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Open Access Research article

Functional deficiency of NBN, the Nijmegen breakage syndrome protein, in a p.R215W mutant breast cancer cell line

Bianca Schröder-Heurich1, Natalia Bogdanova12, Britta Wieland1, Xiaoxi Xie1, Monika Noskowicz1, Tjoung-Won Park-Simon1, Peter Hillemanns1, Hans Christiansen2 and Thilo Dörk1*

Author Affiliations

1 Clinics of Obstetrics and Gynaecology, Hannover Medical School, Carl-Neuberg Straße 1, D-30625 Hannover, Germany

2 Clinics of Radiation Oncology, Hannover Medical School, Carl-Neuberg Straße 1, D-30625 Hannover, Germany

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BMC Cancer 2014, 14:434  doi:10.1186/1471-2407-14-434

Published: 13 June 2014

Abstract

Background

Mutations in NBN, the gene for Nijmegen Breakage Syndrome (NBS), are thought to predispose women to developing breast cancer, but a breast cancer cell line containing mutations in NBN has not yet been described. The p.R215W missense mutation occurs at sub-polymorphic frequencies in several populations. We aimed to investigate its functional impact in breast cancer cells from a carrier of this NBN mutation.

Methods

Breast cancer cell lines were screened by immunoblotting for NBN protein levels, and the NBN coding region was sequenced for mutation analysis. Radiosensitivity assays and functional studies were performed through immunocytochemistry and immunoblotting, and flow cytometry was employed to assess cell cycle progression. Impedance measurements were used to study the consequences of PARP1 inhibition. Statistical comparisons between cell lines were performed using t-tests.

Results

HCC1395 breast cancer cells exhibited reduced NBN protein levels. Direct sequencing identified the NBN p.R215W mutation in the hemizygous state, in addition to a truncation in BRCA1. Mutations in both genes were already present in the heterozygous state in the patient’s germline. HCC1395 cells were highly radiosensitive, susceptible to apoptosis and were deficient in the formation of NBN foci. There was also evidence for some impairment in the formation of γH2AX, MDC1, and 53BP1 foci after irradiation; these foci appeared smaller and irregular compared with repair foci in wild-type cells, although ATM signalling was largely unaffected. In line with their deficiency in NBN and BRCA1, HCC1395 cells were particularly sensitive to PARP1 inhibition.

Conclusion

Our results indicate that the p.R215W mutation in the HCC1395 breast cancer cell line impairs NBN function, making this cell line a potentially useful cellular model for studying defective NBN protein within a mutant BRCA1 background.

Keywords:
Breast carcinoma; DNA damage repair; DNA double strand break repair disorder; Ionising radiation sensitivity; MRN complex