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

成人T细胞白血病多步白血病发生的分子机制

基本信息

批准号：
11671006
负责人：
MATSUOKA Masao
金额：
$ 2.3万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11671006/
关键词：
ATL HTLV-I retrovirus immunodeficiency DNA methylation naive T-cell leukemogenesis 腫瘍化機構ヒトレトロウイルス

项目摘要

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

1) 成人 T 细胞白血病细胞中 CDKNZA(p16) 基因启动子区的进行性 DNA 甲基化：在本研究中，我们使用不同形式的成人 T 细胞白血病 (ATL) 患者检查了 CDKNZA 基因的甲基化状态Southem 印迹分析、甲基化特异性 PCR (MSPCR) 和核苷酸测序我们发现 CDKNZA 基因在从患者分离的新鲜肿瘤细胞中更频繁地甲基化。急性ATL（47%）或淋巴瘤型ATL（73%）的比例高于恶性程度较低的慢性ATL（17%）和冒烟型ATL（17%）。此外，24%的急性ATL中发现CDKNZA基因缺失。 ATL患者；因此，急性ATL患者中CDKNZA基因异常的比例为71%，相比之下，无症状携带者中未发现CDKNZA基因甲基化。亚硫酸氢钠处理基因组 DNA 后，在 36 名 ATL 患者的任何样本中均未发现 p15 基因甲基化，结果显示 32 名 ATL 病例中的 24 名发生了 CpG 位点甲基化。 (75%) 包括慢性和闷烧性 ATL，即使 MSPCR 和 Southem 印迹未能检测到新鲜 ATL 中的 CDKNZA 基因甲基化。在有甲基化的样本中，24 例中的 17 例在启动子区域和外显子中检测到甲基化，在 24 例中的 7 例中检测到外显子无甲基化，其中 1 例的甲基化模式被证明是不同的。外周血细胞和淋巴结细胞，表明尽管相同的 HTLV-I 整合位点显着减少，但甲基化模式存在多个亚克隆。 CDKNZA mRNA 在具有甲基化 CDKNZA 基因的细胞中表达，特别是在启动子区域甲基化的情况下。这些发现表明，CpG 甲基化会降低 CDKNZA 表达，是 ATL 疾病进展的关键因素。2) 幼稚 T 淋巴细胞的产生受损。人类 T 细胞白血病病毒 I 型感染个体：其对免疫缺陷状态的影响：机会性感染经常发生在成人 T 细胞患者中然而，此类感染的潜在免疫学和病毒学机制仍不清楚，为了阐明在 HTLV-I 感染者中观察到的免疫缺陷机制。使用三色荧光分析了 HTLV-I 携带者以及 HAM/TSP 和 ATL 患者的 T 细胞亚群，其中 CD62L 和 CD45RA 共表达为 CD4 或 CD8 阳性与未感染的对照个体相比，ATL 患者中的幼稚 T 淋巴细胞数量明显受到抑制。50 岁以下的 HTLV-I 感染个体中幼稚 T 细胞数量较低。与未感染个体相比，HTLV-I 感染个体的记忆 T 淋巴细胞数量较多，尽管记忆 T 淋巴细胞的增加与 HTLV-I 原病毒载量相关。未发现幼稚 T 细胞计数与 T 细胞受体重排切除环 (TREC) 之间的关系，TREC 是在早期 T 淋巴细胞生成过程中由 DNA 重组产生的，可用于评估 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 70 岁以下的 HTLV-I 携带者中，Epstein-Barr 病毒增加。在 16 例检查中的 6 例（38%）中观察到外周血单核细胞中的 DNA，而在 11 例未感染的对照中仅检测到 1 例，我们的结果强烈表明，幼稚 T 淋巴细胞数量低是由于 T 细胞的产生受到抑制。 -胸腺中的淋巴细胞，这可能是 HTLV-I 感染个体中观察到的免疫缺陷的原因。