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. 2002 May;22(9):2906-17.
doi: 10.1128/MCB.22.9.2906-2917.2002.

"VSports手机版" DNA methyltransferase deficiency modifies cancer susceptibility in mice lacking DNA mismatch repair

Binh N Trinh  1 , Tiffany I Long, Andrea E Nickel, Darryl Shibata, Peter W Laird
Affiliations

DNA methyltransferase deficiency modifies cancer susceptibility in mice lacking DNA mismatch repair

Binh N Trinh et al. Mol Cell Biol. 2002 May.

Abstract

We have introduced DNA methyltransferase 1 (Dnmt1) mutations into a mouse strain deficient for the Mlh1 protein to study the interaction between DNA mismatch repair deficiency and DNA methylation. Mice harboring hypomorphic Dnmt1 mutations showed diminished RNA expression and DNA hypomethylation but developed normally and were tumor free. When crossed to Mlh1(-/-) homozygosity, they were less likely to develop the intestinal cancers that normally arise in this tumor-predisposed, mismatch repair-deficient background. However, these same mice developed invasive T- and B-cell lymphomas earlier and at a much higher frequency than their Dnmt1 wild-type littermates. Thus, the reduction of Dnmt1 activity has significant but opposing outcomes in the development of two different tumor types. DNA hypomethylation and mismatch repair deficiency interact to exacerbate lymphomagenesis, while hypomethylation protects against intestinal tumors. The increased lymphomagenesis in Dnmt1 hypomorphic, Mlh1(-/-) mice may be due to a combination of several mechanisms, including elevated mutation rates, increased expression of proviral sequences or proto-oncogenes, and/or enhanced genomic instability. We show that CpG island hypermethylation occurs in the normal intestinal mucosa, is increased in intestinal tumors in Mlh1(-/-) mice, and is reduced in the normal mucosa and tumors of Dnmt1 mutant mice, consistent with a role for Dnmt1-mediated CpG island hypermethylation in intestinal tumorigenesis VSports手机版. .

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Figures

FIG. 1.
FIG. 1.
Hypomorphic alleles of the mouse Dnmt1 gene. (A) Schematic map (drawn approximately to scale) showing the positions of the Dnmt1R and Dnmt1N mutations in the 5′ region of the Dnmt1 genomic locus. The first four exons shown are represented as numbered black boxes, and intervening introns are represented as solid lines. The 320-bp insertion (43, 44) containing three copies of the lac operator (lacO) sequence from E. coli is located just upstream of an EcoRV site (R) in intron 3 and is depicted as an open box (O). H, HindIII. The Dnmt1N allele, which has been described previously (49, 52), contains a neomycin cassette that replaces a 900-bp NaeI (N) fragment. This mutation deletes part of exon 4 and causes an almost full disruption of Dnmt1 expression. (B) Dnmt1 expression in mutant mice as determined by real-time RT-PCR analysis. RNA was isolated from two thymus tissues of each genotype, and expression was normalized to expression of Gapdh, histone H4, and Pcna. The bars represent the mean value obtained for each of the normalized measurements shown on a relative scale. The error bars represent the standard error of the mean. The slope of the solid triangle below the graph schematically represents the relative Dnmt1 expression for each genotype. (C) DNA methylation of centromeric minor satellite repeats in Dnmt1 mutant mice. Genomic DNA from tail biopsies of Mlh1−/− mice was digested with methylation-sensitive HpaII (H) or with a methylation-insensitive isoschizomer, MspI (M), as a control and then probed with a fragment derived from pMR150 (8). Results for DNA from a Dnmt1C/C complete-knockout embryonic stem cell line are shown as a control (49). Lower-molecular-weight bands in the HpaII lanes indicate DNA hypomethylation. The slope of the solid triangle above the gels schematically represents the relative Dnmt1 expression for each genotype.
FIG. 2.
FIG. 2.
Effect of Dnmt1 mutations on the tumor incidence in Mlh1 deficient mice. (A) Effect of Dnmt1 genotype on the cumulative frequency of intestinal tumors in Mlh1-deficient mice by 9 months of age. The number of mice in each cohort is shown at the bottom. The statistical significance of the difference in tumor frequency for each mutant genotype versus the wild type was tested using chi-square analysis, with the P values shown at the bottom. (B) Effect of Dnmt1 genotype on the cumulative frequency of lymphomagenesis in Mlh1-deficient mice by 9 months of age. All F1 Mlh1-deficient and wild-type mice were sacrificed when they were moribund or when they reached9 months. The statistical significance of the difference in tumor frequency for each mutant genotype versus the wild type was tested using chi-square analysis, with the P values shown at the bottom. (C) Effect of Dnmt1 genotype on the age of onset of lymphomas. The horizontal lines represent the mean age at diagnosis for each genotype. The statistical significance of the difference in age of onset of each mutant genotype versus wild-type mice was tested using an unpaired t test, with the P values shown at the bottom.
FIG. 3.
FIG. 3.
Kaplan-Meier survival curves of Mlh1-deficient, Dnmt1 mutant mice up to 9 months. n, number of mice in each genotype cohort. Each group was monitored closely for 9 months, at which time mice were euthanized and examined on autopsy. Each point represents a death event for a mouse, which is then censored from further survival analysis. Mice removed from cohorts for any other reasons were also censored so that only the survival data for the remaining cohort is monitored.
FIG. 4.
FIG. 4.
MethyLight analysis of five CpG islands. (A) CpG density maps of five CpG islands analyzed by MethyLight. Individual CpG sites are indicated by vertical lines. Locations of MethyLight amplicons are indicated by solid boxes. Sequences are aligned relative to their respective transcriptional starts at position 0, indicated by the arrow pointing to the right. GenBank accession numbers are indicated to the right of the CpG density maps. (B) MethyLight analysis of CpG island hypermethylation in normal mucosal tissue. Genomic DNA from jejunum mucosal tissue was bisulfite converted and then used to measure CpG island hypermethylation. CpG island hypermethylation is expressed as percentage of methylated reference (PMR) value (19). The calculation of PMR values is described in Materials and Methods. Geometric means of the PMR values were calculated, since the methylation data are not normally distributed. The error bars represent the standard errors of the means. The statistical significance of the difference in CpG island hypermethylation for each gene of each mutant genotype versus the wild type was tested using the Mann-Whitney U test, with the P values shown at the bottom.
FIG. 5.
FIG. 5.
MethyLight analysis of CpG island hypermethylation at five sites in normal mucosa and intestinal tumors of Mlh1−/− mice. Genomic DNA from jejunum mucosal tissue or tumor tissue was bisulfite converted and then used to measure CpG island hypermethylation. CpG island hypermethylation is expressed as percentage of methylated reference (PMR) value (19). The calculation of PMR values is described in Materials and Methods. Geometric means of the PMR values were calculated, since the methylation data are not normally distributed. The error bars represent the standard errors of the means. The statistical significance of the difference in CpG island hypermethylation between normal and tumor tissue was tested separately for each gene and for each genotype using the Mann-Whitney U test, with the P values shown at the bottom.
FIG. 6.
FIG. 6.
Recombination of lymphoma DNA in Mlh1-deficient, Dnmt1 mutant mice. (A) Dβ1Jβ2 and Dβ1Jβ2 recombinations at the TCRβ locus and DJ recombination at the IgH locus were determined using PCR of genomic DNAs derived from various tissues, and products were separated on an agarose electrophoresis gel (85). Representative DNA samples from four Mlh1 wild-type thymus samples (lanes 1 to 4, left panels) and four lymphoma samples (lanes 5 to 8, left panels) at each Dnmt1 genotype and their matched control tail DNAs are shown. Lanes with thymus DNA show different possible DJ configurations, while some lanes with lymphoma DNA show one or two predominant configurations (indicated by a closed circle below the lane). Recombined product bands seen in the tail DNA lanes (right panel) are due to DNA from blood lymphocytes that contaminated the samples. GL, unrearranged, germ line configuration; PD, primer dimer. (B) Summary of DNA recombination in lymphoma in Mlh1-deficient mice for each Dnmt1 genotype.
FIG. 7.
FIG. 7.
Summary of genes induced (white boxes), repressed (black boxes), or unchanged (gray boxes) in lymphoid tumors from Mlh1-deficient mice. Gene expression levels of 17 transcripts were analyzed using real-time RT-PCR in lymphomas (the number is indicated at the bottom of the chart) and compared to the mean values for eight age-matched normal thymus tissues from Mlh1 wild-type mice. Gene expression was normalized to both Gapdh and histone H4 expression to correct for RNA input and averaged. The values shown in each box represent the relative increase or decrease in expression level in the lymphomas versus the control tissues.
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