Inhibition of invasion and induction of apoptotic cell death of cancer cell lines by overexpression of TIMP-3

Dysregulation of matrix degrading metalloproteinase enzymes (MMPs) leads to increased extracellular matrix turnover, a key event in the local invasion and metastasis of many tumours. The tissue inhibitors of metalloproteinases (TIMPs) limit the activity of MMPs, which suggests their use in gene therapy. We have previously shown that overexpression of TIMP-1, -2 or -3 inhibits vascular smooth muscle and melanoma cell invasion, while TIMP-3 uniquely promotes apoptosis. We have therefore sought to determine whether TIMP-3 can inhibit invasion and promote apoptosis in other cancer cell types. Adenoviral-mediated overexpression of TIMP-3 inhibited invasion of HeLa and HT1080 cells through artificial basement membrane to similar levels as that achieved by TIMP-1 and -2. However, TIMP-3 uniquely promoted cell cycle entry and subsequent death by apoptosis. Apoptosis was confirmed by morphological analysis, terminal dUTP nick end labelling (TUNEL) and flow cytometry. The apoptotic phenotype was mimicked by addition of exogenous recombinant TIMP-3 to uninfected cultures demonstrating that the death signal is initiated extracellularly and that a bystander effect exists. These results show that TIMP-3 inhibits invasion in vitro and promotes apoptosis in cancer cell type of differing origin. This clearly identifies the potential of TIMP-3 for gene therapy of multiple cancer types. © 1999 Cancer Research Campaig

Analysis of the contribution of the hinge region of human neutrophil collagenase (HNC, MMP-8) to stability and collagenolytic activity by alanine scanning mutagenesis

Knauper V, Docherty AJP, Smith B, Tschesche H, Murphy G. Analysis of the contribution of the hinge region of human neutrophil collagenase (HNC, MMP-8) to stability and collagenolytic activity by alanine scanning mutagenesis. FEBS LETTERS. 1997;405(1):60-64.Analysis of the hinge region of neutrophil collagenase by alanine scanning mutagenesis revealed that this sequence motif has a pronounced effect on the stability and collagenolytic activity of the active enzyme, The mutagenesis of the amino acid residues in the P-1' position of the two autoproteolytically cleaved peptide bonds (Leu(243) and Ile(248)) to Ala showed that the mutant enzymes were more resistant to autoproteolysis, However, these mutants were not completely stable and autoproteolysis occurred mainly at the Ala(239) - Ile(240) peptide bond and the half-life of the active enzyme was increased by 50%, In contrast, mutagenesis of Pro(247) --> Ala (P-1 of the minor cleavage site Pro(247) - Ile(248)) lead to increased susceptibility of the enzyme to autoproteolysis, However,when the other P-1 position Gly(242) was altered to Ala no effect on stability was observed, The analysis of the ability of the mutant active enzymes to hydrolyse C-14-type I collagen was assessed and our results demonstrate that the hinge sequence motif of neutrophil collagenase is important for collagenolytic activity. The alteration of the Gly(242)-Leu-Ser-Ser-Asn-Pro-Ile-Gln-Pro(247) sequence motif to Gly(242)-Ala-Ala-Ala-Ala-Pro-Ala-Ala-Pro(247) showed that the collagenolytic activity was reduced by 68.4%, In addition, mutagenesis of the downstream sequence motif Pro(247)-Thr-Gly-Pro-Ser-Thr-Pro-Lys-Pro(258) to Pro(247)-Ala-Ala-Pro-Ala-Ala-Pro-Ala-Pro(258) had an even more marked effect on the collagenolytic activity, which was reduced by 87.4%, When the Pro residues in the hinge motif (Pro(247), Pro(250), Pro(253) and P-256) were altered to Ala the collagenolytic activity dropped to 1.5% of the value observed for wild-type enzyme. (C) 1997 Federation of European Biochemical Societies

Mutations in the tissue inhibitor of metalloproteinases-3 (TIMP3) in patients with Sorsby's fundus dystrophy

The hereditary macular dystrophies are progressive degenerations of the central retina and contribute significantly to irreversible visual loss in developed countries. Among these disorders, Sorsby's fundus dystrophy (SFD), an autosomal dominant condition, provides an excellent mendelian model for the study of the genetically complex age-related macular degeneration (AMD), the most common maculopathy in the elderly. Recently, we mapped the SFD locus to 22q13−qter. This same region contains the gene for tissue inhibitor of metalloproteinases-3 (TIMP3), which is known to play a pivotal role in extracellular matrix remodeling. We have now identified point mutations in the TIMP3 gene in affected members of two SFD pedigrees. These mutations are predicted to disrupt the tertiary structure and thus the functional properties of the mature protein