Summary Growth of cells in vitro in the presence of fatty acids can alter the membrane composition and hence fluidity and permeability. Exposure of both doxorubicin (2780AD) and cisplatin (2780CP) resistant human ovarian cell lines to non-toxic concentrations of polyunsaturated fatty acids (y-linolenic acid and eicosapentaenoic acid) either before or during exposure to the cytotoxic drug did not modulate drug sensitivity. However, the fatty acids were toxic in their own right. Whilst the ovarian cell lines 2780AD and 2780CP showed a small degree of cross resistance to both fatty acids the doxorubicin resistant breast cell line MCF7/Adr was slightly more sensitive than MCF7. When the interactions between the polyunsaturated fatty acids and cytotoxic drugs were analysed by the isobologram method the toxicities were shown to be additive.

Summary Aerobic sensitivity to indoloquinone E09 has been shown to correlate with cellular levels of the two-electron reducing enzyme DT-diaphorase. However, little is known about the relative roles of one-and two-electron reducing enzymes in the hypoxic cytotoxicity of E09. We have characterised a panel of 23 human tumour cell lines for both bioreductive enzyme activities and aerobic sensitivity to E09. Eight cell lines were then selected for a comparison of aerobic and hypoxic sensitivities. Activities of DT-diaphorase showed a wide range (> 10,000-fold), while activities of the one-electron reducing cytochrome b5 and cytochrome P450 reductases were generally lower and showed only a 15-and 25-fold range respectively. The aerobic cytotoxicity of E09 was clearly related to the cellular levels of DT-diaphorase (r = 0.87), with higher levels giving increased sensitivity, but not to the levels of one-electron reducing enzymes. In contrast, there was no relationship between sensitivity to BCNU, cisplatin or the bioreductive agent SR 4233 (tirapazamine) and activities of any of these reducing enzymes. Under hypoxic conditions sensitivity to E09 was markedly increased in cell lines with low levels of DT-diaphorase activity, while cell lines with high levels show only a small increase in sensitivity. This is reflected by a clear correlation (r = 0.98) between cellular DT-diaphorase activity and the ratio of aerobic to hypoxic sensitivity to E09. However, we have now for the first time demonstrated an inverse correlation (r = 0.93) between the cellular activity of DT-diaphorase and hypoxic sensitivity to E09, that is sensitivity decreases with increasing DT-diaphorase activity. Moreover, this correlation was lost when cells were exposed to drug in the presence of dicoumarol, supporting an involvement of DT-diaphorase in this relationship. These observations question the previously straightforward role for DT-diaphorase in the metabolic activation of E09. Whereas DT-diaphorase is associated with increased toxicity in air, it appears to reduce the cytotoxicity of E09 in hypoxic conditions. This suggests either that the one-electron reduction product of E09 metabolism, the semiquinone, is more toxic than the two-electron reduction product, the hydroquinone, or that the hydroquinone is not cytotoxic and aerobic toxicity is due to the transient appearance of the semiquinone upon back oxidation of the hydroquinone.

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)

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Experiments were undertaken using an ovarian adenocarcinoma cell line (A2780) and a drug-resistant strain (A2780.ad) derived from this line. P-glycoprotein could not be detected in A2780 cells but was essentially ubiquitous in A2780.ad cells, although removing the selective pressure for drug resistance led to reduced expression. However, the amount of P-glycoprotein present was used to predict the capacity of these cells to extrude rhodamine-123 (R-123) and their resistance to adriamycin, a cytotoxic drug. This accords with the role of P-glycoprotein as a drug pump. Although hypotonic solutions increased anion efflux from A2780 and A2780.ad cells, larger responses occurred in the parental line. Moreover, R-123 extrusion and anion efflux appeared to be mutually independent processes and so these data do not support the view that P-glycoprotein is involved in the control of volume-sensitive anion channels. Hypotonic solutions increased intracellular free calcium ([Ca2+]i) in drug-resistant cells but not in the parental line, and so establishing a drug-resistant strain may affect the control of [Ca2+]i during osmotic swelling. This could account for effects that were previously attributed to P-glycoprotein.