Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada.
Cancer Cell Int. 2007 Sep 10;7:14. doi: 10.1186/1475-2867-7-14.
Methylation-mediated silencing of genes is one epigenetic mechanism implicated in cancer. Studies regarding the role of modulation of gene expression utilizing inhibitors of DNA methylation, such as decitabine, in osteosarcoma (OS) have been limited. A biological understanding of the overall effects of decitabine in OS is important because this particular agent is currently undergoing clinical trials. The objective of this study was to measure the response of the OS cell line, U2OS, to decitabine treatment both in vitro and in vivo.
Microarray expression profiling was used to distinguish decitabine-dependent changes in gene expression in U2OS cells, and to identify responsive loci with demethylated CpG promoter regions. U2OS xenografts were established under the sub-renal capsule of immune-deficient mice to study the effect of decitabine in vivo on tumor growth and differentiation. Reduced nuclear methylation levels could be detected in xenografts derived from treated mice by immunohistochemistry utilizing a 5-methylcytidine antibody. Decitabine treatment reduced tumor xenograft size significantly (p < 0.05). Histological analysis of treated U2OS xenograft sections revealed a lower mitotic activity (p < 0.0001), increased bone matrix production (p < 0.0001), and a higher number of apoptotic cells (p = 0.0329). Microarray expression profiling of U2OS cultured cells showed that decitabine treatment caused a significant induction (p < 0.0025) in the expression of 88 genes. Thirteen had a >or=2-fold change, 11 of which had CpG-island-associated promoters. Interestingly, 6 of these 11 were pro-apoptotic genes and decitabine resulted in a significant induction of cell death in U2OS cells in vitro (p < 0.05). The 6 pro-apoptotic genes (GADD45A, HSPA9B, PAWR, PDCD5, NFKBIA, and TNFAIP3) were also induced to >or=2-fold in vivo. Quantitative methylation pyrosequencing confirmed that the tested pro-apoptotic genes had CpG-island DNA demethylationas a result of U2OS decitabine treatment both in vitro and in xenografts.
These data provide new insights regarding the use of epigenetic modifiers in OS, and have important implications for therapeutic trials involving demethylation drugs. Collectively, these data have provided biological evidence that one mode of action of decitabine may be the induction of apoptosis utilizing promoter-CpG demethylation of specific effectors in cell death pathways in OS.
基因的甲基化沉默是癌症中涉及的一种表观遗传机制。关于利用 DNA 甲基化抑制剂（如地西他滨）调节基因表达在骨肉瘤（OS）中的作用的研究有限。全面了解地西他滨在 OS 中的整体作用很重要，因为这种特殊药物目前正在进行临床试验。本研究的目的是测量 OS 细胞系 U2OS 对体外和体内地西他滨治疗的反应。
利用微阵列表达谱来区分 U2OS 细胞中地西他滨依赖性基因表达变化，并鉴定具有去甲基化 CpG 启动子区域的响应基因座。将 U2OS 异种移植物建立在免疫缺陷小鼠的肾下囊中，以研究地西他滨在体内对肿瘤生长和分化的影响。利用 5-甲基胞嘧啶抗体的免疫组化可以检测到来自治疗小鼠的异种移植物中核甲基化水平降低。地西他滨治疗显著降低了肿瘤异种移植物的大小（p < 0.05）。对经处理的 U2OS 异种移植物切片的组织学分析显示，有丝分裂活性降低（p < 0.0001），骨基质产生增加（p < 0.0001），凋亡细胞数量增加（p = 0.0329）。U2OS 培养细胞的微阵列表达谱显示，地西他滨处理导致 88 个基因的表达显著诱导（p < 0.0025）。有 13 个基因的变化倍数大于或等于 2 倍，其中 11 个基因具有 CpG 岛相关启动子。有趣的是，这 11 个基因中有 6 个是促凋亡基因，地西他滨导致体外 U2OS 细胞的细胞死亡显著诱导（p < 0.05）。这 6 个促凋亡基因（GADD45A、HSPA9B、PAWR、PDCD5、NFKBIA 和 TNFAIP3）在体内也被诱导至 > 或等于 2 倍。定量甲基化焦磷酸测序证实，在体外和异种移植物中，经地西他滨处理的测试促凋亡基因的 CpG 岛 DNA 去甲基化。
这些数据为 OS 中使用表观遗传修饰剂提供了新的见解，并对涉及去甲基化药物的治疗试验具有重要意义。总的来说，这些数据提供了生物学证据，表明地西他滨的作用机制之一可能是通过 OS 细胞死亡途径中特定效应物的启动子-CpG 去甲基化诱导细胞凋亡。
