Vitamin D Regulation of the Uridine Phosphorylase 1 Gene and Uridine-Induced DNA Damage in Colon in African Americans and European Americans

Abstract

Background & aims: African Americans have the greatest colorectal cancer (CRC) burden in the United States; interethnic differences in protective effects of vitamin D might contribute to disparities. 1α,25(OH)₂D₃ vitamin D (the active form of vitamin D) induces transcription of the uridine phosphorylase gene (UPP1) in colon tissues of European Americans but to a lesser extent in colon tissues of African Americans. UPP1-knockout mice have increased intestinal concentrations of uridine and Deoxyuridine triphosphate (dUTP), have increased uridine-induced DNA damage, and develop colon tumors. We studied 1α,25(OH)₂D₃ regulation of UPP1 and uridine-induced DNA damage in the colon and differences in these processes between African and European Americans.

Methods: We quantified expression and activity of UPP1 in response to 1α,25(OH)₂D₃ in young adult mouse colonic cells, human CRC cells (LS174T), and organoids (derived from rectosigmoid biopsy samples of healthy individuals undergoing colonoscopies) using quantitative polymerase chain reaction, immunoblot, and immunocytochemistry assays. Binding of the vitamin D receptor to UPP1 was tested by chromatin immunoprecipitation. Uridine-induced DNA damage was measured by fragment-length analysis in repair enzyme assays. Allele-specific 1α,25(OH)₂D₃ responses were tested using luciferase assays.

Results: Vitamin D increased levels of UPP1 mRNA, protein, and enzymatic activity and increased vitamin D receptor binding to the UPP1 promoter in young adult mouse colonic cells, LS174T cells, and organoids. 1α,25(OH)₂D₃ significantly reduced levels of uridine and uridine-induced DNA damage in these cells, which required UPP1 expression. Organoids derived from colon tissues of African Americans expressed lower levels of UPP1 after exposure to 1α,25(OH)₂D₃ and had increased uridine-induced DNA damage compared with organoids derived from tissues of European Americans. Luciferase assays with the T allele of single nucleotide polymorphism rs28605337 near UPP1, which is found more frequently in African Americans than European Americans, expressed lower levels of UPP1 after exposure to 1α,25(OH)₂D₃ than assays without this variant.

Conclusions: We found vitamin D to increase expression of UPP1, leading to reduce uridine-induced DNA damage, in colon cells and organoids. A polymorphism in UPP1 found more frequently in African Americans than European Americans reduced UPP1 expression upon cell exposure to 1α,25(OH)₂D₃. Differences in expression of UPP1 in response to vitamin D could contribute to the increased risk of CRC in African Americans.

Keywords: CRC; Ethnicity; Genetics; Risk Factor.

Figure 1.

Vitamin D induction of UPP1 mRNA expression. 1α,25(OH)₂D₃-induced mRNA expression of UPP1 was examined at 6- and 24-hour time points in (A) YAMC and (B) LS174T cells. YAMC cells showed 36-fold and 46-fold up-regulation after 6 and 24 hours, respectively (P < .001 for both time points). LS174T cells showed and 1.16-fold and 2.32-fold up-regulation at 6 and 24 hours, respectively (6 hours, P = .020 and 24 hours, P < .001).

Figure 2.

Vitamin D induction of Upp1 protein expression. Immunocytochemistry was performed in (A and B) YAMC and (C and D) LS174T cells after 24 hours of treatment with 1α,25(OH)₂D₃ or ethanol. Representative images are shown of (A) YAMC and (C) LS174T cells treated with 1α,25(OH)₂D₃ and ethanol. The columns are nuclear DAPI staining (blue), Upp1 staining (green), and the overlay of DAPI and Upp1. For LS174T cells, maximum intensity projections were used to depict the 3-dimensional structure. Scale bars indicate 32 µm. Localization of Upp1 protein was observed in both cytosol and nucleus in YAMC and LS174T cells. Quantitated mean gray values of Upp1 expression in 1α,25(OH)₂D₃-treated cells were normalized to ethanol and compared using Wilcoxon signed-rank test. Significant Upp1 up-regulation was observed in (B) YAMC (P =.004) and (D) LS174T (P =.004) cells. (E) Western blot confirms Upp1 protein expression at the expected mass of 34 kDa in YAMC and LS174T cells after 24 hours of treatment with ethanol and vitamin D. GAPDH was the loading control. (F) These results were quantitated. In YAMC cells, there was a significant 2.62-fold up-regulation of Upp1 with vitamin D treatment (P < .001), and in LS174T cells, there was a significant 3.56-fold up-regulation of Upp1 (P < .001). DAPI, 4′,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase (phosphorylating).

Figure 3.

VDR binding of the UPP1 promoter region. A VDR binding site located 222 bp upstream of the UPP1 transcription start site was identified using LASAGNA. ChIP was performed in LS174T cells treated with 1 µmol/L 1α,25(OH)₂D₃ or ethanol using VDR antibody or IgG control antibody. A 10.33-fold enrichment was noted in the treated sample compared with ethanol (P < .0001). Ab, antibody.

Figure 4.

Vitamin D suppression of uridine-induced DNA damage. FLARE assay was performed to examine uridine-induced dUTP incorporation in YAMC cells. (A) Electrophoresed cells stained with SYBR green after treatment with uridine plus ethanol and uridine plus vitamin D. Arrowhead points to the tail that results from DNA damage seen with uridine plus ethanol but not with uridine plus vitamin D treatment. (B) DNA damage as estimated by tail moment using ImageJ software. Representative comparisons and P values are shown on the graph for clarity. There was significantly increased tail moment with uridine alone and with uridine plus ethanol treatments (both P < .0001). There was significantly less tail moment in cells treated with the combination of uridine plus vitamin D compared with cells treated with uridine plus ethanol or with uridine alone (both P < .0001). (C) Uridine concentrations by treatment condition. Uridine concentration was significantly increased with uridine treatment (both uridine and uridine plus ethanol) (P < .0001). Uridine concentrations were lower in the cells treated with the combination of uridine plus vitamin D compared with the cells treated with uridine plus ethanol or uridine alone (P < .0001). (D) Representative FLARE results after YAMC cells were transfected with UPP1 siRNA or scrambled siRNA and treated with uridine plus ethanol and uridine plus vitamin D. Arrowheads point to the tails resulting from DNA damage. (E) Tail moments were quantified using ImageJ. Uridine plus vitamin D treatment showed significantly greater tail moment in UPP1 siRNA transfected cells compared with scrambled control (P < .0001).

Figure 5.

Vitamin D up-regulation of UPP1 expression and suppression of uridine induced DNA damage in human colonic organoids. Human colon organoids from 10 individuals were treated with 1α,25(OH)₂D₃ or ethanol, and expression of UPP1 was examined. (A) A 5.32-fold up-regulation of UPP1 mRNA at 6 hours after vitamin D treatment (P < .0005). (B) Representative immunofluorescence images of organoids treated with 1α,25(OH)₂D₃ and ethanol. The columns are nuclear DAPI staining (blue), Upp1 staining (green), and the overlay of DAPI and Upp1. Localization of Upp1 protein was observed in both cytosol and nucleus. Scale bar indicates 64 µm. (C) Quantitated mean gray values of Upp1 expression in 1α,25(OH)₂D₃-treated cells that were normalized to ethanol and showed a 3.42-fold up-regulation of Upp1 protein expression after 24 hours of vitamin D treatment (P = .0020). (D) Representative FLARE images of human colonic organoids treated with uridine plus ethanol and uridine plus vitamin D. The arrowhead points to the tail representing DNA damage. (E) DNA damage as estimated by tail moment using ImageJ is shown. Representative comparisons and P values are shown on the graph for clarity. There was significantly increased tail moment with uridine alone and with uridine plus ethanol treatments compared with control (both P < .0001). There was significantly less tail moment in cells treated with the combination of uridine plus vitamin D compared with cells treated with uridine plus ethanol or with uridine alone (both P < .0001). DAPI, 4′,6-diamidino-2-phenylindole.

Figure 6.

Interethnic differences in UPP1 up-regulation by vitamin D, baseline tissue uridine levels, and uridine-induced DNA damage. Human colonic organoids from African and European Americans were treated with 1α,25(OH)₂D₃ or ethanol. (A) Significantly greater UPP1 mRNA up-regulation in European Americans (n =5) compared with African Americans (n = 6) (P = .0040). (B) Significantly greater Upp1 protein up-regulation in European Americans (n =5) compared with African Americans (n = 5) (P = .0397) by quantification of fluorescence staining using ImageJ. Uridine tissue concentration was compared in colonic biopsy samples from African Americans (n =5) and European Americans (n =5). (C) Significantly higher concentration of uridine in the tissue samples derived from African Americans compared with European Americans (P = .0317). FLARE assay was performed in human colonic organoids from African Americans (n = 8) and European Americans (n = 8). African Americans showed higher relative uridine-induced DNA damage compared with European Americans. (D) Relative DNA damage in ethanol-, uridine-, 1α,25(OH)₂D₃⁻, uridine plus 1α,25(OH)₂D₃⁻, and uridine plus ethanol–treated organoids. All DNA damage values were estimated as tail moments and normalized to ethanol. AA, African American; EA, European American.

Figure 7.

Allele-specific differences in UPP1 expression upon treatment with 1α,25(OH)₂D₃. A region 1500 kb upstream and 1000 kb downstream of UPP1 was examined for candidate SNPs that could be tested using luciferase reporter assays. UPP1 eQTLs in transverse colon from the GTEx database were examined. (A, B) Two candidate regions containing 3 eQTLs, rs1554494 and rs7458962 (region 1) and rs28605337 (region 2), were identified. SNPs in region 1 were located in the promoter region of UPP1 near the VDR binding site validated in our study and a region with a H3K27Ac peak. The SNP in region 2 was located 3′ of UPP1 in a CTCF binding site near a reported VDR binding site from a previous ChIP sequencing study. Enhancer activity ratio was estimated in cells transfected with ancestral or derived allele after being treated with 1α,25(OH)₂D₃ or for 4 hours. (C, D) Enhancer activity ratio (vitamin D:ethanol) for SNPs in regions 1. (E) Enhancer activity ratio for the SNP in region 2. A 2-tailed t test was conducted to compare the ratios between the African- and European-derived alleles for each construct. No significant difference was observed between African- and European-derived alleles in region 1. For rs28605337, a significantly higher enhancer activity ratio was observed for the European allele compared with the African allele after 4-hour treatment with 1α,25(OH)₂D₃ (P = .0005).