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Cytotoxic activity of topotecan in human tumour cell lines and primary cultures of human tumour cells from patients.

The cytotoxic activity and cross-resistance pattern of the novel topoisomerase I inhibitor topotecan (Topo) were investigated in ten cell lines, representing different mechanisms of cytotoxic drug resistance, and in 218 fresh human tumour samples using the fluorometric microculture cytotoxicity assay (FMCA). Resistance to Topo in the cell lines was associated with expression of the multidrug resistance-associated protein (MRP), whereas the cell lines with P-glycoprotein (P-gp), topoisomerase II and glutathione-associated resistance did not show decreased sensitivity to the drug. Topo was more active in haematological than in solid tumour samples, but substantial activity was observed in carcinomas of the ovary and breast, sarcoma and childhood solid tumours. Cross-resistance to standard drugs representing different mechanisms of action was generally low in patient cells. The effect of Topo was better after longer exposure, but this time-dependent effect was largely abolished when adjustment for in vitro exposure was made. Topo showed activity both in proliferative and non-proliferative cell systems. The results indicate that Topo is insensitive to major mechanisms of resistance except for MRP. Proliferation does not seem to be necessary for the effect of Topo, and no superiority for protracted dosing schedules was observed. The results also suggest that, for example, leukaemias, lymphomas, sarcomas and childhood solid tumours may be suitable targets for future phase II trials.

Topotecan (Topo) is a semisynthetic analogue of camptothecin, an alkaloid isolated from certain plant species such as Camptotheca acuminata. Camptothecin was found to be active against tumour cells in the National Cancer Institute (NCI) drug screening programme during the 1960s. Although its supposed mechanism of action was not clarified until the late 1980s, it was introduced into clinical trials during the 1970s. However, because of unexpected toxicity and limited activity in phase II trials further development of this group of drugs was halted until the more water-soluble and less toxic camptothecin analogues, among them Topo, were synthesized (Takimoto and Arbuck, 1996).
Topo is the first cytotoxic drug to be considered for approval for clinical use with the enzyme topoisomerase I as the probable target. At physiological pH the lactone form of Topo is in equilibrium with the open ring form, but only the former is active (Wall et al, 1992). Topoisomerase I is a nuclear enzyme present in all eucaryotic cell types, and is involved in DNA replication and repair. The enzyme unwinds the supercoiled double-stranded DNA by temporarily binding to, and cleaving, one of the strands. In the presence of Topo, the complex of cleaved DNA with topoisomerase I is stabilized, inhibiting religation (Takimoto and Arbuck, 1996). The drug-induced single-strand breaks are reversible, but as the replication goes on and the moving replication fork interacts with the drug-enzyme-DNA complex, doublestrand breaks result that are thought to be responsible for the cytotoxic effects of Topo (Holm et al, 1989). Inhibition of DNA replication with, e.g. a DNA polymerase inhibitor that blocks double-strand break production reduces Topo cytotoxicity (Holm et al, 1989). Topo is therefore considered to be an S phase-specific drug, producing preferential toxicity to proliferating cells.
Topo has shown cytotoxic activity against a broad range of cell types in vitro. Specific activity has been seen against colorectal, breast, non-small-cell lung (NSCLC), ovarian and renal cancers  and acute lymphocytic leukaemia (Uckun et al, 1995) in a clonogenic assay. In mice bearing human tumour xenografts Topo showed activity against rhabdomyosarcoma (Houghton et al, 1995) and central nervous system tumours (Friedman et al, 1994).
The mechanisms possibly responsible for resistance against Topo have been studied in various cell lines in vitro. The results from these studies are not conclusive but have indicated that resistance may be mediated by lowered levels and decreased sensitivity of the target enzyme (topoisomerase I) (Tan et al, 1989;Eng et al, 1990;Sorensen et al, 1995) and decreased drug accumulation unrelated to known mechanisms (Yang et al, 1995). Resistance may also, at least partly, be explained by sensitivity to P-glycoprotein 170 (P-gp)-mediated transport, although to a considerably lesser extent than known P-gp substrates (Hendricks et al, 1992;Mattem et al, 1993). An association between the heat shock protein hsp70 and decreased Topo sensitivity has been found in a murine fibrosarcoma cell line (Sliutz et al, 1996).
As a complement to ongoing clinical studies the present study was undertaken to characterize Topo in vitro with respect to tumour-type specificity, cross-resistance, schedule-dependency and possible mechanisms of resistance. This was carried out in a panel of human tumour cell lines and primary cultures of fresh human tumour cells from a broad spectrum of diagnoses and by use of the fluorometric microculture cytotoxicity assay (FMCA).

MATERIALS AND METHODS Cell lines
To evaluate the resistance pattern of Topo, a human cell line panel of four sensitive parental cell lines, five drug resistant sublines, representing different mechanisms of resistance, and one cell line with primary resistance was used. The cell line panel has been described in detail previously , and the basic information on each cell line together with references are given in Table 1. The cell line RPMI 8226/Dox40 shows the classical MDR phenotype with overexpression of 170 (P-gp). RPMI 8226/LR-5 shows a resistance proposed to be associated with increased levels of glutathione (GSH), whereas the resistance of U-937-Vcr is proposed to be tubulin associated. The H69AR cell line expresses a multidrug-resistant (MDR) phenotype proposed to be mediated by a multidrug resistance-associated protein (MRP) and the CEMNVM-l expresses an atypical MDR, which is associated with altered topoisomerase II (topoll) activity. The exact mechanism of resistance for the primary resistant ACHN cell line is not known and may be multifactorial. The resistance patterns of the cell lines were routinely confirmed in control experiments. Immunocytochemistry P-gp and MRP staining was performed on cytocentrifuge cell preparations with the monoclonal antibodies C219 and QCRL-1 (Centocor, Malvern, PA, USA) fixed in acetone and 70% methanol respectively, essentially as previously described (Cordell et al, 1984). Briefly, the specimens were incubated at a 1:10 dilution for 2 h at room temperature, followed by washing and application of a secondary rabbit anti-mouse antibody (Dako, Copenhagen, Denmark) for 30 min. After washing, a soluble complex of alkaline phosphatase and a mouse monoclonal anti-alkaline phosphatase (APAAP, Dako) was added for 30 min. The slides were developed using 10 mg ml-' Fast red (Sigma, St Louis, MO, USA) dissolved in a 0.5 M Tris buffer containing 2 mg ml' Naphtol-As-Mx-phosphate and 2.4 mg ml' levamisole (Sigma). The specimens were counterstained with Mayers haematoxylin and mounted. The fraction of tumour cells expressing positive staining was estimated after inspection of 100 cells on each slide.

Patient samples
A total of 218 patient tumour samples from the different diagnoses, detailed in Table 2, were used to determine the activity of Topo and, for comparison, six other cytotoxic drugs, as detailed below, chosen to represent different mechanistic classes. However, because of a limited number of cells, all drugs could not be tested in all samples. Thirty solid and 20 haematological tumour samples and five preparations of normal peripheral blood mononuclear cells (PBMCs) from healthy blood donors were used to determine the dose-response relationship for Topo.
The tumour samples were obtained by bone marrow/peripheral blood sampling, routine surgery or diagnostic biopsy, and this sampling was approved by the local ethics committee at the Uppsala University Hospital. Leukaemic cells and PBMCs were isolated from bone marrow or peripheral blood by 1.077 g ml-Ficoll-Paque (Kabi-Pharmacia, Uppsala, Sweden) densitygradient centrifugation . Tumour tissue from solid tumour samples was minced into small pieces and tumour cells were then isolated by collagenase dispersion followed by Percoll (Kabi-Pharmacia) density-gradient centrifugation . Cell viability was determined by trypan blue exclusion test and the proportion of tumour cells in the preparation was judged by inspection of May-Grunwald-Giemsa-stained cytospin preparations by a cytopathologist. In some cases, cells were cryopreserved in a culture medium containing 10% dimethylsulphoxide (DMSO; Sigma) and 50% inactivated fetal calf serum (FCS; HyClone, Cramlington, UK) by initial freezing for 24 h at -70°C, followed by storage in liquid nitrogen or at -150°C. Cryopreservation in this way does not affect drug sensitivity .
Reagents and drugs Fluorescein diacetate (FDA; Sigma) was dissolved in DMSO and kept frozen (-20°C) as a stock solution protected from light. A complete medium consisting of culture medium RPMI-1640 (HyClone) supplemented with 10% inactivated FCS, 2 mm glutamine, 50 ,ug ml-' streptomycin and 60 jg ml-' penicillin was used throughout for both cell lines and patient samples.
Both in the cell lines and in the patient samples the activity of Topo and the standard drugs cisplatin, cytarabine, doxorubicin, etoposide, melphalan and taxol was determined. Additionally, camptothecin was tested in the cell lines. In the cell line panel all drugs were tested at five different drug concentrations, obtained by tenfold serial dilution from the maximum 100 jig ml'. In the patient samples, the concentrations chosen for the comparisons  Nygren and Larsson (1990) were the empirically derived cut-off concentrations (EDCCs), defined as the concentration that produces a significant scatter of survival index (SI) values among haematological tumours. This concentration was used to optimize the conditions for differentiating between sensitive and resistant tumour cell samples. The concentrations 1 jg ml-' and 0.5 jig ml-' were chosen for taxol and Topo respectively and the EDCCs for the other drugs have been described previously . To determine the dose-response relationship for Topo in patient samples, five different drug concentrations were used, obtained by a fivefold serial dilution of the drug from 12.5 to 0.02 jig ml-'. Topo (SmithKline Beecham, King of Prussia, PA, USA) was dissolved and diluted in sterile water, and camptothecin (Sigma) was dissolved in methanol-chloroform and diluted further in phosphate-buffered saline (PBS; HyClone). The other drugs were obtained from commercial sources and were dissolved according to guidelines from the manufacturer and further diluted in PBS or sterile water.
Ninety-six-well microtitre plates (Nunc, Roskilde, Denmark) were prepared with 20 ,ul per well of drug solution at ten times the desired concentration, with the aid of a programmable pipetting robot (Propette, Perkin Elmer, Norwalk, CT, USA). The plates were stored frozen at -70°C for up to 2 months until further use. Under these conditions, no apparent change in drug activity was observed .
The fluorometric microculture cytotoxicity assay procedure The fluorometric microculture cytotoxicity assay (FMCA) is based on measurement of fluorescence generated from hydrolysis of FDA to fluorescein by cells with intact plasma membranes and has been described in detail previously . Briefly, the cells were resuspended in complete medium, and 1 80-gl cell suspension was seeded into the wells of 96-well experimental microtitre plates prepared with drugs as described. Each drug and concentration was tested in triplicate. Six wells, with cells but without drugs, served as control and six wells with only culture medium as blank. The plates were incubated at 37°C for 72 h, followed by aspiration of the medium, one wash in PBS and addition of 100 jl per well of FDA dissolved in PBS (10 jg ml-'). The plates were incubated for 45 min and the generated fluorescence from each well was measured at 538 nm in a 96-well scanning fluorometer (Fluoroscan II, Labsystems Oy, Helsinki, Finland). The fluorescence is proportional to the number of viable cells in the well. The stability of the drugs over the 72 h incubation at 37°C was investigated by a bioassay. Plates prepared with the different drugs were preincubated with 100 gl of medium per well for different time periods, ranging from 0 to 72 h at 37°C before cell suspension (RPMI 8226/S) was added. The activity of the drugs after different preincubation times was evaluated, by comparing the SI values resulting after 72 h incubation and measurement with FMCA as described above.
Quality criteria for a successful analysis included a fluorescence signal in the control wells of more than five times mean blank value, a mean coefficient of variation (CV) in the control wells of less than 30% and more than 70% tumour cells in the cell preparation before incubation.

Quantification of FMCA results
Cell survival is presented as survival index (SI), defined as the fluorescence in experimental wells as a per cent of that in control wells, with blank values subtracted. The IC50 was defined as the Topo concentration giving a SI of 50%.
For the cell lines, the IC50 values were evaluated for each individual cell line and drug with custom-made computer software. A Delta value was calculated as the logarithm of the IC50 of the individual cell line minus the mean of all ten log IC50 values (Boyd and Paul, 1995). The resistance factor for Topo in each subline was defined as the IC 5 of the resistant subline divided by the IC50 of its sensitive parental cell line.
The IC50 values for the haematological and solid patient samples respectively were determined graphically from the mean doseresponse curves. Response rate to Topo was defined as the fraction of samples having a SI below the median SI at 0.5 jig ml-1 for all samples investigated. The relative effect of a drug on solid and haematological tumours was indicated by the S/H ratio, defined as the ratio between the total response rates for the solid and the haematological samples.

Dependency of Topo activity on dosing schedule and cell cycle phase
To evaluate the schedule dependency of Topo activity, cells were exposed to the same AUC (area under the concentration-time curve) of Topo but during different exposure times. CCRF-CEM cells and CLL patient cells were used. The cells were exposed to different Topo concentrations for 8 h, followed by washing with PBS, addition of new culture medium and continuation of incubation up to 72 h, before performing FMCA. In parallel, cells were exposed for the doubled concentrations for 4 h, and for four times the original concentrations for 2 h, before washing the Topo solution away.
British Journal of Cancer (1997) 76(2) To examine if the cells were proliferating during the 72 h incubation in the 96-well plates, bromodeoxyuridine (BrdU) incorporation into cellular DNA was determined with an ELISA kit from Boehringer Mannheim (Mannheim, Germany) essentially according to the protocol from the manufacturer. Briefly, CCRF/CEM cells and CLL patient cells seeded into 96-well plates were incubated for 72 h together with the pyrimidine analogue BrdU, which incorporates into DNA of proliferating cells. Then the cells were fixed and an antibody against BrdU was added. The immune complex was detected by a substrate reaction using tetramethylbenzidine (TMB), giving a product that was measured in a spectrophotometric microplate reader (Dynatech Laboratories, Billingshurst, UK).

RESULTS
The dose-response curves for Topo in the individual cell lines are shown in Figure IA. The SCLC cell line NCI-H69 and its subline H69AR were the most resistant to Topo, together with the renal carcinoma cell line ACHN. The cell lines of haematological origin were more and equally sensitive, except for the myeloma cell line RPMI 8226/S with its sublines 8226/Dox40 and 8226/LR-5, which were of intermediate sensitivity. The experiments were performed twice with similar results and the mean value was used. The greatest difference in sensitivity between a cell line and its subline was seen for NCI-H69 and its MRP-expressing resistant subline H69AR, where the resistance factor was 77 (Table 1). Almost no decreased sensitivity compared with parental cell lines was observed in the sublines with topoIl-, P-gpand tubulin-associated MDR, whereas the 8226/LR-5 subline with GSH-associated MDR showed a slightly increased sensitivity. Figure IB-D shows the Delta values, i.e. the deviation of log IC50 for each cell line from the mean log IC50 of the cell line panel, for Topo (1B) and for comparison for doxorubicin (IC) and camptothecin (ID). Camptothecin showed a sensitivity pattern similar to that of Topo, the solid tumour-and myeloma cell lines being more resistant than the haematological ones. Differences between parental cell line and resistant subline were seen only for NCI-H69AR and its MRP-expressing subline H69AR, and for RPMI 8226/S and its subline 8226/LR-5 with GSH-mediated resistance. A Another pattern was seen for doxorubicin, which was very active 100 against RPMI 8226/S, and which was influenced by all the resistance mechanisms, all the sublines being less sensitive than their 80 parental cell lines.
When examining the immunocytochemical staining of the cell lines, it was found that the only cell line expressing MRP was x 60 H69-AR, with 90% of the cells showing positive staining. P-gp c was highly expressed in the doxorubicin-selected cell line 8226/Dox40 (80% positively stained), but also to a minor extent in the lymphoma cell lines U937 GTB (50%) and U937-Vcr (60%). cn Preliminary data indicate that the MRP inhibitor genistein 20 (Verovski et al, 1996) is able to increase the sensitivity for Topo for the MRP-expressing cell line H69-AR (unpublished data).
The haematological patient samples and the normal PBMCs 0showed quite similar Topo sensitivity, with IC50 values of 0 0.01 0.1 1 10 100 66 ng ml-' and 48 ng ml-' respectively, and were more sensitive than the solid samples in which no IC50 value was obtained ( Figure  2). At the highest Topo concentration the SI was 59% in the solid and 21% in the haematological samples.
In Table 1  \iof 3) and in the haematological tumours (3 out of 3). 20 The relative effect of the drugs in solid, compared with haematological, tumour samples expressed as the S/H ratio is shown in Figure 3. Of the six different drugs compared, taxol showed the 0greatest relative effect on solid tumours (S/H ratio 1.06), followed 0 0.01 0.1 1 10 100 by cisplatin (0.80), etoposide (0.48) and Topo (0.45). Cytarabine was the drug with the most pronounced haematological tumour activity pattern (S/H ratio 0.12).
The correlation coefficients in haematological patient samples between the SI of Topo and the SI of each of the standard cytotoxic drugs at their EDCCs were compared pairwise and listed in Table 3. C Topo and taxol showed low correlations with the standard drugs, 100 with coefficients of correlation under 0.3. The coefficients of correlation between the other standard drugs were higher and varied 80 between 0.31 and 0.68.
When comparing the effect of Topo after different exposure times, a) the longer exposure times gave a better effect than the shorter ones, _ 60 -\ both in CCRF/CEM and in CLL patient cells ( Figure 4A and C). However, when the test concentrations were compensated for the 40 greater AUC resulting from a longer exposure time, the differences CD between the different dosing schedules were diminishing, leaving 20 only a very small tendency towards a preference for prolonged exposure ( Figure 4B

DISCUSSION
The mechanisms of resistance to Topo are not completely understood. Some preclinical cell line data indicate that Topo appears to be part of the P-gp-mediated multidrug-resistance phenotype, but the effect of P-gp-mediated transport on Topo accumulation in cells is substantially smaller compared with classical P-gp substrates (Hendricks et al, 1992;Mattem et al, 1993). According to our findings, the P-gp-mediated resistance does not seem to influence the effect of Topo to any greater extent. However, the MRP-expressing cell line H69AR was found to be 77-fold more   (Yang et al, 1995). It is not known whether the H69AR cells also have similar mechanisms operating in addition to MRP. Investigating the relationship between topoisomerase I and MRP expression and clinical response to Topo therapy would be a highly interesting subject for future studies aiming at elucidating the clinically relevant mechanisms of resistance.
When comparing the sensitivity pattern of Topo in the cell line panel with that of its parent compound camptothecin, a similarity was seen. Topo seems to share the MRP-associated resistance with its parent compound. On the other hand, doxorubicin showed an entirely different sensitivity pattern, and was influenced by all the resistance mechanisms represented in the panel. Comparison of the sensitivity pattern of different drugs in the cell line panel can be used to detect mechanistic similarity between compounds . When the drugs were compared in the patient samples, very low levels of cross-reactivity were observed between Topo and standard drugs representing the major types of mechanisms of action. The standard drugs correlated substantially more with each other, and it has been shown previously that drugs from the same mechanistic classes show even higher correlations (correlation coefficients > 0.70) (Jonsson et al, 1996). This indicates a potential role of Topo in combination therapy.
The activity of Topo has previously been investigated in clonogenic assays of freshly explanted specimens from patients with various solid tumours . In that study, carcinomas of the kidney, lung and breast were found to be most sensitive to Topo. Ovarian carcinoma was found to be less sensitive, giving responses of the same magnitude as, for example, colorectal cancer. These results contrast to the present results in which renal cell carcinoma and colorectal tumours were largely insensitive whereas ovarian carcinoma was sensitive to Topo. The reason for the apparent discrepancy may at least be related partly to the different end points used, i.e. proliferation of clonogenic cells used in the clonogenic assay and cell death used in the FMCA. If prediction of tumour regression in vivo is sought, cell kill may theoretically be a more appropriate and robust end point to measure. In practice, the correlation with known clinical response pattern is the most important argument for the validity of any end point or assay type.
Clinical studies of the anti-tumour activity of Topo have been performed in a variety of tumor types. In ovarian carcinoma, Topo has shown promising phase II activity with reported response rates of around 20% in relapsed patients (Gore et al, 1996). Activity of Topo has also been reported for NSCLC (Perez-Soler et al, 1996), SCLC (Ardizzoni et al, 1994;Shiller et al, 1994), soft tissue sarcoma (Eisenhauer et al, 1994) and breast cancer (Chang et al, 1995). In renal cell cancer (Ilson et al, 1993;Law et al, 1994) and colorectal cancer (Sugarman et al, 1994) the drug appears inactive. Little phase II experience has been reported for haematological tumours, although responses in phase I trials have been observed in patients with relapsed or refractory acute leukaemia (Kantarjian et al, 1993;Rowinsky et al, 1994).
The present 'in vitro phase II' results using cell kill as end point appears therefore to be more in accordance with the reported clinical activity than the clonogenic assay. As for the majority of currently used chemotherapeutic drugs, Topo showed a high degree of differential activity against haematological tumours in the present study. The present results indicate that in addition to various haematological tumours sarcomas, childhood solid tumours and breast cancer may be suitable targets for clinical phase II studies.
The in vitro effect of Topo has previously been shown to be dependent on the exposure time, with longer incubations being more active than shorter ones Cheng et al, 1994;Uckun et al, 1995). However, the drug effects were not adjusted for the total exposures precluding any firm conclusions about superiority for protracted scheduling (true 'time dependency'). In the present study, the apparent time dependency of the effect on both CCRF-CEM and CLL patient cells was largely abolished when adjustments for in vitro exposure was made. Thus, for the different exposure times tested in the present study, the drug effects were best explained by the area under the concentration-time curve (AUC). Furthermore, Topo was clearly highly active against patient CLL cells showing no evidence of DNA synthesis during the 72-h assay period, strongly arguing against a requirement for S-phase progression, at least for this cell type.
In vivo, however, the question of dosing schedule dependency is more complicated and involves both pharmacokinetic factors and the toxic effects on normal host cells. A single optimal schedule could not be determined from the early preclinical in vivo studies (Dancey and Eisenhauer, 1996), although support for protracted schedules was obtained from some tumour model systems (Houghton et al, 1992). Nevertheless, the most commonly used clinical phase II schedule, a daily x five schedule, has been compared with more protracted schedules without any apparent advantage of the latter (Dancey and Eisenhauer, 1996). This clinical experience and the present results may thus suggest that the optimal schedule for Topo is yet to be defined for different tumour types and that additional short-term schedules might be explored. In this context it should be noted that, also from a theoretical point of view, AUC vs time dependency for drugs with short half-lives may vary depending on the sensitivity of the tumour model system used, irrespective of cell cycle phase specificity (Karlsson et al, 1996).
We have previously shown that the FMCA can detect tumourtype specific activity retrospectively for a series of standard drugs  and prospectively for early phase I-LI drugs such as 2-chlorodeoxyadenosine , gemcitabine (Csoka et al, 1995) and taxol (Nygren et al, 1995). The use of a human cell line panel can contribute with information on resistance mechanisms and on mechanistic similarity to other standard and investigational agents . The parallel or sequential application of these model systems may provide important initial information on selectivity and similarity of novel anticancer drugs.