Assessing radiosensitivity through sublethal damage recovery: a comparison of survival-based and molecular repair kinetics

<i>Objective</i>. This study aimed to determine whether the kinetics of sublethal damage recovery after x-ray irradiation, quantified as the repair half time (TrepairSLD) derived from split-dose clonogenic survival, correlates with intrinsic radiosensitivity across four human cancer cell lines: HeLa (cervical), PC3 (prostate), and HCT116 and HT29 (colorectal). In addition, the study compared this survival-based indicator with molecular repair kinetics assessed through<i>γ</i>H2AX and 53BP1 foci clearance.<i>Approach</i>. By using a phenomenological approach, we assessed sublethal damage recovery kinetics, aiming to determine whether this recovery rate could serve as a biomarker for cancer-specific intrinsic radiosensitivity. Cells were subjected to split-dose 4 Gy irradiation delivered in two fractions of 2 Gy across a 0 to 10 h inter-fraction interval range using a Multi-Rad x-ray irradiator with a peak tube voltage of 225 kV. The clonogenic assay was performed following split-dose irradiation of the experimental groups to assess cell survival. Colonies were fixed, stained, and counted (⩾50 cells/colony viable threshold) to calculate survival fractions (SFs) from the four independent experimental runs completed for each cell line. Unirradiated control cells were used to calculate plating efficiency. The measured SF as a function of inter-fraction time was fitted with the Lea-Catcheside modified linear-quadratic model with a half-life of sublethal damage repair,TrepairSLD, as a free parameter. To compare this approach to molecular DNA repair kinetics, immunofluorescence-based ionizing radiation-induced foci (IRIF) clearance experiments were performed following single 2 Gy irradiation using the same x-ray source.<i>γ</i>H2AX and 53BP1 foci were quantified from 0.5 to 24 h post-irradiation, and foci clearance half-lives (TrepairγH2AXandTrepair53BP1) were determined by single-phase exponential decay fitting.<i>Main results</i>. For all measured cell lines, an increase in SF was observed with increasing inter-fraction time. The estimatedTrepairSLDvaried across cell lines, from1.07±0.35 h in HT29, to1.98±0.94 h in HeLa,2.00±0.30 h in PC3, and3.58±1.45 h in HCT116, indicating different capacities for sublethal damage repair. A negative correlation was measured betweenTrepairSLDand clonogenic survival at 2 Gy (SF2Gy) by performing orthogonal distance regression, with a slope of-350±50 min (<i>p</i> = 0.02).TrepairγH2AXandTrepair53BP1ranged from 3 to 11 h, with HT29 showing the fastest foci resolution. However, these molecular repair kinetics times did not significantly correlate withSF2Gy(<i>p</i> > 0.05) or follow the same trend asTrepairSLDacross cell lines. For example, PC3 cells exhibited the slowest foci clearance, whereas HCT116 displayed the slowestTrepairSLD, suggesting that IRIF-based measurements do not reliably reflect functional sublethal damage repair.<i>Significance</i>. Clonogenic survival assays capture the integrated biological outcome of radiation exposure, reflecting not only DNA damage recognition and repair but also downstream processes such as checkpoint activation, chromatin context, and long-term proliferative capacity. In contrast, molecular readouts like<i>γ</i>H2AX and 53BP1 foci clearance, though rapid and widely used, may not fully account for defects in damage response pathways. The observed discrepancy between foci clearance kinetics and survival-based repair rates in this study highlights the limitations of foci quantification as a surrogate for radiosensitivity. These findings underscore the value of survival-based sublethal damage recovery measurements as functionally rich indicators of intrinsic radiosensitivity, which may inform future biomarker development or predictive modeling frameworks.