Suppression of in vivo tumor growth and induction of suspension cell death by tissue inhibitor of metalloproteinases (TIMP)-3

Tissue inhibitor of metalloproteinases-3(TEMP-3), a novel memberof TEMP family genes, has been recently cloned and shown tobe expressed in preneoplastic but not in neoplastic mouse JB6epidermal cells (Sun et al. 1994 Cancer Res., 54, 11139). Thisdown regulation of the gene appears to be attributable at leastin part to alteration of gene methylation (Sun et al. 1995 J.Biol Chem., 270, 19312). Little is known, however, about therole of TEMP-3 in human cancers. We screened several human tumorcell lines for TEMP-3 expression and found that a colon carcinomaline, DLD-1, did not express TEMP-3. If down regulation of TIMP-3is causally related to carcino-genesis, re-expression by transfectionmay reverse the tumor cell phenotype. We therefore overexpressedhuman TEMP-3 in DLD-1 cells. TEMP-3 transfectants showed a serum-dependentgrowth inhibition in monolayer culture and a decreased growthpotential in nude mice in a manner dependent on the level ofTEMP-3 expression. A transfectant expressing a high level ofactive hTEMP-3 completely lost the ability to form tumors followings.c. injection into nude mice. We also tested TEMP-3 expressingcells and neocontrol TEMP-3 negative cells for their abilityto grow in liquid suspension culture, since both cells grewin semi-solid soft agar. As compared to neocontrol cells, TIMP-3over-expressors formed large aggregates, followed by cell death.This effect was not mimicked by BB94, a broad MMP inhibitor.We conclude from this study that (i) TEMP-3 overexpression inhuman colon carcinoma cells induces growth arrest in low serumconditions and inhibits in vivo tumor growth and (ii) the TEMP-3-inducedlarge aggregate formation and subsequent cell death under suspensiongrowth cannot be explained by its MMP inhibitory activity.