The expression of gelatinase B (MMP-9, 92K-GL) has been correlated with the invasive phenotype of cancer cells, including oral-related cancers. 92K-GL is a member of the family of matrix metalloproteinases (MMP), a homologous group of 10 zinc dependent endopeptidases which collectively cleave most components of the extracellular matrix (ECM) and basement membrane (BM). The activation and subsequent degradation of substrate by MMPs are controlled by the tissue inhibitors of metalloproteinases (TIMP- 1, -2, and -3), with TIMP-l having the unique, natural ability to bind to the pro-form of 92K-GL. It has been proposed in the literature that 92K-GL acts cooperatively with other MMP in ECM and BM degradation, and that an imbalance in the TIMP/MMP ratio is related to the invasive potential of cancer cells. The overall objective of this proposal is to understand the role of 92K-GL in relation to other MMP and TIMP-l in the degradation of ECM and BM component proteins by human cancer cells. We have a unique collection of antibodies (Abs) which specifically block each of the metalloproteinases and TIMP-I and -2. We have also developed 2 sublines of fibrosarcoma cells which can be manipulated to produce only 72K-UL (HT- 1080-R) or 92K-GL (HT-1080-B) and serve as a valuable opportunity to compare the effects of these 2 gelatinases by in vitro activity assays and cell-mediated ECM and BM degradation experiments.
In Specific Aim l we propose to determine the effect of 92K-GL in relation to other MMP on the degradation of BM and ECM components. The effect of 92K-GL in pure form and in conditioned media, containing MMP-l,-2, -3, -9, TIMP-l and TIMP-2, on the dissolution of type I collagen, type IV collagen, denatured collagen (gelatin), and a synthetic peptide substrate will be examined. Abs which specifically block 92K-GL activity will be used singly and in combination with similar Abs directed against other MMPs and TIMPs to determine the contribution of 92K-GL to differences in total substrate cleavage and catalytic rates. These experiments should shed light on the level of cooperativity between the MMPs during substrate degradation.
In Specific Aim 2 we propose to determine the effect of 92K-GL on the degradation of ECM and BM molecules by live human cancer cells and to correlate this with the levels of other MMPs and TIMPs produced by these cells. We will examine the manner in which the 2 fibrosarcoma cell lines utilize 92K-GL to cleave type I and IV collagen matrices, again with the use of our MMP- and TIMP-blocking Abs to selectively remove individual MMP and TIMP activities. This data will be examined with respect to the levels of other MPs and TIMP-l and -2, as determined by Northern blot analysis, 2-site (sandwich) ELISA, quantitative PCR, and Western blot analysis. As a long term goal, we plan to use in situ hybridation (with PCR, if necessary) to determine the exact cellular production of 92K-GL, TIMP- l, and other MMPs in human head and neck cancer tissue to investigate to what degree an imbalance in the inhibitor/MMP ratio correlates with the in vivo invasive phenotype. The combination of the studies proposed in this application will provide a better understanding of the role of 92K-GL and TIMP-l, as well as other MMPs, in the invasive phenotype of human cancers of oral and other origins.
| Pickett, K L; Harber, G J; DeCarlo, A A et al. (1999) 92K-GL (MMP-9) and 72K-GL (MMP-2) are produced in vivo by human oral squamous cell carcinomas and can enhance FIB-CL (MMP-1) activity in vitro. J Dent Res 78:1354-61 |
