Download PDF
Nuklearmedizin 2017; 56(01): 47-54
DOI: 10.3413/Nukmed-0842-16-08
Original article
Schattauer GmbH

Comparison of clonogenic cell survival and DNA damage induced by 188Re and X-rays in rat thyroid cells

Vergleich des Zellüberlebens und der DNA-Schädigung nach Bestrahlung von Ratten-Schilddrüsenzellen mit 188Re und Röntgenstrahlung
Roswitha Runge
1   Department of Nuclear Medicine, University Hospital / Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany
,
Jana Arlt
1   Department of Nuclear Medicine, University Hospital / Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany
,
Liane Oehme
1   Department of Nuclear Medicine, University Hospital / Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany
,
Robert Freudenberg
1   Department of Nuclear Medicine, University Hospital / Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany
,
Jörg Kotzerke
1   Department of Nuclear Medicine, University Hospital / Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany
› Author Affiliations
Further Information

Publication History

received: 09 August 2016

accepted in revised form: 14 September 2016

Publication Date:
02 January 2018 (online)

Also available at
    Permissions and Reprints

Summary

Aim: Ionizing radiation produces DNA lesions among which DNA double strand breaks (DSB) are the most critical events. Radiation of various energy types might differ in their biological effectiveness. Here, we compared cell survival and DNA damage induced by 188Re and X-rays using YH2AX foci as a measure of DSB. The correlation between survival and residual foci was also analyzed. Methods: PCCl3 cells were irradiated with 200 kV X-rays (1.2 Gy/min) or 0.5-25 MBq/ml 188Re (1 h irradiation) achieving doses up to 10 Gy. By blocking of sodium iodide sym- porter (NIS) essentially extracellular activity could be guaranteed. Survival fractions (SF) were detected by colony forming assay. Initial and residual YH2AX foci (15 min and 24 h after irradiation) were assessed by immunos- taining. The relationship between SF and residual radiation induced YH2AX foci (RIF) was evaluated by Spearman and Pearson correlation tests. Results: We did not find significant differences between the survival curves in terms of the radiation quality. The D37 values were 4.6 Gy and 4.2 Gy for 188Re or X-ray, respectively. The initial foci numbers were in the same range for 188Re and X-ray, but higher levels of residual foci persisted after X-rays in comparison to 188Re (1 GyX-ray 6.5 ± 0.2; 1 GyRe-188 4.8 ± 0.2 RIF). Accordingly, for 188Re a higher extent of DSB repair was found. The Spearman test revealed a significant (p < 0.01) correlation between SF and residual RIF for both radiation modalities. Conclusion: No differences in terms of radiation were found for SF and initial foci. However, residual foci were lower for 188Re than for X-rays. A prediction of SF by residual foci should consider the properties of the radiation qualities that influence foci removal and DSB repair.

Zusammenfassung

Ziel: Ionisierende Strahlung induziert DNA Schäden. DNA-Doppelstrangbrüche (DSB) sind dabei die kritischsten Ereignisse für das Zellüberleben. In Abhängigkeit von den Strahlungseigenschaften und der Dosis variiert der radiotoxische Effekt. Ziel der Studie war ein quantitativer Vergleich von Zellüberleben und YH2AX Foci als DSB-Marker nach Bestrahlung mit 188Re und Röntgenstrahlung. Weiterhin wurde die Korrelation zwischen Zellüberleben und residualen YH2AX Foci untersucht. Methodik: PCCl3-Schilddrüsenzellen wurden mit Röntgenstrahlung oder 188Re mit 0,5-25 MBq/ml über 1 h im Dosisbereich von 0-10 Gy bestrahlt. Durch Blockade des Natriumiodid- Symporters (NIS) wurde eine Radionuklidakkumulation in die Zellen verhindert. Das Zellüberleben wurde mit dem Koloniebildungstest bestimmt. Die initialen (15 min nach Bestrahlung) und residualen YH2AX Foci (24 h nach Bestrahlung) wurden mit dem YH2AX- Immunfluoreszenz-Assay analysiert. Die Beziehung zwischen Zellüberleben und residualen Foci wurde mit den Korrelationstests nach Spearman und Pearson untersucht. Ergebnisse: Die Dosis-Wirkungs-Kurven für 188Re und Röntgenstrahlung zeigten keine signifikanten Unterschiede. Die D37-Werte betrugen 4,6 Gy für 188Re und 4,3 Gy für Röntgenstrahlung. Zwischen beiden Strahlenmodalitäten wurden keine Unterschiede hinsichtlich initialer YH2AX-Foci gefunden. Die Anzahl der residualen YH2AX-Foci war nach Röntgenbestrahlung höher als nach 188Re-Exposition, z.B. wurden bei 1 Gy Röntgen 6,5 ± 0.2 bzw. 1 Gy 188Re 4,8 ± 0.2 Foci gezählt. Mit dem Spearman-Test wurde eine sehr gute Korrelation (p < 0,01) zwischen dem Zellüberleben und den residualen Foci gefunden. Schlussfolgerung: Mit der Bestimmung von Zellüberleben und Anzahl der initialen YH2AX-Foci konnte kein Unterschied zwischen den Strahlern gefunden werden. Eine Vorhersage des Zellüberlebens - basierend auf der Anzahl residualer YH2AX-Foci - sollte die Eigenschaften der Strahlenqualitäten und deren Einfluss auf das Verschwinden der Foci und somit die Reparatur der DSB in Betracht ziehen.

Keywords

PCCl3 cells - 188Re - X-ray - γH2AX foci - cell survival - correlation

Schlüsselwörter

PCCl3-Zellen - 188Re - Röntgenstrahlung - γH2AX-Foci - Zellüberleben - Korrelation
 

  • References

  • 1 Akudugu JM, Slabbert JP, Serafín A. et al. Frequency of radiation-induced micronuclei in neuronal cells does not correlate with clonogenic survival. Radiat Res 2000; 153: 62-67.
    CrossrefSearch in Google Scholar
  • 2 Avondoglio D, Scott T, Kil WJ. et al. High throughput evaluation of gamma-H2AX. Radiat Oncol 2009; 4: 31.
    CrossrefSearch in Google Scholar
  • 3 Beels L, Werbrouck J, Thierens H. Dose response and repair kinetics of gamma-H2AX foci induced by in vitro irradiation of whole blood and T-lymphocytes with X-and gamma-radiation. Int J Radiat Biol 2010; 86: 760-768.
    CrossrefSearch in Google Scholar
  • 4 Belyaev IY. Radiation-induced DNA repair foci: spatio-temporal aspects of formation, application for assessment of radiosensitivity and biological dosimetry. Mutat Res 2010; 704: 132-141.
    CrossrefSearch in Google Scholar
  • 5 Beyreuther E, Lessmann E, Pawelke J. et al. DNA double-strand break signalling: X-ray energy dependence of residual co-localised foci of gamma-H2AX and 53BP1. Int J Radiat Biol 2009; 85: 1042-1050.
    CrossrefSearch in Google Scholar
  • 6 Bonner WM, Redon CE, Dickey JS. et al. GammaH2AX and cancer. Nat Rev Canc 2008; 8: 957-967.
    CrossrefSearch in Google Scholar
  • 7 Bouquet F, Muller C, Salles B. The loss of gammaH2AX signal is a marker of DNA double strand breaks repair only at low levels of DNA damage. Cell cycle 2006; 5: 1116-1122.
    CrossrefSearch in Google Scholar
  • 8 Burma S, Chen BP, Murphy M. et al. ATM phosphorylates histone H2AX in response to DNA double-strand breaks. J Biol Chem 2001; 276: 42462-42467.
    CrossrefSearch in Google Scholar
  • 9 Cai Z, Chen Z, Bailey KE. et al. Relationship between induction of phosphorylated H2AX and survival in breast cancer cells exposed to 111In-DTPA-hEGF. J Nucl Med 2008; 49: 1353-1361.
    CrossrefSearch in Google Scholar
  • 10 Celeste A, Fernandez-Capetillo O, Kruhlak MJ. et al. Histone H2AX phosphorylation is dispensable for the initial recognition of DNA breaks. Nat Cell Biol 2003; 5: 675-679.
    CrossrefPubMedSearch in Google Scholar
  • 11 Eberlein U, Nowak C, Bluemel C. et al. DNA damage in blood lymphocytes in patients after (177)Lu peptide receptor radionuclide therapy. Eur J Nucl Med Mol Imaging 2015; 42: 1739-1749.
    CrossrefSearch in Google Scholar
  • 12 Eberlein U, Peper M, Fernandez M. et al. Calibration of the gamma-H2AX DNA double strand break focus assay for internal radiation exposure of blood lymphocytes. PloS One 2015; 10: e0123174.
    CrossrefSearch in Google Scholar
  • 13 Eberlein U, Scherthan H, Bluemel C. et al. DNA Damage in Peripheral Blood Lymphocytes of Thyroid Cancer Patients After Radioiodine Therapy. J Nucl Med 2016; 57: 173-179.
    CrossrefSearch in Google Scholar
  • 14 Falk M, Lukasova E, Kozubek S. Higher-order chromatin structure in DSB induction, repair and misrepair. Mutat Res 2010; 704: 88-100.
    CrossrefSearch in Google Scholar
  • 15 Franken NA, Rodermond HM, Stap J. et al. Clonogenic assay of cells in vitro. Nat Protoc 2006; 1: 2315-2319.
    CrossrefSearch in Google Scholar
  • 16 Freudenberg R, Runge R, Maucksch U. et al. On the dose calculation at the cellular level and its implications for the RBE of (99m)Tc and (123)I. Med Physics 2014; 41: 062503.
    CrossrefSearch in Google Scholar
  • 17 Freudenberg R, Wendisch M, Runge R. et al. Reduction in clonogenic survival of sodium-iodide symporter (NIS)-positive cells following intracellular uptake of (99m)Tc versus (188)Re. Int J radiat Biol 2012; 88: 991-997.
    CrossrefSearch in Google Scholar
  • 18 Fusco A, Grieco M, Santoro M. et al. A new oncogene in human thyroid papillary carcinomas and their lymph-nodal metastases. Nature 1987; 328: 170-172.
    CrossrefPubMedSearch in Google Scholar
  • 19 Grudzenski S, Raths A, Conrad S. et al. Inducible response required for repair of low-dose radiation damage in human fibroblasts. PNAS 2010; 107: 14205-14210.
    CrossrefSearch in Google Scholar
  • 20 Kasten-Pisula U, Menegakis A, Brammer I. et al. The extreme radiosensitivity of the squamous cell carcinoma SKX is due to a defect in double-strand break repair. Radiother Oncol 2009; 90: 257-264.
    CrossrefSearch in Google Scholar
  • 21 Khanna KK, Jackson SP. DNA double-strand breaks: signaling, repair and the cancer connection. Nature Gen 2001; 27: 247-254.
    CrossrefSearch in Google Scholar
  • 22 Kinner A, Wu W, Staudt C. et al. Gamma-H2AX in recognition and signaling of DNA double-strand breaks in the context of chromatin. Nucleic Acids Res 2008; 36: 5678-5694.
    CrossrefSearch in Google Scholar
  • 23 Kuo LJ, Yang LX. Gamma-H2AX – a novel biomarker for DNA double-strand breaks. In vivo 2008; 22: 305-309.
    Search in Google Scholar
  • 24 Lassmann M, Hanscheid H, Gassen D. et al. In vivo formation of gamma-H2AX and 53BP1 DNA repair foci in blood cells after radioiodine therapy of differentiated thyroid cancer. J Nucl Med 2010; 51: 1318-1325.
    CrossrefSearch in Google Scholar
  • 25 Lehnert A, Dorr W, Lessmann E. et al. RBE of 10 kV X rays determined for the human mammary epithelial cell line MCF-12A. Radiat Res 2008; 169: 330-336.
    CrossrefSearch in Google Scholar
  • 26 Lobrich M, Jeggo PA. The impact of a negligent G2/M checkpoint on genomic instability and cancer induction. Nature Rev Cancer 2007; 7: 861-869.
    CrossrefSearch in Google Scholar
  • 27 Lobrich M, Kiefer J. Assessing the likelihood of severe side effects in radiotherapy. Int J Cancer 2006; 118: 2652-2656.
    CrossrefPubMedSearch in Google Scholar
  • 28 Lobrich M, Shibata A, Beucher A. et al. gamma-H2AX foci analysis for monitoring DNA doublestrand break repair: strengths, limitations and optimization. Cell Cycle 2010; 9: 662-669.
    CrossrefSearch in Google Scholar
  • 29 MacPhail SH, Banath JP, Yu TY. et al. Expression of phosphorylated histone H2AX in cultured cell lines following exposure to X-rays. Int J radiat Biol 2003; 79: 351-358.
    CrossrefSearch in Google Scholar
  • 30 MacPhail SH, Banath JP, Yu Y. et al. Cell cycle-dependent expression of phosphorylated histone H2AX: reduced expression in unirradiated but not X-irradiated G1-phase cells. Radiat Res 2003; 159: 759-767.
    CrossrefSearch in Google Scholar
  • 31 Markova E, Schultz N, Belyaev IY. Kinetics and dose-response of residual 53BP1/gamma-H2AX foci: co-localization, relationship with DSB repair and clonogenic survival. Int J Radiat Biol 2007; 83: 319-329.
    CrossrefSearch in Google Scholar
  • 32 Maucksch U, Runge R, Wunderlich G. et al. Comparison of the radiotoxicity of the 99mTc-labeled compounds 99mTc-pertechnetate, 99mTc-HMPAO and 99mTc-MIBI. Int J Radiat Biol. 2016: 1-9.
    Search in Google Scholar
  • 33 May MS, Brand M, Wuest W. et al. Induction and repair of DNA double-strand breaks in blood lymphocytes of patients undergoing (1)(8)F-FDG PET/CT examinations. Eur J Nucl Med Mol Imaging 2012; 39: 1712-1719.
    CrossrefSearch in Google Scholar
  • 34 McPhail LT, Vanderluit JL, McBride CB. et al. Endogenous expression of inhibitor of apoptosis proteins in facial motoneurons of neonatal and adult rats following axotomy. Neuroscience 2003; 117: 567-575.
    CrossrefSearch in Google Scholar
  • 35 Menegakis A, Yaromina A, Eicheler W. et al. Prediction of clonogenic cell survival curves based on the number of residual DNA double strand breaks measured by gammaH2AX staining. Int J Radiat Biol 2009; 85: 1032-1041.
    CrossrefPubMedSearch in Google Scholar
  • 36 Meyer B, Voss KO, Tobias F. et al. Clustered DNA damage induces pan-nuclear H2AX phosphorylation mediated by ATM and DNA-PK. Nucleic Acids Res 2013; 41: 6109-6118.
    CrossrefSearch in Google Scholar
  • 37 Oonishi K, Cui X, Hirakawa H. et al. Different effects of carbon ion beams and X-rays on clonogenic survival and DNA repair in human pancreatic cancer stem-like cells. Radiother Oncol 2012; 105: 258-265.
    CrossrefSearch in Google Scholar
  • 38 Rogakou EP, Pilch DR, Orr AH. et al. DNA doublestranded breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem 1998; 273: 5858-5868.
    CrossrefSearch in Google Scholar
  • 39 Rothkamm K, Barnard S, Moquet J. et al. DNA damage foci: Meaning and significance. Environ Mol Mutagen 2015; 56: 491-504.
    CrossrefSearch in Google Scholar
  • 40 Rothkamm K, Lobrich M. Evidence for a lack of DNA double-strand break repair in human cells exposed to very low x-ray doses. PNAS 2003; 100: 5057-5062.
    CrossrefSearch in Google Scholar
  • 41 Runge R, Hiemann R, Wendisch M. et al. Fully automated interpretation of ionizing radiation-induced gammaH2AX foci by the novel pattern recognition system AKLIDES(R). Int J Radiat Biol 2012; 88: 439-447.
    CrossrefPubMedSearch in Google Scholar