Characterization of a DNA topoisomerase IIalpha gene rearrangement in adriamycin-resistant P388 leukemia: expression of a fusion messenger RNA transcript encoding topoisomerase IIalpha and the retinoic acid receptor alpha locus.
Previous studies using cloned lines of Adriamycin-sensitive and -resistant P388 murine leukemia cells have suggested that a reduction in DNA topoisomerase II alpha (topo II alpha) 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 alpha 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 alpha 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 alpha transcript, we have determined that the shorter 4.5-kilobase topo II alpha 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 alpha 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 alpha fusion protein. Of great interest was the finding that the non-topo II alpha 956-base sequence in the shorter transcript encodes the promoter, exon I, and part of the first intron of the murine retinoic acid receptor alpha 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 alpha and retinoic acid receptor alpha.
McPherson
J P
JP
Department of Pharmacology, University of Toronto, Ontario, Canada.
Brown
G A
GA
Goldenberg
G J
GJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004250:DNA Topoisomerases, Type II / Q000235:genetics*; D004317:Doxorubicin / Q000494:pharmacology*; D004351:Drug Resistance; D015321:Gene Rearrangement; D007941:Leukemia P388 / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis*; D018168:Receptors, Retinoic Acid / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D014407:Tumor Cells, Cultured
D012333:RNA, Messenger; D018168:Receptors, Retinoic Acid; D011993:Recombinant Fusion Proteins; D004317:Doxorubicin; D004250:DNA Topoisomerases, Type II