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Biomedical subjects

A J Docherty

Publications and source records attributed to A J Docherty.

At least 37 records · Page 2Linked to original sources
PubMed1996

Combination therapy including a gelatinase inhibitor and cytotoxic agent reduces local invasion and metastasis of murine Lewis lung carcinoma.

The efficacy of combination therapy including an oral gelatinase inhibitor (CT1746) and cytotoxic agent was analyzed using the murine Lewis lung carcinoma model. Primary tumors, pulmonary metastases, and sera from tumor-bearing animals had increased gelatinase B activity that was inhibited by CT1746 levels achievable in vivo. The combination of CT1746 and cyclophosphamide (CTX) was significantly more effective than either single agent in delaying local tumor growth (CT1746/CTX, 30.9 +/- 1.7 days; CT1746, 2.6 +/- 0.3 days; CTX, 19.5 +/- 1.1 days; P < .001) and reducing the number and size of pulmonary metastases [CT1746/CTX, 5 +/- 2 (15% metastases > 3 mm); CT1746, 15 +/- 4 (55% > 3 mm); CTX, 11 +/- 3 (63% > 3 mm); no treatment, 24 +/- 5 (62% > 3 mm); P < .001]. These data support the notion of combining matrix metalloproteinase inhibitors and cytotoxic agents to treat certain epithelial malignancies.

Amides↗
PubMed1996

The C-terminal (haemopexin-like) domain structure of human gelatinase A (MMP2): structural implications for its function.

In common with most other matrix metalloproteinases, gelatinase A has a non-catalytic C-terminal domain that displays sequence homology to haemopexin. Crystals of this domain were used by molecular replacement to solve its molecular structure at 2.6 A resolution, which was refined to an R value of 17.9%. This structure has a disc-like shape, with the chain folded into a beta-propeller structure that has pseudo four-fold symmetry. Although the topology and the side-chain arrangement are very similar to the equivalent domain of fibroblast collagenase, significant differences in surface charge and contouring are observable on 1 side of the gelatinase A disc. This difference might be a factor in allowing the gelatinase A C-terminal domain to bind to natural inhibitor TIMP-2.

Amino Acid Sequence↗
PubMed1995

Plasma assay of gelatinase B: tissue inhibitor of metalloproteinase complexes in cancer.

BACKGROUND: Matrix metalloproteinases (MMPs), especially gelatinase A and gelatinase B (GLB), are believed to be important components of the metastatic process. Tissue Inhibitors of Metalloproteinases (TIMPs) form complexes with MMPs and inhibit cancer dissemination. After local secretion, MMPs and their complexes with TIMPs leach into the blood stream where their concentration can be measured, thereby serving as surrogate markers of disease. Elevated plasma gelatinase B levels have been detected in gastrointestinal cancer and breast cancer. The goal of this study was to determine whether plasma GLB:TIMP complexes also are increased in cancer and whether these tests have potential use as prognostic tumor markers. METHODS: An enzyme-linked immunosorbent assay (ELISA) was developed to measure the plasma concentration of GLB:TIMP complexes in patients with cancer. Correlation between ELISA results and clinical outcome was sought. RESULTS: Plasma GLB:TIMP complexes were significantly increased in patients with gastrointestinal cancer and gynecologic cancer, but not in patients with breast cancer. When results from plasma GLB:TIMP complexes and plasma GLB assays were combined (GLB/complexes), abnormal levels of one or both assays were found in 36% and 65% of patients with gastrointestinal and gynecologic cancer, respectively. In Stage IV gastrointestinal cancer, patient survival was shorter (P < 0.001) in the group with increased plasma GLB/complexes than for those with normal plasma levels (4 months vs. 20 months, respectively). CONCLUSIONS: The assay of plasma gelatinase B and GLB:TIMP complexes may be clinically useful in predicting survival in subsets of patients with cancer. The possibility of using these assays in early stage cancer to predict metastasis should be studied.

Biomarkers, Tumor↗
PubMed1995

Binding of gelatinases A and B to type-I collagen and other matrix components.

Matrix sequestration of matrix metalloproteinases may be important for the facilitation of remodelling events and the migration of cells through the extracellular matrix. Using an ELISA technique we studied the ability of pro and active forms of gelatinases A and B (GLA and GLB) to bind to matrix components and the contribution made by the different enzyme domains. Pro and active forms of GLA and GLB bound to type-I and type-IV collagens, gelatin and laminin films. Binding to collagens occurred exclusively via the N-terminal portion of the molecule in both of the gelatinases; deletion of the fibronectin-like domain in GLA abolished binding. Fibronectin was shown to compete with GLA, confirming that binding occurs through this domain. GLA and GLB competed for binding to collagen type I, whereas collagenase and stromelysin bound to different sites and could be co-localized with the gelatinases. We conclude that gelatinases have different binding specificities from those previously documented for stromelysin and collagenase, which bind through their C-terminal domains to collagen fibrils.

Amino Acid Sequence↗
PubMed1995

Inhibition of bone resorption in vitro by selective inhibitors of gelatinase and collagenase.

Two low-molecular-mass inhibitors of matrix metalloproteinases (MMPs), CT1166, a concentration-dependent selective inhibitor of gelatinases A and B, and Ro 31-7467, a concentration-dependent selective inhibitor of collagenase, were examined for their effects on bone resorption and type-I collagenolysis. The test systems consisted of measuring (1) the release of [3H]proline from prelabelled mouse calvarial explants; (2) the release of 14C from prelabelled type-I collagen films by mouse calvarial osteoblasts; and (3) lacunar resorption by isolated rat osteoclasts cultured on ivory slices. In 24 h cultures, CT1166 and Ro 31-7467 inhibited both interleukin-1 alpha- (IL-1 alpha; 10(-10) M) and 1,25-dihydroxyvitamin D3 (10(-8) M)-stimulated bone resorption in cultured neonatal mouse calvariae at concentration selective for the inhibition of gelatinase (10(-9) M for CT1166) and collagenase (10(-8) M for Ro 31-7467) respectively. For each compound the inhibition was dose-dependent, reversible, and complete at a 10(-7) M concentration. However, CT1166 (10(-9) M) and Ro 31-7467 (10(-8) M) in combination were required to completely abolish IL-1 alpha-stimulated bone resorption in mouse calvariae throughout a 96 h culture period. Neither of the inhibitors affected protein synthesis, DNA synthesis nor the IL-1 alpha-stimulated secretion of the lysosomal enzyme, beta-glucuronidase. Both CT1166 and Ro 31-7467 partially inhibited IL-1 alpha-stimulated lacunar resorption by isolated osteoclasts, but were without effect on unstimulated lacunar resorption. Rodent osteoclasts produced collagenase and gelatinases-A and -B activity. In contrast the substrate used to assess osteoclast lacunar resorption contained no detectable collagenase or gelatinase activity. Both compounds dose-dependently inhibited 1,25-dihydroxyvitamin D3 (10(-8) M)-stimulated degradation of type-I collagen by mouse calvarial osteoblasts; however, complete inhibition of collagenolysis was only achieved at concentrations at which CT1166 and Ro 31-7467 act as general MMP inhibitors. This study demonstrates that collagenase and gelatinases A and/or B participate in bone resorption. While these MMPs may be primarily involved in osteoid removal, we conclude that they may also be released by osteoclasts, where they participate in bone collagen degradation within the resorption lacunae.

Amides↗
PubMed1995

An in vivo model for screening peptidomimetic inhibitors of gelatinase A.

Gelatinase A, a matrix metalloproteinase, is frequently associated with human solid tumors, and its secretion and activation in the tumor milieu is considered important in the process of angiogenesis, invasion, and metastasis. Consequently, metalloproteinase inhibitors may be of value in the therapy of cancer as well as other disease states involving tissue remodeling and release of biologically active peptide/protein by proteolytic cleavage. Here we describe the development of a rapid screening assay for in vivo activity of peptidomimetic inhibitors of gelatinase A that involves assessment of inhibition of an enzyme-substrate reaction in a circumscribed body compartment, the mouse pleural cavity. As examples of the utility of this assay, in vivo activity of the aryl sulfonamide, sulfamyl urea, morpholino and carboxylic acid functionality at the P3' position of a series of hydroxamic acid inhibitors was examined after administration both intraperitoneally (ip) (to approximate systemic administration) and orally. For up to 2 h after ip administration, all inhibitors tested showed marked activity (> 90% inhibition) at 17 mumol/kg (approximately 10 mg/kg). This activity declined in a dose-responsive manner to insignificant levels at 0.67 mumol/kg (approximately 0.4 mg/kg). Aryl sulfonamides showed significant inhibition (> 50%) for up to 7 h after administration. A higher dosage (136 mumol/kg, approximately 80 mg/kg) was required to reveal oral activity, which was observed only with morpholino compounds (> 50% inhibition). Thus, the model described may be of value in the identification of orally active gelatinase A inhibitors.

Administration, Oral↗
PubMed1995

Kupffer cell-derived 95-kd type IV collagenase/gelatinase B: characterization and expression in cultured cells.

Release of 92-kd type IV collagenase/gelatinase, also known as gelatinase B, by inflammatory and tumor cells is increasingly recognized and is believed to facilitate cellular migration across basement membranes. It has been implicated in the pathogenesis of many diseases, but little is known of its cellular origin(s) and function in liver. In this study we have demonstrated synthesis and release of gelatinase B by human and rat Kupffer cells in primary culture. Northern analysis of RNA extracted from Kupffer cells stimulated with phorbol ester demonstrated a 2.8 kb transcript for gelatinase B. Immunoblotting and zymography of serum-free Kupffer cell-conditioned media demonstrated extracellular release of immunoreactive enzyme and gelatinase activity, Mr 92,000 (95,000 from rat cells). The organomercurial 4-aminophenyl mercuric acetate (APMA) activated the enzyme in vitro, indicating secretion primarily as a proenzyme. Stimulation of Kupffer cells by phorbol ester markedly induced gelatinase B release, which was inhibited by cycloheximide. In contrast, cycloheximide had no effect on constitutive secretion in culture, suggesting that there is some intracellular storage. Kupffer cell-derived gelatinase B was also partially purified and characterized. After separation by gelatin sepharose and gel filtration chromatogrpahy, gelatin-degrading activities of 95, 88, 75, and 65 kd were detected, the three lower-molecular-weight species probably representing activated forms. Enzyme activity was inhibited by ethyl-enediaminetetra-acetic acid (EDTA), but not by serine- and thiol-protease inhibitors, and was restored by zinc. Activity was also inhibited by tissue inhibitor of metalloproteinase-1 (TIMP-1) and alpha-2 macroglobulin. The partially purified enzyme rapidly degraded denatured collagens (gelatin) as well as native types III, IV, and V collagens, but had no activity against casein, types I and VI collagens.

Animals↗
PubMed1994

Reciprocated matrix metalloproteinase activation: a process performed by interstitial collagenase and progelatinase A.

Gelatinase A, a member of the matrix metalloproteinase (MMP) family, is secreted possessing an 80 amino acid N-terminal propeptide that must be removed in order to generate the active enzyme. Purified progelatinase A was activated to 38% of maximum by a 6 h incubation at 37 degrees C with equimolar concentrations of trypsin-activated interstitial collagenase (another MMP). The increase in activity was accompanied by cleavage of the M(r) 72,000 progelatinase A to the M(r) 66,000 active enzyme that has Y81 as its N-terminus. At low concentrations, progelatinase A was processed via an inactive intermediate, suggesting that its activation is a biphasic process. This was confirmed by the action of collagenase on proE375-->A (a mutant of progelatinase A that cannot become active) because, in this instance, only an M(r) 68,000 species with L38 as the N-terminus was produced. The remaining propeptide amino acids to Y81 could be readily removed by added active gelatinase A, indicating that collagenase works by generating an intermediate that is susceptible to autolytic activation. Although relatively slow, the rate of activation could be increased approximately 10-fold by the addition of 100 micrograms/mL heparin. This binds to the C-terminal domain of collagenase and progelatinase A and presumably acts as a template that positions the reactants close to one another. Collagenase activated by trypsin retains 8 or 14 amino acids of its propeptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Solution structure of the active domain of tissue inhibitor of metalloproteinases-2. A new member of the OB fold protein family.

Homonuclear two-dimensional and three-dimensional 1H nuclear magnetic resonance spectroscopy has been used to obtain essentially complete sequence-specific assignments for 123 of the 127 amino acid residues present in the truncated form of tissue inhibitor of metalloproteinases-2 (delta TIMP-2), the active N-terminal domain of the protein. Analysis of the through-space nuclear Overhauser effect data obtained for delta TIMP-2 allowed determination of both the secondary structure of the domain and also a low-resolution tertiary structure defining the protein backbone topology. The protein contains a five-stranded antiparallel beta-sheet that is rolled over on itself to form a closed beta-barrel, and two short helices which pack close to one another on the same barrel face. A comparison of the delta TIMP-2 structure with other known protein folds reveals that the beta-barrel topology is homologous to that seen in proteins of the oligosaccharide/oligonucleotide binding (OB) fold family. The common structural features include the number of beta-strands and their arrangement, the beta-barrel shear number, an interstrand hydrogen bond network, the packing of the hydrophobic core, and a conserved beta-bulge. Superpositions of the beta-barrels from delta TIMP-2 and two previously known members of the OB protein fold family (staphylococcal nuclease and Escherichia coli heat-labile enterotoxin) confirmed the similarity in beta-barrel topology. The three-dimensional structure of delta TIMP-2 has allowed a more detailed interpretation than was previously possible of the functional significance of available protein sequence and site-directed mutagenesis data for the TIMP family. Furthermore, the structure has revealed conserved surface regions of potential functional importance.

Amino Acid Sequence↗
PubMed1994

Mutation of the active site glutamic acid of human gelatinase A: effects on latency, catalysis, and the binding of tissue inhibitor of metalloproteinases-1.

Human gelatinase A, a member of the matrix metalloproteinase family, is secreted from cells as the M(r) 72,000 latent precursor, progelatinase A. The autolytic removal of an N-terminal propeptide generates the M(r) 66,000 active form. Mutants of recombinant progelatinase A, altered such that the proposed active site glutamic acid residue (E375) was replaced by either an aspartic acid (proE375-->D), an alanine (proE375-->A) or a glutamine (proE375-->Q), were purified from medium conditioned by transfected NS0 mouse myeloma cells. Like wild-type progelatinase A, the mutant proenzymes were inactive and could bind tissue inhibitor of metalloproteinases (TIMP)-2 but not TIMP-1 to their C-terminal domains. Their rates of autolytic processing induced by the organomercurial (4-aminophenyl) mercuric acetate, however, were markedly slower and, of the three M(r) 66,000 forms so produced, only E375-->D displayed any proteolytic activity against either a synthetic substrate (kcat/Km = 10% that of the wild-type enzyme) or denatured type I collagen (specific activity = 0.9% that of the wild-type enzyme). ProE375-->A and proE375-->Q could be more rapidly processed to their M(r) 66,000 forms by incubation with a deletion mutant of gelatinase A that has full catalytic activity but lacks the C-terminal domain [delta (418-631) gelatinase A]. These two M(r) 66,000 forms displayed low activity on a gelatin zymogram (approximately 0.01% that of the wild-type enzyme) but, like E375-->D were able to bind TIMP-1 with an affinity equal to that of the activated wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Analysis of the role of the COOH-terminal domain in the activation, proteolytic activity, and tissue inhibitor of metalloproteinase interactions of gelatinase B.

Recombinant human progelatinase B and a COOH terminally truncated version, pro-delta426-688 gelatinase B have been prepared from a myeloma cell expression system. Both proenzymes could be processed to active forms by stromelysin-1 to give an NH2 terminus of Phe88, or by treatment with 4-aminophenylmercuric acetate resulting in an NH2-terminal Met75. The kinetics of activation using either treatment was not affected by removal of the enzyme COOH-terminal domain. The specific activities of both gelatinase B and delta426-688 gelatinase B, activated using either method, were found to be similar using either a quenched fluorescent peptide or gelatin as the substrate. Fibroblast monolayers were shown to mediate processing of both progelatinases at similar rates in the presence of either plasminogen or prostromelysin-1. Active wild-type gelatinase B was inhibited by tissue inhibitor of metalloproteinase (TIMP) -1 at a much faster rate than TIMP-2. COOH-terminal truncation of either enzyme or inhibitor gave a marked reduction in the rate constant for TIMP-1 inhibition but had no effect on the rate of TIMP-2 binding. It can be concluded that the COOH-terminal domain of progelatinase B is not involved in autolytic or cellular activation and does not affect the catalytic activity of the enzyme. However, COOH-terminal domain interactions between active gelatinase B and TIMP-1 significantly enhance the rate of complex formation.

Amino Acid Sequence↗
PubMed1994

Assessment of the role of the fibronectin-like domain of gelatinase A by analysis of a deletion mutant.

The properties of a deletion mutant delta V191-Q364 of gelatinase A, which represents the removal of the fibronectin-like type II repeats defined by exons 5-7, were compared with those of full-length gelatinase A. Both enzymes underwent self-activation over a similar time course in the presence of 4-aminophenylmercuric acetate. The fully active enzymes had similar kcat/Km values for the cleavage of an octapeptide substrate, but the deletion mutant had 50% of the activity of wild type gelatinase A against beta-casein and 10% of the activity against gelatin. The cleavage pattern for gelatin was similar for both enzymes but differed for type IV collagen. Comparison of the rates of association of the tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 and their N-terminal domains to both forms of gelatinase indicated that the fibronectin-like domain plays little role in TIMP binding. The deletion mutant failed to bind to collagen, while the wild type gelatinase bound tightly, indicating that the fibronectin-like domain is the sole site of collagen binding. Both gelatinases could be activated by concanavalin A-activated fibroblasts, suggesting that the fibronectin-like domain is not required for the membrane-mediated activation process.

Animals↗
PubMed1994

Different domain interactions are involved in the binding of tissue inhibitors of metalloproteinases to stromelysin-1 and gelatinase A.

The matrix metalloproteinases gelatinase A and stromelysin-1 have definable N-terminal (catalytic) and C-terminal domains. In order to analyze their interactions with the N- and C-terminal domains of the tissue inhibitors of metalloproteinases TIMP-1 and -2, mutants of both the enzymes and the inhibitors were prepared in which the C-terminal domains had been deleted. Since the Ki values for TIMP inhibition of the matrix metalloproteinases are in the picomolar range, it was not possible to measure these accurately within the sensitivity of available activity assays. Rate constants for the association of the wild-type proteins were therefore determined and systematically compared with those for the deletion mutants. It was found that TIMP-1 binds more rapidly than TIMP-2 to stromelysin-1 and that the C-terminal domain of the enzyme does not affect the rate of association of enzyme and inhibitor. This is in contrast to gelatinase A, where the C-terminal domain has been shown to play an important role in increasing the rate of complex formation with the TIMPs (Willenbrock et al., 1993). The TIMPs are also comprised of an N- and C-terminal domain. By deletion mutagenesis, we found that the C-terminal domain of both TIMPs contributed less to the rate of complex formation with stromelysin-1 than to that with gelatinase A. Hybrids of the N- and C-terminal domains of gelatinase A and stromelysin-1 were prepared and used to analyze further the differences in domain interactions with the TIMPs. They demonstrated that the interactions between the C-terminal domains of enzyme and inhibitor can occur irrespective of the nature of the N-terminal domain. We can conclude that the TIMPs have two major binding regions which associate in different ways with the domains of the enzymes gelatinase A and stromelysin-1. The N-terminal domains of the TIMPs bind to the enzyme catalytic domains to inhibit activity. The TIMP C-terminal domain acts to increase the association rate constant by binding to the N-terminal domain of stromelysin or the C-terminal domain of gelatinase A.

Amino Acid Sequence↗
PubMed1994

The effects of selective inhibitors of matrix metalloproteinases (MMPs) on bone resorption and the identification of MMPs and TIMP-1 in isolated osteoclasts.

We have compared the effects of a general matrix metalloproteinase (MMP) inhibitor (CT435) with those of a concentration-dependent specific gelatinase inhibitor (CT543; Ki < 20 nM) on bone resorption in vitro. The test systems consisted of measuring: (i) the release of 45Ca2+ from prelabelled mouse calvarial explants; (ii) the release of 45Ca2+ from prelabelled osteoid-free calvarial explants co-cultured with purified chicken osteoclasts; and (iii) lacunar resorption by isolated rat osteoclasts cultured on ivory slices. Both CT435 and CT543 dose-dependently inhibited the release of 45Ca2+ from neonatal calvarial bones stimulated by either parathyroid hormone or 1,25-dihydroxyvitamin D3. Moreover, CT543 produced a 40% inhibition at a concentration (10(-8) M) selective for the inhibition of human gelatinases A and B. CT435 (10(-5) M) and CT543 (10(-5) M) partially inhibited the release of 45Ca2+ from osteoid-free calvarial explants by chicken osteoclasts with a maximum of approximately 25% for unstimulated cultures, and approximately 36% for cultures stimulated by interleukin-1 alpha (IL-1 alpha; 10(-10) M). Neither inhibitor prevented lacunar resorption on ivory by unstimulated rat osteoclasts, but the compounds produced a partial reduction in both the number and total surface area of lacunae in IL-1 alpha-stimulated cultures, with maximal action at 10(-5) M. Neither of the inhibitors affected protein or DNA synthesis, nor the IL-1 alpha-stimulated secretion of the lysosomal enzyme beta-glucuronidase. Immunocytochemistry demonstrated that isolated rabbit osteoclasts constitutively expressed gelatinase A and synthesized gelatinase B, collagenase and stromelysin, as well as the tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) following IL-1 alpha stimulation. These experiments have shown that in addition to collagenase, gelatinases A and B are likely to play a significant role in bone resorption. They further suggest that MMPs produced by osteoclasts are released into the sub-osteoclastic resorption zone where they participate in bone collagen degradation.

Animals↗
PubMed1994

Elevated plasma stromelysin levels in arthritis.

OBJECTIVE: To determine whether plasma concentrations of stromelysin-1 and gelatinase A are increased in patients with various forms of arthritis. METHODS: A sensitive and specific sandwich enzyme linked immunosorbent assay (ELISA), which employs a murine monoclonal antibody and a rabbit polyclonal antibody to human stromelysin-1, was used to measure plasma stromelysin-1 in 53 healthy subjects, 113 patients with various forms of arthritis and connective tissue diseases, and 65 patients with cancer. Gelatinase A was also measured in these patients using specific polyclonal and monoclonal antibodies to gelatinase A in an ELISA: RESULTS: The plasma concentration of stromelysin-1 (X +/- SEM) was significantly increased (p < 0.001) in patients with rheumatoid arthritis (RA) (187 +/- 14 ng/ml) and systemic lupus erythematosus (SLE) (258 +/- 35 ng/ml) as compared to both healthy control subjects (50 +/- 4 ng/ml) or patients with cancer (61 +/- 20 ng/ml). Plasma stromelysin-1 was also significantly increased in smaller groups of men with osteoarthritis (OA) and gout. In contrast, plasma concentrations of gelatinase A were not significantly increased in patients with RA, OA or gout. In healthy subjects, the concentration of stromelysin-1 was significantly higher in men than women. No correlation was noted between plasma stromelysin-1 levels and age. CONCLUSION: The detection of elevated plasma levels of stromelysin-1 in patients with RA is consistent with increased stromelysin-1 concentrations in inflamed synovial tissues in this disease. The origin of increased plasma stromelysin-1 in SLE is speculative. Measurement of plasma stromelysin-1 may be useful in the diagnosis and management of patients with various forms of arthritis.

Adult↗