Skip to main content

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

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

References

  1. Kanaar, R, Hoeijmakers, J.H.J. and van Gent, D.C. Molecular mechanisms of DNA double-strand break repair. Trends Cell Biol. 8 (1998) 483–489.

    PubMed  Article  CAS  Google Scholar 

  2. Franken, N.A., ten Cate, R., Krawczyk, P.M., Stap, J., Haveman, J., Aten, J. and Barendsen, G.W. Comparison of RBE values of high-LET α-particles for the induction of DNA-DSBs, chromosome aberrations and cell reproductive death. Radiat. Oncol. 6 (2011) 64.

    PubMed Central  PubMed  Article  Google Scholar 

  3. Bergs, J.W., Krawczyk, P.M., Borovski, T., ten Cate, R., Rodermond, H.M., Stap, J., Medema, J.P., Haveman, J., Essers, J., van Bree, C., Stalpers, L.J., Kanaar, R., Aten, J.A. and Franken NA. Inhibition of homologous recombination by hyperthermia shunts early double strand break repair to non-homologous end-joining. DNA Repair (Amst) 12 (2013) 38–45.

    PubMed  Article  CAS  Google Scholar 

  4. Korlimarla, A., Bhandary, L., Prabhu, J.S., Shankar, H., Sankaranarayanan, H., Kumar, P., Remacle, J., Natarajan, D. and Sridhar, T.S. Identification of a non-canonical nuclear localization signal (NLS) in BRCA1 that could mediate nuclear localization of splice variants lacking the classical NLS. Cell. Mol. Biol. Lett. 18 (2013) 284–296.

    PubMed  Article  CAS  Google Scholar 

  5. Rogakou, E.P., Pilch, D.R., Orr, A.H., Ivanova, V.S. and Bonner, W.M. DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J. Biol. Chem. 273 (1998) 5858–5868.

    PubMed  Article  CAS  Google Scholar 

  6. Aten, J.A., Stap, J., Krawczyk, P.M., van Oven, C.H., Hoebe, R.A., Essers, J. and Kanaar, R. Dynamics of DNA double-strand breaks revealed by clustering of damaged chromosome domains. Science 303 (2004) 92–95.

    PubMed  Article  CAS  Google Scholar 

  7. Rogakou, E.P., Boon, C., Redon, C. and Bonner, W.M. Megabase chromatin domains involved in DNA double-strand breaks in vivo. J. Cell Biol. 146 (1999) 905–915.

    PubMed Central  PubMed  Article  CAS  Google Scholar 

  8. Vandersickel, V., Depuydt, J., Van Bockstaele, B., Perletti, G., Philippe, J., Thierens, H. and Vral, A. Early increase of radiation-induced γH2AX foci in a human Ku70/80 knockdown cell line characterized by an enhanced radiosensitivity. J. Radiat. Res. 51 (2010) 633–641.

    PubMed  Article  Google Scholar 

  9. Van Oorschot, B., Hovingh, S.E., Rodermond, H., Güçlü, A., Losekoot, N., Geldof, A.A., Barendsen, G.W., Stalpers, L.J. and Franken, N.A. Decay of γ-H2AX foci correlates with potentially lethal damage repair in prostate cancer cells. Oncol. Rep. 29 (2013) 2175–2180.

    PubMed  Google Scholar 

  10. Banáth, J.P., Klokov, D., MacPhail, S.H., Banuelos, C.A. and Olive, P.L. Residual gammaH2AX foci as an indication of lethal DNA lesions. BMC Cancer 10 (2010) 4.

    PubMed Central  PubMed  Article  CAS  Google Scholar 

  11. Olive, P.L. and Banáth, J.P. Phosphorylation of histone H2AX as a measure of radiosensitivity. Int. J. Radiat. Oncol. Biol. Phys. 58 (2004) 331–335.

    PubMed  Article  CAS  Google Scholar 

  12. Fujii, Y., Genet, M.D., Roybal, E.J., Kubota, N., Okayasu, R., Miyagawa, K., Fujimori, A. and Kato, T.A. Comparison of the bromodeoxyuridine-mediated sensitization effects between low-LET and high-LET ionizing radiation on DNA double-strand breaks. Oncol. Rep. 29 (2013) 2133–2139.

    PubMed  CAS  Google Scholar 

  13. Hublarova, P., Greplova, K., Holcakova, J., Vojtesek, B. and Hrstka, R. Switching p53-dependent growth arrest to apoptosis via the inhibition of DNA damage-activated kinases. Cell. Mol. Biol. Lett. 15 (2010) 473–484.

    PubMed  Article  CAS  Google Scholar 

  14. Franken, N.A., van Bree, C. and Haveman, J. Differential response to radiation of TP53-inactivated cells by overexpression of dominant-negative mutant TP53 or HPVE6. Radiat. Res. 161 (2004) 504–510.

    PubMed  Article  CAS  Google Scholar 

  15. Kumala, S., Niemiec, P., Wideł, M., Hancock, R. and Rzeszowska-Wolny, J. Apoptosis and clonogenic survival in three tumour cell lines exposed to gamma rays or chemical genotoxic agents. Cell. Mol. Biol. Lett. 8 (2003) 655–665.

    PubMed  Google Scholar 

  16. Hikisz, P. and Kiliańska, Z.M. PUMA, a critical mediator of cell death—one decade on from its discovery. Cell. Mol. Biol. Lett. 17 (2012) 646–669.

    PubMed  Article  CAS  Google Scholar 

  17. Schwartz, J.L., Rasey, J., Wiens, L., Jordan, R. and Russell, K.J. Functional inactivation of p53 by HPV-E6 transformation is associated with a reduced expression of radiation-induced potentially lethal damage. Int. J. Radiat. Biol. 75 (1999) 285–291.

    PubMed  Article  CAS  Google Scholar 

  18. Schwartz, J.L., Jordan, R., Kaufmann, W.K., Rasey, J., Russel, K.J. and Weichselbaum, R.R. Evidence for the expression of radiation-induced potentially lethal damage being a p53-dependent process. Int. J. Radiat. Biol. 76 (2000) 1037–1043.

    PubMed  Article  CAS  Google Scholar 

  19. Franken, N.A., van Bree, C, ten Cate, R, van Oven, C.H. and Haveman J. Importance of TP53 and RB in the repair of potentially lethal damage and induction of color junctions after exposure to ionizing radiation. Radiat. Res. 158 (2002) 707–714.

    PubMed  Article  CAS  Google Scholar 

  20. Iliakis, G., Wang, Y, Pantelias, G.E. and Metzger, L. Mechanism of radiosensitization by halogenated pyrimidines: effect of BrdU on repair of DNA breaks, interphase chromatin breaks, and potentially lethal damage in plateau-phase CHO cells. Radiat. Res. 129 (1992) 202–211.

    PubMed  Article  CAS  Google Scholar 

  21. Franken, N.A., van Bree, C., Kipp, J.B. and Barendsen, G.W. Modification of potentially lethal damage in irradiated Chinese hamster V79 cells after incorporation of halogenated pyrimidines. Int. J. Radiat. Biol. 72 (1997) 101–109.

    PubMed  Article  CAS  Google Scholar 

  22. Franken, N.A., van Bree, C., Veltmaat, M.A., Rodermond, H.M., Haveman, J. and Barendsen, G.W. Radiosensitization by bromodeoxyuridine and hyperthermia: analysis of linear and quadratic parameters of radiation survival curves of two human tumor cell lines. J. Radiat. Res. 42 (2001) 179–190.

    PubMed  Article  CAS  Google Scholar 

  23. Franken, N.A.P., Van Bree, C., Streefkerk, J., Kuper, I., Rodermond, H., Kipp, J.B., Haveman, J. and Barendsen, G.W. Radiosensitization by iododeoxyuridine in cultured SW-1573 human lung tumor cells: Effects on alpha and beta of the linear-quadratic model. Oncol. Rep. 4 (1997) 1073–1076.

    PubMed  CAS  Google Scholar 

  24. Franken, N.A., ten Cate, R., van Bree, C. and Haveman, J. Induction of the early response protein EGR-1 in human tumour cells after ionizing radiation is correlated with a reduction of repair of lethal lesions and an increase of repair of sublethal lesions. Int. J. Oncol. 24 (2004) 1027–1031.

    PubMed  CAS  Google Scholar 

  25. Franken, N.A., Oei, A.L., Kok, H.P., Rodermond, H.M., Sminia, P., Crezee J., Stalpers, L.J. and Barendsen, G.W. Cell survival and radiosensitisation: modulation of the linear and quadratic parameters of the LQ model (Review). Int. J. Oncol. 42 (2013) 1501–1515.

    PubMed  CAS  Google Scholar 

  26. Barendsen, G.W. Parameters of linear-quadratic radiation dose-effect relationships: dependence on LET and mechanisms of reproductive cell death. Int. J. Radiat. Biol. 71 (1997) 649–655.

    PubMed  Article  CAS  Google Scholar 

  27. Barendsen, G.W., van Bree, C. and Franken, N.A. Importance of cell proliferative state and potentially lethal damage repair on radiation effectiveness: implications for combined tumor treatments (review). Int. J. Oncol. 19 (2001) 247–256.

    PubMed  CAS  Google Scholar 

  28. Bergs, J.W., Franken, N.A.P, ten Cate, R., van Bree, C., Haveman, J. Effects of cisplatin and gamma-irradiation on cell survival, the induction of chromosomal aberrations and apoptosis in SW-1573 cells. Mutat. Res. 594 (2006) 148–154.

    PubMed  Article  CAS  Google Scholar 

  29. Franken, N.A. and Barendsen, G.W. In Regard to Ohri N et al. Int. J. Radiat. Oncol. Biol. Phys. 86 (2013) 598.

    PubMed  Article  CAS  Google Scholar 

  30. Danielsen, T., Smith-Sørensen, B., Grønlund, H.A., Hvidsten, M, Børresen-Dale, A.L. and Rofstad, E.K. No association between radiosensitivity and TP53 status, G1 arrest or protein levels of p53, myc, ras or raf in human melanoma lines. Int. J. Radiat. Biol. 75 (1999) 1149–1160.

    PubMed  Article  CAS  Google Scholar 

  31. Van Bree, C., Franken, N.A.P., Rodermond, H.M., Stalpers, L.J. and Haveman, J. Repair of potentially lethal damage does not depend on functional TP53 in human glioblastoma cells. Radiat. Res. 161 (2004) 511–516.

    PubMed  Article  Google Scholar 

  32. Kessis, T.D., Slebos, R.J., Nelson, W.G., Kastan, M.B., Plunkett, B.S., Han, S.M., Lorincz, A.T. Hedrick, L. and Cho, K.R. Human papillomavirus 16 E6 expression disrupts the p53-mediated cellular response to DNA damage. Proc. Natl. Acad. Sci. USA 90 (1993) 3988–3992.

    PubMed Central  PubMed  Article  CAS  Google Scholar 

  33. Franken, N.A., Rodermond, H.M., Stap, J., Haveman, J. and van Bree C. Clonogenic assay of cells in vitro. Nat. Protoc. 1 (2006) 2315–2319.

    PubMed  Article  CAS  Google Scholar 

  34. Franken, N.A.P., Hovingh, S., ten Cate, R., Krawczyk, P., Stap, J., Hoebe, R., Aten, J. and Barendsen, G.W. Relative biological effectiveness of high linear energy transfer α-particles for the induction of DNA-double-strand breaks, chromosome aberrations and reproductive cell death in SW-1573 lung tumour cells. Oncol. Rep. 27 (2012) 769–774.

    PubMed  CAS  Google Scholar 

  35. Van Bree, C., Franken, N.A., Rodermond, H.M., Stalpers, L.J. and Haveman, J. Repair of potentially lethal damage does not depend on functional TP53 in human glioblastoma cells. Radiat. Res. 161 (2004) 511–516.

    PubMed  Article  Google Scholar 

  36. Van Bree, C., Savonije, J.H., Franken, N.A.P., Haveman, J., Bakker, P.J. The effect of p53-function on the sensitivity to paclitaxel with or without hyperthermia in human colorectal carcinoma cells. Int. J. Oncol. 16 (2000) 739–744.

    PubMed  Google Scholar 

  37. Tolde, O. and Folk, P. Stress-induced expression of p53 target genes is insensitive to SNW1/SKIP downregulation. Cell. Mol. Biol. Lett. 16 (2011) 373–384.

    PubMed  Article  CAS  Google Scholar 

  38. Ngok-Ngam, P., Watcharasit, P., Thiantanawat, A., Satayavivad, J. Pharmacological inhibition of GSK3 attenuates DNA damage-induced apoptosis via reduction of p53 mitochondrial translocation and Bax oligomerization in neuroblastoma SH-SY5Y cells. Cell. Mol. Biol. Lett. 18 (2013) 58–74.

    PubMed  Article  CAS  Google Scholar 

  39. MacPhail, S.H., Banáth, J.P., Yu, Y., Chu, E. and Olive, P.L. Cell cycledependent expression of phosphorylated histone H2AX: reduced expression in unirradiated but not X-irradiated G1-phase cells. Radiat. Res. 159 (2003) 759–767.

    PubMed  Article  CAS  Google Scholar 

  40. Taneja, N., Davis, M., Choy, J.S., Becket, M.A., Singh, R., Kron, D.J. and Weichselbaum, R.R. Histone H2AX phosphorylation as a predictor of radiosensitivity and target for radiotherapy. J. Biol. Chem. 279 (2004) 2273–2280.

    PubMed  Article  CAS  Google Scholar 

  41. Mahrhofer, H., Bürger, S., Oppitz, U., Flentje, M. and Djuzenova, C.S. Radiation induced DNA damage and damage repair in human tumor and fibroblast cell lines assessed by histone H2AX phosphorylation. Int. J. Radiat. Oncol. Biol. Phys. 64 (2006) 573–580.

    PubMed  Article  CAS  Google Scholar 

  42. Yoshikawa, T., Kashino, G., Ono, K. and Watanabe, M. Phosphorylated H2AX foci in tumor cells have no correlation with their radiation sensitivities. J. Radiat. Res. 50 (2009) 151–160.

    PubMed  Article  Google Scholar 

  43. Yu, T., MacPhail, S.H., Banáth, J.P., Klokov, D. and Olive, P.L. Endogenous expression of phosphorylated histone H2AX in tumors in relation to DNA double-strand breaks and genomic instability. DNA Repair (Amst) 5 (2006) 935–946.

    PubMed  Article  CAS  Google Scholar 

  44. Suzuki, M., Suzuki, K., Kodoma, S. and Watanabe, M. Phosphorylated histone H2AX foci persist on rejoined mitotic chromosomes in normal human diploid cells exposed to ionizing radiation. Radiat. Res. 165 (2006) 269–276.

    PubMed  Article  CAS  Google Scholar 

  45. Markova, E., Schultz, N., Belyaev, I.Y. Kinetics and dose-response of residual 53BP1/gamma-H2AX foci:Co-localization, relationship with DSB repair and clonogenic survival. Int. J. Radiat. Biol. 83 (2007) 319–329.

    PubMed  Article  CAS  Google Scholar 

  46. Belyaev, I.Y. Radiation-induced DNA repair foci: Spatio-temporal aspects of formation, application for assessment of radiosensitivity and biological dosimetry. Mutat. Res. 704 (2010) 132–141.

    PubMed  Article  CAS  Google Scholar 

  47. Kuefner, M.A, Brand, M., Engert, C., Kappey, H., Uder, M., Distel, L.V. The effect of calyculin A on the dephosphorylation of the histone gamma-H2AX after formation of X-ray-induced DNA double-strand breaks in human blood lymphocytes. Int. J. Radiat. Biol. 89 (2013) 424–432.

    PubMed  Article  CAS  Google Scholar 

  48. Franken, N.A., Ruurs, P., Ludwików, G., van Bree, C., Kipp, J.B., Darroudi, F. and Barendsen, G.W. Correlation between cell reproductive death and chromosome aberrations assessed by FISH for low and high doses of radiation and sensitization by iodo-deoxyuridine in human SW-1573 cells. Int. J. Radiat. Biol. 75 (1999) 293–299.

    PubMed  Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Nicolaas A. P. Franken.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.2478/s11658-013-0113-0

Key words

  • p53
  • Radiation sensitivity
  • Potentially lethal damage repair (PLDR)
  • Linear-quadratic model
  • Clonogenic assay
  • Colon cancer cells
  • RKO cells, RC10.1 cells
  • γ-H2AX foci
  • Flow cytometry