Characterization of a DNA Topoisomerase Ila Gene Rearrangement in Adriamycin-resistant P388 Leukemia: Expression of a Fusion Messenger RNA Transcript Encoding Topoisomerase Ha and the Retinoic Acid Receptor a Locus1

Previous studies using cloned lines of Adriamycin-sensitive and -resis tant P3S8 murine leukemia cells have suggested that a reduction in DNA topoisomerase Ila (topo Ila) enzyme activity and protein levels in drugresistant cell lines (A. M. Deffie, J. K. Batra, and G. J. Goldenberg, Cancer Res., 49: 58-62, 1989) may be due to an allelic mutation in the topo Ila gene (A. M. Deffie, D. J. Bosnian, and G. J. Goldenberg, Cancer Res., 49: 6879-6882, 1989). The drug-resistant cell lines P388/ADR/3 and P388/ ADR/7 express a shortened topo Ha mRNA transcript in addition to the native transcript present in the drug-sensitive P388/4 cell line. Using complementary DNA probes derived from the coding sequence and 3' untranslated region of the native mouse topo Ila transcript, we have determined that the shorter 4.5-kilobase topo Ila transcript expressed in the drug-resistant cell lines contains only 3.5-kilobases of topo II sequence from the 5'-terminus onwards. Using a 3'-rapid amplification of cDNA ends strategy, we have cloned cDNAs representing the 3'-termini of both the native and mutant transcripts from both P388/ADR/3 and P388/ ADR/7 cells. DNA sequence analysis revealed that the shorter 4.5-kilobase transcript: (a) encodes topoisomerase Ha until nucleotide position 3494, at which point the sequence diverges for the remaining 956 bases; (/>} contains a polyadenylation signal distinct from the native transcript; and (c) contains an open reading frame predicting a truncated topo Ila fusion protein. Of great interest was the finding that the non-topo Ila 956-base sequence in the shorter transcript encodes the promoter, exon I, and part of the first ¡ninniof the murine retinole acid receptor a gene locus in the antisense orientation, suggesting that a rearrangement on chromosome 11 in the drug-resistant cells led to a gene fusion event between the loci encoding topo Ila and retinole acid receptor a. Introduction DNA topo Ila3 is a common target for many antineoplastic agents, including anthracyclines, epipodophyllotoxins, and acridines. Altered activity and/or reduced levels of topo Ila have been associated with cell lines selected for resistance to topo II-interactive drugs (1). In duction of resistance by topo Il-derived genetic suppressor elements (2) and reversion of drug resistance by transfection oiDrosophila topo II (3) have provided direct evidence that resistance can be mediated by Received 9/24/93; accepted 11/10/93. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This research was supported by operating grants from the National Cancer Institute of Canada, Bristol-Myers Squibb Pharmaceutical Research Institute, and the Max Shore Cancer Research Fund of the Jewish Foundation of Manitoba. The nucleotide sequences reported in this paper have been submitted to the GENBANK/EMBL Data Bank with accession numbers U01915 and U01919. 2 To whom requests for reprints should be addressed, at the Interdepartmental Division of Oncology, University of Toronto, 92 College Street, Toronto, Ontario M5G 1L4, Canada. 3 The abbreviations used are: topo Ila, DNA topoisomerase Ila; ADR, Adriamycin; cDNA, complementary DNA; UTR, untranslated region; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; RARa, retinoic acid receptor a; poly(A)+, polyadenylated; oligo(dT), oligodeoxthymidylic acid. quantitative and qualitative changes in topo II enzyme activity. Sev eral recent reports have proposed that point mutations in the topo ¡la gene are responsible for the appearance of drug-resistant forms of the enzyme (1, 4). Other studies (5-8) have suggested that allelic rear rangements in the topo Ila gene may be responsible for decreased expression of native topo Ila or the expression of drug-resistant forms of the enzyme. Previous studies have shown that both ADR-resistant P388/ADR/3 and P388/ADR/7 cell lines contain reduced topo II en zyme activity and reduced a-isoform gene product (9). ADR-resistant P388/ADR/3 and P388/ADR/7 cell lines contain allelic rearrange ments of the topo ¡la gene that may be responsible for decreased native mRNA encoding topo Ila as well as the appearance of a novel shorter transcript in P388/ADR/3 and P388/ADR/7 cells (5). Here we report that rearrangement of the topo ¡¡a alÃ-elein the drug-resistant cells occurs in the 3'-terminal portion of the gene and that the corre sponding mutant transcript is capable of encoding a novel fusion protein of predicted Mr 127,000 containing topo Ila amino acid se quence from residues 1 to 1148 followed by 31 residues of novel amino acid sequence. Of great interest was the additional finding that the 3'-terminus of the shorter fusion mRNA was transcribed from the promoter, exon I, and part of the first intron of the murine RARa gene locus (10) in an antisense orientation. Taken together, these findings suggest that a rearrangement on mouse chromosome 11 resulted in a topo IlaIRARa gene fusion event that has disrupted one alÃ-eleof both the topo Ila and RARa genes in ADR-resistant murine leukemia P388 cell lines. Materials and Methods Cell Lines and Cultures. Cloned cell lines of ADR-sensitive (P388/4) and -resistant (P388/ADR/3 and P388/ADR/7) leukemia cells were maintained as described previously (5, 9). Cloning of cDNAs Encoding Murine Topo Ha 3'UTR. A 5'-stretch cDNA library (Clontech, Palo Alto, CA) constructed in Àgtll from cDNA derived from the B-cell lymphoblastoid cell line A-20 was screened using standard methodologies (11); probes were derived from the 5.6-kilobase cDNA insert from pBShTOP2 encoding a full-length cDNA of human topo Ila which was kindly provided by Dr. J. C. Wang, Harvard University (12). Purification of positive plaques and subsequent DNA preparation from liquid lysates as well as restriction endonuclease and Southern blot analysis were performed according to standard techniques (11). DNA sequencing from cDNA fragments subcloned into pBluescript II KS+ (Stratagene, La Jolla, CA) was performed with a Sequenase v2.0 kit (United States Biochemical, Cleveland, OH) ac cording to the manufacturer's directions using vector-driven and sequencespecific primers (Biotechnology Service Centre, Hospital for Sick Children, Toronto, Canada). Sequence data was compiled and analyzed using the Uni versity of Wisconsin Genetic Computer Group program (13). RNA Isolation and Amplification of Topo Ha from P388/4 Cells. Poly(A)+ RNA was isolated using the FastTrack mRNA isolation kit (Invitrogen, San Diego, CA) from exponentially growing drug-sensitive P388/4 cells. Mu rine topo II cDNAs spanning the entire coding region and 3' UTR region