Identification and analysis associated with the onset and progression of adult T-cell leukemia

与成人 T 细胞白血病发病和进展相关的鉴定和分析

基本信息

批准号：
09671125
负责人：
MATSUOKA Masao
金额：
$ 1.98万
依托单位：
Kumamoto University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

Adult T-cell leukemia (ATL) is an aggressive neoplasm of helper T-lymphocytes, which is etiologically associated with human T-cell leukemia virus type I (HTLV-I). Siince its long latent period from infection of HTLV-I to onset of ATL, multti-step mechanism of leukemogenesis is considered in ATL.We are trying to identify the genes responsible for the onset and the progression of ATL.Although leukemic cells in most ATL cases expressed Fas antigen, we found Fas-negative cases. Neutrophil from this patient had Fas antigen on the surface, showing that Fas-negative phenotype is specific to leukemie cells. Sequences of RT-PCR products revealed that skipping of exon 4, and small deletion (5bp) caused premature termination of Fas protein synthesis, resulting in Fas negative phenotype. This Fas negative ATL cells were resistant to doxorubicin -induced apoptosis in vitro. Fas negative phenotype is considered to be associated with drug resistance.Methylation of the p16^<INK4A> gene has been recogn … More ized as another mechanism, in addition to somatic DNA changes such as deletion or mutation, which can inactivate the pl6^<INK4A> gene in various malignancies including melanoma, bladder cancer and malignant lymphoma. We analyzed the methylation of the p16^<INK4A> gene in patients with different subtypes of adult T-cell leukemia (ATL). Using Southern blot analysis and methylation-specific PCR (MSPCR), we detected methylation of the p16^<INK4A> gene was more frequently in acute ATL (49%) or lymphoma-type ATL (73%) than in lowgrade malignant stage, chronic (17%) and smoldering types (17%). In contrast, no methylation of the pl6^<INK4A> gene was found in asymptomatic HTLV-I carriers and uninfected control, and methylation of the p15 gene, which encode another inhibitor of CDK4 and 6, could not be found in any ATL samples : methylation of the p16^<INK4A> gene is highly specific for ATL.Deletion of the the p16^<INK4A> gene was found in another 20% of acute ATL patients by Southern blot analysis, and therefore, abnormalities of the p16^<INK4A> gene in acute ATL total to 69%. Furthermore, direct sequencing of the pl6^<INK4A> gene after sodium bisulfite treatment of genomic DNA revealed that the methylation of CpG sites occurred in most (23 out of 29) of ATL cases including chronic or smoldering ATL, even in the cases which methylation could not be detected by MSPCR or the Southern blot method. Semi-quantitative PCR showed markedly decreased p16^<INK4A> mRNA levels in the samples having a methylated p16^<INK4A> gene. These findings show that a level of CpG methylation plays an important role in the progression of ATL by further decreasing the expression of p16. Less

成年T细胞白血病（ATL）是辅助T淋巴细胞的侵袭性肿瘤，在病因上与人类T细胞白血病病毒I型（HTLV-I）相关。从HTLV-I感染到ATL的发作，其长期潜在的时期，在ATL中考虑了白血病的多-TI步骤机制。我们试图识别导致ATL的发病和进展的基因。在大多数ATL病例中，在大多数ATL ATL病例中都表达了FAS抗原，我们发现了Fas antl antl antl fas-nef fas-necy fas-necy案例。该患者的嗜中性粒细胞在表面上有FAS抗原，表明FAS阴性表型是白血对细胞的特异性。 RT-PCR产物的序列表明，外显子4和小缺失（5bp）的跳过导致FAS蛋白合成的过早终止，从而导致FAS负面表型。该FAS阴性ATL细胞在体外对阿霉素诱导的细胞凋亡具有抗性。 FAS负表型被认为与耐药性有关。p16^<ink4a>基因的甲基化已被识别出……除了诸如缺失或突变之外的体细胞DNA变化之外，还可以将PL6^<ink4a>基因灭活，包括其他各种恶性肿瘤，包括黑色素瘤，蓝晶质癌和成叶氏菌。我们分析了成人T细胞白血病（ATL）不同亚型的患者中p16^<ink4a>基因的甲基化。使用Southern印迹分析和甲基化特异性PCR（MSPCR），我们在急性ATL（49％）或淋巴瘤型ATL（73％）中检测到p16^<ink4a>基因的甲基化比低级恶性阶段，慢性（17％）和（17％）（17％）更频繁。相比之下，在不对称的HTLV-I载体和未感染的对照中未发现PL6^<ink4a>基因的甲基化，p15基因的甲基化编码CDK4和6的另一种抑制剂，在任何ATL样品中都找不到p16^<ink4a>基因的甲基化的pa。通过Southern印迹分析在另外20％的急性ATL患者中发现，因此，急性ATL的p16^<ink4a>基因的异常总数为69％。此外，基因组DNA钠治疗后PL6^<ink4a>基因的直接测序表明，即使在29个ATL病例中，大多数（29）中，大多数（23个）（包括慢性或雾化的ATL）发生了CpG位点的甲基化，即使在MSPCR或MSPCR或Southern Blot方法也无法检测到甲基化的情况。半定量PCR显示出具有甲基化P16^<ink4a>基因的样品中的P16^<ink4a> mRNA水平明显降低。这些发现表明，通过进一步降低p16的表达，一定水平的CpG甲基化在ATL的发展中起着重要作用。较少的