Elevated glucose increases genomic instability by inhibiting nucleotide excision repair

(A) Expression of DNA repair genes from 293T WT and XPC cells grown in LG or HG assessed using the NanoString DNA Damage and Repair panel. Expression is presented as the average of each group (triplicate) with high relative expression depicted in red and low relative expression in green. Genes were hierarchically grouped by agglomerative clustering. (B) Significant (P < 0.05) changes in mRNA expression of repair genes induced by elevated glucose are displayed by volcano plot. (C, D, E) Pathway scores were calculated for specific DNA repair pathways using NanoString nSolver Advanced Analysis software including: (C) nucleotide excision repair, (D) homology directed repair, and (E) non-homologous end joining.

(A) NER gene expression was assessed by qRT-PCR following culture of 293T cells in LG or HG (n = 3), normalized to tubulin. Statistical analysis by two-way ANOVA with Sidak’s multiple comparisons test. (A, B) NER gene expression measured as in (A) for MCF10A cells (n = 4). (C) Protein levels of key NER factors were measured by metal-assisted protein quantification with ICP–MS detection in 293T cells grown in LG or HG (normalized to actin). Antibody metal labels: XPA ¹⁷²Yb, XPC ¹⁶³Dy, XPG ¹⁵⁵Gd, and actin ¹⁷¹Yb. (D) Metal-assisted protein quantification of HIF-1α (¹⁶¹Dy) and NER proteins in livers extracted from non-diabetic (Lepr^wt/wt) and diabetic (Lepr^db/db) mice, normalized to actin (n = 5; multiple t tests with Holm-Sidak multiple comparisons correction).

(A) 293T WT cells were exposed to chronic HG, 24 h HG, 4 h MG (50 μM), or 4 h etoposide (ET; 10 μM) and γH2AX foci were analyzed by immunofluorescence. Locations of enlarged insets are depicted by white boxes (scale bar = 20 μm). (B) 293T WT and nucleotide excision repair-deficient cells were grown in LG and treated with 500 or 50 μM MG and 10 or 1 μM etoposide (ET) for 4 h. Subsequent phosphorylation of H2AX (17 kD), ATM (350 kD), and ATR (300 kD) was assessed by Western blot. (C) 293T WT and XPC cells were subjected to an alkaline comet assay after various MG or glucose treatments. Comet tail moment was quantified and graphed as mean ± SEM. (D) Representative comet images from treated WT cells; scale bar = 100 μm. (E) Non-homologous end joining and HDR activity were measured via EJ7 and DR-GFP repair assays, respectively (60 μg transfections). DSBs were induced in GFP reporter plasmids by a CRISPR-Cas9 targeting system, and repaired GFP+ cells were detected by flow cytometry. Activity is presented as %GFP+ cells normalized to a transfection control (n = 10; three independent transfections, unpaired t test).

(A) The expression profile of metabolic genes in 293T WT and XPC cells maintained in LG or HG was assayed using the NanoString Cancer Metabolism panel. Expression is presented as an average of each group (triplicate) with high relative expression depicted in red and low relative expression in green. Genes were hierarchically grouped by agglomerative clustering. (B) Significant (P < 0.05) changes in mRNA expression of metabolism genes induced by elevated glucose are displayed as a volcano plot. (C, D, E) Pathway scores were calculated for specific metabolic pathways using NanoString nSolver Advanced Analysis software, including: (C) HIF-1, (D) glycolysis, and (E) glucose metabolism. (F) Correlation between glycolysis and HIF-1 scores across all samples.

(A) 293T WT cells in LG were treated with the indicated doses of PHD inhibitors CoCl₂, daprodustat (Dap), and DMOG for 6 h to stabilize HIF-1α protein. (B) 293T WT cells in LG or HG were treated with 100 μM CoCl₂ or 50 μM daprodustat and HIF-1α was assessed by Western blot. Relative changes in HIF-1α expression, normalized to β-actin by densitometry, are shown below the blot. (C) HRE-luciferase plasmid was transfected into 293T WT cells exposed to acute or chronic glucose and/or treated with 100 μM CoCl₂ for 24 h. Changes in HIF-1 transcriptional activity were measured via relative luminescence (normalized to transfection control) and analyzed by two-way ANOVA with Sidak’s multiple comparisons. (D) Metal-assisted protein quantification of HIF-1α and downstream target proteins from WT cells grown in LG or HG. Antibody metal labels: HIF-1α ¹⁶¹Dy, PDGFA ¹⁶⁵Ho, VEGFA ¹⁶⁴Dy, PHD3 ¹⁵¹Eu, p-AKT ¹⁵⁹Tb, and actin ¹⁷¹Yb. (E) qRT-PCR analysis of IDH1 mRNA in 293T WT cells grown in LG, 24 h HG, or chronic HG (n = 3, one-way ANOVA). (F) Corresponding IDH1 (47 kD) protein analysis. (G) ELISA quantification of 2-KG in 293T WT LG or HG cells. (H) Schematic showing the up-regulation of IDH1 and 2-KG production in HG, leading to PHD-mediated hydroxylation and degradation of HIF-1α.

(A) 293T WT HG cells were treated with 50 μM daprodustat for 0–24 h to stabilize HIF-1α. NER genes were monitored via qRT-PCR (n = 2). (B) 293T WT cells in HG were treated with 100 μM CoCl₂ for 6 h. HIF-1α, XPD, XPA, and VEGFA proteins were detected by Western blot and quantified by densitometry, normalized to β-actin. (C) qRT-PCR measurement of NER gene expression in cells treated for 6 h with DMSO (vehicle) or 0.1 μM echinomycin, an inhibitor of HIF-1α binding to HREs (n = 3). (D) 293T WT cells in LG or HG were treated with increasing doses of echinomycin and XPA protein was assessed by Western blot. (E) 293T HG cells were transfected with CEdG-modified pM1-luc (366 CEdG/10⁵ dG) and treated with CoCl₂ 6 h before measuring luminescence (paired t test). (F) WT cells in HG and XPC cells in LG were treated with CoCl₂ for 24 h before measurement of CEdG in genomic DNA by LC–MS/MS (one-way ANOVA with Tukey’s multiple comparisons, technical triplicate).