Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The purpose of this study was to correlate the presence of matrix metalloproteinase (MMP)-9 and MMP-2 and tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 mRNAs, detected in serial sections using the reverse transcriptase in situ PCR technique, with prognosis in 23 cases of cervical carcinoma. PCR-amplified MMP and TIMP cDNA were restricted to the invasive cancers cells and the surrounding stromal cells. The ratios of cancer and stromal cells expressing MMP-9 and MMP-2 to those expressing TIMP-1 and TIMP-2 were approximately 1 in those cancers with a good prognosis. This MMP:TIMP ratio in the cancer and stromal cells with a poor prognosis was significantly increased to 5.4 and 3.4 (P < 0.0001), respectively, reflecting a marked reduction in the TIMP detection rate in cancers with a poor prognosis. In cervical cancer cell lines SiHa and HeLa, the MMP:TIMP ratio was also close to 1 and, interestingly, these cell lines are invasive but rarely metastatic in nude mice. These data suggest that the balance of MMP-9 and MMP-2 to TIMP-1 and TIMP-2 expression is an essential factor in the aggressiveness of cervical cancer.
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PMID:Correlation of the in situ detection of polymerase chain reaction-amplified metalloproteinase complementary DNAs and their inhibitors with prognosis in cervical carcinoma. 781 56

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

We reported earlier that the levels of Ca2+-dependent metalloproteinases are increased in Alzheimer's disease (AD) specimens, relative to control specimens. Here we show that these enzymes are forms of the matrix metalloproteinase MMP-9 (EC3.4.24. 35) and are expressed in the human hippocampus. Affinity-purified antibodies to MMP-9 labeled pyramidal neurons, but not granular neurons or glial cells. MMP-9 mRNA is expressed in pyramidal neurons, as determined with digoxigenin-labeled MMP-9 riboprobes, and the presence of this mRNA is confirmed with reverse transcriptase PCR. The cellular distribution of MMP-9 is altered in AD because 76% of the total 100 kDa enzyme activity is found in the soluble fraction of control specimens, whereas only 51% is detectable in the same fraction from AD specimens. The accumulated 100 kDa enzyme from AD brain is latent and can be converted to an active form with aminophenylmercuric acetate. MMP-9 also is detected in close proximity to extracellular amyloid plaques. Because a major constituent of plaques is the 4 kDa beta-amyloid peptide, synthetic Abeta1-40 was incubated with activated MMP-9. The enzyme cleaves the peptide at several sites, predominantly at Leu34-Met35 within the membrane-spanning domain. These results establish that neurons have the capacity to synthesize MMP-9, which, on activation, may degrade extracellular substrates such as beta-amyloid. Because the latent form of MMP-9 accumulates in AD brain, it is hypothesized that the lack of enzyme activation contributes to the accumulation of insoluble beta-amyloid peptides in plaques.
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PMID:Matrix metalloproteinase-9 (MMP-9) is synthesized in neurons of the human hippocampus and is capable of degrading the amyloid-beta peptide (1-40). 898 19

Thyroid cancer can degrade basement membranes and invade tissues. This depends on a cascade of matrix metalloproteinases involving membrane-type 1 matrix metalloproteinase (MT1-MMP), MMP-2, and MMP-9. We analyzed the expression and role of these MMPs and their specific inhibitors TIMP-2 and TIMP-3 in human highly purified thyroid epithelial, C 643, HTh 74, SW 1736, and 8505 C thyroid carcinoma and thyroid-derived fibroblast cell cultures. The effect of phorbol-myristate acetate (PMA), and of the inflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) on MMP and TIMP mRNA levels were monitored by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) including an internal homologous competitor fragment. The highest MT1-MMP mRNA levels were found in thyroid-derived fibroblasts. The MT1-MMP mRNA expression was increased up to 10-fold by PMA, while all other growth factors tested had only negligible effects. The thyroid carcinoma cells themselves did not seem to play a crucial role in the production of MT1-MMP in thyroid tumors. Higher MMP-2 mRNA levels were found in all cell types investigated. The highest MMP-2 mRNA levels were determined in thyroid-derived fibroblasts and HTh 74 cells. We found a lack of MMP-2 response to IL-1, TNF-alpha, and phorbol esters. In unstimulated cells, MMP-9 mRNA was found near the detection limit or at low levels. In nearly all cell types, treatment with PMA, IL-1, and TNF-alpha caused an increase of the MMP-9 mRNA levels. The results of basal and stimulated MMP-2 and MMP-9 mRNA expression were confirmed at the protein level by gelatin zymography. TIMP-2 and TIMP-3 mRNAs were expressed at high levels. In contrast to the basal TIMP-3 mRNA levels, which varied over a great range, there were no striking differences the cell types from analyzing TIMP-2 mRNA. There were no or only slight stimulatory effects on TIMP-2 and TIMP-3 mRNA expression by IL-1, TNF-alpha, and PMA. Taken together, most enzymes of the MT-MMP/MMP class of proteases facilitating invasion of thyroid tumor cells seem to have been produced by fibroblasts, not by the tumor cells themselves. However, some dedifferentiated thyroid tumor cell lines may be capable of secreting some of these enzymes, as in the case of HTh 74 cells.
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PMID:mRNA levels of membrane-type 1 matrix metalloproteinase (MT1-MMP), MMP-2, and MMP-9 and of their inhibitors TIMP-2 and TIMP-3 in normal thyrocytes and thyroid carcinoma cell lines. 954 6

Matrix metalloproteinases (MMPs) comprise a group of proteolytic enzymes that are implicated in the pathogenesis of inflammatory diseases of the nervous system such as multiple sclerosis. However, the exact function and expression pattern of MMPs in the inflamed nervous system are not known. In the present study we investigated the expression of 92-kDa gelatinase (MMP-9) in spinal cord from animals with adoptive transfer experimental autoimmune encephalomyelitis (AT-EAE), using a semiquantitative competitive reverse transcriptase-polymerase chain reaction assay. Increased levels of MMP-9 mRNA were found with peak values at times of maximum disease severity. Increased mRNA expression was associated with enhanced proteolytic activity of this enzyme, as demonstrated by gelatin zymography. Immunohistochemistry revealed immunoreactivity along the meninges, around blood vessels and within the parenchyma, in diseased but not in normal spinal cord. Furthermore, the expression pattern of five other MMPs was investigated. Matrilysin (MMP-7) was also found to be upregulated with maximum mRNA levels at the peak of the disease. In contrast, mRNAs for collagenase-3, 72-kDa gelatinase, and stromelysin-1 and -3 were not changed. Our findings indicate that 92-kDa gelatinase and matrilysin are selectively upregulated during AT-EAE and thus may contribute to the pathogenesis of inflammatory diseases of the CNS.
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PMID:Matrix metalloproteinase-9 and -7 are regulated in experimental autoimmune encephalomyelitis. 954 96

We have previously documented that adoptively transferred IL-2-activated NK (A-NK) cells can accumulate within cancer metastases. Electron microscopic studies of pulmonary metastases have revealed that adoptively transferred A-NK cells that accumulate within metastases bind to endothelial cells and are able to traverse basement membranes. We have now extended these morphologic studies. We report that rat A-NK cells produce two matrix metalloproteinases: MMP-2 and MMP-9, as determined by SDS-PAGE gelatin zymography. These activities are inhibited following incubation with BB-94 (batimastat), a specific inhibitor of matrix metalloproteinases but not with 3,4-dichloroisocoumarin, an inhibitor of neutral serine proteases. The identity of MMP-2 was confirmed by Western blots using a polyclonal Ab against human MMP-2, whereas reverse transcriptase-PCR analysis of mRNA extracts of A-NK cells has confirmed the presence of MMP-9. In addition, we report for the first time that A-NK cells can migrate through a model basement membrane-like extracellular matrix. Moreover, the ability of A-NK cells to migrate through this model basement membrane was partially inhibited by BB-94; however, BB-94 has no effect on A-NK cell-mediated cytotoxicity, suggesting that matrix metalloproteinases do not contribute to cytolytic function of A-NK cells. In sum, our studies show that A-NK cells employ BB-94-inhibitable matrix metalloproteinases to degrade extracellular matrices. This suggests that matrix metalloproteinases may play a role in the accumulation of A-NK cells within cancer metastases.
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PMID:Matrix metalloproteinases produced by rat IL-2-activated NK cells. 957 26

Degradation of extracellular matrix, especially elastin, within the aortic wall is a hallmark of abdominal aortic aneurysms (AAAs). Normal turnover of matrix proteins is mediated by a family of enzymes called matrix metalloproteinases (MMPs). MMP activity is regulated by proteins called tissue inhibitors of metalloproteinases (TIMPs). We analyzed the expression of all known MMPs with established elastolytic activity and TIMPs in human AAA and control tissue. mRNA coding for MMP-9, MMP-2, human macrophage metalloelastase, MMP-7, TIMP-1, and TIMP-2 were amplified by reverse transcriptase-PCR in control and AAA tissue. A Northern blot assay was used to measure the levels of mRNA coding for MMP-2, MMP-9, TIMP-1, and TIMP-2. Control aortic tissue was obtained from patients with occlusive disease and from organ donors. The expression of MMP-7 and human macrophage metalloelastase was not detected in any aortic specimens. By Northern blot analysis the mean level of MMP-2 mRNA was not significantly different between control groups and AAAs (normalized values: occlusive, 1.5 +/- 0.8, n = 3; donor, 4.5 +/- 2.2, n = 6; AAA, 4.0 +/- 0.95, n = 15). There was a significant increase in the level of MMP-9 mRNA in AAA specimens (occlusive, 16.8 +/- 3, n = 3; donor, 5.7 +/- 1.2, n = 6; AAA, 56.7 +/- 11, n = 15, p = 0.0069). The levels of mRNA coding for TIMP-1 were not significantly different. There was a small but statistically significant increase in TIMP-2 mRNA in AAA tissue. These data support the hypothesis that increased activity of MMP-9, but not MMP-2, is an important factor in the etiology of AAAs. This enhanced MMP-9 activity could then result in degradation of the ECM, leading to aneurysmal dilatation.
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PMID:Expression of matrix metalloproteinases and TIMPs in human abdominal aortic aneurysms. 1007 71

In the present study, we have employed a unique breast cancer cell line (Met-1, which was derived from a high metastatic potential tumor in transgenic mice expressing polyomavirus middle T oncogene) to study the role of CD44 variant isoform(s) in the regulation of metastatic breast tumor cell behavior. The results of reverse transcriptase-polymerase chain reaction, Southern blot, nucleotide sequencing, immunoprecipitation, and immunoblot analyses indicated that these cells express a major CD44 isoform (molecular weight approximately 260 kDa) containing a v3,8-10 exon insertion (designated as CD44v3,8-10). In addition, we have determined that CD44v3,8-10 binds specifically to the cytoskeletal proteins such as ankyrin. Biochemical analyses, using competition binding assays and a synthetic peptide identical to NGGNGTVEDRKPSEL (a sequence located between aa480 and aa494 of CD44v3,8-10) indicate that this 15-amino acid peptide binds specifically to the cytoskeletal protein ankyrin (but not to fodrin or spectrin). This peptide competes effectively for ankyrin binding to CD44v3,8-10. Therefore, we believe that the sequence 480NGGNGTVEDRKPSE494L, located at the cytoplasmic domain of CD44v3,8-10, is required for the ankyrin binding. We have also detected that CD44v3,8-10-containing Met-1 cells are capable of forming membrane spikes or "invadopodia" structures and undergo active migration processes. Treatments of Met-1 cells with certain agents including anti-CD44v3 antibody, cytochalasin D (a microfilament inhibitor), and W-7 (a calmodulin antagonist), but not colchicine (a microtubule disrupting agent) effectively inhibit "invadopodia" formation and subsequent tumor cell migration. Further analyses using zymography assays and double immunofluorescence staining indicated that CD44v3,8-10 is closely associated with the active form of matrix metalloproteinase, MMP-9, in a complex within "invadopodia" structures. These findings suggest that CD44v3,8-10 plays an important role in linking ankyrin to the membrane-associated actomyosin contractile system required for "invadopodia" formation (coupled with matrix degradation activities) and tumor cell migration during breast cancer progression.
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PMID:CD44v(3,8-10) is involved in cytoskeleton-mediated tumor cell migration and matrix metalloproteinase (MMP-9) association in metastatic breast cancer cells. 961 60

Vascular endothelial growth factor (VEGF) is a critical regulator of angiogenesis that stimulates proliferation, migration, and proteolytic activity of endothelial cells. Although the mitogenic activity of VEGF is endothelial cell specific, recent reports indicate VEGF is able to stimulate chemotaxis and tissue factor production in monocytes. VEGF-stimulated activity in monocytes is mediated by the VEGF receptor flt-1. The purpose of the present study was to investigate the effects of VEGF on another major cell type in the vascular wall, namely, the vascular smooth muscle cell (SMC). Using cultured cells, we showed that VEGF has a minimal mitogenic effect on SMCs, which is in accordance with published data. However, VEGF treatment significantly enhanced production of matrix metalloproteinase (MMP)-1, -3, and -9 by human SMCs. The upregulation of MMP-1 and MMP-9 was pronounced, and the stimulation for MMP-3 was less prominent. Stimulation could be demonstrated at both protein and mRNA levels, as reflected by ELISA, zymography, and Northern blot analysis. To explore the signal transduction pathway for the effect of VEGF on SMCs, we studied the expression of 2 high-affinity VEGF receptors, the kinase insert domain-containing receptor (KDR) and flt-1, in human SMCs. Both reverse transcriptase-polymerase chain reaction and immunoblotting revealed the expression of flt-1. Immunoprecipitation followed by immunoblotting illustrated phosphorylation of the flt-1 receptor after VEGF treatment. Similar methodology failed to detect expression of KDR in human SMCs. These data suggest the role of flt-1 in mediating VEGF-stimulated MMP expression of SMCs. The physiological relevance of MMP upregulation was studied by examining VEGF-stimulated SMC migration through 2 synthetic extracellular matrix barriers, Matrigel and Vitrogen. Our results indicate that VEGF treatment accelerated SMC migration through both barriers, and that this response was blocked by MMP inhibition in Matrigel, which supports a permissive role of MMP in SMC migration. These data are the first to show a direct effect of VEGF on SMCs. SMC-derived MMPs may be an additional source of proteases to digest vascular basement membrane, which is a crucial step in the initial stage of angiogenesis. The MMPs may also contribute to SMC migration in angiogenesis and atherogenesis.
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PMID:Vascular endothelial growth factor upregulates the expression of matrix metalloproteinases in vascular smooth muscle cells: role of flt-1. 977 30

Degradation of the extracellular matrix is necessary for invasion and metastasis by cancer cells. Two gelatinolytic matrix metalloproteinase enzymes, MMP-2 and MMP-9, are supposed to be key enzymes in this process. The purpose of this study was to correlate the presence of MMP-2, MMP-9 and their inhibitors with the tissue inhibitors of metalloproteinases TIMP-1 and TIMP-2 RNA using reverse transcriptase PCR technique with tumor stage in 17 samples of renal cell carcinoma. The ratio of tissues expressing MMP-2 and MMP-9 to those expressing TIMP-1 and TIMP-2 was defined to be 1 in normal kidney tissue. This MMP:TIMP ratio was significantly increased to 2.43 (standard deviation, SD = 0.8) in locally confined renal cell carcinoma and to 4.86 (SD = 1.1) in advanced carcinoma (p <0.01). In primary tumor cell lines the ratio of MMP:TIMP expression was 3.44 (SD = 0.6). These data suggest that the balance of MMP-2 and MMP-9 to TIMP-1 and TIMP-2 expression is an essential factor in the aggressiveness of renal cell carcinoma.
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PMID:Expression of metalloproteinase 2 and 9 and their inhibitors in renal cell carcinoma. 978 85


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