Etoposide-resistance in the multidrug-resistant LZ-8 cells.

The multidrug-resistant LZ-8 cells were found to exhibit marked resistance to etoposide compared to wild-type, parental V79 cells. The multidrug resistant phenotype did not significantly contribute to this etoposide-resistance. Following exposure of LZ-8 cells and V79 cells to equivalent concentrations of etoposide, there was a dramatic reduction in the number of etoposide-induced stabilized DNA-topoisomerase II complexes in the LZ-8 cells compared to V79 cells, however, this reduction was not found when nuclei isolated from LZ-8 and V79 cells were exposed to equivalent concentrations of etoposide. These results suggest that cytoplasmic factors are involved in the etoposide-resistance of LZ-8 cells.

Sequestration of doxorubicin in vesicles in a multidrug-resistant cell line (LZ-100).

A multidrug-resistant Chinese hamster cell line, LZ-8, was subcultured in increasing levels of doxorubicin (DOX) until capable of growth in 100 micrograms/mL DOX. This new derivative, designated LZ-100, is the most DOX-resistant line in the LZ series, based on a comparison of Ki-1 values from cell survival studies. This increased level of drug resistance in LZ-100 cells did not result from (i) higher levels of P-glycoprotein (P-gp) in the plasma membrane compared with LZ-8 cells, since this protein constitutes approximately 20% of the total plasma membrane protein in both cell lines, or (ii) more efficient drug pumping by the same amount of P-gp, since efflux of rhodamine 123 and DOX was comparable in the two cell lines. However, an altered drug distribution was observed in LZ-100 cells compared with wild-type V79 cells; in LZ-100 cells DOX was largely excluded from the nucleus and was sequestered in vesicles in the cytoplasm. The number of vesicles per cell seen after DOX exposure corresponded with the level of drug resistance achieved by the LZ cell lines studied. DOX concentration-response experiments revealed that vesicle formation exhibited a biphasic relationship, with an initial rapid increase followed by a plateau where no further increase was observed. Time-course studies in LZ-100 cells revealed that the maximum number of DOX-containing vesicles per cell occurred 3-4 hr following initiation of DOX treatment. Radiation exposure (10 Gy) immediately preceding DOX treatment decreased the number of vesicles formed in LZ-100 cells by more than one-half and altered the subcellular distribution of DOX from an almost exclusively cytoplasmic to a homogeneous nuclear/cytoplasmic distribution. This redistribution was not a result of radiation inhibition of P-gp efflux. The inhibitory effect of radiation on vesicle formation increased with increasing radiation dose up to 10 Gy. Drug-containing vesicles were also observed in LZ-100 cells following exposure to mitoxantrone or daunorubicin (to which LZ-100 cells are also resistant), but fewer vesicles were observed than with DOX. These studies demonstrate that the drug sequestration phenomenon (i) occurs in cells exhibiting widely different levels of drug resistance, (ii) correlates with the level of drug resistance in LZ cell lines, (iii) occurs rapidly following exposure to DOX, mitoxantrone, or daunorubicin, and (iv) can be inhibited by irradiation.(ABSTRACT TRUNCATED AT 400 WORDS)

Use of chromosome microdissection, the polymerase chain reaction, and dot blot hybridization to analyze double minute chromosomes.

The potential usefulness of chromosome microdissection, the polymerase chain reaction (PCR), and dot blot hybridization as a quick screening method for determining the genetic composition of double minute chromosomes (DMs) was evaluated. DMs or abnormally banding regions (ABRs) were microdissected from multidrug-resistant hamster cell lines and amplified with PCR using primers specific for the hamster multidrug-resistance (MDR) gene, pgp 1. The microdissected-PCR-amplified products were shown to (a) hybridize to a 32P-labeled pCHP1 probe for the hamster MDR gene by using dot blot or Southern blot analysis and also (b) hybridize back to the chromosome region from which they were originally dissected by using fluorescent in situ hybridization. Microdissected/PCR-amplified DMs were also shown to hybridize to ABRs. When microdissected DMs and ABRs were amplified using hamster specific Alu primers, the resulting material was shown to hybridize with probes for hamster MDR and Alu. These results suggest that the DMs contained in these MDR hamster cell lines contain Alu-like sequences and the chromosome microdissection-PCR-hybridization approach might be used as a quick screening method for identifying genes amplified in DMs and ABRs in cell lines and human tumor samples.

Changes in intra- or extracellular pH do not mediate P-glycoprotein-dependent multidrug resistance.

P-glycoprotein (Pgp)-mediated multidrug resistance (MDR) is thought to result from active extrusion of lipid-soluble, titratable chemotherapeutic agents. Given the lack of demonstration of coupling between ATP hydrolysis and drug transport, the resistance to chemically unrelated compounds, and findings of elevated intracellular pH (pHi), it has been proposed that reduced intracellular accumulation of drugs in MDR is due to changes in the pH difference across the plasma membrane. Elevation of pHi or decrease in local extracellular pH (pHo) could reduce the intracellular accumulation of the protonated chemotherapeutic drugs and account for Pgp-mediated MDR. Alternatively, changes in pHi or pHo could increase drug efflux by other mechanisms, such as coupled transport involving H+ or OH-, or allosteric effects on Pgp or other proteins. Both mechanisms could operate independently of the charge of the substrate. The possibility of a role of pHi in drug efflux is important to test because of the clinical significance of the phenomenon of MDR of tumors. We tested this hypothesis and found that MDR can occur in cells with low, normal, or high pHi. Further, resistant cells exhibited reduced steady-state drug accumulation and increased efflux without changes in local pHo. Finally, acute changes in pHi had no appreciable effect on Pgp-mediated drug efflux. We conclude that Pgp-mediated MDR is not a consequence of changes in pHi or pHo.

Modulation of doxorubicin resistance in multidrug-resistant cells by liposomes.

In this study, we have confirmed the ability of liposome-encapsulated doxorubicin to modulate drug resistance, as previously observed in CH LZ cells (Thierry et al., Cancer Commun. 1, 311-316, 1989), in two human multidrug-resistant (MDR) cell lines, the breast cancer MCF-7/ADR cell line, and the ovarian carcinoma SKVLB cell line. This effect was specific to MDR cells, as liposomally encapsulated doxorubicin did not enhance cell sensitivity to the drug in the parental cell lines. Cytotoxicity assays demonstrated that empty liposomes in the presence of free doxorubicin (Dox) reversed resistance to the drug at a level that may be higher than that observed when liposome-encapsulated Dox is used. This effect seems to be due to the high affinity of Dox for cardiolipin, one of the liposome components, which leads to the association of the drug and the cardiolipin-containing liposomes in the culture medium before entry into the cells. Neither pretreatment of empty liposomes before drug treatment nor combined incubation of vincristine and empty liposomes alter MDR in CH LZ cells, suggesting that the drug must be encapsulated or complexed to the liposomes to overcome MDR. Because MDR in CH LZ cells does not seem to be related to GSH level, MDR modulation by liposome-encapsulated Dox apparently may not be effected by altering the GSH function. These results suggest that the enhancement of sensitivity of MDR cells using Dox encapsulated in liposomes or complexed with liposomes may be explained by an increase in cell drug incorporation and by an intracellular drug redistribution. Fluorescence confocal microscopy study indicated that Dox is transported and distributed mainly in intracytoplasmic vesicles in SKVLB and MCF-7/ADR cells, whereas in parental cells the drug is located mainly in the nucleus. In addition, presentation of Dox in liposomes modifies the drug distribution pattern in MDR cells by partially shifting the drug to nuclear compartments. Thus, liposome-associated Dox may bypass the vesicular drug transport in MDR cells, resulting in the enhancement of the drug biological activity.

Characterization of adriamycin-resistant and radiation-sensitive Chinese hamster cell lines.

A series of cell lines derived from Chinese hamster V79 cells by selection in increasing concentrations of Adriamycin (ADRM) was developed to study the mechanisms of drug resistance and its relationship to radiation response. Survival studies revealed that selection in increasingly higher concentrations of ADRM positively correlated with increased cellular drug resistance. Increased cellular resistance correlated positively with amplification of the hamster multidrug-resistance gene (pgp 1) as detected with dot blot analysis using the pCHP1 probe. Southern blot analysis of restriction endonuclease digested DNA (Eco RI, Hind III, Pst I, or Bam HI) showed that (1) some fragments were preferentially amplified compared to others in the ADRM-resistant lines; and (2) no major gene rearrangement appeared to have occurred during the selection for greater ADRM resistance. Levels of pgp 1 gene expression assayed with dot blot and Northern analysis showed a parallel increase of mRNA with gene amplification and increased ADRM resistance. The amounts of the pgp 1 gene product, P-glycoprotein (P-gp), in the cell membrane of the ADRM-resistant cells correlated with the amount of gene amplification/expression. However, levels of P-gp only correlated with degree of drug resistance as measured by cell survival in earlier selection stages (77A and LZ-3). In later selection stages (LZ-8 and LZ-24), higher levels of ADRM resistance were achieved but levels of P-gp did not increase beyond approximately 20% of plasma membrane proteins. These results suggest that (1) the LZ cell plasma membrane may have a physical limit as to the amount of P-gp it can accommodate and/or there is a cellular mechanism for regulating the amount of P-gp in the plasma membrane, and (2) additional resistance mechanisms are present in LZ-8 and LZ-24 cells. Microscopic observations of intracellular drug distribution in these cell lines revealed that (1) ADRM appeared to be sequestered in cytoplasmic vesicles, and (2) the amount of sequestration (number of vesicles) exhibited correlated with the degree of drug resistance attained by the cell lines. These results suggest that drug sequestration is another mechanism of resistance in LZ cells in addition to P-gp-mediated drug efflux.

An enhanced ability for transforming adriamycin into a noncytotoxic form in a multidrug-resistant cell line (LZ-8).

Multidrug-resistant LZ-8 cells are 9000-fold more resistant to Adriamycin (ADRM) exposure than wild-type V79 cells. To understand more about the mechanisms producing such high level resistance, we tested whether LZ-8 cells inactivate ADRM toxicity to a greater extent than wild-type V79 cells. ADRM was recovered from (1) culture media of wild-type V79 and ADRM-resistant LZ-8 cells; (2) V79 and LZ-8 cells; and (3) LZ-8 cell plasma membrane, and the cytotoxicity was determined by treating V79 cells for 1 hr with a known concentration of the recovered ADRM. ADRM obtained from LZ-8 cells or its culture medium exhibited less cytotoxicity than that recovered from V79 cells or its culture medium. ADRM extracted from LZ-8 cell plasma membrane was noncytotoxic. HPLC analysis revealed that the extracted ADRM was structurally changed compared to stock ADRM. The retention time in the column was 7 min for stock ADRM, and 23 min for the recovered ADRM. Thus, LZ-8 cells have an increased ability to transform ADRM into a noncytotoxic form compared to wild-type V79 cells. This transformation involves structural conversion into a previously unidentified ADRM metabolite. The greatly increased survival of LZ-8 cells compared to V79 cells after ADRM treatment is due to at least two mechanisms: (1) an enhanced ability to inactivate the cytotoxicity of ADRM, and (2) increased drug efflux resulting from the amplification and overexpression of the pgp 1 gene in these cells. Our results suggest the possibility that P-glycoprotein participates in drug binding/inactivation in addition to serving as a drug efflux pump.

Interaction between radiation and drug damage in mammalian cells. VI. Radiation and doxorubicin age-response function of doxorubicin-sensitive and -resistant Chinese hamster cells.

A comparative study of the radiation and/or doxorubicin (DOX) survival response for synchronous populations of Chinese hamster V79 cells and two DOX-resistant variants (77A and LZ-8) was performed. The greatest cellular radiation sensitivity was observed in mitosis, while the greatest resistance was observed during late S phase for the three cell lines. The variation in radiation response throughout the cell cycle was expressed as a change in the width of the shoulder of the survival curves (Dq) with little change in D0. This suggests that each phase of the cell cycle has a different capacity for accumulation of radiation injury. The radiation age-response function for the three cell lines revealed that 77A and LZ-8 cells were more radiosensitive than V79 cells throughout the cell cycle. Exposure of synchronous populations to DOX (1.84 microM for V79, 9.21 microM for 77A, and 921 microM for LZ-8) for 1 h as a function of cell cycle phase revealed that V79, 77A, and LZ-8 cells exhibited the greatest sensitivity to DOX in mitosis and the most resistance to DOX during S phase, as indicated by the differences in the slope of the initial component of the survival curve. Levels of P-glyco-protein (P-gp) are probably not a factor contributing to DOX age-response function since P-gp levels remain constant throughout the cell cycle in all three cell lines. Synchronous populations of V79, 77A, and LZ-8 cells sequentially treated with DOX and radiation at various cell cycle phases were also analyzed. The results showed that the interaction between radiation and DOX damage resulted in a reduced cellular capacity for the accumulation of radiation damage throughout the cell cycle, as indicated by a decrease in the width of the shoulder of the survival curve. Overall, both DOX-sensitive V79 cells and DOX-resistant 77A and LZ-8 cells exhibited (1) a similar age-response function for radiation or DOX, and (2) no differences in the effects of DOX on radiation-induced damage throughout the cell cycle. These results indicate that acquired resistance to DOX associated with increased levels of P-gp in the cell membrane did not appear to affect the age-response function for radiation or DOX, and the nature of the interaction between damage caused by radiation and DOX was also not affected.

The relationship of doxorubicin binding to membrane lipids with drug resistance.

Doxorubicin (DOX, Adriamycin) binds with high affinity to cellular membranes inflicting multiple lesions which are believed to be important in DOX-mediated neoplastic cell death. Using fluorescence and radioactive [14-14C-14]DOX assays for DOX, we have measured the partitioning of DOX between the cytosolic and membrane fractions of erythrocytes and of DOX-sensitive (V-79) and -resistant (LZ) Chinese hamster lung fibroblast cells. In both erythrocytes and fibroblasts, a significant fraction of DOX was associated with the membrane fraction. More significantly, the quantity of lipid-bound DOX in the fibroblasts correlated with the cell's susceptibility to DOX. The significance of these findings in the context of existing knowledge about DOX-membrane interactions is discussed.

Dot-blot hybridization: quantitative analysis with direct beta counting.

The suitability of using direct beta counting (DBC) for quantitating radioactivity of the probe:target complex in dot-blot hybridization was evaluated using a Packard Matrix 96. A comparison of blots analyzed using autoradiography followed by densitometry scanning (film/densitometry) with those analyzed using direct beta counting revealed similar data trends with the two methods. However, direct beta counting quantitated the amount of radioactivity in the dot blots directly (without film exposure or additional sample preparation), which significantly reduced the time required to obtain results. Blots analyzed first with direct beta counting and then liquid scintillation counting exhibited similar data trends with both methods. Despite a decreased counting efficiency, analysis with direct beta counting has the following advantages compared with liquid scintillation counting: 1) no additional sample preparation is required (no vials or cocktail are used), 2) no sample destruction occurs due to analysis and 3) quantitative results are obtained more rapidly (since the radioactivity for all 96 samples in a dot blot is simultaneously determined in real time). Analysis with direct beta counting was also shown not to interfere with the successful reprobing of stripped dot blots with either unique sequence or total genomic probes. Overall, direct beta counting provides quick, quantitative results for dot blots while saving considerable time and effort.

Interaction between radiation and drug damage in mammalian cells. V. DNA damage and repair induced in LZ cells by adriamycin and/or radiation.

A multi-drug-resistant cell line selected in increasing concentrations of Adriamycin and designated LZ (J. A. Belli, Radiat. Res. 119, 88-100, 1989) is shown to exhibit a survival response characterized by radiation sensitivity and Adriamycin resistance. To determine if this response is due to alterations in either the initial levels of damage induced or the repair of DNA damage, LZ cells and the parental V79 cells were exposed to either radiation or Adriamycin and the damage and repair were measured with alkaline or nondenaturing filter elution. After exposure to radiation, induction and repair of both single-strand and double-strand breaks were equivalent. LZ cells exposed to 100 micrograms/ml Adriamycin for 1 h contained no measurable damage while the same treatment induced breaks and crosslinks in V79 cells. Pretreatment of LZ cells for 1 h with Adriamycin before irradiation did not alter either the initial levels of induced damage or the repair of strand breakage. These results suggest that (1) mechanisms other than differential induction and repair of strand breaks are responsible for the increased radiation sensitivity in LZ, and (2) the lack of Adriamycin-induced DNA damage in LZ is at least partially responsible for the increased cell survival after treatment.

Treatment plan optimization using linear programming.

Linear programming is a versatile mathematical tool for optimizing radiation therapy treatment plans. For planning purposes, dose constraint points, possible treatment beams, and an objective function are defined. Dose constraint points are specified in and about the target volume and normal structures with minimum and maximum dose values assigned to each point. A linear objective function is designed that defines the goal of optimization. A list of potential treatment beams is defined by energy, angle, and wedge selection. Then, linear programming calculates the relative weights of all the potential beams such that the objective function is optimized and doses to all constraint points are within the prescribed limits. Historically, linear programming has been used to improve conventional treatment techniques. It can also be used to create sophisticated, complex treatment plans suitable for delivery by computer-controlled therapy techniques.

Comparison of hprt variant frequencies and chromosome aberration frequencies in lymphocytes from radiotherapy and chemotherapy patients: a prospective study.

The autoradiographic 6-thioguanine-resistant mutant lymphocyte assay and a chromosome aberration assay were used to determine the time-course of appearance and persistence of elevated frequencies of hprt variants and dicentric chromosomes in patients receiving x-irradiation therapy. Twelve cancer patients, treated with 180-200 cGy/day, 5 days/wk, for 3-7 wk, were studied before treatment, at various weekly intervals during treatment, and after treatment. The hprt mutation assays were done with frozen/thawed lymphocytes isolated from aliquots of the same blood samples used for the chromosome aberration assays. The hprt variant frequencies (Vfs) of only 4 of the 7 patients assayed at 2 wk of treatment were elevated over pre-treatment Vfs, but during the 3rd and 4th weeks of treatment there were significant (P less than 0.01) 5- to 15-fold increases in all Vfs. By 6-32 wk after treatment Vfs had fallen to levels only slightly higher than the mean pre-treatment Vf. The frequencies of cells with dicentric chromosomes were significantly increased (P less than 0.01) after 1 wk of radiotherapy, continued to increase during therapy, and remained elevated after treatment. Five multiple sclerosis patients were also studied before and at 2 and 4 wk intervals after treatment with monthly i.v. doses of 750 mg/m2 of cyclophosphamide (CP). There were no significant elevations in chromosome aberrations at these post-treatment sample times. Previous assays for hprt mutants, done with aloquots of the same blood samples (Ammenheuser et al.: Mutat Res 204:509-520, 1988), had shown 8- to 20-fold increases in Vfs 2 wk after the 1st CP treatment. Our results demonstrate the complementary nature of these two human monitoring assays and emphasize the importance of careful selection of optimal sampling times.

The influence of dose constraint point placement on optimized radiation therapy treatment planning.

To efficiently use linear and quadratic programming for treatment planning optimization on a routine basis, automated methods are needed for placing dose constraint points. We have investigated, for linear programming optimization, the minimum number of constraint points needed to achieve an acceptable approximation to the desired (ideal) solution. Seven different constraint point placement algorithms were evaluated for a given objective function. One of these algorithms was chosen for routine clinical use at our institution. This algorithm places constraint points on the perimeter of the target volume and on the perimeter and in the interior of each normal structure. Additional points are placed on the perimeter of a constant thickness buffer region surrounding the target volume. Excellent optimization results are obtained with 40-70 constraint points per treatment planning slice.

Glutathione S-transferase and P-glycoprotein in multidrug resistant Chinese hamster cells.

Glutathione S-transferases (GSTs) have been reported to be elevated in some forms of hepatic carcinogenesis, in multidrug resistant (MDR) cells exhibiting elevated P-glycoprotein, and in cells resistant to alkylating agents independent of the MDR phenotype. The reported elevation of GST in association with the MDR phenotype and the overexpression of P-glycoprotein along with induction of GST in hepatic carcinogenesis suggest a correlation in the two mechanisms of cellular detoxification. To evaluate this hypothesis we examined the expression of GSTs in an MDR Chinese hamster fibroblast cell line overexpressing P-glycoprotein. We were unable to demonstrate concordant elevation of GST in these MDR cells. We conclude that GST expression is independent of P-glycoprotein expression in MDR Chinese hamster fibroblasts. The overexpression of GSTs in certain cells may provide an alternative mechanism for the development of drug resistance, either in association with or independent of P-glycoprotein overexpression, but is not essential for the MDR phenotype.

Transfer of adriamycin resistance by fusion of Mr 170,000 P-glycoprotein to the plasma membrane of sensitive cells.

One of the characteristics of multidrug-resistant mammalian cells is the presence of a glycoprotein of approximately Mr 170,000 in their cell membrane. Termed P-glycoprotein, this glycoprotein is thought to be the gene product of an amplified gene cloned and sequenced from a cell line (LZ) which is highly resistant to Adriamycin and cross-resistant to actinomycin D, colchicine, and vincristine. Resistance to Adriamycin has been induced in sensitive cells by chromosome or gene transfer. We now show that P-glycoprotein isolated from LZ cells and fused to sensitive V79 Chinese hamster cells renders the latter transiently resistant to Adriamycin. Incorporation of P-glycoprotein was confirmed by immunoperoxidase staining of fusion products following treatment with antibody to P-glycoprotein and by Western blots of membrane preparations from fusion products. These results suggest that P-glycoprotein is one of the important factors in the expression of Adriamycin resistance and provide added confirmation that it may be the important product of gene amplification in multidrug-resistant cells. The results also suggest that the cell membrane may be one of several targets for Adriamycin cytotoxicity and that P-glycoprotein may be a binding site for Adriamycin, rendering the latter ineffective in registering sufficient membrane damage for cell killing.

Twice-daily anesthesia in infants receiving hyperfractionated irradiation.

Four children ranging in age from 6-30 months were treated with twice-daily (BID) radiation therapy under general anesthesia with a 5-6 hr treatment interval. Anesthesia was accomplished with sodium thiopental administered intravenously (IV) by bolus injection. This as followed by continuous drip infusion of sodium thiopental in a few instances where more prolonged anesthesia was required. Children received an initial formula feeding 6 hr before their first treatment and were subsequently kept NPO (nothing by mouth) until they recovered from their second anesthesia. Recovery from thiopental was rapid and children were ready for a normal feeding within 1-1 1/2 hr of the second treatment. No parenteral feedings were required in any of these patients. Children maintained their weight during courses of radiation therapy which ranged between 19 and 43 elapsed days. There were no radiation-related treatment breaks. One child experienced two hypotensive episodes during anesthesia which responded rapidly to intravenous atropine. No other anesthetic complications occurred. This experience demonstrates that hyperfractionated radiation therapy can be safely delivered in infants requiring general anesthesia for immobilization. We feel that sodium thiopental is the anesthetic of choice in this setting because of the short duration of action and consequently rapid post-anesthesia recovery which makes it possible to achieve adequate nutrition with oral feedings alone.

Interaction between radiation and drug damage in mammalian cells. IV. Radiation response of adriamycin-resistant V79 cells.

Adriamycin-resistant variants derived from V79 Chinese hamster cells were examined for their radiation response properties. A stable resistant cell line (77A) demonstrated a significant reduction in the extrapolation number of the single-dose radiation survival curve. Second-step mutants from 77A cells exhibited a spectrum of radiation response states including decreased D0 values and large extrapolation numbers. A highly Adriamycin-resistant line (LZ) was found to be radiation sensitive with increased capacity for the accumulation of sublethal radiation injury. LZ cells are known to contain double-minute chromosomes and an amplified gene for the multidrug phenotype and to exhibit multidrug resistant properties. These cells require the presence of Adriamycin in their growth medium to maintain their pleiotropic characteristics. LZ cells became more resistant to radiation following reversion to an intermediate Adriamycin response as the consequence of growth in Adriamycin-free medium. Reverted cells also lost their large capacity for sublethal damage. It is suggested that detailed study of these mutants may provide insight into the identification of radiation-sensitive sites and their relationship to the genetic changes characterizing Adriamycin-resistant cell lines.

Modulation of multidrug resistance in Chinese hamster cells by liposome-encapsulated doxorubicin.

A Chinese hamster cell line (LZ), selected for multidrug resistance (MDR), exhibits a 3,000-fold resistance to doxorubicin, compared to parental V-79 cells. These drug resistant cells have amplified MDR genes, overexpress P-glycoprotein, and in the presence of doxorubicin show reduced intracellular drug accumulation. Using liposome-encapsulated doxorubicin (Rahman et al. Cancer Res. 45:796-803; 1985), we observed partial reversal of the resistance of LZ cells to this drug and a higher intracellular drug accumulation, compared to free drug. Parental V-79 cells, however, did not exhibit differences in survival or in drug accumulation when treated with encapsulated or free doxorubicin. Comparison of the effect of liposome-encapsulated doxorubicin with that of verapamil in reversing drug resistance showed that the liposomal preparation was as effective as verapamil used at its maximum clinically relevant concentration (1.5 microM). These results suggest that the use of liposomes as carriers of anticancer drugs may offer a strategy for overcoming MDR in tumor cells.