Contribution of serum and cellular semicarbazide-sensitive amine oxidase to amine metabolism and cardiovascular toxicity.

Semicarbazide-sensitive amine oxidase (SSAO) plays a role in the in vivo and in vitro toxicity of several environmental and endogenous amines. We investigated the role of SSAO as a component of cell culture medium (through addition of fetal calf serum (FCS)) compared to intracellular SSAO in the in vitro cytotoxicity of three amines and metabolites. Smooth muscle cells and beating cardiac myocytes were grown in 96-well plates and exposed to various concentrations and combinations of FCS in medium, amines (allylamine, AA; benzylamine, BZA; and methylamine, MA), and amine metabolites (aldehydes: acrolein, benzaldehyde, and formaldehyde; hydrogen peroxide, H2O2; ammonia, NH3). Amine and amine metabolite cytotoxicity was quantified by monitoring cell viability. SSAO activity was measured in FCS, cardiovascular cells, or rat plasma by a radioenzymatic assay using [14C]BZA. Our data show that AA and its aldehyde metabolite, acrolein, were the most toxic compounds to both cell types. However, AA toxicity was FCS-dependent in both cell types, while BZA, MA, and amine metabolite (i.e., aldehydes, H2O2, and NH3) cytotoxicity showed little FCS dependence. In these experiments, medium containing 10% FCS had a calculated amine metabolic capacity that was 30- to 50-fold that of the cultured smooth muscle cellular content in a single well of a 96-well plate. Our study demonstrates that SSAO in FCS contributes to amine metabolism and cytotoxicity to rat cardiovascular cells in vitro and how critical it is to evaluate serum for its role in mechanisms of amine toxicity in vitro and in vivo.     

The antiproliferative action of deoxyspergualin is different from that induced by amine oxidase

The amine oxidase activities contained in calf serum and human serum were detected at levels of 90.8 and less than 0.1 nmol O2/minute/ml serum, respectively, by measuring oxygen consumption coupled with spermidine oxidation. Deoxyspergualin (NKT-01) and spergualin (SGL) containing spermidine in their structure were also oxidized in calf serum at the rate of 3.6 and 11.6 nmol O2/minute/ml serum, respectively. To investigate whether amine oxidase is essential for NKT-01 and SGL to exhibit their antiproliferative activities or not, the in vitro activities of NKT-01, SGL and polyamines against L1210 cells were examined in the presence of calf or human serum. Polyamines exhibited antiproliferative activity only in the presence of calf serum, while NKT-01 and SGL inhibited cell growth in the presence of both calf and human serum. In the presence of calf serum the activity of NKT-01 was inhibited by aminoguanidine, an amine oxidase inhibitor. Aminoguanidine did not inhibit the activity of NKT-01 in the presence of human serum. The activity of NKT-01 was shown at much lower concentrations in the presence of human serum than that in the presence of calf serum, and was strongly dependent on incubation time. The in vivo activities of NKT-01, SGL and SGL derivatives correlated with their in vitro activities in the presence of human serum. These results suggest that the in vivo antitumor activities of NKT-01, SGL and SGL derivatives may be attributed to a mechanism different from those of amine oxidase-oxidized product and represent a novel growth inhibitory action.     

Allylamine-induced vascular toxicity in vitro: prevention by semicarbazide-sensitive amine oxidase inhibitors

The present studies were designed to evaluate the role that metabolic activation plays in allylamine (AAM)-induced vascular toxicity. The effects of AAM were evaluated in primary cultures of rat vascular endothelial (VEC) and smooth muscle cells (SMC). Semicarbazide (SC) and diethyldithiocarbamate (DDC) were used as inhibitors of semicarbazide-sensitive amine oxidase (SSAO). Clorgyline and pargyline were used as inhibitors of monoamine oxidase (MAO) A and B, respectively. The effect of catalase, a hydrogen peroxide scavenger, on AAM-induced cytotoxicity was also evaluated. Lactate dehydrogenase (LDH) release and morphological alterations were chosen as indicators of cytotoxicity. Confluent cultures of VEC and SMC were exposed to various concentrations of AAM (2-200 microM) in the absence and presence of serum for 4, 12, or 24 hr. High concentrations of AAM (200 microM) alone produced a time-dependent increase in LDH release and morphologic alterations in cultures of both cell types. Lower concentrations of AAM did not compromise the structural integrity of the cells. Semicarbazide (200 microM) or DDC (2 mM), but not clorgyline (10 microM) or pargyline (10 microM), prevented the toxicity of AAM (200 microM). Allylamine-induced cytotoxicity was partially prevented by catalase (2500 U/ml). The presence of fetal bovine serum in the medium was not essential for the manifestation of cytotoxicity. Single cell suspensions of VEC or SMC formed acrolein (ACR) when incubated in the presence of AAM. The formation of ACR mediated by SMC was inhibited by SC (20 microM), but not clorgyline (10 microM). These results support the concept that AAM is oxidatively deaminated by an SSAO present in vascular cells to generate toxic metabolic by-products capable of causing extensive cellular injury.     

The decrease of cytochrome c oxidase activity by 15-deoxyspergualin results in enhancement of XTT reduction in cultured cells

Exposure to immunosuppressant, 15-deoxyspergualin (DSG) induced enhanced formazan producing activity from XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carb oxanilide, sodium salt) in cultured cells, but not from MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). Formazan generation from XTT is known to be stimulated by cytochrome c oxidase (COX) inhibitors such as KCN and NaN3, whereas MTT reduction is not affected by them. So, the effect of DSG on COX was examined. DSG did not directly inhibit the enzyme, but the cellular enzyme activity was decreased by exposure to DSG. It was thought that stimulation of XTT reduction by DSG resulted from the decrease of cellular cytochrome c oxidase activity.     

Differential effects of NAD, nicotinamide and related compounds upon growth and nucleoside incorporation in human cells

Two human melanoma cell lines, MM96 and MM127, were found to be highly sensitive to the toxicity of adenosine (D50 100-150 micrograms/ml) compared with other melanoma lines. HeLa cells and a lymphoblastoid line (D50 greater than 500 micrograms/ml). The MM127 line was also sensitive to NAD (D50 41 micrograms/ml) compared with the other lines (D50 greater than 400 micrograms/ml), and accumulated three-fold more NAD-derived isotopic label. Nicotinamide exhibited little toxicity in any cell type (D50 greater than 400 micrograms/ml); 25-100 micrograms/ml nicotinamide greatly increased the plating efficiency of melanoma cells and fibroblasts when low levels of foetal calf serum were used. The toxicity of DNA-damaging agents (alkylating agents and u.v.) in melanoma cells was not reduced in the presence of NAD, adenosine or nicotinamide. Studies of the effects of the latter compounds upon the incorporation of deoxynucleosides showed that: (a) melanoma cells have lower purine pools than fibroblasts; (b) [3H]deoxyguanosine incorporation was inhibited more than [3H]deoxyadenosine incorporation; (c) incorporation of [3H]deoxyadenosine and [3H]deoxyguanosine into RNA was inhibited by adenosine, thus providing a method for determination of guanine-specific DNA repair; and (d) NAD enhanced thymidine incorporation in intact melanoma cells but not in fibroblasts, in a pattern similar to the release from template restriction previously reported for permeabilised tumour cells.     

Effects of an antitumor agent, ascofuranone, on the macromolecular syntheses of intact cells

Ascofuranone (AF) has antitumor protective property on experimental tumors. We examined the action of AF on lymphoma L5178Y to explore the mechanism of the antitumor activity. AF completely prevented the growth of L5178Y at 25 micrograms/ml cytostatically. The compound exhibited general inhibitory effects on the macromolecular syntheses. Among them, protein synthesis was most severely inhibited by AF and to the same extent as by cycloheximide. AF, however, did not affect protein synthesis by cell-free system even at 2 mg/ml. Although AF inhibited the incorporation of [14C]acetate into total acid precipitable products only slightly, the synthetic pattern of simple lipids from [14C]acetate was significantly changed. Especially, the incorporation of [14C]acetate into squalene was almost completely blocked at 25 micrograms/ml. The incorporation of [14C]acetate into triglyceride was inhibited and that into cholesterol was enhanced. Concerning the diglycerides, the incorporation of [14C]acetate was enhanced and that of [3H]glycerol was inhibited. The incorporation of [3H]glycerol and [3H]mevalonate into the intact cell was significantly inhibited as compared with [14C]acetate. As those effects were not observed with cycloheximide, they were suggested to be characteristic of AF. AF inhibited hypotonic hemolysis. In contrast, hemolysis by deoxycholate was stimulated. Possible mechanism of the antitumor activity of AF is discussed.     

Monoamine oxidase activities in lymphocytes and granulocytes taken from pig blood

The characterisation of monoamine oxidase activities in lymphocytes and granulocytes was studied using cells prepared from pig blood. The specific activities against beta-phenylethylamine, benzylamine, tyramine and 5-hydroxytryptamine as substrates in granulocytes (G) were approximately twice those found in lymphocytes (L). The absence of the semicarbazide-sensitive amine oxidase (SSAO) was confirmed by insensitivity of the latter to semicarbazide as inhibitor with benzylamine as substrate. MAO activity present in (G) and (L) was selectively inhibited by low deprenyl concentrations; this fact, in addition to the simple sigmoid inhibition curves obtained with increasing concentrations of clorgyline with tyramine as substrate, suggests that the MAO activity present both in (G) and (L) is predominantly of the MAO-B form. The absence of any contamination with plasma amine oxidase (EC 1.4.3.6) was confirmed by the fact that activity towards benzylamine (Bz) was insensitive to KCN-induced inhibition. Kinetic constants were determined for each fraction towards beta-phenylethylamine (PEA) and Bz as substrates. MAO-B was titrated with unlabelled pargyline, deprenyl and [3H]-pargyline; the corresponding Kcat values, turnover number and the active concentrations were then determined. The molecular weight of MAO-B present in both cellular fractions was calculated by SDS-electrophoresis and fluorography, after reaction with [3H]-pargyline. Some of these results are compared with those obtained with human blood leucocytes.     

Tyramine antagonistic properties of AGN 1135, an irreversible inhibitor of monoamine oxidase type B.

1 The effects of the irreversible monoamine oxidase (MAO) inhibitors, AGN 1133, AGN 1135 and (-)-deprenyl, on tyramine and noradrenaline responses and uptake of [3H]-metaraminol were investigated in the isolated vas deferens of the rat. Uptake of [3H]-metaraminol and [3H]-octopamine was compared in mouse vas deferens. The modification of tyramine and noradrenaline-induced pressor responses by AGN 1133 and AGN 1135 was examined in anaesthetized rats and cats. 2 AGN 1133 (7.5 x 10(-6)M) greatly potentiated responses to tyramine in the rat isolated vas deferens. Both AGN 1135 and (-)-deprenyl inhibited tyramine responses selectively at concentrations above 10(-5)M (which caused almost complete inhibition of MAO types A and B) but tyramine responses were potentiated on washing out the inhibitors. 3 AGN 1135 (10(-4)M) and (-)-deprenyl (10-5)M) inhibited [3H]-metaraminol uptake by about 20% in rat and mouse vas deferens; neither inhibitor affected [3H]-octopamine uptake in mouse vas deferens. Desmethylimipramine (10(-6)M) inhibited amine uptake by more than 70%. 4 AGN 1133 (1.5 mg/kg) potentiated pressor responses to tyramine in rats and cats whereas AGN 1135 (1.5 mg/kg) did not. 5 AGN 1135 possesses tyramine antagonistic activity which is qualitatively similar to that of (-)-deprenyl but which cannot satisfactorily be explained by inhibition of neuronal or granula amine uptake.     

Mode of action of 3-substituted propylamine cytotoxicity in culture cells

Cytotoxic effects on MDBK cells of various 3-substituted propylamines, including spermine and spermidine, were tested in culture in the presence of calf serum, and the possible mode of action was studied from the viewpoint of oxidative deamination leading to acrolein formation. The compounds were roughly classified in two groups with ic₅₀ values of 0.1 and 0.4mM under the conditions used. Phenyl derivatives of 3-substituted propylamines, 3-benzylaminopropylamine, and polyamines were included in the first group with ic₅₀ values of 0.1 mM, and acrolein was liberated from these compounds after incubation with bovine plasma amine oxidase. Alkyl derivatives of 3-substituted propylamines (with ic₅₀ values of 0.4mM), on the other hand, were unable to release acrolein after the oxidative deamination, which was further confirmed by a lack of liberation of acrolein from the authentic 3-butoxypropanal. These observations indicated that both the acrolein originating from the 3-substituted propanals, and the propanal as such, were possibly involved in manifestation of the cytotoxicity of the 3-substituted propylamines. Thus, spermine and spermidine may exert their cytotoxic effects on the cells in vitro by a combination of two mechanisms involving acrolein and oxidized polyamines.     

The effects of serum, lithium, ethacrynic acid, and a low external concentration of potassium on specific [3H]-ouabain binding to human lymphocytes after incubation for 3 days.

We have quantified specific [3H]-ouabain binding sites in normal human lymphocytes, and have measured the changes in the numbers of those sites which occur in response to various stimuli. We have confirmed previous findings that incubation for 72 h in the presence of fetal calf serum causes an increase in [3H]-ouabain binding, and that this does not occur if the cells are incubated in fetal calf serum which has first been dialysed. During incubation of the lymphocytes for 3 days in the presence of dialysed fetal calf serum each of the following stimuli caused an increase in specific [3H]-ouabain binding: addition of ethacrynic acid (1 mumol l-1), addition of lithium (1 mmol l-1), and reduction of the external potassium concentration (to 0.75 mmol l-1). By analogy with the similar results in HeLa cells reported by others, we suggest that the increase in [3H]-ouabain binding may, in the case of ethacrynic acid and the reduction of the external potassium concentration, be initiated by an increase in the intracellular sodium concentration. The mechanisms whereby fetal calf serum and lithium cause an increase in [3H]-ouabain binding are not clear.     

The effect of 2-ethyl-3-(4-hydroxybenzoyl)-benzofuran (benzarone) on the metabolism of arterial tissue and cultured arterial smooth muscle cells

Studies on the action of 2-ethyl-3-(4-hydroxybenzoyl)-benzofuran (benzarone) on the metabolism of cultured arterial smooth muscle cells and arterial tissue had the following results: 1. On incubation of calf arterial tissue in the presence of 0.03--0.1 mmol/l benzarone (8--26 micrograms/ml medium) the metabolic transformation of [14C]-glucose to [14C]-lactate and 14CO2 and the incorporation of 14C radioactivity into the total lipids is not significantly altered as compared with control values. In cultured human arterial smooth muscle cells 0.03 mmol/l benzarone stimulates the incorporation of [14C]-acetate and [3H]-palmitate into the cellular lipids while the receptor mediated uptake of homologous low-density lipoproteins (LDL) by the cells and their release are not influenced. 2. In concentrations greater than 0.2 mmol/l benzarone the glucose utilisation of arterial tissue is enhanced, while the labelling of lipids, in particular the labelling of the triglyceride fraction, is depressed. Under the same conditions the protein biosynthesis and the incorporation of [14C]-acetate and [3H]-palmitate into the total lipids of cultured arterial smooth muscle cells are decreased.     

Reversal of multidrug resistance by kopsiflorine isolated from Kopsia dasyrachis.

Kopsiflorine, an indole alkaloid of the aspidofractinine-type isolated from Kopsia dasyrachis, was examined for its effect in enhancing drug cytotoxicity in multidrug-resistant tumor cells. The cytotoxicity of vincristine was enhanced in a concentration-dependent manner by kopsiflorine in drug-resistant KB cells (VJ-300). Kopsiflorine alone had no effect on the growth of drug sensitive or resistant cells, but the intracellular accumulation of vincristine was enhanced by kopsiflorine in VJ-300 cells. Kopsiflorine (10 micrograms/ml) significantly inhibited the binding of [3H]azidopine to P-glycoprotein in VJ-300 cells. The results suggest that kopsiflorine interacts directly with P-glycoprotein and inhibits the efflux of antitumor agents in drug-resistant cells.     

Chromosomal aberrations in V79 cells induced by superoxide radical generated by the hypoxanthine-xanthine oxidase system

The effect of the superoxide radical, generated by the hypoxanthine-xanthine oxidase system, on chromosomal mutation was examined in Chinese hamster V79 cells. When cells were treated with this system for 1 h in Hanks' solution, the incidence of metaphases with chromosomal aberrations was increased with hypoxanthine at concentrations of 2.5 to 10 micrograms/ml. On the other hand, in Eagle's minimum essential medium (MEM) or MEM supplemented with 10% fetal bovine serum, only hypoxanthine at 5 micrograms/ml plus xanthine oxidase induced chromosomal aberrations and higher concentrations of hypoxanthine were cytotoxic to V79 cells. The increased frequency of chromosomal aberrations and the cytotoxicity of hypoxanthine plus xanthine oxidase were not affected by superoxide dismutase, but were strongly inhibited by catalase.     

Modulation of doxorubicin-induced chromosomal damage by calmodulin inhibitors and its relationship to cytotoxicity in progressively doxorubicin-resistant tumor cells

Modulation of doxorubicin (DOX) cytotoxicity by the calmodulin inhibitor trifluoperazine (TFP) in progressively doxorubicin-resistant L1210 mouse leukemia cells is unrelated to effects on drug accumulation. Based on the clastogenic activity of DOX, the effects of TFP and the selective calmodulin inhibitor 1,3-dihydro-1-[1-[4-methyl-4H,6H-pyrrolo[1,2-a][4,1]- benzoxazepin-4-yl-methyl]-4-piperidinyl]-2H-benzimidazol-2-o ne(1:1) maleate (CGS9343B) on DOX-induced chromosomal damage and its relationship to cytotoxicity were evaluated in sensitive and progressively DOX-resistant L1210 cells. Potentiation of DOX cytotoxicity by CGS9343B (a potent inhibitor of calmodulin which does not inhibit protein kinase C) was related to the level of resistance. Further, for equivalent cytotoxicity, cellular DOX levels in the absence versus the presence of TFP or CGS9343B were markedly higher. Exposure to calmodulin inhibitors following DOX treatment enhanced chromosomal aberrations and cytotoxicity. Maximal effects of calmodulin inhibitors were apparent when used during and after DOX treatment, and potentiation of cytotoxicity was related to modulation of DOX-induced chromosomal aberrations. Results suggest that inhibition of calmodulin-regulated processes is a potential target in the modulation of DNA damage/repair, and could play a pivotal role in the expression of "acquired resistance" to DOX.     

Formation of spermidine or norspermidine from synthetic diacetylpolyamines by acetylpolyamine oxidase in cultured cells

Prodrugs that can readily release polyamine into cells without the problem of generating cytotoxic compound by serum amine oxidase would be extremely useful for elucidation of polyamine function. As linear polyamines with acetamide groups on both sides are thought to be stable in the presence of serum amine oxidase and produce polyamines by the catalytic reaction of acetylpolyamine oxidase (PAO), a series of diacetyltetraamines, diacetylpentaamines and diacetylhexaamines was prepared as prodrugs and tested for substrate activity against PAO, partially purified from rat liver. Of the compounds, N(1),N(15)-diacetyl-1,15-diamino-4,8,12-triazapentadecane (DA3333) and N(1),N(16)-diacetyl-1,16-diamino-4,8,13-triazahexadecane (DA3343) were found to be stable in culture medium containing newborn bovine serum, and to produce reasonable amounts of norspermidine and spermidine, respectively. DA3333 and DA3343 were then applied to 1-aminooxy-3-aminopropane (AOAP)-treated HTC cells with depleted putrescine and spermidine, and arrested growth. Cell growth recovered with DA3333 and DA3343, but growth rate was reduced in cells with added DA3333 compared with growth rates in cells with added DA3343 and control cells untreated with AOAP. Significant amounts of norspermidine and spermidine were found in cells with added DA3333 and DA3343, respectively. These results show the potential use of diacetylpolyamines in introducing polyamines into cells.     

The long-lasting antiproliferative effect of 15-deoxyspergualin through its spermidine moiety

15-Deoxyspergualin (DSG) inhibited growth of mouse EL-4 lymphoma cells with an IC50 0.02 microg/ml. Even though the cells were treated with DSG for only 4 hours and then washed, the antiproliferative effect lasted long with an IC50 0.4 microg/ml. DSG has spermidine and guanidine moieties in its structure. One decomposed element containing guanidine moiety inhibited the growth at higher doses than DSG, but the effect did not last long unlike DSG. While another element containing spermidine moiety did not affect the growth, it diminished the long-lasting antiproliferative effect of DSG by pretreatment of the cells. Pretreatment with polyamines such as putrescine, spermidine, and spermine also diminished the effect of DSG. Furthermore, N-alkylation of spermidine moiety in DSG abolished the antiproliferative effect. These results suggested that DSG binds to the cells through its spermidine moiety and exerts its long-lasting antiproliferative effect.     

Studies on antineoplastic activity of naphthomycin, a naphthalenic ansamycin, and its mode of action

An antibiotic, identical with naphthomycin, was isolated from a soil Streptomyces. The antibiotic displayed significant therapeutic activity by ip administration against murine tumors: Ehrlich carcinoma and IMC carcinoma implanted ip. The maximum increase of life-span was more than 169% in Ehrlich carcinoma, and 128% in IMC carcinoma. The antibiotic exhibited a potent cytotoxicity against murine leukemic cells: P388, L1210, and L5178Y. IC50 was 0.4-1.3 microgram/ml in culture. The activity of naphthomycin was reversed by SH compounds: 2-mercaptoethanol, dithiothreitol, and glutathione. DNA and RNA syntheses were more markedly inhibited by naphthomycin than protein synthesis in L5178Y cells. Approximately 50% inhibition of nucleic acid syntheses was observed at an antibiotic concentration of 2 micrograms/ml. Naphthomycin blocked alkaline phosphodiesterase obtained from L5178Y cells: IC50 was ca. 7.6 micrograms/ml. The antibiotic neither caused metaphase arrest nor prevented tubulin polymerization. The results suggest that the mechanism of cytotoxicity of naphthomycin is the inhibition of various SH enzymes, particularly those involved in nucleic acid biosynthesis. The mode of action is unique in the ansamycin group of antibiotics.     

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.     

Spermine toxicity and glutathione depletion in BHK-21/C13 cells

Spermine, a polycationic amine, produced a dose-dependent inhibition of BHK-21/C13 cell growth. This response was not due to the extracellular metabolism of spermine by an amine oxidase found in bovine serum, as the toxicity was observed when the cells were grown in medium supplemented with horse serum. Three indices were used to monitor cell growth, cell number, protein content and [3H]thymidine incorporation into DNA. Spermine (2mM) caused significant reductions in all three measurements after a 6-8 hr exposure. The amine was rapidly taken up into the cells reaching levels 15-16-fold greater than in control cells within 2 hr. There was a rapid loss of intracellular reduced glutathione following exposure to toxic concentrations of spermine, which occurred before any effect on cell growth. Three methods for the determination of intracellular glutathione content were compared in this system. The effect on both cell growth and glutathione was reversible following removal of spermine from the extracellular medium. The possible mechanisms involved in this toxic response are discussed with particular reference to the depletion in intracellular reduced glutathione.     

Factors governing the modulation of vinca-alkaloid resistance in doxorubicin-resistant cells by the calmodulin inhibitor trifluoperazine

Calmodulin inhibitors enhance the cytotoxic effects of doxorubicin (DOX) in DOX-resistant (P388/DOX) P388 mouse leukemia cells by augmenting cellular accumulation and retention of drug. In P388/DOX cells which are cross-resistant to vinblastine (VLB) and vincristine (VCR), cell kill following treatment with VLB and VCR alone was evident only after 12 hr of treatment. Additionally, the 2- to 10-fold increase in cytotoxicity of the vinca alkaloids in the presence of 2 and 4 microM trifluoperazine (TFP) was observed only in P388/DOX cells treated for 12 hr, but not for 3 or 6 hr. However, in DOX-sensitive (P388/S) P388 mouse leukemia cells, cytotoxic effects of VCR but not VLB were apparent after treatment for 3 hr, and cell kill with VLB and VCR was enhanced 2- to 20-fold in the presence of 2 and 4 microM TFP following treatment for 12 hr. Cellular accumulation of [3H]VLB in P388/DOX cells was 12-fold lower than in similarly treated P388/S cells and, in the presence of 2 and 4 microM TFP, cellular VLB levels were enhanced 1.3- to 2.0-fold in P388/S cells and 2- to 8-fold in P388/DOX cells. The effect of TFP in increasing cellular retention of [3H]VLB was more apparent with P388/DOX cells, and retention of [3H]VLB in the presence of 4 microM TFP was enhanced less than 1.5-fold and greater than 4-fold in P388/S and P388/DOX cells respectively. Results from this study and our earlier observations with DOX and TFP in P388/DOX cells demonstrate that: (1) TFP potentiates the cytotoxicity of VLB and VCR in P388/S and P388/DOX cells by augmenting drug accumulation and retention; (2) enhanced cell kill in the presence of TFP with P388/DOX cells is apparent at 1 hr for DOX vs 12 hr for VLB and VCR; and (3) in P388/S cells, TFP has a more striking effect on the cellular accumulation, retention and cytotoxicity of VLB and VCR rather than DOX.