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

Jong Baik,Kelvin Lee,Hye-Jin Han

doi:10.3390/pharmaceutics13030376

Jong Baik, Kelvin Lee + Show 1 more

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https://doi.org/10.3390/pharmaceutics13030376

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Abstract

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.

Highlights

To test whether double-strand breaks (DSBs) lead to chromosomal rearrangements in Chinese hamster ovary (CHO) cells, we treated
Understanding chromosomal rearrangement formation in CHO cells is important because chromosomal instability and heterogeneity can affect cell linestability, such as the productivity and product quality of recombinant proteins
We tested the effect of MTX- and bleomycin-mediated DSBs on chromosomal rearrangement formation in CHO cells

Summary

Introduction

Methotrexate (MTX)-mediated gene amplification approaches have been widely used in Chinese hamster ovary (CHO) cells to achieve high productivity of recombinant proteins [1,2]. Co-transfecting Dhfr and a product gene, such as a gene encoding a monoclonal antibody or cytokine, to Dhfr-deficient CHO cells, followed by MTX treatment with a step-wise increase in concentration, provides an effective way to amplify the gene of interest, or the product gene [5,6,7,8]. Dhfr amplification occurs mostly on the same chromosome arm as the original locus [9], and sequence analyses of Dhfr-amplified regions in the CHO genome have revealed a large palindromic structure along with short inverted repeats (head-to-head or tail-to-tail arrays), suggesting bridge–break–fusion cycles presumably resulting from sister chromatid exchange [10,11,12]. The causes and underlying mechanisms of MTX-mediated gene amplification are not fully understood

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