A multidrug resistance transporter from human MCF-7 breast cancer cells

MCF-7/AdrVp is a multidrug-resistant human breast cancer subline that displays an ATP-dependent reduction in the intracellular accumulation of anthracycline anticancer drugs in the absence of overexpression of known multidrug resistance transporters such as P glycoprotein or the multidrug resistance protein. RNA fingerprinting led to the identification of a 2.4-kb mRNA that is overexpressed in MCF-7/AdrVp cells relative to parental MCF-7 cells. The mRNA encodes a 655-aa [corrected] member of the ATP-binding cassette superfamily of transporters that we term breast cancer resistance protein (BCRP). Enforced expression of the full-length BCRP cDNA in MCF-7 breast cancer cells confers resistance to mitoxantrone, doxorubicin, and daunorubicin, reduces daunorubicin accumulation and retention, and causes an ATP-dependent enhancement of the efflux of rhodamine 123 in the cloned transfected cells. BCRP is a xenobiotic transporter that appears to play a major role in the multidrug resistance phenotype of MCF-7/AdrVp human breast cancer cells.     

Expression of the antisense cDNA for protein kinase C alpha attenuates resistance in doxorubicin-resistant MCF-7 breast carcinoma cells

Multidrug resistance is functionally associated with the expression of a plasma membrane energy-dependent drug efflux pump termed P-glycoprotein, the product of the mdr1 gene. Transfection of P-glycoprotein-expressing doxorubicin-resistant MCF-7 cells with an expression vector containing the cDNA for protein kinase C alpha in the antisense orientation reduces protein kinase C alpha levels and decreases total protein kinase C activity by 75%. This is accompanied by reduced phosphorylation of P-glycoprotein, a 2-fold increase in drug retention, and a 3-fold increase in doxorubicin cytotoxicity. These results provide further evidence that protein kinase C alpha can positively regulate multidrug resistance in MCF-7 cells through posttranslational phosphorylation of P-glycoprotein.     

A new anthracycline with potent anti-leukemic activity overcomes P-glycoprotein multidrug resistance

In this study, we assessed the ability of a new anthracycline, moflomycin, to circumvent multidrug resistance. Moflomycin showed superior anti-proliferative activity compared to daunorubicin and doxorubicin on two resistant cell lines: leukemic HL-60 cell line resistant to daunorubicin (HL-60/DR) and breast cancerous cell line resistant to doxorubicin (MCF-7/AR). The effect of moflomycin on cell proliferation was correlated with an increased uptake and a decreased cellular efflux. The data obtained in the presence of the P-gp inhibitor, verapamil, confirmed the absence of interaction between P-gp and moflomycin. Our results indicate that moflomycin exhibits an important reduction in cross-resistance with daunorubicin and doxorubicin resulting from its ability to circumvent P-gp.     

Characterization of the PP2A alpha gene mutation in okadaic acid-resistant variants of CHO-K1 cells

Okadaic acid (OA)-resistant variants of Chinese hamster ovary cells, clones CHO/OAR6-6 and CHO/OAR2-3, were isolated from a CHO-K1 culture. These variant cells were 17- to 26-fold more resistant to OA than the parental cells. The phosphorylase phosphatase activity of the variant cell extracts was 2- to 4-fold more resistant to OA than that of the parental cells in the presence of inhibitor 2, a specific inhibitor of type 1 protein serine/threonine phosphatase (PP1). Nucleotide sequencing of PP2A alpha (an isotype of PP2A catalytic subunit) cDNA demonstrated that both variants have a T-->G transversion at the first base of codon 269 (805 nt), which results in substitution of glycine for cysteine. We expressed in COS-1 cells a mutant PP2A alpha tagged with the influenza hemagglutinin epitope. The recombinant mutant PP2A alpha protein immunoprecipitated with an anti-influenza hemagglutinin antibody was more resistant than the wild type to OA, their IC50 values being 0.65 nM and 0.15 nM, and their IC80 values being 4.0 nM and 0.45 nM, respectively. The cysteine at residue 269 present only in highly OA-sensitive protein serine/threonine phosphatase catalytic subunit isozymes, PP2A alpha, PP2A beta, and PPX, is suggested to be involved in the binding of OA. CHO/OAR6-6 and CHO/OAR2-3 cells also overexpressed the P-glycoprotein, and the efflux of OA was more rapid. It is suggested that the PP2A alpha mutation in cooperation with a high level of P-glycoprotein makes the CHO-K1 variants highly resistant to OA.     

Resistance to tomudex (ZD1694): multifactorial in human breast and colon carcinoma cell lines

ZD1694 (Tomudex; TDX) is a quinazoline antifolate that, when polyglutamated, is a potent inhibitor of thymidylate synthase (TS), the enzyme that converts dUMP to dTMP. Continuous exposure of MCF-7 breast and NCI H630 colon cells to TDX, with stepwise increases in TDX up to 2.0 microM, resulted in stably resistant cell lines (MCFTDX and H630TDX) that were highly resistant to TDX. Initial studies revealed 34-fold increase in TS protein levels in MCFTDX and a 52-fold increase in TS levels in H630TDX cell lines. Despite continued exposure of these cells to 2.0 microM TDX, TS protein and TS mRNA expression decreased to parental levels in H630TDX cells, whereas in MCFTDX cells TS mRNA expression and TS protein levels remained elevated. Southern blot analysis revealed a 20-fold TS gene amplification in the MCFTDX cell line. TDX uptake was 2-fold higher in resistant MCFTDX cells than in parental MCF-7 cells, whereas in H630TDX cells TDX uptake was 50-fold less than that observed in parental H630 cells. In contrast, no change in the transport of either leucovorin or methotrexate into H630TDX cells was noted when compared with the H630 parental cells. In H630TDX cells, folylpolyglutamate synthetase (FPGS) activity was 48-fold less compared to parent H630 cells; however, FPGS mRNA expression was similar in both lines. H630TDX cells were also highly resistant to ZD9331, a novel quinazoline TS inhibitor that does not require polyglutamation, suggesting that defective transport by the reduced folate carrier was also an important mechanism of resistance in these cells. In MCFTDX and H630TDX resistant cells, several mechanisms of resistance are apparent: one increased TS expression; the others evolved over time from increased TS expression to decreased FPGS levels and decreased TDX transport.     

Inverse relationship between galactokinase activity and 2-deoxygalactose resistance in Chinese hamster ovary cells

Galactokinase activity is reduced in 12 independent clones of Chinese hamster ovary cells resistant to 2-deoxygalactose. The frequency of resistant colonies is increased with chemical mutagens. The resistant phenotype is stable in the absence of selection. There is an inverse correlation between the levels of galactokinase activity and the cloning efficiency in deoxygalactose. Cells with high resistance have 1% or less of the enzyme activity observed in the parental cells; while cells with low resistance have 10-30% galactokinase activity. Studies with tetraploid hybrid cells reveal that resistance to deoxygalactose is a recessive trait and that cells with high resistance do not complement those with low resistance. In cell lines with low resistance, the Km for galactose, Ki for deoxygalactose, Km for ATP, and thermolability were not significantly altered compared to sensitive parental cells. Although the possibility of mutation at the structural gene locus has not been ruled out, the reduced enzyme activity may also be due to mutation at a regulatory site which affects the number of galactokinase molecules per cell.     

Synthesis of tissue factor messenger RNA and procoagulant activity in breast cancer cells in response to serum stimulation

The procoagulant activity observed in many types of tissue and cultured cells is due to tissue factor, a 30 kd transmembrane protein. The mRNA for tissue factor is a 2.2-kb species, which in some non-cancer cells can be up-regulated or induced by cytokines or by serum stimulation. In this study, induction of procoagulant activity in cancer cells was evaluated using the breast cancer cell line, MCF-7, and an adriamycin resistant subline, AdrRMCF-7, which has increased tumorigenicity in nude mice compared to the parental cell line. Procoagulant activity was factor VIIa dependent and was inhibited by an anti-tissue factor antibody. MCF-7 cells had minimal tissue factor activity, while AdrRMCF-7 cells had an 10-fold increase compared to the parental line. This increase was not observed in MCF-7 cells transfected with the multi-drug resistant gene, which is associated with adriamycin resistance. Serum stimulation of quiescent MCF-7 cells increased tissue factor activity 5-fold over baseline level, but did not increase activity in cells grown in serum-replete medium. Tissue factor activity of AdrRMCF-7 quiescent cells and AdrMCF-7 cells grown in serum-replete medium was enhanced 2-fold by serum stimulation. The predominant tissue factor mRNA species in MCF-7 cells was a 3.2 to 3.4-kb band, which increased in response to serum stimulation of cells grown in serum-replete medium. The mature 2.2-kb tissue factor mRNA band was detected in quiescent MCF-7 cells within six hours of serum stimulation and remained present 24 hours after stimulation. Synthesis of the 2.2-kb tissue factor mRNA species in MCF-7 and AdrRMCF-7 cells correlated with appearance of procoagulant activity. Thus, while procoagulant activity correlates with the level of the 2.2-kb tissue factor mRNA species in these cancer cells, there are inherent differences in tissue factor activity, antigen, and mRNA levels, as well as in regulation of its synthesis between these cells.     

Activity of P-glycoprotein in B-cell chronic lymphocytic leukemia determined by a flow cytometric assay

BACKGROUND: Chemoresistance in some hematologic malignancies has been associated with overexpression of P-glycoprotein, which is encoded by the MDR1 gene (also known as PGY1). However, inconsistencies in data on frequency and clinical relevance of multidrug resistance in B-cell chronic lymphocytic leukemia (B-CLL) may reflect a need for improved techniques to detect this overexpression. PURPOSE: Our purpose was to measure P-glycoprotein activity in peripheral blood cells of B-CLL patients and to analyze possible clinical correlations (disease duration, prior treatment, Rai disease stage, lymphocyte counts, and disease progression). METHODS: P-glycoprotein activity was assayed in peripheral blood cells of 42 consecutive B-CLL patients (22 treated and 20 untreated). We used dual fluorescence in a flow cytometric assay that detects efflux of the fluorescent dye rhodamine 123, which is transported from the cell by the P-glyprotein pump. Leukemia cells were costained with monoclonal antibody Leu12/CD19, and rhodamine 123 efflux was measured. Expression of MDR1 and MDR3 (also known as PGY3) messenger RNA (mRNA) was quantitatively evaluated by polymerase chain reaction (PCR) in 26 cases. RESULTS: Marked rhodamine 123 efflux was observed in 34 (81%) of the 42 cases and was abolished in the presence of multidrug resistance inhibitors. Rhodamine 123 efflux was not associated with Rai stage, lymphocyte counts, duration of disease, or disease progression. Although rhodamine 123-negative cases were about equally distributed among untreated and previously treated patients, the percentage of cells with rhodamine 123 efflux was significantly lower for untreated patients than for those treated with chemotherapy regimens including at least one multidrug resistance-associated drug. MDR1 mRNA was detected in 25 of 26 cases and MDR3 mRNA in all 26. MDR1 mRNA expression was significantly correlated with rhodamine 123 efflux, whereas MDR3 mRNA expression was not significantly correlated; MDR1 and MDR3 mRNA expression was not significantly associated with Rai stage, prior treatment, or disease progresssion. CONCLUSIONS: These findings suggest that P-glycoprotein overexpression in B-CLL is intrinsic rather than acquired and that P-glycoprotein activity is enhanced after exposure to multidrug resistance-associated drugs. This enhanced activity does not seem to be associated with more aggressive disease. Our results also indicate that an assay of P-glycoprotein function combined with PCR is suitable for clinical multidrug resistance screening. IMPLICATIONS: Additional studies are needed to determine whether functional activity of P-glycoprotein, measured by rhodamine 123 efflux, is directly related to clinical drug resistance.     

Doxoform and Daunoform: anthracycline-formaldehyde conjugates toxic to resistant tumor cells

The recent discovery that the clinically important antitumor drugs doxorubicin and daunorubicin alkylate DNA via catalytic production of formaldehyde prompted the synthesis of derivatives bearing formaldehyde. Reaction of the parent drugs with aqueous formaldehyde at pH 6 produced in 40-50% yield conjugates consisting of two molecules of the parent drug as oxazolidine derivatives bound together at their 3'-nitrogens by a methylene group. The structures were established as bis(3'-N-(3'-N,4'-O-methylenedoxorubicinyl)) methane (Doxoform) and bis(3'-N-(3'-N,4'-O-methylenedaunorubicinyl))methane (Daunoform) from spectroscopic data. Both derivatives are labile with respect to hydrolysis to the parent drugs. 3'-N,4'-O-Methylenedoxorubicin and 3'-N,4'-O-methylenedaunorubicin are intermediates in the hydrolysis. Daunoform reacts with the self-complementary deoxyoligonucleotide (GC)4 faster than the combination of daunorubicin and formaldehyde at an equivalent concentration to given drug-DNA adducts. In spite of hydrolytic instability, Doxoform is 150-fold more toxic to MCF-7 human breast cancer cells and 10000-fold more toxic to MCF-7/ADR resistant cells. Toxicity to resistant cancer cells is interpreted in terms of higher lipophilicity of the derivatives and circumvention of catalytic formaldehyde production.     

Mechanisms of resistance in a human cell line exposed to sequential topoisomerase poisoning

Camptothecins are a new class of anticancer drugs that target DNA topoisomerase I; current efforts are directed toward elucidating optimal combinations of these drugs with other antineoplastic agents. A rationale for the use of sequential therapy involving the combination of camptothecins with topoisomerase II-targeting drugs, such as etoposide, has arisen from observations of increased topoisomerase II protein levels in cell lines resistant to camptothecin. In an effort to understand potential mechanisms of resistance to this strategy, we developed a U-937 cell subline, denoted RERC, that is capable of surviving exposure to sequential topoisomerase poisoning. The RERC cells are 200-fold resistant to camptothecin, 8-fold resistant to etoposide, and 10-fold hypersensitive to cisplatin compared to the parental U-937 cells. Biochemical analyses indicate that the resistant phenotype involves alterations in both topoisomerase I and topoisomerase IIalpha. Topoisomerase I catalytic activity in the resistant cells is similar to that of the parental line but is resistant to camptothecin. Moreover, the resistant cells express a single mRNA species of topoisomerase I that codes for a mutation in codon 533. In addition, topoisomerase IIalpha protein levels are decreased 10-fold in the resistant line, coincident with a two-fold decrease in the expression of topoisomerase IIalpha mRNA. Collectively, these results indicate that resistance to sequential topoisomerase poisoning may involve a reduction in total cellular topoisomerase activity.     

Cellular adaptation to drug exposure: evolution of the drug-resistant phenotype

The efficacy of all chemotherapeutic agents is limited by the occurrence of drug resistance. For etoposide (VP-16), increased expression of MDR-1 or MRP and alterations in topoisomerase IIalpha have been shown to confer tolerance. To further understand resistance to VP-16, three sublines, designated MCF-7-VP17, ZR-75B-VP13, and MDA-MB-231-VP7, were initially isolated as single clones from parental cells by exposure to VP-16. Subsequently, a population of cells from each subline was exposed to 3-fold higher drug concentrations, allowing stable sublines to be established at higher extracellular drug concentrations. Characterization of the resistant sublines demonstrates the adaptation that occurs with advancing drug concentrations during in vitro selections. Reduced topoisomerase II mRNA and protein levels were observed in the initial isolates. This reduction was accompanied by a decrease in topoisomerase II activity and cellular growth rate and was associated with 6-314-fold resistance to topoisomerase II poisons. With advancing resistance, MRP expression increased and VP-16 accumulation decreased. This adaptation allowed for partial restoration of topoisomerase II activity as a result of increased expression (MCF-7-VP17 and ZR-75B-VP13) or hyperphosphorylation (MDA-MB-231-VP7), with a resultant increase in growth rate. In MDA-MB-231-VP7 cells, hyperphosphorylation coincided with increased casein kinase II mRNA and protein levels, suggesting a role for this kinase in the acquired hyperphosphorylation. In this cell line, hyperphosphorylation mediated the increased activity despite a fall in topoisomerase IIalpha protein levels secondary to an acquired 600-bp deletion in one topoisomerase IIalpha allele, which resulted in reduced protein levels. In all three sublines, high levels of resistance were attained as a result of synergism between the reduced topoisomerase IIalpha levels and MRP overexpression. These studies demonstrate how cellular adaptation to increasing drug pressure occurs and how more than one mechanism can contribute to the resistant phenotype when increasing selecting pressure is applied. Reduced expression of topoisomerase II is sufficient to confer substantial resistance early in the selection process, with synergy from MRP overexpression helping to confer high levels of resistance.     

Decreased melphalan accumulation in a human breast cancer cell line selected for resistance to melphalan

An in vitro model of acquired melphalan resistance was developed by serial incubation of an MCF-7 human breast cancer cell line in increasing concentrations of melphalan. The resulting derivative cell line, Me1R MCF-7, was 30-fold resistant to melphalan. Uptake studies demonstrated decreased initial melphalan accumulation in Me1R MCF-7 cells. Inverse-reciprocal plots of initial melphalan uptake revealed a 4-fold decrease in the apparent Vmax of Me1R MCF-7 compared with WT MCF-7 (516 amol cell-1 min-1 vs 2110 amol cell-1 min-1 respectively) as well as a decrease in the apparent Kt (36 microM vs 70 microM respectively). Two amino acid transporters have previously been identified as melphalan transporters: system L, which is sodium-independent and inhibited by 2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid (BCH), and system ASC which is sodium dependent and unaffected by BCH. At low concentrations of melphalan (3-30 microM), 1mM BCH competition eliminated the differences between the two cell lines, thus implicating an alteration of the system L transporter in the transport defect in the resistant cells. Me1R MCF-7 cells were also evaluated for glutathione-mediated detoxification mechanisms associated with melphalan resistance. There was no difference between Me1R MCF-7 and WT MCF-7 in glutathione content, glutathione-S-transferase activity and expression of pi class glutathione S-transferase RNA. In addition, buthionine sulfoximine did not reverse melphalan resistance in Me1R MCF-7 cells. Therefore, Me1R MCF-7 cells provide an in vitro model of transport-mediated melphalan resistance in human breast cancer cells.     

P-glycoprotein expression in Ehrlich ascites tumour cells after in vitro and in vivo selection with daunorubicin

Fluctuation analysis experiments were performed to assess whether selection or induction determines expression of P-glycoprotein and resistance in the murine Ehrlich ascites tumour cell line (EHR2) after exposure to daunorubicin. Thirteen expanded populations of EHR2 cells were exposed to daunorubicin 7.5 x 10(-9) M or 10(-8) M for 2 weeks. Surviving clones were scored and propagated. Only clones exposed to daunorubicin 7.5 x 10(-9) M could be expanded for investigation. Drug resistance was assessed by the tetrazolium dye (MTT) cytotoxicity assay. Western blot was used for determination of P-glycoprotein. Compared with EHR2, the variant cells were 2.5- to 5.2-fold resistant to daunorubicin (mean 3.6-fold). P-glycoprotein was significantly increased in 11 of 25 clones (44%). Analysis of variance supported the hypothesis that spontaneous mutations conferred drug resistance in EHR2 cells exposed to daunorubicin 7.5 x 10(-9) M. At this level (5 log cell killing) of drug exposure, the mutation rate was estimated at 4.1 x 10(-6) per cell generation. In contrast, induction seemed to determine resistance in EHR2 cells in vitro exposed to daunorubicin 10(-8) M. The revertant EHR2/0.8/R was treated in vivo with daunorubicin for 24 h. After treatment, P-glycoprotein increased in EHR2/0.8/R (sevenfold) and the cell line developed resistance to daunorubicin (12-fold), suggesting that in EHR2/0.8/R the mdr1 gene was activated by induction. In conclusion, our study demonstrates that P-glycoprotein expression and daunorubicin resistance are primarily acquired by selection of spontaneously arising mutants. However, under certain conditions the mdr1 gene may be activated by induction.     

Decreased activity of the multidrug resistance P-glycoprotein in acquired aplastic anaemia: possible pathophysiologic implications

To address a possible impairment of multidrug resistance mechanisms in acquired aplastic anaemia (AA), the functions of P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP) were respectively assessed by rhodamine 123 (Rh123) and daunorubicin (DNR) efflux in peripheral blood lymphocytes from AA patients. The proportion of Rh123-effluxing T cells was significantly decreased in AA, relative to controls. Interestingly, these changes were also present in patients with AA in remission. Conversely, Rh123 efflux in B and natural killer (NK) cells and DNR efflux in peripheral blood lymphocytes were unchanged. These data indicated that P-gp activity was decreased in AA not only during the development of the disease, but also after remission, introducing a new concept on the pathophysiology of AA by suggesting that it may contribute to drug-induced injury to haemopoietic cells in some cases of AA, by increasing the proportion of susceptible cells.     

Development and characterisation of novel human multidrug resistant mammary carcinoma lines in vitro and in vivo

Clinical chemotherapy of breast carcinomas must be considered insufficient, mainly due to the appearance of drug resistance. The multidrug resistance (MDR) phenotype, either intrinsically occurring or acquired, e.g., against a panel of different antineoplastic drugs, is discussed in relation to several MDR-associated genes such as the MDR-gene mdr1 encoding the P-glycoprotein (PGP), the MRP gene (multidrug resistance protein) encoding an MDR-related protein or the LRP gene encoding the lung resistance protein. Numerous experimental and clinical approaches aiming at reversing resistance require well-characterised in vitro and in vivo models. The aim of our work was to develop multidrug resistant sublines from human xenotransplanted breast carcinomas, in addition to the broadly used line MCF-7 and its multidrug resistant subline MCF-7/AdrR. MDR was induced in vitro with increasing concentrations of Adriablastin (ADR) for several weeks, resulting in a 3.5- to 35-fold increase in IC50 values using the MTT-test. Cell lines were cross-resistant toward another MDR-related drug, vincristine, but remained sensitive to non-MDR-related compounds such as cisplatin and methotrexate. The resistance toward Adriamycin and vincristine was confirmed in vivo by a lack of tumour growth inhibition in the nude mouse system. Gene expression data for the mdr1/PGP, MRP/MRP and LRP/LRP on both the mRNA (RT-PCR) and the protein levels (immunoflow cytometry) demonstrated that induction of mdr1 gene expression was responsible for the acquired MDR phenotype. Rhodamine efflux data, indicated by PGP overexpression, underlined the development of this MDR mechanism in the newly established breast carcinoma lines MT-1/ADR, MT-3/ADR and MaTu/ADR.     

Na,K-ATPase subunit isoforms in human reticulocytes: evidence from reverse transcription-PCR for the presence of alpha1, alpha3, beta2, beta3, and gamma

The objective of this study has been to determine which Na,K-ATPase isoforms are expressed in red blood cells and whether kinetic differences in the uncoupled sodium efflux mode between the human red blood cell Na,K-ATPase and other preparations can be explained by differences in the underlying subunit composition. To this end, human reticulocyte RNA was isolated, reverse transcribed, amplified by PCR and appropriate primers, and sequenced. Primers from highly conserved areas as well as isoform-specific primers were used. The alpha1 and alpha3 isoforms of the alpha subunit, and the beta2 and beta3 isoforms of the beta subunit were found. The complete coding regions of the cDNAs for the reticulocyte subunits were sequenced from overlapping PCR fragments. No difference was found between the reticulocyte isoforms and the ones already known. The fact that we found beta2 but not beta1 in reticulocyte single-stranded cDNA, and beta1 but not beta2 in a leukocyte library indicates that leukocyte contamination of our reticulocyte preparation was negligible. Analysis of a human bone marrow library showed that alpha1, alpha2, and alpha3 as well as all three beta isoforms were present. The extent to which the kinetic properties of uncoupled sodium efflux might depend on different isoform combinations is not yet known.     

Topoisomerase II as a target of VM-26 and 4'-(9-acridinylamino)methanesulfon-m-aniside in atypical multidrug resistant human small cell lung carcinoma cells

The Adriamycin-resistant small cell lung carcinoma cell line, GLC4/ADR, showed large differences in cross-resistance to drugs such as Adriamycin, etoposide (VP-16), teniposide (VM-26), 4'-(9-acridinylamino)-methanesulfon-m-anisidide (m-AMSA), and mitoxantrone, which stimulate the formation of topoisomerase (Topo) II-DNA complexes. GLC4/ADR cells demonstrated a reduced Topo II activity and no detectable levels of the P-glycoprotein compared to the parental GLC4 cells (S. De Jong et al., Cancer Res., 50: 304-309, 1990). In the present study, the resistance to VM-26 (59.5-fold) and to m-AMSA (4-fold) of GLC4/ADR after a 1-h incubation was further analyzed. Using the K(+)-sodium dodecyl sulfate precipitation assay, a reduction in VM-26- and m-AMSA-induced cleavable complex formation was found in GLC4/ADR cells compared to GLC4 cells that was related to the degree of resistance to each drug. Cellular accumulation of the VM-26 analogues VP-16 was 3- to 8-fold less and the accumulation of m-AMSA 1- to 2-fold less in GLC4/ADR cells than in the parental cells. Following the removal of VM-26, the cleavable complexes in GLC4/ADR cells disappeared at least 2-fold faster than in GLC4 cells, while the efflux of VP-16 was also enhanced in the resistant cells. On the contrary, no differences in cleavable complex disappearance or drug efflux between these cell lines were observed with m-AMSA. Efflux of both drugs, however, occurred at a much higher rate than cleavable complex disappearance. Using isolated nuclei, a reduction in cleavable complexes in GLC4/ADR was still observed with VM-26 as well as m-AMSA compared to GLC4. The resistant nuclei and nuclear extracts showed a 3-fold decrease in M(r) 170,000 Topo II by immunoblotting. No differences in cleavable complex formation were found between nuclear extracts of both cell lines, when the Topo II activities were equalized. These findings suggest that the cross-resistance to m-AMSA is due to a decreased amount of Topo II and decreased drug accumulation, while in addition to these mechanisms an increased rate of cleavable complex disappearance is involved in the cross-resistance to VM-26 of the GLC4/ADR cell line.     

Functional multidrug resistance phenotype associated with combined overexpression of Pgp/MDR1 and MRP together with 1-beta-D-arabinofuranosylcytosine sensitivity may predict clinical response in acute myeloid leukemia

Overexpression of P-glycoprotein (Pgp) or MDR1 mRNA has been shown to be a negative prognostic factor for clinical outcome in acute myeloid leukemia (AML). However, resistance to chemotherapy also occurs in the absence of Pgp overexpression. Therefore, besides Pgp expression, we have assessed the expression of MRP, a novel drug transporter gene, along with the functional multidrug-resistant (MDR) phenotype of leukemic cells. These MDR parameters are correlated with clinical outcome in individual patients. We found functional changes in fresh leukemic cells from de novo or relapsed patients similar to those reported for tumor cell lines with the MDR phenotype. These changes were reduced drug accumulation as assessed with radiolabeled doxorubicin (factor 1.6), daunomycin (factor 1.13), and vincristine (factor 1.6) in patients who were refractory to the combination treatment of 1-beta-D-arabinofuranosylcytosine (ara-C) and daunomycin or mitoxantrone as opposed to patients who had complete responses. Also, the intracellular distribution of doxorubicin fluorescence (nuclear/cytoplasmic ratio), as assessed with laser scan microscopy, was reduced 1.4-fold in blasts from refractory patients. Based on historically known clinical response to single-agent daunomycin or ara-C in the group of responding de novo AML patients, we have set a threshold level such that a defined part of the samples that had the highest drug accumulation or nuclear to cytoplasmic ratios were above this threshold value. This allowed discrimination between patients responding to daunomycin from those who were refractory to this drug. By using this threshold level, in the refractory group clinical resistance corresponded with high sensitivity with a resistant phenotype. A similar threshold was set for the data of the in vitro ara-C sensitivity test. By combining both assays for all individual patients, clinical refractoriness as well as sensitivity could be predicted with high accuracy. There appeared to be no stringent relationship between the functional MDR phenotype with expression of either Pgp (fluorescence-activated cell sorting analysis) or MRP mRNA (RNase protection). However, by combining both parameters the functional MDR phenotype correlated with the overexpression of either one or both of the parameters in 94% of the samples studied. It is concluded that this combined overexpression in conjunction with functional changes for MDR drugs and ara-C reveal a correlation of MDR phenotype with clinical resistance to combination chemotherapy in AML patients and hereby may adequately predict clinical MDR in individual AML patients.     

Is rhodamine 123 an appropriate fluorescent probe to assess P-glycoprotein mediated multidrug resistance in vinblastine-resistant CHO cells?

Cellular drug resistance, which involves several mechanisms such as P-glycoprotein (P-gp) overexpression, kinetic and metabolic quiescence, or the increase in the intracellular levels of glutathione, limits the effectiveness of cancer treatment. It has been reported that functional assessment of the cationic dye rhodamin 123 (Rho123) efflux reveals accurately the drug-resistant phenotype. To study cellular drug resistance, we have obtained a CHO-K1 derived cell line resistant to vinblastine by means of multistep selection. This cell line (CHOVBR) displays high reactivity with a monoclonal antibody (MAb) (C219) directed against an internal domain of P-gp, and an active Rho123 efflux, as shown by parallel flow cytometric and fluorometric assays. However, under similar experimental conditions, the drug-sensitive parental cell line CHO-K1 (as well as the myeloblastic KG1 and KG1a cell lines), was also able to pump Rho123 out. These parental CHO-K1 cells had a very low reactivity against the C219 Mab, as confirmed by Western blot analysis. Both vinblastine and verapamil inhibited Rho123 efflux in CHO-K1 cells, but had no effect on CHOVBR cultures. Also, deprivation of vinblastine for one month did not affect Rho123 efflux in these cells. Our results suggest that the activity of P-gp appears to be essential, but not sufficient to confer drug resistance, and that Rho123-based functional assays of drug resistance should be evaluated for each cellular experimental model.