Prediction of clonogenic cell survival curves based on the number of residual DNA double strand breaks measured by γH2AX staining

Abstract

Purpose: To assess the potential of using the residual phosphorylation of histone H2AX (γH2AX) after irradiation as a marker of radiosensitivity in vitro.

Material and methods: Confluent cell cultures of FaDu and SKX human squamous cell carcinoma lines were irradiated with graded single doses. Twenty-four hours after irradiation cells were seeded for standard colony forming assay (CFA). In parallel, staining for γH2AX was performed to visualise the residual foci.

Results: In the CFA, FaDu showed a higher radioresistance than SKX. After analysis of the residual foci data, we constructed ‘predicted’ survival curves using two different methods. First, the proportion of nuclei with <3 foci was found to correlate closely with the observed surviving fraction (SF) in FaDu, with a slight overestimation of the true SF in SKX. Second, there was a strong linear correlation of the mean number of residual foci and observed −lnSF. Based on regression analysis, we calculated the SF for both cell lines based on the mean number of residual γH2AX foci. This second approach again led to a good correlation of predicted and observed SF values in FaDu and a (slight) overestimation in SKX.

Conclusion: In the two cell lines investigated the mean number of residual foci of γH2AX can be used to predict differences in the radiation dose response relationship in vitro.

Keywords:

• clonogenic cell survival
• irradiation
• γH2AX
• DNA repair
• double strand breaks
• prediction

Acknowledgement

The authors wish to thank Ms Sigrid Balschukat and Ms Dorothee Pfitzmann for excellent technical assistance. This work was supported in part by the German Federal Ministry of Education and Research (BMBF 03ZIK/OncoRay), the Deutsche Forschungsgemeinschaft (Ba1433) and the 6th framework EU-project ‘BioCare’, proposal #505785. This publication does not necessarily reflect the views of the EC. The community is not liable for any use that may be made of the information contained herein. AM holds a scholarship of the State Scholarships Foundation of Greece (IKY).

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.