SELECTIVE USE OF AN ALTERNATIVE STOP CODON AND POLYADENYLATION SIGNALWITHIN INTRON SEQUENCES LEADS TO A TRUNCATED TOPOISOMERASE II-ALPHA MESSENGER-RNA AND PROTEIN IN HUMAN HL-60 LEUKEMIA-CELLS SELECTED FOR RESISTANCE TO MITOXANTRONE

Topoisomerase II alpha is an essential nuclear enzyme involved in DNA replication and a target for many of the clinically useful antineoplas tic agents. In a mitoxantrone-selected human leukemia cell line, HL-60 /MX2, cellular topoisomerase II (topo II) catalytic activity is decrea sed, in association with the finding of reduced nuclear topo II alpha and beta protein levels. In addition, HL-60/MX2 cells contain a novel M(r) 160,000 topo II alpha-related protein that localizes predominantl y to the cell cytoplasm (W. G. Harker et al., Biochemistry, 30: 9953-9 961, 1991). In these studies, we have investigated the molecular mecha nisms underlying the altered expression of the topo II alpha protein(s ) in these cells. Three topo II alpha mRNAs, 7.2, 6.3, and 4.8 kb, wer e identified in the HL-60/MX2 cells, with the 6.3 and 4.8 kb transcrip ts being present in roughly equivalent amounts, while the 7.2-kb mRNA represents <7% of the total topo II(alpha-specific mRNA. Portions of t he 3'-coding and 3'-untranslated regions were found to be missing from the 7.2- and 4.8-kb topo II alpha mRNAs by Northern blot analysis. Se quences encoding the 3' regions of the normal and truncated forms of t he topo II alpha enzyme were obtained from the HL-60/MX2 cells through the use of a 3'-rapid amplification of cDNA ends strategy. Approximat ely 1321 nucleotides are missing from the 3'-coding and 3'-untranslate d regions of the 4.8-kb mRNA and are replaced by 122 nucleotides that contain an in-frame stop codon and consensus polyadenylation signal. T he translation product of the truncated 4388-bp topo II alpha transcri pt would have a predicted M(r) of 157,850, with 108 COOH-terminal amin o acids being replaced by 13 novel residues. Immunoblot analysis confi rmed that amino acids in the COOH-terminal region of topo II alpha wer e missing from the M(r) 160,000 HL-60/MX2 protein, and antisera genera ted to a synthetic peptide representing the 13 unique amino acids iden tified a M(r) 160,000 protein in nuclear extracts from these cells. PC R evaluation of the organization of the 3' region of the topo II alpha 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 s ite. The 122-bp novel nucleotides identified in the truncated transcri pt appear to originate from an adjacent intron as a result of altered RNA processing. These studies suggest that as a result of the disrupti on of the carboxy terminus of the topo II alpha protein and the putati ve nuclear targeting sequences identified therein, cellular localizati on of the protein is altered, which may confer a growth advantage for the HL-60/MX2 cells in the presence of mitoxantrone.