Functional assay of multidrug resistant cells using JC-1, a carbocyanine fluorescent probe

Multidrug resistance (MDR) is characterized by a decrease in the efficiency of chemotherapeutic agents correlated with the expression and activity of a membrane protein: the permeability-glycoprotein (Pgp 170). Clinically, detection of MDR can be performed by functional tests based on the accumulation of fluorescent compounds such as rhodamine 123. With the aim of improving the sensitivity of such analysis, we have evaluated JC-1, a fluorescent lipophilic carbocyanine dye. Above a critical concentration, JC-1 aggregates in a 'liquid crystal' form. Aggregates display a specific red emission band centered at 597 nm whereas the monomers display a green emission band centered at 540 nm. JC-1 was avidly accumulated in sensitive K562 cells where it displayed both a green cytoplasmic and red mitochondrial fluorescence. In contrast, JC-1 was poorly accumulated in resistant K562 cells, which displayed only a slight green fluorescence. The level of JC-1 accumulation was correlated with the level of Pgp expression detected by MRK16 and UIC2 antibodies on a set of K562 subclones with increasing resistance levels. The specific fluorescence properties of JC-1 allow accurate discrimination between low-level resistant cells and sensitive cells. Chemosensitizers such as verapamil, cyclosporine A or S9788 restored JC-1 accumulation in resistant cells. The fluorescence properties of JC-1 could therefore be used for monitoring the effects of reversing agents.     

Human MDR 1 protein overexpression delays the apoptotic cascade in Chinese hamster ovary fibroblasts

Several laboratories have reported that overexpression of the multidrug resistance (MDR) protein is associated with intracellular alkalinization, and several investigators have reported that cells induced to undergo programmed cell death (apoptosis) acidify quite significantly. Because it is difficult to fully explain the resistance to apoptosis-inducing chemotherapeutic drugs that is exhibited by MDR tumor cells solely via altered drug transport alone [Hoffman et al. (1996) J. Gen. Physiol. 108, 295-313], we have investigated whether overexpression of the hu MDR 1 protein alters progression of the apoptotic cascade. LR73 fibroblasts induced to undergo apoptosis either via treatment with the chemotherapeutic drug colchicine or by serum withdrawal exhibit cellular volume changes, intracellular acidification, nuclear condensation, and chromosomal digestion ("ladder formation"), characteristic of apoptosis, in a temporally well-defined pattern. However, multidrug resistant LR73/20E or LR73/27 hu MDR 1 transfectants recently created in our laboratory without selection on chemotherapeutic drug are significantly delayed in the onset of apoptosis as defined by the above criteria, regardless of whether apoptosis is induced by colchicine treatment or by serum withdrawal. Thus, the delay cannot simply be due to the well-known ability of MDR protein overexpression to lower chemotherapeutic drug accumulation in MDR cells. LR73/27V500 "selectants", exhibiting similar levels of MDR protein overexpression but higher multidrug resistance due to selection with the chemotherapeutic drug vincristine, exhibit a slightly longer delay in the progression of apoptosis. Normal apoptotic cascade kinetics are partially restored by pre-treatment of the MDR cells with the MDR protein inhibitor verapamil. Untransfected LR73 cells not expressing MDR protein but elevated in pHi via manipulation of CO2/HCO3- as described [Hoffman et al. (1996) J. Gen. Physiol. 108, 295-313] are inhibited in DNA ladder formation, similar to LR73/hu MDR 1 transfectants. These results uncover an additional mechanism whereby MDR protein overexpression may promote the survival of tumor cells and further support the notion that in some systems intracellular acidification may be either causal or permissive for proper progression of the apoptotic cascade.     

Comparison between technetium-99m-sestamibi and hydrogen-3-daunomycin myocardial cellular retention in vitro

This study was undertaken to verify whether 99mTc-sestamibi uptake parallels that of 3H-daunomycin in cells treated with multidrug resistance (MDR) reversing agents. Since we have detected in a previous work a moderate typical MDR phenotype in rat cardiac cells, a model of cultured myocardial cells was used. METHODS: Newborn-rat cultured myocardial cells were incubated 120 min with the MDR-reversing agent verapamil 50 microM, PSC833 1 microM or S9788 10 microM alone or in combination, and the cellular retention of 3H-daunomycin and 99mTc-sestamibi was counted. RESULTS: Hydrogen-3-daunomycin cellular accumulation was never modified by more than 15% when compared to control values, while 99mTc-sestamibi decreased to 75% +/- 32% (m +/- s.d.) of controls in the presence of S9788 and to 44% +/- 19% when S9788 was associated with verapamil. CONCLUSION: The variations of 99mTc-sestamibi and 3H-daunomycin cellular accumulation induced by MDR-reversing agents in cultured myocardial cells can be dramatically different. While some MDR-reversing agents can significantly increase the 3H-daunomycin retention in cardiac cells, they have unexpected effects on that of 99mTc-sestamibi.     

The relationship between modulation of MDR and glutathione in MRP-overexpressing human leukemia cells

Multidrug resistance-associated protein (MRP) causes multidrug resistance (MDR) involving the anthracyclines and epipodophyllotoxins. Many studies show modulation of anthracycline levels and cytotoxicity in MRP-overexpressing cells, but there is limited data on the modulation of etoposide levels and cytotoxicity in MRP-overexpressing or in P-glycoprotein-expressing cells. Etoposide accumulation was 50% reduced in both the CEM/E1000 MRP-overexpressing subline and the CEM/VLB100 P-glycoprotein-expressing subline compared to the parental CEM cells, correlating with similar resistance to etoposide (200-fold) of the two sublines. For the CEM/VLB100 subline, the P-glycoprotein inhibitor SDZ PSC 833, but not verapamil, was able to increase etoposide accumulation and cytotoxicity. For the CEM/E1000 subline, neither SDZ PSC 833 nor verapamil had any effect on etoposide accumulation. However, verapamil caused a 4-fold sensitization to etoposide in this subline, along with an 80% decrease in cellular glutathione (P < 0.05). Buthionine sulfoximine (BSO), which depletes glutathione, also caused a 2.5-fold sensitization to etoposide with no effect on accumulation in the CEM/E1000 subline. In contrast, SDZ PSC 833 was able to increase daunorubicin accumulation in the CEM/E1000 subline (P < 0.05), but had no effect on daunorubicin cytotoxicity, or cellular glutathione. These results show that modulation of etoposide cytotoxicity in MRP-overexpressing cells may be through changes in glutathione metabolism rather than changes in accumulation and confirm that changes in drug accumulation are not related to drug resistance in MRP-overexpressing cells.     

The ability of verapamil to restore intracellular accumulation of anthracyclines in multidrug resistant cells depends on the kinetics of their uptake

The basic distinguishing feature of all cells expressing functional P-glycoprotein-multidrug resistance is a decrease of steady state drug levels as compared to those in drug-sensitive controls. A variety of small molecules, such as verapamil and cyclosporin A, bind to P-glycoprotein and inhibit its ability to pump out antitumor drugs. The kinetics of P-glycoprotein-mediated efflux of various anthracycline derivatives was measured in multidrug-resistant (MDR) K562 cells in the presence of verapamil. Used for the purpose were daunorubicin, idarubicin and 8-S-fluoro-idarubicin which have the same pKa of deprotonation equal to 8.4, but different lipophilicity, 4'-epi-2'-bromo-daunorubicin which has a lipophilicity which is comparable to that of daunorubicin but a pKa equal to 6.3, pirarubicin with pKa equal to 7.7 and lipophilicity different from that of these derivatives were used. Our data show (1) that verapamil is unable to completely block the P-glycoprotein-mediated efflux of anthracyclines and that 10% of its functionality remains even with high verapamil concentrations, (2) that the ability of verapamil to restore intracellular accumulation of anthracyclines in MDR cells depends on the kinetics of their uptake. With fast kinetics uptake, as is the case for idarubicin, 8-S-fluoro-idarubicin, 4'-epi-2'-bromo-daunorubicin and pirarubicin (which have either a low pKa and/or high lipophilicity), verapamil can restore in multidrug resistant cells an intracellular drug level which is comparable to that observed in sensitive cells. This is not possible when the kinetics of uptake is low as is the case for daunorubicin. Cyclosporin A is a more potent modulator and is able to fully restore daunorubicin accumulation in multidrug resistant cells.     

Are altered pHi and membrane potential in hu MDR 1 transfectants sufficient to cause MDR protein-mediated multidrug resistance?

Multidrug resistance (MDR) mediated by overexpression of the MDR protein (P-glycoprotein) has been associated with intracellular alkalinization, membrane depolarization, and other cellular alterations. However, virtually all MDR cell lines studied in detail have been created via protocols that involve growth on chemotherapeutic drugs, which can alter cells in many ways. Thus it is not clear which phenotypic alterations are explicitly due to MDR protein overexpression alone. To more precisely define the MDR phenotype mediated by hu MDR 1 protein, we co-transfected hu MDR 1 cDNA and a neomycin resistance marker into LR73 Chinese hamster ovary fibroblasts and selected stable G418 (geneticin) resistant transfectants. Several clones expressing different levels of hu MDR 1 protein were isolated. Unlike previous work with hu MDR 1 transfectants, the clones were not further selected with, or maintained on, chemotherapeutic drugs. These clones were analyzed for chemotherapeutic drug resistance, intracellular pH (pHi), membrane electrical potential (Vm), and stability of MDR 1 protein overexpression. LR73/hu MDR 1 clones exhibit elevated pHi and are depolarized, consistent with previous work with LR73/mu MDR 1 transfectants (Luz, J.G. L.Y. Wei, S. Basu, and P.D. Roepe. 1994. Biochemistry. 33:7239-7249). The extent of these perturbations is related to the level of hu MDR 1 protein that is expressed. Cytotoxicity experiments with untransfected LR73 cells with elevated pHi due to manipulating percent CO2 show that the pHi perturbations in the MDR 1 clones can account for much of the measured drug resistance. Membrane depolarization in the absence of MDR protein expression is also found to confer mild drug resistance, and we find that the pHi and Vm changes can conceivably account for the altered drug accumulation measured for representative clones. These data indicate that the MDR phenotype unequivocally mediated by MDR 1 protein overexpression alone can be fully explained by the perturbations in Vm and pHi that accompany this overexpression. In addition, MDR mediated by MDR protein overexpression alone differs significantly from that observed for MDR cell lines expressing similar levels of MDR protein but also exposed to chemotherapeutic drugs.     

Dietary iron overload as a risk factor for hepatocellular carcinoma in Black Africans

We analyzed the effect of high temperature (a 1-h incubation at 43 degrees C) on the accumulation and cytotoxicity of vinblastine and docetaxel in two model cell lines, K562 and MESSA, and their multidrug resistance (MDR) counterparts, K562/R7 and MESSA/Dx5. High temperature increased the amount of intracellular vinblastine and docetaxel significantly in MESSA cell and, to a much lesser extent, in K562 cells. MDR-positive cells retained a profound drug accumulation defect at 43 degrees C. Hyperthermia enhanced the cytotoxic effect of vinblastine (but not docetaxel) in both K562 and MESSA cells, but not in the MDR-positive variants. PSC833, a potent modulator of P-glycoprotein, induced high levels of drug accumulation in the two MDR-positive cell lines at both 37 degrees C and at 43 degrees C. PSC833 also significantly reduced the resistance levels of the two MDR-positive lines at both 37 degrees C and at 43 degrees C. The effect of hyperthermia on drug accumulation thus seems to depend on the cell line, whereas the effect on cytotoxicity depends on the type of compound. The MDR phenotype remains a therapeutic obstacle at 43 degrees C but is accessible to modulation.     

Role of chaos in trial-and-error problem solving by an artificial neural network

The effect of heparin as a reversing agent of multidrug resistance (MDR) was tested on normal mononuclear cells from 24 healthy volunteers and leukaemic cells from 12 acute myeloid leukaemia, five chronic myeloid leukaemia, five acute lymphoid leukaemia and three chronic lymphoid leukaemia patients. Two cell lines were used as controls, the human erythroleukaemia K562 and its vincristine-resistant derivative K562-Lucena 1. Heparin was not cytotoxic by itself as determined using a MTT assay and cell counts. MDR modulation was assessed by Rhodamine 123 extrusion using flow-cytometry. Modulation of the resistant cell line was produced by the classical reversing agent verapamil and also by heparin, the same being observed in normal and leukaemic cells and being independent of the type of leukaemia. Our work suggests that heparin may be considered a potential MDR modulator.     

The multidrug-resistant human lung tumour cell line, DLKP-A10, expresses novel drug accumulation and sequestration systems

Drug accumulation studies with the anticancer agents adriamycin and vincristine were carried out on the MDR variant of the human lung cell lines DLKP, DLKP-A10 which overexpresses the MDR associated P-glycoprotein efflux pump. Reduced cellular accumulation of both agents was observed in the resistant variant. The subsequent addition of verapamil and cyclosporin A resulted in partial restoration of cellular accumulation of both drugs in the DLKP-A10 resistant variant while complete restoration of cellular drug levels was observed in the SKMES-1/ADR cell line. These results suggested that the accumulation defect observed in the SKMES-1/ADR cell line was P-glycoprotein mediated and that accordingly, the cells exhibited characteristics consistent with the classical MDR phenotype. In contrast, while P-glycoprotein also appears to mediate a reduction in cellular drug accumulation in the DLKP-A10 cells, an alternative transport mechanism may also be present. No significant increase in the expression of either the MRP or LRP transport proteins was observed in the resistant cells. Metabolic inhibition by antimycin A (but not sodium azide or 2-deoxy-D-glucose) resulted in complete restoration of drug accumulation suggesting the presence of an alternative energy dependent transport mechanism. Fluorescent microscopy studies indicated different cellular localisation of the drug within the parental and resistant cells despite equivalent intracellular concentrations. These studies also revealed the presence of an ATP-dependent, vesicular sequestration mechanism which may be involved in the reduction of nuclear adriamycin accumulation in the DLKP-A10 cell line. This was indicated by observation of the disruption of cytoplasmic vesicles by antimycin A and also inhibition of cytoplasmic drug sequestration by the carboxylic ionophores, monensin and nigericin, accompanied by increased adriamycin accumulation and redistribution of the drug from the cytoplasm to the nucleus.     

Water quality in rural Australia

The comet test is a reported method for measuring DNA damage in individual mammalian cells. In the present report, the ability of this test to detect multidrug resistance (MDR) was evaluated. For this purpose, two human leukemia, well-characterized parental cell lines, HL60 and CEM, and their derived multidrug-resistant cells, HL60/DNR and CEM/VBL, were cultured with or without different anti-cancer agents. To evaluate the comet test, two DNA-damaging agents were used: daunorubicin (DNR), which is involved in MDR, and ambamustine (AMBA), which is independent from MDR. Moreover, in order to evaluate the specificity of the comet test, the activity of vinblastine (VBL), an MDR-related, DNA-independent anti-cancer drug, was also tested. Finally, the specificity of the comet test in detecting MDR was confirmed by culturing parental or resistant cells with DNR with or without the revertant agent verapamil (VER). Results confirm that the comet test is able to predict cellular chemoresistance when DNA damaging agents are tested. Finally, experiments on the role of the comet test in evaluating certain aspects of DNA repair are discussed.     

Flavonoid-related modulators of multidrug resistance: synthesis, pharmacological activity, and structure-activity relationships

A series of 28 flavonoid derivatives containing a N-benzylpiperazine chain have been synthesized and tested for their ability to modulate multidrug resistance (MDR) in vitro. At 5 microM, most compounds potentiated doxorubicin cytotoxicity on resistant K562/DOX cells. They were also able to increase the intracellular accumulation of JC-1, a fluorescent molecule recently described as a probe of P-glycoprotein-mediated MDR. This suggests that these compounds act, at least in part, by inhibiting P-glycoprotein activity. As in other studies, lipophilicity was shown to influence MDR-modulating activity but was not the only determinant. Diverse di- and trimethoxy substitutions on N-benzyl were examined and found to affect the activity differently. The most active compounds had a 2,3, 4-trimethoxybenzylpiperazine chain attached to either a flavone or a flavanone moiety (13, 19, 33, and 37) and were found to be more potent than verapamil.     

Semi-automated PCR method for quantitating MDR1 expression

The MDR1 gene is involved in drug resistance in many hematopoietic and solid tumors. The Quantitative PCR System 5000 (QPCR-5000; Perkin-Elmer) is a new instrument system that uses electrochemiluminescence to automatically quantitate polymerase chain reaction (PCR) products. A comparative study between radioactively labeled PCR (32P-PCR) and QPCR was performed to analyze the MDR1 gene expression in the drug-resistant (Doxorubicin) cell lines Dox40, Dox6, the parental cell line 8226/S, CEM Dox1 and three acute myeloid leukemia (AML) patient samples. Using the Dox40 and Dox6 resistant cell lines, we compared the sensitivities of QPCR and 32P-PCR. A strong signal was obtained from QPCR at 20 to 25 cycles (which is in the linear range for quantitation), while a weak signal was obtained using 32P-PCR at the same cycle number. Dilution experiments gave better precision with the QPCR than with the radioactive method. AML samples were studied with the MDR1-specific MAbs MRK16 and 4E3, and the efflux function was analyzed using Rh-123 retention in the absence or presence of verapamil. The three samples showed high (D = 0.79), medium (D = 0.52) and negative (D = 0.08) p-glycoprotein (P-gp) levels and correlated with efflux function. The MDR1/beta 2-M mRNA ratios for 32P-PCR were 0.41, 0.40 and 0.12, respectively, and were 0.127, 0.097 and 0.028, respectively, for QPCR. There were significant differences between the samples with high and medium P-gp levels comparing the two methods. Very low levels of MDR1 in CEM Dox1 cells could be detected only by QPCR. In conclusion, QPCR was found to be more reproducible, accurate and sensitive than 32P-PCR.     

Pirarubicin nuclear uptake does not correlate with its induced cell death effect during reversal of multidrug resistance by quinine in human K562 and CEM leukemic cells

A number of small and lipophilic cations are able to reverse in vitro the resistance to anthracyclines and other natural products through their interaction with P-glycoprotein or P-gp. However, some modulators do not interact with P-gp. We have demonstrated in a previous a work, using confocal laser microspectrofluorometry, that quinine does not increase nuclear anthracycline uptake in multidrug-resistant Chinese hamster ovary LR73 cells. In this case the LR73 cells were transfected with the mdr1 gene. Moreover, quinine induced in these cells an increase of mdr1 gene expression. In the present study, we investigated verapamil and quinine for their ability to increase nuclear pirarubicin uptake in multidrug-resistant K562R and CEMR human leukemic cell lines. These two cell lines resist, respectively, to doxorubicin and vinblastine and both overexpress the P-gp. Verapamil was able to restore nuclear pirarubicin in both cell lines. On the other hand, quinine was unable to significantly increase nuclear pirarubicin uptake. Both modulators were able to restore pirarubicin sensitivity in both resistant cell lines. After treatment with quinine, mdr1 gene and P-gp expression was not significantly altered as observed previously in the LR73 cells. This suggest that the effect of quinine on mdr1 gene expression is dependent on the cell line studied. These data suggest that quinine could modify the molecular environment of anthracyclines and/or its binding to a possible cytoplasmic target, and that the mechanisms by which anthracyclines induce cell death, and ways by which chemotherapy fails in multidrug-resistant leukemic cells remain complex and are related to more than one target.     

Gbetagamma stimulates phosphoinositide 3-kinase-gamma by direct interaction with two domains of the catalytic p110 subunit

Multidrug resistance mediated by the drug efflux protein, P-glycoprotein (P-gp), is one mechanism that tumor cells use to escape death induced by chemotherapeutic agents. However, the mechanism by which P-gp confers resistance to a large variety of structurally diverse molecules has remained elusive. In this study, classical multidrug resistant human CEM and K562 tumor cell lines expressing high levels of P-gp were less sensitive to multiple forms of caspase-dependent cell death, including that mediated by cytotoxic drugs and ligation of Fas. The DNA fragmentation and membrane damage inflicted by these stimuli were defined as caspase dependent by various soluble peptide fluoromethylketone caspase inhibitors. Inhibition of P-gp function by the anti-P-gp mAb MRK-16 or verapamil could reverse resistance to these forms of cell death. Inhibition of P-gp function also enhanced drug or Fas-mediated activation of caspase-3 in drug-resistant CEM cells. By contrast, caspase-independent cell death events in the same cells, including those mediated by pore-forming proteins or intact NK cells, were not affected by P-gp expression. These observations suggest that, in addition to effluxing drugs, P-gp may play a specific role in regulating some caspase-dependent apoptotic pathways.     

Effect of S9788 on the efficiency of doxorubicin in vivo and in vitro in medullary thyroid carcinoma xenograft

In medullary carcinoma of the thyroid (MTC), drug resistance remains the major obstacle to effective chemotherapy. In this work, we studied the effect of S9788 on doxorubicin (DOX) efficiency in a MTC cell line (TT cells) injected in nude mice. After two passages, TT cells were injected in 40 nude mice divided into four groups [controls and groups receiving DOX alone (10 mg/kg), S9788 alone (50 mg/kg) or both DOX + S9788]. The weight of the mice, tumoral volume (TV), doubling time (DT) of the tumor and survival time of mice were evaluated in each group. In addition, the efficiency of DOX with or without S9788 was assessed by the inhibition of tumoral growth and specific growth delay. In vitro, glycoprotein P 170 (P-gp) was detected on tissular sections and on tumoral cells by immunocytochemistry or flow cytometry with several monoclonal antibodies: JSB1, MRK 16, C219 and UIC2. In vivo the weight of the mice decreased slightly with DOX and dropped dramatically with DOX + S9788. The DT of the tumors increased with DOX over controls (22.5 +/- 8.5/12.7 +/- 3.9 days) and showed a higher value with DOX + S9788 (29.2 +/- 11.4 days). Inhibition of tumoral growth, 89% with DOX, fell to 47.6% with DOX + S9788. Specific growth delay increased with the double treatment (130 versus 75% with DOX alone). In vitro, P-gp was not detected on tissular sections and cells whatever the method and the antibody used. In conclusion, S9788 potentiates the efficiency of DOX treatment in vivo. The absence of P-gp may result from the absence of translation of the MDR1 gene. The reversal effect of S9788 may involve another resistance mechanism such as the MDR Sister of MRP.     

The actin-based motility of the facultative intracellular pathogen Listeria monocytogenes

Drug resistance is a major obstacle to successful cancer chemotherapy. P-glycoprotein, which transports various antitumor agents outside the resistant tumor cells, plays a key role in multidrug resistance. We found that MRK-16, a monoclonal antibody against P-glycoprotein, and cyclosporine, synergistically enhanced the antitumor effects of vincristine and adriamycin in multidrug-resistant K562/ADM cells. On the other hand, the combined use of MRK-16 with verapamil or FK-506 did not show such synergistic effects. Drug accumulation studies revealed that MRK-16 remarkably increased the accumulation of cyclosporine, but not verapamil, in K562/ADM cells. This increased accumulation of cyclosporine by MRK-16 in K562/ADM cells directly resulted in the enhanced accumulation of vincristine and adriamycin in the cells. The synergistic effect of MRK-16 and cyclosporine was further confirmed by isobologram analysis in three different highly multidrug-resistant tumor cells. Moreover, while MRK-16 alone did not enhance the sensitivity of the KB-8-5 cells moderately resistant to vincristine, it increased two-fold the reversing effect of cyclosporine at 1 microM, an achievable blood concentration. Since MRK-16 alone showed therapeutic effects against multidrug-resistant tumors, the combined use of MRK-16, cyclosporine and antitumor agents would provide therapeutic benefits for the treatment of resistant tumors.     

Cellular pharmacology of mitoxantrone in p-glycoprotein-positive and -negative human myeloid leukemic cell lines

Previous reports suggest that resistance to mitoxantrone in different tumor cell lines is unrelated to the overexpression of p-glycoprotein. In order to determine the role of p-glycoprotein in the cellular pharmacology of mitoxantrone flow cytometry and confocal microscopy were used to study two human myeloid leukemia cell lines selected for resistance to mitoxantrone (HL-60MX2) and doxorubicin (HL-60DOX). To optimize the detection of intracellular mitoxantrone, we determined the maximum excitation (607 nm) and emission (684 nm) wavelength by fluorescence spectroscopy. The modified flow cytometric conditions using 568.2 nm laser emission for excitation and a 620 nm long pass filter for fluorescence collection resulted in a 1-log increase in sensitivity, compared with standard 488-nm laser excitation. Uptake and retention of mitoxantrone in the presence of verapamil, a calcium channel blocker known to inhibit p-glycoprotein, were analyzed. Our results showed no change in uptake and retention of the drug in p-glycoprotein-negative mitoxantrone-resistant HL-60MX2 cells and in its sensitive parental line, HL-60s. In contrast, 3.1- and 2.4-fold increases were found in uptake and retention of mitoxantrone in p-glycoprotein-positive cells (HL-60DOX) incubated with verapamil. Confocal microscopy of intracellular drug distribution demonstrated reduced nuclear uptake, which could be reversed by verapamil, in HL-60DOX. A characteristic punctate pattern was observed for the intracytoplasmic drug distribution in HL-60DOX and HL-60MX2 cells and was partially modified by the presence of verapamil in HL-60DOX cells. Verapamil increased cytotoxicity of mitoxantrone two-fold in HL-60DOX cells, 1.4-fold in HL-60MX2, and had no effect in HL-60s. Our study demonstrates that the cellular pharmacology of mitoxantrone is affected by p-glycoprotein and can be reversed at least in part by verapamil. Other mechanisms of resistance however, seem to play a determinant role in the modulation of mitoxantrone cytotoxicity.