Download MICRONUCLEUS FORMATION AND CELL PROLIFERATION IN A

MICRONUCLEUS FORMATION AND CELL PROLIFERATION IN A BRCA1 AND 2
KNOWCKDOWN BREAST EPITHELIAL CELL LINE AFTER EXPOSURE TO
GAMMA RAYS AND NEUTRONS.
J. Depuydt 1, P. Beukes 2, V. Vandersickel 1, H. Thierens 1, J. Slabbert 2 and A. Vral 1
1 Department of Basic Medical Sciences, University of Ghent, De Pintelaan 185, 9000 Gent,
Belgium
2 iThemba LABS, PO box 722, 7129 Somerset West, South Africa
[email protected]
Background: A better understanding of the underlying mechanisms of DNA repair after lowand high-LET radiations is needed to improve the outcome of clinical radiotherapy making
e.g. use of high-LET new particle beams like carbon ions. 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. In this study we investigated
the role of BRCA1 and 2, both involved in DNA double strand break (DSB) repair after
exposure of breast epithelial cells to either low-LET 60Co -rays or high-LET p(66)+Be(40)
neutrons.
Methods: To investigate the role of the DSB-repair proteins BRCA1 and BRCA2, breastepithelial cell lines (MCF10A) deficient in these proteins were generated using RNA
interference. To analyze the radiation response of these repair-deficient cell lines, cells were
exposed to different doses of both 60Co -rays and p(66)+Be(40) neutrons and this while
being in G2-S phase or G0-G1 phase of the cell cycle. After radiation-exposure, the role of
the 2 genes in the repair of DNA damage was analyzed by investigating the cellular outcome
using the micronucleus assay (MN) and the crystal violet (CV) cell proliferation assay.
Results: Downregulation of the BRCA-proteins resulted in an increase in the number of
micronuclei and a decrease in the proliferative capacity of the cells compared to the reference
cell line, and this after exposure of cells to both radiation qualities. The extent of the increase
in MN number and decrease in cell proliferation depends on the protein down-regulated, the
cell-cycle phase at the moment of irradiation and the radiation quality used.
Conclusion: Both the MN-assay and the CV-proliferation assay show that the BRCA1 and
BRCA2 knock-down cell lines are more sensitive to ionizing radiation than the reference cell
line, irrespective of the radiation-quality used. Knowledge about the radiosensitivity in
BRCA1 and 2 deficient cells is important for the clinical management of patients carrying a
BRCA1 or 2 mutation.