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DNA Double-Strand Breaks Affect Chromosomal Rearrangements during Methotrexate-Mediated Gene Amplification in Chinese Hamster Ovary Cells

Methotrexate (MTX)-mediated gene amplification has been widely used in Chinese hamster ovary (CHO) cells for the biomanufacturing of therapeutic proteins. Although many studies have reported chromosomal instability and extensive chromosomal rearrangements in MTX-mediated gene-amplified cells, which...

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Autores principales: Baik, Jong Youn, Han, Hye-Jin, Lee, Kelvin H.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8000239/
https://www.ncbi.nlm.nih.gov/pubmed/33809068
http://dx.doi.org/10.3390/pharmaceutics13030376
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author Baik, Jong Youn
Han, Hye-Jin
Lee, Kelvin H.
author_facet Baik, Jong Youn
Han, Hye-Jin
Lee, Kelvin H.
author_sort Baik, Jong Youn
collection PubMed
description Methotrexate (MTX)-mediated gene amplification has been widely used in Chinese hamster ovary (CHO) cells for the biomanufacturing of therapeutic proteins. Although many studies have reported chromosomal instability and extensive chromosomal rearrangements in MTX-mediated gene-amplified cells, which may be associated with cell line instability issues, the mechanisms of chromosomal rearrangement formation remain poorly understood. We tested the impact of DNA double-strand breaks (DSBs) on chromosomal rearrangements using bleomycin, a DSB-inducing reagent. Bleomycin-treated CHO-DUK cells, which are one of the host cell lines deficient in dihydrofolate reductase (Dhfr) activity, exhibited a substantial number of cells containing radial formations or non-radial formations with chromosomal rearrangements, suggesting that DSBs may be associated with chromosomal rearrangements. To confirm the causes of DSBs during gene amplification, we tested the effects of MTX treatment and the removal of nucleotide base precursors on DSB formation in Dhfr-deficient (i.e., CHO-DUK) and Dhfr-expressing (i.e., CHO-K1) cells. Immunocytochemistry demonstrated that MTX treatment did not induce DSBs per se, but a nucleotide shortage caused by the MTX-mediated inhibition of Dhfr activity resulted in DSBs. Our data suggest that a nucleotide shortage caused by MTX-mediated Dhfr inhibition in production cell lines is the primary cause of a marked increase in DSBs, resulting in extensive chromosomal rearrangements after gene amplification processes.
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spelling pubmed-80002392021-03-28 DNA Double-Strand Breaks Affect Chromosomal Rearrangements during Methotrexate-Mediated Gene Amplification in Chinese Hamster Ovary Cells Baik, Jong Youn Han, Hye-Jin Lee, Kelvin H. Pharmaceutics Article Methotrexate (MTX)-mediated gene amplification has been widely used in Chinese hamster ovary (CHO) cells for the biomanufacturing of therapeutic proteins. Although many studies have reported chromosomal instability and extensive chromosomal rearrangements in MTX-mediated gene-amplified cells, which may be associated with cell line instability issues, the mechanisms of chromosomal rearrangement formation remain poorly understood. We tested the impact of DNA double-strand breaks (DSBs) on chromosomal rearrangements using bleomycin, a DSB-inducing reagent. Bleomycin-treated CHO-DUK cells, which are one of the host cell lines deficient in dihydrofolate reductase (Dhfr) activity, exhibited a substantial number of cells containing radial formations or non-radial formations with chromosomal rearrangements, suggesting that DSBs may be associated with chromosomal rearrangements. To confirm the causes of DSBs during gene amplification, we tested the effects of MTX treatment and the removal of nucleotide base precursors on DSB formation in Dhfr-deficient (i.e., CHO-DUK) and Dhfr-expressing (i.e., CHO-K1) cells. Immunocytochemistry demonstrated that MTX treatment did not induce DSBs per se, but a nucleotide shortage caused by the MTX-mediated inhibition of Dhfr activity resulted in DSBs. Our data suggest that a nucleotide shortage caused by MTX-mediated Dhfr inhibition in production cell lines is the primary cause of a marked increase in DSBs, resulting in extensive chromosomal rearrangements after gene amplification processes. MDPI 2021-03-12 /pmc/articles/PMC8000239/ /pubmed/33809068 http://dx.doi.org/10.3390/pharmaceutics13030376 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) ).
spellingShingle Article
Baik, Jong Youn
Han, Hye-Jin
Lee, Kelvin H.
DNA Double-Strand Breaks Affect Chromosomal Rearrangements during Methotrexate-Mediated Gene Amplification in Chinese Hamster Ovary Cells
title DNA Double-Strand Breaks Affect Chromosomal Rearrangements during Methotrexate-Mediated Gene Amplification in Chinese Hamster Ovary Cells
title_full DNA Double-Strand Breaks Affect Chromosomal Rearrangements during Methotrexate-Mediated Gene Amplification in Chinese Hamster Ovary Cells
title_fullStr DNA Double-Strand Breaks Affect Chromosomal Rearrangements during Methotrexate-Mediated Gene Amplification in Chinese Hamster Ovary Cells
title_full_unstemmed DNA Double-Strand Breaks Affect Chromosomal Rearrangements during Methotrexate-Mediated Gene Amplification in Chinese Hamster Ovary Cells
title_short DNA Double-Strand Breaks Affect Chromosomal Rearrangements during Methotrexate-Mediated Gene Amplification in Chinese Hamster Ovary Cells
title_sort dna double-strand breaks affect chromosomal rearrangements during methotrexate-mediated gene amplification in chinese hamster ovary cells
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8000239/
https://www.ncbi.nlm.nih.gov/pubmed/33809068
http://dx.doi.org/10.3390/pharmaceutics13030376
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