The drug resistance proteins, multidrug resistance-associated protein and P-glycoprotein, do not confer resistance to Fas-induced cell death

BACKGROUND: Multidrug resistance (MDR) is mediated by the drug resistance proteins, the multidrug resistance-associated protein (MRP) and P-glycoprotein, both of which confer resistance by the active efflux of chemotherapeutic drugs from the cell. Reduced Fas (CD95/APO-1) expression and resistance to Fas-mediated apoptosis have also been correlated with P-glycoprotein-mediated MDR. METHODS: We investigated cell surface Fas expression (using anti-Fas monoclonal antibody DX2.1) in a series of MRP-expressing drug-resistant leukemia sublines, and P-glycoprotein-expressing leukemia sublines, and their susceptibility to apoptosis induced by anti-Fas treatment (CH-11 monoclonal antibody). Caspase-3 activation was detected by Western blot and apoptosis was determined by flow cytometry with 7-aminoactinomycin D (7-AAD) staining of cells. RESULTS: Fas expression was not reduced in either the MRP- or P-glycoprotein-expressing drug-resistant cell lines, although expression was reduced by 15% in one low-level drug-resistant subline. Expression of MRP or P-glycoprotein did not confer resistance to caspase-3 activation or to anti-Fas-induced cell death. CONCLUSIONS: MDR mediated by the drug transport proteins MRP and P-glycoprotein does not correlate with resistance to Fas-mediated cell death or resistance to caspase-3 activation.     

Protein kinases and multidrug resistance

The role of protein kinases in the multidrug resistance phenotype of cancer cell lines is discussed with an emphasis on protein kinase C and protein kinase A. Evidence that P-glycoprotein is phosphorylated by these kinases is summarised and the relationship between P-glycoprotein phosphorylation and the multidrug-resistant phenotype discussed. Results showing that protein kinase C, particularly the alpha subspecies, is overexpressed in many MDR cell lines are described: this common but by no means universal finding seems to be drug- and cell line-dependent and in only in a few cases is there a direct correlation between protein kinase C activity and multidrug resistance. From co-immunoprecipitation results it is suggested that P-glycoprotein is a specific protein kinase C receptor, as well as being a substrate. Revertant experiments provide conflicting results as to a direct relationship between expression of P-glycoprotein and protein kinase C. Evidence that protein kinase A influences P-glycoprotein expression at the gene level is well documented and the mechanisms by which this occurs are becoming clarified. Results on the relationship between protein kinase C and multidrug resistance using many inhibitors and phorbol esters are difficult to interpret because such compounds bind to P-glycoprotein. In spite of huge effort, a direct involvement of protein kinase C in regulating multidrug resistance has not yet been firmly established. However, evidence that PKC regulates a Pgp-independent mechanism of drug resistance is accumulating. This revised version was published online in August 2006 with corrections to the Cover Date.     

Expression of hamster P-glycoprotein and multidrug resistance in DNA-mediated transformants of mouse LTA cells.

The overexpression of a plasma membrane glycoprotein, P-glycoprotein, is strongly correlated with the expression of multidrug resistance. This phenotype (frequently observed in cell lines selected for resistance to a single drug) is characterized by cross resistance to many drugs, some of which are used in cancer chemotherapy. In the present study we showed that DNA-mediated transformants of mouse LTA cells with DNA from multidrug-resistant hamster cells acquired the multidrug resistance phenotype, that the transformants contained hamster P-glycoprotein DNA sequences, that these sequences were amplified whereas the recipient mouse P-glycoprotein sequences remained at wild-type levels, and that the overexpressed P-glycoprotein in these cells was of hamster origin. Furthermore, we showed that the hamster P-glycoprotein sequences were transfected independently of a group of genes that were originally coamplified and linked within a 1-megabase-pair region in the donor hamster genome. These data indicate that the high expression of P-glycoprotein is the only alteration required to mediate multidrug resistance.     

Epigenetic loss of CDH1 correlates with multidrug resistance in human hepatocellular carcinoma cells

Promoter CpG hypermethylation of tumor suppressor genes is an essential step in cancer progression but little is known about its effect on cancer multidrug resistance. In this study, we showed that CDH1 promoter was hypermethylated in drug resistance of a doxorubicin-induced multidrug resistant hepatocellular carcinoma cell line R-HepG2. Transfection of CDH1 cDNA into R-HepG2 cells led to increased amount of doxorubicin uptake, decreased cell viability, decreased P-glycoprotein expression and increased apoptotic population of cells exposed to doxorubicin. Proto-oncogene tyrosine-protein kinase FYN was over-expressed in R-HepG2 cells which displayed a negative correlation with the expression of CDH1. FYN was knocked down in R-HepG2 cells, leading to less drug resistance by increased cell viability, increased doxorubicin uptake and attenuated P-glycoprotein expression. Our findings identified epigenetic silencing of CDH1 in cancer cells might be a new molecular event of multidrug resistance.     

Identification of a membrane glycoprotein overexpressed in murine lymphoma sublines resistant to cis-diamminedichloroplatinum(II)

A series of murine thymic lymphoma cell sublines was selected in vitro for resistance to cis-diamminedichloroplatinum(II) (CDDP). The level of CDDP resistance correlated with reduced drug accumulation in these cells. A rabbit antiserum was raised against the plasma membrane of a CDDP-resistant subline and used in Western blot analyses. Increased expression of a surface antigen of approximately 200 kDa was observed and found to correlate with the degree of resistance. Further biochemical and immunological studies demonstrated that this is a plasma membrane glycoprotein. However, it is different from the multidrug resistance-associated P-glycoprotein with a molecular weight of about 170,000. We have called this unique CDDP resistance-associated membrane protein CPR-200.     

Multidrug resistance (MDR) genes in haematological malignancies

The emergence of drug resistant cells is one of the main obstacles for successful chemotherapeutic treatment of haematological malignancies. Most patients initially respond to chemotherapy at the time of first clinical admission, but often relapse and become refractory to further treatment not only to the drugs used in the first treatment but also to a variety of other drugs. Laboratory investigations have now provided a cellular basis for this clinical observation of multidrug resistance (MDR). Expression of a glycoprotein (referred to as P-glycoprotein) in the membrane of cells made resistantin vitro to naturally occurring anticancer agents like anthracyclines, Vinca alkaloids and epipodophyllotoxins, has been shown to be responsible for the so-called classical MDR phenotype. P-glycoprotein functions as an ATP-dependent, unidirectional drug efflux pump with a broad substrate specificity, that effectively maintains the intracellular cytotoxic drug concentrations under a non-cytotoxic threshold value. Extensive clinical studies have shown that P-glycoprotein is expressed on virtually all types of haematological malignancies, including acute and chronic leukaemias, multiple myelomas and malignant lymphomas. Since in model systems for P-glycoprotein-mediated MDR, drug resistance may be circumvented by the addition of non-cytotoxic agents that can inhibit the outward drug pump, clinical trials have been initiated to determine if such an approach will be feasible in a clinical situation. Preliminary results suggest that some haematological malignancies, among which are acute myelocytic leukaemia, multiple myeloma and non-Hodgkin's lymphoma, might benefit from the simultaneous administration of cytotoxic drugs and P-glycoprotein inhibitors. However, randomised clinical trials are needed to evaluate the use of such resistance modifiers in the clinic.Abbreviations ALL acute lymphocytic leukaemia - AML acute myelocytic leukaemia - BM bone marrow - CAT chloramphenicol acetyltransferase - CLL chronic lymphocytic leukaemia - CML chronic myelocytic leukaemia - CR complete remission - HCL hairy cell leukaemia - MDR multidrug resistance - MDS myelodysplastic syndrome - MM multiple myeloma - MoAb monoclonal antibody - NHL non-Hodgkin's lymphoma - PB peripheral blood - PCR polymerase chain reaction - PLL prolymphocytic leukaemia - RMA resistance modifying agent - VAD vincristine, doxorubicin, dexamethasone     

P-glycoproteins: mediators of multidrug resistance

Multidrug resistance represents a major obstacle to successful chemotherapy of metastatic disease. Elevated levels in cancer cells if the product of the multidrug resistance gene, P-glycoprotein or the multidrug transporter, have been associated with the development of simultaneous resistance to a great variety of amphiphilic cytotoxic drugs. P-glycoproteins is an integral plasma membrane protein which contains 12 putative transmembrane regions and two ATP binding sites. It confers multidrug resistance by functioning as an energy-dependent drug efflux pump. Here we describe recent studies on the biosynthesis, structure, function, and mechanism of action of P-glycoprotein which have provided insights into the complexity of this multifunctional transport system and revealed an additional chloride channel activity. The physiological role of P-glycoprotein, however, still remains to be elucidated.     

Effects of phosphorylation of P-glycoprotein on multidrug resistance

Cells expressing elevated levels of the membrane phosphoprotein P-glycoprotein exhibit a multidrug resistance phenotype. Studies involving protein kinase activators and inhibitors have implied that covalent modification of P-glycoprotein by phosphorylation may modulate its biological activity as a multidrug transporter. Most of these reagents, however, have additional mechanisms of action and may alter drug accumulation within multidrug resistant cells independent of, or in addition to their effects on the state of phosphorylation of P-glycoprotein. The protein kinase(s) responsible for P-glycoprotein phosphorylation has(ve) not been unambiguously identified, although several possible candidates have been suggested. Recent biochemical analyses demonstrate that the major sites of phosphorylation are clustered within the linker region that connects the two homologous halves of P-glycoprotein. Mutational analyses have been initiated to confirm this finding. Preliminary data obtained from phosphorylation- and dephosphorylation-defective mutants suggest that phosphorylation of P-glycoprotein is not essential to confer multidrug resistance.     

Expression of a drug resistance gene in human neuroblastoma cell lines: modulation by retinoic acid-induced differentiation.

Expression of a multidrug resistance gene (mdr1) and its protein product, P-glycoprotein (Pgp), has been correlated with the onset of multidrug resistance in vitro in human cell lines selected for resistance to chemotherapeutic agents derived from natural products. Expression of this gene has also been observed in normal tissues and human tumors, including neuroblastoma. We therefore examined total RNA prepared from human neuroblastoma cell lines before and after differentiation with retinoic acid or sodium butyrate. An increase in the level of mdr1 mRNA was observed after retinoic acid treatment of four neuroblastoma cell lines, including the SK-N-SH cell line. Western blot (immunoblot) analysis demonstrated concomitant increases in Pgp. However, studies of 3H-vinblastine uptake failed to show a concomitant Pgp-mediated decrease in cytotoxic drug accumulation. To provide evidence that Pgp was localized on the cell surface, an immunotoxin conjugate directed against Pgp was added to cells before and after treatment with retinoic acid. Incorporation of [3H]leucine was decreased by the immunotoxin in the retinoic acid-treated cells compared with the undifferentiated cells. These results demonstrate that whereas expression of the mdr1 gene can be modulated by differentiating agents, increased levels of expression are not necessarily associated with increased cytotoxic drug accumulation.     

bfr1+, a novel gene of Schizosaccharomyces pombe which confers brefeldin A resistance, is structurally related to the ATP-binding cassette superfamily.

We have isolated a Schizosaccharomyces pombe gene, bfr1+, which on a multicopy plasmid vector, pDB248', confers resistance to brefeldin A (BFA), an inhibitor of intracellular protein transport. This gene encodes a novel protein of 1,531 amino acids with an intramolecular duplicated structure, each half containing a single ATP-binding consensus sequence and a set of six transmembrane sequences. This structural characteristic of bfr1+ protein resembles that of mammalian P-glycoprotein, which, by exporting a variety of anticancer drugs, has been shown to be responsible for multidrug resistance in tumor cells. Consistent with this is that S. pombe cells harboring bfr1+ on pDB248' are resistant to actinomycin D, cerulenin, and cytochalasin B, as well as to BFA. The relative positions of the ATP-binding sequences and the clusters of transmembrane sequences within the bfr1+ protein are, however, transposed in comparison with those in P-glycoprotein; the bfr1+ protein has N-terminal ATP-binding sequence followed by transmembrane segments in each half of the molecule. The bfr1+ protein exhibited significant homology in primary and secondary structures with two recently identified multidrug resistance gene products of Saccharomyces cerevisiae, Snq2 and Sts1/Pdr5/Ydr1. The bfr1+ gene is not essential for cell growth or mating, but a delta bfr1 mutant exhibited hypersensitivity to BFA. We propose that the bfr1+ protein is another member of the ATP-binding cassette superfamily and serves as an efflux pump of various antibiotics.     

Purification of the 170- to 180-kilodalton membrane glycoprotein associated with multidrug resistance. 170- to 180-kilodalton membrane glycoprotein is an ATPase

170-180-kDa membrane glycoprotein (P-glycoprotein) associated with multidrug resistance is involved in drug transport mechanisms across the plasma membrane of resistant cells. From sequence analysis of cDNAs of the P-glycoprotein gene, it is postulated that the active drug-efflux pump function may be attributable to the protein. However, purification of the P-glycoprotein while preserving its enzymatic activity has not been reported. In this study, we have purified the P-glycoprotein from the human myelogenous leukemia K562 cell line resistant to adriamycin (K562/ADM) by means of one-step immunoaffinity chromatography using a monoclonal antibody against P-glycoprotein. The procedure was simple and efficiently yielded an electrophoretically homogeneous P-glycoprotein sample. By solubilization with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, the purified P-glycoprotein was found to have ATPase activity. This ATP hydrolysis may be coupled with the active efflux of anticancer drugs across the plasma membrane of multidrug-resistant cells.     

Altered plasma membrane ultrastructure in multidrug-resistant cells

Multidrug resistance is mediated by P-glycoprotein, an integral plasma membrane component which is thought to function as a drug export pump. This model can explain drug resistance, but fails to account for the broader pleiotropy of the multidrug resistance phenotype. We report here a freeze-fracture study revealing increases in the densities of protoplasmic face intramembrane particles in multidrug-resistant Chinese hamster ovary (CHO) and human leukemic cells. The intramembrane particle density in a CHO cell revertant which had lost the characteristics of the multidrug resistance phenotype was indistinguishable from that of the drug-sensitive parental cell line. This demonstration of a global multidrug resistance-linked change in plasma membrane architecture may have significant implications for understanding the variety of concurrent membrane-related changes which are not easily explained by the current model for multidrug resistance.     

P-糖蛋白在多药耐药的K562细胞转运苯妥英纳与卡马西平中的作用

研究证实,多药转运体与难治性癫痫耐药机制密切相关,P-糖蛋白在其中起重要作用．主要研究P-糖蛋白拮抗剂维拉帕米对P-糖蛋白过表达的K562细胞耐药性及细胞内苯妥英纳与卡马西平浓度的影响．首先建立了P-糖蛋白高表达的K562/Dox(阿霉素诱导)耐药细胞株,比较耐药细胞株和P-糖蛋白表达阴性的K562细胞株对苯妥英纳和卡马西平的耐药性,并观察给予维拉帕米后,耐药细胞内抗癫痫药物的浓度变化．结果发现,苯妥英纳和卡马西平对K562/Dox细胞株的半数抑制浓度(IC₅₀)明显高于K562细胞株,加入维拉帕米后,苯妥英纳和卡马西平对K562/Dox 细胞的IC₅₀明显下降,逆转倍数分别为2.5和1.5．进一步研究发现,K562/Dox细胞内苯妥英纳和卡马西平的浓度均显著少于其药敏K562细胞,仅分别为正常K562细胞的23.6%和32.2%．当加入维拉帕米后,K562/Dox细胞内抗癫痫药物浓度明显升高(P < 0.05)．由此证明,高表达的P-糖蛋白参与了细胞的药物转运,在难治性癫痫的耐药机制中扮演重要角色．     

Evaluating limited specificity of drug pumps reduced relative resistance in human MDR phenotypes.

In the parallel paper, we developed a property to characterize drug efflux pumps, i.e. the reduced relative resistance (RRR). Using this RRR, we here investigate whether the observed diversity in human multidrug resistance (MDR) phenotypes might be due to variable levels of P-glycoprotein encoded by MDR1. We analyzed resistance phenotypes of various human cell lines in which either one, or both, classical human multidrug resistance genes, MDR1 and MDR3, are overexpressed. In addition, RRR values were calculated for MDR phenotypes presented in the literature. The results suggest that more than a single mechanism is required to account for the observed phenotypic diversity of classical multidrug resistance. This diversity is only partly due to differences in plasma membrane permeabilities between cell line families. It is discussed whether the alternative MDR phenotypes might be MDR1 phenotypes modified by other factors that do not themselves cause MDR. The method we here apply may also be useful for other nonspecific enzymes or pumps.     

Heat shock and arsenite increase expression of the multidrug resistance (MDR1) gene in human renal carcinoma cells

The multidrug transporter, initially identified as a multidrug efflux pump responsible for resistance of cultured cells to natural product cytotoxic drugs, is normally expressed on the apical membranes of excretory epithelial cells in the liver, kidney, and intestine. This localization suggests that the multidrug transporter may have a normal physiological role in transporting cytotoxic compounds or metabolites. In the liver, hepatectomy or treatment with chemical carcinogens increases expression of the MDR1 gene which encodes the multidrug transporter. To evaluate conditions which increase MDR1 gene expression, we have investigated the induction of the MDR1 gene by physical and chemical environmental insults in the renal adenocarcinoma cell line HTB-46. There are two strong heat shock consensus elements in the major MDR1 gene promoter. Exposure of HTB-46 cells to heat shock, sodium arsenite, or cadmium chloride led to a 7- to 8-fold increase in MDR1 mRNA levels. MDR1 RNA levels did not change following glucose starvation or treatment with 2-deoxyglucose and the calcium ionophore A23187, conditions which are known to activate the expression of another family of stress proteins, the glucose-regulated proteins. The levels of the multidrug transporter, P-glycoprotein, as measured by immunoprecipitation, were also increased after heat shock and sodium arsenite treatment. This increase in the level of the multidrug transporter in HTB-46 cells correlated with a transient increase in resistance to vinblastine following heat shock and arsenite treatment. These results suggest that the MDR1 gene is regulatable by environmental stress.     

Novel approaches to reversing anti-cancer drug resistance using gene-specific therapeutics.

One of the underlying mechanisms of multidrug resistance (MDR) is cellular overproduction of P-glycoprotein (P-gp), which acts as an efflux pump for various anti-cancer drugs. P-gp is encoded by a group of related genes termed MDR; only MDR1 is known to confer the drug resistance, and its overexpression in cancer cells has been a therapeutic target to circumvent the resistance. To overcome P-gp-mediated drug resistance, we have developed six anti-MDR1 hammerhead ribozymes and delivered them to P-gp-overproducing human leukemia cell line by a retroviral vector containing RNA polymerase III promoter. These ribozyme-transduced cells became vincristine-sensitive, concomitant with the decreases in MDR1 expression, P-gp amount and efflux pump function. Among the ribozymes tested, the anti-MDR1 ribozyme against the translation-initiation site exhibited the highest efficacy. The retrovirus-mediated transfer of this most potent anti-MDR1 ribozyme into a human lymphoma cell line, which was made resistant by infection of pHaMDR1/A retroviral vector and thus possessed a low degree of MDR due to P-gp expression relevant to clinical MDR, resulted in a complete reversal of MDR phenotype. In addition to retrovirus-mediated transfer of ribozymes, we evaluated the efficacy of cationic liposome-mediated transfer of ribozyme. Treatment of a P-gp-producing human breast cancer cell line with the liposome-ribozyme complex resulted in reversal of resistance, concomitant with the decreases in both MDR1 expression and P-gp amount. Confocal microscopic imaging of the cells after treatment with liposome/FITC-dextran showed cytoplasmic fluorescence that was abolished by cytochalasin B, indicating a high endocytotic activity in these cells. The endocytotic activity was well correlated with the success of cationic liposome-mediated transfer of MDR1 ribozyme. These distinct approaches using either retrovirus- or liposome-mediated transfer of anti-MDR1 ribozyme may be selectively applicable to the treatment of MDR cells with different properties such as endocytotic activity as a specific means to reverse resistance.     

Using purified P-glycoprotein to understand multidrug resistance

Since P-glycoprotein was discovered almost 20 years ago, its causative role in multidrug resistance has been established, but central problems of its biochemistry have not been definitively resolved. Recently, major advances have been made in P-glycoprotein biochemistry with the use of purified and reconstituted P-glycoprotein, as well as membranes from nonmammalian cells containing heterologously expressed P-glycoprotein. In this review we describe recent findings using these systems which are elucidating the molecular mechanism of P-glycoprotein-mediated drug transport.     

Multidrug resistance increment in a human colon carcinoma cell line by colchicine

The most important mechanism in drug resistance is the multidrug resistance (MDR) phenomenon. It is possible to select MDR cells by in vitro exposure to cytotoxic agents. The resistance is due to the hyperexpression of the P-glycoprotein (P-Gp) that take drugs out from the cells. In this study, a colchicine resistant subline (HCA-2/1cch) was selected from a human colon adenocarcinoma after a short period of drug exposure, as an in vitro model of drug resistance selection. These cells showed cross-resistance to other drugs, which were not present in the medium during selection. The relative resistance was 3.32 for colchicine, 3.15 for vinblastine, 2.62 for vincristine and 5.22 for mitomycin C. P-glycoprotein levels were assayed by flow cytometry. It was found that a significant increase of 2.35 and 1.59 had occurred in the peak and mean channel of fluorescence, respectively, indicating an increment of P-glycoprotein expression in relation to the parental line. Moreover, verapamil (10 microg/ml) produced a partial reversion of multidrug resistance. The sensitisation rates were 7.41 for colchicine, 1.25 for vinblastine, 2.36 for vincristine and 1.17 for mitomycin C. The data obtained suggest that colchicine exposure period (10 weeks) and dose (0.5 microg/ml) assayed were sufficient to produce an increment in multidrug resistance. This resistance could be due to higher level of P-Gp expression.     

Molecular mechanism of multidrug resistance in tumor cells

The ability of tumor cells to develop simultaneous resistance to multiple lipophilic cytotoxic compounds represents a major problem in cancer chemotherapy. This review describes recent molecular biological studies which resulted in the identification and cloning of the gene responsible for multidrug resistance in human tumor cells. This gene, designated mdr1, is overexpressed in all and amplified in many of the multidrug-resistant cell lines analyzed. Gene transfer and expression assays have indicated that the mdr1 gene is both necessary and sufficient for multidrug resistance. The product of the mdr1 gene is P-glycoprotein, a transmembrane protein which shares homology with several bacterial proteins involved in active membrane transport. P-glycoprotein appears to function as an energy-dependent efflux pump responsible for the removal of drugs from multidrug-resistant cells. The functions of the mdr system in normal cells and its potential clinical implications are discussed.