Multidrug Resistance Phenomenon Research Articles

Fluoxetine and reversal of multidrug resistance

This review centers on recent findings with respect to modulating cancer multidrug resistance (MDR) with the well-known antidepressant fluoxetine (prozac). The MDR phenomena and mechanisms are discussed, including the roles of ABC transporters as MDR-pumps and the potential involvement of cancer stem cells. The three generations of MDR reversal agents (chemosensitizers) are reviewed, introducing the concept of single-pump and multi-pump agents. The current status of chemosensitization is summarized, pointing-out the need for additional agents and outlining experimental criteria for testing novel candidates. Major in vitro and in vivo findings are summarized showing that fluoxetine is a chemosensitizer of the multi-pump type, and proposing it be considered a fourth-generation chemosensitizer. In concluding, we contemplate future prospects of modulating MDR in the clinic.

ATP‐binding cassette superfamily transporter gene expression in human soft tissue sarcomas

The phenomenon of multidrug resistance (MDR) in various malignant neoplasms has been reported as being caused by one or multiple expressions of ATP-binding cassette (ABC) superfamily protein, including P-glycoprotein/multidrug resistance (MDR) 1 and the MDR protein (MRP) family. However, their expression levels and distribution within soft tissue sarcomas remain controversial. In 86 cases of surgically resected soft tissue sarcoma, intrinsic mRNA levels of MDR1, MRP1, MRP2 and MRP3 were assessed using a quantitative reverse transcriptase-PCR (RT-PCR) method. Moreover, immunohistochemical protein expressions of P-glycoprotein (P-gp), MRP1, MRP2, MRP3 and p53 protein were evaluated in concordant paraffin-embedded material. The mRNA expression and immunohistochemical expression of ABC superfamily transporters were compared to clinicopathologic parameters and proliferative activities as evaluated by the MIB-1-labeling index (LI). Among the various histologic types, malignant peripheral nerve sheath tumor (MPNST) showed significantly high levels of MDR1 (p=0.017) and MRP3 (p=0.0384) mRNA expression, compared to the other tumor types. When the immunohistochemical method was compared to the RT-PCR technique to assess ABC transported expression at the protein and mRNA levels, a significantly close relationship was found between the 2 methods (p<0.05). P-gp expression was significantly correlated with large tumor size (> or =5 cm, p=0.041) and high AJCC stage (stages III and IV) (p=0.0365). Furthermore, cases with nuclear expression of p53 revealed significantly higher levels of MDR1 mRNA expression, compared to those with negative immunoreaction for p53 (p=0.0328). Our results suggest that MDR1/P-gp expression may have an important role to play in tumor progression in the cases of soft tissue sarcoma, and p53 may be one of the active regulators of the MDR1 transcript. In addition, the high levels of both MDR1 and MRP3 mRNA expression in MPNST may help to explain the poor response of this tumor to anticancer-drugs.

In vitro detection of mdr1 mRNA in murine leukemia cells with 111In-labeled oligonucleotide.

The feasibility of intracellular mdr1 mRNA expression detection with radiolabeled antisense oligonucleotide (ODN) was investigated in the murine leukemia cell line, P388/S, and its subclonal, adriamycin-resistant cell line, P388/R. The expression level of mdr1 mRNA was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). Existence of the multidrug resistance (MDR) phenomenon was assessed via cellular uptake of 99mTc-sestamibi (MIBI), a known substrate for P-glycoprotein. A 15-mer phosphorothioate antisense ODN complementary to the sequences located at -1 to 14 of mdr1 mRNA and its corresponding sense ODN were conjugated with the cyclic anhydride of diethylene triamine penta-acetic acid (cDTPA) via an amino group linked to the terminal phosphate at the 5' end at pH 8-9. The DTPA-ODN complexes at concentrations of 0.1-17.4 microM were reacted with 111InCl3 at pH 5 for 1 h. The hybridization affinity of labeled ODN was evaluated with size-exclusion high-performance liquid chromatography following incubation with the complementary sequence. Cellular uptake of labeled ODN was examined in vitro. Furthermore, enhancing effects of synthetic lipid carriers (Transfast) on transmembrane delivery of ODN were assessed. P388/R cells displayed intense mdr1 mRNA expression in comparison with P388/S cells. 99mTc-MIBI uptake in P388/S cells was higher than that in P388/R cells. Specific radioactivity up to 1,634 MBq/nmol was achieved via elevation of added radioactivity relative to ODN molar amount. The hybridization affinity of antisense 111In-ODN was preserved at approximately 85% irrespective of specific activity. Cellular uptake of antisense 111In-ODN did not differ from that of sense 111In-ODN in either P388/S cells or P388/R cells. However, lipid carrier incorporation significantly increased transmembrane delivery of 111In-ODN; moreover, specific uptake of antisense 111In-ODN was demonstrated in P388/R cells. Radiolabeling of ODN at high specific radioactivity and specific uptake of antisense 111In-ODN in drug-resistant cells may facilitate future gene imaging of mdr1 mRNA.

The Reduction of P-Glycoprotein Expression by Small Interfering RNAs Is Improved in Exponentially Growing Cells

Small interfering RNAs (siRNAs) are powerful tools in specifically silencing gene expression. Nevertheless, their efficiency can be limited when targeting proteins with an unusually long half-life, such as P-glycoprotein (P-gp), which is involved in the multidrug resistance phenomenon. P-gp is characterized by a long half-life, which may vary depending on the cell line and, for some of them, on serum deprivation or high cell density. In the present paper, involvement of an exponential cell growth phase in the improvement of siRNA efficiency has been suggested. The doxorubicin-selected human line MCF7-R was shown to be a more adapted model than NIH-MDR-G185 cells stably transfected with human mdr1. Nonspecific effects occurring at moderate (100 nM) siRNA concentration have been shown. Two efficient siRNAs led to a very satisfactory P-gp extinction (only 20% P-gp expression remaining) with siRNA concentration as low as 20 nM.

Significant co-expression of WT1 and MDR1 genes in acute myeloid leukemia patients at diagnosis.

A high expression of Wilms' tumor gene (WT1) in acute myeloid leukemia (AML) seems to correlate with a poor outcome and its increased levels can be predictive of an impending relapse. WT1 has been shown in vitro to interact with the promoter of the MDR1, a gene involved in the multidrug resistance phenomenon. The aim of this study was to measure, by real-time polymerase chain reaction, levels of WT1 and MDR1 expression, in order to find a possible association between these genes, in a series of 50 newly diagnosed AML cases. Twenty-five percent of patients carried very high (>75 degrees percentile) MDR1- and 23.3%WT1-mRNA levels. Interestingly, high levels of WT1 were significantly correlated with correspondent high levels of MDR1 gene. Nevertheless, the co-expression of these genes did not significantly influence the complete response rate to the induction therapy. Reported data confirm the existence of a co-expression of WT1 and MDR1 genes even in vivo; this may be relevant because one consequence could be the positive selection by chemotherapeutic regimens of cells with higher MDR1 levels already present before treatment. Thus, the association between these genes could suggest avoiding the use of drugs involved in the multidrug resistance (MDR) phenomenon in patients carrying high levels of WT1 at diagnosis.

Competitive Promoter Occupancy by Two Yeast Paralogous Transcription Factors Controlling the Multidrug Resistance Phenomenon

Highly flexible gene expression programs are required to allow cell growth in the presence of a wide variety of chemicals. We used genome-wide expression analyses coupled with chromatin immunoprecipitation experiments to study the regulatory relationships between two very similar yeast transcription factors involved in the control of the multidrug resistance phenomenon. Yrm1 (Yor172w) is a new zinc finger transcription factor, the overproduction of which decreases the level of transcription of the target genes of Yrr1, a zinc finger transcription factor controlling the expression of several membrane transporter-encoding genes. Surprisingly, the absence of YRR1 releases the transcriptional activity of Yrm1, which then up-regulates 23 genes, 14 of which are also direct target genes of Yrr1. Chromatin immunoprecipitation experiments confirmed that Yrm1 binds to the promoters of the up-regulated genes only in yeast strains from which YRR1 has been deleted. This sophisticated regulatory program can be associated with drug resistance phenotypes of the cell. The program-specific distribution of paired transcription factors throughout the genome may be a general mechanism by which similar transcription factors regulate overlapping gene expression programs in response to chemical stress.

Synthesis of a Library of 3-Oxopiperazinium and Perhydro-3-oxo-1,4-diazepinium Derivatives and Identification of Bioactive Compounds

The design and synthesis of a library of novel families of 3-oxopiperazinium and perhydro-3-oxo-1,4-diazepinium derivatives is reported. The library was composed of 44 3-oxopiperazinium derivatives (11 of these compounds had a spiranic skeleton) and 22 perhydro-3-oxo-1,4-diazepinium compounds. The synthetic procedure involved a 6-step sequence carried out in solution, along with the use of solid-phase linked scavengers and microwave activation for the rapid removal of the excess of amine reagents. A final cyclization step performed under mild conditions led to the charged heterocyclic moiety. Screening of this library in two biological assays identified active compounds that inhibit the activity of the vanilloid receptor TRPV1 and modulators of the multidrug resistance phenomenon. Thus, this synthetic sequence represents a facile and convenient entry to unprecedented libraries of this sort of tetraalkylammonium derivatives that may be of use for identification of novel scaffolds of diverse biological activity.

Drug delivery to resistant tumors: the potential of poly(alkyl cyanoacrylate) nanoparticles

Simultaneous cellular resistance to multiple lipophilic drugs represents a major problem in cancer chemotherapy. This drug resistance may appear clinically either as a lack of tumor size reduction or as the occurrence of clinical relapse after an initial positive response to antitumor treatment. The resistance mechanism can have different origins either directly linked to specific mechanisms developed by the tumor tissue or connected to the more general problem of distribution of a drug towards its targeted tissue. The purpose of this paper is to summarize the results of the use of poly(alkyl cyanoacrylate) nanoparticles to overcome multidrug resistance (MDR) phenomena at both the cellular and the non-cellular level.

Lipids as a target for drugs modulating multidrug resistance of cancer cells.

In this review we focus on the role of the membrane lipids in multidrug resistance and its modulation. Results of the research performed in recent years indicate the importance of lipid phase playing active role in many membrane processes. Along with the alterations of lipid membrane composition of cancer cells (with respect to the normal ones) the resulting changes of the biophysical membrane properties are discussed. Next we describe the general features of multidrug resistance phenomenon paying a special attention to the role of lipids and alterations of lipid membrane composition in MDR cells. Taking into account the phase separation properties of sphingolipids the importance of membrane heterogeneity (presence of caveole and lipid rafts) is emphasised. On the basis of vacuum cleaner hypothesis of drug transport proteins action we discuss the importance of lipid bilayer as medium in which diffusion of drugs takes place. Considering the membrane fluidity and its influence on the integral proteins activity, we underline the role of balance between the passive cellular influx and active efflux of the drug molecules. Finally the effects exerted on membranes by different kinds of multidrug resistance modulators (chemosensitizers) are described. In this part we discuss the influence of verapamil, phenothiazine derivatives, tamoxifen and chosen flavonoids on the biophysical properties of membrane lipids. Some further consequences of the alteration of membrane state are also considered.

Multidrug resistance genes and p-glycoprotein in the testis of the rat, mouse, Guinea pig, and human.

Study of the multidrug resistance phenomenon in tumor cell lines has led to the discovery of the product of the multidrug resistance (MDR) type 1 genes, the plasma membrane P-glycoprotein (P-gp) that functions as an energy-dependent pump for the efflux of diverse anticancer drugs. P-gp was also recently identified in normal epithelial cells with secretory/excretory functions and in the endothelial cells of the capillary blood vessels in the brain and the testis. These endothelial cells are key elements of the blood-brain and blood-testis barriers, respectively. The aim of this study, in the rat, mouse, guinea pig, and human, was to determine whether testicular cells other than the capillary endothelial cells could express MDR type I genes. Immunohistochemistry on testicular sections revealed that P-gp is present in interstitial cells in the mouse, rat, and human testes, in early and late spermatids in guinea pig testis, and in late spermatids in the rat, mouse, and human. Reverse transcription-polymerase chain reaction analysis on isolated mouse, rat, and human cells showed that all somatic testicular cells (Leydig cells, macrophages, peritubular cells, and Sertoli cells) and the cytoplasmic lobes from rat late spermatids expressed MDR type I mRNAs, whereas spermatogonia, pachytene spermatocytes, and early spermatids did not. An ontogenesis study in the mouse reveals that type I MDR gene expression begins at 13.5 days postcoitum at the time when the seminiferous cords and the blood vessels appear and are maintained thereafter. Finally, two functional tests on isolated rat cells, the doxorubicin and rhodamine uptake assays, demonstrated that rat testicular macrophages, Leydig cells, peritubular cells, and Sertoli cells displayed a multidrug-resistance activity, whereas spermatogonia, pachytene spermatocytes, and early spermatids did not. Western blot experiments have revealed that a P-gp of 175 kDa is present in the human testis as well as in the rat Leydig cells, testicular macrophages, peritubular cells, and Sertoli cells, but is absent in spermatogonia, spermatocytes, and early spermatids. We conclude that P-gp is involved in the self-protection of the somatic cells and is most probably one of the molecules that confers its functionality to the blood-testis barrier. The absence of expression of MDR type I genes in mitotic and meiotic germ cells probably explains their particular vulnerability to various anticancer drugs. In contrast, expression of the P-gp in the haploid cells most likely reflects the ability of spermatozoa to assume their own antidrug defense.

CPT-11-induced cell death in leukemic cells is not affected by the MDR phenotype

CPT-11 is a topoisomerase I (Topo I) inhibitor which was initially described as active in multi-drug resistance (MDR) tumors. The MDR phenomenon is characterized by the overexpression of efflux pumps which are able to extrude a range of drugs non-related chemical or functionally. In this work, we treated leukemic cells with CPT-11 300 μM at 24 h and compared its cytotoxicity with the activity of efflux pumps and with cell cycle phase. Our findings show that CPT-11 has a potent anti-tumor activity in leukemic cells regardless MDR phenotype and the cell cycle phase, suggesting new avenues to be explored in leukemia treatment.

Monoclonal antibodies as a tool for structure-function studies of the MDR1-P-glycoprotein.

P-glycoprotein is considered one of the most important member of the rapidly growing superfamily of integral proteins known as the ATP-binding cassette (ABC) which in human also include several other multidrug resistance membrane proteins (i.e., MRP), the product of the cystic fibrosis gene, the TAP-1/TAP2 peptide transporters encoded by the major histocompatibility complex genes and the gene encoding for breast cancer resistance protein (BCRP) also known as MXR1 (mitoxantrone resistance protein). Many monoclonal antibodies (MAbs) reacting with distinct P-glycoprotein domains have been isolated and used to study the molecular organization and cellular functions of this ABC protein. MAbs have been used for multidrug resistance (mdr) gene cloning, delineation of the secondary and tertiary structure of P-glycoprotein and molecular analysis of the mechanisms involved in substrate recognition and transport. The immunodetection of the distinct products of the mdr gene family in normal and malignant cells and tissues has greatly contributed to the understanding of the physiological role of P-glycoprotein and its possible involvement in the refractory of tumors to chemotherapy. The present article deals with the immunological methods used for the structure-function studies of the P-glycoprotein. After introducing the basic structural features of this ABC transporter, the antibody based-approach is discussed with aiming to furnishing methodological perspectives for further investigations of the physiological role of P-glycoprotein and the multidrug resistance phenomenon.

Modulation of the atypical multidrug-resistant phenotype by a hammerhead ribozyme directed against the ABC transporter BCRP/MXR/ABCG2

The phenomenon of multidrug resistance (MDR) in human cancers is one of the major causes of failure of chemotherapy. A recently identified new member of the superfamily of ATP-binding cassette transporters, breast cancer resistance protein (BCRP), was demonstrated to confer an atypical multidrug-resistant phenotype to tumor cells. To overcome the BCRP-mediated drug resistance, a specific anti-BCRP hammerhead ribozyme was introduced into the human gastric carcinoma cell line, EPG85-257RNOV, exhibiting an atypical MDR phenotype. By this approach, the expression levels of the targeted BCRP-encoding mRNA and the BCRP transport protein were decreased to the low constitutive expression level that was observed in highly drug-sensitive parental gastric carcinoma cells. In addition, in the anti-BCRP ribozyme-treated cells, the cellular drug accumulation was dramatically increased to the level measured in drug-sensitive cells. These effects were accompanied by an extensive reversal of the drug-resistant phenotype of more than 80%. Because additional mechanisms contribute to the multimodal-mediated MDR phenotype exhibited by this gastric carcinoma cell line, the data suggest that the BCRP-mediated contingent to the drug resistance was overcome nearly completely. Moreover, the data indicate that ribozyme-based gene therapy may be clinically applicable in preventing and reversing BCRP-mediated atypical MDR.

The multidrug resistance transporters of the major facilitator superfamily, 6 years after disclosure of Saccharomyces cerevisiae genome sequence

The emergence of multidrug resistance (MDR) plays a crucial role in the failure of treatments of tumors and infectious diseases and in the control of plant pathogens, weeds and food-poisoning and food-spoilage microorganisms. Among the mechanisms underlying the MDR phenomenon in various organisms is the action of transmembrane transport proteins that presumably catalyse the active expulsion of structurally and functionally unrelated cytotoxic compounds out of the cell or their intracellular partitioning. On the basis of the complete genome sequence of Saccharomyces cerevisiae, numerous established and putative multidrug transporters were identified in this non-pathogenic, easy to manipulate eukaryotic model system. In yeast, the putative drug:H +-antiporters belong to the major facilitator superfamily; they comprise at least 23 proteins that have largely escaped characterisation by classical approaches. Other MDR determinants are membrane transporters belonging to the ATP binding cassette (ABC) superfamily, that utilize the energy of ATP hydrolysis for activity, and factors for transcriptional regulation of all the MDR transporters. This work reviews the current status of knowledge on the poorly characterized H +-antiporters, with 12 and 14 predicted spans, DHA12 and DHA14, drug efflux families. Consideration is given to the inventory and phylogenetic characterization, role as MDR determinants, regulation of gene expression, subcellular localisation and activity as solute transporters. Most of the present knowledge on these putative drug:H +-antiporters was driven by disclosure of S. cerevisiae genome sequence, in April 1996, being a paradigm of post-genomic research.

Monitoring daunorubicin-induced alterations in protein expression in pancreas carcinoma cells by two-dimensional gel electrophoresis.

Tumors of the pancreas are characterized by a high intrinsic potency to develop chemoresistance towards cytotoxic drugs, which is the main cause of ineffective treatment. The phenomenon of multidrug resistance is known to be a multifactorial event in which several mechanisms act simultaneously. We investigated the response of pancreas tumor cells after exposure to the anthracycline daunorubicin (DRC), a well-known antitumor agent in chemotherapy, by two-dimensional gel electrophoresis (2-DE). DRC is known to cause DNA damage and to affect tumor cell growth. Importantly, we aimed at investigating alterations in the protein expression pattern after first contact of the tumor cells with DRC, thus simulating a situation close to clinical chemotherapy and elucidating cell survival strategies following initial drug exposure. A concentration dependent up-regulation of a variety of proteins was observed, indicating that cell response to DRC involves multiple signaling events. Since the p53 tumor suppressor is essentially involved in the regulation of cell growth and controlled cell death (apoptosis) after cellular stress (like DNA damage), we investigated the role of p53 in DRC-resistant and -sensitive pancreas carcinoma cells by measuring p53 transcriptional transactivation activities. No differences in p53 activities were observed in response to DRC treatment in both pancreas cell lines, whereas mamma carcinoma cells (MCF-7), possessing wild-type p53, demonstrated the expected increase in p53 transcriptional transactivation activity. Hence, the tested pancreas carcinoma cells harbor a mutant, nonfunctional p53. We additionally analyzed the steady state protein levels of the cyclin dependent kinase inhibitor p21(CIP1), which is known to be involved in cell cycle control. Interestingly, p21(CIP1 )was induced by DRC in sensitive cells in a concentration dependent manner and was highest in resistant cells. In conclusion, our results suggest that the induction of proteins by DRC in pancreas carcinoma cells, as observed by 2-DE, occurs independently from p53 signaling events, but is probably associated with increased levels of p21(CIP1).

Anthrapyridones, a novel group of antitumour non-cross resistant anthraquinone analogues. Synthesis and molecular basis of the cytotoxic activity towards K562/DOX cells.

1. Multidrug resistance (MDR) to antitumour agents, structurally dissimilar and having different intracellular targets, is the major problem in cancer therapy. MDR phenomenon is associated with the presence of membrane proteins which belong to the ATP-binding cassette family transporters responsible for the active drug efflux leading to the decreased intracellular accumulation. 2. The search of new compounds able to overcome MDR is of prime importance. 3. Recently we have synthesized a new family of anthrapyridone compounds. The series contained derivatives modified with appropriate hydrophobic or hydrophylic substituents at the side chain. 4. The interaction of these derivatives with erythroleukemia K562 sensitive and K562/DOX resistant (overexpressing P-glycoprotein) cell lines has been examined. The study was performed using a spectrofluorometric method which allows to continuously follow the uptake and efflux of fluorescent molecules by living cells. 5. It was demonstrated that the increase in the lipophilicity of anthrapyridones favoured the very fast cellular uptake exceeding the rate of P-gp dependent efflux out of the cell. For these derivatives, very high accumulation (the same for sensitive and resistant cells) was observed and the in vitro biological data confirmed that these compounds exhibited comparable cytotoxic activity towards sensitive and P-gp resistant cell line. In contrast, anthrapyridones modified with hydrophylic substituents exhibited relatively low kinetics of cellular uptake. 6. For these derivatives decreased accumulation in resistant cells was observed and the in vitro biological data demonstrated that they were much less active against P-gp resistant cells in comparison to sensitive cells. 7. We also studied, using confocal microscopy, the intracellular distribution of anthrapyridones in NIH-3T3 cells. Our data showed that these compounds were strongly accumulated in the nucleus and lysosomes.

Bromocriptine reverses P-glycoprotein-mediated multidrug resistance in tumor cells.

One of the most important causes of anticancer treatment failure is the development of multidrug resistance (MDR). The main characteristics of tumor cells displaying the MDR phenomena are cross‐resistance to structurally unrelated cytotoxic drugs having different mechanisms of action and the overexpression of the MDR1 gene, which encodes a transmembrane glycoprotein named P‐ glycoprotein (P‐gp). This study evaluated whether bromocriptine, a D2 dopaminergic receptor agonist, influenced anticancer drug cytotoxicity and P‐gp activity in a P‐gp‐expressing cell line compared to a non‐expressing subline. The Ki values for P‐gp of cyclosporine and verapamil were 1.09 and 540 μM, respectively, and that of bromocriptine was 6.52 μM in a calcein‐AM efflux assay using porcine kidney epithelial LLC‐PK1 and L‐MDR1 cells, overexpressing human P‐gp. Bromocriptine at 10 μM reduced the IC50 of doxorubicin (DXR) in K562‐DXR from 9000 to 270 ng/ml and that of vincristine (VCR) in K562‐VCR from 700 to 0.30 ng/ml, whereas the IC50 values of DXR and VCR in the K562 subline were only marginally affected by these drugs. Bromocriptine restored the anticancer effect of DXR, VCR, vinblastine, vinorelbine and etoposide on MDR‐tumor cells overexpressing P‐gp. These observations suggest that bromocriptine has the potential to reverse tumor MDR involving the efflux protein P‐gp in the clinical situation.

Clinical impact of multidrug resistance in acute leukemia

The phenomenon of multi-drug resistance is well-described in the experimental literature and can be produced in in vitro cell cultures of leukemic cells. The classic approach to the induction of drug resistance is by the growth of leukemic cells in increasing concentrations of the agent to which resistance develops. The most likely species of drugs to induce resistance is the group known as natural products, those derived from plants and bacteria. Important to the treatment of leukemia is the development of resistance to anthracyclines and to the vinca alkaloids and epipodophyllotoxins, all natural products. P-glycoprotein has been identified as the most ubiquitous mediator of resistance. This protein forms a transmembrane pump which reduces the concentration of natural products within the cell by increasing drug efflux, an active, energy-requiring pumping mechanism [1–3]. P-glycoprotein, Pgp, is also found in the membrane of normal tissues with major excretory functions, such as bowel and kidney [4]. However, its presence in the membrane of leukemic blasts is key to the extrusion of natural products such as the anthracyclines, and this has been studied extensively in in vitro systems [1–5]. In addition, numerous clinical correlative trials have been reported in which expression and function of P-glycoprotein have been evaluated as a possible prognostic factor in terms of remission and survival [6–13]. There appears to be considerable consensus that the demonstration in de novo leukemia of increased Pgp function confers a poorer prognosis in terms of achieving remission, but the degree of independence of this expression as a prognostic factor continues to be debated [6,8–11,14]. This has also led to experimental studies designed to inhibit or reverse the function of the efflux pump. Several agents have been identified which in the laboratory have the capability of inhibiting the effectiveness of the efflux pump and thus promoting increased anthracycline concentration within the cells [15,16]. This has led to clinical trials of Pgp modulating agents, such as cyclosporine and PSC 833, administered with either daunorubicin or mitoxantrone plus other induction agents in poor risk patients in an attempt to overcome drug resistance. Despite some evidence of improved retention of the anthracycline or anthraquinone within the leukemic cells by use of this combination approach with reversal agents [17,18], the clinical outcome has not been improved [18–21]. During efforts to better understand mechanisms of drug resistance, other proteins which mediate resistance have been identified and are being evaluated for their impact on in vitro and in vivo resistance. Three of these being studied in acute leukemia are major vault transporter protein/lung-resistance protein (LRP), MDR-related protein (MRP) and breast cancer resistance protein (BCRP). Of importance to leukemia treatment, all of these proteins reduce the retention of anthracyclines in malignant cells. MRP mediates resistance to numerous natural products [4]. It has also been shown to limit anthracycline accumulation and produce resistance in the absence of Pgp [22]. LRP also appears to evolve in cells which lose their expression of P-glycoprotein, but which maintain their resistance phenotype [4]. BCRP was initially demonstrated in the membrane of cells from solid tumors and mediates resistance to natural products such as mitoxantrone, topotecan and doxorubicin [23,24]. However, Ross et al. have also demonstrated the presence of this protein in the membrane of up to 30% of leukemic blasts [22,25]. The expression of this protein was associated with reduced accumulation of daunorubicin in leukemic blasts [26]. * Corresponding author. Tel.: +1-718-920-1100; fax: +1-718-9201123. E-mail address: jpd4401@aol.com (J.P. Dutcher).

Drug resistance in yeasts — an emerging scenario

In view of the increasing threat posed by fungal infections in immunocompromised patients and due to the non-availability of effective treatments, it has become imperative to find novel antifungals and vigorously search for new drug targets. Fungal pathogens acquire resistance to drugs (antifungals), a well-established phenomenon termed multidrug resistance (MDR), which hampers effective treatment strategies. The MDR phenomenon is spread throughout the evolutionary scale. Accordingly, a host of responsible genes have been identified in the genetically tractable budding yeast Saccharomyces cerevisiae, as well as in a pathogenic yeast Candida albicans. Studies so far suggest that, while antifungal resistance is the culmination of multiple factors, there may be a unifying mechanism of drug resistance in these pathogens. ABC (ATP binding cassette) and MFS (major facilitator superfamily) drug transporters belonging to two different superfamilies, are the most prominent contributors to MDR in yeasts. Considering the abundance of the drug transporters and their wider specificity, it is believed that these drug transporters may not exclusively export drugs in fungi. It has become apparent that the drug transporters of the ABC superfamily of S. cerevisiae and C. albicans are multifunctional proteins, which mediate important physiological functions. This review summarizes current research on the molecular mechanisms underlying drug resistance, the emerging regulatory circuits of MDR genes, and the physiological relevance of drug transporters.

Microvillar cell surface as a natural defense system against xenobiotics: a new interpretation of multidrug resistance

The phenomenon of multidrug resistance (MDR) is reinterpreted on the basis of the recently proposed concept of microvillar signaling. According to this notion, substrate and ion fluxes across the surface of differentiated cells occur via transporters and ion channels that reside in membrane domains at the tips of microvilli (MV). The flux rates are regulated by the actin-based cytoskeletal core structure of MV, acting as a diffusion barrier between the microvillar tip compartment and the cytoplasm. The expression of this diffusion barrier system is a novel aspect of cell differentiation and represents a functional component of the natural defense system of epithelial cells against environmental hazardous ions and lipophilic compounds. Because of the specific organization of epithelial Ca(2+) signaling and the secretion, lipophilic compounds associated with the plasma membrane are transferred from the basal to the apical cell surface by a lipid flow mechanism. Drug release from the apical pole occurs by either direct secretion from the cell surface or metabolization by the microvillar cytochrome P-450 system and efflux of the metabolites and conjugation products through the large multifunctional anion channels localized in apical MV. The natural microvillar defense system also provides a mechanistic basis of acquired MDR in tumor cells. The microvillar surface organization is lost in rapidly growing cells such as tumor or embryonic cells but is restored during exposure of tumor cells to cytotoxins by induction of a prolonged G(0)/G(1) resting phase.