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Decay of γ-H2AX foci correlates with potentially lethal damage repair and P53 status in human colorectal carcinoma cells

Cellular & Molecular Biology Letters volume 19, pages 37–51 (2014)Cite this article

Abstract

The influence of p53 status on potentially lethal damage repair (PLDR) and DNA double-strand break (DSB) repair was studied in two isogenic human colorectal carcinoma cell lines: RKO (p53 wild-type) and RC10.1 (p53 null). They were treated with different doses of ionizing radiation, and survival and the induction of DNA-DSB were studied. PLDR was determined by using clonogenic assays and then comparing the survival of cells plated immediately with the survival of cells plated 24 h after irradiation. Doses varied from 0 to 8 Gy. Survival curves were analyzed using the linear-quadratic formula: S(D)/S(0) = exp-(αD+βD2). The γ-H2AX foci assay was used to study DNA DSB kinetics. Cells were irradiated with single doses of 0, 0.5, 1 and 2 Gy. Foci levels were studied in non-irradiated control cells and 30 min and 24 h after irradiation. Irradiation was performed with gamma rays from a 137Cs source, with a dose rate of 0.5 Gy/min. The RKO cells show higher survival rates after delayed plating than after immediate plating, while no such difference was found for the RC10.1 cells. Functional p53 seems to be a relevant characteristic regarding PLDR for cell survival. Decay of γ-H2AX foci after exposure to ionizing radiation is associated with DSB repair. More residual foci are observed in RC10.1 than in RKO, indicating that decay of γ-H2AX foci correlates with p53 functionality and PLDR in RKO cells.

Abbreviations

BrdUrd:

bromodeoxyuridine

Cs:

cesium

DAPI:

4′,6-diamidino-2-phenylindole

dp:

delayed plating

DSB:

double-strand breaks

FACS:

fluorescent activated cell sorter

FCS:

fetal bovine calfserum

HEPES:

2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid

HPV:

human papilloma virus

ip:

immediate plating

PBS:

phosphate buffered saline

PLD:

potentially lethal damage

PLDR:

potentially lethal damage repair

PVDF:

polyvinylidene fluoride

RIPA:

radio immunoprecipitation assay

SDS PAGE:

sodium dodecyl sulfate polyacrylamide gel electrophoresis

TNBS:

PBS containing 0.1% Triton X-100 and 1% FCS

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Authors and Affiliations

  1. Laboratory for Experimental Oncology and Radiobiology (LEXOR), Center for Experimental Molecular Medicine, Department of Radiation Oncology, University of Amsterdam, P.O. Box 22700, 1100 DE, Amsterdam, The Netherlands

    Bregje Van Oorschot, Arlene L. Oei, Anna C. Nuijens, Hans Rodermond, Lukas J. Stalpers & Nicolaas A. P. Franken

  2. van Leeuwenhoek Center for Advanced Microscopy AMC, Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, P.O. Box 22700, 1100 DE, Amsterdam, The Netherlands

    Ron Hoeben & Jan Stap

Authors
  1. Bregje Van Oorschot
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  2. Arlene L. Oei
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  3. Anna C. Nuijens
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  4. Hans Rodermond
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  5. Ron Hoeben
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  6. Jan Stap
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  7. Lukas J. Stalpers
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  8. Nicolaas A. P. Franken
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Correspondence to Nicolaas A. P. Franken.

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Van Oorschot, B., Oei, A.L., Nuijens, A.C. et al. Decay of γ-H2AX foci correlates with potentially lethal damage repair and P53 status in human colorectal carcinoma cells. Cell Mol Biol Lett 19, 37–51 (2014). https://doi.org/10.2478/s11658-013-0113-0

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Cellular & Molecular Biology Letters

ISSN: 1689-1392

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