Nitrative and oxidative DNA damage caused by K-ras mutation in mice

https://doi.org/10.1016/j.bbrc.2011.08.076Get rights and content

Abstract

Ras mutation is important for carcinogenesis. Carcinogenesis consists of multi-step process with mutations in several genes. We investigated the role of DNA damage in carcinogenesis initiated by K-ras mutation, using conditional transgenic mice. Immunohistochemical analysis revealed that mutagenic 8-nitroguanine and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) were apparently formed in adenocarcinoma caused by mutated K-ras. 8-Nitroguanine was co-localized with iNOS, eNOS, NF-κB, IKK, MAPK, MEK, and mutated K-ras, suggesting that oncogenic K-ras causes additional DNA damage via signaling pathway involving these molecules. It is noteworthy that K-ras mutation mediates not only cell over-proliferation but also the accumulation of mutagenic DNA lesions, leading to carcinogenesis.

Highlights

► Mutated K-ras in transgenic mice caused nitrative DNA damage, 8-nitroguanine. ► The mutagenic 8-nitroguanine seemed to be generated by iNOS via Ras-MAPK signal. ► Mutated K-ras produces additional mutagenic lesions, as a new oncogenic role.

Introduction

Activating mutations of ras family genes have been frequently found in human cancers [1]. It is reported that the activation of ras oncogene leads to cancer in mouse models [1], [2]. Therefore ras mutations have been thought important for carcinogenesis. On the other hand, it is considered to be necessary for carcinogenesis to occur mutations in multiple genes. It can be supposed to accumulate additional mutations via DNA damage, even in the case of carcinogenesis initiated by ras mutation. These findings raised an idea that ras mutation mediates DNA damage and resulting additional mutations, which contribute to tumor development.

Guanine is most easily oxidized among the four DNA bases, because the oxidation potential of guanine is lower than the other three DNA bases. 8-Oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) and 8-nitroguanine are considered to be mutagenic DNA lesions. It is generally accepted that misincorporation of adenine occurs opposite 8-oxodG during DNA synthesis, leading to transversions from guanine to thymine, which may promote carcinogenesis [3]. 8-Nitroguanine is also can cause transversions from guanine to thymine via formation of an apurinic site [4], [5]. 8-OxodG and 8-nitroguanine can be formed by reactive oxygen (ROS) and nitrogen species (RNS). ROS are generated from multiple sources, including inflammatory cells, carcinogenic chemicals and their metabolites, and electron transport chain in mitochondria.

In various cases of inflammation-related carcinogenesis, we have reported the formation of 8-oxodG and 8-nitroguanine, using tissue specimens obtained from patients with inflammatory diseases and animals under inflammatory conditions [6]. Moreover, we have reported that the induction of inducible nitric oxide synthase (iNOS). In the case of carcinogenesis initiated by ras mutation, iNOS may be induced via inflammation through abnormal cell-proliferation caused by oncogenic activation of ras.

To investigate the role of DNA damage in carcinogenesis initiated by ras mutation, we analyzed the formation of DNA damage after oncogenic activation of K-ras in the transgenic mice with Cre/LoxP system.

Section snippets

Transgenic mice and oncogenic K-ras activation

There are some reports about preparation of transgenic mice with oncogenic K-ras, as summarized by Schubbert et al. [1]. In this study, we prepared conditional transgenic mice, with C57BL/6 genetic background. The mutated K-rasVal12 has a point mutation in codon 12 with change of amino acid glycine to valine. We introduced a 5′-CAG promoter-LoxP-STOP-LoxP-K-rasVal12-IRES-LacZ-pA-Neo within the first intron of ryanodine receptor type 2 (Ryr2) gene by homologous recombination (Fig. 1A). All mice

Formation of 8-nitroguanine and 8-oxodG in adenocarcinomas

Adenocarcinomas were developed in the lung of the transgenic mice infected with Cre-adenovirus to airway. Strong immunoreactivities of 8-nitroguanine and 8-oxodG were observed in lung tissues of all mice at 9 weeks after K-rasVal12 activation (n = 3). Three mice showed similar tendency for immunoreactivities. Representative images were shown in Fig. 2. 8-Nitroguanine and 8-oxodG were co-localized in adenocarcinomas and bronchioles (Fig. 2A). The strong expression of K-rasVal12 was also

Discussion

In this study, we demonstrated that mutagenic DNA lesions, 8-oxodG and 8-nitroguanine were apparently formed in adenocarcinoma initiated by K-rasVal12 in mice. The formation of 8-nitroguanine was co-localized with iNOS. NO, which is generated through iNOS expression, reacts with O2- to produce ONOO, forming not only 8-oxodG but also 8-nitroguanine [11], [12]. It is noteworthy that oncogenic K-rasVal12 activation mediated iNOS-dependent DNA damage in mice, which is considered to participate in

Acknowledgment

This work was supported by a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan.

References (22)

  • N. Suzuki et al.

    Miscoding events during DNA synthesis past the nitration-damaged base 8-nitroguanine

    Biochemistry

    (2005)
  • View full text