Hofmann W-K, Takeuchi S, Takeuchi N, Thiel E, Hoelzer D, Koeffler H P
Department of Hematology, University Hospital Benjamin Franklin, Berlin, Germany.
Leuk Res. 2006 Nov;30(11):1347-53. doi: 10.1016/j.leukres.2006.03.015. Epub 2006 May 8.
Hypermethylation of CpG islands within the promoter region is one of the mechanisms by which genes are inactivated and may be one of the reason for silencing of cell cycle control or DNA-mismatch repair genes in myelodysplastic syndrome (MDS). Since the function of cell cycle control genes including the cyclin-dependent kinase inhibitors known as p15(INK4b) and p16(INK4a), as well as p14(ARF) which blocks MDM-2 (an inhibitor of p53), the retinoblastoma (RB1) protein and the mismatch repair gene MGMT is critical for hematopoietic proliferation and differentiation, we performed methylation specific polymerase chain reaction (MSP) in low-density, non-adherent bone marrow cells from 49 patients with MDS. In addition, expression of p15(INK4b) and RB1 was analysed by quantitative real-time PCR. From selected patients, we analyzed the methylation pattern of cell cycle control genes in CD34+ bone marrow cells. Thirty-nine of 49 cases (80%) had at least one of five genes methylated in our MDS samples by analysing low-density non-adherent bone marrow cells. The frequency of p15(INK4b) methylation was 34 of 49 samples (69%). The incidence of methylation of both p14(ARF) and p16(INK4a) was four of 49 (8%). RB1 gene was methylated in seven samples (14%) and each patient had RA. Interestingly, none of these genes were methylated in the purified CD34+ hematopoietic stem cells from the MDS patients. Furthermore, all our RARS patients had a methylated p15(INK4b) promoter correlating with non-detectable expression of this gene in bone marrow cells from those patients. These results indicate that hypermethylation of cell cycle control genes in MDS may occur late during the differentiation of myelodysplastic stem cells.
启动子区域CpG岛的高甲基化是基因失活的机制之一，可能是骨髓增生异常综合征（MDS）中细胞周期调控或DNA错配修复基因沉默的原因之一。由于包括被称为p15（INK4b）和p16（INK4a）的细胞周期蛋白依赖性激酶抑制剂以及阻断MDM-2（p53的抑制剂）的p14（ARF）、视网膜母细胞瘤（RB1）蛋白和错配修复基因MGMT在内的细胞周期调控基因的功能对造血增殖和分化至关重要，我们对49例MDS患者的低密度、非贴壁骨髓细胞进行了甲基化特异性聚合酶链反应（MSP）。此外，通过定量实时PCR分析了p15（INK4b）和RB1的表达。从选定的患者中，我们分析了CD34+骨髓细胞中细胞周期调控基因的甲基化模式。通过分析低密度非贴壁骨髓细胞，49例病例中有39例（80%）在我们的MDS样本中至少有五个基因中的一个发生甲基化。p15（INK4b）甲基化频率为49个样本中的34个（69%）。p14（ARF）和p16（INK4a）两者甲基化的发生率为49个中的4个（8%）。RB1基因在7个样本（14%）中发生甲基化，且每位患者均患有RA。有趣的是，这些基因在MDS患者纯化的CD34+造血干细胞中均未发生甲基化。此外，我们所有的RARS患者都有p15（INK4b）启动子甲基化，这与这些患者骨髓细胞中该基因的不可检测表达相关。这些结果表明，MDS中细胞周期调控基因的高甲基化可能发生在骨髓增生异常干细胞分化的后期。
