Use of CRISPR/Cas9 with homology-directed repair to silence the human topoisomerase IIα intron-19 5' splice site: Generation of etoposide resistance in human leukemia K562 cells.
Use of CRISPR/Cas9 with homology-directed repair to silence the human topoisomerase IIα intron-19 5' splice site: Generation of etoposide resistance in human leukemia K562 cells.
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DOI：
10.1371/journal.pone.0265794
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发表时间：
2022
期刊：
影响因子：
3.7
通讯作者：
Elton, Terry S.
中科院分区：
文献类型：
--
作者：
Hernandez, Victor A.;Carvajal-Moreno, Jessika;Wang, Xinyi;Pietrzak, Maciej;Yalowich, Jack C.;Elton, Terry S.
DNA拓扑异构酶IIα(α/170)是细胞增殖所必需的一种酶。对于快速繁殖的恶性肿瘤，这使得TOP2α/170成为依托泊苷和其他临床活性抗癌药物的重要靶点。这些药物的疗效往往受到与TOP2α/170表达水平改变相关的化疗耐药性的限制。我们实验室最近证实，由于内含子多聚腺苷化(内含子19)，在获得的依托泊苷耐药的K562克隆细胞系K/VP中，TOP2α/170的水平降低，而C末端截短的90 kDa亚型TOP2α/90过表达。我们先前报道，这种异构体与TOP2α/170异源二聚体，是对依托泊苷获得性耐药的决定因素。通过基因编辑优化耐药K/VP细胞TOP2α外显子19/内含子19 5‘剪接位点，恢复TOP2α/170水平，降低TOP2α/90表达，避免耐药。相反，在本研究中，利用同源定向修复技术沉默亲本K562细胞的19号外显子/19号内含子5‘剪接点，从而迫使内含子19保留，从而通过中断正常的核糖核酸加工(即基因敲除)来诱导抗性，并进一步评价TOP2α/170和TOP2α/90亚型作为抗性决定因素的作用。基因编辑克隆经定量聚合酶链式反应(QPCR)鉴定，并经Sanger测序验证。TOP2α/170mRNA/蛋白的表达水平在TOP2DNA编辑克隆中减弱，这导致了对依托泊苷的抗性，通过减少依托泊苷诱导的α损伤(γH 2AX，彗星试验)和生长抑制来评估。RNAseq和qPCR研究表明，内含子19的保留导致TOP2α/170mRNA转录产物的降解，从而导致TOP2α/170的表达下降。TOP2DNA/90在基因编辑的K562细胞中的强制表达进一步减少了依托泊苷诱导的α损伤，以支持这种截短的异构体的显性负向作用。综上所述，研究结果支持TOP2α/170和TOP2α/90作为TOP2α靶向药物敏感性/耐药性决定因素的重要作用。
DNA Topoisomerase IIα (TOP2α/170) is an enzyme essential for proliferating cells. For rapidly multiplying malignancies, this has made TOP2α/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 TOP2α/170 expression levels. Our laboratory recently demonstrated reduced levels of TOP2α/170 and overexpression of a C-terminal truncated 90-kDa isoform, TOP2α/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 TOP2α/170 and was a determinant of acquired resistance to etoposide. Optimization of the weak TOP2α exon 19/intron 19 5′ splice site in drug-resistant K/VP.5 cells by gene-editing restored TOP2α/170 levels, diminished TOP2α/90 expression, and circumvented drug resistance. Conversely, in the present study, silencing of the exon 19/intron 19 5′ 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 TOP2α/170 and TOP2α/90 isoforms as resistance determinants. Gene-edited clones were identified by quantitative polymerase chain reaction (qPCR) and verified by Sanger sequencing. TOP2α/170 mRNA/protein expression levels were attenuated in the TOP2α gene-edited clones which resulted in resistance to etoposide as assessed by reduced etoposide-induced DNA damage (γH2AX, Comet assays) and growth inhibition. RNA-seq and qPCR studies suggested that intron 19 retention leads to decreased TOP2α/170 expression by degradation of the TOP2α edited mRNA transcripts. Forced expression of TOP2α/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 TOP2α/170 and TOP2α/90 as determinants of sensitivity/resistance to TOP2α-targeting agents.
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影响因子：
5.3
作者：
Hicks, Martin J.;Mueller, William F.;Hertel, Klemens J.
通讯作者：
Hertel, Klemens J.
影响因子：
7
作者：
Cho SW;Kim S;Kim Y;Kweon J;Kim HS;Bae S;Kim JS
通讯作者：
Kim JS
影响因子：
10.5
作者：
Boutz PL;Bhutkar A;Sharp PA
通讯作者：
Sharp PA
DOI：
10.1042/bcj20160583
发表时间：
2018-01-23
期刊：
The Biochemical journal
影响因子：
--
作者：
Delgado JL;Hsieh CM;Chan NL;Hiasa H
通讯作者：
Hiasa H
影响因子：
5.6
作者：
Ganapathi RN;Ganapathi MK
通讯作者：
Ganapathi MK