GENES DIFFERENTIALLY EXPRESSED DURING TUMOR PROMOTION AND PROGRESSION

mRNA的差异显示已用于鉴定 表达可以驱动或阻止肿瘤细胞表型的进展。 一个这样的基因，金属蛋白酶组织抑制剂（TIMP-3）， 在肿瘤前而非肿瘤性JB 6细胞中一致表达 (Sun例如，Cancer Res，1994; Sun等人，生物化学杂志，1995年） 提示TIMP-3可能的肿瘤抑制作用。 TIMP-3 在缺乏表达的人DLD结肠癌细胞中表达 内源性基因在皮下注射后确实抑制了肿瘤生长， 注射（Sun等人，致癌作用，1996年）。 因此，我们感兴趣 TIMP-3转录抑制的分子基础。 的 TIMP-3启动子中的6个AP-1结合位点显示差异结合 和转录活性表明，只有一个子集的作用， AP-1位点在该基因的正调控中（Kim等人， 提交）。 TIMP-3启动子在肿瘤性JB 6中高甲基化 甲基化酶抑制剂5-氮杂胞苷上调TIMP-3 表情 TIMP-3启动子-荧光素酶上HpaII位点的甲基化 而不是胶原酶-荧光素酶报告基因构建体沉默启动子 转录活性 基于PCR的HpaII甲基化位点定位 被执行。 两个HpaII位点显示差异甲基化 在肿瘤和癌前JB 6细胞中，表明 序列特异性甲基化调控TIMP-3转录。 进行单独的差异显示分析以鉴定 肿瘤启动子诱导的基因表达可能介导或抑制 肿瘤启动子依赖性进展阶段。 全长cDNA 已经分离出两种基因的克隆，这两种基因优先表达于 促进抗性小鼠JB 6细胞（Cmarik等，正在筹备中）。 一个是新的，另一个是已知的定位在细胞核中。 的 这两种基因在预防癌症发生中的可能作用是 正在评估。 最后，我们发现， 染色质蛋白HMG I（Y）优先被肿瘤诱导 启动子在促进敏感细胞，并正在研究 因果关系的重要性。

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.