Inhibition of MT1-MMP activity using functional antibody fragments selected against its hemopexin domain.

The membrane associated MMP, MT1-MMP, is a critical pericellular protease involved in tumour cell invasion and angiogenesis and is highly up-regulated in numerous human cancers. It therefore represents an exciting new therapeutic cancer-specific target. We have generated recombinant human scFv antibodies against the non-catalytic, hemopexin domain of MT1-MMP that modulate its interactions with collagen. One of these is an effective inhibitor of the invasive capacity of cancer cells and of angiogenesis in model systems. This demonstrates that targeting sites outside the catalytic domain presents a potential novel approach to proteinase inhibition that could have applications in cancer therapeutics.

Absence of p300 induces cellular phenotypic changes characteristic of epithelial to mesenchyme transition.

p300 is a transcriptional cofactor and prototype histone acetyltransferase involved in regulating multiple cellular processes. We generated p300 deficient (p300-) cells from the colon carcinoma cell line HCT116 by gene targeting. Comparison of epithelial and mesenchymal proteins in p300- with parental HCT116 cells showed that a number of genes involved in cell and extracellular matrix interactions, typical of 'epithelial to mesenchyme transition' were differentially regulated at both the RNA and protein level. p300- cells were found to have aggressive 'cancer' phenotypes, with loss of cell-cell adhesion, defects in cell-matrix adhesion and increased migration through collagen and matrigel. Although migration was shown to be metalloproteinase mediated, these cells actually showed a downregulation or no change in the level of key metalloproteinases, indicating that changes in cellular adhesion properties can be critical for cellular mobility.

Characterization of the role of the "MT-loop": an eight-amino acid insertion specific to progelatinase A (MMP2) activating membrane-type matrix metalloproteinases.

Progelatinase A (proGLA) activation is thought to be initiated almost exclusively by the type I transmembrane members of the membrane type matrix metalloproteinase family (MT-MMP): MT1, -2, -3, and -5-MMP (MMP14, -15, -16, and -24). One difference between these enzymes and the other MMP family members is the insertion of eight amino acids between strands betaII and III in the catalytic domain. In MT1-MMP, the best characterized of these enzymes to date, these residues consist of (163)PYAYIREG(170). To investigate the role of this region of MT1-MMP on its catalytic activities, we have made a variety of mutations and deletions in both soluble and membrane-bound forms of the enzyme. Characterization of the activity of the soluble forms toward peptides and fibrinogen revealed that neither mutation nor deletion of residues 163-170 significantly impaired catalytic function, suggesting these residues have little influence on conformation of the active site cleft. Equally none of the mutants showed significant differences in K(I)(app) for the N-terminal inhibitory domain of TIMP2, again indicating that mutation or deletion of resides 163-170 has no major effect on the overall topology of the active site of MT1-MMP. However, characterization of the kinetics of activation of proGLA with and without its gelatin binding region by the mutants generated have shown that efficient activation of proGLA is, at least in part, through an interaction with residues 163-170 of MT1-MMP. The expression, localization, and processing from the 63- to the 60/45-kDa forms of wild-type and key mutant forms of MT1-MMP were also examined by transient transfection in Chinese hamster ovary cells, but no differences were observed. Processing and activation of proGLA was also examined in transiently transfected cells. All the mutants examined were able process proGLA but, as found with the soluble forms, were kinetically impaired when compared with wild-type MT1-MMP.

Catalytic activities of membrane-type 6 matrix metalloproteinase (MMP25).

This study describes the biochemical characterisation of the catalytic domain of membrane-type 6 matrix metalloproteinase (MT6-MMP, MMP25, leukolysin). Its activity towards synthetic peptide substrates, components of the extracellular matrix and inhibitors of MMPs was studied and compared with MT1-MMP, MT4-MMP and stromelysin-1. We have found that MT6-MMP is closer in function to stromelysin-1 than MT1 and MT4-MMP in terms of substrate and inhibitor specificity, being able to cleave type-IV collagen, gelatin, fibronectin and fibrin. However, it differs from stromelysin-1 and MT1-MMP in its inability to cleave laminin-I, and unlike stromelysin-1 cannot activate progelatinase B. Our findings suggest that MT6-MMP could play a role in cellular migration and invasion of the extracellular matrix and basement membranes and its activity may be tightly regulated by all members of the TIMP family.

Membrane type 4 matrix metalloproteinase (MMP17) has tumor necrosis factor-alpha convertase activity but does not activate pro-MMP2.

Membrane type 4 matrix metalloproteinase (MT4-MMP) shows the least sequence homology to the other MT-MMPs, suggesting a distinct function for this protein. We have isolated a complete cDNA corresponding to the mouse homologue which includes the signal peptide and a complete pro-domain, features that were lacking from the human form originally isolated. Mouse MT4-MMP (mMT4-MMP) expressed in COS-7 cells is located at the cell surface but does not show ability to activate pro-MMP2. The pro-catalytic domain was expressed in Escherichia coli as insoluble inclusions and active enzyme recovered after refolding. Activity of the isolated catalytic domain against synthetic peptides commonly used for MMP enzyme assays could be inhibited by TIMP1, -2, and -3. The recombinant mMT4-MMP catalytic domain was also unable to activate pro-MMP2 and was very poor at hydrolyzing components of the extracellular matrix with the exception of fibrinogen and fibrin. mMT4-MMP was able to hydrolyze efficiently a peptide consisting of the pro-tumor necrosis factor alpha (TNFalpha) cleavage site, a glutathione S-transferase-pro-TNFalpha fusion protein, and was found to shed pro-TNFalpha when co-transfected in COS-7 cells. MT4-MMP was detected by Western blot in monocyte/macrophage cell lines which in combination with its fibrinolytic and TNFalpha-converting activity suggests a role in inflammation.

Individuality of human palatal rugae.

Investigators have implied that palatal rugae are unique to each individual. However, these researchers have disagreed as to whether or not legal identification could be based solely on palatal rugae. Previous studies used poor duplicating materials and may not have considered the effects of growth, extractions, palatal expansion, or some combination of these. The inadvertent use of other features of the cast, such as teeth, edentulous ridge morphology, muscle attachments, vestibular depth, or some combination of these, to aid in the identification, may have influenced their results. The purpose of this investigation was to determine if palatal rugae can be relied upon for identification. Twenty-five orthodontic cases were selected with pre- and post-treatment casts; orthodontic treatment had taken eighteen to sixty months. One hundred casts were randomly selected as variables. All casts were trimmed so that all areas except for the rugae area of the hard palate were removed. The twenty-five post-orthodontic casts were dispersed within 100 randomly selected casts. Nine investigators were given the twenty-five pre-orthodontic casts and asked to compare them to the other one hundred and twenty-five casts for possible matches. Data were collected as to percentage of correct matches and time required for comparison. Each set of pre- and post-orthodontic casts was properly identified (100%) by 8 investigators, and the remaining investigator correctly matched 22 out of 25 casts (88%). These conclusions were drawn.(ABSTRACT TRUNCATED AT 250 WORDS)