Molecular mechanism of multi-step leukemogenesis in adult T-cell leukemia

1)成人T细胞白血病细胞中CDKNZA(P16)基因启动子区域的DNA甲基化：本研究采用Southern印迹分析、甲基化特异性聚合酶链式反应(MSPCR)和核苷酸测序等方法检测了不同类型成人T细胞白血病(ATL)患者CDKNZA基因的甲基化状态。我们发现CDKNZA基因在从急性ATL患者(47%)或淋巴瘤类型ATL患者(73%)分离的新鲜肿瘤细胞中甲基化频率高于那些恶性程度较低的慢性ATL患者(17%)和阴燃ATL患者(17%)。此外，24%的急性ATL患者存在CDKNZA基因缺失；因此，急性ATL患者中CDKNZA基因的异常总数为71%。相反，在无症状携带者或未感染的个体中未发现CDKNZA基因甲基化。在36例ATL患者的任何样本中均未发现p15基因甲基化。亚硫酸氢钠处理后的CDKNZA基因直接测序显示…的甲基化在32例ATL中有24例(75%)出现了更多的CpG位点，包括慢性和阴燃的ATL，即使在MSPCR和Souhim印迹未能检测到CDKNZA基因甲基化的情况下。在发生甲基化的新鲜ATL标本中，24例中有17例在启动子区和外显子发生甲基化，7例在无上游的外显子发生甲基化。在一个病例中，外周血细胞和淋巴结细胞的甲基化模式被证明是不同的，这表明尽管HTLV-I整合位点相同，但存在关于甲基化模式的多个亚克隆。定量聚合酶链式反应显示带有CDKNZA基因甲基化的细胞中CDKNZA mRNA的表达显著降低，尤其是当启动子区域发生甲基化时。这些发现表明，CpG甲基化降低了CDKNZA的表达，并在ATKNZA的疾病进展中是一个关键因素。在人类T细胞白血病病毒I型感染者中，CpG甲基化抑制了原始T淋巴细胞的产生：其在免疫缺陷状态中的意义：机会感染经常发生在成人T细胞白血病(ATL)和人类T细胞白血病病毒I型(HTLV-1)携带者中。然而，这种感染的潜在免疫学和病毒学机制仍不清楚。为了阐明HTLV-I感染者免疫缺陷的机制，我们用CD62L和CD45RA与CD4或CD8阳性T细胞共表达的三色荧光分析了HTLV-I携带者和HAM/TSP和ATL患者的T细胞亚群。与未感染的对照组相比，ATL患者，尤其是急性期患者的幼稚T淋巴细胞数量明显受到抑制。50岁以下HTLV-I感染者的幼稚T细胞数量低于非感染者，而记忆性T淋巴细胞数量高于非感染者。尽管记忆T淋巴细胞的增加与HTLV-I前病毒载量有关，但未见幼稚T细胞计数与前病毒载量之间的关系。对HTLV-I感染者的T细胞受体重排切除环路(TREC)进行了定量检测，以评估HTLV-I感染者的甲状腺功能。HTLV-I感染者的TREC水平低于非感染者。在小于70岁的HTLV-I携带者中，16例受检者中有6例(38%)外周血单个核细胞中EB病毒DNA含量升高，而11例未感染对照中仅1例可检测到EB病毒DNA。我们的结果有力地表明，幼稚T淋巴细胞数量的减少是由于胸腺中T淋巴细胞的产生受到抑制，这可能是HTLV-I感染者免疫缺陷的原因。较少

1)Progressive DNA methylation in the promoter region of CDKNZA(p16)gene in adult T-cell luekemia cells : In this study we examined the methylation ststus of the CDKNZA gene in patients with different forms of adult T-cell leukemia(ATL)using Southem blot analysis, methylation-specific PCR(MSPCR), and nucleotide sequencing. We found that the CDKNZA gene was more frequently methylated in fresh tumor cells isolated from patients with acute ATL(47%)or lymphoma-type ATL(73%)than in those with less malignant chronic(17%)and smoldering ATL(17%). In addition, deletions of the CDKNZA gene were found in 24% of acute ATL patients ; thus abnormalities of the CDKNZA gene totaled 71% in acute ATL patients. In contrast, no CDKNZA gene methylation was found in asymptomatic carriers or uninfected individuals. Methylation of the p15 gene was not found in any samples from 36 ATL patients. Direct sequencing of the CDKNZA gene after sodium bisulfite treatment of genomic DNA revealed that the methylation of … More CpG sites had occurred in 24 of 32 ATL cases(75%)including chronic and smoldering ATL, even when MSPCR and the Southem blot had failed to detect CDKNZA gene methylation. Among fresh ATL samples with methylation, methylation was detected in the promoter region and exon in 17 out of 24 casee, and methylation in the exon wihout upstream was detected in 7 cases out of 24 cases. In one case' the pattern of methylation proved to be different between peripheral blood cells and lymph node cells, suggesting the presence of multiple subclones with regard to methyhlation patterns despite the same HTLV-I integration site. Quantitative PCR showed a marked decrease in CDKNZA mRNA expression in the cells with a methylated CDKNZA gene, especially if promoter region was methylated. These findings suggest that CpG methylation decreases CDKNZA expression and represents a critical factor in the disease progression of ATL.2)Impaired prduction of naive T-lymphocytes in human T-cell leukemia virus type I infected individuals : its implications in the immunodeficient state : Opportunustic infections frequently occur in patients with adult T-cell leukemia(ATL), and human T-cell leukemia virus type I(HTLV-1)carriers. However, the underlying immunological and virological mechanisms of such infections remain unknown. To clarify the mechanism of immunodeficiency observed in HTLV-I infected individuals, we analyzed the T-cell subsets in HTLV-I carriers and patients with HAM/TSP and ATL using three color fluorescence with CD62L and CD45RA coexpression either with CD4 or CD8 positive T-cells. The number of naive T-lymphocytes was markedly suppressed in patients with ATL, particularly in those with acute form, compared with uninfected control individuals. The number of naive T-cells was low in HTLV-I infected individuals under 50 year-old compared with uninfected individuals whereas the number of memory Tlymphocytes was greater in HTLV-I infected individuals. Although the increase of memory T-lymphocytes correlated wih HTLV-I provirus loads, no relationship was found between naive T-cell counts and provirus loads. T-cell receptor rearrangement excision circels(TREC), which are generated by DNA recombination during early T-lymphopoiesis, were quantified to evaluate thyrnic function in HTLV-I infected individuals. TREC levels were lower in HTLV-I infected individuals than in uninfected individuals. In less than 70 years old HTLV-I carriers, an increase of Epstein-Barr virus DNA in peripheral blood mononuclear cells was observed in 6 of 16(38%) examined whereas it was detectable in only one case of 11 uninfected controls. Our results strongly sugggested that the low number of naive T-lymphocytes was due to suppressed production of T-lymphocytes in the thymus, which might account for immunodeficiency observed in HTLV-I infected individuals. Less