Comparative analysis of basal and etoposide-induced alterations in gene expression by DNA-PKcs kinase activity

Abstract

Background: Maintenance of the genome is essential for cell survival, and impairment of the DNA damage response is associated with multiple pathologies including cancer and neurological abnormalities. DNA-PKcs is a DNA repair protein and a core component of the classical nonhomologous end-joining pathway, but it also has roles in modulating gene expression and thus, the overall cellular response to DNA damage. Methods: Using cells producing either wild-type (WT) or kinase-inactive (KR) DNA-PKcs, we assessed global alterations in gene expression in the absence or presence of DNA damage. We evaluated differential gene expression in untreated cells and observed differences in genes associated with cellular adhesion, cell cycle regulation, and inflammation-related pathways. Following exposure to etoposide, we compared how KR versus WT cells responded transcriptionally to DNA damage. Results: Downregulated genes were mostly involved in protein, sugar, and nucleic acid biosynthesis pathways in both genotypes, but enriched biological pathways were divergent, again with KR cells manifesting a more robust inflammatory response compared to WT cells. To determine what major transcriptional regulators are controlling the differences in gene expression noted, we used pathway analysis and found that many master regulators of histone modifications, proinflammatory pathways, cell cycle regulation, Wnt/β-catenin signaling, and cellular development and differentiation were impacted by DNA-PKcs status. Finally, we have used qPCR to validate selected genes among the differentially regulated pathways to validate RNA sequence data. Conclusion: Overall, our results indicate that DNA-PKcs, in a kinase-dependent fashion, decreases proinflammatory signaling following genotoxic insult. As multiple DNA-PK kinase inhibitors are in clinical trials as cancer therapeutics utilized in combination with DNA damaging agents, understanding the transcriptional response when DNA-PKcs cannot phosphorylate downstream targets will inform the overall patient response to combined treatment.

Keywords: DNA damage repair; DNA-PKcs; etoposide; gene expression; non-homologous end-joining; transcriptome.

FIGURE 1

DNA-PKcs protects cells from etoposide toxicity via its kinase domain. (A) Evaluation of DNA-PKcs proteins in CHO variants by Western blot. (B, C) Cell survival profiles of CHO cells assessed using the CellTiter-Glo (CTG) assay with increasing doses (0.003–1 μM) of etoposide at 72 h post-exposure with the associated area under the curve analysis. (D, E) Clonogenic survival of DNA-PKcs WT or KR cells following exposure to etoposide (0.003–1 μM) with the associated area under the curve analysis, ****p < 0.0001.

FIGURE 2

Comparison of shared versus discreet differentially regulated genes in DNA-PK wild-type (WT) versus kinase-inactive (KR) cells. (A) Shared or exclusively upregulated genes in KR and WT cells after etoposide exposure. (B) Shared or exclusively downregulated genes in KR and WT cells after etoposide exposure.

FIGURE 3

Diverse biological pathways regulated by the DEGs from the mRNA seq data found from KEGG pathway analysis. Upregulated genes are represented with red (highest positive fold change) and blue (lowest positive fold change) dots, and downregulated genes are represented with blue (lowest negative fold change) and green (highest negative fold change) dots. Corresponding pathways regulated by DEGs are represented with a solid tangerine circle. The size of the correlates with the number of genes. (A, B) Upregulated and downregulated, respectively, genes in KR cells compared to WT; (C, D) upregulated and downregulated, respectively, genes in WT cells after etoposide exposure; (E, F) upregulated and downregulated, respectively, genes in KR cells after etoposide exposure.

FIGURE 4

Comparison of shared versus divergent etoposide-induced biological pathways altered by differential gene regulation in DNA-PKcs wild-type (WT) compared to kinase-inactive (KR) cells. Etoposide-induced upregulated biological pathways in WT and KR cells. Etoposide-induced downregulated biological pathways in WT and KR cells.

FIGURE 5

Comparative assessment of etoposide-induced biological pathways altered by differential gene regulation in DNA-PKcs wild-type (WT) and kinase-inactive (KR) cells. (A) Upregulated biological pathways in etoposide-treated KR cells compared to WT genotype-specific upregulated biological processes. (B) Downregulated biological pathways in etoposide-treated KR cells compared to WT.

FIGURE 6

Diverse biological pathways regulated by the DEGs from the mRNA seq data found from KEGG pathway analysis in etoposide-treated WT and KR cells. Upregulated and downregulated genes are represented with blue and red dots and blue and green dots, respectively. Pathways are shown as a solid tangerine circle. (A, B) Upregulated and downregulated, respectively, genes in etoposide-treated KR cells compared to WT.

FIGURE 7

Relative expressions of selected genes detected by qPCR and immunoblotting showed a similar trend with mRNA sequencing data. (A–Q) Seventeen DEGs were selected to perform qPCR to detect their relative expression in WT and KR cells with or without etoposide treatment. The mean relative expression of a DEG was analyzed by Cq values of the target and control genes using the 2^−ΔΔCq method after normalizing with the Cq value of GAPDH for any specific sample. (R, S) Two DEGs were selected for immunoblotting to detect their protein expression in WT and KR cells with or without etoposide treatment. Two-way ANOVA with Fisher’s LSD test was used to detect the significant differences among the mean relative expression levels. ns: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.