Campo Dell'Orto Marta, Banelli Barbara, Giarin Emanuela, Accordi Benedetta, Trentin Luca, Romani Massimo, te Kronnie Geertruy, Basso Giuseppe
Department of Paediatrics, University of Padova, I-35128 Padova, Italy.
Oncol Rep. 2007 Aug;18(2):417-23.
Hypermethylation of CpG islands is the most well defined epigenetic change in neoplasia and plays an important role in the inactivation or silencing of cancer related genes. DLX genes (1-7), with large CpG islands in their 5' region, are implicated in a number of processes among which haematopoiesis. They are characterized by highly dynamic spatio-temporal expression and supposed to be involved in resistance to apoptosis of several tumor cell lines. In acute lymphoblastic leukemia (ALL) hypermethylation is a common phenomenon frequently associated with poor prognosis in specific genetic childhood leukemia subgroups. These data together with the presence of large CpG islands in the up-stream regions of the DLX genes make them attractive candidates for methylation regulated gene expression and leukemia related aberrancies. To validate the role of DLX genes in paediatric B-ALL cells, we studied two cell lines and two groups of patients with paediatric chromosomal rearrangements: MLL-AF4 and TEL-AML1, respectively. Analysis of methylation and gene expression patterns of DLX3 in 64 specimens of B-lineage ALL revealed that DLX3 presents aberrant methylation in paediatric B-ALL patients. In vitro experiments with 5-Aza-2'dC on leukemia cell lines, confirmed by Western blot analysis, indicated that the methylation of DLX3 CpG islands has a functional role and interferes with the DLX3 gene and DLX3 protein expression in B-ALL cells. Importantly, hypermethylation of DLX3 significantly reduces its expression in MLL-AF4 rearranged leukemias while methylation is almost absent in TEL-AML1 positive ALL specimens. These results show that differential DLX3 methylation could be a new epigenetic marker for genotypic B-cell leukemia subgroup with high-risk features.
CpG岛的高甲基化是肿瘤形成中最明确的表观遗传变化，在癌症相关基因的失活或沉默中起重要作用。DLX基因（1 - 7）在其5'区域有大的CpG岛，参与多种过程，其中包括造血作用。它们的特点是具有高度动态的时空表达，并且被认为参与了几种肿瘤细胞系对凋亡的抵抗。在急性淋巴细胞白血病（ALL）中，高甲基化是一种常见现象，在特定的儿童白血病基因亚组中常与预后不良相关。这些数据以及DLX基因上游区域存在大的CpG岛，使得它们成为甲基化调节基因表达和白血病相关异常的有吸引力的候选基因。为了验证DLX基因在儿童B-ALL细胞中的作用，我们研究了两种细胞系以及两组患有儿童染色体重排的患者：分别是MLL-AF4和TEL-AML1。对64例B系ALL标本中DLX3的甲基化和基因表达模式分析显示，DLX3在儿童B-ALL患者中存在异常甲基化。用5-氮杂-2'-脱氧胞苷对白血病细胞系进行的体外实验，经蛋白质免疫印迹分析证实，表明DLX3 CpG岛的甲基化具有功能作用，并干扰B-ALL细胞中DLX3基因和DLX3蛋白的表达。重要的是，DLX3的高甲基化显著降低了其在MLL-AF4重排白血病中的表达，而在TEL-AML1阳性ALL标本中几乎不存在甲基化。这些结果表明，DLX3甲基化差异可能是具有高危特征的基因型B细胞白血病亚组的一种新的表观遗传标志物。
