Abstract
We aimed to elucidate the mechanism underlying carcinogenesis by comparing normal and BRCA1/2-mutated ovarian epithelial cells established via Sendai virus-based immortalization. Ovarian epithelial cells (normal epithelium: Ovn; with germline BRCA1 mutation: OvBRCA1; with germline BRCA2 mutation: OvBRCA2) were infected with Sendai virus vectors carrying three immortalization genes (Bmi-1, hTERT, and SV40T). The immunoreactivity to anti-epithelial cellular adhesion molecule (EpCAM) antibodies in each cell line and cells after 25 passages was confirmed using flow cytometry. Chromosomes were identified and karyotyped to detect numerical and structural abnormalities. Total RNA extracted from the cells was subjected to human transcriptome sequencing. Highly expressed genes in each cell line were confirmed using real-time polymerase chain reaction. Immortalization techniques allowed 25 or more passages of Ovn, OvBRCA1, and OvBRCA2 cells. No anti-EpCAM antibody reactions were observed in primary cultures or after long-term passages of each cell line. Structural abnormalities in the chromosomes were observed in each cell line; however, the abnormal chromosomes were successfully separated from the normal structures via cloning. Only normal cells from each cell line were cloned. MMP1, CCL2, and PAPPA were more predominantly expressed in OvBRCA1 and OvBRCA2 cells than in Ovn cells. Immortalized ovarian cells derived from patients with germline BRCA1 or BRCA2 mutations showed substantially higher MMP1 expression than normal ovarian cells. However, the findings need to be validated in the future.
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Acknowledgements
The authors are grateful to Mitsuhiko Ozaki, Ph.D., from the Department of Experimental Pathology, Department of Life Sciences, Faculty of Medicine, Tottori University, for providing guidance on the research methods. This work was supported by a research grant from the Tottori University Hospital and a Grant-in-Aid for Scientific Research (21K16770) in 2021.
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This work was supported by a research grant from the Tottori University Hospital and a Grant-in-Aid for Scientific Research (Grant No. 21K16770) in 2021.
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Hiroaki Komatsu, Yasuhiro Kazuki, and Masayo Okawa conceived the study. Hiroaki Komatsu and Masayo Okawa developed the statistical analysis plan and conducted statistical analyses. All authors contributed to the interpretation of the results. Hiroaki Komatsu drafted the original manuscript. Yasuhiro Kazuki and Masayo Okawa supervised the conduct of this study. All authors reviewed the manuscript draft and revised it critically for intellectual content. All authors approved the final version of the manuscript to be published.
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Komatsu, H., Okawa, M., Kazuki, Y. et al. Characterization of immortalized ovarian epithelial cells with BRCA1/2 mutation. Human Cell (2024). https://doi.org/10.1007/s13577-024-01064-z
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DOI: https://doi.org/10.1007/s13577-024-01064-z