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To study the evolution of camptothecin (CPT) resistance, we have established two small-cell lung cancer cell lines with low (3.2-fold, NYH/CAM15) and high (18-fold, NYH/CAM50) resistance to CPT by stepwise drug exposure. NYH/CAM50 cells had reduced topoisomerase I (topo I) content and activity, and consequently CPT-induced DNA single strand breaks (SSBs) were reduced, as measured by alkaline elution. In contrast, NYH/CAM15 cells had identical topo I content and activity as compared with wild-type (wt) cells. CPT-mediated SSBs and the rate of their reversal after drug removal were also equal in wt and NYH/CAM15 cells, as were doubling time, the fraction of cells in S-phase and DNA synthesis rate in response to CPT. As the conversion of DNA SSBs to DNA double strand breaks (DSBs) is thought to represent a critical event leading to cell death, we measured DNA DSBs by neutral elution. In contrast to DNA SSBs, CPT induced fewer DNA DSBs in NYH/CAM15 than in wt cells. DNA flow cytometry showed that, in CPT-treated cells, the G1 phase was emptied as cells accumulated in late S- and G2M phase. A Spearman rank correlation showed that depletion of G1 and accumulation in late S and G2M correlated to CPT sensitivity in these three cell lines. In conclusion, acquired resistance to CPT can occur without a reduction in either topo I enzyme or CPT-induced cleavable complex formation, while a decrease in the level of CPT-induced DNA DSBs may be of major importance in the early stages of CPT resistance.

Non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC) differ significantly in their clinical response to topoisomerase IIalpha (topo-IIalpha)-directed drugs, such as etoposide and teniposide, as NSCLC is virtually insensitive to single-agent therapy, while SCLC responds in two-thirds of cases. Preclinical studies have indicated that resistance to topo-IIalpha drugs depends on topo-IIalpha content and/or activity, the altered-topo-II multidrug resistance phenotype (at-MDR) and/or one of two different drug efflux pumps, P-glycoprotein (P-gp) and the multidrug resistance protein (MRP). Immunohistochemical analysis on paraffin-embedded tissue from 27 cases of untreated NSCLC and 29 cases of untreated SCLC (of which additional tumour biopsies after treatment with topo-IIalpha-directed drugs were available in ten cases) yielded the following results: NSCLC had significantly less topo-IIalpha than SCLC (P < 0.0001), as only 5 out of 27 NSCLC cases had > 5% positive cells compared with 28 out of 29 SCLC, and 0 out of 27 NSCLC had > 25% positive cells compared with 26 out of 29 SCLC. P-gp was detected in > 5% of cells in only 3 out of 27 NSCLC and in 6 out of 29 SCLC, and MRP in 5 out of 27 of NSCLC and 9 out of 29 SCLC. After treatment of patients with SCLC with either etoposide or teniposide, which are topo-IIalpha-directed drugs, there was an increase in MRP (P < 0.1) and P-gp (P < 0.05) positivity, while topo-IIalpha decreased (P < 0.05). In conclusion, the major difference between untreated NSCLC and SCLC was in topo-IIalpha content. In the small series of ten patients treated for SCLC, all three MDR phenotypes appeared to increase.

The acquisition of drug-resistant tumour cells is the main problem in the medical treatment of a range of malignant diseases. In recent years, three new classes of anti-cancer agents, each with a novel mechanism of action, have been brought forward to clinical trials. These are the topoisomerase I (topo I) poisons topotecan and irinotecan, which are both camptothecin derivatives, the taxane tubulin stabilizers taxol and taxotere and, finally, the antimetabolite gemcitabin, which is active in solid tumours. The process of optimizing their use in a combination with established agents is very complex, with numerous possible drug and schedule regimens. We describe here how a broad panel of drug-resistant small-cell lung cancer (SCLC) cell lines can be used as a model of tumour heterogeneity to aid in the selection of non-cross-resistant regimens. We have selected low-fold (3-10x) drug-resistant sublines from a classic (NCI-H69) and a variant (OC-NYH) SCLC cell line. The resistant cell lines include two sublines with different phenotypes towards alkylating agents (H69/BCNU and NYH/CIS), two sublines with different phenotypes against topo I poisons (NYH/CAM and NYH/TPT) and three multidrug resistant (MDR) sublines (H69/DAU, NYH/VM, and H69/VP) with combinations of mdr1 and MRP overexpression as well as topoisomerase II (topo II) down-regulation or mutation. Sensitivity to 20 established and new agents was measured in a standardized clonogenic assay. Resistance was highly drug specific. Thus, none of the cell lines was resistant to all drugs. In fact, all resistant cell lines exhibited patterns of collateral sensitivity to various different classes of drugs. The most intriguing pattern was collateral sensitivity to gemcitabin in two cell lines and to ara-C in five drug-resistant cell lines, i.e. in all lines except the lines resistant to topo I poisons. Next, all sensitivity patterns in the nine cell lines were compared by correlation analysis. A high correlation coefficient (CC) for a given pair of compounds indicates a similar pattern in response in the set of cell lines. Such data corroborate the view that there is cross-resistance among the drugs. A numerically low coefficient indicates that the two drugs are acting in different ways, suggesting a lack of cross-resistance between the drugs, and a negative correlation coefficient implies that two drugs exhibit collateral sensitivity. The most negative CCs (%) to the new drug leads were: taxotere-carmustine (BCNU) (-75), taxol-cisplatin (-58), ara-C-taxol (-25), gemcitabin-doxorubicin (-32), camptotecin-VM26 (-41) and topotecan-VP16 (-17). The most negative correlations to the clinically important agent VP-16 were: cisplatin (-70); BCNU (-68); camptothecin (-38); bleomycin (-33), gemcitabin (-32); ara-C (-21); topotecan (-17); melphalan (-3); and to the other main drug in SCLC treatment cisplatin were: doxorubicin (-70); VP-16 (-70); VM-26 (-69); mAMSA (-64); taxotere (-58); taxol (-58). Taxol and taxotere were highly correlated (cross-resistant) to VP-16 (0.76 and 0.81 respectively) and inversely correlated to cisplatin (both -0.58). Similarly, camptothecin and topotecan were correlated to cisplatin but inversely correlated to VP-16 and other topo II poisons. From the sensitivity data, we conclude that collateral sensitivity and lack of cross-resistance favours a cisplatin-taxane or topo I-topo II poison combination, whereas patterns of cross-resistance suggest that epipodophyllotoxin-taxane or topo I poison-cisplatin combinations may be disadvantageous.

Camptothecins are DNA topoisomerase I-directed anti-tumour drugs with a novel mechanism of action. Topotecan (TPT), a hydrophilic derivative of camptothecin, is currently undergoing phase II clinical trials in small-cell lung cancer (SCLC). Human SCLC OC-NYH cells were made more than 6-fold resistant to topotecan by stepwise drug exposure and resistance was stable for 70 passages without drug. NYH/TPT cells had half the topoisomerase I level and activity of wild-type cells. However, no difference in camptothecin or topotecan inhibition of topoisomerase I-mediated DNA relaxation was found, indicating that the enzyme itself was unchanged in the resistant cell. In NYH/TPT cells, topoisomerase II alpha and beta levels were increased approximately 2-fold. Accordingly, the topoisomerase II-directed drug etoposide (VP-16) induced an increased number of DNA single-strand breaks in NYH/TPT cells. However, sensitivity to different topoisomerase II-targeting agents in NYH/TPT cells varied from increased to decreased, indicating a role for as yet unidentified factors acting on the pathway to cell death after topoisomerase II-induced DNA damage has occurred. Of 20 anti-cancer agents tested, only hydroxyurea showed marked collateral hypersensitivity in NYH/TPT cells.

A panel of six 'wild type' and three VP-16 resistant small cell lung cancer (SCLC) cell lines is used to evaluate to what extent in vitro sensitivity testing using a clonogenic assay can contribute to combine cytotoxic drugs to regimens with improved efficacy against SCLC. The resistant lines include (a) H69/DAU4, which is classical multidrug resistant (MDR) with a P-glycoprotein efflux pump (b) NYH/VM, which exhibits an altered topoisomerase II (topo II) activity and (c) H69/VP, which is cross-resistant to vincristine, exhibits a reduced drug accumulation as H69/DAU4 but is without P-glycoprotein. 19 anticancer agents were compared in the panel. The MDR lines demonstrated, as expected, cross-resistance to all topo II drugs, but also different patterns of collateral sensitivity to BCNU, cisplatin, ara-C, hydroxyurea, and to the topo I inhibitor camptothecin. The complete panel of nine cell lines clearly demonstrated diverse sensitivity patterns to drugs with different modes of action. Correlation analysis showed high correlation coefficients (CC) among drug analogues (e.g. VP-16/VM-26 0.99, vincristine/vindesine 0.89), and between drugs with similar mechanisms of action (e.g. BCNU/Cisplatin 0.89, VP-16/Doxorubicin 0.92), whereas different drug classes demonstrated low or even negative CC (e.g. BCNU/VP-16 -0.21). When the CC of the 19 drug patterns to VP-16 were plotted against the CC to BCNU, clustering was observed between drugs acting on microtubules, on topo II, alkylating agents, and antimetabolites. In this plot, camptothecin and ara-C patterns were promising by virtue of their lack of cross-resistance to alkylating agents and topo II drugs. Thus, the differential cytotoxicity patterns on this panel of cells can (1) give information about drug mechanism of action, (2) enable the selection and combination of non-cross-resistant drugs, and (3) show where new drugs 'fit in' among established agents.

Eight anthracycline analogs that have been shown to modulate multidrug resistance (Friche et al., Biochem. Pharmacol., 39, 1721-1726; 1990) were tested for their inhibitory effect on the photolabelling of P-glycoprotein. We photoaffinity labelled P-glycoprotein in daunorubicin (DNR) resistant Ehrlich ascites tumour cells (EHR2/DNR +) with a [125I]iodinated Bolton-Hunter derivative of daunorubicin ([125I]iodomycin) and with [3H]azidopine. The photolabelling of P-glycoprotein by [125I]iodomycin was inhibited more than 50% by 10 microM (1000-fold molar excess) of DNR (52%), N,N-dibenzyl-DNR (52%), and N-benzyladriamycin-14-valerate (AD-198) (85%). Vincristine at 10 microM inhibited [125I]iodomycin labelling of P-glycoprotein by 95%. Thus vincristine was more potent than any of the eight anthracyclines tested, despite its relatively low lipophilicity. Increasing the concentration of DNR, AD-198 and N,N-dibenzyl-DNR to 40 microM resulted in 90, 99.5 and 99.5% inhibition of P-glycoprotein labelling by [125I]iodomycin, respectively. In comparison with the other anthracycline analogs, N,N-dibenzyl-DNR and Ad-198 were also found to exert the greatest inhibition of [3H]azidopine labelling of P-glycoprotein (about 90% at 100-fold molar excess). The solvents Cremophor EL and Tween 80 (30 micrograms ml-1; 0.003% v/v), which are modulators of multidrug resistance in EHR2/DNR + cells, also inhibited [125I]iodomycin labelling > 90%. We showed earlier that there is a correlation between the lipid solubility within the anthracycline group of MDR-associated drugs and their ability to enhance DNR accumulation in EHR2/DNR + cells but a corresponding correlation to lipophilicity when it comes to the inhibitory effect on the specific photolabelling of Pgp ligand binding sites could not be demonstrated. Neither could a correlation between the modulating effect of the analogs on DNR accumulation and inhibition on the labelling of Pgp be demonstrated. With increasing lipophilicity of the analogs it seems that the chemical structure plays a lesser role, and the degree of lipophilicity becomes a more important feature.

Verapamil has been proposed to modulate the multidrug resistance phenotype by competitive inhibition of an energy dependent efflux of cytotoxic drug. However, the accumulation of both 14C-verapamil and 3H-verapamil was similar in wild type EHR2 and multidrug resistant EHR2/DNR+ Ehrlich ascites cells, and was much less in both cell lines in energy deprived medium than in medium containing glucose. Azidopine accumulation was also similar in both EHR2 and EHR2/DNR+ cells but, in contrast to verapamil, did not differ significantly with changes in cellular energy levels. Azidopine photolabelled a 170 kDa protein in EHR2/DHR+ plasma membrane vesicles which was immunoprecipitated by monoclonal antibody towards P-glycoprotein. Azidopine increased daunorubicin accumulation and modulated vincristine resistance in EHR2/DNR+ cells in a similar fashion to verapamil. Azidopine photolabelling was inhibited by vincristine and verapamil, but not by daunorubicin. Vincristine, but not daunorubicin, was able to increase both azidopine and verapamil accumulation in EHR2/DNR+ cells only. Finally, though both verapamil and azidopine are a substrate for P-glycoprotein in EHR2/DNR+ cells, they do not themselves appear to be transported by the multidrug resistance efflux mechanism to any significant extent in these cells.

Numerous studies have indicated that the plasma membrane plays an important role in the development of resistance to anthracycline and vinca alkaloid drugs (pleiotropic resistance). We have previously shown that pleiotropically resistant Ehrlich ascites tumour cells, which are of epithelial origin, have a significantly increased plasma membrane traffic (endo/exocytosis) to the endosomal compartment compared to sensitive cells. The present study, using the same ultrastructural morphometric technique, has demonstrated a similar significant difference in plasma membrane traffic between daunorubicin resistant and sensitive P388 cell lines (which are of lymphoid origin). Furthermore, we have shown that this difference between the P388 sublines is accompanied by an approximately 4 fold increase in the plasma membrane area participating in recycling together with an increased endosomal volume, number and membrane area in resistant cells. Plasma membrane traffic in resistant cells was significantly inhibited by the calcium channel blocker verapamil, a well known modulator of anthracycline resistance, but unaffected by daunorubicin itself. The confirmation of this phenotype in an additional pleiotropically resistant cell type with a different histogenesis further supports a hypothesis of endosomal drug trapping and vesicular extrusion as a possible resistance mechanism.

The sensitivity of eight cell lines established from treated and untreated patients with small cell carcinoma of the lung (SCCL) was tested in the clonogenic assay with 1 h and continuous exposure to aclarubicin (ACLA), adriamycin (ADR), daunorubicin (DAU) and mitoxantrone (MITO). The sensitivity to ADR, DAU and MITO covariated, and varied with a factor of five. The sensitivity to ACLA was independent of the sensitivity to ADR and varied only within a factor of two. Only ACLA showed pronounced increased potency with continuous incubation, and ACLA was the most potent drug in the three cell lines least sensitive to ADR. Two resistant cell lines were selected by treating NCI-H69 in vitro with DAU. One cell line (9-fold resistant to DAU) expressed large amounts of P-glycoprotein, the other cell line (4-fold resistant to DAU) had barely detectable glycoprotein. Both lines acquired resistance to ADR, ACLA and MITO. The cross-resistance to ACLA and MITO was only partial and ACLA was still the most potent drug on these lines. The sensitivity to ACLA of the cell lines least sensitive to ADR suggest that ACLA partially circumvents mechanisms of multidrug resistance. Together with the pronounced increase in potency with prolonged exposure, these results suggest that ACLA has a mechanism of action different from the 'classical' anthracyclines. In this context mitoxantrone is more similar to the classical anthracyclines although its structure is more dissimilar.

The multidrug resistance (MDR) phenotype is presumed to be mostly dependent on changes in the resistant cell plasma membrane, notably the emergence of a 170 kDa glycoprotein called P-glycoprotein, which facilitate increased drug efflux. We have previously demonstrated that ATP-enhanced binding of vincristine (VCR) to plasma membrane vesicles is much greater in MDR than in wild type cells. The present study has shown that VCR binding to MDR Ehrlich ascites tumour cell plasma membrane vesicles is inhibited 50% most efficiently by quinidine (0.5 microM) followed by verapamil (4.1 microM) and trifluoperazine (23.2 microM). This is the reverse order of the effect on whole cells where a ranking of efficiency in terms of enhancement of VCR accumulation, inhibition of VCR efflux, DNA perturbation and modulation of resistance in a clonogenic assay, was trifluoperazine greater than or equal to verapamil much greater than quinidine. The detergent Tween 80 inhibited VCR binding to plasma membrane vesicles at 0.001% v/v which agreed with the level which modulated resistance and increased VCR accumulation in whole cells. No effect was observed on daunorubicin binding to MDR plasma membrane vesicles after incubation with either Tween 80 (up to 0.1% v/v) or verapamil (up to 25 microM). We conclude that the effect of a modulating drug in reversing resistance to VCR correlates with its ability to raise intracellular VCR levels but not with its capability to inhibit VCR binding to the plasma membrane. Thus, enhancement of VCR accumulation in MDR cells is hardly solely due to competition for a drug binding site on P-glycoprotein. Furthermore, the lack of a demonstrable effect on daunorubicin binding to the plasma membrane by modulators points to transport mechanisms which do not utilise specific drug binding to the plasma membrane.