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. 2023 Jan 5;14(1):144.
doi: 10.3390/genes14010144.

High Glucose Increases DNA Damage and Elevates the Expression of Multiple DDR Genes

Mai A Rahmoon  1   2 , Reem A Elghaish  1   3 , Aya A Ibrahim  1   3 , Zina Alaswad  1   3 , Mohamed Z Gad  4 , Sherif F El-Khamisy  1   5   6 , Menattallah Elserafy  1   3
Affiliations

High Glucose Increases DNA Damage and Elevates the Expression of Multiple DDR Genes

Mai A Rahmoon et al. Genes (Basel). .

Abstract

The DNA Damage Response (DDR) pathways sense DNA damage and coordinate robust DNA repair and bypass mechanisms. A series of repair proteins are recruited depending on the type of breaks and lesions to ensure overall survival. An increase in glucose levels was shown to induce genome instability, yet the links between DDR and glucose are still not well investigated. In this study, we aimed to identify dysregulation in the transcriptome of normal and cancerous breast cell lines upon changing glucose levels. We first performed bioinformatics analysis using a microarray dataset containing the triple-negative breast cancer (TNBC) MDA-MB-231 and the normal human mammary epithelium MCF10A cell lines grown in high glucose (HG) or in the presence of the glycolysis inhibitor 2-deoxyglucose (2DG). Interestingly, multiple DDR genes were significantly upregulated in both cell lines grown in HG. In the wet lab, we remarkably found that HG results in severe DNA damage to TNBC cells as observed using the comet assay. In addition, several DDR genes were confirmed to be upregulated using qPCR analysis in the same cell line. Our results propose a strong need for DDR pathways in the presence of HG to oppose the severe DNA damage induced in cells.

Keywords: DNA damage; DNA damage response (DDR); cancer; diabetes mellitus; hyperglycemia (HG); metabolic diseases.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Schematic representation of the workflow for this project “Created with BiRender.com”.
Figure 2
Figure 2
Hierarchical clustering of the samples analyzed. HG, high glucose; LG, 50 mM 2-DG added to inhibit glucose uptake.
Figure 3
Figure 3
WGCNA analysis to identify the key modules associated with different glucose levels. (a) The module–trait relationships were demonstrated using correlation values and p-values with a range of colors; the degree of correlation between modules and glucose levels is shown. HG, high glucose; LG, 50 mM 2-DG added to inhibit glucose uptake. (b) Median rank and Z-summary statistics in the module preservation tests. Left plot shows the module position in the test dataset based on the median rank. Right plot illustrates the analysis of the Z-summary between different modules.
Figure 4
Figure 4
Pathway enrichment analysis for the brown module. The ReactomePA package [38] was used to identify the pathways enriched in the brown modules. The pathways represented are statistically significant (adjusted p-value < 0.05) as indicated by the color of the nodes (right panel). The number of genes retrieved in our analysis and identified in the pathways are represented using the size of the circles as indicated in the right panel.
Figure 5
Figure 5
Pathway enrichment analysis for the green module. The Reactome tool [38] was used to identify the pathways enriched in the green modules. The pathways represented are statistically significant (adjusted p-value < 0.05) as indicated by the color of the nodes (right panel). The number of genes retrieved in our analysis and identified in the pathways is represented using the size of the circles as indicated in the right panel.
Figure 6
Figure 6
Alkaline comet assay representing significant DNA damage in HG- compared to LG-cultured cells. MDA-MB-231 cells were cultured in high glucose (25 mM glucose, HG) or low glucose (5 mM glucose, LG). Each bar represents the mean ± SEM of three repeats. Results were analyzed using an unpaired Student’s t-test where each error bar represents SEM. ** = p < 0.01.
Figure 7
Figure 7
Normalized expression of the differentially expressed DDR genes in MDA-MB-231 analyzed using the microarray dataset. The genes analyzed are represented in (a–i). Boxplots represent normalized counts in LG (grey) and HG (red). HG, high glucose; LG, 50 mM 2-DG added to inhibit glucose uptake.
Figure 8
Figure 8
Heatmap of DNA repair genes identified in high- and low-glucose levels in MDA-MB-231 cells. Expression of the genes was normalized and log2 transformed. Both rows and columns are clustered using correlation distance and average linkage. The color intensity reflects the expression levels where positive values indicate upregulation and negative values indicate downregulation. HG, high glucose; LG, 50 mM 2-DG added to inhibit glucose uptake.
Figure 9
Figure 9
qPCR analysis of DDR genes in HG- vs. LG-treated MDA-MB-231 cells. MDA-MB-231 cells cultured in LG conditions (5.55 mM glucose) resulted in a significant downregulation of genes presented in (a–i) in comparison to HG (25 mM glucose)-cultured cells. GAPDH was used as a housekeeping gene for the RQ calculations. Data from three or four independent biological replicates of each treatment are presented. Each bar represents the mean ± SEM. Results were analyzed using a paired Student’s t-test where *** = p < 0.001, ** = p < 0.01, * = p < 0.05.
Figure 10
Figure 10
A concluding model showing the HG impact on DNA damage. In the presence of high glucose, the DNA accumulates high damage which requires an increase in the recruitment of DNA damage response genes. This is in contrast to LG conditions that showed a decrease in DNA damage “Created with BioRender.com”.
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References

    1. Molinaro C., Martoriati A., Cailliau K. Proteins from the DNA Damage Response: Regulation, Dysfunction, and Anticancer Strategies. Cancers. 2021;13:3819. doi: 10.3390/cancers13153819. - DOI - PMC - PubMed
    1. Ingram S.P., Warmenhoven J.-W., Henthorn N.T., Chadiwck A.L., Santina E.E., McMahon S.J., Schuemann J., Kirkby N.F., Mackay R.I., Kirkby K.J., et al. A computational approach to quantifying miscounting of radiation-induced double-strand break immunofluorescent foci. Commun. Biol. 2022;5:700. doi: 10.1038/s42003-022-03585-5. - DOI - PMC - PubMed
    1. Polo S.E., Jackson S.P. Dynamics of DNA damage response proteins at DNA breaks: A focus on protein modifications. Genes Dev. 2011;25:409–433. doi: 10.1101/gad.2021311. - DOI - PMC - PubMed
    1. Pascal J.M. The comings and goings of PARP-1 in response to DNA damage. DNA Repair. 2018;71:177–182. doi: 10.1016/j.dnarep.2018.08.022. - DOI - PMC - PubMed
    1. Chatterjee N., Walker G.C. Mechanisms of DNA damage, repair, and mutagenesis. Environ. Mol. Mutagen. 2017;58:235–263. doi: 10.1002/em.22087. - DOI - PMC - PubMed

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This research was funded and supported by the Science and Technology Development Fund (STDF 33376 and also a contribution from STDF 12694) in addition to Zewail City for Science and Technology.

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