Replication stress induces tumor-like microdeletions in FHIT/FRA3B
Replication stress induces tumor-like microdeletions in FHIT/FRA3B
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DOI：
10.1073/pnas.0708097105
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发表时间：
2008-01-08
影响因子：
11.1
通讯作者：
Glover, Thomas W.
中科院分区：
文献类型：
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作者：
Durkin, Sandra G.;Ragland, Ryan L.;Glover, Thomas W.
常见脆性位点（CFS）是在DNA合成部分抑制后优先表现出中期染色体间隙和断裂的基因座。脆性位点FRA 3B位于FHIT肿瘤抑制基因内，是许多癌细胞和癌前病变中常见的杂合和纯合缺失位点。肿瘤中绝大多数FHIT和其他CFS相关基因重排是亚显微的，数百千碱基的基因座内缺失，通常导致相关基因的失活。虽然CFS不稳定性导致染色体间隙和断裂和易位，但没有直接证据表明CFS不稳定性或复制应激可以产生癌细胞中所见的大的亚显微缺失。在这里，我们已经产生了FHIT/FRA 3B缺失密切类似于那些在肿瘤中暴露的人-小鼠3号染色体体细胞杂交细胞aphidicolin介导的复制压力。通过使用PCR、阵列比较基因组杂交（aCGH）和FISH分析克隆细胞群体的缺失。13%至23%的克隆表现出FRA 3B内跨越约200-600 kb的亚显微FHIT缺失。与FRA 3B缺失的染色体表现出显着降低的脆性，与2- 12倍减少中期差距和断裂相比，控制。序列分析显示在断裂点处没有同源性区域，并表明NHEJ参与产生缺失。我们的研究结果表明，复制应激诱导了一个显着的高频率的肿瘤样微缺失，减少在CFS在培养细胞的脆性，并表明，在肿瘤形成过程中的类似条件导致基因座内删除和基因在CFS的失活，也许在基因组的其他地方。
Common fragile sites [CFSs) are loci that preferentially exhibit metaphase chromosome gaps and breaks after partial inhibition of DNA synthesis. The fragile site FRA3B, which lies within the FHIT tumor-suppressor gene, is a site of frequent heterozygous and homozygous deletions in many cancer cells and precancerous lesions. The great majority of FHIT and other CFS-associated gene rearrangements in tumors are submicroscopic, intralocus deletions of hundreds of kilo-bases that often result in inactivation of associated genes. Although CFS instability leads to chromosome gaps and breaks and translocations, there has been no direct evidence showing that CFS instability or replication stress can generate large submicroscopic deletions of the type seen in cancer cells. Here, we have produced FHIT/FRA3B deletions closely resembling those in tumors by exposing human-mouse chromosome 3 somatic hybrid cells to aphidicolin-mediated replication stress. Clonal cell populations were analyzed for deletions by using PCR, array comparative genomic hybridization (aCGH), and FISH. Thirteen percent to 23% of clones exhibited submicroscopic FHIT deletions spanning approximate to 200-600 kb within FRA3B. Chromosomes with FRA3B deletions exhibited significantly decreased fragility of this locus, with a 2- to 12-fold reduction in metaphase gaps and breaks compared with controls. Sequence analysis showed no regions of homology at breakpoints and suggests involvement of NHEJ in generating the deletions. Our results demonstrate that replication stress induces a remarkably high frequency of tumor-like microdeletions that reduce fragility at a CFS in cultured cells and suggests that similar conditions during tumor formation lead to intralocus deletion and inactivation of genes at CFSs and perhaps elsewhere in the genome.