Kinetics and dose-response of residual 53BP1/gamma-H2AX foci: co-localization, relationship with DSB repair and clonogenic survival.
OBJECTIVE
Recent studies revealed that some foci produced by phosphorylated histone 2A family member X (gamma-H2AX) and tumor suppressor p53 binding protein 1 (53BP1) that co-localize with radiation-induced DNA double-strand breaks (DSB) remain in cells at relatively long times after irradiation and indicated a possible correlation between cellular radiosensitivity and residual foci. In this study, we investigated dose-responses and kinetics for radiation-induced 53BP1/gamma-H2AX foci formation in relation to their co-localization, DSB repair and cell survival.


METHODS
Cell survival, DSB and foci were analyzed by clonogenic assay, pulsed field gel electrophoresis (PFGE), and confocal laser microscopy, respectively, in normal human fibroblasts (VH-10) and in a cancer cell line (HeLa). Computer analysis was used to determine both the number and the area of foci.


RESULTS
We show that even at doses down to 1 cGy a statistically significant induction of 53BP1 foci is observed. While the number of foci was found to constantly decrease with post-irradiation time, the per-cell normalized area of foci does not change within a time window of approximately 4 h post-irradiation. Co-localization of gamma-H2AX and 53BP1 foci is shown to depend on dose and post-irradiation time. No clear correlations were established between radiosensitivity and foci formation because the dose response for 53BP1/gamma-H2AX foci may depend on time after irradiation and duration of the cell cycle. We show that the kinetics of foci disappearance within 24 h post-irradiation do not coincide with those of DSB repair.


CONCLUSIONS
The data suggest that the post-irradiation time used for estimation of radiosensitivity at therapeutically relevant low doses (e.g., <3 Gy) in proliferating cells by scoring residual foci should be limited by the duration of the cell cycle, and that direct comparison of the kinetics of DSB repair and disappearance of DSB-co-localizing foci is not possible. Therefore, results obtained from the counting of foci should be interpreted with caution in terms of DSB repair.
Marková
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E
Department of Genetics, Microbiology and Toxicology, Stockholm University, Stockholm, Sweden.
Schultz
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N
Belyaev
I Y
IY
D002470:Cell Survival / Q000528:radiation effects; D002478:Cells, Cultured; D019457:Chromosome Breakage; D003114:Colony-Forming Units Assay; D004247:DNA / Q000378:metabolism* / Q000528:radiation effects*; D004260:DNA Repair / Q000528:radiation effects; D004307:Dose-Response Relationship, Radiation; D016521:Electrophoresis, Gel, Pulsed-Field; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism / Q000528:radiation effects; D005720:Gamma Rays; D006367:HeLa Cells; D006657:Histones / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D007700:Kinetics; D018613:Microscopy, Confocal; D013997:Time Factors; D000071857:Tumor Suppressor p53-Binding Protein 1
C468783:H2AX protein, human; D006657:Histones; D047908:Intracellular Signaling Peptides and Proteins; C498301:TP53BP1 protein, human; D000071857:Tumor Suppressor p53-Binding Protein 1; D004247:DNA