{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,27]],"date-time":"2026-09-27T11:12:55Z","timestamp":1790507575556,"version":"4.1.0"},"reference-count":92,"publisher":"Public Library of Science (PLoS)","issue":"5","license":[{"start":{"date-parts":[[2022,5,26]],"date-time":"2022-05-26T00:00:00Z","timestamp":1653523200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000054","name":"National Cancer Institute","doi-asserted-by":"publisher","award":["R01 CA226906-01A1"],"award-info":[{"award-number":["R01 CA226906-01A1"]}],"id":[{"id":"10.13039\/100000054","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:p>DNA Topoisomerase II\u03b1 (TOP2\u03b1\/170) is an enzyme essential for proliferating cells. For rapidly multiplying malignancies, this has made TOP2\u03b1\/170 an important target for etoposide and other clinically active anticancer drugs. Efficacy of these agents is often limited by chemoresistance related to alterations in TOP2\u03b1\/170 expression levels. Our laboratory recently demonstrated reduced levels of TOP2\u03b1\/170 and overexpression of a C-terminal truncated 90-kDa isoform, TOP2\u03b1\/90, due to intronic polyadenylation (IPA; within intron 19) in an acquired etoposide-resistant K562 clonal cell line, K\/VP.5. We previously reported that this isoform heterodimerized with TOP2\u03b1\/170 and was a determinant of acquired resistance to etoposide. Optimization of the weak TOP2\u03b1 exon 19\/intron 19 5\u2032 splice site in drug-resistant K\/VP.5 cells by gene-editing restored TOP2\u03b1\/170 levels, diminished TOP2\u03b1\/90 expression, and circumvented drug resistance. Conversely, in the present study, silencing of the exon 19\/intron 19 5\u2032 splice site in parental K562 cells by CRISPR\/Cas9 with homology-directed repair (HDR), and thereby forcing intron 19 retention, was used to induce resistance by disrupting normal RNA processing (i.e., gene knockout), and to further evaluate the role of TOP2\u03b1\/170 and TOP2\u03b1\/90 isoforms as resistance determinants. Gene-edited clones were identified by quantitative polymerase chain reaction (qPCR) and verified by Sanger sequencing. TOP2\u03b1\/170 mRNA\/protein expression levels were attenuated in the TOP2\u03b1 gene-edited clones which resulted in resistance to etoposide as assessed by reduced etoposide-induced DNA damage (\u03b3H2AX, Comet assays) and growth inhibition. RNA-seq and qPCR studies suggested that intron 19 retention leads to decreased TOP2\u03b1\/170 expression by degradation of the TOP2\u03b1 edited mRNA transcripts. Forced expression of TOP2\u03b1\/90 in the gene-edited K562 cells further decreased etoposide-induced DNA damage in support of a dominant negative role for this truncated isoform. Together results support the important role of both TOP2\u03b1\/170 and TOP2\u03b1\/90 as determinants of sensitivity\/resistance to TOP2\u03b1-targeting agents.<\/jats:p>","DOI":"10.1371\/journal.pone.0265794","type":"journal-article","created":{"date-parts":[[2022,5,26]],"date-time":"2022-05-26T17:46:21Z","timestamp":1653587181000},"page":"e0265794","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":5,"title":["Use of CRISPR\/Cas9 with homology-directed repair to silence the human topoisomerase II\u03b1 intron-19 5\u2019 splice site: Generation of etoposide resistance in human leukemia K562 cells"],"prefix":"10.1371","volume":"17","author":[{"given":"Victor 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