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

Departments of Pharmacology [J. P. M., G. A. B., G. J. G.] and Medicine [G. J. G.], and the Interdepartmental Division of Oncology [G. J. G.], University of Toronto, Toronto, Ontario M5G 1L4, Canada

Previous studies using cloned lines of Adriamycin-sensitive and -resistant P388 murine leukemia cells have suggested that a reduction in DNA topoisomerase II (topo II) enzyme activity and protein levels in drug-resistant 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 II gene (A. M. Deffie, D. J. Bosman, 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 II 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 II transcript, we have determined that the shorter 4.5-kilobase topo II 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 II until nucleotide position 3494, at which point the sequence diverges for the remaining 956 bases; (b) contains a polyadenylation signal distinct from the native transcript; and (c) contains an open reading frame predicting a truncated topo II fusion protein. Of great interest was the finding that the non-topo II 956-base sequence in the shorter transcript encodes the promoter, exon I, and part of the first intron of the murine retinoic acid receptor 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 II and retinoic acid receptor .

¹ This 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.

Received 9/24/93. Accepted 11/10/93.

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