GENES DIFFERENTIALLY EXPRESSED DURING TUMOR PROMOTION AND PROGRESSION

MRNA的差异显示已被用来鉴定其基因 表达可能驱动或阻止进展为肿瘤细胞表型。 其中一个基因，金属蛋白酶组织抑制因子(TIMP-3)，是 在癌前病变细胞中持续表达，但在肿瘤JB6细胞中不表达 (Sun等人，癌症资源，1994；Sun等人，J Biol Chem，1995) 提示TIMP-3可能具有肿瘤抑制作用。TIMP-3 基因在人DLD结肠癌细胞中的表达 皮下注射内源性基因确实抑制了肿瘤的生长 注射(Sun等人，致癌，1996)。因此，我们很感兴趣 在转录抑制TIMP-3的分子基础上。这个 TIMP-3启动子中6个AP-1结合位点显示差异结合 以及转录活动表明只有一小部分 AP-1位点在该基因的正调控中(Kim等人， 已呈交)。TIMP-3启动子在JB6肿瘤中的高甲基化 细胞和甲基酶抑制剂5-氮杂胞苷上调TIMP-3 表情。TIMP-3启动子-荧光素酶HpaII位点甲基化的研究 而不是胶原酶-荧光素酶报告构建沉默启动子 转录活性。应用聚合酶链式反应定位HpaII甲基化位点 被执行了。其中两个HpaII位点显示出差异甲基化 在肿瘤和癌前病变的JB6细胞中，表明了 序列特异性甲基化在调节TIMP-3转录中的作用。 进行了单独的差异显示分析以确定 肿瘤启动子诱导的可能调节或抑制的基因表达 肿瘤促进剂依赖的进展阶段。全长c DNA 已分离到两个优先表达的基因克隆 具有促进抗性的小鼠JB6细胞(Cmarik等人，正在准备中)。 一种是新奇的；另一种是已知的定位于细胞核。这个 这两个基因在预防癌症发生中的可能作用是 正在接受评估。最后，我们发现 染色质蛋白(S)HMG I(Y)优先受肿瘤诱导 启动子在促进敏感细胞中，并正在研究 这件事的因果意义。

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.