Characterisation of multidrug-resistant Ehrlich ascites tumour cells selected in vivo for resistance to etoposide.

An Ehrlich ascites tumour cell line (EHR2) was selected for resistance to etoposide (VP16) by in vivo exposure to this agent. The resulting cell line (EHR2/VP16) was 114.3-, 5.7-, and 4.0-fold resistant to VP16, daunorubicin, and vincristine, respectively. The amount of salt-extractable immunoreactive topoisomerase IIalpha and beta in EHR2/VP16 was reduced by 30-40% relative to that in EHR2. The multidrug resistance-associated protein (MRP) mRNA was increased 20-fold in EHR2/VP16 as compared with EHR2, whereas the expression of P-glycoprotein was unchanged. In EHR2/VP16, the steady-state accumulation of [(3)H]VP16 and daunorubicin was reduced by 64% and 17%, respectively, as compared with EHR2. Deprivation of energy by addition of sodium azide increased the accumulation of both drugs to the level of sensitive cells. When glycolysis was restored by the addition of glucose to EHR2/VP16 cells loaded with drug in the presence of sodium azide, extrusion of [(3)H]VP16 and daunorubicin was induced. Addition of verapamil (25 microM) decreased the efflux of daunorubicin to the level of sensitive cells, but had only a moderate effect on the efflux of [(3)H]VP16. The resistant cells showed moderate sensitisation to VP16 on treatment with verapamil, whereas cyclosporin A had no effect. Compared with that of sensitive cells, the ATPase activity of plasma membrane vesicles prepared from EHR2/VP16 cells was very low. Vanadate inhibited the ATPase activity of EHR2/VP16 microsomes with a K(i) value of 30 microM. ATPase activity was slightly stimulated by daunorubicin, whereas vinblastine, verapamil, and cyclosporin A had no effect. In conclusion, development of resistance to VP16 in EHR2 is accompanied by a significant reduction in topoisomerase II (alpha and beta) and by increased expression of MRP mRNA (20-fold). MRP displays several points of resemblance to P-glycoprotein in its mode of action: 1) like P-glycoprotein, MRP causes resistance to a range of hydrophobic drugs; 2) MRP decreases drug accumulation in the cells and this decrease is abolished by omission of energy; and 3) MRP increases efflux of drug from cells. However, compared with that of P-glycoprotein-positive cells, the ATPase activity of MRP-positive cells is found to be low and not able to be stimulated by verapamil.

Characterisation of non-P-glycoprotein multidrug-resistant Ehrlich ascites tumour cells selected for resistance to mitoxantrone.

An Ehrlich ascites tumour cell line (EHR2) was selected in vivo for resistance to mitoxantrone (MITOX). The resistant cell line (EHR2/MITOX) was 6123-, 33-, and 30-fold-resistant to mitoxantrone, daunorubicin, and etoposide, respectively, but retained sensitivity to vincristine. The resistant cells showed moderate sensitisation to mitoxantrone on treatment with verapamil or cyclosporin A. Compared with EHR2, the multidrug resistance-associated protein mRNA was increased 13-fold in EHR2/MITOX. Western blot analysis showed an unchanged, weak expression of P-glycoprotein. Topoisomerase IIalpha was reduced to one-third in EHR2/MITOX relative to EHR2 cells, whereas topoisomerase IIbeta was present in EHR2 but could not be detected in EHR2/MITOX. In the resistant subline, net accumulation of MITOX (120 min) and daunorubicin (60 min) was reduced by 43% and 27%, respectively, as compared with EHR2. The efflux of daunorubicin from preloaded EHR2/MITOX cells was significantly increased. EHR2/MITOX microsomes had a significant basal unstimulated ATPase activity. The apparent K(i) value for vanadate inhibition of the ATPase activity in EHR2/MITOX microsomes was not significantly different from the K(i) value for P-glycoprotein-positive cells. However, whereas verapamil (50 microM) inhibited the ATPase activity of EHR2/MITOX microsomes, it stimulated the ATPase activity of microsomes derived from P-glycoprotein-positive cells. In conclusion, the resistance in EHR2/MITOX was multifactorial and appeared to be associated with: 1) a quantitative reduction in topoisomerase IIalpha and beta protein; 2) reduced drug accumulation, probably as a result of increased expression of a novel transport protein with ATPase activity; and 3) increased expression of MRP mRNA.

Kinetics of anthracycline accumulation in multidrug-resistant tumor cells: relationship to drug lipophilicity and serum albumin binding.

A multidrug-resistant Ehrlich ascites tumor cell line (EHR2/DNR+) was used to examine the membrane transport kinetics of lipophilic anthracycline derivatives in the presence of serum albumin. We present a model for theoretical data analysis with consideration of drug-albumin complex formation. For a set of five derivatives (doxorubicin, daunorubicin, 4-demethoxydaunorubicin, 4'-deoxy-4'-iododoxorubicin, and 13-dihydro-4'-deoxy-4'-iododoxorubicin), data were given on the rates of diffusional drug uptake, and membrane permeability coefficients of the noncharged molecules were estimated. Both the initial rates and the steady-state levels of drug uptake were found to decrease by addition of BSA at concentrations ranging from 5 to 75 mg/mL. For each drug, this effect of serum albumin could be accounted for by the altered distribution between free and protein-bound drug molecules in the bulk aqueous medium. A good fit of theoretical accumulation curves to the experimental data was obtained. It was concluded that a mathematical simulation method makes it possible to predict the uptake characteristics of lipophilic anthracycline compounds into tumor cells under serum conditions.

Equilibrium binding of anthracycline cytostatics to serum albumin and small unilamellar phospholipid vesicles as measured by gel filtration.

A Sephadex G-200 gel filtration method was used to measure directly the equilibrium binding of five important anthracycline analogs to serum albumin. The order of the overall binding constant (K) in a 150 mM NaCl, 20 mM Hepes buffer (pH 7.45) was doxorubicin < daunorubicin < 4-demethoxydaunorubicin approximately 13-dihydro-4'-deoxy-4'-iododoxorubicin < 4'-deoxy-4'-iododoxorubicin for human serum albumin (K = 2.67 +/- 0.07 mM(-1) to 24.5 +/- 3.1 mM[-1]) and bovine serum albumin (K = 1.36 +/- 0.25 mM(-1) to 48.4 +/- 5.2 mM[-1]). Data were given on the pH-dependence of K. The anthracycline-albumin association reaction was compared with measurements of drug partitioning into unilamellar phospholipid membranes and octanol. The results provide important new data required for a systematic kinetic analysis of anthracycline transport in tumor cells under serum conditions in a biological system.

Method for determination of intracellular sodium in perfused cancer cells by 23Na nuclear magnetic resonance spectroscopy.

Changes of intracellular sodium concentrations are often an indication of disease or malfunction. In this work, shift reagent-aided 23Na NMR spectroscopic determination of intracellular sodium was adapted to measurements with perfused cells embedded in agarose gel threads. Ehrlich ascites tumor cells (EHR2) and their multidrug-resistant counterparts (EHR2/DNR+) were immobilized and perfused until the metabolic steady state had been reached as shown by 31P NMR spectroscopy. Subsequent addition of 5 mM dysprosium(III) bis(tripolyphosphate) to the perfusion medium caused a separation of extracellular and intracellular 23Na NMR signals, making quantification of the intracellular sodium possible. The dysprosium shift reagent was apparently nontoxic to the cells, as shown by the unchanged level of ATP and other intracellular phosphates. NMR visibility of the intracellular sodium was determined in suspensions of EHR2 and EHR2/DNR+ cells by treatment with digitonin; the increase of intensity of the extracellular sodium resonance observed after the digitonin treatment corresponded well (97 +/- 3%) to the sum of intracellular and extracellular sodium observed with intact cells prior to the digitonin treatment. The resistant EHR2/DNR+ cells contained a moderately higher intracellular sodium level than the wild-type EHR2 cells, 1.02 +/- 0.10 and 0.77 +/- 0.07 mumol Na/mg protein, respectively. Closely similar levels of intracellular sodium were found by flame photometry. Thus, 23Na NMR offers a reliable method for noninvasive quantification of intracellular sodium in perfused cancer cells.

Antagonistic effect of the cardioprotector (+)-1,2-bis(3,5-dioxopiperazinyl-1-yl)propane (ICRF-187) on DNA breaks and cytotoxicity induced by the topoisomerase II directed drugs daunorubicin and etoposide (VP-16).

The effect of the bisdioxopiperazine cardioprotector ICRF-187 (ADR-529, dexrazoxan) on drug-induced DNA damage and cytotoxicity was studied. Using alkaline elution assays, ICRF-187 in a dose-dependent manner inhibited the formation of DNA single strand breaks (SSBs) as well as DNA-protein cross-links induced by drugs such as VP-16 (etoposide), m-AMSA [4'-(9-acridinylamino)-methanesulfon-m-anisidide], daunorubicin and doxorubicin (Adriamycin) which are known to stimulate DNA-topoisomerase II cleavable complex formation. Thus, 50% inhibition of DNA SSBs induced by 5 microM doxorubicin occurred already at equimolar ICRF-187. In contrast, ICRF-187 did not affect DNA SSBs induced by H2O2. In clonogenic assay, ICRF-187 in non-toxic doses antagonized both VP-16 and daunorubicin cytotoxicity in a dose-dependent manner. Our results indicate that the previously described acute in vivo protection by ICRF-187 against anthracycline toxicity may be due to inhibition of topoisomerase II activity. The antagonistic effect of ICRF-187 on daunorubicin cytotoxicity should be taken into consideration when planning clinical trials.

Different modes of anthracycline interaction with topoisomerase II. Separate structures critical for DNA-cleavage, and for overcoming topoisomerase II-related drug resistance.

In contrast to the classic anthracyclines (doxorubicin and daunorubicin), aclarubicin (ACLA) does not stimulate topoisomerase II (topo II) mediated DNA-cleavage. This distinction may be important with respect to topo II-related drug resistance, and the aim of this study was to clarify drug-structures responsible for this difference. Various ACLA analogs were tested for: (a) interaction with purified topo II, (b) induction of DNA cleavage in cells, (c) cellular uptake and (d) cytotoxicity. A remarkable distinction was seen between analogs containing the chromophore aklavinone (AKV) (e.g. ACLA) which have a carboxymethyl group (COOCH3) at C-10 and drugs with a beta-rhodomycinone (RMN) chromophore with hydroxyl groups at C-10 and at C-11. Thus, RMN-containing analogs, including the aglycone RMN itself, effectively stimulated topo II-mediated DNA cleavage. In contrast, AKV-containing drugs inhibited DNA cleavage and antagonized cytotoxicity mediated by RMN-containing drugs. In OC-NYH/VM cells, exhibiting multidrug resistance due to an altered topo II phenotype (at-MDR), cross-resistance was only seen to the RMN-containing drugs whereas no cross-resistance was seen to the non-DNA cleaving AKV-containing compounds. Thus, our data show that one domain in the anthracycline is of particular importance for the interaction with topo II, namely the positions C-10 and C-11 in the chromophore, and further that at-MDR was circumvented by a COOCH3 substitution at position C-10. These findings may provide guidance for the synthesis and development of new analogs with activity in at-MDR cells.

Single-strand conformational polymorphism analysis of the M(r) 170,000 isozyme of DNA topoisomerase II in human tumor cells.

Five cell lines selected for resistance to the cytotoxicity of inhibitors of DNA topoisomerase II have point mutations in the gene that codes for the M(r) 170,000 form of this enzyme. In each case, the mutation results in an amino acid change in or near an ATP binding sequence of the M(r) 170,000 isozyme of topoisomerase II. We used single-strand conformational polymorphism analysis to screen for similar mutations in other drug-resistant cell lines or in leukemic cells from patients previously treated with etoposide or teniposide. We also analyzed the region of the gene that codes for amino acids adjacent to the tyrosine at position 804 of topoisomerase II which binds covalently to DNA. CEM/VM-1, CEM/VM-1-5, and HL-60/AMSA human leukemic cell lines were used as controls; 3 of 3 known mutations were detected by migration differences of polymerase chain reaction products from the RNA extracted from these three lines. A previously unknown mutation was found in the tyrosine 804 region of the M(r) 170,000 topoisomerase II expressed by CEM/VM-1 and CEM/VM-1-5 cells. Sequence analysis showed that substitution of a T for a C at nucleotide 2404 resulted in an amino acid change of a serine for a proline at amino acid 802. No mutations in any of the ATP binding sequences or in the tyrosine 804 region were detected in polymerase chain reaction products from RNA extracted from human leukemia HL-60/MX2 or CEM/MX1 cells (both cell lines selected for resistance to mitoxantrone) or in human myeloma 8226/Dox1V cells (selected for resistance by simultaneous exposure to doxorubicin and verapamil). No mutations were detected in polymerase chain reaction products from RNA extracted from blasts of 15 patients with relapsed acute lymphocytic leukemia, previously treated with etoposide or teniposide. We conclude that: (a) single-strand conformational polymorphism analysis is useful for screening for mutations in topoisomerase II; (b) resistance to the cytotoxicity of inhibitors of DNA topoisomerase II is not always associated with mutations in ATP binding sequences or the active site tyrosine region of M(r) 170,000 topoisomerase II; and (c) mutations similar to those detected in drug resistant cells selected in culture have not been identified in blast cells from patients with relapsed acute lymphocytic leukemia, previously treated with etoposide or teniposide.

Effect of anthracycline analogs on photolabelling of p-glycoprotein by [125I]iodomycin and [3H]azidopine: relation to lipophilicity and inhibition of daunorubicin transport in multidrug resistant cells.

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.

Altered DNA topoisomerase II in multidrug resistance.

The characteristic feature of multidrug resistance (MDR) associated with drugs that interact with DNA topoisomerase II (topo II) is alterations in topo II activity or amount (at-MDR). We have characterized the at-MDR phenotype in human leukemic CEM cells selected for resistance to the topo II inhibitor, VM-26. Compared to drug-sensitive cells, the key findings are that at-MDR cells exhibit (i) decreased topo II activity; (ii) decreased drug sensitivity, activity and amount of nuclear matrix topo II; (iii) increased ATP requirement of topo II; (iv) a single base mutation in topo II resulting in a change of Arg to Gln at position 449, at the start of the motif B/nucleotide binding site; and (v) decreased topo II phosphorylation, suggesting decreased kinase or increased phosphatase activities. Recent results using single-stranded conformational polymorphism analysis reveals the presence of a mutation in the motif B/nucleotide binding site of the topo II alpha gene in CEM at-MDR cells and in another leukemic cell line selected for resistance to m-AMSA. Finally, we have observed marked changes in the nuclear distribution of topo II in cells treated with anti-topo II drugs and have also found these changes to be attenuated in drug-resistant cells. We postulate that traditional inhibitors of topo II alter the equilibrium of the strand-passing reaction such that the number of enzyme-DNA covalent complexes increases. We further suggest that when the enzyme is bound to DNA it is protected from proteolysis, thus allowing more topo II molecules to be detected.(ABSTRACT TRUNCATED AT 250 WORDS)

31P and 13C NMR spectroscopic study of wild type and multidrug resistant Ehrlich ascites tumor cells.

Steady-state 31P NMR spectra of wild type EHR2 Ehrlich ascites tumor cells and multidrug resistant EHR2/DNR+ cells, immobilized in agarose threads, and continuously perfused with medium, showed temperature-dependent differences in the levels of intracellular phosphate metabolites. At 37 degrees C, the EHR2/DNR+ cells contained four times more phosphocreatine (PCr) than the EHR2 cells. At 20 degrees C, the EHR2 cells contained 80% more of phosphodiesters (PDE), the levels of PCr being equal. The quantitative metabolite level data are based on T1 relaxation times data and are normalized for the protein content of the cells. Perfusion of the cells with azide, an inhibitor of mitochondrial respiration, had no effect on the ATP level, and caused no changes in glucose consumption and lactate production. Azide perfusion, combined with glucose depletion, caused rapid drop in the ATP content, which was reestablished after renewed perfusion with glucose. Similarly, perfusion with 2,4-dinitrophenol, an uncoupler of the respiration chain, had no effect on the phosphate metabolites. These results demonstrate that aerobic glycolysis is the main route by which glucose is metabolised under the conditions used (glucose concentration in medium 2 g/L). Rates of uptake and phosphorylation of 2-deoxy-D-glucose were measured by following the formation of intracellular [6-13C]2-deoxy-D-glucose-6-phosphate by 13C NMR; at 37 degrees C the observed rates for EHR2 and EHR2/DNR+ cells were equal, about 10 nmol/(min x mg protein), whereas at 20 degrees C the wild type cells produced the 6-phosphate at an approximately twice the rate found for the resistant cells [about 4 and 2 nmol/(min x mg protein), respectively].(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of tumor cell resistance to 4'-deoxy-4'-iododoxorubicin developed in Ehrlich ascites cells in vivo.

Reduced drug accumulation is the most common functional change accompanying development of P-glycoprotein-associated multidrug resistance. One of our laboratories showed earlier that the anthracycline analogue 4'-deoxy-4'-iododoxorubicin (DIDOX) was accumulated to identical levels in Ehrlich ascites tumor (EHR2) and daunorubicin (DNR)-resistant EHR2/DNR+ cells (E. Friche, P. B. Jensen, T. Skovsgaard, and N. I. Nissen, J. Cell. Pharmacol., 1:57-65, 1990). In this communication, we show that weekly treatment of EHR2-bearing mice with 4, 8, or 12 mg of DIDOX/kg/week led to the development of three DIDOX-resistant cell lines, EHR2/DIDOX-1, EHR2/DIDOX-2, and EHR2/DIDOX-3. The levels of DIDOX accumulation and retention and its outward transport were similar in the drug-sensitive and three drug-resistant cell lines. By contrast, the accumulation of the active DIDOX metabolite, 13-dihydro-DIDOX (13-OH-DIDOX), the parent compound doxorubicin, and daunorubicin were all decreased in proportion to the resistance of the cells. In EHR2/DIDOX-3 cells, the reduction in daunorubicin accumulation coincided with the development of P-glycoprotein as demonstrated by Western blot and flow cytometry with C219 antibody. DIDOX had no effect on the photolabeling of P-glycoprotein by [3H]azidopine, whereas 13-OH-DIDOX inhibited this labeling in a concentration-dependent manner. Subsequent analysis of topoisomerase II activities and amounts in EHR2/DIDOX-3 cells revealed decreased DNA topoisomerase II catalytic activity. The amounts of immunoreactive DNA topoisomerase II from EHR2/DIDOX-1, EHR2/DIDOX-2, and EHR2/DIDOX-3 cells were about 89%, 73%, and 52%, respectively, of that seen in the drug-sensitive cells. We also found that teniposide stabilized DNA-protein complexes in EHR2/DIDOX-3 but they never reached the level seen in EHR2 cells. Because it has been reported that DIDOX is rapidly metabolized to 13-OH-DIDOX, we postulate that the development of resistance to DIDOX in vivo is due in part to its metabolite, 13-OH-DIDOX, which is a substrate for plasma membrane glycoprotein, and in part to DIDOX, which is an inhibitor of topoisomerase II.

Relationship of VP-16 to the classical multidrug resistance phenotype.

The classical multidrug resistance (MDR) phenotype is characterized by cross-resistance between a number of chemically unrelated drugs due to an increased efflux across the plasma membrane via a P-glycoprotein-mediated mechanism. The epipodophyllotoxin derivatives etoposide (VP-16) and teniposide (VM-26) are usually included among the drugs recognized by this MDR phenotype, and the MDR EHR2/DNR cell line is greater than 50-fold cross-resistant to VP-16. The steady-state accumulation of VP-16 in EHR2/DNR cells is only half that of wild-type EHR2 cells, and deprivation of energy by sodium azide surprisingly increased accumulation to a similar extent in both sublines. Efflux was rapid (halflife of 32-35 s) and similar in both sublines, while initial influx was markedly lower in the resistant cells. The temperature coefficients over 10 degrees C for VP-16 in- and efflux indicated passive transport in both sublines. In agreement with this finding, up to 10-fold molar excess (50 microM) VM-26 had no effect on VP-16 accumulation in MDR cells. VP-16 at a 100-fold molar excess inhibited azidopine photoaffinity labeling of P-glycoprotein by only 30% and vincristine binding to plasma membrane vesicles from EHR/DNR cells by 45%. However, VP-16 itself did not differentially bind to plasma membrane vesicles from EHR2 and EHR2/DNR cells. Finally, neither VP-16 accumulation nor cytotoxicity in EHR2/DNR cells were increased to the same degree as for daunorubicin and vincristine by verapamil, and the modulation was similar in wild-type and resistant cells. Thus, although VP-16 may be a substrate for P-glycoprotein, its other transport characteristics such as rapid diffusion and sensitivity to membrane perturbation in wild-type cells lessen any effect of P-glycoprotein-mediated efflux, resulting in a lack of differential modulation by verapamil. These results may be considered when planning clinical trials involving MDR modulators and epipodophyllotoxin derivatives.

Comparison of cyclosporin A and SDZ PSC833 as multidrug-resistance modulators in a daunorubicin-resistant Ehrlich ascites tumor.

Recent studies by Boesch et al. have demonstrated that a nonimmunosuppressive cyclosporin analog, SDZ PSC 833 (an analog of cyclosporin D), is an active multidrug-resistance modifier that is at least 10 times more potent than cyclosporin A. In vitro accumulation and cytotoxicity experiments using daunorubicin (DNR) and vincristine (VCR) under the influence of SDZ PSC 833 and cyclosporin A were performed in wild-type (EHR2) and the corresponding highly DNR-resistant (about 80-fold) Ehrlich ascites tumor cells (EHR2/DNR+). In accumulation experiments, both SDZ PSC 833 and cyclosporin A were found to reverse the multidrug-resistant (MDR) phenotype, but to the same degree at equimolar concentrations. Thus, in EHR2/DNR+ cells, both cyclosporins at 5 micrograms/ml enhanced DNR and VCR accumulation to sensitive levels, but only a negligible effect on DNR accumulation in the drug-sensitive cells was seen. In the clonogenic assay, the cytotoxicity of the two modulators was equal. The lethal dose for 50% of the cell population (LD50) was approx. 7 micrograms/ml for both compounds, and no toxicity was observed at concentrations below 2 micrograms/ml. At nontoxic doses, both cyclosporins effectively increased the cytotoxicity of DNR and VCR in a concentration-dependent manner. The dose-response curves were nearly identical and did not demonstrate differences in modulator potency. These data permit the conclusion that cyclosporin A and SDZ PSC 833 do raise the intracellular accumulation of DNR and VCR to the same levels and that SDZ PSC 833 does not potentiate cytotoxicity better than cyclosporin A in EHR2/DNR+ cells. However, since the new compound is nonimmunosuppressive and causes less organ toxicity, clinical studies of its MDR modulating effect seem highly relevant.

Antagonistic effect of aclarubicin on daunorubicin-induced cytotoxicity in human small cell lung cancer cells: relationship to DNA integrity and topoisomerase II.

The effect of combinations of the anthracyclines aclarubicin and daunorubicin was investigated in a clonogenic assay using the human small cell lung cancer cell line OC-NYH and a multidrug-resistant (MDR) murine subline of Ehrlich ascites tumor (EHR2/DNR+). It was found that the cytotoxicity of daunorubicin in OC-NYH cells was antagonized by simultaneous exposure to nontoxic concentrations of aclarubicin. Coordinately, aclarubicin inhibited the formation of daunorubicin-induced protein-concealed DNA single-strand breaks and DNA-protein cross-links in OC-NYH cells when assayed by the alkaline elution technique. Aclarubicin had no influence on the accumulation of daunorubicin in these cells. In contrast, the accumulation of daunorubicin in EHR2/DNR+ cells was enhanced by more than 300% when the cells were simultaneously incubated with the MDR modulator verapamil, aclarubicin, or the two agents combined. Yet the cytotoxicity of daunorubicin was potentiated significantly only by verapamil. The increased cytotoxicity of daunorubicin in the presence of verapamil was completely antagonized when aclarubicin was used together with the MDR modulator. Finally, the effect of daunorubicin on the DNA cleavage activity of purified topoisomerase II in the presence and absence of aclarubicin was examined. It was found that daunorubicin stimulated DNA cleavage by topoisomerase II at specific DNA sites. The addition of aclarubicin completely inhibited the daunorubicin-induced stimulation of DNA cleavage. Taken together, these data indicate that aclarubicin-mediated inhibition of daunorubicin-induced cytotoxicity is due mainly to a drug interaction with the nuclear enzyme topoisomerase II. This antagonism at the nuclear level explains why aclarubicin is a poor modulator of daunorubicin resistance even though aclarubicin is able to increase the intracellular accumulation of daunorubicin in a MDR cell line.

Decreased DNA topoisomerase II in daunorubicin-resistant Ehrlich ascites tumor cells.

We have previously shown that the multidrug-resistant EHR2/DNR+ cells, which overexpress P-glycoprotein, accumulate only about 20-30% of daunorubicin at steady state compared to the sensitive cells. These cells have been thought to be a "pure" P-glycoprotein cell line. We now report that the EHR2/DNR+ cells exhibit decreased DNA topoisomerase II catalytic activity. We also found that the amount of immunoreactive DNA topoisomerase II from these cells is about one-third that seen in the drug-sensitive cell line. In agreement with the decreased activity and amount of topoisomerase II, the number of DNA-protein complexes stabilized by teniposide (VM-26) was reduced by about 50% in nuclear extracts from EHR2/DNR+ cells. Furthermore, using an intact cell assay for DNA protein complexes, we found that the VM-26-stimulated complexes formed in the drug-resistant cells never reached the level seen in the drug-sensitive cells. Verapamil and Cremophor EL block P-glycoprotein-mediated efflux of "natural product" drugs and increase their accumulation in resistant cells. Coincubation of the EHR2/DNR+ cells with VM-26 and either of these modulators increased the number of complexes formed in the resistant cells. However, neither modulator increased the number of topoisomerase II-DNA complexes in the drug-resistant cells to the level seen in the EHR2 cells. We conclude that the resistance of EHR2/DNR+ cells is due in part to reduced amounts of DNA topoisomerase II. Furthermore, we note that a single cell line can express features of both P-glycoprotein-associated multidrug resistance and altered topoisomerase II-associated multidrug resistance.

Cytosolic free Ca2+ in daunorubicin and vincristine resistant Ehrlich ascites tumor cells. Drug accumulation is independent of intracellular Ca2+ changes.

The possible role of intracellular calcium on daunorubicin (DNR) accumulation in wild-type (EHR2) and multi-drug resistant (MDR) Ehrlich ascites tumor cell subline was investigated. DNR accumulation was not enhanced either by increasing the concentration of cellular calcium with the calcium ionophore ionomycin nor by chelating the cytosolic free Ca2+ by the membrane permeable Ca2(+)-buffering agents BAPTA or MAPTAM. No effect was observed in the presence of extremely low extracellular calcium concentration that prevent transmembrane calcium influx or when the cells were calcium depleted using EGTA and ionomycin. Using the fluorescent Ca2+ indicator fura-2 it is further shown that both drug-resistant daunorubicin (EHR2/DNR+) and vincristine (EHR/VCR+) sublines had lower (50-80 nM) concentration of cytosolic free calcium ([Ca2+]i) compared to their corresponding wild-type parenteral tumors (140-180 nM). In calcium free medium, however, no significant difference was found, all cell lines having a [Ca2+]i of 60-80 nM. Furthermore, the total amount of Ca2+ released to the cytosol with 10 microM ionomycin and 5 mM EGTA was 3-4-fold higher in EHR2 than in EHR2/DNR+ or EHR2/VCR+. Mobilization of Ca2+ with 1 microM ionomycin was almost identical in the presence and absence of Ca2+ in the extracellular medium in EHR2 as well as in EHR2/DNR+ suggesting that the increase in [Ca2+]i is mainly due to discharge of Ca2+ from intracellular stores. Furthermore, the total cell calcium [Ca2+]t concentration was slightly higher in EHR2/DNR+ and EHR2/VCR+ cells compared to EHR2. Incubation of the cells with the Ca2(+)-channel blocker verapamil or the intracellular Ca2(+)-antagonist TMB-8 causes depression of the Ca2(+)-response in terms of rise in [Ca2+]i caused by ionomycin. Sorcin, a major calcium-binding protein (Mr 22 kDa), is shown to be overproduced in EHR2/DNR+ cells. The overproduction of this protein in resistant cells may be related to the difference in the intracellular calcium observed in this study. Thus, though handling of Ca2+ is different in wild-type and MDR cell lines, our data suggest that calcium is not involved directly in drug transport processes and the level of Ca2+ per se have no influence on drug accumulation.

Transport of the multidrug resistance modulators verapamil and azidopine in wild type and daunorubicin resistant Ehrlich ascites tumour cells.

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.

In vitro circumvention of anthracycline--resistance in Ehrlich ascites tumour by anthracycline analogues.

In previously reported studies, acquired experimental resistance and cross resistance to anthracyclines are related to decreased drug accumulation and retention. The decreased accumulation seems to depend on a cellular mechanism for active drug efflux. N-Acetyl-daunorubicin (N-acetyl-DNR) has demonstrated the ability to increase drug accumulation and to overcome experimental resistance to daunorubicin (DNR) in resistant cells. In the present in vitro study 25 different anthracycline analogues were tested for their influence on [3H]DNR accumulation in resistant cells. At equimolar concentrations (5 microM) four of the analogues enhanced [3H]DNR accumulation more than 200%. Increasing the concentration of the analogues 3-20-fold, 12 of the compounds could enhance [3H]DNR accumulation above 200%. No specific structural changes separated those 12 compounds from the 13 analogues with no or minor effect. The lipid solubility of the 25 analogues was examined by measuring the partition coefficient in octanol/phosphate and pentanol/phosphate buffer (pH 7.45). A good correlation was demonstrated between increased lipid solubility of the analogues and their effect on [3H]DNR accumulation in resistant cells. Further studies demonstrated that N,N-dibenzyl-DNR was able to potentiate cytotoxicity of DNR in resistant cells. It is concluded that several anthracycline analogues are able to reverse resistance, but it is not possible from the chemical structure to predict which analogue results in enhanced [3H]DNR accumulation in resistant cells.

The solvents cremophor EL and Tween 80 modulate daunorubicin resistance in the multidrug resistant Ehrlich ascites tumor.

Cremophor EL (polyoxyethylene castor oil) and Tween 80, used as solvents for cyclosporin A and VP-16, respectively, were found to reverse the multidrug resistant (MDR) phenotype. In daunorubicin (DNR) resistant Ehrlich ascites tumor cells (EHR2/DNR+), both solvents at percentages of 0.01% (v/v) enhanced DNR accumulation to sensitive levels. Cremophor EL and Tween 80 did not influence DNR accumulation in drug-sensitive cells (EHR2). The concentration of cyclosporin A alone that enhanced DNR accumulation in EHR2/DNR+ cells to sensitive levels was 5 micrograms/mL whereas 0.2 micrograms/mL of cyclosporin A dissolved in 0.001% (v/v) Cremophor EL enhanced DNR accumulation to sensitive levels, thus indicating synergy between Cremophor EL and cyclosporin A. Cyclosporin A had a negligible effect on DNR accumulation in the drug-sensitive cells. In clonogenic assays, the LD10 of DNR was 1 microM in EHR2/DNR+ cells. Combining 1 microM DNR with non-toxic amounts of Cremophor EL (0.001% and 0.002%, v/v) potentiated the cytotoxicity of DNR and resulted in a cell kill of 77% and 86%, respectively, in the resistant cells. In non-toxic amounts, CrEL and Tween 80 acted synergistically with reduced concentrations of verapamil, resulting in DNR accumulation approaching close to the sensitive level. Azidopine photoaffinity labeling of P-glycoprotein in plasma membrane vesicles from EHR2/DNR+ cells was inhibited 100% and 80%, by 0.003% (v/v) Cremophor EL or Tween 80, respectively. These data permit the conclusion that non-toxic amounts of CrEL and Tween 80 modulated DNR resistance by raising intracellular DNR levels, due to their abilities to bind to the plasma membrane P-glycoprotein.