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Expression of topoisomerase II alpha and beta in an adenocarcinoma cell line carrying amplified topoisomerase II alpha and retinoic acid receptor alpha genes.

Human topoisomerase II enzymes are targets for a number of widely used anticancer agents. We have analysed a lung adenocarcinoma cell line CALU3, which has co-amplified topoisomerase II alpha and ERBB2 sequences, for the structure of the amplicon and for expression of both topoisomerase II alpha and beta. The region of chromosome 17q amplified in CALU3 also includes the retinoic acid receptor alpha locus and is therefore similar to the amplicon observed in breast cancers carrying amplified topoisomerase II alpha and retinoic acid receptor sequences. The use of fluorescence in situ hybridisation localises the amplified topoisomerase II alpha sequences to a cluster on one chromosome with single copies localised to others. CALU3 express high levels of topoisomerase II alpha is determined by Western blot, immunofluorescence and enzyme activity. The enzyme activity extracted from CALU3 is sensitive to inhibition by the topoisomerase II poison etoposide. Topoisomerase II beta expression was observed in three lung cancer cell lines including CALU3 and was confined to the nucleoli. Thus, the CALU3 cell line is an ideal model to study the amplification and expression of topoisomerase II alpha in adenocarcinomas. ImagesFigure 1Figure 2Figure 3Figure 4Figure 5Figure 6

chromosome 17q amplified in CALU3 also includes the retinoic acid receptor a locus and is therefore similar to the amplicon observed in breast cancers carrying amplified topoisomerase IIa and retinoic acid receptor sequences. The use of fluorescence in situ hybridisation localises the amplified topoisomerase Ila sequences to a cluster on one chromosome with single copies localised to others. CALU3 expresses high levels of topoisomerase Ila as determined by Western blot, immunofluorescence and enzyme activity. The enzyme activity extracted from CALU3 is sensitive to inhibition by the topoisomerase II poison etoposide.
Topoisomerase Ip expression was observed in three lung cancer cell lines including CALU3 and was confined to the nucleoli. Thus, the CALU3 cell line is an ideal model to study the amplification and expression of topoisomerase Ila in adenocarcinomas.
Topoisomerase II enzymes localised in the nucleus catalyse the breakage and rejoining of DNA (Takano et al., 1992). The enzyme cuts a gate in the DNA through which a second molecule of DNA can pass. This DNA manipulation is vital to a number of cellular processes such as chromosome condensation, replication and segregation as well as gene transcription and topoisomerase II activity is associated with all these processes (Anderson & Roberge, 1992).
Topoisomerase II activity in human cells can be attributed to the expression of two distinct genes. Topoisomerase II alpha is localised to chromosome 17q bands 21-22 (Tsai-Pflugfelder et al., 1988) whereas topoisomerase II beta is localised to chromosome 3p24 Jenkins et al., 1992). Both genes are functional but their products differ in sequence and biochemical activity (Jenkins et al., 1992;Drake et al., 1989). Expression of the topoisomerase II alpha gene is cell cycle regulated in contrast to the relatively constant expression of the beta gene product Woessner et al., 1991). Recent studies have also indicated that the alpha and beta enzymes are sublocalised within the nucleus to the nucleoplasm and nucleoli respectively (Negri et al., 1992).
Interest in topoisomerase II is due both to its essential catalytic activity in normal cells and that it is a key target for a group of anticancer agents including etoposide, doxorubicin and mAMSA (Takano et al., 1992;Liu, 1989). Topoisomerase II interactive drugs interfere with the normal catalytic cycle of the enzyme resulting in cell death. The quantity of topoisomerase II within a cell will determine its sensitivity to topoisomerase inhibitors with high levels conferring sensitivity to the cytotoxic effects of the drugs (Takano et al., 1992). Indeed, one cell line selected for sensitivity to doxorubicin has increased expression of topoisomerase II compared to its parental line (Davies et al., 1988). In addition, yeast cells which over express topoisomerase II encoded by a plasmid are hypersensitive to topoisomerase II inhibitors (Nitiss et al., 1992). In contrast, cell lines selected for resistance to the cytotoxic actions of topoisomerase inhibitors express low levels of topoisomerase or produce a mutated protein with altered catalytic activity (Takano et al., 1992). Due to the clinical use of topoisomerase II inhibitors (Muggia & Gill, 1991) the levels of expression in tumours may be important in determining the success of the treatment. Molecular changes at topoisomerase loci which result in altered expression are therefore important and recently it has been shown that the topoisomerase II alpha gene is co-amplified along with ERBB2 in a subset of breast adenocarcinomas (Keith et al., 1993).
We have previously shown that the lung adenocarcinoma cell line CALU3, has co-amplification of ERBB2 and topoisomerase Ilcc, (Keith et al., 1992). This cell line therefore provides a model to examine the role of topoisomerase Ila amplification and expression in drug sensitivity. We have now investigated the expression, localisation and enzymatic activity of both topoisomerase Ila and P in CALU3 and further characterised the amplicon containing the topoisomerase Ila gene. We show here that the amplified topoisomerase II alpha gene in CALU3 is expressed at a high level and that enzyme activity is inhibited by a topoisomerase II interactive drug. Immunofluorescence studies using antibodies against topoisomerase II alpha and beta show the alpha product to be expressed heterogeneously within the cell population. In contrast, the beta isoform is expressed in all cells and localised to the nucleolus. In addition, we show by fluorescence in situ hybridisation (FISH), that the topoisomerase II alpha amplification in CALU3 is intrachromosomal and clustered in one region with single gene copies also detectable on other chromosomes. In agreement with the observations in breast cancer, the chromosome 17q amplicon in CALU3 contains at least three genes, namely ERBB2, topoisomerase IIa and RARa and is a good model for studying molecular alterations around the topoisomerase IIa locus in breast cancer and their effects on gene expression.

Materials and methods
Cell lines and cytotoxicity assays The human non-small cell lung carcinoma cell lines CALU3 and SK-MES were obtained from the American Type Culture Collection (ATCC Rockville, MD). L-DAN is a squamous lung cancer cell line established in our own laboratory. Cells were grown in a mixture of Hams F10 and Dulbecco's modified Eagles medium (50:50, Life Technologies, Paisley, Scotland) supplemented with glutamine (2 mM) and foetal calf serum (10%). The three lines CALU3, SK-MES and L-DAN, have ICs values for etoposide of 2.3 x 10-8M, 81-120 x 10-8 and 92-110 x 10-8M respectively and IC5o values for doxorubicin of 2.7 x 10-9M, 48-53 x 10-9M and 34-70 x 10-9M respectively (Merry et al., 1987). Confirmation of the sensitivity of CALU3 to topoisomerase II inhibitory drugs was determined by a tetrazolium dye based microtitration assay as described previously (Plumb et al., 1989).
Topoisomerase II alpha probe and probe labelling The topoisomerase II alpha probe used for in situ hybridisation was a 35 kilobase cosmid (Hochhauser et al., 1992) kindly provided by Dr Ian Hickson, (ICRF, Oxford). Cosmid DNA was labelled with digoxigenin-ll-dUTP by nick translation according to the manufacturer's instructions, (Boehringer Mannheim). Labelled cosmid was precipitated in the presence of a 50-fold excess of human COTI DNA and resuspended in hybridisation buffer consisting of 50% formamide, 2 x SSC (1 x SSC is 0.15 M NaCl, 0.015 M sodium citrate pH 7), 500 fg ml-' salmon sperm DNA, 5% dextran sulphate, at a concentration of 2 ng LI-'.
In situ hybridisation (Pinkel et al., 1986, Nederlof et al., 1992 Metaphase spreads were fixed in 3: 1 methanol, acetic acid for 1 h and then treated with RNAse (1I00 tg ml-') for 1 h. Slides were then fixed in 1% paraformaldehyde in PBS and dehydrated. Chromosomes were denatured prior to hybridisation in 70% formamide/2 x SSC at 70°C for 2 min and dehydrated. Probe was denatured at 70°C for 5 min and allowed to reanneal at 37°C for 1 h. Ten microlitres of probe was applied to denatured chromosomes and hybridisation was carried out under a sealed coverslip overnight in a humidified chamber.
Slides were washed for 20 min in 0.1 M maleic acid, 0.15 M NaCl, 0.05% Tween 20, pH 7.5. The second antibody, FITC-conjugated donkey anti-sheep (Jackson ImmunoResearch), diluted in the above blocking buffer at 3 lig ml1 l was applied to slides for 30 min, 37°C. Slides were then washed for 30 min, counterstained with propidium iodide (0.4 yg ml-') and mounted in anti-fade medium (Vectashield, Vector Labs). Fluorescence was analysed on a Bio-Rad MRC-600 laser scanning confocal microscope equipped with a krypton/ argon ion laser using 488/568 nm line excitation and dual channel 522 nm and 585 nm emission filters.
Immunofluorescence Cells were cultured on multi-chamber slides at low cell density, rinsed in PBS and fixed in acetone for I min at room temperature. Slides were blocked in 3% BSA in PBS, 0.05% Tween 20 for 10 min prior to the addition of antibodies. Polyclonal antiserum against topoisomerase II alpha was obtained from TopoGen (Columbus Ohio), the monoclonal antibody to topoisomerase II beta was kindly provided by Dr G.C.B. Astaldi Ricotti (Negri et al., 1992). Both antibodies were diluted 1:20 in 3% BSA in PBS, 0.05% Tween. Incubations were for 2 h at room temperature. FITC-conjugated donkey anti-rabbit and FITC-conjugated sheep anti-mouse antibodies (Jackson Immunoresearch), at 30 tgml1' in 3% BSA, PBS, 0.05% Tween 20, were applied for 30 min at room temperature to detect the polyclonal alpha antiserum and the monoclonal beta antibody respectively. Slides were washed for 15 min in PBS, 0.05% Tween 20, counterstained, mounted and analysed by confocal microscopy as described above. In order to gain quantitative, comparative data, image acquisition parameters were set and remained unaltered during image collection by confocal microscopy. Pre-incubation of the alpha polyclonal antiserum with its complimentary peptide (TopoGen, Columbus Ohio) abolishes antigen binding and can be used to calibrate the image detection parameters.
Topoisomerase II assays Topoisomerase activity was extracted from cell lines as described by van der Zee et al. (1991). Topoisomerase II activity was quantified using a kit obtained from TopoGen (Columbus, Ohio). Decatenation assays were carried out according to the manufacturer's instructions. Inhibition of topoisomerase II activity by etoposide was carried out by addition of the drug to the decatenation assays in 2 l.t of DMSO, (final concentration of DMSO is 2%, also added to controls). Assay products were resolved by agarose gel electrophoresis and stained with 0.5 ytg ml-' ethidium bromide. The gels were then photographed and the negatives contact print-

Western blot analysis
Cell extracts containing topoisomerase II activity were resolved on 6% polyacrylamide gels and transferred to Immobilon-P membranes. To detect topoisomerase II alpha by Western blot analysis a polyclonal antibody obtained from Cambridge Research Biochemicals (Cheshire, UK) was used as previously described (Keith et al., 1993). Densitometry was performed using a Molecular Dynamics laser scanning densitometer.
Southern blot analysis Genomic DNA was isolated from cell lines as previously described (Keith et al., 1992), and digested with restriction enzymes according to the manufacturer's directories. Hybridisations were carried out as described previously (Keith et al., 1992). The probe SPI was used to detect topoisomerase IIa sequences (Chung et al., 1989), pHPKC-alpha 7 to detect PKCcx (Parker et al., 1986), p63 to detect RARx and pHJi was used as a control for DNA loading (Keith et al., 1992). The RARo and PKCx probes were obtained from the ATCC.

Results
Detection of RARa and topoisomerase II a gene amplification by Southern blot analysis Previously, we have shown ERBB2 and topoisomerase IIa to be co-amplified in CALU3 (Keith et al., 1992). We have now expanded this analysis to a number of other chromosome 17q loci.
DNA extracted from CALU3 cell line was analysed for amplifcation of genes on chromosome 17q. Figure 1 shows a Southern blot of DNA extracted from the lung cancer cell lines, SK-MES (lane 1); L-DAN (lane 2); CALU3 (lane 3). The filter was sequentially hybridised to analyse the copy number of (a) topoisomerase IME; (b) RARa; (c) Protein kinase Co (PKCa); (d) pHJi loading control. The topoisomerase IIa and RARa loci have previously been localised to chromosome 17q 21-22 (Tsai-Pflugfelder et al., 1988;Mattei et al., 1988). The PKCa locus has been mapped to chromosome 17q 22-24, (Parker et al., 1986). Figure 1 shows that in addition to the topoisomerase IIa locus the RARa locus is amplified in CALU3. Hybridisation of the filter to detect PKCa shows that the increased copy number of RARa and topoisomerase IIa is not due to aneuploidy but to amplification, as CALU3 shows no evidence of increased copy numbers of PKCo sequences. Densitometry of the autoradiographs in Figure 1 shows topoisomerase IIa and RARa sequences to be amplified four fold in CALU3 compared to the other lines.
Analysis of topoisomerase II alpha amplification by fluorescence in situ hybridisation (FISH) In order to further characterise the nature of amplified topoisomerase Ila sequences in CALU3 we have used fluorescence in situ hybridisation. As shown in Figure 2a, hybridisation of the topoisomerase II alpha cosmid to chromosomes prepared from control lymphocytes detects the two alleles as expected. In the metaphase spread shown all four chromatids are labelled. Figure 2b shows hybridisation of topoisomerase II alpha to a metaphase spread prepared from CALU3. Fluorescent signal is observed on several chromosomes with a cluster of signals localised to one chromosome suggesting that the amplified sequences are intra-chromosomal. There was no evidence for extra-chromosomal sequences. The pattern of hybridisation was consistent between nuclei within the cell line. Each nucleus had five copies of topoisomerase IIa, three of which were clustered on one chromosome. Occas-  sionally cells were found with seven regions of hybridisation of which the two additional hybridisations may represent a low level of hybridisation to telomeric repeat sequences. The regions of hybridisation marked by arrows in Figure 2b represent topoisomerase II alpha sequences in CALU3, the remaining two regions of hybridisation are telomeric and due to the complex karyotype of the cell line, cannot be unambiguously assigned as topoisomerase alpha as the probe used contains repeat sequences which can hybridise to telomeric sequences. However, under the conditions of hybridisation used, the intensity of any telomeric hybridisation in lymphocyte controls is much lower than the topoisomerase Ilo signal (data not shown).

Analysis of topoisomerase II expression by Western blot and immunofluorescence
The CALU3 cell line is relatively sensitive to topoisomerase II inhibitory agents etoposide and doxorubicin when compared 'to other lung cancer cell lines (see Materials and methods, Merry et at., 1987;Carmichael et al., 1985: Giaccone et at., 1992. In order to investigate whether expression of the amplified topoisomerase II alpha gene is responsible for the sensitivity to topoisomerase inhibitors, expression of the alpha and beta genes was examined by Western blot analysis and immunofluorescence. For comparison with CALU3, two other non-small cell lung cancer cell lines, L-DAN and SK-MES, which do not have amplified topo-isomerase II genes (Keith et al., 1992) were included in the analysis. Figure 3 shows a Western blot of topoisomerase II alpha expression in L-DAN (lane 1), CALU3 (lane 2) and Figure 3 it can be seen that CALU3 expresses topoisomerase II alpha at higher levels than the other cell lines. Densitometric analysis of the autoradiographs shows CALU3 to express 10-15 times more topoisomerase II alpha than the other lines. Therefore amplification of the topoisomerase II alpha gene in CALU3 is accompanied by high levels of expression.

SK-MES (lane 3). From
The high levels of topoisomerase II alpha expression in CALU3 was confirmed by immunofluorescence using a second polyclonal antibody as described in the Materials and methods. Figure 4a, c, and e, shows topoisomerase II alpha expression in optical section of L-DAN, CALU3 and SK-MES respectively. The nuclei are counterstained red with propidium iodide and the topoisomerase II alpha expression in CALU3, as visualised by the green fluorescence, is confined to the nucleus as expected. In contrast to Western blot analysis, immunofluorescent detection of the alpha protein in CALU3 highlights the heterogeneity in alpha expression between cells (Figure 4c). Topoisomerase II alpha expression is known to be cell cycle regulated but whether the observed heterogeneity in expression is due to this or sub-population diversity is untested. The lower levels of topoisomerase II alpha expression observed in L-DAN and SK-MES by Western blot analysis were also confirmed by immunofluorescence. However, in order to maintain the optimal conditions for visualisation of the alpha protein in CALU3, alpha expression in the other two lines shown in Figure 4a and e is almost undetectable due to the settings for digital image analysis used for this experiment (see Materials and methods). The topoisomerase II beta enzyme is also a target for topoisomerase interactive drugs . We therefore analysed the three cell lines for beta expression to complete their topoisomerase II isoenzyme profiles. We were unable to generate consistent results on beta expression by Western blot analysis due to the instability of the protein during extraction. The instability of the beta protein has been reported on several occasions (Woessner et al., 1990;Negri et al., 1992;Holden et al., 1992). However, there were no major differences in the levels of the 150 kD protein between the three cell lines. There is no information on how the P protein degrades and so different cell lines may show varying stability of the P protein. In order to circumvent this problem, beta expression was analysed by immunofluorescence. Figure 4b, d and f shows expression of topoisomerase II beta in L-DAN, CALU3 and SK-MES respectively. Beta expression is reported to be largely confined to the nucleolus (Negri et al., 1992) and the data presented in Figure 4 confirms this. All three lines express the beta isozyme and it is expressed in every cell in the population. Immunocytochemistry is only semi-quantitative and due to the intense localised fluorescence pattern obtained when analysing expression of the beta protein, differences in expression levels between the three lines is difficult to assess. Figure 4e shows that using the polyclonal antiserum against the alpha protein, low levels of fluorescence can be detected in the nucleoli. Whether this represents a weak cross-reactivity of this antiserum with the beta protein or co-localisation of the alpha enzyme is not known.
Biochemical analysis of topoisomerase II activity In order to determine whether the high levels of topoisomerase II alpha protein observed in CALU3 has enzyme activity, decatenation assays were carried out. Topoisomerase II activity can be measured due to its ability to monomerise covalently linked kinetoplast DNA circles. Decatenation of kinetoplast DNA requires both the DNA breakage and strand passage activities of topoisomerase II. The decatenation assay does not however distinguish between the alpha and beta isoforms. Figure 5 shows that CALU3 has at least eight-fold more topoisomerase II activity than the other two  Figure 5 are the same as those used for the Western analysis of topoisomerase II alpha expression shown in Figure 3. The results of both analyses are in agreement with each other and show CALU3 to express high levels of topoisomerase II. Western blot analysis of the extracts used in the biochemical analysis for beta expression detected degraded beta protein of 150 kD (data not shown). The 150 kD breakdown product of the beta enzyme has been observed previously and shown to be active in the biochemical assays (Negri et al., 1992). Thus, some of the topoisomerase II activity detected may be due to the Ji isoform.
The topoisomerase II activity in protein extracts from CALU3 is largely due to alpha enzyme expression (Figures 3,  4 and 5). The alpha gene is amplified in CALU3 (Figures 1  and 2) and so it is possible that it might contain genetic alterations rendering its protein insensitive to inhibition by topoisomerase II interactive drugs (Takano et al., 1992). We therefore examined the ability of the topoisomerase II interactive drug etoposide to inhibit topoisomerase II activity in decatenation assays. Figure 6 shows that etoposide can inhibit topoisomerase II activity in extracts from all three cell lines, including CALU3. Due to the higher levels of topoisomerase II activity in CALU3, only 1.25 jg of extract was used per reaction in comparison to 2.5 jig for L-DAN and SK-MES.

Discussion
The adenocarcinoma cell line CALU3 has the co-amplification of topoisomerase II alpha and ERBB2 sequences characteristic of a subset of breast adenocarcinomas (Keith et al., 1992(Keith et al., , 1993. We therefore examined CALU3 as a suitable model for the study of expression of topoisomerase II alpha from an amplified locus and its consequences for cellular sensitivity to topoisomerase II inhibitory drugs. The amplified topoisomerase II alpha gene in CALU3 is expressed at high levels as determined by both Western blot analysis and immunofluorescence, (Figures 3 and 4). Two distinct antibodies were used to examine alpha expression by Western and immunofluorescence therefore confirming by independent methods that the isoform detected is indeed the alpha product. Neither of the other two cell lines tested had such high levels of alpha expression (Figures 3 and 4). CALU3 has five copies of the topoisomerase IIa gene ( Figure  2b) yet a 10-15-fold increase in expression over the other two cell lines. This suggests there may be further complexity in the regulation of the topoisomerase IIa gene at the transcriptional or post-transcriptional level. The human genome has the potential to express a second topoisomerase II isozyme encoded by the topoisomerase II beta gene. There is still a paucity of data on the beta enzyme but since its cloning, recent chromosomal mapping and the generation of monoclonal antibodies against it, progress should now become more rapid (Jenkins et al., 1992;Negri et al., 1992). It is known however that the beta enzyme is expressed, sensitive to inhibition by topoisomerase II interactive drugs and likely to be confined to the nucleolus (Jenkins et al., 1992;Drake et al., 1989;Negri et al., 1992). Any model system used to study topoisomerase II inhibitors would therefore benefit from the analysis of both alpha and beta expression. In contrast to the alpha locus in CALU3, none of the three lung cancer cell lines used in this study have gross chromosomal changes at the beta locus (Keith et al., 1992). By immunofluorescence there is little detectable difference in beta expression between the cell lines, with all lines expressing the beta isozyme in every cell (Figure 4). The localisation of the beta product to the nucleoli confirms the data of Negri et al., 1992 and suggests a role for the beta product in ribosomal gene expression or organisation (Kim & Wang, 1989). The data presented in Figure 4 on beta expression suggest that the beta product represents a target for inhibition and may be a target for topoisomerase II poisons in the absence of alpha isozyme expression.
In addition to the correlation between alpha gene amplification in CALU3 and high levels of alpha expression, biochemical assays for topoisomerase II activity confirmed a concomitant elevated level of activity which could be inhibited by etoposide (Figures 4 and 5). Taken together, the Western blot analysis, immunofluorescence studies and the enzyme activity assays suggest that the sensitivity of CALU3 to topoisomerase inhibitors (Merry et al., 1987) is due to an amplified and over-expressed topoisomerase II alpha gene. The finding that in CALU3, expression of an amplified topoisomerase II alpha gene can confer relative sensitivity to the cytotoxic effects of topoisomerase II inhibitors is of importance to tumours where topoisomerase II alpha gene amplification has occurred, as these cases may benefit preferentially from treatment with topoisomerase II inhibitors.
We have previously shown that the region of amplification found in primary breast tumours encompasses at least three loci including ERBB2, RARa and topoisomerase IIa. The PKCa locus was found to be outside the region of amplification showing that the increased copy number of the three genes is due to amplification rather than aneuploidy (Keith et al., 1993). We have also previously shown that the lung adenocarcinoma cell line, CALU3, has co-amplification ERBB2 and topoisomerase IIa (Keith et al., 1992). As part of our efforts to characterise similarities between the genetic events found around the topoisomerase IIa locus in breast cancer biopsies and the CALU3 cell line, we have shown that the RARa locus is also amplified in CALU3 (Figure 1). By Southern analysis, topoisomerase Ila and RARa are amplified around 4-fold. The finding that the locus for PKCoa is not within the amplicon, is again consistent with the observations in breast cancer (Keith et al., 1992) and demonstrates that the increased copy number of ERBB2, RARa and topoisomerase IIa in CALU3 is not due to aneuploidy. The RARx receptor regulates normal cellular proliferation and differentiation through activation by retinoids, (Bollag & Holdener, 1992) and can, when expressed as a fusion protein in acute promyelocytic leukaemia act as an oncogene. (Clarkson, 1991). Gebert et al. (1991) have shown the RARa gene to be expressed at high levels in CALU3 and so CALU3 may be a suitable model to study retinoids in cancer therapy, (Bollag & Holdener, 1992).
The finding that topoisomerase II alpha can be amplified and expressed in adenocarcinomas opens the question as to whether there is intra-tumour heterogeneity in topoisomerase Ila gene amplification and its consequences for the sensitivity of subpopulations within the tumour to cytotoxic agents. Fluorescence in situ hybridisation is an ideal approach to study this heterogeneity. Indeed, it has recently been shown by FISH that in breast cancer biopsies there is considerable intra-tumour variation in ERBB2 copy number (Kallioniemi et al., 1992). Our demonstration that amplification of topoisomerase II alpha can be studied by FISH (Figure 2), opens up the possibility of examining the importance of topoisomerase gene amplification and allele imbalance at the single cell level.