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）基因启动子区进行性DNA甲基化：本研究采用Southem印迹分析、甲基化特异性PCR（MSPCR）和核苷酸序列测定法检测了不同类型成人T细胞白血病（ATL）患者CDKNZA基因甲基化状态。我们发现CDKNZA基因在急性ATL（47%）或淋巴瘤型ATL（73%）患者的新鲜肿瘤细胞中的甲基化频率高于恶性程度较低的慢性ATL（17%）和阴燃型ATL（17%）患者。此外，在24%的急性ATL患者中发现了CDKNZA基因的缺失;因此在急性ATL患者中CDKNZA基因的异常总计为71%。相比之下，在无症状携带者或未感染个体中未发现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基因组DNA的CDKNZA基因的直接测序显示， ...更多信息 32例ATL病例中有24例（75%）出现CpG位点，包括慢性和阴燃型ATL，即使MSPCR和Southem印迹未能检测到CDKNZA基因甲基化。在24例有甲基化的新鲜ATL标本中，17例在启动子区和外显子区均检测到甲基化，7例在外显子区无甲基化。在一种情况下，甲基化模式被证明在外周血细胞和淋巴结细胞之间是不同的，这表明尽管HTLV-1整合位点相同，但存在多个甲基化模式的亚克隆。定量PCR显示，在CDKNZA基因甲基化的细胞中，CDKNZA mRNA表达显著降低，尤其是如果启动子区域甲基化。这些发现表明，CpG甲基化降低CDKNZA表达，并代表ATL疾病进展的关键因素。2）I型人T细胞白血病病毒感染个体中幼稚T淋巴细胞的产生受损：其在免疫缺陷状态中的意义：成人T细胞白血病（ATL）患者和I型人T细胞白血病病毒（HTLV-1）携带者经常发生幼稚T淋巴细胞感染。然而，这种感染的潜在免疫学和病毒学机制仍然未知。为了阐明HTLV-I感染个体中观察到的免疫缺陷机制，我们使用三色荧光分析了HTLV-I携带者和HAM/TSP和ATL患者中的T细胞亚群，其中CD 62 L和CD 45 RA与CD 4或CD 8阳性T细胞共表达。与未感染的对照个体相比，ATL患者的幼稚T淋巴细胞数量明显受到抑制，特别是在急性形式的患者中。与未感染个体相比，50岁以下HTLV-I感染个体中幼稚T细胞的数量较低，而HTLV-I感染个体中记忆T淋巴细胞的数量较多。虽然记忆T淋巴细胞的增加与HTLV-Ⅰ型前病毒载量相关，但未发现幼稚T细胞计数与前病毒载量之间存在相关性。T细胞受体重排切除环（TREC），这是在早期T淋巴细胞生成过程中通过DNA重组产生的，定量评估HTLV-I感染个体的胸腺功能。HTLV-I感染者的TREC水平低于未感染者。在小于70岁的HTLV-I携带者中，在16例检查中的6例（38%）中观察到外周血单个核细胞中EB病毒DNA的增加，而在11例未感染的对照中仅一例中可检测到。我们的研究结果强烈地表明，幼稚T淋巴细胞的数量低是由于抑制T淋巴细胞在胸腺中的生产，这可能是解释在HTLV-I感染个体中观察到的免疫缺陷。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