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Research ArticleChemotherapy, Antibiotics, and Gene Therapy

Alternative RNA Processing of Topoisomerase IIα in Etoposide-Resistant Human Leukemia K562 Cells: Intron Retention Results in a Novel C-Terminal Truncated 90-kDa Isoform

Ragu Kanagasabai, Lucas Serdar, Soumendrakrishna Karmahapatra, Corey A. Kientz, Justin Ellis, Mary K. Ritke, Terry S. Elton and Jack C. Yalowich
Journal of Pharmacology and Experimental Therapeutics January 2017, 360 (1) 152-163; DOI: https://doi.org/10.1124/jpet.116.237107
Ragu Kanagasabai
Division of Pharmacology, College of Pharmacy, The Ohio State University, Columbus, Ohio (R.K., L.S., S.K., C.A.K., J.E., T.S.E., J.C.Y.); James Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio (J.C.Y.); and Department of Biology, University of Indianapolis, Indianapolis, Indiana (M.K.R.)
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Lucas Serdar
Division of Pharmacology, College of Pharmacy, The Ohio State University, Columbus, Ohio (R.K., L.S., S.K., C.A.K., J.E., T.S.E., J.C.Y.); James Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio (J.C.Y.); and Department of Biology, University of Indianapolis, Indianapolis, Indiana (M.K.R.)
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Soumendrakrishna Karmahapatra
Division of Pharmacology, College of Pharmacy, The Ohio State University, Columbus, Ohio (R.K., L.S., S.K., C.A.K., J.E., T.S.E., J.C.Y.); James Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio (J.C.Y.); and Department of Biology, University of Indianapolis, Indianapolis, Indiana (M.K.R.)
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Corey A. Kientz
Division of Pharmacology, College of Pharmacy, The Ohio State University, Columbus, Ohio (R.K., L.S., S.K., C.A.K., J.E., T.S.E., J.C.Y.); James Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio (J.C.Y.); and Department of Biology, University of Indianapolis, Indianapolis, Indiana (M.K.R.)
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Justin Ellis
Division of Pharmacology, College of Pharmacy, The Ohio State University, Columbus, Ohio (R.K., L.S., S.K., C.A.K., J.E., T.S.E., J.C.Y.); James Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio (J.C.Y.); and Department of Biology, University of Indianapolis, Indianapolis, Indiana (M.K.R.)
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Mary K. Ritke
Division of Pharmacology, College of Pharmacy, The Ohio State University, Columbus, Ohio (R.K., L.S., S.K., C.A.K., J.E., T.S.E., J.C.Y.); James Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio (J.C.Y.); and Department of Biology, University of Indianapolis, Indianapolis, Indiana (M.K.R.)
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Terry S. Elton
Division of Pharmacology, College of Pharmacy, The Ohio State University, Columbus, Ohio (R.K., L.S., S.K., C.A.K., J.E., T.S.E., J.C.Y.); James Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio (J.C.Y.); and Department of Biology, University of Indianapolis, Indianapolis, Indiana (M.K.R.)
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Jack C. Yalowich
Division of Pharmacology, College of Pharmacy, The Ohio State University, Columbus, Ohio (R.K., L.S., S.K., C.A.K., J.E., T.S.E., J.C.Y.); James Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio (J.C.Y.); and Department of Biology, University of Indianapolis, Indianapolis, Indiana (M.K.R.)
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Abstract

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 19/intron 19 read-through. Immunoassays, utilizing antisera raised against these unique amino acids, confirmed that TOP2α/90 is expressed in both cell types, with overexpression in K/VP.5 cells. Immunodetection of complex of enzyme-to-DNA and single-cell gel electrophoresis (Comet) assays demonstrated that K562 cells transfected with a TOP2α/90 expression plasmid exhibited reduced etoposide-mediated TOP2α–DNA covalent complexes and decreased etoposide-induced DNA damage, respectively, compared with similarly treated K562 cells transfected with empty vector. Because TOP2α/90 lacks the active site tyrosine (Tyr805) of full-length TOP2α, these results strongly suggest that TOP2α/90 exhibits dominant-negative properties. Further studies are underway to characterize the mechanism(s) by which TOP2α/90 plays a role in acquired resistance to etoposide and other TOP2α targeting agents.

Footnotes

    • Received August 9, 2016.
    • Accepted November 4, 2016.
  • dx.doi.org/10.1124/jpet.116.237107.

  • Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics: 360 (1)
Journal of Pharmacology and Experimental Therapeutics
Vol. 360, Issue 1
1 Jan 2017
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Research ArticleChemotherapy, Antibiotics, and Gene Therapy

Intron Retention Results in a Novel TOP2α/90-kDa Isoform

Ragu Kanagasabai, Lucas Serdar, Soumendrakrishna Karmahapatra, Corey A. Kientz, Justin Ellis, Mary K. Ritke, Terry S. Elton and Jack C. Yalowich
Journal of Pharmacology and Experimental Therapeutics January 1, 2017, 360 (1) 152-163; DOI: https://doi.org/10.1124/jpet.116.237107

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Research ArticleChemotherapy, Antibiotics, and Gene Therapy

Intron Retention Results in a Novel TOP2α/90-kDa Isoform

Ragu Kanagasabai, Lucas Serdar, Soumendrakrishna Karmahapatra, Corey A. Kientz, Justin Ellis, Mary K. Ritke, Terry S. Elton and Jack C. Yalowich
Journal of Pharmacology and Experimental Therapeutics January 1, 2017, 360 (1) 152-163; DOI: https://doi.org/10.1124/jpet.116.237107
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