GENES DIFFERENTIALLY EXPRESSED DURING TUMOR PROMOTION AND PROGRESSION

mRNA的差异显示已被用于鉴定其表达可驱动或阻止进展为肿瘤细胞表型的基因。一种这样的基因，金属蛋白酶组织抑制剂TIMP-3），在肿瘤前而非肿瘤性JB 6细胞中表达（Sun等人，Cancer Res，1994; Sun等人，J Biol Chem，1995），并且发现当在人DLD结肠癌细胞中表达时充当肿瘤抑制因子（Bian等人，致癌作用，1996年）。因此，我们对TIMP-3转录抑制的分子基础感兴趣（Kim et al.，生物。J，1997和Pennie等人，Cell Growth and Diff，1999）。TIMP-3启动子在肿瘤性JB 6细胞中高甲基化，甲基化酶抑制剂5-氮杂胞苷上调TIMP-3表达。基于PCR的HpaII甲基化位点定位显示，三个HpaII位点在肿瘤和癌前JB 6细胞中显示差异甲基化，表明序列特异性甲基化在调节TIMP-3转录中的重要性。反义DNA甲基转移酶的表达恢复了肿瘤细胞中TIMP-3的表达，并重现了与肿瘤前JB 6细胞中TIMP-3表达相关的相同位点的未甲基化状态（Pennie等，Cell Growth and Diff，1999）。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，其可能介导或抑制肿瘤启动子依赖性进展阶段。已经分离了在促进抗性小鼠JB 6细胞中优先表达的两个基因的全长cDNA克隆（Cmarik等人，PNAS in press，1999，Cmarik等人in prep，1999）。一个命名为pdcd 4是新的;另一个是plekstrin家族的成员。新型pdcd 4基因的反义表达将P-细胞转化为P+细胞，这表明与预防肿瘤启动子诱导的转化存在因果关系。目前的研究主要集中在pdcd 4蛋白的分子功能和定位。最后，我们已经发现，在促进敏感细胞中，染色质蛋白HMG I（Y）的表达优先由肿瘤促进剂诱导（Cmarik等，Oncogene，1998），并且正在研究这一点的因果意义以及HMG I（Y）与转化所需活化的其他分子的可能相互作用。- mRNA差异显示，基因表达，TIMP-3，肿瘤启动子诱导基因，HMG-I（Y），pdcd 4，

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 expressed in preneoplastic but not neoplastic JB6 cells (Sun et al., Cancer Res, 1994; Sun et al., J Biol Chem, 1995) and was found to act as a tumor suppressor when expressed in human DLD colon carcinoma cells (Bian et al., Carcinogenesis, 1996). We are thus interested in the molecular basis for transcriptional repression of TIMP-3(Kim et al.,Bioch. J, 1997 and Pennie et al., Cell Growth and Diff, 1999). 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 revealed that three 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, Cell Growth and Diff, 1999). 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., PNAS in press, 1999, Cmarik et al in prep, 1999). One designated pdcd4 is novel; the other is a member of the plekstrin family. Antisense expression of the novel pdcd4 gene converts P- to P+ cells suggesting a causal relationship to prevention of tumor promoter induced transformation. Current studies focus on the molecular function and localization of pdcd4 protein. 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, 1998), and are investigating the causal significance of this as well as the possible interaction of HMG I(Y) with other molecules whose activation is required for transformation. - Differential Display of mRNA, gene expression, TIMP-3, Tumor Promoter Induced Genes, HMG-I(Y), pdcd4,