The radiosensitizing effect of Ku70/80 knockdown in MCF10A cells irradiated with X-rays and p(66)+Be(40) neutrons
Open Access
- 27 April 2010
- journal article
- Published by Springer Science and Business Media LLC in Radiation Oncology
- Vol. 5 (1), 30
- https://doi.org/10.1186/1748-717x-5-30
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.Keywords
This publication has 38 references indexed in Scilit:
- Repair of ionizing radiation-induced DNA double-strand breaks by non-homologous end-joiningBiochemical Journal, 2009
- Mutations to Ku reveal differences in human somatic cell linesDNA Repair, 2008
- Regulation of DNA repair throughout the cell cycleNature Reviews Molecular Cell Biology, 2008
- Ku protein targeting by Ku70 small interfering RNA enhances human cancer cell response to topoisomerase II inhibitor and γ radiationMolecular Cancer Therapeutics, 2005
- Mechanisms of DNA double strand break repair and chromosome aberration formationCytogenetic and Genome Research, 2004
- In vitro micronucleus-centromere assay to detect radiation-damage induced by low doses in human lymphocytesInternational Journal of Radiation Biology, 1997
- Effect of Radiation Quality on Lesion Complexity in Cellular DNAInternational Journal of Radiation Biology, 1994
- RBE—LET Relationships for Different Types of Lethal Radiation Damage in Mammalian Cells: Comparison with DNA Dsb and an Interpretation of Differences in RadiosensitivityInternational Journal of Radiation Biology, 1994
- Micronuclei Induced by Fast Neutrons Versus60Co γ-rays in Human Peripheral Blood LymphocytesInternational Journal of Radiation Biology, 1994
- Direct Comparison of Biological Effectiveness of Protons and Alpha-particles of the Same LET. III. Initial Yield of DNA Double-strand Breaks in V79 CellsInternational Journal of Radiation Biology, 1992