Genomic analysis of a homozygously deleted region in neuroblastoma cell lines on human chromosome 1p36

人染色体 1p36 上神经母细胞瘤细胞系纯合缺失区域的基因组分析

• 批准号: 13670857
• 负责人: OHIRA Miki
• 金额: $ 0.9万
• 依托单位: Chiba Cancer Center Research Institute
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670857/
• 关键词: 1p36; Neuroblastoma; homozygous deletion; positional cloning; tumor suppressor gene

Loss of heterozygosity of the distal region of chromosome 1p where tumor suppressor gene(s)might harbor is frequently observed in many human cancers including neuroblastoma (NBL) with MYCN amplification and poor prognosis. We have found a homozygously deleted (HD)region within the smallest region of overlap at 1p36.2-p36.3 in two NBL cell lines. The 800-kb PAC contig covering the entire region of homozygous deletion was made and sequenced (about 85%). The estimated length of the deleted region was 500 kb. We have, thus far, identified six genes (DFF45, PGD, CORT, HDNB1/UFD2, KIAA0591F/KIF1B-beta, and PEX14) within the region. They include the genes related to apoptosis, glucose metabolism, ubiquitin-proteasome pathway, a neuronal microtubule-associated motor molecule and biogenesis of peroxisome. At least three genes (HDNB1/UFD2, KIAA0591F/KIF1B-beta, and PEX14) were differentially expressed at high levels in favorable and at low levels in unfavorable subsets of primary neuroblastoma. … More RT-PCR-SSCP analysis has demonstrated infrequent mutation of the genes so far identified. Since the 1p distal region is reported to be imprinted, those differentially expressed genes could be the new members of the candidate NBL suppressor. To assess this possibility, we investigated these genes' transcriptional expression before and after the treatment with demethylation reagent to eight neuroblatoma cell lines. All cell lines, when treated or not treated with the reagent, showed certain level of expression, suggesting that these genes were not suppressed by genomic methylation in these cell lines. However, one of them exhibited slight increase of expression after the de-methylation, we therefore started to analyze its promoter region. To further identify novel genes in this region and also detect a promoter region of the gene, we isolated 4 mouse BAC clones covering a synthenic portion of the HD region. Approximately 660-kb of genomic sequencing has been accomplished. Full-sequencing and gene prediction for the region of homozygous deletion would elucidate more detailed structure of this region and might lead to discovery of additional candidate genes. Less

在包括神经母细胞瘤在内的多种人类肿瘤中，常可观察到可能含有肿瘤抑制基因(S)的染色体1p远端区域杂合性缺失，其扩增产物为MYCN，且预后不良。我们在两个NBL细胞系的1p36.2-p36.3的最小重叠区域内发现了一个纯合缺失(HD)区。制作了覆盖整个纯合缺失区域的800kb的PAC重叠群并对其进行了测序(约85%)。该缺失区域的估计长度为500kb。到目前为止，我们已经在该区域鉴定了6个基因(DFF45、PGD、CORT、HDNB1/UFD2、KIAA0591F/KIF1B-BETA和PEX14)。这些基因包括与细胞凋亡、葡萄糖代谢、泛素-蛋白酶体途径、神经元微管相关的运动分子和过氧化物酶的生物发生有关的基因。至少有三个基因(HDNB1/UFD2、KIAA0591F/KIF1B-beta和PEX14)在原发神经母细胞瘤中的高水平和低水平差异表达。…更多的RT-PCR-SSCP分析表明，到目前为止已鉴定的基因很少发生突变。由于1p远端区域被报道为印迹，这些差异表达的基因可能是候选NBL抑制子的新成员。为了评估这种可能性，我们研究了这些基因在去甲基化试剂处理前后对八个神经母细胞瘤细胞系的转录表达。无论用试剂处理与否，所有细胞株都表现出一定程度的表达，这表明这些基因在这些细胞系中并没有受到基因组甲基化的抑制。然而，其中一个基因在去甲基化后表达略有增加，因此我们开始分析其启动子区域。为了进一步确定该区域的新基因并检测该基因的启动子区域，我们分离了4个覆盖HD区域合成部分的小鼠BAC克隆。目前已完成约660kb的基因组测序。纯合子缺失区域的全序列测定和基因预测将阐明该区域更详细的结构，并可能导致发现更多的候选基因。较少

项目成果

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