BRCA2 heterozygosity delays cytokinesis in primary human fibroblasts

Article type: Research Article

Authors: Jonsdottir, Asta Björk^{; ;} | Vreeswijk, Maaike P.G. | Wolterbeek, Ron | Devilee, Peter^; | Tanke, Hans J. | Eyfjörd, Jorunn E.^{; ;} | Szuhai, Karoly

Affiliations: Faculty of Medicine, University of Iceland, Reykjavik, Iceland | The University of Iceland and Icelandic Cancer Society Molecular and Cell Biology Research Laboratory, University of Iceland, Reykjavik, Iceland | Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands | Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands | Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands | Department of Medical Statistics, Leiden University Medical Center, Leiden, The Netherlands

Note: [] Corresponding author: Jorunn Erla Eyfjörd, Faculty of Medicine, University of Iceland, The University of Iceland and Icelandic Cancer Society Molecular and Cell Biology Research Laboratory, Vatnsmyrarvegi 16, 101 Reykjavik, Iceland. Tel.: +354 5255825; Fax: +354 5254884; E-mail: [email protected].

Abstract: Background: Inherited mutations in the tumour suppressor gene BRCA2 greatly increase the risk of developing breast, ovarian and other types of cancers. So far, most studies have focused on the role of BRCA-pathways in the maintenance of genomic stability. In this study we investigated the potential role of the BRCA2 protein in cytokinesis in unmodified primary human fibroblast carrying a heterozygous mutation in the BRCA2 gene. Methods: Cell divisions were monitored with time lapse live-cell imaging. BRCA2 mRNA expression levels in BRCA2+/− and BRCA2+/+ cells were quantified with quantitative real-time polymerase chain reaction (qRT-PCR). To investigate the localization of the BRCA2 protein during cytokinesis, immunofluorescence staining using antibody directed against BRCA2 was carried out. Immunofluorescence staining was performed directly after live-cell imaging and cells with delayed cytokinesis, of which the co-ordinates were saved, were automatically repositioned and visualized. Results: We demonstrate that unmodified primary human fibroblasts derived from heterozygous BRCA2 mutation carriers show significantly prolonged cytokinesis. A Subset of the BRCA2+/− cells had delayed cytokinesis (40 min or longer) making the mean cell division time 6 min longer compared with BRCA2+/+ cells, 33 min versus 27 min, respectively. Lower BRCA2 mRNA expression levels were observed in the BRCA2 heterozygous samples compared with the BRCA2 wild type samples. The BRCA2 protein localizes and accumulates to the midbody during cytokinesis, and no difference was detected in distribution and localization of the protein between BRCA2+/− and BRCA2+/+ samples or cells with delayed cytokinesis and normal division time. Conclusions: The delayed cytokinesis phenotype of the BRCA2 heterozygous cells and localization of the BRCA2 protein to the midbody confirms that BRCA2 plays a role in cytokinesis. Our observations indicate that in a subset of cells the presence of only one wild type BRCA2 allele is insufficient for efficient cytokinesis.

Keywords: BRCA2, cytokinesis, live-cell imaging, primary human fibroblasts, heterozygous phenotype

DOI: 10.3233/CLO-2009-0465

Journal: Analytical Cellular Pathology, vol. 31, no. 3, pp. 191-201, 2009