Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0027651 (tumor)
 685,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Members of the matrix metalloproteinase (MMP) family have been implicated in disease states such as arthritis, periodontal disease, and tumor cell invasion and metastasis. Stromelysin 1 (MMP-3) has a broad substrate specificity and participates in the activation of several MMP zymogens. We examined known sequences of MMP-3 cleavage sites in natural peptides and proteins and compared sequence specificities of MMP-3 and interstitial collagenase (MMP-1) in order to design fluorogenic substrates that (i) would be hydrolyzed rapidly by MMP-3, (ii) would discriminate between MMP-3 and MMP-1, and (iii) could be monitored continuously without interference from MMP amino acid residues. Designed substrates were then screened for activity toward MMP-1, gelatinase A (MMP-2), MMP-3, and gelatinase B (MMP-9). The first of these substrates, NFF-1 (Mca-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Lys-(Dnp)-Gly, where Mca is (7-methoxycoumarin-4-yl)acetyl and Dnp is 2,4-dinitrophenyl), was hydrolyzed equally well by MMP-3 and MMP-2 (kcat/Km approximately 11,000 s-1 M-1). MMP-1 had 25% of the activity of MMP-3 toward NFF-1. The second substrate, NFF-2 (Mca-Arg-Pro-Lys-Pro-Tyr-Ala-Nva-Trp-Met-Lys(Dnp)-NH2, where Nva is norvaline), was hydrolyzed 60 times more rapidly by MMP-3 (kcat/Km = 59,400 s-1 M-1) than MMP-1. Unfortunately, NFF-2 showed little discrimination between MMP-3, MMP-2 (kcat/Km = 54,000 s-1 M-1), and MMP-9 (kcat/Km = 55,300 s-1 M-1). The third substrate, NFF-3 (Mca-Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Lys(Dnp)-NH2), was hydrolyzed rapidly by MMP-3 (kcat/Km = 218,000 s-1 M-1) and very slowly by MMP-9 (kcat/Km = 10,100 s-1 M-1), but there was no significant hydrolysis by MMP-1 and MMP-2. NFF-3 is the first documented synthetic substrate hydrolyzed by only certain members of the MMP family and thus has important application for the discrimination of MMP-3 activity from that of other MMPs. Although NFF-3 was designed by assuming that substrate subsites were independent and hence free energy changes derived from single mutation experiments were additive, we found discrepancies between predicted and experimental kcat/Km values, one on the order of 2000-5000. Thus, the design of additional discriminatory MMP substrates may require approaches other than assuming additive free energy changes, such as screening synthetic libraries and consideration of secondary and tertiary structures of substrates and the enzyme.
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PMID:Design and characterization of a fluorogenic substrate selectively hydrolyzed by stromelysin 1 (matrix metalloproteinase-3). 806 13

The biochemical events associated with tumor invasion involve localized degradation of the basement membrane by tumor-associated proteinases. In this study, we have characterized the proteinase secretion profiles of 5 ovarian epithelial carcinoma cell lines (DOV 13, OVCA 420, OVCA 429, OVCA 432, OVCA 433) as well as normal ovarian epithelial cells. Immunocapture assays demonstrated that all 5 carcinoma cell lines produce both secreted and surface-associated plasminogen activator. Urinary-type plasminogen activator (u-PA) production was one order of magnitude greater than production of tissue-type plasminogen activator (t-PA). Furthermore, t-PA secretion by normal ovarian epithelial cells was not detectable, whereas u-PA production was 17- to 38-fold lower than in ovarian carcinoma cells. Western-blotting analysis demonstrated that u-PA was secreted as the single chain form (scu-PA) when cells were cultured in serum-free medium. Incubation of plasminogen with ovarian carcinoma cell-conditioned medium resulted in direct activation of the zymogen to plasmin. Furthermore, following incubation of cells with plasminogen, plasmin was eluted from the cell surface, indicating that ovarian carcinoma cells contain binding sites for plasminogen/plasmin which are accessible to surface-associated plasminogen activators. In addition to plasminogen activators, metalloproteinases were also produced by DOV 13, OVCA 429 and OVCA 433 cells. DOV 13 cells produce a 68-kDa metalloproteinase similar to matrix metalloproteinase 2 (MMP-2) whereas a 92-kDa enzyme similar to MMP-9 is secreted by OVCA 429 and 433. Together, ovarian carcinoma-associated plasminogen activators and metalloproteinases catalyze the hydrolysis of the major basement membrane protein components, type-IV collagen, type-IV gelatin, laminin and fibronectin. The enhanced proteolytic capability of ovarian carcinoma cells relative to normal ovarian epithelium suggests a biochemical mechanism by which invasion and spread of ovarian epithelial carcinoma may be mediated.
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PMID:Secretion of extracellular matrix-degrading proteinases is increased in epithelial ovarian carcinoma. 811 91

We examined the in vitro regulation of the production of two type IV collagenases, MMP-2 and MMP-9, by A431 human epidermoid carcinoma cells. The A431 cells were cultured under sparse or confluent conditions. The addition of transforming growth factor-beta (TGF-beta) or phorbolester-TPA to sparse cultures induced low levels of MMP-9 secretion, whereas in confluent cultures only TGF-beta produced this effect. Neither treatment altered the level of constitutive secretion of MMP-2. Treatment of sparse, actively growing cultures but not confluent stationary cultures with both TGF-beta and TPA produced synergistic induction of MMP-9 but did not affect MMP-2. A431 cells were grown as discrete large monolayer colonies. Radiolabeling with [3H]leucine or [3H]thymidine followed by autoradiography revealed that all the A431 cells in the colonies were metabolically active and only those on the periphery were dividing. Only these dividing A431 cells stained positive by anti-MMP-9 antibodies. Our results demonstrate that the synergistic induction of MMP-9 secretion in A431 cells occurs subsequent to stimulation by external signals in only noncontact-inhibited dividing tumor cells. These regulatory mechanisms may account for the in vivo finding that many proteinases are localized at the invasion front of a neoplasm where tumor cells are dividing and accessible to various environmental signals.
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PMID:Density-dependent induction of 92-kd type IV collagenase activity in cultures of A431 human epidermoid carcinoma cells. 817 29

Members of the matrix metalloproteinase (MMP) family have been implicated in the metastasis of tumor cells, but no direct evidence linking any given member of the MMP family to metastatic behavior has been presented. Rat embryo cells transformed by the Ha-ras and v-myc oncogenes or by Ha-ras alone are metastatic in nude mice and release the 92-kDa gelatinase/collagenase (MMP-9), whereas those transformed by Ha-ras plus the adenovirus E1A gene are not metastatic and do not release MMP-9. Here we demonstrate that MMP-9 expression can be induced in these tumorigenic but nonmetastatic rat cells by transfection with an MMP-9 expression vector. Transfection of a MMP-9 expression vector, but not control DNAs, conferred metastatic capacity on the nonmetastatic cells. The majority of colonies isolated after continued passage either in vivo or in vitro had lost the MMP-9 expression vector. However, occasional cells were isolated from metastases which retained MMP-9 expression after passage. These cells retained metastatic capacity. In contrast, cells isolated after losing MMP-9 expression also lost the ability to metastasize. These results provide direct evidence that MMP-9 has a role in tumor metastasis.
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PMID:Direct evidence linking expression of matrix metalloproteinase 9 (92-kDa gelatinase/collagenase) to the metastatic phenotype in transformed rat embryo cells. 818 3

The role of matrix metalloproteinases (MMP's) and their inhibitor, tissue inhibitor of metalloproteinases-1 (TIMP-1), in human brain tumor invasion was investigated. Gelatinolytic activity was assayed via gelatin zymography, and four MMP's (MMP-1, MMP-2, MMP-3, and MMP-9) and TIMP-1 were immunolocalized in human brain tumors and in normal brain tissues using monoclonal antibodies. The tissue was surgically removed from 44 patients: glioblastoma (five cases), anaplastic astrocytoma (six cases), astrocytoma (four cases), metastatic tumor (six cases), neurinoma (10 cases), meningioma (10 cases), and normal brain tissue (three cases). Glioblastomas, anaplastic astrocytomas, and metastatic tumors showed high gelatinolytic activity and positive immunostaining for MMP's; TIMP-1 was also expressed in these tumors, but some tumor cells were negative for the antibody. Astrocytomas had low gelatinolytic activity and the tumor cells showed no immunoreactivity for MMP's and TIMP-1. Although neurinomas and meningiomas had only moderate proteinase activity and exhibited positive immunoreactivity for MMP-9, intense expression of TIMP-1 was simultaneously observed in these tumor cells. These findings suggest that MMP's play an important role in human brain tumor invasion, probably due to an imbalance between the production of MMP's and TIMP-1 by the tumor cells.
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PMID:Production of matrix metalloproteinases and tissue inhibitor of metalloproteinases-1 by human brain tumors. 820 29

We have used quantitative zymography to measure levels of the type IV collagenases matrix metalloproteinase (MMP)-9 and MMP-2 in 42 biopsies of transitional cell carcinoma and in 7 biopsies of normal bladder. Mean levels of MMP-9 were significantly higher in tumor compared with normal samples (P = 0.08). Levels of MMP-9 and active MMP-2 increased with tumor grade (test for trend, P = 0.002 and P = 0.05, respectively). Levels of MMP-9 and activated MMP-2 were also higher in invasive tumors than in superficial ones (P = 0.001 and P = 0.008, respectively). In situ hybridization studies showed that the mRNAs for both MMP-2 and MMP-9 were located chiefly in the stroma rather than epithelial tumor cells and were concentrated at the interface between the two tissues.
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PMID:Levels of matrix metalloproteases in bladder cancer correlate with tumor grade and invasion. 822 72

The expression of metalloproteinases, such as type IV collagenase/gelatinase, enables tumor cells to degrade type IV collagen present in the basement membrane and correlates with metastatic potential of several tumor types. We found that increased levels of rat serum type IV collagenolytic activity are associated with increased 13762NF mammary adenocarcinoma metastases in lungs and lymph nodes of syngeneic rats. To investigate serum metalloproteinases responsible for type IV collagenolysis, we performed zymography and Western blot analysis of rat sera. A M(r) 92,000 progelatinase (progelatinase B, M(r) 92,000 type IV procollagenase, MMP-9) was detected on zymograms of rat sera within 16 days after intramammary fat pad inoculation of highly metastatic MTLn3 cells. The activated serum M(r) 92,000 progelatinase degraded sodium dodecyl sulfate-denatured type I and IV collagens but was not active on casein, albumin, hemoglobin, and immunoglobulin. Sera from rats with fat pad tumors and lung metastases formed from highly metastatic clones possessed greater than 7 times higher levels of serum M(r) 92,000 progelatinase than control sera or sera from rats bearing similar size fat-pad tumors of low or nonmetastatic clones. The results were confirmed by Western blot analysis of rat sera using rabbit anti-human M(r) 92,000 progelatinase antibodies. Similar results were obtained from the analysis of rat plasma samples. The serum and plasma M(r) 92,000 progelatinase levels correlated with the extent of metastases in the lung and lymph nodes. The results indicate that high levels of serum and plasma M(r) 92,000 progelatinase are associated with the presence of disseminated metastatic adenocarcinoma cells and suggest that this enzyme may facilitate the invasion of blood-borne tumor cells through vascular basement membranes.
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PMID:Serum and plasma M(r) 92,000 progelatinase levels correlate with spontaneous metastasis of rat 13762NF mammary adenocarcinoma. 824 39

Two members of the matrix metalloproteinase (MMP)1 family of enzymes are expressed at elevated levels in highly aggressive human tumor cells and have been implicated in the catalytic functions of extracellular proteolysis. The zymogen forms of these enzymes are designated proMMP-2 and proMMP-9, also known as 72kDa and 92kDa type IV collagenases/gelatinases, respectively. The MMP family of enzymes can be activated in vitro by a number of compounds including the organomercurial 4-aminophenylmercuric acetate (APMA). The natural or in vivo activators of MMP-2 and MMP-9 are at present unknown. A partially purified preparation of MMP-9 was used to immunize mice for the isolation of monoclonal antibodies (mAbs). Three IgG1 mAbs were identified by immunoreactivity with purified MMP-9 and are designated 6-6B, 7-11C, and 8-3H. These mAbs react specifically with MMP-9 by ELISA and Western blot. Additionally, these mAbs react with N-glycanase treated 92kDa protein. These mAbs were tested for their ability to inhibit enzyme activation in a radio-labeled gelatin assay. The 6-6B mAb inhibited the activation of MMP-9, but had no effect on MMP-2. These mAbs are highly specific to human MMP-9 and the 6-6B mAb will be extremely useful for examining the autolytic and catalytic activity of MMP-9 in normal and abnormal biological processes.
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PMID:Inhibition of matrix metalloproteinase 9 activation by a specific monoclonal antibody. 824 15

Chick embryo has been used as a model system for evaluating the metastatic potential of tumor cells. We have previously demonstrated that expression of the tissue inhibitor of matrix metalloproteinase-I (TIMP-I) gene can suppress liver colonization of tumor cels in chick embryo, probably by inhibiting the activity of matrix metalloproteinases (MMP) produced by tumor cells. In an attempt to identify MMP associated with liver colonization, we examined 24 human tumor cell lines for their potential to form metastatic colonies in chick-embryo liver after the cells had been inoculated into the chorioallantoic membrane (CAM) vein. We compared the results with the mRNA expression of MMP (MMP-I, MMP-2, MMP-3, MMP-9) studied previously. Three of 8 cell lines from mesenchymal tumors (fibrosarcoma HT1080, osteosarcomas SK-ES and MNNG/HOS) and 2 of 16 cell lines from epithelial tumors (gastric carcinoma KKLS and bladder carcinoma T24) proliferated in the livers. MMP-2 and MMP-9 were the enzymes whose transcripts were more frequently expressed in these 5 metastatic cell lines (MMP-1; 2/5, MMP-2; 4/5, MMP-3; 0/5, MMP-9; 3/5), but other cell lines that did not form liver colonies expressed the transcripts at lower frequency (MMP-2; 7/19, MMP-9; 3/19). Although either or both MMP-2 and MMP-9 transcripts were expressed in 4 of the 5 metastatic cell lines, they were undetectable in T24 cells. However, induced expression of both enzymes was detected by immunostaining in the T24 cells colonized in the liver. Thus, type-IV collagenases expressed by tumor cells may play a role in facilitating colonization in chick embryos.
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PMID:Expression of type-IV collagenases in human tumor cell lines that can form liver colonies in chick embryos. 826 76

The matrix metalloproteinase enzymes have been implicated in tumor invasion and metastasis by a series of correlative immunohistochemical studies. In addition, direct evidence for the role of these enzymes in this pathologic process comes from studies using specific metalloproteinase inhibitors to block tumor invasion and metastasis formation, both in vitro and in vivo. Synthetic oligonucleotide primers for four metalloproteinases (MMP-1, MMP-2, MMP-9, MMP-10) and their tissue inhibitors (TIMP-1, TIMP-2) were selected, synthesized, and optimized in the reverse transcriptase-polymerase chain reaction (RT-PCR) to study the qualitative profile of these enzymes and inhibitors in cultured human tumor cells and tumor tissues. These primers are specific and generate unique amplification products for each appropriate enzyme and inhibitor. Slight enhancement in the amplification of cDNA products was achieved by adding dimethylsulfoxide to the reaction mixture, but commercial enhancement reagents were ineffective. Using this RT-PCR method, cDNA amplification was successful with RNA from as few as 20 cultured tumor cells. The RT-PCR analysis was done on three invasive human colon adenocarcinomas and their paired adjacent normal mucosa. The results show MMP-1 and MMP-2 products in all three tumors, and MMP-2 detected in one of the three normal mucosa samples; TIMP-2 expression was present in two of three patients and awaits quantitative assessment of RT-PCR products.
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PMID:Reverse transcription-polymerase chain reaction phenotyping of metalloproteinases and inhibitors involved in tumor matrix invasion. 826 80


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