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Open Access 01-12-2010 | Research

The radiosensitizing effect of Ku70/80 knockdown in MCF10A cells irradiated with X-rays and p(66)+Be(40) neutrons

Authors: Veerle Vandersickel, Monica Mancini, Jacobus Slabbert, Emanuela Marras, Hubert Thierens, Gianpaolo Perletti, Anne Vral

Published in: Radiation Oncology | Issue 1/2010

Abstract

Background

A better understanding of the underlying mechanisms of DNA repair after low- and high-LET radiations represents a research priority aimed at improving the outcome of clinical radiotherapy. To date however, our knowledge regarding the importance of DNA DSB repair proteins and mechanisms in the response of human cells to high-LET radiation, is far from being complete.

Methods

We investigated the radiosensitizing effect after interfering with the DNA repair capacity in a human mammary epithelial cell line (MCF10A) by lentiviral-mediated RNA interference (RNAi) of the Ku70 protein, a key-element of the nonhomologous end-joining (NHEJ) pathway. Following irradiation of control and Ku-deficient cell lines with either 6 MV X-rays or p(66)+Be(40) neutrons, cellular radiosensitivity testing was performed using a crystal violet cell proliferation assay. Chromosomal radiosensitivity was evaluated using the micronucleus (MN) assay.

Results

RNAi of Ku70 caused downregulation of both the Ku70 and the Ku80 proteins. This downregulation sensitized cells to both X-rays and neutrons. Comparable dose modifying factors (DMFs) for X-rays and neutrons of 1.62 and 1.52 respectively were obtained with the cell proliferation assay, which points to the similar involvement of the Ku heterodimer in the cellular response to both types of radiation beams. After using the MN assay to evaluate chromosomal radiosensitivity, the obtained DMFs for X-ray doses of 2 and 4 Gy were 2.95 and 2.66 respectively. After neutron irradiation, the DMFs for doses of 1 and 2 Gy were 3.36 and 2.82 respectively. The fact that DMFs are in the same range for X-rays and neutrons confirms a similar importance of the NHEJ pathway and the Ku heterodimer for repairing DNA damage induced by both X-rays and p(66)+Be(40) neutrons.

Conclusions

Interfering with the NHEJ pathway enhanced the radiosensitivity of human MCF10A cells to low-LET X-rays and high-LET neutrons, pointing to the importance of the Ku heterodimer for repairing damage induced by both types of radiation. Further research using other high-LET radiation sources is however needed to unravel the involvement of DNA double strand break repair pathways and proteins in the cellular response of human cells to high-LET radiation.

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Barendsen GW: The relationships between RBE and LET for different types of lethal damage in mammalian cells: Biophysical and molecular mechanisms. Radiat Res 1994, 139: 257-270. 10.2307/3578823CrossRefPubMed

Barendsen GW: RBE-LET relationships for different types of lethal radiation damage in mammalian cells: Comparison with DNA dsb and an interpretation of differences in radiosensitivity. Int J Radiat Biol 1994, 66: 433-436. 10.1080/09553009414551411CrossRefPubMed

Britten RA, Peters LJ, Murray D: Biological factors influencing the RBE of neutrons: implications for their past, present and future use in radiotherapy. Radiat Res 2001,56(2):125-135. 10.1667/0033-7587(2001)156[0125:BFITRO]2.0.CO;2CrossRef

Jenner TJ, Belli M, Goodhead DT, Ianzini F, Simone G, Tabocchini MA: Direct comparison of biological effectiveness of protons and alpha-particles of the same LET. III. Initial yield of DNA doublestrand breaks in V79 cells. Int J Radiat Biol 1992, 61: 631-637. 10.1080/09553009214551441CrossRefPubMed

Kysela BP, Arrand JE, Michael BD: Relative contributions of levels of initial damage and repair of double-strand breaks to the ionizing radiation-sensitive phenotype of the Chinese hamster cell mutant, XR-V15B. Part II. Neutrons. Int J Radiat Biol 1993,64(5):531-538. 10.1080/09553009314551741CrossRefPubMed

Prise KM, Folkard M, Davies S, Michael BD: The irradiation of V79 mammalian cells by protons with energies below 2 MeV. Part II. Measurement of oxygen enhancement ratios and DNA damage. Int J Radiat Biol 1990, 58: 261-277. 10.1080/09553009014551611CrossRefPubMed

Prise KM, Folkard M, Newman HC, Michael BD: Effect of radiation quality on lesion complexity in cellular DNA. Int J Radiat Biol 1994, 66: 537-542. 10.1080/09553009414551581CrossRefPubMed

Hill MA: Radiation damage to DNA: the importance of track structure. Radiat Meas 1999,31(1-6):15-23. 10.1016/S1350-4487(99)00090-6CrossRefPubMed

Terato H, Tanaka R, Nakaarai Y, Nohara T, Doi Y, Iwai S, Hirayama R, Furusawa Y, Ide H: Quantitative analysis of isolated and clustered DNA damage induced by gamma-rays, carbon ion beams, and iron ion beams. J Radiat Res 2008,49(2):133-146. 10.1269/jrr.07089CrossRefPubMed

10.

Branzei D, Foiani M: Regulation of DNA repair throughout the cell cycle. Nat Rev Mol Cell Bio 2008,9(4):297-308. 10.1038/nrm2351CrossRef

11.

Iliakis G, Wang H, Perrault AR, Boecker W, Rosidi B, Windhofer F, Wu W, Guan J, Terzoudi G, Pantelias G: Mechanisms of DNA double strand break repair and chromosome aberration formation. Cytogenet Genome Res 2004, 104: 14-20. 10.1159/000077461CrossRefPubMed

12.

Mahaney BL, Meek K, Lees-Miller SP: Repair of ionizing radiation-induced DNA double-strand breaks by non-homologous end-joining. Biochem J 2009,417(3):639-650. 10.1042/BJ20080413PubMedCentralCrossRefPubMed

13.

Weterings E, Chen D: The endless tale of non-homologous end-joining. Cell Res 2008, 18: 114-124. 10.1038/cr.2008.3CrossRefPubMed

14.

Singleton BK, Priestley A, Steingrimsdottir H, Gell D, Blunt T, Jackson SP, Lehmann AR, Jeggo PA: Molecular and biochemical characterization of xrs mutants defective in Ku80. Mol Cell Biol 1997,7(3):1264-1273.CrossRef

15.

Ayene IS, Ford LP, Koch CJ: Ku protein targeting by Ku70 small interfering RNA enhances human cancer cell response to topoisomerase II inhibitor and gamma radiation. Mol Cancer Ther 2005, 4: 529-536. 10.1158/1535-7163.MCT-04-0130CrossRefPubMed

16.

Belenkov AI, Paiement JP, Panasci LC, Monia BP, Chow TY: An antisense oligonucleotide targeted to human Ku86 messenger RNA sensitizes M059K malignant glioma cells to ionizing radiation, bleomycin, and etoposide but not DNA cross-linking agents. Cancer Res 2002, 62: 5888-5896.PubMed

17.

Fattah KR, Ruis BL, Hendrickson EA: Mutations to Ku reveal differences in human somatic cell lines. DNA Repair 2008, 7: 762-774. 10.1016/j.dnarep.2008.02.008PubMedCentralCrossRefPubMed

18.

He F, Li L, Kim D, Wen B, Deng X, Gutin P, Ling CC, Li GC: Adenovirus-mediated expression of a dominant negative Ku70 fragment radiosensitizes human tumor cells under aerobic and hypoxic conditions. Cancer Res 2007, 67: 634-642. 10.1158/0008-5472.CAN-06-1860CrossRefPubMed

19.

Li G, Nelsen C, Hendrickson EA: Ku86 is essential in human somatic cells. Proc Natl Acad Sci USA 2002, 99: 832-837.PubMedCentralCrossRefPubMed

20.

Li GC, He F, Shao X, Urano M, Shen L, Kim D, Borelli M, Leibel SA, Gutin PH, Ling CC: Adenovirus-mediated heat-activated antisense Ku70 expression radiosensitizes tumor cells in vitro and in vivo. Cancer Res 2003, 63: 3268-3274.PubMed

21.

Marangoni E, Le Romancer M, Foray N, Muller C, Douc-Rasy S, Vaganay S, Abdulkarim B, Barrois M, Calsou P, Bernier J, Salles B, Bourhis J: Transfer of Ku86 RNA antisense decreases the radioresistance of human fibroblasts. Cancer Gene Therapy 2000, 7: 339-346. 10.1038/sj.cgt.7700111CrossRefPubMed

22.

Omori S, Takiguchi Y, Suda A, Sugimoto T, Miyazawa H, Takiguchi Y, Tanabe N, Tatsumi K, Kimura H, Pardington PE, Chen F, Chen DJ, Kuriyama T: Suppression of a DNA double-strand break repair gene, Ku70, increases radio- and chemosensitivity in a human lung carcinoma cell line. DNA Repair 2002, 1: 299-310. 10.1016/S1568-7864(02)00006-XCrossRefPubMed

23.

Vandersickel V, Mancini M, Marras E, Willems P, Slabbert J, Philippé J, Deschepper E, Thierens H, Perletti G, Vral A: Lentivirus-mediated RNA interference of Ku70 to enhance radiosensitivity of human mammary epithelial cells. Int J Radiat Biol 2010, 86: 144-124. 10.3109/09553000903419940CrossRef

24.

Britten RA, Murray D: Constancy of the relative biological effectiveness of 42 MeV (p-->Be+) neutrons among cell lines with different DNA repair proficiencies. Radiat Res 1997, 148: 308-316. 10.2307/3579515CrossRefPubMed

25.

Thacker J, Stretch A: Responses of 4 X-ray-sensitive CHO cell mutants to different radiations and to irradiation conditions promoting cellular recovery. Mutat Res 1985, 146: 99-108.PubMed

26.

Nagasawa H, Little JB, Inkret WC, Carpenter S, Raju MR, Chen DJ, Strniste GF: Response of X-ray-sensitive CHO mutant cells (xrs-6c) to radiation. Radiat Res 1991, 126: 280-288. 10.2307/3577917CrossRefPubMed

27.

Wang H, Wang X, Zhang P, Wang Y: The Ku-dependent non-homologous end-joining but not other repair pathway is inhibited by high linear energy transfer ionizing radiation. DNA Repair (Amst) 2008,7(5):725-733. 10.1016/j.dnarep.2008.01.010CrossRef

28.

Okayasu R, Okada M, Okabe A, Noguchi M, Takakura K, Takahashi S: Repair of DNA damage induced by accelerated heavy ions in mammalian cells proficient and deficient in the non-homologous end-joining pathway. Radiat Res 2006,165(1):59-67. 10.1667/RR3489.1CrossRefPubMed

29.

Amaldi U, Kraft G: Radiotherapy with beams of carbon ions. Rep Prog Phys 2005, 68: 1861-1882. 10.1088/0034-4885/68/8/R04CrossRef

30.

Jones DTL, Wambersie A: Radiation therapy with fast neutrons: a review. Nucl Instr Meth Phys Res A 2007, 580: 522-525. 10.1016/j.nima.2007.05.220CrossRef

31.

Jones DTL, Schreuder AN, Symons JE, Yudelev M: The NAC particle therapy facilities. In Hadrontherapy in Oncology. Edited by: Amaldi U, Larsson B. Amsterdam: Elsevier Science BV; 1994:307-328.

32.

Slabbert JP, Theron T, Serafin A, Jones DT, Böhm L, Schmitt G: Radiosensitivity variations in human tumor cell lines exposed in vitro to p(66)/Be neutrons or ⁶⁰Co gamma-rays. Strahlenther Onkol 1996,172(10):567-572.PubMed

33.

Baeyens A, Thierens H, Vandenbulcke K, De Ridder L, Vral A: The use of EBV-transformed cell lines of breast cancer patients to measure chromosomal radiosensitivity. Mutagenesis 2004,19(4):285-290. 10.1093/mutage/geh029CrossRefPubMed

34.

Vral A, Verhaegen F, Thierens H, De Ridder L: Micronuclei induced by fast neutrons versus ⁶⁰Co γ-rays in human pheripheral blood lymphocytes. Int J Radiat Biol 1994,65(3):321-328. 10.1080/09553009414550381CrossRefPubMed

35.

Slabbert JP, Theron T, Zolzer F, Streffer C, Bohm L: A comparison of the potential therapeutic gain of p(66)/Be neutrons and d(14)/Be neutrons. Int J Radiat Oncol Biol Phys 2000,47(4):1059-1065.CrossRefPubMed

36.

Vral A, Thierens H, De Ridder L: The in vitro micronucleus-centromere assay to detect radiation damage induced by low doses in human lymphocytes. Int J Radiat Biol 1997,71(1):61-68. 10.1080/095530097144427CrossRefPubMed

37.

Begg EC: Molecular targeting and patient individualization. In Basic clinical radiobiology. 4th edition. Edited by: Joiner M, van der Kogel A. London: Hodder Arnold; 2009:316-331.CrossRef

38.

Belzile JP, Choudhury SA, Cournoyer D, Chow TY: Targeting DNA repair proteins: a promising avenue for cancer gene therapy. Curr Gene Ther 2006,6(1):111-123. 10.2174/156652306775515538CrossRefPubMed

39.

Lieberman HB: DNA damage repair and response proteins as targets for cancer therapy. Curr Med Chem 2008,15(4):360-367. 10.2174/092986708783497328CrossRefPubMed

Title: The radiosensitizing effect of Ku70/80 knockdown in MCF10A cells irradiated with X-rays and p(66)+Be(40) neutrons
Authors: Veerle Vandersickel
Monica Mancini
Jacobus Slabbert
Emanuela Marras
Hubert Thierens
Gianpaolo Perletti
Anne Vral
Publication date: 01-12-2010
Publisher: BioMed Central
Published in: Radiation Oncology / Issue 1/2010
Electronic ISSN: 1748-717X
DOI: https://doi.org/10.1186/1748-717X-5-30

Keynote webinar | Spotlight on medication adherence

Springer Medicine

The radiosensitizing effect of Ku70/80 knockdown in MCF10A cells irradiated with X-rays and p(66)+Be(40) neutrons

Abstract

Background

Methods

Results

Conclusions

Keynote webinar | Spotlight on medication adherence

Springer Medicine

Abstract

Background

Methods

Results

Conclusions

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