ras oncogene mutations in diethylnitrosamine-induced hepatic tumors in medaka (Oryzias latipes), a teleost fish
ras oncogene mutations in diethylnitrosamine-induced hepatic tumors in medaka (Oryzias latipes), a teleost fish
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DOI：
10.1016/s1383-5718(03)00133-5
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发表时间：
2003-08-05
影响因子：
1.9
通讯作者：
Hinton, DE
中科院分区：
文献类型：
--
作者：
Liu, Z;Kullman, SW;Hinton, DE
青鳉鱼是一种已建立的非哺乳动物研究模型，用于研究肝癌发生和暴露于环境污染物。研究强调青鳉暴露于模型致癌物后会发生肝肿瘤。然而，迄今为止，很少有信息是已知的机制，在这个物种的肝肿瘤的启动。本研究的目的是将我们对二乙基亚硝胺（DEN）诱导的肿瘤形成的理解与暴露青鳉肝肿瘤中ras基因激活联系起来。初步研究进行了确定青鳉ras外显子1和2的逆转录聚合酶链反应（RT-PCR）。ras外显子1和2的扩增未经处理的青鳉肝脏导致三个多态性的ras序列变异体的鉴定，表现出高度的同源性，其他硬骨鱼和哺乳动物的ras基因。青鳉暴露于159 ppm的DEN导致了广泛的肝肿瘤，包括：肝细胞腺瘤，肝细胞癌，胆管瘤和混合性肝胆管细胞癌。通过用特异于激活点突变的探针探测基因组DNA或通过使用RT-PCR直接克隆和测序ras转录物来检查个体肝肿瘤的致癌激活ras突变。使用等位基因特异性寡核苷酸（阿索）分析，在8/25（32%）检查的肿瘤中检测到密码子12位置2的单点突变。突变ras等位基因，另外检测到12 39（30%）青鳉肝肿瘤序列分析。12个突变中有10个在密码子12处含有一个点突变，导致Gly替换为Asp。在密码子16处鉴定了两个独特的突变，导致Lys至Asn或Lys至Thr氨基酸取代。我们的研究结果表明，ras基因突变是由DEN诱导的，并且存在于超过30%的发生肿瘤的鱼中。ras突变发生率为30%，与暴露于DEN的哺乳动物物种中报告的相似。虽然在密码子12突变以前已被报道，本研究是第一次在体内报告的ras点突变密码子16。(C)2003 Elsevier B. V.保留所有权利。
Medaka fish are an established non-mammalian research model for the study of liver carcinogenesis and exposure to environmental pollutants. Studies have emphasized the development of hepatic neoplasms in medaka following exposure to model carcinogens. To date however, little information is known regarding the mechanisms underlying initiation of hepatic tumors in this species. The aim of this study was to relate our understanding of diethylnitrosamine (DEN)-induced tumor formation to ras gene activation in hepatic neoplasms of exposed medaka. Initial studies were conducted to identify medaka ras exons 1 and 2 by reverse transcriptase polymerase chain reaction (RT-PCR). Amplification of ras exons 1 and 2 from untreated medaka liver resulted in the identification of three polymorphic ras sequence variants exhibiting a high degree of homology to other teleost and mammalian ras genes. Exposure of medaka to 159 ppm of DEN resulted in a wide range of hepatic neoplasms including: hepatocellular adenomas, hepatocellular carcinomas, cholangiomas, and mixed hepatocholangiocellular carcinomas. Individual liver tumors were examined for oncogenically activating ras mutations by probing genomic DNA with probes specific for activating point mutations or by direct cloning and sequencing of ras transcripts using RT-PCR. Using allele-specific oligonucleotide (ASO) analysis, a single point mutation was detected in codon 12 position two in 8/25 (32%) tumors examined. Mutated ras alleles were additionally detected in 12 of 39 (30%) medaka liver tumors by sequence analysis. Ten of the 12 mutations identified contained a single point mutation at codon 12 resulting in a Gly to Asp amino acid substitution. Two unique mutations were identified at codon 16 resulting in either Lys to Asn or Lys to Thr amino acid substitutions. Our results show that ras mutations are induced by DEN and are present in over 30% of the fish that developed tumors. A ras mutation incidence of 30% is similar to that reported in mammalian species exposed to DEN. While mutations at codon 12 have previously been reported, the present study is the first in vivo report of ras point mutations at codon 16. (C) 2003 Elsevier B.V. All rights reserved.