Chaetocin induced chromatin condensation: effect on DNA repair signaling and survival

Abstract

Purpose

The aim of the present study was to evaluate the effect of the histone lysine-methyltransferase (HKMT) inhibitor chaetocin on chromatin structure and its effect on ionizing radiation (IR) induced DNA damage response.

Methods

Concentration and time-dependent effects of chaetocin on chromatin clustering and its reversibility were analyzed by immunofluorescent assays in the non-small cell lung carcinoma (NSCLC) cell lines H460 and H1299Q4 and in human skin fibroblasts. In addition, IR induced damage response (γH2AX, 53BP1, and pATM foci formation) was studied by immunofluorescent assays. The effect on survival was determined by performing single-cell clonogenic assays.

Results

Chaetocin significantly increased the radiation sensitivity of H460 (F test on nonlinear regression, p < .0011) and of H1299 (p = .0201). In addition, treatment with 15 nM chaetocin also decreased the total radiation doses that control 50% of the plaque monolayers (TCD50) from 17.2 ± 0.3 Gy to 7.3 ± 0.4 Gy (p < .0001) in H1299 cells and from 11.6 ± 0.1 Gy to 6.5 ± 0.3 Gy (p < .0001). Phenotypically, chaetocin led to a time and concentration-dependent clustering of the chromatin in H1299 as well as in fibroblasts, but not in H460 cells. This phenotype of chaetocin induced chromatin clustering (CICC) was reversible and depended on the expression of the HKMTs SUV39H1 and G9a. Treatment with siRNA for SUV39h1 and G9a significantly reduced the CICC phenotype. Immunofluorescent assay results showed that the CICC phenotype was enriched for the heterochromatic marker proteins H3K9me3 and HP1α. γH2AX foci formation was not affected, neither in cells with normal nor with CICC phenotype. In contrast, repair signaling with 53BP1 and pATM foci formation was significantly reduced in the CICC phenotype.

Conclusions

Treatment with chaetocin increased the radiation sensitivity of cells in vitro and DNA damage response, especially of 53BP1 and ATM-dependent repair by affecting chromatin structure. The obtained results support the potential use of natural HKMT inhibitors such as chaetocin or other bioactive compounds in improving radiosensitivity of cancer cells.

Keywords:

• Radiation sensitivity
• chaetocin
• histone methyl transferases
• DNA repair
• chromatin structure
• ATM

Acknowledgements

We thank George Iliakis for valuable discussions, reagents and technical advice. We thank Sabine Levegrün for language editing and proofreading. This work is funded by the Deutsche Forschungsgemeinschaft (DFG) as part of the Graduate School (GRK1739) and is part of a PhD thesis of Kristina Bannik.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Notes on contributors

A. Sak

Ali Sak, PhD, is a biologist and head of the working group ‘molecular radiation oncology’ at the Department of Radiotherapy, Faculty of Medicine, University of Duisburg Essen, Essen, Germany. He works on biological effects of radiation, in vivo and in vitro effects of combined radio-chemotherapy, modulation of epigenetic factors and their effect on the radiation response of tumor cell lines.

K. Bannik

Kristina Bannik, PhD, was a biophysicist at the Department of Radiotherapy, Faculty of Medicine, University of Duisburg Essen, Essen, Germany. The presented work was part of her PhD thesis prepared in the working group ‘molecular radiation oncology’. She was responsible for the planning and processing of the experiments and for data analysis.

M. Groneberg

Michael Groneberg is a research assistant and an expert in cell culture and microscopic analysis at the Department of Radiotherapy, Faculty of Medicine, University of Duisburg Essen, Essen, Germany. He is involved in the planning and processing of the experimental settings.

M. Stuschke

Prof. Dr. med. Martin Stuschke is the Chair of the Department of Radiation Therapy, Faculty of Medicine, University of Duisburg Essen, Essen, Germany. His research focusses on clinical studies with combined therapy schedules involving chemotherapy and radiotherapy and preclinical studies on the role of DNA repair pathways and checkpoint activation in the response of NSCLC to IR.