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

Pharmaceutics ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 376
Author(s):  
Jong Youn Baik ◽  
Hye-Jin Han ◽  
Kelvin H. Lee
Keyword(s):  
Cell Lines ◽  
Gene Amplification ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chromosomal Rearrangements ◽  
Chinese Hamster ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
The Impact

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.

scholarly journals Inducible expression and cytogenetic effects of the EcoRI restriction endonuclease in Chinese hamster ovary cells.

1988 ◽  
Vol 8 (10) ◽  
pp. 4204-4211 ◽  
Author(s):  
W F Morgan ◽  
M L Fero ◽  
M C Land ◽  
R A Winegar
Keyword(s):  
Restriction Endonuclease ◽  
Chromosome Aberrations ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Gene Promoter ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Bacterial Gene ◽  
Strand Breaks

The cytogenetic endpoints sister chromatid exchange (SCE) and chromosome aberrations are widely used as indicators of DNA damage induced by mutagenic carcinogens. Chromosome aberrations appear to result directly from DNA double-strand breaks, but the lesion(s) giving rise to SCE formation remains unknown. Most compounds that induce SCEs induce a spectrum of lesions in DNA. To investigate the role of double-strand breakage in SCE formation, we constructed a plasmid that gives rise to one specific lesion, a staggered-end ("cohesive") DNA double-strand break. This plasmid, designated pMENs, contains a selectable marker, neo, which is a bacterial gene for neomycin resistance, and the coding sequence for the bacterial restriction endonuclease EcoRI attached to the mouse metallothionein gene promoter. EcoRI recognizes G decreases AATTC sequences in DNA and makes DNA double-strand breaks with four nucleotides overhanging as staggered ends. Cells transfected with pMENS were resistant to the antibiotic G418 and contained an integrated copy of the EcoRI gene, detectable by DNA filter hybridization. The addition of the heavy metal CdSO4 resulted in the intracellular production of EcoRI, as measured by an anti-EcoRI antibody. Cytogenetic analysis after the addition of CdSO4 indicated a dramatic increase in the frequency of chromosome aberrations but very little effect on SCE frequency. Although there was some intercellular heterogeneity, these results confirm that DNA double-strand breaks do result in chromosome aberrations but that these breaks are not sufficient to give rise to SCE formation.

Inducible expression and cytogenetic effects of the EcoRI restriction endonuclease in Chinese hamster ovary cells

1988 ◽  
Vol 8 (10) ◽  
pp. 4204-4211
Author(s):  
W F Morgan ◽  
M L Fero ◽  
M C Land ◽  
R A Winegar
Keyword(s):  
Restriction Endonuclease ◽  
Chromosome Aberrations ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Gene Promoter ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Bacterial Gene ◽  
Strand Breaks

The cytogenetic endpoints sister chromatid exchange (SCE) and chromosome aberrations are widely used as indicators of DNA damage induced by mutagenic carcinogens. Chromosome aberrations appear to result directly from DNA double-strand breaks, but the lesion(s) giving rise to SCE formation remains unknown. Most compounds that induce SCEs induce a spectrum of lesions in DNA. To investigate the role of double-strand breakage in SCE formation, we constructed a plasmid that gives rise to one specific lesion, a staggered-end ("cohesive") DNA double-strand break. This plasmid, designated pMENs, contains a selectable marker, neo, which is a bacterial gene for neomycin resistance, and the coding sequence for the bacterial restriction endonuclease EcoRI attached to the mouse metallothionein gene promoter. EcoRI recognizes G decreases AATTC sequences in DNA and makes DNA double-strand breaks with four nucleotides overhanging as staggered ends. Cells transfected with pMENS were resistant to the antibiotic G418 and contained an integrated copy of the EcoRI gene, detectable by DNA filter hybridization. The addition of the heavy metal CdSO4 resulted in the intracellular production of EcoRI, as measured by an anti-EcoRI antibody. Cytogenetic analysis after the addition of CdSO4 indicated a dramatic increase in the frequency of chromosome aberrations but very little effect on SCE frequency. Although there was some intercellular heterogeneity, these results confirm that DNA double-strand breaks do result in chromosome aberrations but that these breaks are not sufficient to give rise to SCE formation.

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