurs on the surface of specialized membrane extensions termed "invadopodia," which are sites of cell invasion into the extracellular matrix. Here we show the localization of the M(r) 72,000 type IV collagenase of the matrix metalloproteinase family at invadopodia. When added exogenously, latent M(r) 72,000 collagenase binds to invadopodia of chicken embryo fibroblasts transformed by Rous sarcoma virus, whereupon the bound collagenase loses its propeptide. The collagenase binds to a component contained within the detergent extract of transformed cells, and increased levels of the active M(r) 62,000 form of the collagenase are seen here. Such an association is not detected in the detergent extract derived from normal cells. Using a recently developed cell fractionation procedure to collect cell surfaces enriched in invadopodia, we show that the M(r) 72,000 collagenase associates with the invadopodial fraction and active forms of the enzyme become immobilized on the collagenous surface. Thus, invadopodia direct intense localized degradation of the extracellular matrix by concentrating active membrane-associated collagenases at sites of cellular invasion.
Assuntos
Colagenases/metabolismo , Proteínas de Membrana/metabolismo , Sequência de Aminoácidos , Animais , Vírus do Sarcoma Aviário , Sítios de Ligação , Biotina , Membrana Celular/enzimologia , Membrana Celular/ultraestrutura , Transformação Celular Viral/fisiologia , Células Cultivadas , Embrião de Galinha , Colágeno/metabolismo , Ativação Enzimática , Matriz Extracelular/enzimologia , Fibroblastos/enzimologia , Gelatina/metabolismo , Humanos , Metaloproteinase 9 da Matriz , Microscopia de Fluorescência , Dados de Sequência Molecular , Proteínas de Neoplasias/metabolismo , Rodaminas , Inibidor Tecidual de Metaloproteinase-2RESUMO
Cancer cells elaborate metalloproteinases which may play a role in invasion and metastasis. The serum level of the M(r) 72,000 type IV collagenase (MMP-2) was measured in 87 lung cancer patients. Stage IV cancer levels were significantly elevated (P less than 0.0001) compared to normal sera. A significant difference (P less than 0.01) was found between enzyme levels in the presence versus the absence of distant metastasis. For 29 patients treated with combination chemotherapy, a positive relationship was noted between response failure and elevated enzyme levels. Serum metalloproteinase levels may provide information relevant to prognosis as well as treatment decisions.
Assuntos
Biomarcadores Tumorais/sangue , Neoplasias Pulmonares/enzimologia , Colagenase Microbiana/sangue , Carcinoma de Células Pequenas/sangue , Carcinoma de Células Pequenas/tratamento farmacológico , Carcinoma de Células Pequenas/patologia , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Metaloproteinase 9 da Matriz , Metástase NeoplásicaRESUMO
Vasculogenic mimicry describes a process where aggressive tumor cells in three-dimensional matrices mimic embryonic vasculogenesis by forming extracellular matrix (ECM)-rich, patterned tubular networks. Microarray gene chip analyses revealed significant increases in the expression of laminin 5 (Ln-5, gamma2 chain) and matrix metalloproteinases (MMP)-1, -2, -9, and MT1-MMP (MMP-14) in aggressive compared with poorly aggressive melanoma cells. These components colocalized with developing patterned networks and antisense oligonucleotides to the Ln-5 gamma2 chain (but not sense oligonucleotides), and antibodies to MMP-2 or MT1-MMP (but not MMP-9) inhibited the formation of these networks. Cultures which did not receive antibodies to either MMPs-2 or -14 contained the Ln-5 gamma2 chain promigratory cleavage fragments. Poorly aggressive melanoma cells seeded on collagen I matrices preconditioned by the aggressive cells formed tubular networks along the Ln-5 gamma2 chain-enriched tracks deposited by the aggressive cells. These results suggest that increased expression of MMP-2 and MT1-MMP, along with matrix deposition of the Ln-5 gamma2 chain and/or its cleavage fragments, are required for vasculogenic mimicry by aggressive melanoma cells. Furthermore, the apparent recapitulation of laminin-rich, patterned networks observed in aggressive melanoma patients' tissue sections by aggressive melanoma tumor cells in three-dimensional culture may also serve as a model to help identify specific molecular targets which could function as templates for the coordinated migration of aggressive tumor cells and their proteolytic remodeling of the ECM and may have profound implications for the development of novel therapies directed at the ECM to alter tumor progression.
Assuntos
Moléculas de Adesão Celular/fisiologia , Metaloproteinase 2 da Matriz/fisiologia , Melanoma/irrigação sanguínea , Melanoma/patologia , Metaloendopeptidases/fisiologia , Neovascularização Patológica/metabolismo , Moléculas de Adesão Celular/biossíntese , Moléculas de Adesão Celular/genética , Humanos , Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinase 2 da Matriz/genética , Metaloproteinases da Matriz Associadas à Membrana , Melanoma/genética , Melanoma/metabolismo , Metaloendopeptidases/biossíntese , Metaloendopeptidases/genética , Mimetismo Molecular , Neovascularização Patológica/genética , Análise de Sequência com Séries de Oligonucleotídeos , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/farmacologia , Neoplasias Cutâneas/irrigação sanguínea , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Células Tumorais Cultivadas , Neoplasias Uveais/irrigação sanguínea , Neoplasias Uveais/genética , Neoplasias Uveais/metabolismo , Neoplasias Uveais/patologia , CalininaRESUMO
Proteolytic enzymes, such as type IV collagenase, play an important role in tumor invasion and metastasis. To examine Mr 72,000 type IV collagenase expression in human colon carcinoma, blot hybridization of total RNA from 19 primary colon tumors were performed. These filters were probed with complementary DNA probes encoding the Mr 72,000 type IV collagenase metalloenzyme. The results were expressed as the ratio of the messenger RNA (mRNA) levels in the tumor tissue to that in the adjacent normal mucosa (R). The level of the 3.1-kilobase type IV collagenase mRNA was higher in the primary tumor than in the normal adjacent colonic mucosa in 13 of 18 (72%) cases with a diagnosis of adenocarcinoma. These cases were divided into high expression (R, 4.50 to 29.34) and intermediate expression (R, 2.54 to 3.31) subgroups. Both groups showed statistically significant (P less than 0.05) elevations when compared with the five cases showing the lowest levels of Mr 72,000 type IV collagenase mRNA expression (low expression subgroup; R, 0.96 to 1.48). With this demonstrated elevation of Mr 72,000 type IV collagenase mRNA in colorectal adenocarcinoma we examined concomitant expression at the protein level using immunohistochemical techniques. Immunohistochemical examination of 70 cases of colon tumors, including 30 benign adenomas, using anti-Mr 72,000 type IV collagenase antibodies demonstrated a significant correlation with Duke's classification (P less than 0.001). Our results suggest that enhanced expression of the Mr 72,000 type IV collagenase enzyme may be a marker of human colorectal tumor invasiveness.
Assuntos
Adenocarcinoma/enzimologia , Neoplasias Colorretais/enzimologia , Colagenase Microbiana/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Clonagem Molecular , Colágeno/metabolismo , Neoplasias Colorretais/genética , DNA/genética , Expressão Gênica , Humanos , Imuno-Histoquímica , Mucosa Intestinal/enzimologia , Metaloproteinase 9 da Matriz , Colagenase Microbiana/genética , Dados de Sequência Molecular , Peso Molecular , RNA Mensageiro/genética , RNA Neoplásico/genética , Transcrição GênicaRESUMO
We have studied the capacity of two human breast adenocarcinoma cells, MDA-MB231 and MCF-7, to bind exogenous M(r) 72,000 type IV collagenase by both morphological and radioreceptor binding assays. By indirect immunofluorescence, staining with a specific anti-M(r) 72,000 type IV collagenase antibody was strongly induced when cells were preincubated with the purified enzyme. Scatchard plot analysis indicated the existence of a binding site for the M(r) 72,000 type IV collagenase with high affinity for both cell lines (Kd = 2 x 10(-9) M). These results are the first demonstration of the existence of a tumor cell membrane-associated putative receptor for a member of the matrix metalloproteinase family, as previously evidenced for the urokinase-type plasminogen activator.