Phenotypic alterations in Caki-1 Cells as a consequence of TIMP-1 overexpression H.M. Reid a , A.M. McElligott b , H. McGlynn a, * a Cancer & Ageing Research Group, School of Biomedical Sciences, University of Ulster, Cromore Road, Coleraine, County Londonderry, Northern Ireland BT52 1SA, UK b Department of Biochemistry, Trinity College Dublin, Dublin 2, Ireland Received 31 January 2001; received in revised form 26 March 2001; accepted 29 March 2001 Abstract Maintenance of the extracellular matrix (ECM) is important for tissue integrity and cellular physiology. Normal ECM turnover is regulated by a balance between matrix metalloproteinases and their inhibitors, the tissue inhibitors of metallopro- teinases (TIMPs). In metastasis, this balance favours increased ECM degradation. The objective of this study was to determine the effects of TIMP-1 overexpression on the metastatic process. To this end, we stably transfected a renal carcinoma cell line, Caki-1, with TIMP-1, using a pRc/CMV expression plasmid and LIPOFECTAMINE e transfection reagent. The resultant clones displayed increased adhesion on the ECM substratum, including collagen type IV and laminin, and altered invasive capacity through ®bronectin and Matrigel w , dependent upon the level of TIMP-1 expression. These changes were not due to altered integrin expression, as assessed by ¯ow cytometry. As well as protease inhibitory activity, TIMPs can in¯uence cell proliferation and cell survival. The TIMP-1 clones displayed no changes in proliferation under normal growth conditions, compared with Caki-1 cells. However, under reduced serum conditions, the TIMP-1 clones had a greater percentage of cells in both S (P , 0:05) and G 2 /M (P , 0:005) phases and less cells in G 0 /G 1 (P , 0:001) of the cell cycle than Caki-1 cells. The results con®rm a dual role for TIMP-1 in invasion and metastasis, and provide further clues behind the molecular mechanisms in these processes. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Tissue inhibitor of metalloproteinase-1; Cell adhesion; Cell cycle; Invasion 1. Introduction Maintenance of the extracellular matrix (ECM) is important for normal tissue integrity and cell physiol- ogy. The ECM provides a framework to anchor cells and an environment that plays an integral role in cell proliferation, differentiation and cell survival [1,2]. ECM degradation and turnover are required for normal physiological processes, such as wound heal- ing and in¯ammation, but disturbances in ECM home- ostasis may have signi®cant consequences in the aetiology of many diseases, including arthritis, ather- osclerosis, and tumour progression [3,4]. The primary enzymes responsible for normal ECM turnover are the matrix metalloproteinase (MMP) family of zinc-dependent endopeptidases. At present, there are 24 members of the MMP family identi®ed and cloned [5±12], with speci®c and overlapping speci®city for the numerous components of the ECM, including ®bronectin, collagen and laminin [13]. They are synthesized as latent zymogens, Cancer Letters 169 (2001) 189±198 0304-3835/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S0304-3835(01)00520-1 www.elsevier.com/locate/canlet * Corresponding author. Tel.: 144-28-7032-4058; fax: 144-28- 7032-4965. E-mail address: h.mcglynn@ulst.ac.uk (H. McGlynn).