Summary A panel of doxorubicin-resistant sublines of the human small-cell lung carcinoma cell line GLC4 displays decreasing DNA topoisomerase Ila (TopoIla) mRNA levels with increasing resistance. In the present study we describe how this decrease may be regulated. No significant differences in TopoIla mRNA stability or gene arrangement were found, using mRNA slot-blotting and Southern blotting, in the most resistant cell line compared with the parental cell line. To investigate if Topolla gene copy loss contributed to the mRNA decrease, fluorescence in situ hybridisation using a TopoIllx-specific probe was performed. During doxorubicin resistance development, the composition of the population in each cell line shifted with increasing resistance, from a population in which most cells contain three TopoIla gene copies (GLC4) to a population in which most cells contain only two copies. A partial revertant of the most resistant cell line displayed a shift back to the original situation. We conclude that the Topolla gene copy number decrease per cell line is in good agreement with the decreased TopolIa mRNA and protein levels, and Topoll activity levels in these cell lines which were described previously.

Breast cancer is a genetically complex disease. Fluorescence in situ hybridisation can be used to analyse the genetics of breast-cancer progression in interphase cytogenetics. We have analysed the histological distribution of erbB2 and topoll alpha co-amplification in paraffin sections of invasive breast cancer and show that the co-amplified loci share the same histological distribution in the tumour and have a similar nuclear distribution within individual nuclei. Regions of the tumours without amplification are easily recognized and tumours with erbB2 and topoll alpha co-amplification can be distinguished from those with erbB2 amplification alone. In addition, FISH was used to show polysomy of chromosome 17 in non-invasive ductal carcinoma in situ of the breast and erbB2 amplification in both the invasive and non-invasive components of a breast cancer biopsy. This report of an interphase cytogenetic analysis of non-invasive breast carcinoma in situ demonstrates the usefulness of FISH for the genetic study of breast cancer progression.

Summary 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 IIax and ERBB2 sequences, for the structure of the amplicon and for expression of both topoisomerase Ila and P. The al., 1989;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 (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)

Summary The molecular cytogenetic techniques of comparative genomic hybridization (CGH) and reverse in situ hybridization (REVISH) allow the entire genomes of tumours to be screened for genetic changes without the requirement for specific probes or markers. In order to define the ability of REVISH to detect and map regions of amplification associated with drug resistance, we investigated a panel of cell lines selected for resistance to doxorubicin and intrinsic sensitivity to topoisomerase 11-inhibitory drugs. We have defined a modified REVISH protocol, which involves double hybridizations with genomic DNA from the test cell lines and chromosome-specific whole chromosome paints to identify the chromosomes to which the amplicons localize. Sites of amplification are then mapped by fractional length measurements (Flpter), using published genome databases. Our findings show that amplification of the topoisomerase lla gene is readily detected and mapped, as is amplification of the MDR and MRP loci. Interestingly, REVISH detected a new amplicon in the doxorubicin-resistant lung cancer cell line, GLC4-ADR, which mapped to chromosome 1 q. REVISH is therefore ideally suited to characterize genetic changes specific for drug resistance within a background of genetic anomalies associated with tumour progression.Keywords: molecular cytogenetics; fluorescence in situ hybridization; chromosome painting; drug resistance; gene amplification; gene mapping; reverse in situ hybridization Many tumours respond to a range of cytotoxic agents. However, resistance often develops (van der Zee et al, 1995;Harrison, 1995). Understanding the mechanisms of resistance may provide new therapeutic options (Kastan et al, 1995;FroelichAmmon and Osheroff, 1995). At the cellular level, a number of resistance mechanisms can potentially operate. These mechanisms include drug efflux via membrane pumps, such as p-glycoprotein or multidrug resistance protein (MRP), drug metabolism, including inactivation or failure to activate a prodrug, an alteration in abundance of the target protein, for example topoisomerase II (topoll) enzyme, mutation of target protein and inactivation of pathways leading to cell death, such as apoptotic signalling (Booser and Hortobagyi, 1994;Harrison, 1995;Kastan et al, 1995;van der Zee et al, 1995). It is likely that in any one particular tumour, response to therapy is dependent on concurrent expression of multiple mechanisms. Even for extensively studied drugs, such as etoposide, which is a known topoll inhibitor, resistance is a complex issue (Su et al, 1992;Takano et al, 1992;Booser and Hortobagyi, 1994;Chen and Liu, 1994;Pommier et al, 1994;Sinha, 1995). The recent association of reduced kinesin expression with etoposide resistance, as identified by a genetic suppressor element approach, highlights the usefulness and requirement for new approaches to define potential components of the drug resistance repertoire (Gudkov et al, 1994;Roninson et al, 1995).A major drawback to many of the conventional approaches used to investig...