Tumour specific promoter region methylation of the human homologue of the Drosophilia Roundabout gene DUTT1 (ROBO1) in human cancers
Tumour specific promoter region methylation of the human homologue of the Drosophilia Roundabout gene DUTT1 (ROBO1) in human cancers
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DOI：
10.1038/sj.onc.1205421
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发表时间：
2002-05-02
期刊：
影响因子：
8
通讯作者：
Latif, F
中科院分区：
文献类型：
--
作者：
Dallol, A;Forgacs, E;Latif, F
果蝇回旋基因DUTT 1（在U 2020年）或ROBO 1（基因座链接ID 6091），受体的NCAM家族的成员，最近从肺癌肿瘤抑制基因区域2（LCTSGR 2或U 2020区域）在3 p12克隆的人类同源物。DUTT 1在小细胞肺癌细胞系（SCLC）和乳腺癌细胞系中特征性重叠纯合缺失区域内作图。在本报告中，我们（a）定义了DUTT 1基因的基因组结构，（B）进行了肺癌、乳腺癌和肾癌中DUTT 1的突变和表达分析，（c）鉴定了人类癌症中DUTT 1的肿瘤特异性启动子区域甲基化。发现该基因含有29个外显子，并且跨越至少240 kb的基因组序列。5'区含有CpG岛，并且poly（A）（+）尾具有非典型的5'-GATAAA-3'信号。我们分析了DUTT 1在肺癌、乳腺癌和肾癌中的突变，PCR-SSCP未检测到失活突变。然而，发现并表征了7个种系错义变化。DUTT 1的表达是没有检测到的18个乳腺肿瘤细胞系通过RTPCR分析。HTB-19乳腺肿瘤细胞系（不表达DUTT 1）中DUTT 1基因启动子区的亚硫酸氢盐测序显示，DUTT 1基因启动子区内的CpG位点完全超甲基化（相对于翻译起始位点为-244至+27）。用去甲基化剂5-氮杂-2 '-脱氧胞苷处理后，HTB-19中DUTT 1基因的表达被重新激活。在32例原发性浸润性乳腺癌中有6例（19%）、44例原发性透明细胞肾细胞癌（CC-RCC）中有8例（18%）和26例原发性NSCLC肿瘤中有1例（4%）中也发现了相同区域的高甲基化。此外，显示DUTT 1甲基化的80%的乳腺癌和75%的CC-RCC肿瘤具有3 p12标记物的等位基因丢失，因此服从Knudson的两次击中假设。我们的研究结果表明，DUTT 1值得进一步分析作为肿瘤抑制基因（TSG）在3 p12，半和纯合缺失和功能分析定义的区域的候选人。
The human homologue of the Drosophila Roundabout gene DUTT1 (Deleted in U Twenty Twenty) or ROBO1 (Locus Link ID 6091), a member of the NCAM family of receptors, was recently cloned from the lung cancer tumour suppressor gene region 2 (LCTSGR2 or U2020 region) at 3p12. DUTT1 maps within a region of overlapping homozygous deletions characterized in both small cell lung cancer lines (SCLC) and in a breast cancer line. In this report we (a) defined the genomic organization of the DUTT1 gene, (b) performed mutation and expression analysis of DUTT1 in lung, breast and kidney cancers, (c) identified tumour specific promoter region methylation of DUTT1 in human cancers. The gene was found to contain 29 exons and spans at least 240 kb of genomic sequence. The 5' region contains a CpG island, and the poly(A)(+) tail has an atypical 5'-GATAAA-3' signal. We analysed DUTT1 for mutations in lung, breast and kidney cancers, no inactivating mutations were detected by PCR-SSCP. However, seven germline missense changes were found and characterized. DUTT1 expression was not detectable in one out of 18 breast tumour lines analysed by RTPCR. Bisulfite sequencing of the promoter region of DUTT1 gene in the HTB-19 breast tumour cell line (not expressing DUTT1) showed complete hypermethylation of CpG sites within the promoter region of the DUTT1 gene (-244 to +27 relative to the translation start site). The expression of DUTT1 gene was reactivated in HTB-19 after treatment with the demethylating agent 5-aza-2'-deoxycytidine. The same region was also found to be hypermethylated in six out of 32 (19%) primary invasive breast carcinomas and eight out of 44 (18%) primary clear cell renal cell carcinomas (CC-RCC) and in one out of 26 (4%) primary NSCLC tumours. Furthermore 80% of breast and 75% of CC-RCC tumours showing DUTT1 methylation had allelic losses for 3p12 markers hence obeying Knudson's two hit hypothesis. Our findings suggest that DUTT1 warrants further analysis as a candidate for the tumour suppressor gene (TSG) at 3p12, a region defined by hemi and homozygous deletions and functional analysis.