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.     

HepG2/mdr1稳定细胞系的建立及特性研究

将已构建好的含有人多药耐药（multidrug resistance, MDR）全长基因的真核表达质粒pCI-neo-mdr1,应用脂质体导入人肝癌HepG2细胞,应用G418筛选出人肝癌多药耐药细胞株HepG2/mdr1。通过对HepG2/mdr1细胞形态学的观察和生物学特性的研究,成功地建立了高效、稳定的HepG2/mdr1细胞系;为深入研究肝癌的MDR及其逆转提供了理想的细胞模型,并为探索建立肝癌MDR细胞株提供新的方法和思路,同时为研究肝癌细胞胰岛素抵抗与MDR的关系提供了模型细胞。 将已构建好的含有人多药耐药（multidrug resistance, MDR）全长基因的真核表达质粒pCI-neo-mdr1,应用脂质体导入人肝癌HepG2细胞,应用G418筛选出人肝癌多药耐药细胞株HepG2/mdr1。通过对HepG2/mdr1细胞形态学的观察和生物学特性的研究,成功地建立了高效、稳定的HepG2/mdr1细胞系;为深入研究肝癌的MDR及其逆转提供了理想的细胞模型,并为探索建立肝癌MDR细胞株提供新的方法和思路,同时为研究肝癌细胞胰岛素抵抗与MDR的关系提供了模型细胞。     

Peptide transport by the multidrug resistance pump.

The membrane P-glycoprotein (P170) is an ATP-hydrolyzing transmembrane pump, and elevated levels of P170, due to higher expression with or without amplification of the multidrug resistance gene (mdr1), result in resistance to a variety of chemotherapeutic agents in mammalian cells. The function of the P170 pump has been proposed as a protection against toxic substances present in animal diets. Here we describe a Chinese hamster ovary cell line that was selected for resistance to a synthetic tripeptide, N-acetyl-leucyl-leucyl-norleucinal (ALLN). This ALLN-resistant variant shows the classical multidrug resistance (MDR) phenotype, including overexpression and amplification of the mdr1 gene. Additionally, a mouse embryo cell line overexpressing the transfected mdr1 gene is likewise resistant to ALLN. Our results demonstrate that P170 is capable of transporting peptides and raise the possibility that the mdr1 gene product or other MDR-like genes, present in the genome of mammalian cells, may be involved in secretion of peptides or cellular proteins as is the case with the structurally similar hylB and ste6 gene products of Escherichia coli and yeast, respectively.     

Downregulation of mdr1 and abcg2 genes is a mechanism of inhibition of efflux pumps mediated by polymeric amphiphiles

The ability of cells to acquire resistance to multiple pharmaceuticals, namely multidrug resistance (MDR), is often mediated by the over-expression of efflux transporters of the ATP-binding cassette (ABC) superfamily; for example P-glycoprotein (P-gp or MDR1), breast cancer resistance protein (BCRP or ABCG2), and multidrug resistance-associated protein MRP1. ABCs pump drug molecules out of cells against a concentration gradient, reducing their intracellular concentration. The ability of polymeric amphiphiles to inhibit ABCs as well as the cellular pathways involved in the inhibition has been extensively investigated. This work investigated for the first time the effect of branched poly(ethylene oxide)-poly(propylene oxide) block copolymers (poloxamines) on the levels of mRNA encoding for MDR1, BCRP and MRP1, in a human hepatoma cell line (Huh7). Copolymers with a broad range of molecular weights and hydrophilic-lipophilic balances were assayed. Results confirmed the down-regulation of mdr1 and abcg2 genes. Conversely, the mrp1 gene was not affected. These findings further support the versatility of these temperature- and pH-responsive copolymers to overcome drug resistance in cancer and infectious diseases.     

Increased mdr gene expression and decreased drug accumulation in a human colonic cancer cell line resistant to hydrophobic drug

An adriamycin-resistant human colonic cancer cell line was characterized. This clone exhibits the classical multidrug resistance (MDR) phenotype, being cross-resistant to hydrophobic drugs such as colchicine, and vinblastine. In contrast, this clone shows a normal response to DNA-damaging agents. The appearance of MDR in these cells was linked to a decreased accumulation of the drug [3H]colchicine as compared to the drug-sensitive cells. This MDR line expressed 80-100 fold increased levels of the specific 4.5-kb mdr mRNA, and a gene amplification. Our results indicate that MDR in human colonic cancer cells can result from increased expression of at least one member of the mdr gene family.     

Two members of the mouse mdr gene family confer multidrug resistance with overlapping but distinct drug specificities.

We report the cloning and functional analysis of a complete clone for the third member of the mouse mdr gene family, mdr3. Nucleotide and predicted amino acid sequence analyses showed that the three mouse mdr genes encode highly homologous membrane glycoproteins, which share the same length (1,276 residues), the same predicted functional domains, and overall structural arrangement. Regions of divergence among the three proteins are concentrated in discrete segments of the predicted polypeptides. Sequence comparison indicated that the three mouse mdr genes were created from a common ancestor by two independent gene duplication events, the most recent one producing mdr1 and mdr3. When transfected and overexpressed in otherwise drug-sensitive cells, the mdr3 gene, like mdr1 and unlike mdr2, conferred multidrug resistance to these cells. In independently derived transfected cell clones expressing similar amounts of either MDR1 or MDR3 protein, the drug resistance profile conferred by mdr3 was distinct from that conferred by mdr1. Cells transfected with and expressing MDR1 showed a marked 7- to 10-fold preferential resistance to colchicine and Adriamycin compared with cells expressing equivalent amounts of MDR3. Conversely, cells transfected with and expressing MDR3 showed a two- to threefold preferential resistance to actinomycin D over their cellular counterpart expressing MDR1. These results suggest that MDR1 and MDR3 are membrane-associated efflux pumps which, in multidrug-resistant cells and perhaps normal tissues, have overlapping but distinct substrate specificities.     

Membrane homoeostasis and multidrug resistance in yeast

The development of MDR (multidrug resistance) in yeast is due to a number of mechanisms. The most documented mechanism is enhanced extrusion of drugs mediated by efflux pump proteins belonging to either the ABC (ATP-binding cassette) superfamily or MFS (major facilitator superfamily). These drug-efflux pump proteins are localized on the plasma membrane, and the milieu therein affects their proper functioning. Several recent studies demonstrate that fluctuations in membrane lipid composition affect the localization and proper functioning of the MDR efflux pump proteins. Interestingly, the efflux pumps of the ABC superfamily are particularly susceptible to imbalances in membrane-raft lipid constituents. This review focuses on the importance of the membrane environment in functioning of the drug-efflux pumps and explores a correlation between MDR and membrane lipid homoeostasis.     

Analysis of DNA content in multidrug-resistant cells by image and flow cytometry

Abstract. Nuclear DNA content was assessed in multidrug-resistant (MDR) cells by image and flow cytometry. Two human MDR cell lines (K562-Dox and CEM-VLB) obtained by in vitro drug selection and overexpressing mdr1 gene were compared to their respective sensitive counterparts (K562 and CCRF-CEM) and to the MDR hamster LR73-R cell line obtained by transfection of mouse mdr1 cDNA. Both cell lines obtained by selection displayed a decreased DNA content, as measured by image cytometry after Feulgen staining, or by flow cytometry after staining with propidium iodide, ethidium bromide, or Hoechst 33342. This decrease was not accompanied by changes in cell cycle phase distribution of cells. Moreover, image cytometry of cells stained after various hydrolysis times in 5 M HCl indicated that MDR cells displayed the same hydrolysis kinetics and sensitivity as drug-sensitive cells with a well-preserved stoichiometry of the Feulgen reaction. LR73-R cells transfected with mdr1 cDNA exhibited only a very limited change in propidium iodide staining as compared with sensitive LR73 cells, suggesting that mdr1 gene overexpression alone could not account for the alterations in DNA content observed in the selected MDR cells.     

Co-amplification and over-expression of two mdr genes in a multidrug-resistant human colon carcinoma cell line.

The human P-glycoprotein gene family contains the mdr1 and the mdr3 gene. The mdr1 P-glycoprotein is over-expressed in multidrug resistant (MDR) tumor cells and is believed to play a role in the elimination of certain cytotoxic drugs used in the chemotherapy of cancer. The mdr3 gene has not been found to be amplified or over-expressed in MDR cells. In this study, gene-specific mdr gene probes were developed for the detection of the gene and the total mRNA level. Southern and Northern hybridization analyses showed that the mdr genes and the mRNA levels were increased 30--40-fold in a MDR human colon cancer cell line. In addition, this MDR cell line had an altered growth rate and morphology and detectable double minute chromosomes.     

Discrete mutations introduced in the predicted nucleotide-binding sites of the mdr1 gene abolish its ability to confer multidrug resistance.

In cells stably transfected and overexpressing the mouse mdr1 gene, multidrug resistance is associated with an increased ATP-dependent drug efflux. Analysis of the predicted amino acid sequence of the MDR1 protein revealed the presence of two putative nucleotide-binding sites (NBS). To assess the functional importance of these NBS in the overall drug resistance phenotype conferred by mdr1, we introduced amino acid substitutions in the core consensus sequence for nucleotide binding, GXGKST. Mutants bearing the sequence GXAKST or GXGRST at either of the two NBS of mdr1 and a double mutant harboring the sequence GXGRST at both NBS were generated. The integrity of the two NBS was essential for the biological activity of mdr1, since all five mutants were unable to confer drug resistance to hamster drug-sensitive cells in transfection experiments. Conversely, a lysine-to-arginine substitution outside the core consensus sequence had no effect on the activity of mdr1. Failure to reduce intracellular accumulation of [3H]vinblastine paralleled the loss of activity in cell clones expressing mutant MDR1 proteins. However, the ability to bind the photoactivatable ATP analog 8-azido ATP was retained in the five inactive MDR1 mutants. This result implies that an essential step subsequent to ATP binding is impaired in these mutants, possibly ATP hydrolysis or secondary conformational changes induced by ATP-binding or hydrolysis. Our results suggest that the two NBS function in a cooperative fashion, since mutations in a single NBS completely abrogated the biological activity of mdr1.     

P-glycoprotein as multidrug transporter: a critical review of current multidrug resistant cell lines.

MDR has been studied extensively in mammalian cell lines. According to usual practice, the MDR phenotype is characterized by the following features: cross resistance to multiple chemotherapeutic agents (lipophilic cations), defective intracellular drug accumulation and retention, overexpression of P-gp (often accompanied by gene amplification), and reversal of the phenotype by addition of calcium channel blockers. An hypothesis for the function of P-gp has been proposed in which P-gp acts as a carrier protein that actively extrudes MDR compounds out of the cells. However, basic questions, such as what defines the specificity of the pump and how is energy for active efflux transduced, remain to be answered. Furthermore, assuming that P-gp acts as a drug transporter, one will expect a relationship between P-gp expression and accumulation defects in MDR cell lines. A review of papers reporting 97 cell lines selected for resistance to the classical MDR compounds has revealed that a connection exists in most of the reported cell lines. However, several exceptions can be pointed out. Furthermore, only a limited number of well characterized series of sublines with different degrees of resistance to a single agent have been reported. In many of these, a correlation between P-gp expression and transport properties can not be established. Co-amplification of genes adjacent to the mdr1 gene, mutations [122], splicing of mdr1 RNA [123], modulation of P-gp by phosphorylation [124] or glycosylation [127], or experimental conditions [26,78] could account for some of the complexity of the phenotype and the absence of correlation in some of the cell lines. However, both cell lines with overexpression of P-gp without increased efflux [i.e., 67,75] and cell lines without P-gp expression and accumulation defects/increased efflux [i.e., 25,107] have been reported. Thus, current results from MDR cell lines contradict--but do not exclude--that P-gp acts as multidrug transporter. Other models for the mechanism of resistance have been proposed: (1) An energy-dependent permeability barrier working with greater efficacy in resistant cells. This hypothesis is supported by studies of influx which, although few, all except one demonstrate decreased influx in resistant cells; (2) Resistant cells have a greater endosomal volume, and a greater exocytotic activity accounts for the efflux.(ABSTRACT TRUNCATED AT 400 WORDS)     

P-glycoprotein-mediated multidrug resistance phenotype of L1210/VCR cells is associated with decreases of oligo- and/or polysaccharide contents

Multidrug resistance of murine leukaemic cell line L1210/VCR (obtained by adaptation of parental drug-sensitive L1210 cells to vincristine) is associated with overexpression of mdr1 gene product P-glycoprotein (Pgp)-the ATP-dependent drug efflux pump. 31P-NMR spectra of L1210 and L1210/VCR cells (the latter in the presence of vincristine) revealed, besides the decrease of ATP level, a considerable lower level of UDP-saccharides in L1210/VCR cells. Histochemical staining of negatively charged cell surface binding sites (mostly sialic acid) by ruthenium red (RR) revealed a compact layer of RR bound to the external coat of sensitive cells. In resistant cells cultivated in the absence or presence of vincristine, the RR layer is either reduced or absent. Consistently, resistant cells were found to be less sensitive to Concanavalin A (ConA). Moreover, differences in the amount and spectrum of glycoproteins interacting with ConA-Sepharose were demonstrated between sensitive and resistant cells. Finally, the content of glycogen in resistant cells is lower than in sensitive cells. All the above facts indicate that multidrug resistance of L1210/VCR cells mediated predominantly by drug efflux activity of Pgp is accompanied by a considerable depression of oligo- and/or polysaccharides biosynthesis.     

Reversal MDR in breast carcinoma cells by transfection of ribozyme designed according the secondary structure of mdr1 mRNA

Multidrug resistance (MDR) is a major obstacle in cancer chemotherapy. The present study aims to investigate whether the ribozyme could reverse MDR in breast carcinoma cells. In this study, two GUC sites (GUC106 and GUC135) on the surface of mdr1 mRNA were selected according to the secondary structure of the 5'-region of mdrl mRNA. The ribozyme gene RZ106 and RZ135 complementary to two sides bases of the target GUC were synthesized and cloned into the plasmid pEGFP -C1 which has EGFP (Enhanced Green Fluorescence Protein) as report gene and Kan/Neo as selection gene. After transfection with the recombinant plasmid and selected by G418, the stable cell clones were produced and used for detection. The alteration of mdr1 mRNA and P-gp in the treated cells was detected by RT-PCR, flow cytometry and Rh123 retention. The reversal efficiency of the drug resistance for adriamycin was determined by MTT assay. The results showed that after transfection with RZ106 and RZ135, the amount of the mdr1 mRNA and P-gp decreased significantly and the efflux function of P-gp was inhibited accordingly. Nine-fold and 16-fold reduction of resistance for adriamycin was observed in the two groups of treated cells. These results suggested that both ribozymes can reverse the MDR phenotype by inhibiting the expression of mdr1 mRNA and P-gp, and the RZ135 showed the better cleavage efficiency. The ribozyme strategy designed according the secondary structure of the target RNA could be a useful therapy for reversal of MDR.     

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.     

Increased ABCB1 Expression in TP-110-Resistant RPMI-8226 Cells

TP-110, a novel proteasome inhibitor, has been found to possess potent growth inhibition in human multiple myeloma cells. To enhance its therapeutic effects, we established TP-110-resistant RPMI-8226 (RPMI-8226/TP-110) cells and elucidated their resistance mechanisms. The IC₅₀ value for TP-110 cytotoxicity in the RPMI-8226/TP-110 cells was about 10-fold higher than that of the parental sensitive cells. The RPMI-8226/TP-110 cells exhibited distinct drug resistance to other proteasome inhibitors. Furthermore, they showed high cross-resistance to the cytotoxic effects of doxorubicin, etoposide, taxol, and vincristine. P-glycoprotein (MDR1), encoded by ABCB1, was elevated in the RPMI-8226/TP-110 cells, and the MDR1 inhibitor verapamil overcame their resistance to TP-110. The results of DNA microarray and RT-PCR suggested that the expression of ABCB1 is significantly elevated in RPMI-8226/TP-110 cells. This indicates that resistance in RPMI-8226/TP-110 cells is involved in the expression of P-glycoprotein, a drug-efflux pump.     

The reversion effect of the RNAi-silencing mdr1 gene on multidrug resistance of the leukemia cell HT9

Overexpression of P-glycoprotein (P-gp), the mdr1 gene product, confers multidrug resistance (MDR) to tumor cells and often limits the efficacy of chemotherapy. This study evaluated RNAi for specific silencing of the mdr1 gene and reversion of multidrug resistance. Three different short hairpin RNAs (shRNAs) were designed and constructed in a pSilencer 3.1-H1 neo plasmid. The shRNA recombinant plasmids were transfected into HT9 leukemia cells. The RNAi effect was evaluated by real-time PCR, Western blotting and cell cytotoxicity assay. In the cell, shRNAs can specifically down-regulate the expression of mdr1, mRNA and P-gp. Resistance against harringtonine, doxorubicin and curcumin was decreased. The study indicated that shRNA recombinant plasmids could modulate MDR in vitro.     

Multiple resistance of eukaryotic cells caused by P-glycoprotein]

Recent data concerning cloning and sequencing of mdr genes involved in multiple drug resistance in higher eukaryotes are reviewed. Structures of ABC-superfamily members, including the mdr products as well as mechanisms of their superproduction at various levels are considered. The possible role of MDR-transporter in normal tissues and various approaches to overcoming the MDR phenotype are discussed. Non-P-glycoprotein mechanisms of drug resistance capable to modify MDR phenotype and applications of mdr in biotechnology are provided.