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.

Targeting the cytotoxicity of topoisomerase II-directed epipodophyllotoxins to tumor cells in acidic environments.

The epipodophyllotoxins etoposide and teniposide are probably the most important drugs in the treatment of small cell lung cancer. The drugs are used in maximally tolerated doses, and the toxicity of the drugs precludes significant dose increments. The cellular target is the nuclear enzyme topoisomerase II which, in the presence of these drugs, causes an extensive fragmentation of DNA. The cell kill can be antagonized by distinct drug types. We have demonstrated previously that the intercalating drug aclarubicin and the cardioprotecting agent ICRF-187 antagonize the cytotoxicity of etoposide in vitro. We have studied possible ways of using this antagonism as a means of differentially protecting normal tissue. Here we demonstrate that the intercalating agent chloroquine prevents the introduction of topoisomerase II-mediated DNA breaks and thereby antagonizes the cytotoxicity of etoposide. This interaction depends on the extracellular pH. Chloroquine, in contrast to etoposide, is a weak base and therefore does not enter the cell if the extracellular fluid is acidic, as is the case in most solid tumors. We propose that such a pH-dependent drug interaction may be useful in directing topoisomerase II drug effects toward solid tumors. Thus, by lowering the extracellular pH (pHe) from neutral (pHe = 7.4) to acidic (pHe = 6.0), [3H]chloroquine accumulation was decreased 5-fold in a human small cell lung cancer cell line, OC-NYH, and in murine leukemia L1210 cells. In parallel, the antagonism exhibited by chloroquine on etoposide cytotoxicity was pHe dependent. Thus, no protection by chloroquine was observed at pHe = 6.5, whereas at pHe = 7.4, etoposide cytotoxicity was almost completely antagonized with a 460-fold protection or more than eight doublings of the cell population. This exploitation of antagonist extracellular trapping by acidic pH is a novel model for regulation of anticancer drug effects.

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.

Antagonistic effect of aclarubicin on the cytotoxicity of etoposide and 4'-(9-acridinylamino)methanesulfon-m-anisidide in human small cell lung cancer cell lines and on topoisomerase II-mediated DNA cleavage.

The effect of combinations of the anthracycline aclarubicin and the topoisomerase II targeting drugs 4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidene-beta-D-glucopyra noside) (VP-16) and 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) was investigated in a clonogenic assay. The cytotoxicity of VP-16 was almost completely antagonized by preincubating cells with nontoxic concentrations of aclarubicin. The inhibition of cytotoxicity was not seen when the cells were exposed to aclarubicin after exposure to VP-16. The inhibition was significant over a wide range of aclarubicin concentrations (3 nM to 0.4 microM), above which the toxicity of aclarubicin became apparent. A similar effect was seen on the toxicity of m-AMSA. In contrast to aclarubicin, preincubation with Adriamycin did not antagonize the effect of VP-16. With purified topoisomerase II and naked DNA, aclarubicin did not stimulate the formation of cleavable complexes between topoisomerase II and DNA. Aclarubicin concentrations above 1 microM inhibited the baseline formation of cleavable complexes elicited with the enzyme alone. Low (1 to 10 nM) aclarubicin concentrations increased the formation of cleavable complexes obtained with VP-16 and m-AMSA; however, at aclarubicin concentrations above 1 microM an antagonistic effect was obtained. In cells, the m-AMSA- and VP-16-induced, protein-concealed DNA strand breaks were completely inhibitable by aclarubicin preincubation with no synergic dose levels. Our results suggest that aclarubicin inhibits topoisomerase II-mediated DNA cleavage. This inhibition could represent the mechanism of action of the drug and explain the lack of cross-resistance to the classical anthracyclines. The observed antagonism could have consequences for scheduling of aclarubicin with topoisomerase II-active anticancer drugs.

Covalent complexes between serum albumin and 7-hydroxycoumarin-4-acetic acid: synthesis and applications in the spectrophotometric detection of long-chain fatty acids.

Using a hydrophobic 8-aminooctanoic acid cross-linker, the pH-indicator dye 7-hydroxycoumarin-4-acetic acid (7-HCA) is covalently bound to bovine serum albumin (BSA) at the positions of reactive amino groups. A highly stable and water-soluble complex (BSA-HCA) with a 1:4 molar stoichiometry is synthesized. Appearance of a strong absorption band at gamma max = 372 nm is associated to ionization of the 7-HCA chromophore when it is transferred from water into a basic microenvironment on the BSA surface. This particular surface site is related to the region(s) for high-affinity binding of long-chain fatty acids (FA). BSA-HCA responds to binding of FA (14-20 carbons) with immediate spectral changes and a decrease in 372 nm absorption. BSA-HCA provides an indicator-protein having a range of practical applications for the quantitative determination of long-chain FA in biochemical studies. The lower detection limit in a spectrophotometric method is approximately 1 microM FA. BSA-HCA is usable both in various buffers and in the presence of detergents such as n-octylglucoside, Triton X-100 and CHAPS. A novel method for continuous assay of phospholipase A2 activity with BSA-HCA and a mixed phosphatidylcholine/CHAPS micellar substrate is reported.

Incorporation of large quantities of phospholipid by net de novo synthesis in cytoplasmic membrane vesicles of Escherichia coli.

De novo phospholipid synthesis has been studied in cytoplasmic membrane vesicles of Escherichia coli in the presence of sn-glycerol 3-phosphate, CTP, serine and acyl-CoA. Continuous addition of acyl-CoA was found to result in phospholipid biosynthesis during more than 4 h of incubation and phospholipid corresponding to 3--4-times the total amount of lipid originally present was synthesized under these conditions. The major biosynthetic products were phosphatidylglycerol, cardiolipin and phosphatidic acid. The composition of the accumulated lipid could to some extent be varied by adding the acyl-CoA to the vesicles at different rates. The newly synthesized lipid was found to be present in a population of membrane vesicles (80% of the total) which had a decreased density in comparison to the starting material. Electron microscopy of negatively stained preparations before and after incubation indicated that the vesicular nature of the cytoplasmic membrane fragments was preserved throughout incubation. A minor population of vesicles, the density of which was unchanged during incubation, contained little de novo synthesized lipid. The activity of the sn-glycerol-3-phosphate acyltransferase decreased during incubations to 25% in vesicles which had accumulated phospholipid. Conversion of phosphatidic acid into phosphatidylglycerol and cardiolipin was uneffected by the lipid content of the vesicles.

Recognition of anthracycline binding domains in bovine serum albumin and design of a free fatty acid sensor protein.

The mode of binding of N-acylated doxorubicin derivatives to bovine serum albumin (BSA) has been determined by spectrophotometric analysis. A water-insoluble derivative containing a N-hydroxysuccinimide ester moiety on the sugar amino group side-chain is found to react very rapidly with a specific domain (BSAA600) located in the NH2-terminal half of the BSA molecule. A stable covalent protein-anthracycline complex with 1:1 molar stoichiometry and containing the albumin monomer is formed. This specific association between albumin and doxorubicin derivative is accompanied by large changes in the spectral characteristics of the anthracycline chromophore. A new strong absorption band at 600 nm is associated to ionization of the chromophore phenolic groups. Titration experiments indicate that the pKa of the protein bound anthracycline is about 3 pH units lower than the pKa of free doxorubicin in aqueous buffer indicating chromophore localization in a basic microenvironment on the albumin molecule. For N-acylated doxorubicin derivatives which associate non-covalently to the BSAA600 domain, the strength of binding is found to be controlled by ionic as well as hydrophobic protein-anthracycline interactions. Water-soluble derivatives containing a side-chain carboxylic group bind with Kd approximately 10 microM, which is at least 100-fold more strongly than doxorubicin. The anthracycline chromophore is displaced from the BSAA600 domain in a non-competitive manner by fatty acids ranging in chain length from C6 to C18 and at a fatty acid/BSA molar ratio < 2. We therefore propose a model for the anthracycline binding domain in which the chromophore resides near the opening of the hydrophobic channel into which the fatty acid hydrocarbon chain is inserted. The clusters of basic amino acid residues located at this site may form the basic anthracycline microenvironment. Our results demonstrate that doxorubicin derivatives with a sugar amino group side-chain are well suited as probes for investigations on protein-anthracycline interactions. The practical application of the covalent BSA-DOX complex as a free fatty acid sensor protein for detection of enzymatic release of fatty acids in liposomal and cell membranes is suggested.

A model for computer simulation of P-glycoprotein and transmembrane delta pH-mediated anthracycline transport in multidrug-resistant tumor cells.

Anthracycline resistance in multidrug-resistant (MDR) tumor cells is due in part to a reduced cellular drug accumulation. Using a simple kinetic model and numerical computer simulations, we have analyzed mathematically the following possible mechanisms controlling fluxes of the membrane permeable anthracyclines in MDR cells: (1) active outward transport via a specific drug transporter (P-glycoprotein), (2) exocytotic drug export via the endosomal vesicle system, and (3) pH-gradients across the plasma membrane. Model calculations were based on morphometric and kinetic data previously presented in the literature for daunorubicin transport in wild-type Ehrlich ascites tumor cells (EHR2) and the corresponding daunorubicin (DNR)-resistant cell line EHR2/DNR+. The results confirm the possible importance of the cell-surface pH in controlling DNR accumulation in the cells. With P-glycoprotein as the main efflux pump, a catalytic constant of the protein greater than 40 mol DNR transported/mol protein per min is predicted by the model calculations. Changes in the drug binding affinity of P-glycoprotein (Km = 10(-9)-10(-6) M) is of little importance in influencing its effectiveness to reduce DNR accumulation, which could explain the broad substrate specificity of the MDR efflux pump system. The conditions to evaluate unidirectional fluxes of DNR across the plasma membrane in cells with active P-glycoprotein are defined. An efflux mechanism which relies solely on pH-dependent drug trapping in a pH 5 endosomal compartment by a simple diffusion process followed by exocytosis, appears inadequate to account for the high rate of DNR efflux in EHR2/DNR+ cells.

Characterization of the cooperative cross-linking of doxorubicin N-hydroxysuccinimide ester derivatives to water soluble proteins.

Protein-anthracycline interactions have been examined by using reactive N-hydroxysuccinimide ester derivatives of doxorubicin. These compounds cross-link to lysine epsilon-amino groups with high efficiency and offer the possibility for structural studies of protein-anthracycline complex formation by using gel filtration, ultracentrifugation and spectrophotometric methods. The results are in accordance with association of anthracycline to the hydrophobic ligand binding cavities of serum albumin. The results for proteins not having hydrophobic domains (IgG, serum transferrin, lactotransferrin, ovotransferrin) suggest that complex formation is cooperative and involves two steps: initial self-association of anthracycline into aggregated structures and subsequent binding of protein at the aggregate surface. With serum transferrin, anthracycline self-association makes possible the assembly of stable nanometer-sized protein-anthracycline particles held together by non-covalent bonds. This reaction, which is highly reproducible and efficient, may have applications in the field of development of anthracycline carrier systems.

Covalent modification of serum transferrin with phospholipid and incorporation into liposomal membranes.

A method is described for incorporation of water-soluble proteins into liposomal membranes using covalent protein-phospholipid conjugates in detergent solution. A disulfide derivative of phosphatidylethanolamine containing a reactive N-hydroxysuccinimide ester group is synthesized, and the derivative is reacted with serum transferrin in deoxycholate-containing buffer. Disulfide-linked transferrin-phosphatidylethanolamine conjugates containing up to 6 mol phospholipid/mol protein are prepared. The amphiphilic conjugates have solubility properties very similar to integral membrane proteins. The conjugates self-associate to form protein micelles of narrow size distribution (Stokes radii 6-7 nm), and in the presence of excess phospholipid (egg phosphatidylcholine), they readily incorporate into liposomal membranes upon removal of detergent. Stable incorporation into liposomes requires the introduction of two molecules of phosphatidylethanolamine into the transferrin. Using the disulfide linker to release transferrin from the liposomes, evidence is presented for a function of the phosphatidylethanolamine as an anchor-molecule into the liposomal lipid. Optimal conditions for preparation of homogeneous liposomes with diameters in the range 30-125 nm and with a varying content of transferrin are defined. The liposomes appear well suited for studies on liposome-cell membrane interactions.

Transfer of ferritin-bound iron to adriamycin.

Interactions of adriamycin with ferritin-bound iron have been investigated. It is demonstrated (i) that adriamycin stimulates an iron-dependent lipid peroxidation in submitochondrial particles in the presence of ferritin, and (ii) that incubation of adriamycin with ferritin results in a slow transfer of iron to adriamycin with formation of an adriamycin-iron complex. The results are discussed in relation to the possible role for intracellular iron in adriamycin toxicity.

Binding of transferrin-iron by adriamycin at acidic pH.

It is shown that adriamycin is able to chelate iron released from iron-loaded serum transferrin in the pH range from 6.5-4.1. The kinetics of iron transfer to free adriamycin and to adriamycin covalently attached to the transferrin has been determined. The results show that adriamycin, if introduced into intracellular acidic compartments, could function as acceptor for transferrin-iron.

NADH oxidation in phospholipid-enriched cytoplasmic membrane vesicles from Escherichia coli.

NADH oxidation in Escherichia coli cytoplasmic membrane vesicles enriched in anionic phospholipids by de novo synthesis of lipid in the vesicles from acyl-CoA esters and sn-glycerol 3-phosphate has been studied. NADH-oxidase but not NADH-dehydrogenase activity was found to decrease during synthesis and accumulation of phospholipid in the vesicles. Density gradient fractionation showed that NADH-oxidase activity was reduced to approximately 30% in vesicles with a 3-6 fold increase in anionic phospholipid, whereas vesicles with a greater than 10-fold increase in phospholipid had virtually no NADH oxidase activity.

Inactivation of cytochrome c oxidase activity in mitochondrial membranes during redox cycling of doxorubicin.

Interactions of doxorubicin (DX) with the cardiolipin-dependent cytochrome c oxidase have been examined by using pig heart submitochondrial particles (SMP). A progressive and irreversible loss of oxidase activity is demonstrated in 2 hr incubations of the SMP with 10-100 microM DX in air-equilibrated medium with excess NADH to support redox-cycling of the drug. This oxidative mechanism for oxidase inactivation occurs in connection with a peroxidation process in the bulk membrane lipid, and is independent on turnover of the enzyme. It is related in a complex manner to the electron flux in the respiratory chain with antioxidant properties, and is maximal at the high reduction level of respiratory chain Complex I obtained in the presence of rotenone. Reduction of DX per se plays a minor role, and trace concentrations of chelatable metal ions (iron) are required to catalyse the reaction. Iron in the iron storage protein ferritin is released by DX, and at physiological low O2 concentrations ([O2] less than 20 microM), this iron is a better promoter of oxidase inactivation than is endogenous iron in the SMP. Kinetic analysis of inactivation data indicates the interaction of DX with low affinity (Km 35-55 microM) binding sites in the SMP membranes. Overall, the results point to the possible role of ferritin-iron in the mechanism of DX mitochondrial toxicity and argue against site specific effects of the DX-reduction/oxidation cycle on the cytochrome c oxidase or on its essential phospholipid (cardiolipin) environment.

Doxorubicin induced alterations in lipid metabolism of cultured myocardial cells.

Doxorubicin (DX) was found to inhibit the incorporation of [1-14C]linoleic acid and [1(3)-3H]glycerol into the major membrane phosphoglycerides, phosphatidylcholine and phosphatidylethanolamine of cultured myocardial cells in a dose-dependent manner (0.16-16 microM). It is suggested that DX affects de novo biosynthesis of these lipids. In contrast, DX-treatment of the cells stimulated incorporation of [1-14C]linoleic acid into triacylglycerol. The effects of DX on lipid metabolism were only demonstrable 20-24 hr after a 1 hr exposure of the cells to the drug indicating that DX exerts little or no direct effect on the enzymes participating in lipid synthesis and that the alterations in lipid metabolism induced by DX probably are secondary to inhibition of protein synthesis and progressive cell injury. Extensive peroxidative decomposition of membrane lipids appeared not to take place in the DX-treated cells as judged from fatty acid analysis of total membrane phosphoglyceride.

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.

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.

Daunorubicin and vincristine binding to plasma membrane vesicles from daunorubicin-resistant and wild type Ehrlich ascites tumor cells.

Tumor cell resistance to anthracyclines, epipodophyllotoxins and vinca alkaloids, called multi-drug resistance (MDR) is intimately linked to changes in the plasma membrane which facilitate an increased energy dependent drug extrusion in the resistant cell compared to the wild type cell. Isolated plasma membrane vesicles from wild type Ehrlich ascites tumor cells (EHR2) and the daunorubicin (DNR) resistant subline EHR2/DNR+ were utilised to study binding and possible transport of DNR and vincristine (VCR). A significant ATP enhanced increase in VCR binding to vesicles from EHR2/DNR+ compared to EHR2 was demonstrated. Furthermore, an increase in ATP enhanced VCR binding in proportion to content of the MDR associated P-glycoprotein was seen in plasma membrane vesicles prepared from various benign human endocrine tumors. VCR binding to EHR2/DNR+ vesicles was inhibited by other vinca alkaloids greater than actinomycin D greater than colchicine greater than anthracyclines, with 35-75 microM concentrations of anthracyclines needed for 50% inhibition. VCR binding to EHR2/DNR+ vesicles was pH and temperature dependent with an activation energy of -30 kJ/mol and was decreased by replacement of Na+ with K+ and by addition of Ca2+. Preincubation of vesicles with monoclonal antibody against the C terminal of P-glycoprotein had no effect on VCR binding and osmolality tests failed to show genuine transmembranal transport of VCR. DNR binding was similar in plasma membrane vesicles from both cell lines, and showed none of the characteristics mentioned for VCR. Furthermore, a radiolabeled N-hydroxysuccinimide ester derivative of doxorubicin, which inhibited VCR binding to EHR2/DNR+ membranes to an even greater extent than doxorubicin, labeled plasma membrane proteins from EHR2 and EHR2/DNR+ identically and did not demonstrate any binding to P-glycoprotein. Therefore, even though the study confirms the close link between vinca alkaloid binding and P-glycoprotein, it could not detect a similar association between anthracyclines and P-glycoprotein thus attesting to the complexity of the MDR phenotype.

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.