Fig 1: Deletion of Msh2 in medium-spiny neurons delays nuclear huntingtin phenotypes. A, B.Nuclear mutant huntingtin immunostaining is decreased in the striata of five-month old HdhQ111/+ mice with deletion of Msh2 in MSNs. A. Fluorescent micrographs of striata double-stained with anti-huntingtin mAb5374 and anti-histone H3 antibodies for three CAG repeat length-matched mice (Msh2+/+ CAG 113, Msh2Δ/Δ CAG 112, Msh2−/− CAG 113). B. Box plot showing upper and lower quartiles, median and range for the normalized mAb5374 immunostaining intensity (total mAb5374 staining intensity normalized to the number of H3-positive nuclei). Outlier (circle) is defined by a standard interquartile method and is included in the analysis. Multiple regression analysis was used to determine the effect of Msh2 genotype on mAb5374 staining using normalized mAb5374 intensity (continuous variable) as a dependent variable and Msh2 genotype (discrete variable), constitutive CAG length (continuous variable) and position (medial versus lateral, discrete variable) as independent variables. Both constitutive CAG length (P<0.05) and medial versus lateral position (P<0.001) were significantly associated with normalized mAb5374 intensity. Asterisks above the bars indicate a significant difference from Msh2+/+ at a p-value cut-off of p<0.05(*), p<0.01 (**), p<0.001 (***) in the regression analysis. Msh2Δ/− was not significantly different from Msh2+/− (p = 0.18). The five-month mice used in the quantitative analysis are as follows: Msh2+/+ (n = 6, CAG 113, 118, 119, 121, 123, 125), Msh2+/− (n = 4, CAG 114, 114, 120, 123), Msh2Δ/Δ (n = 5, CAG 113, 121, 121, 126, 129), Msh2Δ/− (n = 7, CAG 113, 121, 121, 122, 125, 125, 133) and Msh2−/− (n = 3, CAG 112, 120, 123). Note that the relatively “weak” effect of the Msh2−/− genotype likely reflects the small number of mice of this genotype and hence the least accurate estimate of the relationship of mAb5374 intensity to CAG length in the regression analysis. C, D. Intranuclear inclusions are decreased in the striata of ten-month old HdhQ111/+ mice with deletion of Msh2 in MSNs. C. Fluorescent micrographs of striata stained with mAb5374 from mice with Msh2+/+ (CAG 121), Msh2+/− (CAG 123), Msh2Δ/Δ (CAG 133), Msh2Δ/− (CAG 123) and Msh2−/− (CAG 132) genotypes. D. Quantification of the percentage of cells containing an inclusion (more than one inclusion per cell was rarely observed). The total number of cells was determined by co-staining with histone H3 (not shown). The ten-month mice used in the quantitative analysis are as follows: Msh2+/+ (n = 6, CAG 118, 121, 121, 123, 126, 134), Msh2+/− (n = 4, CAG 116, 118, 123, 131), Msh2Δ/Δ (n = 1, CAG 133), Msh2Δ/− (n = 7, CAG 115, 115, 117, 120, 121, 122, 123) and Msh2−/− (n = 1, CAG 132). Bars represent mean ±S.D.
Fig 2: AZD5153-resistant tumors acquires sensitivity to αPD-L1 (A) Schematic diagram shows CT26 and CT26R cells were injected into BALB/c mice to establish the in vivo models. (B) CT26 and CT26R cells were injected into BALB/c mice. Three days later, mice were randomized into treatment cohorts: Vehicle (n=5, 0.5% hydroxypropylmethylcellulose and 0.2% Tween 80), AZD5153 (n=5, 1.25 mg/kg per day, oral gavage), αPD-L1 antibody (n=5, 200 µg/mouse every 3 days for six times), or a combination of AZD5153 and αPD-L1 antibody (n=5). Average tumor volumes±SEM for each cohort were displayed. (C) Tumor volumes on day 21 of (B). Data represent mean±SEM. Analysis of variance (ANOVA) was used to compare differences among multiple groups: *, p<0.05; **, p<0.01; ***, p<0.001; n.s., not significant. (D) Western blotting of MLH1, MSH2, MSH6, and PMS2 expression levels in CT26 and CT26R mice treated with vehicle. (E–F) Representative flow cytometry plots (left) and quantification of effector CD8 T cells (CD8+ IFN-γ+) (right) (E), exhausted CD8 T cells (TIM3+PD-1+) (F). T cells proportions of all CD8+ T cells in tumors from each group, respectively (n=3). Data represent mean±SEM. P values were determined by ANOVA. *, p<0.05; **, p<0.01; ***, p<0.001. IFN, interferon; TIM-3, T cell immunoglobulin and mucin domain 3; MLH1, mutL homologue 1; MSH2, mutS homologue 2; MSH6, mutS homologue 6; n.s., not significant; PD-1, programmed death; PD-L1, programmed death ligand 1; PMS2, PMS1 homolog 2, mismatch repair system component.
Fig 3: Plasma acetaldehyde concentrations in ethanol‐ (black bar) and water‐treated (white bar) induced Msh2‐LS mice and non‐induced Msh2‐LS mice; Mann–Whitney U‐test, **p = 0.0019 (data shown as mean ± SD error bars, n = 4–6).
Fig 4: Tumour formation in Msh2‐LS mice. Bar chart of the numbers of Msh2‐LS mice that developed large intestinal tumours (both adenomas and adenocarcinomas) after receiving either 20% ethanol or standard drinking water. 15/23 (65%) ethanol‐treated induced Msh2‐LS mice developed large intestinal tumours compared with 1/23 (4%) water‐treated induced Msh2‐LS mice; Fisher's exact test, ****p < 0.0001. In both groups of non‐induced Msh2‐LS mice (water‐treated and ethanol‐treated), 1/12 (8.4%) non‐induced Msh2‐LS control mice developed colonic neoplasms; Fisher's exact test, no significant differences observed. Comparison of the tumour‐bearing ethanol‐treated induced Msh2‐LS mice with ethanol‐treated non‐induced Msh2‐LS mice showed a significant difference in the development of large intestinal tumours; Fisher's exact test, ΔΔ p = 0.0016.
Fig 5: Schematic diagram of the proposed model of the MMR/ethanol/acetaldehyde gene–environment interactions in both MMR‐proficient (upper panel) and MMR‐deficient (lower panel) colonic epithelial cells. Upon ethanol/acetaldehyde exposure, in some colonic epithelial stem cells there is DNA base damage that normally would be recognised and repaired by the MMR system, or if unrepaired this base damage may induce replication errors, such as base mismatches or insertion/deletion loops (InDels), during S‐phase of the cell cycle. Here, the MMR‐proficient cell is able to activate DNA mismatch repair of the (MMR‐recognised) base damage, bringing about either cell cycle arrest in the context of mild DNA damage to allow DNA repair or cell death by apoptosis for more severe DNA damage. By contrast, the MMR‐deficient cell (dMMR due to mutant Msh2) is unable to activate the MMR signalling pathway and so there is neither cell cycle arrest nor apoptosis, resulting in aberrant survival of DNA‐damaged cells that can undergo ethanol‐induced subsequent proliferation. The proliferating dMMR stem cells populate the colonic crypt and expand further to form dMMR crypt foci. Stimulated by ethanol to undergo increased proliferation, these cells form hyperproliferative crypts whilst remaining subject to ongoing DNA damage from continued exposure to ethanol/acetaldehyde. These dMMR cells can accumulate mutations reflecting dMMR genomic instability and are consequently at increased risk of tumour formation, thus explaining the acceleration of colonic adenoma formation and increased probability of evolution to adenocarcinoma.
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