Multiple molecular mechanisms for multidrug resistance transporters.

The acquisition of multidrug resistance is a serious impediment to improved healthcare. Multidrug resistance is most frequently due to active transporters that pump a broad spectrum of chemically distinct, cytotoxic molecules out of cells, including antibiotics, antimalarials, herbicides and cancer chemotherapeutics in humans. The paradigm multidrug transporter, mammalian P-glycoprotein, was identified 30 years ago. Nonetheless, success in overcoming or circumventing multidrug resistance in a clinical setting has been modest. Recent structural and biochemical data for several multidrug transporters now provide mechanistic insights into how they work. Organisms have evolved several elegant solutions to ridding the cell of such cytotoxic compounds. Answers are emerging to questions such as how multispecificity for different drugs is achieved, why multidrug resistance arises so readily, and what chance there is of devising a clinical solution.

Membrane phosphatidylserine distribution as a non-apoptotic signalling mechanism in lymphocytes.

Phosphatidylserine (PS) exposure is normally associated with apoptosis and the removal of dying cells. We observed that PS is exposed constitutively at high levels on T lymphocytes that express low levels of the transmembrane tyrosine phosphatase CD45RB. CD45 was shown to be a negative regulator of PS translocation in response to various signals, including activation of the ATP receptor P2X(7). Changes in PS distribution were shown to modulate several membrane activities: Ca(2+) and Na(+) uptake through the P2X(7) cation channel itself; P2X(7)-stimulated shedding of the homing receptor CD62L; and reversal of activity of the multidrug transporter P-glycoprotein. The data identify a role for PS distribution changes in signal transduction, rapidly modulating the activities of several membrane proteins. This seems to be an all-or-none effect, coordinating the activity of most or all the molecules of a target protein in each cell. The data also suggest a new approach to circumventing multidrug resistance.

Role for centromeric heterochromatin and PML nuclear bodies in the cellular response to foreign DNA.

Nuclear spatial positioning plays an important role in the epigenetic regulation of eukaryotic gene expression. Here we show a role for nuclear spatial positioning in regulating episomal transgenes that are delivered by virus-like particles (VLPs). VLPs mediate the delivery of plasmid DNA (pDNA) to cell nuclei but lack viral factors involved in initiating and regulating transcription. By tracking single fluorescently labeled VLPs, coupled with luciferase reporter gene assays, we found that VLPs transported pDNA to cell nuclei efficiently but transgenes were immediately silenced by the cell. An investigation of the nuclear location of fluorescent VLPs revealed that the pDNAs were positioned next to centromeric heterochromatin. The activation of transcription by providing viral factors or inhibiting histone deacetylase activity resulted in the localization to euchromatin regions. Further, the activation of transcription induced the recruitment of PML nuclear bodies (PML-NBs) to the VLPs. This association did not play a role in regulating transgene expression, but PML protein was necessary for the inhibition of transgene expression with alpha interferon (IFN-alpha). These results support a model whereby cells can prevent foreign gene expression at two levels: by positioning transgenes next to centromeric heterochromatin or, if that is overcome, via the type I IFN response facilitated by PML-NB recruitment.

The ATP switch model for ABC transporters.

ABC transporters mediate active translocation of a diverse range of molecules across all cell membranes. They comprise two nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs). Recent biochemical, structural and genetic studies have led to the ATP-switch model in which ATP binding and ATP hydrolysis, respectively, induce formation and dissociation of an NBD dimer. This provides an exquisitely regulated switch that induces conformational changes in the TMDs to mediate membrane transport.

Ability to acquire drug resistance arises early during the tumorigenesis process.

Resistance to chemotherapy is one of the principal causes of cancer mortality and is generally considered a late event in tumor progression. Although cellular models of drug resistance have been useful in identifying the molecules responsible for conferring drug resistance, most of these cellular models are derived from cell lines isolated from patients at a late stage in cancer progression. To ask at which stage in the tumorigenic progression does the cell gain the ability to acquire drug resistance, we generated a series of pre-tumorigenic and tumorigenic cells from human embryonic skin fibroblasts by introducing, sequentially, the catalytic subunit of telomerase, SV40 large T and small T oncoproteins, and an oncogenic form of ras. We show that the ability to acquire multidrug resistance (MDR) can arise before the malignant transformation stage. The minimal set of changes necessary to obtain pre-tumorigenic drug-resistant cells is expression of telomerase and inactivation of p53 and pRb. Thus, the pathways inactivated during tumorigenesis also confer the ability to acquire drug resistance. Microarray and functional studies of drug-resistant pre-tumorigenic cells indicate that the drug efflux pump P-glycoprotein is responsible for the MDR phenotype in this pre-tumorigenic cell model.

Production of P-glycoprotein from the MDR1 upstream promoter is insufficient to affect the response to first-line chemotherapy in advanced breast cancer.

Multidrug resistance, the phenomenon by which cells treated with a drug become resistant to the cytotoxic effect of a variety of other structurally and functionally unrelated drugs, is often associated with the expression of P-glycoprotein, an efflux membrane pump coded by the MDR1 (ABCB1) gene. Transcription from MDR1 can start at 2 promoters: a well-characterized downstream promoter and an as yet uncharacterized upstream promoter (USP). We have previously determined that the USP is activated in some drug-resistant cell lines, in primary breast tumors and in metastatic epithelial cells isolated from the lymph nodes of breast cancer patients. In this study, we report the cloning and characterization of the MDR1 USP and studied its association with chemotherapy response in breast cancer patients. Deletion analysis indicated that a nearby endogenous retroviral long terminal repeat is not responsible for promoter activation, and that the region within the first 400 nucleotides upstream from the transcription start point contained all the elements necessary for promoter activity in drug-resistant cells. We identified an element recognized by the transcription factor NF-IL6 (activated upon interleukin-6 exposure) which is necessary for promoter activity in drug-resistant cells and plays a role in the activation of the promoter in response to interleukin-6 in breast cancer MCF-7 cells. Although transcripts from this promoter are associated with translating polyribosomes, their low abundance makes the amount of synthesized P-glycoprotein insufficient to affect the response to first-line chemotherapy in patients with advanced breast cancer.

Role of the highly structured 5'-end region of MDR1 mRNA in P-glycoprotein expression.

Overexpression of P-glycoprotein, encoded by the MDR1 (multidrug resistance 1) gene, is often responsible for multidrug resistance in acute myeloid leukaemia. We have shown previously that MDR1 (P-glycoprotein) mRNA levels in K562 leukaemic cells exposed to cytotoxic drugs are up-regulated but P-glycoprotein expression is translationally blocked. In the present study we show that cytotoxic drugs down-regulate the Akt signalling pathway, leading to hypophosphorylation of the translational repressor 4E-BP [eIF (eukaryotic initiation factor) 4E-binding protein] and decreased eIF4E availability. The 5'-end of MDR1 mRNA adopts a highly-structured fold. Fusion of this structured 5'-region upstream of a reporter gene impeded its efficient translation, specifically under cytotoxic stress, by reducing its competitive ability for the translational machinery. The effect of cytotoxic stress could be mimicked in vivo by blocking the phosphorylation of 4E-BP by mTOR (mammalian target of rapamycin) using rapamycin or eIF4E siRNA (small interfering RNA), and relieved by overexpression of either eIF4E or constitutively-active Akt. Upon drug exposure MDR1 mRNA was up-regulated, apparently stochastically, in a small proportion of cells. Only in these cells could MDR1 mRNA compete successfully for the reduced amounts of eIF4E and translate P-glycoprotein. Consequent drug efflux and restoration of eIF4E availability results in a feed-forward relief from stress-induced translational repression and to the acquisition of drug resistance.

ClC-3 expression enhances etoposide resistance by increasing acidification of the late endocytic compartment.

Resistance to anticancer drugs and consequent failure of chemotherapy is a complex problem severely limiting therapeutic options in metastatic cancer. Many studies have shown a role for drug efflux pumps of the ATP-binding cassette transporters family in the development of drug resistance. ClC-3, a member of the CLC family of chloride channels and transporters, is expressed in intracellular compartments of neuronal cells and involved in vesicular acidification. It has previously been suggested that acidification of intracellular organelles can promote drug resistance by increasing drug sequestration. Therefore, we hypothesized a role for ClC-3 in drug resistance. Here, we show that ClC-3 is expressed in neuroendocrine tumor cell lines, such as BON, LCC-18, and QGP-1, and localized in intracellular vesicles co-labeled with the late endosomal/lysosomal marker LAMP-1. ClC-3 overexpression increased the acidity of intracellular vesicles, as assessed by acridine orange staining, and enhanced resistance to the chemotherapeutic drug etoposide by almost doubling the IC(50) in either BON or HEK293 cell lines. Prevention of organellar acidification, by inhibition of the vacuolar H(+)-ATPase, reduced etoposide resistance. No expression of common multidrug resistance transporters, such as P-glycoprotein or multidrug-related protein-1, was detected in either the BON parental cell line or the derivative clone overexpressing ClC-3. The probable mechanism of enhanced etoposide resistance can be attributed to the increase of vesicular acidification as consequence of ClC-3 overexpression. This study therefore provides first evidence for a role of intracellular CLC proteins in the modulation of cancer drug resistance.

The human ClC-4 protein, a member of the CLC chloride channel/transporter family, is localized to the endoplasmic reticulum by its N-terminus.

Despite considerable similarity in their amino acid sequences and structural features, the mammalian members of the CLC chloride channel/transporter family have different subcellular locations. The subcellular location and function of one of these members, hClC-4, is controversial. To characterize its cellular function, we investigated its tissue distribution and subcellular location. Expression was high in excitable tissues such as the nervous system and skeletal muscle. When heterologously expressed in HEK293 cells and in skeletal muscle fibers, hClC-4 localizes to the endoplasmic/sarcoplasmic reticulum (ER/SR) membranes, in contrast to hClC-3, which localizes to vesicular structures. This location was confirmed by identification of endogenous ClC-4 in membrane fractions from mouse brain homogenate enriched for the sarco-endoplasmic reticulum ATPase SERCA2, an ER/SR marker. To identify the motif responsible for ER localization of hClC-4, we generated hClC-4 truncations and chimeras between hClC-4 and hClC-3 or the unrelated plasma membrane protein Ly49E. A stretch of amino acids, residues 14-63, at the N-terminus constitutes a novel motif both necessary and sufficient for targeting hClC-4 and other membrane proteins to the ER.

Cytoplasmic domains of the transporter associated with antigen processing and P-glycoprotein interact with subunits of the proteasome.

The proteasome is a multi-protein complex that degrades cellular proteins as well as foreign proteins destined for antigen presentation. The latter function involves the immunoproteasome, in which several proteasome subunits are exchanged for gamma-interferon-induced subunits. The transporter associated with antigen processing (TAP) transports proteasome-generated peptides across the membrane of the endoplasmic reticulum (ER) prior to presentation on the plasma membrane. We demonstrate interactions between the cytoplasmic domains of TAP subunits and subunits of both the proteasome and the immunoproteasome, suggesting direct targeting of antigenic peptides to the ER via a TAP-proteasome association. We also show interaction between one of the cytoplasmic domains of P-glycoprotein and a proteasome subunit, but not the corresponding immunoproteasome subunit, suggesting a possible role for P-glycoprotein in the transport of proteasome-derived peptides.

Cell volume regulation and swelling-activated chloride channels.

Maintenance of a constant volume is essential for normal cell function. Following cell swelling, as a consequence of reduction of extracellular osmolarity or increase of intracellular content of osmolytes, animal cells are able to restore their original volume by activation of potassium and chloride conductances. The loss of these ions, followed passively by water, is responsible for the homeostatic response called regulatory volume decrease (RVD). Activation of a chloride conductance upon cell swelling is a key step in RVD. Several proteins have been proposed as candidates for this chloride conductance. The status of the field is reviewed, with particular emphasis on ClC-3, a member of the ClC family which has been recently proposed as the chloride channel involved in cell volume regulation.

Two novel missense mutations in ABCA1 result in altered trafficking and cause severe autosomal recessive HDL deficiency.

Extremely low concentrations of high density lipoprotein (HDL)-cholesterol and apolipoprotein (apo) AI are features of Tangier disease caused by autosomal recessive mutations in ATP-binding cassette transporter A1 (ABCA1). Less deleterious, but dominantly inherited mutations cause HDL deficiency. We investigated causes of severe HDL deficiency in a 42-year-old female with progressive coronary disease. ApoAI-mediated efflux of cholesterol from the proband's fibroblasts was less than 10% of normal and nucleotide sequencing revealed inheritance of two novel mutations in ABCAI, V1704D and L1379F. ABCA1 mRNA was approximately 3-fold higher in the proband's cells than in control cells; preincubation with cholesterol increased it 5-fold in control and 8-fold in the proband's cells, but similar amounts of ABCA1 protein were present in control and mutant cells. When transiently transfected into HEK293 cells, confocal microscopy revealed that both mutant proteins were retained in the endoplasmic reticulum, while wild-type ABCA1 was located at the plasma membrane. Severe HDL deficiency in the proband was caused by two novel autosomal recessive mutations in ABCA1, one (V1704D) predicted to lie in a transmembrane segment and the other (L1379F) in a large extracellular loop. Both mutations prevent normal trafficking of ABCA1, thereby explaining their inability to mediate apoA1-dependent lipid efflux.

Major histocompatibility complex class I shedding and programmed cell death stimulated through the proinflammatory P2X7 receptor: a candidate susceptibility gene for NOD diabetes.

It has been hypothesized that type 1 diabetes is initiated by neonatal physiological pancreatic beta-cell death, indicating that the early stages of this autoimmune response may reflect a dysregulated response to immune "danger" signals. One potential danger signal is ATP, high concentrations of which stimulate the purinergic receptor P2X7 on hematopoietic cells. We compared the sensitivity of lymphocytes from model type 1 diabetic (NOD) and control (C57BL/10) mice to activation of this pathway. Stimulation of the P2X7 receptor of NOD mice resulted in more pronounced shedding of the lymphocyte homing receptor CD62L and in increased programmed cell death. Levels of major histocompatibility complex class I molecules, which have previously been reported to be poorly expressed on NOD lymphocytes, were initially normal, but the molecules were shed preferentially from NOD cells after P2X7 receptor stimulation. Thus, although NOD lymphocytes have been considered resistant to programmed cell death, they are highly sensitive to that stimulated through the P2X7 receptor. Because NOD mice express a low activation threshold allele of the P2X7 receptor and the P2X7 gene maps to a locus associated with disease, P2X7 is a good candidate susceptibility gene for NOD diabetes.

H-NS oligomerization domain structure reveals the mechanism for high order self-association of the intact protein.

H-NS plays a role in condensing DNA in the bacterial nucleoid. This 136 amino acid protein comprises two functional domains separated by a flexible linker. High order structures formed by the N-terminal oligomerization domain (residues 1-89) constitute the basis of a protein scaffold that binds DNA via the C-terminal domain. Deletion of residues 57-89 or 64-89 of the oligomerization domain precludes high order structure formation, yielding a discrete dimer. This dimerization event represents the initial event in the formation of high order structure. The dimers thus constitute the basic building block of the protein scaffold. The three-dimensional solution structure of one of these units (residues 1-57) has been determined. Activity of these structural units is demonstrated by a dominant negative effect on high order structure formation on addition to the full length protein. Truncated and site-directed mutant forms of the N-terminal domain of H-NS reveal how the dimeric unit self-associates in a head-to-tail manner and demonstrate the importance of secondary structure in this interaction to form high order structures. A model is presented for the structural basis for DNA packaging in bacterial cells.

An atomic detail model for the human ATP binding cassette transporter P-glycoprotein derived from disulfide cross-linking and homology modeling.

The multidrug resistance P-glycoprotein mediates the extrusion of chemotherapeutic drugs from cancer cells. Characterization of the drug binding and ATPase activities of the protein have made it the paradigm ATP binding cassette (ABC) transporter. P-glycoprotein has been imaged at low resolution by electron cryo-microscopy and extensively analyzed by disulphide cross-linking, but a high resolution structure solved ab initio remains elusive. Homology models of P-glycoprotein were generated using the structure of a related prokaryotic ABC transporter, the lipid A transporter MsbA, as a template together with structural data describing the dimer interface of the nucleotide binding domains (NBDs). The first model, which maintained the NBD:transmembrane domain (TMD) interface of MsbA, did not satisfy previously published cross-linking data. This suggests that either P-glycoprotein has a very different structure from MsbA or that the published E. coli MsbA structure does not reflect a physiological state. To distinguish these alternatives, we mapped the interface between the two TMDs of P-glycoprotein experimentally by chemical cross-linking of introduced triple-cysteine residues. Based on these data, a plausible atomic model of P-glycoprotein could be generated using the MsbA template, if the TMDs of MsbA are reoriented with respect to the NBDs. This model will be important for understanding the mechanism of P-glycoprotein and other ABC transporters.

Activation of the MDR1 upstream promoter in breast carcinoma as a surrogate for metastatic invasion.

PURPOSE: Activation of the MDR1 upstream promoter (USP) has been described previously in four lymphoblastic leukemia patients, where it is the major MDR1 promoter associated with P-glycoprotein overexpression. We asked whether MDR1 USP-derived transcripts were also present in breast carcinoma and assessed their potential as a biomarker. EXPERIMENTAL DESIGN: We developed a sensitive method for detecting transcripts derived from the MDR1 USP and used it to identify MDR1 USP-derived transcripts in cell model systems, in 61 breast carcinoma biopsies of the primary tumor, and in isolated malignant epithelial cells both from the primary tumor and from the associated invaded lymph nodes. RESULTS: The MDR1 USP was not active in several independent leukemic and breast cancer cell lines or nucleated peripheral blood cells (n = 9). However, transcripts derived from the MDR1 USP were detected in some drug-resistant cell lines and a high proportion of primary breast tumors (71.6%; n = 61), whereas they were present at low frequency in normal breast tissue (10%; n = 10). Activation of MDR1 USP was not due to chromosomal amplifications or rearrangements at the MDR1 locus. Transcription from the MDR1 USP correlated with metastatic node invasion [N = 0-3 versus N > 3 (N = number of lymph nodes invaded); Fisher's exact test, P = 0.011] and was detected in malignant epithelial cells from the primary tumor and those that metastasized to the lymph nodes. CONCLUSIONS: MDR1 USP activation is a surrogate marker for breast carcinoma progression and can be used as a marker to study breast cancer susceptibility.

Multidrug transporter activity in lymphocytes.

Multidrug transporters play a dual role in haematopoietic cells, mediating the efflux of xenobiotics and regulating cell migration. For several reasons including the lack of specific antibodies, reports of multidrug transporter distribution on lymphocytes conflict. Murine B cells have been reported to completely lack transporter activity. Through analysis of parental and 'knockout' mice we show that, contrary to previous studies, murine B and T lymphocytes possess at least three active multidrug transporters and also a hitherto unrecognised drug-specific import activity. Surprisingly, the drug specificity of P-glycoprotein appears cell type dependent. The data indicate that a range of developmentally regulated, multidrug transporters can impose a barrier to treatment of immune disorders.

Sequential shrinkage and swelling underlie P2X7-stimulated lymphocyte phosphatidylserine exposure and death.

Patterns of change in cell volume and plasma membrane phospholipid distribution during cell death are regarded as diagnostic means of distinguishing apoptosis from necrosis, the former being associated with cell shrinkage and early phosphatidylserine (PS) exposure, whereas necrosis is associated with cell swelling and consequent lysis. We demonstrate that cell volume regulation during lymphocyte death stimulated via the purinergic receptor P2X7 is distinct from both. Within seconds of stimulation, murine lymphocytes undergo rapid shrinkage concomitant with, but also required for, PS exposure. However, within 2 min shrinkage is reversed and swelling ensues ending in cell rupture. P2X7-induced shrinkage and PS translocation depend upon K+ efflux via KCa3.1, but use a pathway of Cl- efflux distinct from that previously implicated in apoptosis. Thus, P2X7 stimulation activates a novel pathway of cell death that does not conform to those conventionally associated with apoptosis and necrosis. The mixed apoptotic/necrotic phenotype of P2X7-stimulated cells is consistent with a potential role for this death pathway in lupus disease.