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

成人T细胞白血病(ATL)是一种与人类T细胞白血病病毒I型(HTLV-I)相关的侵袭性辅助性T淋巴细胞肿瘤。由于其从HTLV-I感染到ATL发病的潜伏期很长，因此我们认为ATL是多步骤的白血病发生机制，我们正在努力寻找与ATL发病和进展有关的基因。尽管大多数ATL患者的白血病细胞表达Fas抗原，但我们发现了Fas阴性的病例。患者中性粒细胞表面有Fas抗原，表明Fas阴性表型是白血病细胞所特有的。RT-PCR产物序列分析显示，外显子4的跳跃和小片段缺失(5bp)导致Fas蛋白合成提前终止，导致Fas阴性表型。Fas阴性的ATL细胞在体外对阿霉素诱导的细胞凋亡具有抵抗力。Fas阴性表型被认为与耐药有关。p16^-lt；ink4a-gt；基因甲基化已被识别为…更多的是作为另一种机制，除了体细胞DNA的变化，如缺失或突变，可以在包括黑色素瘤、膀胱癌和恶性淋巴瘤在内的各种恶性肿瘤中使pl6^lt；ink4a和gt；基因失活。我们分析了不同亚型成人T细胞白血病(ATL)患者的p16^&lt；ink4a&gt；基因甲基化情况。采用Southern杂交和甲基化特异性聚合酶链式反应(MSPCR)检测p16^&lt；Ink4a&gt；基因甲基化在急性ATL(49%)和淋巴瘤类型ATL(73%)中的甲基化频率高于低度恶性、慢性(17%)和阴燃类型(17%)。相反，在无症状的HTLV-I携带者和未感染的对照中没有发现p6^；lt；ink4a&gt；基因的甲基化，在任何ATL样本中都没有发现编码CDK4和6的另一种抑制物的p15基因的甲基化：p16^&lt；ink4a&gt；基因的甲基化是ATL的高度特异性基因，Southern杂交分析发现另有20%的急性ATL患者p16^&lt；ink4a&gt；基因异常，因此，在急性ATL中p16^&lt；ink4a&gt；基因的异常总数为69%。此外，亚硫酸氢钠处理基因组DNA后对pl6^&lt；ink4a&gt；基因进行直接测序表明，大多数(23/29)ATL病例(包括慢性或阴燃ATL)都发生了CpG位点的甲基化，即使在MSPCR或Southern印迹法无法检测到甲基化的病例中也是如此。半定量聚合酶链式反应显示具有p16^lt；ink4a&gt；基因甲基化的样本中p16^lt；ink4a&gt；基因表达水平显著降低。这些发现表明，一定程度的CpG甲基化通过进一步降低p16的表达，在ATL的进展中起着重要作用。较少

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