ACTN4-dependent regulation of double-strand DNA break repair is independent of NF-Kb activity

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Abstract

α-Actinin-4 is an actin-binding protein that is involved in a wide range of cellular processes. Along with actin and other proteins of the actin cytoskeleton, α-actinin-4 was found not only in the cytoplasm, but also in the nucleus of various cells. As a nuclear protein, it is involved in regulation of certain transcription factors. In particular, it can regulate transcriptional activity of NF-kB, which largely determines the resistance of cancer cells to apoptosis and anticancer therapy. During our previous studies, it was found that α-actinin-4 can influence resistance of cancer cells to topoisomerase II inhibitors and determine the efficiency of DNA double-strand break repair. We have demonstrated that α-actinin-4 interferes with the assembly of complexes involved in DNA repair via NHEJ and HRR, which in turn leads to an imbalance between these pathways. In this study, we were answering to the question of how α-actinin-4 is involved in the regulation of the DNA double-strand breaks repair following genotoxic stress. Our results indicate that the effect of α-actinin-4 on repair progression in H1299 non-small cell lung cancer cells does not depend on the transcription factor NF-kB activity. We found that in the nucleus of H1299 cells, α-actinin-4 is localized not only in the nucleoplasm, but also reveals close association with chromatin.

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About the authors

D. V. Kriger

Institute of Cytology, Russian Academy of Sciences

Author for correspondence.
Email: daryamalikova@gmail.com
Russian Federation, St. Petersburg

G. V. Vasileva

Institute of Cytology, Russian Academy of Sciences

Email: daryamalikova@gmail.com
Russian Federation, St. Petersburg

E. V. Lomerta

Institute of Cytology, Russian Academy of Sciences

Email: daryamalikova@gmail.com
Russian Federation, St. Petersburg

D. G. Tentler

Institute of Cytology, Russian Academy of Sciences

Email: daryamalikova@gmail.com
Russian Federation, St. Petersburg

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2. Fig. 1. Comparison of expression levels of NF-kB-dependent genes in the control line H1299 (ACTN4WT) and in two ACTN4 gene knockout lines (ACTN4KO_cl1 and ACTN4KO_cl2). Real-time PCR. * - Differences with the control line are reliable at p < 0.05 (Mann-Whitney U-test); n/r - not reliable

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3. Fig. 2. Expression levels of DNA damage response genes. Real-time PCR: a - comparison of gene expression levels in control H1299 cells (ACTN4WT) and in H1299 cell lines knockout for the ACTN4 gene (ACTN4KO_cl1 and ACTN4KO_cl2) before (control) and after etoposide treatment (Eto); b - comparison of gene expression levels in a control H1299 cell line (ACTN4WT) and an H1299 cell line overexpressing the ACTN4 gene (ACTN4OE). * - differences are reliable at p < 0.05 (Mann-Whitney U-test)

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4. Fig. 3. Relative resistance of H1299 (curve 1) and H1299 RelAOE (curve 2) cells to doxorubicin (a), etoposide (b) and cisplatin (c). Cytotoxicity (MTT test) was evaluated after treatment of cells for 72 h. For each substance, a dose-effect curve was plotted using a logistic regression model. Vertical bars are errors of the mean value

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5. Fig. 4. Evaluation of DNA break repair efficiency in H1299 and H1299 RelAOE cell lines: a - average number (and its error) of histone γH2AX focuses in the nucleus. Cells were treated with etoposide (Eto) or doxorubicin (Doxo) for 40 min, after which the drug was removed and cultured for another 4 h; b - results of DNA Comet test under alkaline conditions. Cells were treated with etoposide as described previously, followed by culturing in the absence of the drug for 8 h. Control - intact cells

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6. Fig. 5. Distribution of ACTN4 in H1299 cells: a - electrophoresis in SDS-gel of proteins obtained by subcellular fractionation from H1299 cells. Protein detection by Coomassie staining; b - Western blot with antibodies to ACTN4. Histone H1 was used as a protein loading control. M - protein marker; Cyt - cytoplasm; NucPl - nucleoplasm; Chrom - chromatin

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