Related Experiment Video
Updated: Aug 11, 2026
One-step Protocol for Evaluation of the Mode of Radiation-induced Clonogenic Cell Death by Fluorescence Microscopy
Published on: October 23, 2017
Persistent decrease in viability as a function of X irradiation of human bladder carcinoma cells in G1 or S phase
E A Leonhardt1, M Trinh, H B Forrester
1Radiation Oncology Research Laboratory, University of California San Francisco, 94103, USA.
Abstract:
A persistent decrease in viability after treatment with a variety of mutagenic agents has been observed previously, but the dependence of the decrease on the phase of the cell cycle in which the cells are treated has not been fully explored. Synchronous human bladder carcinoma cells (EJ30-15) were obtained by mitotic selection (88-96% in or near mitosis). As monitored by microscopy and pulse labeling with [3H]dThd, approximately 98% of the cells were in G1 phase when they were irradiated after 3 h of incubation, and approximately 80% were in S phase when they were irradiated after 14 h of incubation. The initial plating efficiencies demonstrated no difference in cell survival when cells were irradiated in G1 or S phase, with normalized clonogenic survival and standard error of 60+/-6% for 3 Gy and 13+/-2% for 6 Gy. However, when the cell populations were allowed to incubate and were replated 5 to 33 days later (5.5 to 36 doublings), a difference between the populations irradiated in G1 and S phase became clear. Cells that were irradiated with 6 Gy regained and maintained the high plating efficiencies (67.9+/-3.6%) of the unirradiated populations much sooner when they were irradiated in S phase compared with irradiation in G1 phase, i.e. 11 days (12 cell doublings) for S phase compared to approximately 20 days (22 cell doublings) for G1 phase. During these periods when the plating efficiencies were increasing, the populations irradiated in G1 phase were multiplying at rates lower than those for the populations irradiated in S phase. Furthermore, after 6 Gy, more giant cells and multinucleated cells were seen in the populations irradiated in G1 phase than in the populations irradiated in S phase. These results indicate that, although the clonogenic survival was the same for cells irradiated in G1 or S phase, the residual damage in progeny of the irradiated cells persisted longer (approximately 20 days compared to 11 days) when cells were irradiated in G1 phase than when they were irradiated in S phase.
Insights
Radiation damage in human bladder cancer cells persists longer when cells are irradiated in the G1 phase compared to the S phase, affecting long-term cell viability and recovery rates.
Area of Science:
- Cell Biology
- Radiation Oncology
- Cancer Research
Background:
- Cell cycle phase influences cellular response to DNA damage.
- Previous studies noted decreased viability after mutagenic treatment, but cell cycle phase dependence was underexplored.
Purpose of the Study:
- To investigate the impact of cell cycle phase (G1 vs. S) on human bladder carcinoma cell survival and recovery after radiation exposure.
- To determine if initial cell survival differences correlate with long-term progeny viability.
Main Methods:
- Synchronized human bladder carcinoma cells (EJ30-15) were irradiated in G1 or S phase.
- Cell survival was assessed via initial plating efficiency and clonogenic survival assays.
- Long-term recovery and viability were monitored over 33 days (up to 36 cell doublings).
Main Results:
- Initial clonogenic survival was similar for cells irradiated in G1 or S phase.
- Cells irradiated in S phase recovered plating efficiency faster (11 days) than those irradiated in G1 (20 days).
- G1-irradiated cells exhibited slower multiplication rates and increased multinucleated/giant cells post-irradiation.
Conclusions:
- While initial survival is unaffected, radiation-induced damage persists longer in progeny of G1-irradiated cells.
- Cell cycle phase is a critical determinant of long-term radiation damage persistence and cellular recovery.
- Targeting cell cycle for radiation therapy may optimize treatment outcomes by minimizing residual damage effects.
