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

Veerle Vandersickel UGent, Monica Mancini, Jacobus Slabbert, Emanuela Marras, Hubert Thierens UGent, Gianpaolo Perletti and Anne Vral UGent (2010) RADIATION ONCOLOGY. 5.
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 non-homologous 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 Xrays 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.
Please use this url to cite or link to this publication:
author
organization
year
type
journalArticle (original)
publication status
published
subject
keyword
ALPHA-PARTICLES, GAMMA-RAYS, TUMOR-CELLS, BIOLOGICAL EFFECTIVENESS, DNA-DAMAGE, MAMMALIAN-CELLS, IONIZING-RADIATION, DOUBLE-STRAND BREAKS, IN-VITRO, REPAIR
journal title
RADIATION ONCOLOGY
Radiat. Oncol.
volume
5
article_number
30
pages
7 pages
Web of Science type
Article
Web of Science id
000278326800001
JCR category
RADIOLOGY, NUCLEAR MEDICINE & MEDICAL IMAGING
JCR impact factor
2.409 (2010)
JCR rank
44/111 (2010)
JCR quartile
2 (2010)
ISSN
1748-717X
DOI
10.1186/1748-717X-5-30
language
English
UGent publication?
yes
classification
A1
copyright statement
I have retained and own the full copyright for this publication
id
1053132
handle
http://hdl.handle.net/1854/LU-1053132
date created
2010-10-04 14:58:10
date last changed
2010-10-05 08:54:14
@article{1053132,
  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 non-homologous 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 Xrays 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.},
  articleno    = {30},
  author       = {Vandersickel, Veerle and Mancini, Monica and Slabbert, Jacobus and Marras, Emanuela and Thierens, Hubert and Perletti, Gianpaolo and Vral, Anne},
  issn         = {1748-717X},
  journal      = {RADIATION ONCOLOGY},
  keyword      = {ALPHA-PARTICLES,GAMMA-RAYS,TUMOR-CELLS,BIOLOGICAL EFFECTIVENESS,DNA-DAMAGE,MAMMALIAN-CELLS,IONIZING-RADIATION,DOUBLE-STRAND BREAKS,IN-VITRO,REPAIR},
  language     = {eng},
  pages        = {7},
  title        = {The radiosensitizing effect of Ku70/80 knockdown in MCF10A cells irradiated with X-rays and p(66)+Be(40) neutrons},
  url          = {http://dx.doi.org/10.1186/1748-717X-5-30},
  volume       = {5},
  year         = {2010},
}

Chicago
Vandersickel, Veerle, Monica Mancini, Jacobus Slabbert, Emanuela Marras, Hubert Thierens, Gianpaolo Perletti, and Anne Vral. 2010. “The Radiosensitizing Effect of Ku70/80 Knockdown in MCF10A Cells Irradiated with X-rays and p(66)+Be(40) Neutrons.” Radiation Oncology 5.
APA
Vandersickel, V., Mancini, M., Slabbert, J., Marras, E., Thierens, H., Perletti, G., & Vral, A. (2010). The radiosensitizing effect of Ku70/80 knockdown in MCF10A cells irradiated with X-rays and p(66)+Be(40) neutrons. RADIATION ONCOLOGY, 5.
Vancouver
1.
Vandersickel V, Mancini M, Slabbert J, Marras E, Thierens H, Perletti G, et al. The radiosensitizing effect of Ku70/80 knockdown in MCF10A cells irradiated with X-rays and p(66)+Be(40) neutrons. RADIATION ONCOLOGY. 2010;5.
MLA
Vandersickel, Veerle, Monica Mancini, Jacobus Slabbert, et al. “The Radiosensitizing Effect of Ku70/80 Knockdown in MCF10A Cells Irradiated with X-rays and p(66)+Be(40) Neutrons.” RADIATION ONCOLOGY 5 (2010): n. pag. Print.