Evidence for the transforming activity of a truncated Int6 gene, in vitro
Evidence for the transforming activity of a truncated Int6 gene, in vitro
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DOI：
10.1038/sj.onc.1204624
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发表时间：
2001-08-30
期刊：
影响因子：
8
通讯作者：
Smith, GH
中科院分区：
文献类型：
--
作者：
Rasmussen, SB;Kordon, E;Smith, GH
Int6/eIF3-P48首次被鉴定为MMTV在小鼠乳腺肿瘤中的共同整合位点。在所有情况下，MMTV整合事件导致一个等位基因的正常Int6转录中断，而第二个等位基因完好无损。我们推测，将MMTV插入到Int6中会导致一个突变的等位基因，该等位基因编码一个缩短的Int6 mRNA和蛋白(Int6sh)，它要么改变正常的Int6功能，要么具有新的独立功能。为了确定突变的转化潜力及其主导功能，我们在延伸因子-1a(EEF1A)启动子的控制下，将Int6sh导入两个乳腺上皮细胞系MCF10A(人)和HC11(小鼠)。Int6sh在MCF10A和HC11乳腺上皮细胞中的表达导致软琼脂中锚定无关的生长，表明有转化的表型。经RT-PCR和Northern印迹分析，从琼脂中筛选到的菌落具有高水平的突变Int6sh和野生型Int6RNA转录本。此外，在体内，Int6sh转化的MCF10A和HC11细胞在免疫受损宿主中形成结节生长。NIH3T3细胞，小鼠胚胎成纤维细胞，也通过Int6sh的表达，在体外转化为非贴壁生长。这些观察结果提供了直接证据，证明在MMTV诱导的肿瘤和增生中观察到的Int6突变有助于乳腺上皮细胞的恶性转化。
Int6/eIF3-p48 was first identified as a common integration site for MMTV in mouse mammary tumors. In all cases, the MMTV integration event resulted in an interruption of the normal Int6 transcript from one allele leaving the second allele intact and operative. We hypothesize that insertion of MMTV into Int6 results in a mutated allele that encodes a shortened Int6 mRNA and protein (Int6sh), which either modifies normal Int6 function or possesses a new independent function. To confirm the transforming potential of the mutation and its dominant function, we transfected two mammary epithelial cell lines, MCF10A (human), and HC11 (mouse), with Int6sh under the control of the elongation factor-la (eEF1A) promoter. Expression of Int6sh in MCF10A and HC11 mammary epithelial cells leads to anchorage-independent growth in soft agar indicative of a transformed phenotype. Colonies selected from agar exhibited high levels of mutated Int6sh and wild type Int6 RNA transcripts by RT-PCR and Northern blot analysis. In addition, Int6sh transformed MCF10A and HC11 cells formed nodular growths, in vivo, in immune compromised hosts. NIH3T3 cells, mouse embryo fibroblasts, were also transformed to anchorage-independent growth in vitro by Int6sh expression. These observations provide direct evidence that the Int6 mutations observed in MMTV-induced tumors and hyperplasia contribute to the malignant transformation of the mammary epithelial cells.