Differential display of mRNA has been used to identify genes whose expression may drive or prevent progression to tumor cell phenotype. One such gene, tissue inhibitor of metalloproteinases TIMP-3), was consistently expressed in preneoplastic but not neoplastic JB6 cells (Sun et al., Cancer Res, 1994; Sun et al., J Biol Chem, 1995) suggesting a possible tumor suppression role for TIMP-3. TIMP-3 expression in human DLD colon carcinoma cells which lack expression of the endogenous gene did suppress tumor growth following subcutaneous injection (Bian et al., Carcinogenesis, 1996). We are thus interested in the molecular basis for transcriptional repression of TIMP-3. The 6 AP-1 binding sites in the TIMP-3 promoter show differential binding and transcriptional activities suggesting a role for only a subset of AP-1 sites in the positive regulation of this gene (Kim et al.,Bioch. J, 1997). The TIMP-3 promoter is hypermethylated in neoplastic JB6 cells and the methylase inhibitor 5-azacytidine upregulates TIMP-3 expression. PCR based mapping of HpaII methylation sites was carried out. Two of the HpaII sites show differential methylation in neoplastic and preneoplastic JB6 cells, suggesting the importance of sequence specific methylation in regulating TIMP-3 transcription. Expression of antisense DNA methyl transferase restored expression of TIMP-3 to tumor cells and recapitulated the unmethylated status of the same sites associated with TIMP-3 expression in preneoplastic JB6 cells (Pennie et al, submitted to PNAS). A separate differential display analysis was carried out to identify tumor promoter-induced gene expression that may mediate or inhibit tumor promoter dependent stages of progression. Full length cDNA clones have been isolated for two genes preferentially expressed in promotion resistant mouse JB6 cells (Cmarik et al., in preparation). One is novel; the other is known to localize in the nucleus. Antisense expression of one gene converts P- to P+ cells suggesting a causal relationship to prevention of tumor promoter induced transformation. Finally, we have discovered that expression of the chromatin protein(s)HMG I(Y)is preferentially induced by tumor promoters in promotion sensitive cells (Cmarik et al, Oncogene, in press), and are investigating the causal significance of this.
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