GENES DIFFERENTIALLY EXPRESSED DURING TUMOR PROMOTION AND PROGRESSION

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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 (Sun 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., submitted). The TIMP-3 promoter is hypermethylated in neoplastic JB6 cells and the methylase inhibitor 5-azacytidine upregulates TIMP-3 expression. Methylation of HpaII sites on TIMP-3 promoter-luciferase but not collagenase-luciferase reporter constructs silences promoter transcriptional activity. 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. 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. The possible role of both genes in the prevention of carcinogenesis is being assessed. Finally, we have discovered that expression of the chromatin protein(s) HMG I(Y) is preferentially induced by tumor promoters in promotion sensitive cells, and are investigating the causal significance of this.