Selective Use of an Alternative Stop Codon and Polyadenylation Signal within Intron Sequences Leads to a Truncated Topoisomerase IIα Messenger RNA and Protein in Human HL-60 Leukemia Cells Selected for Resistance to Mitoxantrone

Topoisomerase IIα is an essential nuclear enzyme involved in DNA replication and a target for many of the clinically useful antineoplastic agents. In a mitoxantrone-selected human leukemia cell line, HL-60/MX2, cellular topoisomerase II (topo II) catalytic activity is decreased, in association with the finding of reduced nuclear topo IIα and β protein levels. In addition, HL-60/MX2 cells contain a novel M r 160,000 topo IIα-related protein that localizes predominantly to the cell cytoplasm (W. G. Harker et al. , Biochemistry, 30: 9953–9961, 1991). In these studies, we have investigated the molecular mechanisms underlying the altered expression of the topo IIα protein(s) in these cells. Three topo IIα mRNAs, 7.2, 6.3, and 4.8 kb, were identified in the HL-60/MX2 cells, with the 6.3 and 4.8 kb transcripts being present in roughly equivalent amounts, while the 7.2-kb mRNA represents <7% of the total topo IIα-specific mRNA. Portions of the 3′-coding and 3′-untranslated regions were found to be missing from the 7.2- and 4.8-kb topo IIα mRNAs by Northern blot analysis. Sequences encoding the 3′ regions of the normal and truncated forms of the topo IIα enzyme were obtained from the HL-60/MX2 cells through the use of a 3′-rapid amplification of cDNA ends strategy. Approximately 1321 nucleotides are missing from the 3′-coding and 3′-untranslated regions of the 4.8-kb mRNA and are replaced by 122 nucleotides that contain an inframe stop codon and consensus polyadenylation signal. The translation product of the truncated 4388-bp topo IIα transcript would have a predicted M r of 157,850, with 108 COOH-terminal amino acids being replaced by 13 novel residues. Immunoblot analysis confirmed that amino acids in the COOH-terminal region of topo IIα were missing from the M r 160,000 HL-60/MX2 protein, and antisera generated to a synthetic peptide representing the 13 unique amino acids identified a M r 160,000 protein in nuclear extracts from these cells. PCR evaluation of the organization of the 3′ region of the topo IIα gene revealed that the 4.8-kb mRNA found in HL-60/MX2 cells diverges from that of the 6.3-kb mRNA at a consensus exon-intron splice donor site. The 122-bp novel nucleotides identified in the truncated transcript appear to originate from an adjacent intron as a result of altered RNA processing. These studies suggest that as a result of the disruption of the carboxy terminus of the topo IIα protein and the putative nuclear targeting sequences identified therein, cellular localization of the protein is altered, which may confer a growth advantage for the HL-60/MX2 cells in the presence of mitoxantrone.