Lentivirus-mediated RNA interference of Ku70 to enhance radiosensitivity of human mammary epithelial cells

Abstract

Purpose: To investigate the radiosensitising effect of Ku autoantigen 70 (Ku70) and Ku autoantigen 80 (Ku80) knockdown by lentivirus-mediated RNA interference (RNAi) in the MCF10A immortalised human mammary epithelial cell line.

Materials and methods: MCF10A cells were infected with lentiviral vectors for RNAi of Ku70. The Ku70-knockdown cell line (Ku70i) and a mock-infected control cell line (LVTHM) were used to perform radiation experiments. For the in vitro Micronucleus (MN) assay, both cell lines were irradiated with doses of 2 and 4 Gy ⁶⁰Co γ-rays. For cell survival experiments, doses ranging between 0 and 8 Gy were used.

Results: Western blot analysis showed that the Ku70 lentiviral vector was effective in silencing the expression of both Ku70 and Ku80. A significantly higher radiation-induced MN yield was obtained in the Ku70i cell line compared to the control LVTHM cell line. RNAi of Ku70 also resulted in a lower survival yield after irradiation compared to the control cell line. Analysis of cell death mechanisms showed that MCF10A cells (Ku70i and LVTHM) do not undergo apoptosis, but undergo post-irradiation cellular senescence.

Conclusion: RNAi of Ku70 resulted in increased chromosomal and cellular radiosensitivity in the MCF10A human mammary cell line after irradiation with ⁶⁰Co γ-rays. These results further strengthen the role of the Ku protein in correct DNA double strand break (DSB) repair.

Keywords:

• RNA interference
• Ku heterodimer
• radiosensitivity
• DNA DSB repair
• micronuclei
• cell survival

Acknowledgements

We thank Jutho Haegeman for additional statistical evaluation. The work was supported by a grant of the ‘Bijzonder Onderzoeksfonds’ (Ghent University, No 01D30105), a grant of the Research Foundation Flanders (FWO, No 1.5.080.08), a ‘VLIR Own Initiative Programme’ between Belgium and South Africa (ZEIN2005PR309) and a ‘FAR’ grant, University of Insubria, Italy.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.