Genes Differentially Expressed During Tumor Promotion and Progression

MRNA的差异显示已被用来确定其表达可能驱动或阻止肿瘤细胞表型进展的基因。其中一个这样的基因，金属蛋白酶组织抑制因子TIMP-3，在癌变前的JB6细胞中表达，但不在肿瘤性JB6细胞中表达(Sun等人，癌症Res，1994；Sun等人，J Biol Chem，1995)，并被发现在人DLD结肠癌细胞中表达时起到肿瘤抑制作用(Bian等人，癌症发生，1996)。因此，我们对TIMP-3转录抑制的分子基础感兴趣(Kim等人，Bioch。J，1997和Pennie等人，Cell Growth and Diff，1999)。TIMP-3启动子在肿瘤JB6细胞中高甲基化，甲基酶抑制剂5-氮杂胞苷上调TIMP-3表达。基于聚合酶链式反应的定位显示，三个HpaII甲基化位点在肿瘤和癌前病变的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TIMP-3的表达，并概括了与癌前JB6细胞中TIMP-3表达相关的相同位点的未甲基化状态(Pennie等人，Cell Growth and Diff，1999)。进行了单独的差异显示分析，以确定可能调节或抑制肿瘤启动子依赖的进展阶段的基因表达。已分离到两个在抗启动子的小鼠JB6细胞中优先表达的基因的全长cDNA克隆。一个命名为pdcd4(Cmarik等人，PNAS 1999，是新的；另一个是plekstrin(Cmarik等人基因组学2000)。Pdcd4基因的反义表达可将P-转化为P+细胞，pdcd4正义表达可将P+转化为P-细胞，从而建立了阻止肿瘤启动子诱导转化的因果关系。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的可能抑制作用的研究表明，pdcd4的表达抑制了转录因子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，我们发现染色质蛋白(S)HMG I(Y)的表达是由肿瘤促进剂在促进敏感细胞中优先诱导的(Cmarik等，Oncogene，1998)，并正在研究这一原因的意义以及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.