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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Southem印迹分析、甲基化特异性PCR(MSPCR)和核苷酸测序检查了不同形式的成人T细胞白血病(ATL)患者中CDKNZA基因的甲基化状态。我们发现，与恶性程度较低的慢性 ATL（17%）和冒烟型 ATL（17%）患者相比，从急性 ATL（47%）或淋巴瘤型 ATL（73%）患者分离的新鲜肿瘤细胞中，CDKNZA 基因更频繁地甲基化。此外，24%的急性ATL患者发现CDKNZA基因缺失；因此，急性 ATL 患者中 CDKNZA 基因异常的比例为 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 名 ATL 患者的任何样本中均未发现 p15 基因甲基化。对基因组 DNA 进行亚硫酸氢钠处理后，对 CDKNZA 基因进行直接测序，结果显示，32 例 ATL 病例中，有 24 例 (75%) 发生了 CpG 位点甲基化，包括慢性和闷烧性 ATL，即使 MSPCR 和 Southem blot 未能检测到 CDKNZA 基因甲基化。新鲜ATL甲基化样本中，24例中有17例检测到启动子区和外显子甲基化，24例中7例检测到外显子上游无甲基化。在一个案例中，外周血细胞和淋巴结细胞之间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，但在甲基化模式方面存在多个亚克隆。定量 PCR 显示，具有甲基化 CDKNZA 基因的细胞中 CDKNZA mRNA 表达显着降低，尤其是启动子区域甲基化时。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) 人 T 细胞白血病病毒 I 型感染个体中幼稚 T 淋巴细胞的产生受损：其对免疫缺陷状态的影响：机会性感染经常发生在成人 T 细胞白血病 (ATL) 和人 T 细胞白血病病毒患者中 I 型（HTLV-1）载体。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 感染个体中观察到的免疫缺陷机制，我们使用 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 携带者以及 HAM/TSP 和 ATL 患者的 T 细胞亚群。与未感染的对照个体相比，ATL 患者的初始 T 淋巴细胞数量明显受到抑制，尤其是急性患者。与未感染个体相比，50岁以下HTLV-I感染个体的幼稚T细胞数量较低，而HTLV-I感染个体中记忆T淋巴细胞数量较多。尽管记忆T淋巴细胞的增加与HTLV-1原病毒载量相关，但未发现幼稚T细胞计数和原病毒载量之间存在关系。 T 细胞受体重排切除环 (TREC) 是在早期 T 淋巴细胞生成过程中由 DNA 重组产生的，通过量化来评估 HTLV-I 感染个体的胸腺功能。 HTLV-I 感染个体的 TREC 水平低于未感染个体。在 70 岁以下的 HTLV-I 携带者中，16 名检查者中的 6 名（38%）观察到外周血单核细胞中 Epstein-Barr 病毒 DNA 的增加，而在 11 名未感染对照中仅 1 名病例中检测到这种增加。我们的结果强烈表明，幼稚 T 淋巴细胞数量较少是由于胸腺中 T 淋巴细胞的产生受到抑制，这可能是在 HTLV-I 感染个体中观察到的免疫缺陷的原因。较少的