Shelagh E. L. Mirski

Structural organization of the human TOP2A and TOP2B genes.

Eucaryotic topoisomerase II is an essential nuclear enzyme involved in processes such as chromosome condensation, chromatid separation, and in the relief of torsional stress that occurs during DNA transcription and replication. In cells from vertebrate species, there are two forms of the enzyme, designated alpha and beta. Human topoisomerase IIalpha (TOP2A) is encoded by the TOP2A gene on chromosome 17q21-22, and human topoisomerase IIbeta (TOP2B) is encoded by the TOP2B gene on chromosome 3p24. The protein products of these two genes are important cellular targets of several drugs widely used in the treatment of many human cancers, and a variety of mutations in TOP2A have been associated with the development of drug resistance. In the present study, we have defined the intron-exon structures of TOP2A and TOP2B. TOP2A is approx. 30kb whereas TOP2B is at least 49kb. TOP2A and TOP2B contain 35 and 36 exons, respectively, and both genes contain a high proportion of class 0 introns. Alignment of the amino-acid sequences of the two proteins indicates that the intron-exon organization of the two genes is highly conserved, except for the regions encoding the extreme NH2 and COOH termini of the proteins. These findings suggest strongly that the vertebrate isoforms evolved by duplication of an ancestral gene. Mutations in TOP2A associated with drug resistance show clustering in exons 12, 13, 19-21 and 34-35. Knowledge of the genomic organization of TOP2A and TOP2B will be useful for detection of mutations in clinical samples from patients with drug-resistant malignant disease.

Identification of functional nuclear export sequences in human topoisomerase IIα and β

Abstract Nuclear localization of topoisomerase IIα and β is important for normal cell function as well as being a determinant of tumour cell sensitivity to topoisomerase II-targeting chemotherapeutic agents. However, topoisomerase II is cytoplasmic under certain circumstances, indicating that it may undergo active nuclear export. We have examined the ability of Leu-rich potential nuclear export signal (NES) sequences present in human topoisomerase IIα and β to direct the export of a green fluorescent protein–glutathione- S -transferase fusion protein following microinjection into HeLa cell nuclei. Of 12 sequences tested, only one potential NES sequence from the comparable location in each isoform (αNES 1018–1028 and βNES 1034–1044 ) was active. Mutation of hydrophobic residues in αNES 1018–1028 and βNES 1034–1044 substantially reduced their nuclear export activity as did leptomycin B treatment of microinjected cells. Our results provide the first evidence of active nuclear export of topoisomerase II and suggest it is mediated by a CRM1-dependent pathway.

Reduced levels of topoisomerase IIα and IIβ in a multidrug-resistant lung-cancer cell line

We have previously shown that the doxorubicinselected multidrug-resistant small-cell lung-cancer cell line H69AR is resistant to VP-16-induced single-strand DNA breaks as compared with its parental H69 cell line. Levels of immunoreactive topoisomerase IIα are also reduced in H69AR cells. In the present study, we found that cleaved complex formation in the presence of VP-16 was decreased in H69AR cells as compared with H69 cells. In addition, the resistant cells contained lower levels of both topoisomerase IIα and topoisomerase IIβ protein and mRNA. However, these changes were not accompanied by a decrease in the P4-unknotting (strand-passing) activity of 0.67M NaCl nuclear extracts of H69AR cells, nor was there any difference in VP-16 inhibition of unknotting activity in the H69 and H69AR nuclear extracts. These data suggest that reduced levels of topoisomerase IIα and IIβ may contribute to the resistance of H69AR cells to VP-16 and other drugs that target these isoenzymes.

Nuclear interactions of topoisomerase II α and β with phospholipid scramblase 1

DNA topoisomerase (topo) II modulates DNA topology and is essential for cell division. There are two isoforms of topo II (α and β) that have limited functional redundancy, although their catalytic mechanisms appear the same. Using their COOH-terminal domains (CTDs) in yeast two-hybrid analysis, we have identified phospholipid scramblase 1 (PLSCR1) as a binding partner of both topo II α and β. Although predominantly a plasma membrane protein involved in phosphatidylserine externalization, PLSCR1 can also be imported into the nucleus where it may have a tumour suppressor function. The interactions of PLSCR1 and topo II were confirmed by pull-down assays with topo II α and β CTD fusion proteins and endogenous PLSCR1, and by co-immunoprecipitation of endogenous PLSCR1 and topo II α and β from HeLa cell nuclear extracts. PLSCR1 also increased the decatenation activity of human topo IIα. A conserved basic sequence in the CTD of topo IIα was identified as being essential for binding to PLSCR1 and binding of the two proteins could be inhibited by a synthetic peptide corresponding to topo IIα amino acids 1430-1441. These studies reveal for the first time a physical and functional interaction between topo II and PLSCR1.

A truncated cytoplasmic topoisomerase IIα in a drug-resistant lung cancer cell line is encoded by a TOP2A allele with a partial deletion of exon 34

To study the problem of acquired resistance to widely used anti‐cancer drugs that target the 170 kDa topoisomerase IIα (topo IIα), a drug‐resistant human small‐cell lung cancer cell line, H209/VP, was selected in VP‐16. H209/VP cells express reduced levels of the 170 kDa topo IIα that is localized normally in the nucleus and also express lower levels of a 160 kDa topo IIα‐related protein that is located predominantly in the cytoplasm. Band depletion immunoblotting experiments suggest that the H209/VP nuclear 170 kDa topo IIα is able to form ternary complexes with DNA and VP‐16 in intact cells, but the ability of the cytoplasmic 160 kDa protein to do so is greatly diminished. Sequence analysis of the 3` end of the H209/VP mutant topo IIα mRNA and the TOP2A gene indicates that the mRNA is missing 200 nt that corresponds to exon 34 because the partial loss of the minimal 3` splice‐acceptor sequence at the beginning of exon 34 results in splicing of exon 33 to exon 35. The protein predicted to be encoded by this mutant mRNA does not contain the COOH‐terminal 109 amino acids of the wild‐type enzyme that we have demonstrated contain a strongly functional nuclear localization signal sequence. Consequently, our data explain both the size and the cytoplasmic localization of the H209/VP mutant topo IIα. The mutant TOP2A allele in H209/VP cells differs from those in previously characterized cell lines with cytoplasmic topo IIα and extends the number of types of resistance‐associated deletions in this region to 4. These findings indicate that this region of the TOP2A gene may be a hot spot for mutations. Int. J. Cancer 85:534–539, 2000.

Growth of human×human hybridomas in protein-free medium supplemented with ethanolamine

A protein-free medium was developed which supports the growth of human X human hybridomas derived from the fusion partner KR-4. This medium consists of a 1:1 mixture of Iscoves modified Dulbeccos medium and Hams F-12 medium supplemented with 300 microM ethanolamine. The addition of ethanolamine was essential to maintain cell viability and proliferation. Phosphoethanolamine could not substitute for ethanolamine. Although the maximal cell density achieved in protein-free medium was lower than that in medium supplemented with albumin and transferrin, immunoglobulin production was still supported. This protein-free medium may be useful for large-scale cultivation and for metabolic and immunologic studies of human hybridomas.

Multidrug resistance-associated antigens on drug-sensitive and -resistant human tumour cell lines.

In this paper the biochemical properties of the antigens detected by six murine monoclonal antibodies (MAbs) are described. These MAbs react selectively with the multidrug-resistant small cell lung cancer (SCLC) cell line, H69AR, compared to its sensitive parent cell line, H69 (Mirski &Cole, 1989). Because H69AR cells do not overexpress P-glycoprotein, the antigens detected by these MAbs may be markers for non-P-glycoprotein-mediated mechanisms of resistance. We found that the 36 kDa protein precipitated by MAb 3.186 is phosphorylated and has a pI of approximately 6.7. The 55 kDa protein precipitated by MAb 3.50 is also phosphorylated and has a pI of approximately 5.7. Several observations suggest that MAbs 3.80, 3.177 and 3.187 recognise the same 47 kDa molecule and hence only MAb 3.187 was characterised further. This MAb precipitates an acidic protein which runs as a streak on isoelectric focusing gels. The 25 and 22.5 kDa cell surface proteins precipitated by MAb 2.54 both have a pI of approximately 7.6. Treatment of immunoprecipitates with glycosidase F indicated that none of the proteins detected by MAbs 2.54, 3.187, 3.50 and 3.186 have large N-linked carbohydrates. The peptide nature of the epitopes detected by MAbs 2.54 and 3.186 was unequivocally demonstrated by precipitation from in vitro translation products of H69AR RNA. The antigens detected by MAbs 3.50 and 3.187 were not detectable in immunoprecipitates of translation products but the epitopes are probably peptides because they were destroyed by boiling in sodium dodecyl sulphate. When the reaction of the MAbs with a panel of 15 paired drug-sensitive and -resistant cell lines was examined in a cell enzyme-linked immunosorbent assay, only a few resistance associated reactions were observed. Most of the reactions were either negative or not resistance-associated. When tested with three SCLC cell lines, MAb 3.187 reacted in a manner consistent with the relative resistance of the cell lines. Antigens that had similar electrophoretic mobility to those from H69AR cells were precipitated from extracts of five human cell lines of various tumour types. These data indicate that the cross-reactivities of the MAbs are due to antigens shared among the cell lines and not just the expression of common epitopes on different proteins. Resistance-associated proteins with the biochemical properties of the antigens described in this paper have not been reported previously and they remain potential markers for the as yet to be determined mechanisms of drug resistance in SCLC and other human malignancies.

Simultaneous Quantitation of Topoisomerase II α and β Isoform mRNAs in Lung Tumor Cells and Normal and Malignant Lung Tissue

Certain drugs used in the treatment of lung cancer and other human malignancies are cytotoxic because of their ability to interact with the two isoforms of topoisomerase II (topo II), topo IIα and topo IIβ. As part of an effort to evaluate the contribution of topo II alterations to drug sensitivity and resistance in lung cancer, we have developed a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay to measure levels of topo II α and β mRNAs simultaneously using a single pair of primers with sequences common to both isoforms. The PCR products derived from the topo II α and β mRNAs are both 446 bp but have different electrophoretic mobilities in a nondenaturing polyacrylamide gel, allowing sensitive, rapid quantitation when the products are radiolabeled with [35S]-dATP. Using this RT-PCR method, poly(A+) RNA from 13 non-small cell lung cancer (NSCLC) cell lines was analyzed. The results obtained indicated that the cell lines express a wide range of topo IIα mRNA levels (12-fold) and topo IIβ mRNA levels (5.5-fold). Tumor and normal lung tissues from 25 patients with NSCLC were also examined. In the tumor samples, the levels of the topo II α and β mRNAs were similar. However, mean topo IIα mRNA levels in the tumors were approximately 7-fold higher than those of the paired normal lung tissues. In contrast, topo IIβ mRNA levels were similar in both tumor and normal lung. Topo II α and β mRNA levels were both significantly lower in the squamous cell tumors than in the adenocarcinoma samples. Topo IIβ mRNA levels in the squamous cell tumors were also significantly lower than those in paired normal lung tissue. The RT-PCR method described is reliable and convenient, and for the first time, makes the rapid simultaneous direct comparison of topo IIα and topo IIβ mRNA levels feasible in large numbers of clinical samples.