MOLECULAR-CLONING OF MOUSE-TISSUE INHIBITOR OF METALLOPROTEINASES-3 AND ITS PROMOTER - SPECIFIC LACK OF EXPRESSION IN NEOPLASTIC JB6 CELLS MAY REFLECT ALTERED GENE METHYLATION

Mouse tissue inhibitor of metalloproteinases-3 (mTIMP-3), a gene speci fically not expressed in neoplastic JB6 cells, has been isolated recen tly through the use of the mRNA differential display technique (Sun, Y ., Hegamyer, G., and Colburn, N. H. (1994) Cancer Res. 54, 1139-1144). We report here the full-length mTIMP-3 cDNA sequence, the promoter se quence and partial characterization, expression and induction of TIMP- 3, and the possible molecular basis for the lack of mTIMP-3 expression in neoplastic JB6 cells. There are three transcripts arising from alt ernative polyadenylation of mouse TIMP-3 gene, having sizes of 4.6, 2. 8, and 2.3 kilobase pairs, respectively. All three TIMP-3 transcripts are expressed in preneoplastic but not neoplastic JB6 cells. Computer analysis of cloned TIMP-3 promoter revealed six AP-1 binding sites, tw o NF-kappa B sites, a c-Myc site, and two copies of a p53 binding moti f separated by eight base pairs with two mismatches at the second moti f, along with many other cis elements, TIMP-3 gene expression was indu cible by AP-1 and NF-kappa B activators, 12-O-tetradecanoylphorbol-13- acetate, and tumor necrosis factor-alpha only in preneoplastic cells w ith an induction peak at 2 h post-treatment, suggesting classification of mTIMP-3 as a member of the immediate early gene family. Southern b lot, mutational analysis, and transient transcriptional activation exp eriments revealed that the lack of expression of mTIMP-3 in neoplastic JB6 cells was due neither to gross deletion nor to promoter mutation of the gene, nor was there a lack of transcription factors required fo r transcriptional activation. Instead, the lack of TIMP-3 expression i n neoplastic JB6 cells may reflect an abnormal methylation of the gene . Both hyper- and hypomethylation of the mTIMP-3 gene are associated w ith complete down-regulation of gene expression in neoplastic JB6 cell lines. Treatment of neoplastic cells with the methylase inhibitor 5-a zacytidine caused reexpression of the mTIMP-3 gene in a tumor cell lin e that showed hypermethylation but not in another that showed hypometh ylation of the gene, suggesting a complex role for methylation in the silencing of gene expression.