Spontaneous tumorigenesis in mice defective in the MTH1 gene encoding 8-oxo-dGTPase - PubMed (original) (raw)

. 2001 Sep 25;98(20):11456-61.

doi: 10.1073/pnas.191086798.

A Egashira, H Igarashi, T Iwakuma, Y Nakatsuru, Y Tominaga, H Kawate, K Nakao, K Nakamura, F Ide, S Kura, Y Nakabeppu, M Katsuki, T Ishikawa, M Sekiguchi

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Spontaneous tumorigenesis in mice defective in the MTH1 gene encoding 8-oxo-dGTPase

T Tsuzuki et al. Proc Natl Acad Sci U S A. 2001.

Abstract

Oxygen radicals, which can be produced through normal cellular metabolism, are thought to play an important role in mutagenesis and tumorigenesis. Among various classes of oxidative DNA damage, 8-oxo-7,8-dihydroguanine (8-oxoG) is most important because of its abundance and mutagenicity. The MTH1 gene encodes an enzyme that hydrolyzes 8-oxo-dGTP to monophosphate in the nucleotide pool, thereby preventing occurrence of transversion mutations. By means of gene targeting, we have established MTH1 gene-knockout cell lines and mice. When examined 18 months after birth, a greater number of tumors were formed in the lungs, livers, and stomachs of MTH1-deficient mice, as compared with wild-type mice. The MTH1-deficient mouse will provide a useful model for investigating the role of the MTH1 protein in normal conditions and under oxidative stress.

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Figures

Figure 1

Figure 1

Targeted disruption of the MTH1 gene by homologous recombination. (a) Targeting of the MTH1 gene. The upper lines represent targeting vector and wild-type_MTH1_ allele, whereas the lower line shows mutated_MTH1_ allele. The thick lines show genomic sequences with exons (filled boxes), whereas a thin line shows the bacterial plasmid portion. Open boxes, a positive [pol II neo poly (A)] or a negative (HSV-tk) selection cassette; lettered bars, 5′-flanking probe A (0.1-kb _Apa_I-_Eco_RI fragment) and 3′-flanking probe B (0.1-kb _Pst_I-_Bam_HI fragment). The observed sizes of the diagnostic restriction fragments, used to distinguish the wild-type and mutant alleles, correspond to their expected sizes. The restriction enzyme sites are abbreviated as B for _Bam_HI, E for Eco_RI, and X for_Xho_I. The restriction enzyme sites in parentheses are lost in the process of targeting vector construction. (b) Southern blot analysis of_Bam_HI-digested genomic DNA from_MTH1+/+(CCE),MTH1+/−(SK1), and two_MTH1_−/−(DK1, DK7) ES cell clones, using the external 3′ probe. (c) Northern blot analysis of poly(A)+ RNA from MTH1+/+ and two _MTH1_−/− ES cell clones, using the 503-bp _Nco_I/_Bam_HI fragment of mouse cDNA as a probe that detects an ≈1.2-kb band corresponding to the size of MTH1 transcript. Each lane contained 3 μg of poly(A)+ RNA. (d) Genotype analysis of DNAs from tails from MTH1+/− intercrosses by PCR. PCR amplification of the wild-type MTH1 allele produces a 0.65-kb DNA fragment (bottom band), whereas amplification of the mutated MTH1 allele produces a 0.80-kb DNA fragment (upper band). Lane 1, marker DNA fragments (M); lane 2,MTH1+/+; lane 3,MTH1+/−; and lane 4,_MTH1_−/−. Sizes of marker fragments are indicated Left.

Figure 2

Figure 2

Absence of MTH1 protein in MTH1_-deficient mouse. (a) Western blot analysis of extracts of liver from_MTH1+/+ and_MTH1_−/− mice, with Abs against purified MTH1 protein. Lane 1, MTH+/+; lane 2,_MTH1_−/−; and lane 3, purified MTH1 protein (1 ng). Arrows (Right) indicate the positions of normal rabbit IgG heavy-chain and purified MTH1 protein, respectively. (b) Assay of 8-oxo-dGTPase activity in the liver. Crude extracts of liver (1 g) prepared from MTH+/+ and _MTH1_−/− mice loaded on a HiTrap Q column and eluted by a linear gradient of 0–0.5 M NaCl. Radioactivities of 8-oxo-dGMP produced were measured by PSL (photo-stimulated luminescence).

Figure 3

Figure 3

Tumors in lung, liver, and glandular stomach developed in_MTH1_−/− mice. (a) Adenocarcinoma of the lung developed in_MTH1_−/− mouse; 7 mm in maximum diameter protruding on the surface of a lung lobe (arrows). (b) Histologic section of a lung adenoma developed in_MTH1_−/− mice. (c) Histologic section of a lung adenocarcinoma. Papillary/alveolar proliferation of basophilic cells is the common feature of both tumor types. (d) Large carcinoma nodule of the liver developed in _MTH1_−/− mouse; 16 mm in maximum diameter (arrows). (e) Histologic section of an adenoma developed in liver of _MTH1_−/− mice. (f) Histologic section of a hepatocellular carcinoma developed in _MTH1_−/− mouse showing typical trabecular pattern of malignant hepatocytes. (g) Elevated lesion of the pyloric mucosa of_MTH1_−/− mouse (arrows) diagnosed as well differentiated adenocarcinoma. (h) Histologic section of adenocarcinoma of the stomach in MTH1_−/− mouse, cut perpendicular to the mucosal surface, consisted of irregular proliferation of dysplastic cells arranged in tubular structure. [Bar = 10 mm (a, d, and_g).] Paraffin-embedded sections (4 mm) were stained with hematoxylin and eosin. Magnification: b, ×100;c, ×100; e, ×40; f, ×100; h, ×20.

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