Tobe H, Ruether B A, Jerry L M, Tamaoki T
Cancer Biochem Biophys. 1986 Oct;8(4):313-26.
HLA-DR antigens are expressed as differentiation markers in certain human leukemias. To investigate whether DNA methylation plays a role in expression of DR genes in leukemia, we analyzed methylation patterns of the DR-alpha and D/DR-beta genes in the DR antigen-positive and -negative B-cell lines, in normal adults and in chronic lymphocytic leukemia (CLL) patients using Southern blot hybridization of DNA digested with Msp I and Hpa II. The DR-alpha and D/DR-beta genes of a DR antigen positive B-cell line, T5-1, were heavily methylated, while those of DR antigen-negative variant, 6.1.6, were hypomethylated. Blood cells collected from four normal adults contained different levels of DR-alpha and D/DR-beta mRNAs, but their relative amounts were about the same among the individuals. By contrast, the relative amounts of these mRNAs in CLL cells varied widely, indicating aberrant expression of one or both of these genes in CLL. The DR-alpha gene in four normal adults and six CLL patients produced only a 3 kb hybridizable band after Msp I digestion. Normal adult DR-alpha genes were resistant to Hpa II digestion, suggesting that all Hpa II sites are methylated. In contrast, digestion of CLL DNA with Hpa II yielded various bands of larger sizes which differed among the CLL patients, suggesting that Hpa II sites are differentially methylated in the CLL DNA. In the case of D/DR-beta genes, normal adult DNA gave Msp I bands which were slightly polymorphic among four individuals tested. In contrast, CLL DNA showed a high degree of restriction fragment length polymorphism (RFLP) on Msp I digestion. We speculate that the high RFLPs in the CLL DNA may result from differential methylation in CpG clusters in the D/DR-beta genes, and that this characteristic may be of use for diagnosis of CLL.
HLA - DR抗原在某些人类白血病中作为分化标志物表达。为了研究DNA甲基化是否在白血病中DR基因的表达中起作用，我们使用经Msp I和Hpa II消化的DNA进行Southern印迹杂交，分析了DR抗原阳性和阴性B细胞系、正常成年人以及慢性淋巴细胞白血病（CLL）患者中DR - α和D/DR - β基因的甲基化模式。DR抗原阳性的B细胞系T5 - 1的DR - α和D/DR - β基因高度甲基化，而DR抗原阴性变体6.1.6的这些基因则低甲基化。从四名正常成年人采集的血细胞含有不同水平的DR - α和D/DR - β mRNA，但个体之间它们的相对量大致相同。相比之下，CLL细胞中这些mRNA的相对量差异很大，表明这些基因中的一个或两个在CLL中表达异常。四名正常成年人和六名CLL患者的DR - α基因在Msp I消化后仅产生一条3 kb的可杂交带。正常成年人的DR - α基因对Hpa II消化有抗性，表明所有Hpa II位点均被甲基化。相反，用Hpa II消化CLL DNA产生了大小不同的各种条带，这些条带在CLL患者之间有所不同，表明CLL DNA中的Hpa II位点存在差异甲基化。对于D/DR - β基因，正常成年人的DNA经Msp I酶切后产生的条带在四名受测个体中略有多态性。相比之下，CLL DNA在Msp I消化后显示出高度的限制性片段长度多态性（RFLP）。我们推测CLL DNA中的高RFLP可能是由于D/DR - β基因中CpG簇的差异甲基化导致的，并且这种特征可能有助于CLL的诊断。
