Ritter M, de Kant E, Huhn D, Neubauer A
Abteilung für Innere Medizin mit Schwerpunkt Hämatologie/Onkologie, Universitätsklinikum Rudolf Virchow, Berlin, Germany.
Leukemia. 1995 May;9(5):915-21.
One of the earliest events in the multistep process of malignant transformation is a change in the methylation pattern of certain genes. DNA methylation is usually detected by Southern blotting after restriction digest with methylation-sensitive endonucleases. Calcitonin gene hypermethylation has been described in a variety of human malignancies including lymphomas and leukemias. Here we report a technique based on the semi-quantitative differential polymerase chain reaction (PCR) which is capable of detecting subtle changes in the methylation pattern of the human calcitonin gene. This technique is based on two principles: (i) simultaneous coamplification of the target gene (5'-region of the calcitonin gene) and a reference gene for quantitative purposes; and (ii) simultaneous coamplification of a competitor with identical primer-binding sites as the target gene to control for proper restriction digest. Using this technique, we investigated calcitonin gene methylation in a variety of human cell lines, primary leukemias and normal human blood donors. The data revealed good correlation with standard Southern blotting. Weak calcitonin gene methylation was found in all normal blood donors tested (n = 14). In contrast, strong calcitonin gene methylation was detected in most acute leukemias (five of 10 acute myeloid leukemias (AML); six of seven acute lymphoblastic leukemias (ALL)). These data show that this technique can reliably be used to quantitate gene methylation and indicate that there exists heterogeneity with regard to methylation status in different leukemias, suggesting that hypermethylation of the calcitonin gene may play a role in the transformation process of some, but not all, human leukemias. Furthermore, differential PCR may facilitate determination of calcitonin gene methylation in clinical or archival tumor samples.
恶性转化多步骤过程中最早出现的事件之一是某些基因甲基化模式的改变。DNA甲基化通常在用甲基化敏感的核酸内切酶进行限制性消化后通过Southern印迹法检测。降钙素基因高甲基化已在包括淋巴瘤和白血病在内的多种人类恶性肿瘤中被描述。在此，我们报告一种基于半定量差异聚合酶链反应（PCR）的技术，该技术能够检测人类降钙素基因甲基化模式的细微变化。该技术基于两个原理：（i）为了定量目的同时共扩增靶基因（降钙素基因的5'区域）和一个参照基因；（ii）同时共扩增一个与靶基因具有相同引物结合位点的竞争物以控制适当的限制性消化。使用该技术，我们研究了多种人类细胞系、原发性白血病和正常人类献血者中的降钙素基因甲基化。数据显示与标准Southern印迹法有良好的相关性。在所有测试的正常献血者（n = 14）中发现了弱的降钙素基因甲基化。相比之下，在大多数急性白血病中检测到强的降钙素基因甲基化（10例急性髓细胞白血病（AML）中有5例；7例急性淋巴细胞白血病（ALL）中有6例）。这些数据表明该技术可可靠地用于定量基因甲基化，并表明不同白血病在甲基化状态方面存在异质性，提示降钙素基因的高甲基化可能在一些但并非所有人类白血病的转化过程中起作用。此外，差异PCR可能有助于临床或存档肿瘤样本中降钙素基因甲基化的测定。
