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 (original) (raw)
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Molecular Pharmacology, 2021
An essential function of DNA topoisomerase IIα (TOP2α; 170 kDa, TOP2α/170) is to resolve DNA topologic entanglements during chromosome disjunction by introducing transient DNA double-stranded breaks. TOP2α/170 is an important target for DNA damage-stabilizing anticancer drugs, whose clinical efficacy is compromised by drug resistance often associated with decreased TOP2α/170 expression. We recently demonstrated that an etoposide-resistant K562 clonal subline, K/VP.5, with reduced levels of TOP2α/170, expresses high levels of a novel C-terminal truncated TOP2α isoform (90 kDa, TOP2α/90). TOP2α/90, the translation product of a TOP2α mRNA that retains a processed intron 19 (I19), heterodimerizes with TOP2α/170 and is a resistance determinant through a dominant-negative effect on drug activity. We hypothesized that genome editing to enhance I19 removal would provide a tractable strategy to circumvent acquired TOP2α-mediated drug resistance. To enhance I19 removal in K/VP.5 cells, CRISPR...
Cancers
Intronic polyadenylation (IPA) plays a critical role in malignant transformation, development, progression, and cancer chemoresistance by contributing to transcriptome/proteome alterations. DNA topoisomerase IIα (170 kDa, TOP2α/170) is an established clinical target for anticancer agents whose efficacy is compromised by drug resistance often associated with a reduction of nuclear TOP2α/170 levels. In leukemia cell lines with acquired resistance to TOP2α-targeted drugs and reduced TOP2α/170 expression, variant TOP2α mRNA transcripts have been reported due to IPA that resulted in the translation of C-terminal truncated isoforms with altered nuclear-cytoplasmic distribution or heterodimerization with wild-type TOP2α/170. This review provides an overview of the various mechanisms regulating pre-mRNA processing and alternative polyadenylation, as well as the utilization of CRISPR/Cas9 specific gene editing through homology directed repair (HDR) to decrease IPA when splice sites are intri...
Effects of DNA topoisomerase IIα splice variants on acquired drug resistance
Cancer Drug Resistance, 2020
DNA topoisomerase IIα (170 kDa, TOP2α/170) induces transient DNA double-strand breaks in proliferating cells to resolve DNA topological entanglements during chromosome condensation, replication, and segregation. Therefore, TOP2α/170 is a prominent target for anticancer drugs whose clinical efficacy is often compromised due to chemoresistance. Although many resistance mechanisms have been defined, acquired resistance of human cancer cell lines to TOP2α interfacial inhibitors/poisons is frequently associated with a reduction of Top2α/170 expression levels. Recent studies by our laboratory, in conjunction with earlier findings by other investigators, support the hypothesis that a major mechanism of acquired resistance to TOP2α-targeted drugs is due to alternative RNA processing/splicing. Specifically, several TOP2α mRNA splice variants have been reported which retain introns and are translated into truncated TOP2α isoforms lacking nuclear localization sequences and subsequent dysregulated nuclear-cytoplasmic disposition. In addition, intron retention can lead to truncated isoforms that lack both nuclear localization sequences and the active site tyrosine (Tyr805) necessary for forming enzyme-DNA covalent complexes and inducing DNA damage in the presence of TOP2α-targeted drugs. Ultimately, these truncated TOP2α isoforms result in decreased drug activity against TOP2α in the nucleus and manifest drug resistance. Therefore, the complete characterization of the mechanism(s) regulating the alternative RNA processing of TOP2α pre-mRNA may result in new strategies to circumvent acquired drug resistance. Additionally, novel TOP2α splice variants and truncated TOP2α isoforms may be useful as biomarkers for drug resistance, prognosis, and/or direct future TOP2α-targeted therapies.
The Journal of pharmacology and experimental therapeutics, 2017
DNA topoisomerase IIα (TOP2α) is a prominent target for anticancer drugs whose clinical efficacy is often limited by chemoresistance. Using antibody specific for the N-terminal of TOP2α, immunoassays indicated the existence of two TOP2α isoforms, 170 and 90 kDa, present in K562 leukemia cells and in an acquired etoposide (VP-16)-resistant clone (K/VP.5). TOP2α/90 expression was dramatically increased in etoposide-resistant K/VP.5 compared with parental K562 cells. We hypothesized that TOP2α/90 was the translation product of novel alternatively processed pre-mRNA, confirmed by 3'-rapid amplification of cDNA ends, polymerase chain reaction, and sequencing. TOP2α/90 mRNA includes retained intron 19, which harbors an in-frame stop codon, and two consensus poly(A) sites. The processed transcript is polyadenylated. TOP2α/90 mRNA encodes a 90,076-Da translation product missing the C-terminal 770 amino acids of TOP2α/170, replaced by 25 unique amino acids through translation of the exon...
Molecular pharmacology, 2018
DNA topoisomerase II (170 kDa, TOP2/170) is essential in proliferating cells by resolving DNA topological entanglements during chromosome condensation, replication, and segregation. We previously characterized a C-terminally truncated isoform (TOP2/90), detectable in human leukemia K562 cells but more abundantly expressed in a clonal subline, K/VP.5, with acquired resistance to the anticancer agent etoposide. TOP2/90 (786 aa) is the translation product of a TOP2 mRNA that retains a processed intron 19. TOP2/90 lacks the active-site tyrosine-805 required to generate double-strand DNA breaks as well as nuclear localization signals present in the TOP2/170 isoform (1531 aa). Here, we found that TOP2/90, like TOP2/170, was detectable in the nucleus and cytoplasm of K562 and K/VP.5 cells. Coimmunoprecipitation of endogenous TOP2/90 and TOP2/170 demonstrated heterodimerization of these isoforms. Forced expression of TOP2/90 in K562 cells suppressed, whereas siRNA-mediated knockdown of TOP2...
Ectopic expression of human topoisomerase IIα fragments and etoposide resistance in mammalian cells
International Journal of Cancer, 2000
Cellular resistance to etoposide has been correlated both with reduced levels and an aberrant cytoplasmic accumulation of the drug's target, topoisomerase II␣ (topo II␣). It is not known, however, whether a cytoplasmic pool of topo II␣ is sufficient to confer drug resistance on cultured mammalian cells. In our study, we have transfected mouse fibroblasts and human 293 cells with truncated forms of human topo II␣ fused to GFP and have examined the transformants for the subcellular localization of topo II␣ and their resistance to etoposide. Transient transfection resulted in high-level expression of all GFP-topo II␣ fusions tested, whereas in stably transfected cells the levels varied significantly. Transfectants expressing a central or a carboxy-terminal topo II␣ domain (aa 428-1504, 639-1028 or 1028-1504) accumulated high levels of the fusion proteins, while only very low amounts of GFP-topo II␣ proteins were observed in cell lines expressing constructs that retain the amino-terminus of the enzyme (aa 1-1214, aa 1-939, aa 1-611). Our results suggest that the topo II␣ amino-terminus affects the stability of truncated forms of the enzyme in mammalian cells, perhaps due to targeted degradation. Assays that screen for cell vitality and DNA synthesis reveal no significant changes in etoposide sensitivity in transfected cells expressing high levels of cytoplasmic or nuclear localized topo II fusion proteins. Retroviral expression of a cytoplasmically anchored domain of human topo II␣ also failed to confer drug resistance. These results suggest that a cytoplasmic pool of topo II␣ is not sufficient to render cultured mammalian cells drug resistant.
British journal of cancer, 1994
K562 leukaemia cells were selected for resistance using 0.5 microM etoposide (VP-16). Cloned K/VP.5 cells were 30-fold resistant to growth inhibition by VP-16 and 5- to 13-fold resistant to m-AMSA, adriamycin and mitoxantrone. K/VP.5 cells did not overexpress P-glycoprotein; VP-16 accumulation was similar to that in K562 cells. VP-16-induced DNA damage was reduced in cells and nuclei from K/VP.5 cells compared with K562 cells. Topoisomerase II protein was reduced 3- to 7-fold and topoisomerase II alpha and topoisomerase II beta mRNAs were each reduced 3-fold in resistant cells. After drug removal, VP-16-induced DNA damage disappeared 1.7 times more rapidly and VP-16-induced DNA-topoisomerase II adducts dissociated 1.5 times more rapidly in K/VP.5 cells than in K562 cells. ATP (1 mM) was more effective in enhancing VP-16-induced DNA damage in nuclei isolated from sensitive cells than in nuclei from resistant cells. In addition, ATP (0.3-5 mM) stimulated VP-16-induced DNA-topoisomeras...
Clinical Cancer Research, 2004
Purpose: The purpose of the study was to investigate the mechanisms associated with antitumor activity and resistance to F11782, a novel dual catalytic inhibitor of topoisomerases with DNA repair-inhibitory properties. Experimental Design: For that purpose, an F11782resistant P388 leukemia subline (P388/F11782) has been developed in vivo and characterized. Results: Weekly subtherapeutic doses of F11782 (10 mg/kg) induced complete resistance to F11782 after 8 weekly passages. This resistant P388/F11782 subline retained some in vivo sensitivity to several DNA-topoisomerase II and/or I complex-stabilizing poisons and showed marked collateral sensitivity to cisplatin, topotecan, colchicine, and Vinca alkaloids, while proving completely cross-resistant only to merbarone and doxorubicin. Therefore, resistance to F11782 did not appear to be associated with a classic multidrug resistance profile, as further reflected by unaltered drug uptake and no overexpression of resistance-related proteins or modification of the glutathione-mediated detoxification process. In vivo resistance to F11782 was, however, associated with a marked reduction in topoisomerase II␣ protein (87%) and mRNA (50%) levels, as well as a diminution of the catalytic activity of topoisomerase II␣. In contrast, only minor reductions in topoisomerases II and I levels were recorded. However, of major interest, nucleotide excision repair activity was decreased 3-fold in these P388/ F11782 cells and was more specifically associated with a decreased (67%) level of XPG (human xeroderma pigmen-tosum group G complementing protein), an endonuclease involved in this DNA repair system. Conclusions: These findings suggest that both topoisomerase II␣ and XPG are major targets of F11782 in vivo and further demonstrate the original mechanism of action of this novel compound.
British journal of cancer, 1996
Drug resistance to anti-tumour agents often coincides with mutations in the gene encoding DNA topoisomerase II alpha. To examine how inactive forms of topoisomerase II can influence resistance to the chemotherapeutic agent VP-16 (etoposide) in the presence of a wild-type allele, we have expressed point mutations and carboxy-terminal truncations of yeast topoisomerase II from a plasmid in budding yeast. Truncations that terminate the coding region of topoisomerase II at amino acid (aa) 750, aa 951 and aa 1044 are localised to both the cytosol and the nucleus and fail to complement a temperature-sensitive top2-1 allele at non-permissive temperature. In contrast, the plasmid-borne wild-type TOP2 allele and a truncation at aa 1236 are nuclear localised and complement the top2-1 mutation. At low levels of expression, truncated forms of topoisomerase II render yeast resistant to levels of etoposide 2- and 3-fold above that tolerated by cells expressing the full-length enzyme. Maximal resi...
Cancer Research
The human small cell lung cancer NCI-H69 cell line selected for resist ance to etoposide (H69/VP) has been reported previously to sequentially overexpress both the MRP and MDK1 multidrug resistance-conferring genes. In addition, immunocytochemistry of H69/VP cells demonstrated a distinct extranuclear localization of the nuclear enzyme topoisomerase Ha, the target of etoposide. Immunoblots showed a decrease in .17, 170.000 topoisomerase Ha in nuclear extracts in H69/VP but equal amounts of the enzyme in whole-cell extracts. Topoisomerase II catalytic activities in H69 and H69/VP whole-cell extracts were equal, as were their inhibition by etoposide. Sequencing of the entire H69/VP topoisomerase Ila cDNA showed a homozygous 9-nucleotide deletion encompassing nucleotides 4468-76, coding for Lys-Ser-Lys, overlapping two potential bipartite nuclear localization signals. The deletion occurred at the initial nine nucleotides of an exon, suggesting alternative splicing of topoisomerase Ha mRNA. Subsequent sequencing of H69/VP genomic DNA revealed a Gâ€">Tpoint mutation in the 3' acceptor splice site consensus sequence, resulting in the use of an alternate splice site. Comparison with previous reports on three drug-resistant cell lines with large truncations/deletions in the COOH-terminal region of topoisomerase Ha and extranuclear localization point to a pivotal role for the basic cluster U90Lys-Ser-Lys1'"2 in the nuclear import of this enzyme.