Genes Differentially Expressed During Tumor Promotion and Progression

mRNA的差异显示已被用于鉴定可能驱动或阻止肿瘤细胞表型进展的基因。其中一个基因，金属蛋白酶组织抑制剂TIMP-3)，在肿瘤前而非肿瘤JB6细胞中表达（Sun etal ., Cancer Res, 1994; Sun etal ., J Biol Chem, 1995），并且在人DLD结肠癌细胞中表达时被发现具有肿瘤抑制作用（Bian etal ., carcingenesis, 1996）。因此，我们对TIMP-3转录抑制的分子基础感兴趣（Kim et al.,Bioch.）。J， 1997和Pennie et al., Cell Growth and Diff, 1999)。TIMP-3启动子在肿瘤JB6细胞中高甲基化，甲基化酶抑制剂5-氮杂胞苷上调TIMP-3的表达。基于PCR的定位显示，三个HpaII甲基化位点在肿瘤和肿瘤前JB6细胞中表现出不同的甲基化，这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，并重现了肿瘤前JB6细胞中与TIMP-3表达相关的相同位点的未甲基化状态（Pennie et al, Cell Growth and Diff, 1999）。一项单独的差异显示分析被用于鉴定可能介导或抑制肿瘤启动子依赖的进展阶段的基因表达。两个基因在小鼠JB6促进抗性细胞中优先表达，并获得了完整cDNA克隆。一个指定的pdcd4 （Cmarik et al., PNAS 1999）是新颖的；另一个是plekstrin （Cmarik et al Genomics 2000）。新型pdcd4基因的反义表达将P-细胞转化为P+细胞，pdcd4基因的正义表达将P+细胞转化为P-细胞，从而与预防肿瘤启动子诱导的转化建立了因果关系。目前的研究主要集中在pdcd4蛋白的分子功能和定位上。对pdcd4对已知促进肿瘤所需的分子事件可能的抑制作用的研究表明，pdcd4的表达抑制转录因子AP-1的激活，但不抑制NFkappa B或鸟氨酸脱羧酶的激活（Yang等人提交）。通过酵母双杂交分析发现pdcd4结合伙伴有望阐明pdcd4预防癌症的分子机制。最后，我们发现，在促进敏感细胞中，肿瘤启动子优先诱导染色质蛋白HMG I(Y)的表达(s) (Cmarik等人，Oncogene, 1998)，并正在研究这一因果意义以及HMG I(Y)与其他分子的可能相互作用，这些分子的激活是转化所必需的。

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 revealed that three of the HpaII methylation 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 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. One designated pdcd4 (Cmarik et al., PNAS 1999, is novel; the other is plekstrin (Cmarik et al Genomics 2000). Antisense expression of the novel pdcd4 gene converts P- to P+ cells and pdcd4 sense expression converts P+ to P- cells, thus establishing a causal relationship to prevention of tumor promoter induced transformation. Current studies focus on the molecular function and localization of pdcd4 protein. Examination of the possible inhibitory effect of pdcd4 on molecular events known to be required for tumor promotion revealed that pdcd4 expression inhibited the activation of transcription factor AP-1 but not of NFkappa B or of ornithine decarboxylase( Yang et al submitted). Discovery of pdcd4 binding partners by yeast two-hybrid analysis is expected to clarify the molecular mechanism by which pdcd4 prevents cancer. 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.