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

成人t细胞白血病（ATL）是一种辅助性t淋巴细胞的侵袭性肿瘤，其病因学与人t细胞白血病病毒I型（HTLV-I）相关。由于从HTLV-I感染到ATL发病潜伏期较长，ATL的白血病发生机制被认为是多步骤的。我们正试图找出导致ATL发病和发展的基因。虽然大多数ATL病例的白血病细胞表达Fas抗原，但我们发现Fas阴性病例。该患者的中性粒细胞表面有Fas抗原，表明Fas阴性表型是白血病细胞特有的。RT-PCR产物序列显示，外显子4的跳变和小缺失（5bp）导致Fas蛋白合成过早终止，导致Fas阴性表型。Fas阴性ATL细胞对阿霉素诱导的体外凋亡具有抗性。Fas阴性表型被认为与耐药有关。p16^<INK4A>基因的甲基化已被认为是除体细胞DNA改变（如缺失或突变）外的另一种机制，它可以使pl6^<INK4A>基因失活，包括黑色素瘤、膀胱癌和恶性淋巴瘤。我们分析了不同亚型成人t细胞白血病（ATL）患者p16^<INK4A>基因的甲基化。通过Southern blot分析和甲基化特异性PCR (MSPCR)，我们检测到p16^<INK4A>基因的甲基化在急性ATL（49%）或淋巴瘤型ATL（73%）中比在低恶性期、慢性（17%）和阴烧型（17%）中更频繁。相比之下，在无症状HTLV-I携带者和未感染对照中未发现pl6^<INK4A>基因的甲基化，编码另一种CDK4和6抑制剂的p15基因的甲基化在任何ATL样本中均未发现：p16^<INK4A>基因的甲基化对ATL具有高度特异性。Southern blot分析发现，另外20%的急性ATL患者存在p16^<INK4A>基因缺失，因此急性ATL患者p16^<INK4A>基因异常占69%。此外，亚硫酸氢钠处理基因组DNA后，对pl6^<INK4A>基因的直接测序显示，包括慢性或阴燃ATL在内的大多数ATL病例（29例中的23例）发生了CpG位点的甲基化，即使在MSPCR或Southern blot方法无法检测到甲基化的情况下也是如此。半定量PCR显示p16^<INK4A>基因甲基化的样品中p16^<INK4A> mRNA水平显著降低。这些发现表明，CpG甲基化水平通过进一步降低p16的表达在ATL的进展中起重要作用。少

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