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. 2001 Sep 25;98(20):11456-61.
doi: 10.1073/pnas.191086798.

Spontaneous Tumorigenesis in Mice Defective in the MTH1 Gene Encoding 8-oxo-dGTPase

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Free PMC article

Spontaneous Tumorigenesis in Mice Defective in the MTH1 Gene Encoding 8-oxo-dGTPase

T Tsuzuki et al. Proc Natl Acad Sci U S A. .
Free PMC article

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.

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 ApaI-EcoRI fragment) and 3′-flanking probe B (0.1-kb PstI-BamHI 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 BamHI, E for EcoRI, and X for XhoI. The restriction enzyme sites in parentheses are lost in the process of targeting vector construction. (b) Southern blot analysis of BamHI-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 NcoI/BamHI 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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