A novel method, digital genome scanning detects KRAS gene amplification in gastric cancers: involvement of overexpressed wild-type KRAS in downstream signaling and cancer cell growth.
A novel method, digital genome scanning detects KRAS gene amplification in gastric cancers: involvement of overexpressed wild-type KRAS in downstream signaling and cancer cell growth.
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DOI：
10.1186/1471-2407-9-198
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发表时间：
2009-06-23
期刊：
影响因子：
3.8
通讯作者：
Tokino T
中科院分区：
文献类型：
--
作者：
Mita H;Toyota M;Aoki F;Akashi H;Maruyama R;Sasaki Y;Suzuki H;Idogawa M;Kashima L;Yanagihara K;Fujita M;Hosokawa M;Kusano M;Sabau SV;Tatsumi H;Imai K;Shinomura Y;Tokino T
胃癌是影响全世界普通人群的第三大常见恶性肿瘤。 KRAS 的异常激活是多种肿瘤发生的关键因素，然而 KRAS 的致癌突变在胃癌中并不常见。我们开发了一种新的 DNA 拷贝数定量分析方法，称为数字基因组扫描 (DGS)，该方法基于短限制性片段的计数，不涉及 PCR 或杂交。在当前的研究中，我们使用 DGS 来调查胃癌细胞的拷贝数变化。使用5000至15000个限制性片段的序列进行胃癌细胞系的DGS。我们通过定量PCR筛选了20个胃癌细胞系和86个原发性胃肿瘤的KRAS扩增，并通过突变分析、实时PCR、免疫印迹分析、GTP-RAS Pull-down实验和免疫组化分析研究了DNA、mRNA和蛋白质水平的KRAS扩增。分别通过免疫印迹和比色测定检查 KRAS 敲低对 p44/42 MAP 激酶和 AKT 激活以及细胞生长的影响。 HSC45 胃癌细胞系的 DGS 分析揭示了染色体 12p12.1 上 500 kb 区域的扩增，其中包含 KRAS 基因位点。在 15% (3/20) 的胃癌细胞系（8-18 倍扩增）和 4.7% (4/86) 的原发性胃肿瘤（8-50 倍扩增）中检测到 KRAS 基因座扩增。在 KRAS 扩增的三种细胞系中的两种中发现了 KRAS 突变，但在任何原发性肿瘤中均未检测到 KRAS 突变。 KRAS 蛋白的过度表达与 KRAS 拷贝数的增加直接相关。在野生型 KRAS 扩增的细胞中，血清刺激后 GTP 结合的 KRAS 水平升高，但突变型 KRAS 扩增的细胞中则没有升高。在携带扩增的野生型 KRAS 的胃癌细胞中敲低 KRAS 会导致细胞生长受到抑制并抑制 p44/42 MAP 激酶和 AKT 活性。我们的研究强调了 DGS 在鉴定拷贝数改变方面的实用性。使用 DGS，我们确定 KRAS 是在人类胃癌中扩增的基因。我们证明基因扩增可能构成胃癌中 KRAS 过度激活的分子基础。需要使用更多的胃癌样本进行更多研究，以确定 KRAS 扩增和过度表达的诊断和治疗意义。
Gastric cancer is the third most common malignancy affecting the general population worldwide. Aberrant activation of KRAS is a key factor in the development of many types of tumor, however, oncogenic mutations of KRAS are infrequent in gastric cancer. We have developed a novel quantitative method of analysis of DNA copy number, termed digital genome scanning (DGS), which is based on the enumeration of short restriction fragments, and does not involve PCR or hybridization. In the current study, we used DGS to survey copy-number alterations in gastric cancer cells. DGS of gastric cancer cell lines was performed using the sequences of 5000 to 15000 restriction fragments. We screened 20 gastric cancer cell lines and 86 primary gastric tumors for KRAS amplification by quantitative PCR, and investigated KRAS amplification at the DNA, mRNA and protein levels by mutational analysis, real-time PCR, immunoblot analysis, GTP-RAS pull-down assay and immunohistochemical analysis. The effect of KRAS knock-down on the activation of p44/42 MAP kinase and AKT and on cell growth were examined by immunoblot and colorimetric assay, respectively. DGS analysis of the HSC45 gastric cancer cell line revealed the amplification of a 500-kb region on chromosome 12p12.1, which contains the KRAS gene locus. Amplification of the KRAS locus was detected in 15% (3/20) of gastric cancer cell lines (8–18-fold amplification) and 4.7% (4/86) of primary gastric tumors (8–50-fold amplification). KRAS mutations were identified in two of the three cell lines in which KRAS was amplified, but were not detected in any of the primary tumors. Overexpression of KRAS protein correlated directly with increased KRAS copy number. The level of GTP-bound KRAS was elevated following serum stimulation in cells with amplified wild-type KRAS, but not in cells with amplified mutant KRAS. Knock-down of KRAS in gastric cancer cells that carried amplified wild-type KRAS resulted in the inhibition of cell growth and suppression of p44/42 MAP kinase and AKT activity. Our study highlights the utility of DGS for identification of copy-number alterations. Using DGS, we identified KRAS as a gene that is amplified in human gastric cancer. We demonstrated that gene amplification likely forms the molecular basis of overactivation of KRAS in gastric cancer. Additional studies using a larger cohort of gastric cancer specimens are required to determine the diagnostic and therapeutic implications of KRAS amplification and overexpression.
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影响因子：
6.2
作者：
Kusano, M;Toyota, M;Tokino, T
通讯作者：
Tokino, T
影响因子：
14.8
作者：
Leary, Rebecca J.;Cummins, Jordan;Velculescu, Victor E.
通讯作者：
Velculescu, Victor E.
DOI：
10.1111/apm.1965.64.1.31
发表时间：
1965-01-01
期刊：
ACTA PATHOLOGICA ET MICROBIOLOGICA SCANDINAVICA
影响因子：
--
作者：
LAUREN, P
通讯作者：
LAUREN, P
影响因子：
7.5
作者：
Gamboa-Dominguez, A;Dominguez-Fonseca, C;Luber, B
通讯作者：
Luber, B
DOI：
10.1073/pnas.0402932101
发表时间：
2004-06-15
影响因子：
11.1
作者：
Aguirre, AJ;Brennan, C;Chin, L
通讯作者：
Chin, L