Manam S, Storer R D, Prahalada S, Leander K R, Kraynak A R, Ledwith B J, van Zwieten M J, Bradley M O, Nichols W W
Department of Safety Assessment, Merck Sharp and Dohme Research Laboratories, West Point, Pennsylvania 19486.
Cancer Res. 1992 Jun 15;52(12):3347-52.
We compared the profile of ras gene mutations in spontaneous CD-1 mouse liver tumors with that found in liver tumors that were induced by a single i.p. injection of either 7,12-dimethylbenz(a)anthracene (DMBA), 4-aminoazobenzene, N-hydroxy-2-acetylaminofluorene, or N-nitrosodiethylamine. By direct sequencing of polymerase chain reaction-amplified tumor DNA, the carcinogen-induced tumors were found to have much higher frequencies of ras gene activation than spontaneous tumors. Furthermore, each carcinogen caused specific types of ras mutations not detected in spontaneous tumors, including several novel mutations not previously associated with either the carcinogen or mouse hepatocarcinogenesis. For example, the model compound DMBA is known to cause predominantly A to T transversions in Ha-ras codon 61 in mouse skin and mammary tumors, consistent with the ability of DMBA to form bulky adducts with adenosine. Our results demonstrate that the predominant mutation caused by DMBA in mouse liver tumors is a G to C transversion in Ki-ras codon 13 (DMBA is also known to form guanosine adducts), illustrating the influence of both chemical- and tissue-specific factors in determining the type of ras gene mutations in a tumor. 4-Aminoazobenzene and N-hydroxy-2-acetylaminofluorene also caused the Ki-ras codon 13 mutation. In addition, we found that N-nitrosodiethylamine, 4-aminoazobenzene, and N-hydroxy-2-acetylaminofluorene all caused G to T transversions in the N-ras gene (codons 12 or 13). This is the first demonstration of N-ras mutations in mouse liver tumors, establishing a role for the N-ras gene in mouse liver carcinogenesis. Finally, comparison of the ras mutations detected in the direct tumor analysis with those detected after NIH3T3 cell transfection indicates that spontaneous ras mutations (in Ha-ras codon 61) are often present in only a small fraction of the tumor cells, raising the possibility that they may sometimes occur as a late event in CD-1 mouse hepatocarcinogenesis.
我们比较了自发的CD-1小鼠肝肿瘤中ras基因突变情况与单次腹腔注射7,12-二甲基苯并(a)蒽（DMBA）、4-氨基偶氮苯、N-羟基-2-乙酰氨基芴或N-亚硝基二乙胺诱导产生的肝肿瘤中的ras基因突变情况。通过对聚合酶链反应扩增的肿瘤DNA进行直接测序，发现致癌物诱导的肿瘤中ras基因激活频率比自发肿瘤高得多。此外，每种致癌物都会导致自发肿瘤中未检测到的特定类型的ras突变，包括几种以前与致癌物或小鼠肝癌发生均无关联的新突变。例如，模型化合物DMBA已知在小鼠皮肤和乳腺肿瘤中主要导致Ha-ras密码子61处的A到T颠换，这与DMBA与腺苷形成大体积加合物的能力一致。我们的结果表明，DMBA在小鼠肝肿瘤中导致的主要突变是Ki-ras密码子13处的G到C颠换（已知DMBA也会形成鸟苷加合物），这说明了化学和组织特异性因素在决定肿瘤中ras基因突变类型方面的影响。4-氨基偶氮苯和N-羟基-2-乙酰氨基芴也会导致Ki-ras密码子13突变。此外，我们发现N-亚硝基二乙胺、4-氨基偶氮苯和N-羟基-2-乙酰氨基芴都会导致N-ras基因（密码子12或13）中的G到T颠换。这是首次在小鼠肝肿瘤中证明N-ras突变，确立了N-ras基因在小鼠肝癌发生中的作用。最后，将直接肿瘤分析中检测到的ras突变与NIH3T3细胞转染后检测到的ras突变进行比较表明，自发的ras突变（在Ha-ras密码子61处）通常仅存在于一小部分肿瘤细胞中，这增加了它们有时可能是CD-1小鼠肝癌发生后期事件的可能性。
