Abstract
DNA topoisomerase IIβ (TOP2β/180; 180 kDa) is a nuclear enzyme that regulates DNA topology by generation of short-lived DNA double-strand breaks, primarily during transcription. TOP2β/180 can be a target for DNA damage-stabilizing anticancer drugs, whose efficacy is often limited by chemoresistance. Our laboratory previously demonstrated reduced levels of TOP2β/180 (and the paralog TOP2α/170) in an acquired etoposide-resistant human leukemia (K562) clonal cell line, K/VP.5, in part due to overexpression of microRNA-9-3p/5p impacting post-transcriptional events. To evaluate the effect on drug sensitivity upon reduction/elimination of TOP2β/180, a premature stop codon was generated at the TOP2β/180 gene exon 19/intron 19 boundary (AGAA//GTAA→ATAG//GTAA) in parental K562 cells (which contain four TOP2β/180 alleles) by CRISPR/Cas9 editing with homology-directed repair to disrupt production of full-length TOP2β/180. Gene-edited clones were identified and verified by quantitative polymerase chain reaction and Sanger sequencing, respectively. Characterization of TOP2β/180 gene-edited clones, with one or all four TOP2β/180 alleles mutated, revealed partial or complete loss of TOP2β mRNA/protein, respectively. The loss of TOP2β/180 protein correlated with decreased (2-{4-[(7-chloro-2-quinoxalinyl)oxy]phenoxy}propionic acid)-induced DNA damage and partial resistance in growth inhibition assays. Partial resistance to mitoxantrone was also noted in the gene-edited clone with all four TOP2β/180 alleles modified. No cross-resistance to etoposide or mAMSA was noted in the gene-edited clones. Results demonstrated the role of TOP2β/180 in drug sensitivity/resistance in K562 cells and revealed differential paralog activity of TOP2-targeted agents.
SIGNIFICANCE STATEMENT Data indicated that CRISPR/Cas9 editing of the exon 19/intron 19 boundary in the TOP2β/180 gene to introduce a premature stop codon resulted in partial to complete disruption of TOP2β/180 expression in human leukemia (K562) cells depending on the number of edited alleles. Edited clones were partially resistant to mitoxantrone and XK469, while lacking resistance to etoposide and mAMSA. Results demonstrated the import of TOP2β/180 in drug sensitivity/resistance in K562 cells and revealed differential paralog activity of TOP2-targeted agents.
Footnotes
- Received November 21, 2023.
- Accepted February 27, 2024.
This work was supported by the National Institutes of Health Office of Extramural Research [Grant R01-CA226906-01A1].
The authors declare that they have no conflicts of interest with the contents of this article.
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- Copyright © 2024 by The American Society for Pharmacology and Experimental Therapeutics
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