title: 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.
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.
authors.affiliation: 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.).
authors.forename: Ragu
authors.initials: R
authors.lastname: Kanagasabai
authors.affiliation: 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.).
authors.forename: Lucas
authors.initials: L
authors.lastname: Serdar
authors.affiliation: 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.).
authors.forename: Soumendrakrishna
authors.initials: S
authors.lastname: Karmahapatra
authors.affiliation: 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.).
authors.forename: Corey A
authors.initials: CA
authors.lastname: Kientz
authors.affiliation: 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.).
authors.forename: Justin
authors.initials: J
authors.lastname: Ellis
authors.affiliation: 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.).
authors.forename: Mary K
authors.initials: MK
authors.lastname: Ritke
authors.affiliation: 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.) yalowich.1@osu.edu elton.8@osu.edu.
authors.forename: Terry S
authors.initials: TS
authors.lastname: Elton
authors.affiliation: 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.) yalowich.1@osu.edu elton.8@osu.edu.
authors.forename: Jack C
authors.initials: JC
authors.lastname: Yalowich
mesh_terms: D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000951:Antigens, Neoplasm / Q000737:chemistry / Q000235:genetics*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001483:Base Sequence; D004250:DNA Topoisomerases, Type II / Q000737:chemistry / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D005047:Etoposide / Q000494:pharmacology*; D006801:Humans; D007438:Introns / Q000235:genetics*; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics; D020014:K562 Cells; D058990:Molecular Targeted Therapy; D008970:Molecular Weight; D012323:RNA Processing, Post-Transcriptional / Q000187:drug effects*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion
chemical_list: D000951:Antigens, Neoplasm; D000970:Antineoplastic Agents; D004268:DNA-Binding Proteins; D007527:Isoenzymes; D012333:RNA, Messenger; D005047:Etoposide; D004250:DNA Topoisomerases, Type II