Vesicular anthracycline accumulation in doxorubicin-selected U-937 cells: participation of lysosomes

The U-A10 cell line, a doxorubicin-selected variant of human U-937 myeloid leukemia cells, exhibits a redistribution of anthracyclines into a expanded vesicular compartment. The acidic nature of this compartment was confirmed by vital staining with a pH sensitive dye, LysoSensor yellow/blue DND-160. Identification of the vesicular compartment was performed by immunofluorescence analysis. Staining for the LAMP-1 and LAMP-2 antigens showed that the vesicles are enlarged lysosomes that are eccentrically placed near the nucleus of U-A10 cells. By contrast, the expression of the multidrug resistance-associated protein and the P-glycoprotein was observed predominately on the plasma membrane of the drug-resistant cells. The accumulation of daunorubicin into cellular compartments was quantified using radiolabeled drug. Exposing cells to 3[H]-daunorubicin and then isolating intact nuclei showed that nuclei from U-A10 cells accumulated twofold to threefold less anthracycline than nuclei from U-937 cells. However, when nuclei were isolated first and then exposed to 3[H]-daunorubicin, little difference in net nuclear drug accumulation was detected. Cytoplasts prepared from U-A10 and U-937 cells were exposed to 3[H]-daunorubicin to measure cytoplasmic drug accumulation. At external daunorubicin concentrations of 100 ng/mL or higher, cytoplasts from U-A10 cells accumulated significantly more daunorubicin than cytoplasts from U-937 cells. Moreover, studies with the lysosomotropic agent chloroquine showed that U-A10 cells accumulated twofold more chloroquine and showed twofold enhanced sensitivity to this agent as compared with parental U-937 cells. Fluorescence microscopy showed that chloroquine affects vesicular anthracycline sequestration in U-A10 cells with an associated increase in daunorubicin nuclear fluorescence. Although chloroquine did not alter anthracycline cytotoxicity in parental cells, it restored daunorubicin and doxorubicin sensitivity to U-A10 cells. Taken together, these studies demonstrate that U-A10 cells exhibit a redistribution of the lysosomal compartment. The trapping of drug into an expanded acidic vesicular compartment results in decreased nuclear drug accumulation and decreased cytotoxicity. Lysosomotropic agents, such as chloroquine, warrant further study as modulators of this acquired drug-resistance phenotype.     

Cellular and karyotypic characterization of two doxorubicin resistant cell lines isolated from the same parental human leukemia cell line

Separate mechanisms underlying the multidrug resistant (MDR) phenotype were identified in 2 independent approaches to select tumour cells resistant to low concentrations of doxorubicin (Dox) from the sensitive T cell leukemia cell line CCRF-CEM. The CEM/A7 cell line was selected at an initial concentration of 0.005 microgram/ml of Dox and maintained at 0.07 microgram/ml. In contrast, the CEM/A5 line was selected using an initial concentration of 0.01 microgram/ml and maintained in Dox at a concentration of 0.05 microgram/ml. P-glycoprotein expression was demonstrated in the CEM/A7 line but not the CEM/A5 line. Amplification of the mdrI gene was not observed in the CEM/A7 cell line. Both cell lines showed cross-resistance to a number of structurally unrelated cytotoxic drugs including anthracyclines and etoposide (VP-16), although only the CEM/A7 line was cross resistant to Vinca alkaloids. Immunoblots of total cell lysates of the CEM/A5 line have revealed almost undetectable levels of topoisomerase II alpha and beta in this line. Cytogenetic analyses of both lines revealed numerous karyotypic abnormalities which were present in the parental cell line as well as both resistant cell lines. The CEM/A7 line also demonstrated a duplication of part of the long arm of chromosome 7 which included the region containing the mdrI gene, a finding not seen in the parental or CEM/A5 line. CEM/A5, however, demonstrated an abnormality of chromosome 7, outside the region of the mdrI gene, and it also contained a deletion of the short arm of chromosome 2. Abnormalities in this latter region of genome have been associated with non-P-glycoprotein-mediated MDR.     

Rapid recovery of a functional MDR phenotype caused by MRP after a transient exposure to MDR drugs in a revertant human lung cancer cell line

Prior studies have shown that, in some human tumour cells, increased expression of the multidrug resistance gene MDR1 can be induced in response to certain stress conditions such as a transient exposure to cytotoxic agents. Little is known about the possibility of increasing the expression of the recently cloned multidrug resistance-associated protein (MRP) in response to a transient exposure to cytotoxic drugs. In order to examine this possibility, we have used sensitive assays (RT-PCR, flow cytometry) and the sensitive large cell lung cancer cell line, COR-L23/P, and the revertant line (COR-L23/Rev), generated by growing the doxorubicin-selected, MRP-overexpressing resistant variant COR-L23/R without drug exposure for 24-28 weeks. COR-L23/Rev overexpresses MRP, but to a lesser extent than COR-L23/R. COR-L23/Rev rapidly recovered similar levels of MRP mRNA, protein expression, resistance and drug accumulation deficit as COR-L23/R after a 48-72 h exposure to cytotoxic concentrations of doxorubicin or vincristine but not cisplatin. The increase in MRP mRNA could only be detected 3 to 4 days after the transient exposure to drugs. However, when the parental line, COR-L23/P, was exposed to equitoxic doses of doxorubicin, vincristine or cisplatin, no increase in the levels of MRP mRNA could be observed at higher doses (5- to 10-fold the IC50) of doxorubicin or vincristine (but not of cisplatin), we detected a transient increase in the levels of MDR1 mRNA immediately after short-term exposure. In conclusion, we have shown that a human revertant lung cancer cell line (COR-L23/Rev) has the ability to recover quickly, similar levels of MRP expression and resistance as COR-L23/R after a transient exposure to the MDR-drugs doxorubicin and vincristine.     

Confocal scanning microspectrofluorometry reveals specific anthracyline accumulation in cytoplasmic organelles of multidrug-resistant cancer cells

We used confocal microspectrofluorometry to investigate intracellular distribution of pirarubicin or THP-DOX in parental K562, CEM, and LR73 tumor cells and their corresponding multidrug-resistant (MDR) strains. Each spectrum of a recorded image was considered as a combination of cell autofluorescence and fluorescence of the drug. In the cytoplasm of parental K562, CEM, and LR73 cells, THP-DOX fluorescence emission profile was similar to that of free drug in aqueous buffer. The (I550nm/I600nm) ratio was 0. 50 +/- 0.1. However, in the cytoplasm of resistant cells the 550-nm band profile was modified. The I550nm/I600nm ratio was 0.85 +/- 0.2 in MDR K562 cells, which is significantly different from the ratio in sensitive cells (p<0.01). This appeared first to correspond to accumulation and self-oligomerization of THP-DOX in cytoplasmic organelles of MDR cells. Transfection of LR73 cells with the mdr1 gene conferred this characteristic on the resistant LR73R cells. Bodipy-ceramide, a trans-Golgi probe, was co-localized with the typical fluorescence emission peak at 550 nm observed in the cytoplasm of MDR cells. This organelle has been shown to be more acidic in MDR cells. Moreover, this specific pattern was similar to that observed when anthracycline is complexed with sphingomyelin. The typical fluorescence emission peak at 550 nm decreased in MDR cells incubated simultaneously in the presence of the drug and quinine, verapamil, or S9788. Growth inhibitory effect and nuclear accumulation of THP-DOX data obtained on LR73R and LR73D cell lines showed that only during reversion of resistance by verapamil and S9788 was an increase of nuclear THP-DOX accumulation observed. Our data suggest that characteristics of molecular environment, such as higher pH gradient or lipid structures, would be potential mechanisms of multidrug-resistance via the sequestration of anthracyclines.     

Human pharmacokinetic characterization and in vitro study of the interaction between doxorubicin and paclitaxel in patients with breast cancer

PURPOSE: We performed a pharmacologic investigation of paclitaxel (PTX) infused over 3 hours and bolus doxorubicin (DOX) to assess the role of sequence, interval between drugs, and duration of doxorubicin infusion on paclitaxel and anthracycline plasma disposition. We also explored possible mechanisms of pharmacokinetic interference involving the physiologic role of the multidrug resistance phenotype in anthracycline and taxane biliary excretion. PATIENTS AND METHODS: Pharmacokinetics was performed in 80 cycles and 36 women with previously untreated metastatic breast cancer. PTX, DOX, and their metabolites 6 alpha-hydroxyl-PTX (6 alpha OH-PTX) and doxorubicinol (DOL) were measured by high-pressure liquid chromatography (HPLC). Human breast cancer MCF-7 wild-type (WT) and resistant (TH) cell lines were cultured in whole human plasma to study anthracycline retention after treatment with different combinations of PTX, Cremophor EL (CEL) (PEG35 castor oil; BASF, Parsippany, NJ), and DOX. RESULTS: Pharmacokinetic interference between PTX and DOX was responsible for nonlinearity of DOX plasma disposition and increased concentrations of DOX and DOL. These effects were PTX dose-dependent, DOX concentration-dependent, and likely a result of interference at the level of liver elimination. In view of the physiologic role of P-glycoproteins (P-gp) in xenobiotic biliary excretion, retention of DOX was assessed in MCF-7 WT and MCF-7 TH cells. Intracellular was significantly higher in MCF-7 WT than MCF-7 TH (P < .05). However, concomitant exposure to DOX, PTX, and CEL caused similar DOX retention in both MCF-7 WT and TH cells. CONCLUSION: PTX, as clinically formulated in CEL, is responsible for a nonlinear disposition of DOX and DOL. Nonlinearity is PTX- and DOX-dependent, and possibly caused by competition for biliary excretion of taxanes and anthracyclines mediated by P-gp. Nonlinearity indicates that even minor modifications of dose and infusion duration of DOX and PTX may lead to unpredictable pharmacodynamic consequences. The postulated role of P-gp suggests that CEL is clinically active, and advises caution in designing combinations of PTX with other drugs that are substrate for P-gp.     

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.     

The multidrug resistance in human leukemias. Minireview

Leukemia/lymphoma cells, clinically refractory to therapy are often associated with expression of P-glycoprotein (P-gp), which is encoded by the multidrug resistance (MDR) gene, mdr1. Cell lines expressing mdr1 exhibit resistance to several structurally unrelated lipophilic drugs, such as anthracyclines, vinca alkaloids, and epopodophyllotoxins. This MDR can be conferred to drug-sensitive cells mdr1 cDNA transfer. In resistant cells, MDR is characterized by overexpression of P-gp and by the enhanced efflux, and P-gp fluorescence probe, rhodamine 123 (Rh 123). This can be circumvented by addition of certain non-cytotoxic drugs, such as verapamil and cyclosporin A.     

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.     

Extracorporeal shock wave lithotripsy for lower pole nephrolithiasis: efficacy and variables that influence treatment outcome

OBJECTIVES: To clarify the role of the membranous glycoprotein gp-170 in renal cell carcinoma (RCC) cell lines and their multidrug resistant (MDR) sublines. and to correlate gp-170 with the natural and acquired drug resistance of these cell lines to anthracyclines. MATERIALS AND METHODS: The expression of gp-170 in five cultured RCC cell lines and serial RCC8701 MDR sublines was analysed by immunofluorescent flow cytometry. The chemosensitivity of these tumour cells to the anthracycline anticancer drugs adriamycin and epirubicin was measured using the microplate tetrazolium (MTT) cytotoxicity assay, and the results correlated with gp-170 expression. RESULTS: All six natural RCC cell lines showed a variably increased expression of gp-170, with the A704 and Caki-1 cell lines the highest. In contrast, gp-170 expression increased and then was suppressed in acquired MDR sublines of RCC8701 cultured in increasing concentrations of adriamycin. The A704 and Caki-1 cells were much more resistant to adriamycin and epirubicin than the A498, ACHN and RCC8701 cell lines, in parallel with the expression of gp-170. The resistant cell line cultured long-term in 800 ng/mL adriamycin, RCC8701/ADR800, was 122 times more resistant to adriamycin and 238 times more resistant to epirubicin than the parent cell line: the pattern differed from that in native RCC cell lines and was unrelated to the expression of gp-170. CONCLUSION: Membranous gp-170 plays an important role in MDR of native RCC cell lines, while acquired MDR cells have different mechanisms of obtaining drug resistance in addition to gp-170. This phenomenon may be applicable to the clinical treatment of patients newly diagnosed with RCC or those with disease refractory to chemotherapy.     

The expression of P-glycoprotein is causally related to a less aggressive phenotype in human osteosarcoma cells

The relationship between P-glycoprotein expression and malignancy is controversial. We have recently found that, in osteosarcoma, multidrug resistance (MDR) is associated with a less aggressive behavior, both in vitro and in clinical settings. In this study, we evaluated whether P-glycoprotein overexpression has a cause-effect relationship with the reduced metastatic potential of MDR cells, or rather reflects a more complex phenotype. MDR1 gene-transfected osteosarcoma cell clones, showing different levels of P-glycoprotein expression, were analysed for their in vitro characteristics and their tumorigenic and metastatic ability in athymic mice. Apart from the different levels of P-glycoprotein, no significant change in the expression of surface antigens or in the differentiative features were observed in the MDR1 gene transfectants compared to the parental cell lines or control clones, obtained by transfection with neo gene alone. In contrast to controls, however, MDR1 transfectants showed a significantly lower ability to grow in semi-solid medium and were completely unable to grow and give lung metastases in athymic mice. These findings indicate that P-glycoprotein overexpression is causally associated with a low malignant potential of osteosarcoma cells, and open new insights on the role and functions of P-glycoprotein activity.     

Combined effects of buthionine sulfoximine and cepharanthine on cytotoxic activity of doxorubicin to multidrug-resistant cells

We studied the potentiation of doxorubicin (DOX) activity in multidrug-resistant (MDR) cells by buthionine sulfoximine (BSO), a specific inhibitor of gamma-glutamylcysteine synthetase, and by cepharanthine (CE), which interacts with P-glycoprotein. The glutathione (GSH) of MDR cells was approximately 1.5-fold greater than that of the parental cell line. BSO reduced GSH content of MDR cells compared to that of the sensitive ones. The BSO treatment (50 microM) enhanced the effect of DOX by 1.8-fold, while CE caused a greater reversal of drug resistance. The combination of BSO with CE produced further potentiation of DOX activity in an antiproliferative effect. Pretreatment of cells with BSO did not alter the cellular accumulation of DOX in the absence or presence of CE. The addition of BSO (30 mM) to the drinking water of mice reduced the tissue levels of GSH in tumor cells, suggesting that the marked decrease in GSH might diminish the ability of that tumor to resist DOX. Combined administration of CE and DOX resulted in enhancement of DOX antitumor activity and prolongation of survival time. The survival of mice treated with BSO and CE as a supplement to DOX treatment was superior that of mice receiving DOX alone. These studies demonstrated that the combinations of BSO with CE may be useful for killing drug-resistant tumor cells.     

Sensitivity of vincristine-sensitive K562 and vincristine-resistant K562-Lucena 1 cells to anthracyclines and reversal of multidrug resistance

The phenomenon of multidrug resistance (MDR), that involves the efflux pump P-glycoprotein, can be reversed by a number of substances known as MDR modulators or reversing agents. In the present study we investigated the action of three anthracyclines, mitoxantrone and vincristine on short-term (72 h) cultures using 2 methods ([3H] incorporation and MTT (3-[4,5-dimethylthiasol-2-yl]-2,5-diphenyltetrazolium bromide)), on 2 cell lines: K562, a human erythroleukemia, and a vincristine-resistant subline K562-Lucena 1. Using the same culture methods plus flow cytometry analysis, the reversing potentials of cyclosporin A and verapamil were studied in both cell lines. There were differences in the sensitivity and resistance profiles of the two lines to the various drugs but daunorubicin (5 micrograms/ml) and idarubicin (0.035 micrograms/ml) were the most effective when each was used in high concentration. Cyclosporine at 200 ng/ml and verapamil at 5 micrograms/ml reversed MDR in the resistant line, and had a synergistic action with chemotherapeutic agents on the sensitive line. Again differences were demonstrable between combinations of the various drugs and reversal was only clearly shown with the method measuring cell proliferation ([3H] incorporation) but not by the method measuring metabolic activity (MTT). The efflux of rhodamine-123 mimics the functional activity of the pump and cyclosporine was a better reversing agent by this criteria. These data show that the results obtained in in vitro studies attempting to identify treatments for different types of leukemias depend to a large extent on the methods used to measure cell response.     

Characterization and chemosensitivity of a human epithelioid sarcoma cell line (SARCCR 2)

A new cell line was derived from the epithelioid sarcoma of a Caucasian woman who had previously received chemotherapy. The cells grew as an adherent monolayer, with a doubling time of 28 hr and had mainly epithelial morphology, but with areas of mesenchymal-like cytoplasmic extensions. The cells were tumorigenic in nude mice, with a short growth time, and a doubling time of 8 days. The cell line showed over-expression of P-glycoprotein by Western blot analysis, and its sensitivity to doxorubicin and vincristine was low. This sensitivity could be enhanced by reversants of multidrug resistance (MDR), such as cyclosporin or verapamil. This cell line constitutes an excellent model for studying compounds able to reverse MDR.     

Targetable HPMA copolymer-adriamycin conjugates. Recognition, internalization, and subcellular fate

Recognition, internalization, and subcellular trafficking of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugates containing N-acylated galactosamine (GalN) or monoclonal OV-TL16 antibodies (Ab) have been investigated in human hepatocarcinoma HepG2 and ovarian carcinoma OVCAR-3 cells, respectively. The intrinsic fluorescence of fluorescein or adriamycin (ADR) attached to HPMA copolymers permitted us to follow the subcellular fate of HPMA copolymer conjugates by confocal fluorescence microscopy and fluorescence spectroscopy. The pattern of fluorescence during incubation of HPMA copolymer-ADR-GalN conjugate containing lysosomally degradable tetrapeptide (GFLG) side-chains with HepG2 cells was consistent with conjugate recognition, internalization, localization in lysosomes, followed by the release of ADR from the polymer chains and ultimately diffusion via the cytoplasm into the cell nuclei. A similar pattern was observed in OVCAR-3 cells for Ab targeted HPMA copolymer conjugates. To test our hypothesis that HPMA-copolymer-bound anticancer drugs will be inaccessible to the energy-driven P-glycoprotein efflux pump in multidrug resistant (MDR) cells, we have compared the internalization of the HPMA copolymer-ADR conjugates by sensitive (A2780) and ADR-resistant (A2780/AD) ovarian carcinoma cell lines. Preliminary data on relative retention of ADR in MDR (A2780/AD) cells indicate a higher intracellular ADR concentration after incubation with HPMA copolymer-ADR conjugate when compared to incubation with free (unbound) ADR.     

The overall partitioning of anthracyclines into phosphatidyl-containing model membranes depends neither on the drug charge nor the presence of anionic phospholipids

Anthracyclines are potent anticancer agents. Their use is limited by the problem of multidrug resistance (MDR) associated with a decreased intracellular accumulation of drug correlated with the presence, in the membrane of resistant cells, of the P-glycoprotein responsible for an active efflux of the drug. The activity of a drug depends upon its intracellular concentration which itself depends on the kinetics (a) of passive influx (b) of passive efflux and (c) of the P-glycoprotein-mediated efflux of drug across the cell membrane. The ability of an anthracycline to overcome MDR depends largely on the first point. The passive drug uptake is governed by their incorporation into the lipid matrix and both electrostatic and hydrophobic forces seem necessary for the stabilization of anthracyclines into lipid bilayers. The aim of the present study was to determine the relative importance of these two interactions. Using microspectrofluorometry and the observation that the fluorescence of anthracycline is enhanced when the dihydroanthraquinone part is embedded within the lipid bilayer, we have determined the partition coefficient (alternatively, the binding constant) of 12 anthracycline derivatives in large unilamellar vesicles. The anthracyclines were (a) doxorubicin, daunorubicin and idarubicin which, at pH 7.2, bear a single positive charge at the level of the amino group on the sugar, (b) their corresponding neutral 3'-hydroxy derivatives where the amino group in the sugar has been replaced by a hydroxyl, (c) the three 13-hydroxy derivatives, doxorubicinol, daunorubicinol and idarubicinol, (d) pirarubicin and (e) two permanently positively charged derivatives. The large unilamellar vesicles contained phosphatidylcholine with various amounts of phosphatidic acid which is negatively charged and of cholesterol. We came to the conclusion that the efficiency of drug incorporation in the bilayers depends neither on the presence of a positive charge on the drug nor on the presence of anionic phospholipid but on the hydrophobicity of the molecule: the neutral and the positively charged form have the same ability to partition into the bilayer. However, the percentage of each form present should depend on the electrostatic parameters.     

Short-term and long-term memory in elderly patients with NIDDM

The new lipophilic anthracycline N-benzyl-adriamycin-14-valerate (AD198) was evaluated for its activity in comparison to doxorubicin in P-glycoprotein (Pgp)-positive and -negative cell lines. AD198 and doxorubicin showed comparable antitumor activity in the Pgp-negative breast cancer cell line MCF-7 and the Pgp-negative ovarian carcinoma cell line A2780. By contrast, AD198 was significantly more active than doxorubicin in the Pgp-positive breast cancer cell line MCF7AD (IC50 values 2.5 and 0.15 microM for 96 h continuous exposure) and the Pgp-positive ovarian carcinoma cell line A2780 DX5 (IC50 values 0.6 and 0.07 microM, respectively). Unlike doxorubicin, the activity of AD198 was not increased by concommittant application of cyclosporin A in cell line MCF7AD. Flow cytometry studies showed that, in contrast to doxorubicin, AD198 was not transported by Pgp and that verapamil did not change the intracellular pharmacokinetics of this new anthracycline. These data provide evidence that AD198 possesses high activity in human solid tumor cell lines expressing the classical multidrug resistant phenotype. Its further clinical development appears to be warranted.     

Altered methylation of the human MDR1 promoter is associated with acquired multidrug resistance

One of the most important forms of drug resistance in acute myeloid leukemia is the multidrug resistance (MDR) phenotype, which is characterized by the expression of the MDR1 gene product, P-glycoprotein. Although a number of factors affect MDR1 gene expression, the genetic events that "switch on" the human MDR1 gene in tumor cells that were previously P-glycoprotein negative have remained elusive. Here, we report evidence that the methylation status of the human MDR1 promoter may serve as a basis for this "switch." Based on Southern analysis using methylation-sensitive and methylation-insensitive restriction enzymes, a tight correlation was found between MDR phenotype and demethylation of the 5' region of the MDR1 gene in a human T cell leukemia cell line. Similar results were obtained from the analysis of P-glycoprotein-positive and P-glycoprotein-negative samples of chronic lymphocytic leukemia. Treatment of the cell lines with the demethylating agent 5'-azadeoxycytidine altered the methylation pattern of the MDR1 promoter in P-glycoprotein-negative cells to resemble that of P-glycoprotein-positive cells and activated the promoter such that MDR1 mRNA was now detectable. Treatment also resulted in an increased resistance to epirubicin and decreased daunomycin accumulation, both of which were reversible by verapamil, a characteristic of the classical MDR phenotype in cells expressing P-glycoprotein. These results suggest that the MDR phenotype may be acquired as a result of changes in methylation of the MDR1 promoter.     

Fuzzy-logic control of blood pressure through enflurane anesthesia

BACKGROUND: A 190k (190-kilodalton) membrane protein has been identified in several multidrug-resistant (MDR) cell lines that show decreased drug accumulation without expression of P-glycoprotein. It is not clear whether this 190k protein is involved directly in drug efflux. Recently, a gene for a putative transporter protein, MRP (multidrug resistance-associated protein) has been sequenced and localized to chromosome 16. The protein encoded by this gene contains a 7-amino-acid sequence present in the synthetic peptide used to generate the antiserum recognizing the 190k protein. PURPOSE: The study was undertaken to clarify the relationship of the 190k protein to MRP gene expression in non-P-glycoprotein-containing MDR cells of the large-cell and adenocarcinoma lung cancer lines, COR-L23 and MOR. METHODS: Expression of the 190k protein was determined by Western blot analysis and that of the MRP gene by polymerase chain reaction amplification of complementary DNA reverse transcribed from RNA. Abnormalities of chromosome 16 were investigated in chromosome spreads by fluorescence in situ hybridization. RESULTS: The amount of detectable 190k protein is closely associated with degree of drug resistance. Cell lines surviving in higher drug concentrations have greater amounts of protein, and revertant lines grown without drug for up to 28 weeks show reduced expression of the protein together with enhanced drug sensitivity. The 190k protein appears to be one of the major proteins differentially expressed in membranes of drug-resistant cells. The amount of MRP messenger RNA correlates closely with that of the 190k protein. The MDR cells contain amplified chromosome 16 material with many double minutes in the large-cell lung tumor lines and an enlarged chromosome 16 in the adenocarcinoma lines. CONCLUSION: The 190k protein detected immunologically is likely to be the protein, encoded by the MRP gene, which becomes overexpressed in these cells as a consequence of chromosomal amplification and fragmentation. IMPLICATION: Though associated with drug resistance, enhanced drug efflux, and decreased drug accumulation in cell lines, the role of this protein in clinical resistance has yet to be determined.     

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.