Matrix metalloproteinase and tissue inhibitor of metalloproteinase ...

Biochemical Society Transactions (1 997) 25 147s

Matrix metalloproteinase and tissue inhibitor of metalloproteinase regulation of the invasive potential of a metastatic renal cell line.

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ANTHONY M. McELLIGOTT, ANDREW H. BAKER* and HUGH M c G L Y ” School of Biomedical Sciences, University of Ulster at Coleraine BT52 ISA, Northern Ireland. *British Heart Institute, University of Bristol, Bristol BS2 8 H W , U.K. Matrix metalloproteinases (MMPs) are members of a family of zinc atom dependent endopeptidases that can degrade components of the extracellular matrix (ECM) and, along with their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), play the primary role in ECM remodelling [I]. As metastatic tumour cells have to degrade the ECM in order to invade surrounding tissues it is believed that increased expression of MMPs by tumour cells play an important role in tumour cell invasion and metastasis [2]. Increased MMP expression has been reported to correlate positively with an invasive and metastatic phenotype in tumour cells and this invasive and metastatic process has been shown to be inhibited by TIMPs [3,4,51. Here we report on analysis of the relative level of expression of MMP-2 and MMP-9 (the gelatinases), and TIMP- 1, TIMP-2 and TIMP-3 in a metastatic renal carcinoma cell line, caki- 1, using the reverse transcriptase polymerase chain reaction (RT-PCR), and compare this expression profile to profiles obtained from the noninvasive renal cell lines, human proximal tubular (HPT) cells and 293 cell line. Using liposomal mediated gene transfer we transfected caki-1 cells with the genes for MMP-2, MMP-9, TIMPl,TIMP-2 and TIMP-3 and assessed the effect this had on the invasion potential of the caki-l cells in vitro . RT-PCR was preformed as described previously [6]. In summary, total RNA was isolated from the cells using the Promega total RNA isolation system. lug of total RNA was reversed transcribed using AMV reverse transcriptase (Boehringer Mannheim) and 5 2 0 % of the reaction mix was used for subsequent PCR (Figure. 1). Caki-1 cells show a greater level of expression of MMP-2 and a lower level of expression of TIMP-3 than the noninvasive controls. The levels of expression of TIMP-I and TIMP-2 in caki-1 cells, HFT cells and 293 cells are similar. MMP-9 expression was not detected in these cells. The constant intensity of the glyceraldehyde-phosphatedehydrogenase gene band in the caki1, HFT and 293 cells demonstrates that the differences in intensity of the MMP and TIMP genes bands between the three cell types is due to different levels of expression of these genes.

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Figure 1. RT-PCR products using primers specific for the respective genes analysed using electrophoresis in 1.2% agarose gels.

Abbreviations used MMP, matrix metalloproteinase;ECM, extracellular matrix; TIMP, tissue inhibitor of metalloproteinase; HPT, human proximal tubular cells

Control MMP-2 MMP-9 TIMP-I TIMP-2 TIMP-3 Figure 2. The relative in vitro invasion potential of caki-l cells transfected with pRC/CMV (control) and pRC/CMV containing cDNAs for MMPs and TIMPs. ( * p IO.01) Caki-1 cells were transfected with the genes for MMP-2, MMP9, TIMP-I, TIMP-2 and TIMP-3 using liposomal mediated gene transfection. In brief, human cDNAs for these genes were cloned downstream of the cytomegalovirus promoter in the eukaryotic expression vector pRCICMV. Caki- 1 cells were transfected with these plasmids using LipofectAMINE reagent (Gibco BRL) [7]. Following incubation of caki-l cells with the LipofectAMINE-DNA complexes the cells were seeded at constant density in the Matrigel invasion chamber which consisted of an 8 km pore membrane coated with ECM components. Invasive cells can degrade the ECM components and migrate through the membrane and adhere to the underside of the membrane where they can be counted after staining with Mayers Haemalum. The invasive profile of caki-1 cells transfected with the plasmids containing genes for MMP-2, MMP-9, TIMP-I, TIMP-2 and TIMP-3 were compared to the invasion profile obtained for caki- I cells transfected with pRC/CMV vector. Figure 2 shows a significantly increased invasive profile for cells transfected with MMP genes and significantly decreased invasion potential for cells transfected with genes for the TIMPs. These results show that caki-1 cells have a higher relative expression levels of MMP-2 and MMP-9 than the non-invasive controls and lower relative expression levels for TIMP-2 and TIMP3. The in vitro invasion studies show increased expression of MMPs corresponding with an increased invasive profile in caki-1 cells, while increased TIMP expression corresponds with a decreased invasive profile in these cells. These results suggest that MMPs and TIMPs play an important role in the invasive process in caki-1 cells. Human renal cell cancer is highly invasive and refractory to conventional cancer therapies [9]. This study of the role of MMPs and TIMPs in the invasive process in a renal cell carcinoma cell line may add to a greater understanding of the invasive and metastatic processes in renal cell cancer and contribute to the development of new therapies. The authors acknowledge the financial support of the Ulster Cancer Foundation. 1. Matrisian, L.M. (1990) Trends Genet. 6, 121-125 2. Liotta, L.A. (1986) Cancer Res. 46, 1-7 3. Khokhar, R. & Denhardt D.T. (1989) Invasion Metastasis 9, 39 1-405 4 . Naylor M.S., Stamp G.W., Davies B.D. Balkwill F.R. (1994) 1nt.J.Cancer 58, 50-56 5 . Watanabe, M., Takahashi, Y., Ohta, T., Mai, M., Sasaki, T. & Seiki, M. (1996) Cancer 77, 1676-1680 6 . McElligott, A.M., Baker A.H. & McGlynn H. (1996) Clin. Exp. Metastasis (In Press) 7. Felgner P.L. (1991) in Methods in Molecular Biology (Murray E.J., ed.), Vol. 7, pp. 81-89, The Humana Press Inc., Clifton, New Jersey. 8. Dekernion J.B. (1986) in Cambels Urology (Walsh P.C., Gittes R.F., Perlmutter A.D. & Stamey T.A., eds.), pp. 1294-1342 W.B. Sanders Company, Philadelphia.