https://orcid.org/0000-0002-5092-3052
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Abstract
Purpose
Although breast cancer (BC) patients benefit from radiotherapy (RT), some radiosensitive (RS) patients suffer from side effects caused by ionizing radiation in healthy tissues. It is thought that RS is underlaid by a deficiency in the repair of DNA double-strand breaks (DSB). DNA repair proteins such as p53-binding protein 1 (53BP1) and phosphorylated histone H2AX (γH2AX), form DNA repair foci at the DSB locations and thus serve as DSB biomarkers. Peripheral blood lymphocytes (PBL) are commonly believed to be an appropriate cell system for RS assessment using DNA repair foci. The amount of DSB may also be influenced by chemotherapy (CHT), which is often chosen as the first treatment modality before RT. As it is not always possible to analyze blood samples immediately after collection, there is a need for cryopreservation of PBL in liquid nitrogen. However, cryopreservation may potentially affect the number of DNA repair foci. In this work, we studied the effect of cryopreservation and CHT on the amount of DNA repair foci in PBL of BC patients undergoing radiotherapy.
Materials and methods
The effect of cryopreservation was studied by immunofluorescence analysis of 53BP1 and γH2AX proteins at different time intervals after in vitro irradiation. The effect of chemotherapy was analyzed by fluorescent labelling of 53BP1 and γH2AX proteins in PBL collected before, during, and after RT.
Results
Higher number of primary 53BP1/γH2AX foci was observed in frozen cells indicating that cryopreservation affects the formation of DNA repair foci in PBL of BC patients. In CHT-treated patients, a higher number of foci were found before RT, but no differences were observed during and after the RT.
Conclusions
Cryopreservation is the method of choice for analyzing DNA repair residual foci, but only similarly treated and preserved cells should be used for comparison of primary foci. CHT induces DNA repair foci in PBL of BC patients, but this effect disappears during radiotherapy.
Acknowledgements
The authors are thankful to Prof. Gabriel Kralik, St. Elizabeth Cancer Institute, Bratislava, Slovak Republic, for in vitro irradiation of samples.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Additional information
Funding
Notes on contributors
Katarína Vigašová
Mgr. Katarína Vigašová, PhD student, studies oncology at the Faculty of Medicine of the Comenius University in Bratislava, and previously studied genetics at the Faculty of Natural Sciences of Comenius University in Bratislava. She has skills in DNA repair foci measurements using fluorescent microscopy and in chromosomal aberrations/cytokinesis block micronucleus assay.
Matúš Durdík
Mgr. Matúš Durdík, PhD, scientist, finished PhD in Oncology at the Faculty of Medicine, Comenius University and got a master degree in molecular biology at the Faculty of Natural Sciences of Comenius University in Bratislava. He is experienced in standard and imaging flow cytometry, DNA repair foci assay, apoptosis analysis, fluorescent microscopy. He studied the effect of ionizing and non-ionizing radiation on the HSC and was trained at the Cancer Institute, Faculty of Medical Science, University College of London in sorting human hematopoietic cell populations and subsequent PGF analysis by FISH.
Lukáš Jakl
Mgr. Lukáš Jakl, PhD, scientist, studied genetics at the Faculty of Natural Sciences of Comenius University in Bratislava and oncology at the Faculty of Medicine, Comenius University in Bratislava. He has practical skills in molecular cloning, western and southern blotting, isolation and purification of DNA and proteins, immunochemistry, fluorescent microscopy, methods for assessment of DNA repair focus enumeration, detection of PFG using FISH and basic methods of flow cytometry.
Zuzana Dolinská
MUDr. Zuzana Dolinská, head of Department of Radiation Oncology at National Cancer Institute in Bratislava, medical and radiation oncologist
Margita Pobijaková
MUDr. Margita Pobijaková, PhD., deputy head of Department of Radiation Oncology at National Cancer Institute in Bratislava, medical and radiation oncologist
Marta Fekete
MUDr. Marta Fekete, medical and radiation oncologist at Department of Radiation Oncology, National Cancer Institute in Bratislava
Ingrid Závacká
MUDr. Ingrid Závacká, medical and radiation oncologist at Department of Radiation Oncology, National Cancer Institute in Bratislava
Igor Belyaev
Associate Professor, Ing. Igor Belyaev, DrSc., head research scientist, head of the Radiobiological Department, 8 PhD students trained, project leader or co-leader for projects in USA, Russia, Sweden, Austria, Germany and Slovak Republic. Dr. Igor Belyaev has a broad experience on managing the team work and contributed significantly to the knowledge of DNA repair and the role of DNA damage in apoptosis, mutagenesis, carcinogenesis and cancer treatment. He is the member of several international scientific committees. He possesses methodological skills and long-term experience in stem cells biology, techniques for analysis of DNA damage and chromosomal aberrations, confocal laser and fluorescent microscopy, immunological techniques; image acquisition and analysis.
Eva Marková
RNDr. Eva Marková, CSc., senior scientist, she is supervisor of PhD and diploma students, has long-term experience in molecular biology, radiobiology and stem cell biology, and has advanced practical skills in immunocytochemistry, laser confocal and fluorescent microscopy, methods for assessment of DNA damage including DNA repair focus enumeration. She has been a principal investigator of four VEGA grants and project manager of the European Structural Funds project. She worked at Stockholm University in Sweden and cooperated on the grants supported by the Swedish Radiation Protection Institute and The Swedish Council for Work Life Research.