An invitro assay to measure cell sensitivity to ionizing radiation
Sherin Mathew1,*, Pegah Johansson1, Ola Hammarsten1
1Department of Clinical Chemistry and Transfusion Medicine, Gothenburg University, Gothenburg, Sweden
Introduction: Mostly cancer patients receive treatment with DNA damaging agents. Cellular response to DNA damaging agents varies among individuals and therefore presents a major clinical challenge. The tolerance level of normal tissue is derived empirically from population averages and this may result in some patients receiving suboptimal treatment dose while the sensitive patients are relatively overdosed. The Clonogenic survival assay which measures cell sensitivity based on clonogenic cell division is considered as gold standard, but the laborious and slow procedure makes it almost impractical to use in clinical settings.
Objectives: To measure individual sensitivity to radiation, we have optimized a flow cytometry based method that measure incorporation of the thymidine analogue, EdU as a measure of cell proliferation.
Methods: In the optimized experimental setting, cells were allowed to grow more than one cell division after exposure to DNA damaging agents and labeled with EdU for 16 h followed by fixation and staining. Using a flow cytometer, cells that incorporated EdU or proliferating cells are detected and separated from non-proliferating cells.
Results: The radiation sensitivity of primary human skin fibroblasts measured by this method correlated to the sensitivity measured with the colony forming assay. In peripheral blood lymphocytes, we were able to detect DNA-damage induced proliferation arrest and sensitization to ionizing radiation following DNA-PK and ATM inhibition.
Conclusion: Our results indicate that the EdU-based flow cytometry measurement of cell proliferation is a sensitive and reproducible method in predicting cellular sensitivity to DNA damage. This method can therefore be adapted for the clinic to detect the sensitive cancer patients prior to treatment with DNA-damaging agents.
Keywords: Cancer, DNA, Method