¹Division of Medical Oncology, Tochigi Cancer Center, Tochigi, Japan

²Division of Laboratory Medicine, Tochigi Cancer Center, Tochigi, Japan

³Center for Molecular Medicine, Department of Hematology, Jichi Medical School, Tochigi, Japan

⁴Research Department, Nihon Schering Co. Ltd., Osaka, Japan

Correspondence to: Y Kano, Division of Medical Oncology, Tochigi Cancer Center, Yonan 4-9-13, Utsunomiya, Tochigi, 320-0834, Japan; Fax: 011-81-28-658-5488

Fludarabine phosphate (2-F-ara-AMP) is an adenine nucleoside analogue that shows significant activity against chronic lymphocytic leukemia and indolent lymphoma. We assessed the cytotoxic interaction produced by the combination of the active metabolite of fludarabine phosphate, fludarabine (9--D-arabinofuranosyl-2-fluoroadenine, 2-F-ara-A), and some commonly used antileukemic agents against human hairy cell leukemia cell line JOK-1, human chronic lymphocytic leukemia cell line SKW-3, and adult T cell leukemia cell lines ED-40810 (-) and SALT-3. The leukemia cells were exposed simultaneously to 2-F-ara-A and to the other agents for 4 days. Cell growth inhibition was determined using MTT reduction assay. The isobologram method of Steel and Peckham was used to evaluate the cytotoxic interaction. 2-F-ara-A and cytarabine showed synergistic effects in SKW-3 cells, additive and synergistic effects in JOK-1 and SALT-3 cells, and additive effects in ED-40810(-) cells. 2-F-ara-A and doxorubicin showed additive effects in SKW-3, ED-40810(-) and SALT-3 cell lines, and additive and synergistic effects in JOK-1 cells. 2-F-ara-A showed additive effects with etoposide, 4-hydroperoxy-cyclophosphamide, and hydroxyurea in all four cell lines. 2-F-ara-A showed antagonistic effects with methotrexate and vincristine in all four cell lines. Our findings suggest that the simultaneous administration of fludarabine phosphate with cytarabine, doxorubicin, etoposide, cyclophosphamide, or hydroxyurea would be advantageous for cytotoxic effects. Among these agents, cytarabine may be the best agent for the combination with fludarabine phosphate. The simultaneous administration of fludarabine phosphate with methotrexate or vincristine would have little cytotoxic effect, and this combination may be inappropriate. These findings may be useful in clinical trials of combination chemotherapy with fludarabine phosphate and these agents. Leukemia (2000) 14, 379-388.

fludarabine; 2-F-ara-A; isobologram; synergism; antagonism

Introduction

Fludarabine phosphate (2-F-ara-AMP) is an adenine nucleoside analogue. Unlike adenine arabinoside (ara-A), this drug is water soluble and resistant to deamination by adenosine deaminase.¹ Fludarabine phosphate is converted rapidly to fludarabine (9--D-arabinofuranosyl-2-fluoroadenine, 2-F-ara-A) in plasma, which is transported into the cell, where it is converted to 2-F-ara-ATP by deoxycytidine kinase.² The major cytotoxic mechanism of fludarabine phosphate is believed to inhibit DNA synthesis by incorporation of 2-F-ara-ATP into the elongation chain.^3,4,5,6 The inhibitions of RNA, ribonucleotide reductase, DNA and RNA polymerases, DNA primase, DNA ligase, and exonuclease are also considered to contribute to the cytotoxic action of 2-F-ara-ATP. However, the mechanism by which F-ara-A induces apoptosis in cancer cells is not yet fully understood.

Clinical studies have shown that the dose-limiting toxicity of fludarabine phosphate involves myelosuppression, while nausea, vomiting, diarrhea, and liver damage are mild.^7,8 Fludarabine phosphate has promising therapeutic effects in the treatment of chronic lymphocytic leukemia, lymphoma, and acute leukemia.^{9,10,11,12,13,14}

Most clinical regimens incorporate two or more drugs in combination, and fludarabine phosphate has been combined with a variety of agents.^{15,16,17,18,19,20,21,22,23,24,25} Although 2-F-ara-A has been reported to show synergistic effects with cytarabine and cisplatin, there are few experimental data available about the combination of 2-F-ara-A and other anticancer agents which are active against leukemia and lymphoma.^{26,27,28,29,30,31,32,33}

In the present study, we investigated the in vitro effects of 2-F-ara-A in combination with cytarabine, doxorubicin, etoposide, 4-hydroperoxy-cyclophosphamide, hydroxyurea, methotrexate, and vincristine against four human leukemia cell lines. The dose-response curves for the combinations were obtained using the 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assay³⁴ and data were analyzed by the isobologram method (Steel and Peckham).³⁵

The results underline the importance of the design of the combination of fludarabine phosphate with other anticancer agents for clinical therapy.

Materials and methods

Cell lines

Experiments were conducted with the human hairy cell leukemia cell line, JOK-1,³⁶ the human chronic lymphocytic leukemia cell line SKW-3,³⁷ and two human adult T cell leukemia cell lines, ED-40810(-)³⁸ and SALT-3.³⁹ All these cell lines were kindly provided by the Fujisaki Cell Center of Hayashibara Institute of Biochemical Science. p53 mutations were observed in JOK-1 and SKW-3 cells, while no mutations were found in ED-40810(-) and SALT-3 cells. Bcl-2 overexpression and no rearrangements of bcl-2 were observed in all four cell lines.

Cells were maintained in 75-cm³ plastic tissue culture flasks containing RPMI1640 medium (Grand Island Biological Co., Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Grand Island Biological Co.) and antibiotics.

Drugs

Anticancer agents used and their sources were: 2-F-ara-A (Berlex Laboratories, Richmond, CA, USA), cytarabine (Nihon Shinyaku Co. Ltd, Tokyo, Japan), doxorubicin (Meiji Co. Ltd, Tokyo, Japan), etoposide (Nihon Kayaku Co. Ltd), 4-hydroperoxy-cyclophosphamide, and vincristine (Shionogi Co. Ltd, Tokyo, Japan), hydroxyurea (Sigma Chemical, St Louis, MO, USA), and methotrexate (Lederle Japan Ltd, Tokyo, Japan). All drugs were dissolved in RPMI-1640. Appropriate drug concentrations were made by dilution with fresh medium immediately before each experiment.

Inhibition of cell growth by the combination of 2-F-ara-A and other agents

JOK-1, SKW-3, ED-40810(-) and SALT-3 cells in the logarithmic phase were harvested from the media and resuspended to a final concentration of 1 ´ 10⁵ cells/ml of fresh medium containing 10% FCS. Cell suspensions (100 l) were dispensed into individual wells of a 96-well tissue culture plate with a lid (Falcon, Oxnard, CA, USA). Eight plates were prepared for the testing of each drug combination. Each plate had one 8-well control column containing medium alone and one 8-well control column containing cells but no drugs. Cells were incubated in a humidified atmosphere of 95% air/5% CO₂ at 37°C overnight. Drug solutions of 2-F-ara-A and other drugs at different concentrations were then added (50 l) to 8 wells containing cell suspensions and the plates were then incubated under the same conditions for 4 days.

MTT assay

Viable cell growth was determined using a modified MTT assay as described previously.³⁴ For the background control, control (no drug), each drug, or drug combination, the four intermediate data values among the eight data values were used for the analysis, and the two highest and the two lowest values were discarded. In the study of hydroxyurea, each concentration of hydroxyurea was used for the respective background control, since hydroxyurea had influenced the absorbance of 570 nm. For all cell lines examined, we established a linear relation between the MTT assay and cell number within the range of the experiments shown.

Isobologram method of Steel and Peckham

Cytotoxic interactions of 2-F-ara-A with other agents at the point of IC₈₀ were evaluated by the isobologram method of Steel and Peckham.³⁵ The theoretical basis of the isobologram method and the procedure for making isobolograms have been described in detail previously.^35,40,41

Based upon the dose-response curves of 2-F-ara-A and the other agents, three isoeffect curves were constructed (Figure 1). If the agents are acting additively by independent mechanisms, combined data points will lie near the mode I line (hetero-addition). If the agents are acting additively by similar mechanisms, combined data points will lie near the mode II lines (iso-addition).

Since we cannot know in advance whether the combined effects of two agents will be hetero-additive, iso-additive, or an effect intermediate between these extremes, all possibilities should be considered. Thus, when the data points of the drug combination fell within the area surrounded by three lines (envelope of additivity), the combination was regarded as additive. The envelope of additivity should not be considered as a reliable definition of additivity. An expression of the uncertainty is an important concept of the isobologram method of Steel and Peckham.

When the data points fell to the left of the envelope, ie the combined effect was caused by lower doses of the two agents than was predicted, we regarded the drug combination as having a supra-additive effect (synergism). When the points fell to the right of the envelope, ie the combined effect was caused by higher doses of the two agents than was predicted, but within the square or on the line of the square, we regarded the combination as having a sub-additive effect, ie the combination was superior or equal to a single agent but was less than additive. When the data points were outside the square, the combination was regarded as having a protective effect, ie the combination was inferior in cytotoxic action to a single agent. Both sub-additive and protective interactions were regarded as antagonism.

Data analysis

As described, when the observed data points in combination mainly fell within the envelope of additivity, the combination was considered as having an additive effect. The mean value of the observed data was compared with that of the predicted maximum values and that of the predicted minimum values for an additive effect.⁴² If the mean value of the observed data was equal to or smaller than that of the predicted maximum values and equal to or larger than that of the predicted minimum values, the combination was regarded as an additive.

When the observed data points in combinations mainly fell in the area of supra-additivity or in the areas of sub-additivity and protection, ie the mean value of the observed data was smaller than that of the predicted minimum values or larger than that of the predicted maximum values, the combinations were considered to have a synergistic or antagonistic effect, respectively. To determine whether the condition of synergism (or antagonism) truly existed, a statistical analysis was performed. The Wilcoxon signed-rank test was used for comparing the observed data with the predicted minimum (or maximum) values for additive effects, which were closest to the observed data (ie the data on the boundary (mode I or mode II lines) between the additive area and supra-additive area (or sub-additive and protective areas).³⁸ Probability (P) values 0.05 were considered significant. Combinations with P > 0.05 were regarded as indicating additive to synergistic (or additive to antagonistic effects). All statistical analyses were performed using the Stat View 4.01 software program (Abacus Concepts, Berkeley, CA, USA).

Results

The IC₈₀ values of 2-F-ara-A alone against JOK-1, SKW-3, ED-40810(-) and SALT-3 are 2.8 ± 1.6 M, 1.9 ± 0.6 M, 10.6 ± 2.9 M and 17.4 ± 3.6 M, respectively (n = 30). Figure 2 shows the dose-response curves for 2-F-ara-A in combination with cytarabine (a), etoposide (b), and methotrexate (c) in SKW-3 cells. Each isobologram was generated based on such dose-response curves.

Cytotoxic interaction between 2-F-ara-A and cytarabine

Figure 3a-d shows the isobolograms of this combination in JOK-1, SKW-3, ED-40810(-) and SALT-3 cells, respectively. In the ED-40810(-) cells, the combined data points fell within the envelope of additivity (Figure 3c). The mean value of the data (0.67) was larger than that of the predicted minimum values (0.62) and smaller than that of the predicted maximum values for an additive effect (0.80) (Table 1), indicating that the simultaneous exposure to 2-F-ara-A and cytarabine produced an additive effect. In JOK-1, SKW-3 and SALT-3 cells, the data points for the combination fell in the area of supra-additivity and within the envelope of additivity (Figure 3a, b and d). The mean values of the observed data (0.53, 0.55 and 0.53) were smaller than those of the predicted minimum additive values (0.60, 0.62 and 0.61), respectively (Table 1). The observed data and the predicted minimum values were compared by Wilcoxon signed-rank test. The observed data in the SKW-3 cells were significantly smaller than the predicted minimum values (P < 0.05), indicating a synergistic effect of the simultaneous exposure to these two agents. The observed data in the JOK-1 and SALT-3 cells were not significantly smaller than the predicted minimum values (P > 0.05), indicating additive and synergistic effects of the simultaneous exposure to these two agents.

Cytotoxic interaction between 2-F-ara-A and doxorubicin

Figure 4a-d shows the isobolograms of this combination in JOK-1, SKW-3, ED-40810(-) and SALT-3 cells, respectively. In the JOK-1 cells, the combined data points fell in the area of supra-additivity and within the envelope of additivity (Figure 4a). The mean value of the data was smaller than that of the predicted minimum values for an additive effect (Table 1). However, statistical analysis showed that the difference was not significant, indicating that the simultaneous exposure to 2-F-ara-A and doxorubicin produced additive and synergistic effects. In the SKW-3, ED-40810(-) and SALT-3 cells, the combined data points fell within the envelope of additivity (Figure 4b, c and d). The mean values of the data were larger than those of the predicted minimum values and smaller than those of predicted maximum values for an additive effect (Table 1), indicating that the simultaneous exposure to 2-F-ara-A and doxorubicin produced additive effects.

Cytotoxic interaction between 2-F-ara-A and etoposide

Figure 5a-d shows the isobolograms of this combination in JOK-1, SKW-3, ED-40810(-) and SALT-3 cells, respectively. All four cell lines showed similar effects. All data points for the combination fell within the envelope of additivity. The mean values of the data were larger than those of the predicted minimum values and smaller than those of the predicted maximum values for an additive effect (Table 1), indicating that the simultaneous exposure to 2-F-ara-A and etoposide produced additive effects.

Cytotoxic interaction between 2-F-ara-A and 4-hydroperoxy-cyclophosphamide

All four cell lines showed similar effects. All data points for the combination fell within the envelope of additivity (isobolograms not shown). The mean values of the data were larger than those of the predicted minimum values and smaller than those of the predicted maximum values for an additive effect (Table 1), indicating that the simultaneous exposure to 2-F-ara-A and 4-hydroperoxy-cyclophosphamide produced additive effects.

Cytotoxic interaction between 2-F-ara-A and hydroxyurea

All four cell lines showed similar effects. All data points for the combination fell within the envelope of additivity (the isobolograms not shown). The mean values of the data were larger than the those of predicted minimum values and smaller than those of the predicted maximum values for an additive effect (Table 1), indicating that the simultaneous exposure to 2-F-ara-A and hydroxyurea produced additive effects.

Cytotoxic interaction between 2-F-ara-A and methotrexate

Figure 6a-d shows the isobolograms of this combination in JOK-1, SKW-3, ED-40810(-) and SALT-3 cells, respectively. In all four cell lines, all data points for the combination fell in the areas of sub-additivity and protection. The mean values of the observed data (0.81, 1.13, 0.85 and 0.96) were larger than those of the predicted maximum additive values (0.64, 0.75, 0.57 and 0.76), respectively (Table 1). Statistical analysis showed that the difference was significant (P < 0.05, P < 0.02, P < 0.02 and P < 0.01, respectively) indicating antagonistic effects of the simultaneous exposure to these two agents.

Cytotoxic interaction between 2-F-ara-A and vincristine

Figure 7a-d shows the isobolograms of this combination in JOK-1, SKW-3, ED-40810(-) and SALT-3 cells, respectively. In all four cell lines, all or most of data points fell in the areas of sub-additivity and protection. The mean values of the observed data (0.97, 0.91, 0.85 and 0.89) were larger than those of the predicted maximum additive values (0.78, 0.82, 0.71 and 0.74), respectively (Table 1). Statistical analysis showed that the difference was significant (P < 0.02, P < 0.02, P < 0.05 and P < 0.05, respectively), indicating antagonistic effects of the simultaneous exposure to these two agents.

Discussion

The purpose of this study was to assess the cytotoxic effects of 2-F-ara-A in combination with commonly used antileukemic agents against four human lymphoid cell lines. We have been using the Steel and Peckham isobologram method because this method can be applied to agents with unclear cytotoxic mechanisms and most dose-response curves of anticancer agents.³⁵

Fludarabine phosphate and cytarabine are nucleoside analogues with significant activity against leukemia and lymphoma. We observed that the simultaneous exposure to 2-F-ara-A and cytarabine produced synergistic effects in one cell line, synergistic/additive effects in two cell lines, and additive effect in one cell line. 2-F-ara-A and cytarabine are transported into the cells, where they are phosphorylated to form active metabolites, 2-F-ara-ATP or ara-CTP, by a series of kinases. Among these kinases, deoxycytidine kinases appears to be the rate-limiting enzyme. Strong correlations have been observed between cellular ara-CTP levels and the cytotoxicity of cytarabine. Plunkett's group has intensively studied the combination of 2-F-ara-A or fludarabine phosphate with cytarabine at both the preclinical and clinical levels. They reported that the sequential exposure to 2-F-ara-A followed by cytarabine had synergistic effects,²⁷ while the reverse sequence had rather antagonistic effects. The clinical study of the fludarabine phosphate/cytarabine sequence produced a high response rate.^16,17,21 The decreased cellular deoxynucleotide concentration due to the inhibition of ribonucleotide reductase by 2-F-ara-ATP is associated with an increase in the rate of cytarabine phosphorylation by deoxycytidine kinase.^16,27 This is considered to be the major mechanism of the synergistic interaction of sequential exposure to 2-F-ara-A or fludarabine phosphate followed by cytarabine. As we used continuous and simultaneous exposure to 2-F-ara-A and cytarabine, this mechanism would also work in this schedule. Since cytarabine is often administered continuously for few days, simultaneous and continuous administration of fludarabine phosphate and cytarabine may be beneficial.

Topoisomerase-II inhibitors such as doxorubicin, daunorubicin, mitoxantrone and etoposide have been widely used for the treatment of hematological malignancies and solid tumors. Clinical studies of fludarabine phosphate with topo-II inhibitors have been carried out against leukemia and lymphoma.^19,20,21,22 In our study, 2-F-ara-A in combination with doxorubicin showed additive effects for three cell lines and synergistic/additive effects for one cell line, while 2-F-ara-A in combination with etoposide showed additive effects in all four cell lines. These findings suggest that the simultaneous administration of fludarabine phosphate with doxorubicin or etoposide would have the expected activity. Since, however, the dose-limiting toxicity of fludarabine phosphate, doxorubicin and etoposide involves myelosuppression, there must be careful monitoring for myelosuppression during combination treatment with fludarabine phosphate plus doxorubicin or etoposide. Bellosillo et al³² reported that 2-F-ara-A showed an additive effect with mitoxantrone in B-CLL cells. Our data are consistent with their findings.

2-F-ara-A showed additive effects with the active form of cyclophosphamide, 4-hydroperoxycyclophosphamide, in all four cell lines. The combination of 2-F-ara-A and 4-hydroperoxycyclophosphamide has been observed to produce synergistic effects in K562 cells by median effect principle.³³ Differences in the methods for evaluating the effects of the drug combinations might have produced the different results. The isobologram of Steel and Peckham is stricter for synergism and antagonism than median effect principle. In addition, experimental conditions such as the cell lines used, culture schedules, duration of drug exposure, and assay method used to determine viable cells were different. These differences might influence the different results. Cyclophosphamide is one of the most important therapeutic agents not only for malignant lymphoma but also for high-dose chemotherapy for leukemia and lymphoma. Combined therapy with fludarabine phosphate and cyclophosphamide in relapsed/resistant patients with B cell chronic lymphocytic leukemia and non-Hodgkin's lymphomas has been reported.^43,44 Recently, fludarabine phosphate has also been incorporated into the conditioning regimen for high-dose chemotherapy with cyclophosphamide.⁴⁵ The combination of fludarabine and cyclophosphamide would be active as expected.

2-F-ara-A showed additive effects with hydroxyurea in all four cell lines. The major cytotoxic action of hydroxyurea is the inhibition of ribonucleotide reductase, which is also considered to be the major cytotoxic mechanism of 2-F-ara-A. Ribonucleotide reductase is a key enzyme in DNA replication. A non-heme iron containing subunit is the catalytic subunit of ribonucleotide reductase and the site of hydroxyurea inhibition⁴⁶ and gallium nitrate inhibition,⁴⁷ while the effector-binding subunit of this enzyme is the site of 2-F-ara-A inhibition.²⁶ Combinations of drugs directed at the non-heme iron and effector-binding subunits of ribonucleotide reductase have been reported to produce synergistic effects.^47,48 Therefore, the combination of fludarabine phosphate and hydroxyurea is interesting and this combination has been incorporated into clinical trials. However, Sato et al,²⁶ reported that the combination of 2-F-ara-A and hydroxyurea produced only additive effects in L1210 cells in vitro. Our data are consistent with the findings of Sato et al. The discrepancy may be due to the differences in experimental design and analysis between the systems.

2-F-ara-A showed antagonistic effects with methotrexate and vincristine. The many data points of these combinations fell in the area of protection (Figures 6,7), in which the cytotoxic effects of the combinations were inferior to those of each agent. The observed data of 2-F-ara-A in combination with methotrexate and vincristine were more than 0.80 in all cell lines (Table 1). These findings suggest that the simultaneous administration of fludarabine phosphate with methotrexate or vincristine may have almost no cytotoxic advantage over the administration of each agent, and thus may be inappropriate, unless there is greater antagonism of lethal effects for normal cells as compared to cancer cells. The mechanisms of antagonistic interaction of these combinations are unknown. However, the protection observed in these combinations indicates that these two agents interfere with each other. Interestingly, simultaneous exposure to cytarabine and methotrexate or vincristine also produced marked antagonistic effects (Ref. 40 and unpublished data).

Defects of apoptosis are considered as the pathogenic tenet of a variety of cancers including B cell chronic lymphocytic leukemia.⁴⁹ Recent studies suggest that cancer cells treated with most anticancer agents or irradiation die from apoptosis and p53 and bcl-2 families play important roles in regulating apoptosis.^50,51 All four leukemia cell lines used in this study had overexpression of bcl-2 protein and no rearrangement of bcl-2. p53 mutations were observed in JOK-1 and SKW cells, while no mutations were observed in ED- and SALT-3 cells. Clinically, p53 mutations are often associated with poor response to therapy.⁵² However, sensitivity to fludarabine in vitro has been reported not to correlate with the presence of p53 mutations.⁵³ In our study, JOK-1 and SKW cells were sensitive to F-ara-A, while ED- and SALT-3 cells were rather resistant to F-ara-A. Our data are consistent with previous in vitro findings. The cytotoxic effects of fludarabine in combination with cytarabine or doxorubicin showed some difference among four cell lines. The mechanisms of different cytotoxic effects among cell lines are obscure. The differences in intracellular drug metabolisms, inhibition of DNA and RNA synthesis by the drugs, and apoptosis may contribute to the differences in results.

There are a number of difficulties in the translation of results from in vitro to clinical therapy. Pharmacokinetic is significantly different between them. Second, toxic effects of the combination cannot be measured by an in vitro system. Third, cell kinetics and cell biochemistry may be quite different. The mechanism of cytotoxic action of fludarabine phosphate in dividing cells is mainly cell cycle-specific and incorporation of 2-F-ara-A into DNA during S phase is required for apoptosis.⁵⁴ Clinically, fludarabine phosphate is more active for B cell chronic lymphocytic leukemia and indolent lymphoma than acute leukemia and aggressive lymphoma. These suggest that S phase-independent apoptosis may be important for cytotoxicity of fludarabine phosphate. These differences between in vitro and clinical systems may influence the cytotoxic interaction of fludarabine phosphate and other agents.

In conclusion, the present study showed that 2-F-ara-A had synergistic to additive effects with cytarabine, additive effects with doxorubicin, etoposide, cyclophosphamide and hydroxyurea, and antagonistic effects with methotrexate and vincristine. Although in vitro models are not absolutely predictive of clinical activity, our findings suggest that the simultaneous administration of fludarabine phosphate with cytarabine, doxorubicin, etoposide or hydroxyurea may be advantageous, while that with methotrexate or vincristine may be disadvantageous in clinical trials. These findings may be of importance in the design of fludarabine phosphate-based combination chemotherapy. It must be noted that we tested only simultaneous exposure to 2-F-ara-A and other agents. Since cytotoxic effects are often schedule-dependent, sequential exposure to 2-F-ara-A followed by other agents or the reverse sequence may not show the same effects as simultaneous exposure to these agents. Continued preclinical and clinical studies should provide further insights and assist in optimal combination and schedule of fludarabine phosphate in clinical use.

Acknowledgements

This work was supported in part by a Grant-in-Aid for Cancer Research (11-8) from the Ministry of Health and Welfare, Japan.

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Figure 1  Schematic representation of isobologram. Envelope of additivity, surrounded by Mode I (solid line) and Mode II (dotted lines) isobologram lines, was constructed from the dose-response curves of 2-F-ara-A and a combined drug. The concentrations that produced 80% cell growth inhibition were expressed as 1.0 on the ordinate and the abscissa of the isobolograms. Combined data points Pa, Pb, Pc and Pd show supra-additive, additive, sub-additive, and protective effects, respectively.

Figure 2  Dose-response curves for fludarabine (2-F-ara-A) in combination with cytarabine (a), etoposide (b), and methotrexate (c) in SKW-3 cells. Cell growth was measured using the MTT assay after 4 days and was plotted as a percentage of the control (cells not exposed to drugs). 2-F-ara-A concentrations are shown on the abscissa. Each point represents the mean value for at least three independent experiments; the s.e.s of the means were less than 11% and were thus omitted.

Figure 3  Isobolograms of simultaneous exposure to 2-F-ara-A and cytarabine (ara-C) in JOK- 1(a), SKW-3 (b), ED-40810(-) (c) and SALT-3 (d) cells. Data are presented as mean values ± s.e. (bars) for at least three independent experiments (some data points have error bars that are concealed by the symbol). In SKW-3 cells, the data points of the combinations fell mainly in the area of supra-additivity. In ED-40810(-) cells, most data points fell in the area of the envelope of additivity. In JOK-1 and SALT-3 cells, the data points of the combinations fell both in the area of supra-additivity and in the area of the envelope of additivity.

Figure 4  Isobolograms of simultaneous exposure to 2-F-ara-A and doxorubicin (DOX) in JOK-1 (a), SKW-3 (b), ED-40810(-) (c) and SALT-3 (d) cells. Data are presented as mean values ± s.e. (bars) for at least three independent experiments. In JOK-1 cells, the data points of the combinations fell both in the area of supra-additivity and in the area of the envelope of additivity. In SKW-3, ED-40810(-) and SALT-3 cells, all or most data points fell within the area of the envelope of additivity.

Figure 5  Isobolograms of simultaneous exposure to 2-F-ara-A and etoposide (VP-16) in JOK-1 (a), SKW-3 (b), ED-40810(-) (c) and SALT-3 (d) cells. Data are presented as mean values ± s.e. (bars) for at least three independent experiments. In all four cell lines, all or most data points fell within the area of the envelope of additivity.

Figure 6  Isobolograms of simultaneous exposure to 2-F-ara-A and methotrexate (MTX) in JOK-1 (a), SKW-3 (b), ED-40810(-) (c) and SALT-3 (d) cells. Data are presented as mean values ± s.e. (bars) for at least three independent experiments. In all four cell lines, all or most data points fell in the areas of sub-additivity and protection.

Figure 7  Isobolograms of simultaneous exposure to 2-F-ara-A and vincristine (VCR) in JOK-1 (a), SKW-3 (b), ED-40810(-) (c) and SALT-3 (d) cells. Data are presented as mean values ± s.e. (bars) for at least three independent experiments. In all four cell lines, all or most data points fell in the areas of sub-additivity and protection.

Table 1  Mean values of observed data, predicted minimum, and predicted maximum values of 2-F-ara-A in combination with other anticancer agents