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Analysis of the cell membrane of Adriamycin (doxorubicin)-resistant UV-2237 ADMR murine fibrosarcoma cells revealed a 170,000-dalton component that is not found in the drug-sensitive parent or revertant cells. Immunoblot (Western blot) analysis showed that this component is similar to the 170,000-dalton P-glycoprotein found on the surface of Chinese hamster ovary cells that exhibit multidrug resistance. Thus, multidrug resistance and P-glycoprotein expression apparently can occur in a wide variety of cells, including the metastatic murine solid tumor cell line described here. [ABSTRACT FROM AUTHOR]

The a subunit is the largest of 15 different subunits that make up the vacuolar H + -ATPase (V-ATPase) complex, where it functions in proton translocation. In mammals, this subunit has four paralogous isoforms, a 1- a 4, which may encode signals for targeting assembled V-ATPases to specific intracellular locations. Despite the functional importance of the a subunit, its structure remains controversial. By studying molecular mechanisms of human disease-causing missense mutations within a subunit isoforms, we may identify domains critical for V-ATPase targeting, activity and/or regulation. cDNA-encoded FLAG-tagged human wildtype ATP6V0A2 ( a 2) and ATP6V0A4 ( a 4) subunits and their mutants, a 2 P405L (causing cutis laxa), and a 4 R449H and a 4 G820R (causing renal tubular acidosis, dRTA), were transiently expressed in HEK 293 cells. N -Glycosylation was assessed using endoglycosidases, revealing that a 2 P405L , a 4 R449H , and a 4 G820R were fully N -glycosylated. Cycloheximide (CHX) chase assays revealed that a 2 P405L and a 4 R449H were unstable relative to wildtype. a 4 R449H was degraded predominantly in the proteasomal pathway, whereas a 2 P405L was degraded in both proteasomal and lysosomal pathways. Immunofluorescence studies disclosed retention in the endoplasmic reticulum and defective cell-surface expression of a 4 R449H and defective Golgi trafficking of a 2 P405L Co-immunoprecipitation studies revealed an increase in association of a 4 R449H with the V 0 assembly factor VMA21, and a reduced association with the V 1 sector subunit, ATP6V1B1 (B1). For a 4 G820R , where stability, degradation, and trafficking were relatively unaffected, 3D molecular modeling suggested that the mutation causes dRTA by blocking the proton pathway. This study provides critical information that may assist rational drug design to manage dRTA and cutis laxa., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

The a subunit of the V 0 membrane-integrated sector of human V-ATPase has four isoforms, a1-a4, with diverse and crucial functions in health and disease. They are encoded by four conserved paralogous genes, and their vertebrate orthologs have positionally conserved N-glycosylation sequons within the second extracellular loop, EL2, of the a subunit membrane domain. Previously, we have shown directly that the predicted sequon for the a4 isoform is indeed N-glycosylated. Here we extend our investigation to the other isoforms by transiently transfecting HEK 293 cells to express cDNA constructs of epitope-tagged human a1-a3 subunits, with or without mutations that convert Asn to Gln at putative N-glycosylation sites. Expression and N-glycosylation were characterized by immunoblotting and mobility shifts after enzymatic deglycosylation, and intracellular localization was determined using immunofluorescence microscopy. All unglycosylated mutants, where predicted N-glycosylation sites had been eliminated by sequon mutagenesis, showed increased relative mobility on immunoblots, identical to what was seen for wild-type a subunits after enzymatic deglycosylation. Cycloheximide-chase experiments showed that unglycosylated subunits were turned over at a higher rate than N-glycosylated forms by degradation in the proteasomal pathway. Immunofluorescence colocalization analysis showed that unglycosylated a subunits were retained in the ER, and co-immunoprecipitation studies showed that they were unable to associate with the V-ATPase assembly chaperone, VMA21. Taken together with our previous a4 subunit studies, these observations show that N-glycosylation is crucial in all four human V-ATPase a subunit isoforms for protein stability and ultimately for functional incorporation into V-ATPase complexes., (© 2017 Wiley Periodicals, Inc.)

Recombinant human bone morphogenetic protein 2 (rhBMP-2) is used clinically to enhance implant-mediated bone regeneration. However, there are risks associated with the high rhBMP-2 dose that is required in the implant to mitigate diffusional loss over the therapeutic timespan. On-demand, localized control over delivery of rhBMP-2, days after implantation, would therefore be an attractive solution in the area of bone repair and reconstruction, yet this has posed a significant challenge, with little data to support in vivo efficacy to date. To address this, we have developed novel liposome-rhBMP-2 nanocomplexes that release rhBMP-2 in response to non-thermogenic, clinical diagnostic ultrasound exposure. In vitro validation shows that rhBMP-2 release is in proportion to applied ultrasound pressure and duration of exposure. Moreover, here we show in vivo validation of this ultrasound-triggered rhBMP-2 delivery system in a standard mouse bone regeneration model. Implanted into hindleg muscles, the liposome-rhBMP-2 nanocomplexes induced local bone formation only after ultrasound exposure. Such post-implantation control of delivery has potential to improve the safety, efficacy and cost of rhBMP-2 use in bone reconstruction. Furthermore, this first proof-of-concept demonstration of in vivo efficacy for ultrasound-triggered liposomal delivery of rhBMP-2 has broader implications for tunable delivery of a variety of drugs and biologics in medicine and tissue engineering., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The a subunit is the largest of 14 different subunits that make up the V-ATPase complex. In mammalian species this membrane protein has four paralogous isoforms, a1-a4. Clinically, a subunit isoforms are implicated in diverse diseases; however, little is known about their structure and function. The subunit has conserved, predicted N-glycosylation sites, and the a3 isoform has been directly shown to be N-glycosylated. Here we ask if human a4 (ATP6V0A4) is N-glycosylated at the predicted site, Asn489. We transfected HEK 293 cells, using the pCDNA3.1 expression-vector system, to express cDNA constructs of epitope-tagged human a4 subunit, with or without mutations to eliminate the putative glycosylation site. Glycosylation was characterized also by treatment with endoglycosidases; expression and localization were assessed by immunoblotting and immunofluorescence. Endoglycosidase-treated wild type (WT) a4 showed increased relative mobility on immunoblots, compared with untreated WT a4. This relative mobility was identical to that of unglycosylated mutant a4 N489D , demonstrating that the a4 subunit is glycosylated. Cycloheximide pulse-chase experiments showed that the unglycosylated subunit degraded at a higher rate than the N-glycosylated form. Unglycosylated a4 was degraded mostly in the proteasomal pathway, but also, in part, through the lysosomal pathway. Immunofluorescence colocalization data showed that unglycosylated a4 was mostly retained in the ER, and that plasma membrane trafficking was defective. Co-immunoprecipitation studies suggested that a4 N489D does not assemble with the V-ATPase V 1 domain. Taken together, these data show that N-glycosylation plays a crucial role in a4 stability, and in V-ATPase assembly and trafficking to the plasma membrane. J. Cell. Biochem. 117: 2757-2768, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Introduction: Bone loss occurs in many diseases, including osteoporosis, rheumatoid arthritis and periodontal disease. For osteoporosis alone, it is estimated that 75 million people are afflicted worldwide, with high risks of fractures and increased morbidity and mortality. The demand for treatment consumes an ever-increasing share of healthcare resources. Successive generations of antiresorptive bisphosphonate drugs have reduced side effects, minimized frequency of dosing, and increased efficacy in halting osteoporotic bone loss, but their shortcomings have remained significant to the extent that a monoclonal antibody antiresorptive has recently taken a significant market share. Yet this latter, paradigm-shifting approach has its own drawbacks., Areas Covered: This review summarizes recent literature on bone-remodeling cell and molecular biology and the background for existing approaches and emerging therapeutics and targets for treating osteoporosis. The authors discuss vacuolar H(+)-ATPase (V-ATPase) molecular biology and the recent advances in targeting the osteoclast ruffled-border V-ATPase (ORV) for the development of novel antiresorptive drugs. They also cover examples from the V-ATPase-targeted drug discovery literature, including conventional molecular biology methods, in silico drug discovery, and gene therapy in more detail as proofs of concept., Expert Opinion: Existing therapeutic options for osteoporosis have limitations and inherent drawbacks. Thus, the search for novel approaches to osteoporosis drug discovery remains relevant. Targeting the ORV may be one of the more selective means of regulating bone resorption. Furthermore, this approach may be effective without removing active osteoclasts from the finely balanced osteoclast-osteoblast coupling required for normal bone remodeling.

Published topological models of the integral membrane a subunit of the vacuolar proton-translocating ATPase complex have not been in agreement with respect to either the number of transmembrane helices within the integral membrane domain, or their limits and orientations within the lipid bilayer. In the present work we have constructed a predictive model of the membrane insertion of the yeast a subunit, Vph1p, from a consensus of seven topology prediction algorithms. The model was tested experimentally using epitope tagging, green fluorescent protein fusion, and protease accessibility analysis in purified yeast vacuoles. Results suggest that a consensus prediction of eight transmembrane helices with both the amino-terminus and carboxyl-terminus in the cytoplasm is correct. Characterization of two glycosylation sites within the homologous mouse a subunit membrane domain further corroborates this topology. Moreover, the model takes into account published data on cytoplasmic and luminal accessibility of specific amino acids. Changes in the degree of protease accessibility in response to the V-ATPase substrate, MgATP, and the V-ATPase-specific inhibitor, concanamycin A, suggest that functional conformational changes occur in the large cytoplasmic loop between TM6 and TM7 of Vph1p. These data substantially confirm one topological model of the V-ATPase a subunit and support the notion that conformational changes occur within the membrane domain, possibly involving previously proposed axial rotation and/or linear displacement of TM7 in the proton transport cycle., (Copyright © 2013 Wiley Periodicals, Inc.)

V-ATPase-mediated acid secretion is required for osteoclast bone resorption. Osteoclasts are enriched in V-ATPase a3 and d2 subunit isoforms, and disruption of either of their genes impairs bone resorption. Using purified fusion proteins of a3 N-terminal domain (NTa3) and full-length d subunits we determined in a solid-phase binding assay that half-maximal binding of d1 or d2 to immobilized NTa3 occurs at 3.1 ± 0.4 or 3.6 ± 0.6 nM, respectively, suggesting equally high-affinity interactions. A high-throughput modification of this assay was then used to screen chemical libraries for a3-d2 interaction inhibitors, and luteolin, a naturally occurring flavonoid, was identified, with half-maximal inhibition at 2.4 ± 0.9 µM. Luteolin did not significantly affect NIH/3T3 or RAW 264.7 cell viability, nor did it affect cytokine-induced osteoclastogenesis of RAW 264.7 cells or bone marrow mononuclear cells at concentrations ≤ 40 µM. Luteolin inhibited osteoclast bone resorption with an EC(50) of approximately 2.5 µM, without affecting osteoclast actin ring formation. Luteolin-treated osteoclasts produced deeper resorption pits, but with decreased surface area, resulting in overall decreased pit volume. Luteolin did not affect transcription, or protein levels, of V-ATPase subunits a3, d2, and E, or V(1) V(0) assembly. Previous work has shown that luteolin can be effective in reducing bone resorption, and our studies suggest that this effect of luteolin may be through disruption of osteoclast V-ATPase a3-d2 interaction. We conclude that the V-ATPase a3-d2 interaction is a viable target for novel anti-resorptive therapeutics that potentially preserve osteoclast-osteoblast signaling important for bone remodeling., (Copyright © 2012 Wiley Periodicals, Inc.)

Osteopetrosis is a genetic bone disease characterized by increased bone density and fragility. The R444L missense mutation in the human V-ATPase a3 subunit (TCIRG1) is one of several known mutations in a3 and other proteins that can cause this disease. The autosomal recessive R444L mutation results in a particularly malignant form of infantile osteopetrosis that is lethal in infancy, or early childhood. We have studied this mutation using the pMSCV retroviral vector system to integrate the cDNA construct for green fluorescent protein (GFP)-fused a3(R445L) mutant protein into the RAW 264.7 mouse osteoclast differentiation model. In comparison with wild-type a3, the mutant glycoprotein localized to the ER instead of lysosomes and its oligosaccharide moiety was misprocessed, suggesting inability of the core-glycosylated glycoprotein to traffic to the Golgi. Reduced steady-state expression of the mutant protein, in comparison with wild type, suggested that the former was being degraded, likely through the endoplasmic reticulum-associated degradation pathway. In differentiated osteoclasts, a3(R445L) was found to degrade at an increased rate over the course of osteoclastogenesis. Limited proteolysis studies suggested that the R445L mutation alters mouse a3 protein conformation. Together, these data suggest that Arg-445 plays a role in protein folding, or stability, and that infantile malignant osteopetrosis caused by the R444L mutation in the human V-ATPase a3 subunit is another member of the growing class of protein folding diseases. This may have implications for early-intervention treatment, using protein rescue strategies.

Over the last three decades, V-ATPases have emerged from the obscurity of poorly understood membrane proton transport phenomena to being recognized as ubiquitous proton pumps that underlie vital cellular processes in all eukaryotic and many prokaryotic cells. These exquisitely complex molecular motors also engage in diverse specialized roles contributing to development, tissue function and pH homeostasis within complex organisms. Increasingly, mutations and misappropriation of V-ATPase function have been linked to diseases, ranging from sclerosing bone pathologies and renal tubular acidosis to bone-loss disorders and cancer metastasis. Much remains to be learned about the details of V-ATPase cell and molecular biology; nevertheless, interest in V-ATPases as potential therapeutic targets has burgeoned in recent years. In this review, we present a history of our involvement and contributions to the understanding of V-ATPase structure and function and our nascent and ongoing contributions to translating the knowledge gained from basic research on the nature of V-ATPases into tools for drug discovery. We focus here primarily on the treatment of bone-loss pathologies, like osteoporosis, and present proof-of-concept for a drug screening strategy based on targeting a3-B2 subunit interactions within the V-ATPase complex.

Vacuolar H(+)-ATPases (V-ATPases) are highly expressed in ruffled borders of bone-resorbing osteoclasts, where they play a crucial role in skeletal remodeling. To discover protein-protein interactions with the a subunit in mammalian V-ATPases, a GAL4 activation domain fusion library was constructed from an in vitro osteoclast model, receptor activator of NF-κB ligand-differentiated RAW 264.7 cells. This library was screened with a bait construct consisting of a GAL4 binding domain fused to the N-terminal domain of V-ATPase a3 subunit (NTa3), the a subunit isoform that is highly expressed in osteoclasts (a1 and a2 are also expressed, to a lesser degree, whereas a4 is kidney-specific). One of the prey proteins identified was the V-ATPase B2 subunit, which is also highly expressed in osteoclasts (B1 is not expressed). Further characterization, using pulldown and solid-phase binding assays, revealed an interaction between NTa3 and the C-terminal domains of both B1 and B2 subunits. Dual B binding domains of equal affinity were observed in NTa, suggesting a possible model for interaction between these subunits in the V-ATPase complex. Furthermore, the a3-B2 interaction appeared to be moderately favored over a1, a2, and a4 interactions with B2, suggesting a mechanism for the specific subunit assembly of plasma membrane V-ATPase in osteoclasts. Solid-phase binding assays were subsequently used to screen a chemical library for inhibitors of the a3-B2 interaction. A small molecule benzohydrazide derivative was found to inhibit osteoclast resorption with an IC(50) of ∼1.2 μm on both synthetic hydroxyapatite surfaces and dentin slices, without significantly affecting RAW 264.7 cell viability or receptor activator of NF-κB ligand-mediated osteoclast differentiation. Further understanding of these interactions and inhibitors may contribute to the design of novel therapeutics for bone loss disorders, such as osteoporosis and rheumatoid arthritis.

V-ATPases are multimeric proton pumps. The 100-kDa "a" subunit is encoded by four isoforms (a1-a4) in mammals and two (Vph1p and Stv1p) in yeast. a3 is enriched in osteoclasts and is essential for bone resorption, whereas a4 is expressed in the distal nephron and acidifies urine. Mutations in human a3 and a4 result in osteopetrosis and distal renal tubular acidosis, respectively. Human a3 (G405R and R444L) and a4 (P524L and G820R) mutations were recreated in the yeast ortholog Vph1p, a3 (G424R and R462L), and a4 (W520L and G812R). Mutations in a3 resulted in wild type vacuolar acidification and growth on media containing 4 mM ZnCl2, 200 mM CaCl2, or buffered to pH 7.5 with V-ATPase hydrolytic and pumping activity decreased by 30-35%. Immunoblots confirmed wild type levels for V-ATPase a, A, and B subunits on vacuolar membranes. a4 G812R resulted in defective growth on selective media with V-ATPase hydrolytic and pumping activity decreased by 83-85% yet with wild type levels of a, A, and B subunits on vacuolar membranes. The a4 W520L mutation had defective growth on selective media with no detectable V-ATPase activity and reduced expression of a, A, and B subunits. The a4 W520L mutation phenotypes were dominant negative, as overexpression of wild type yeast a isoforms, Vph1p, or Stv1p, did not restore growth. However, deletion of endoplasmic reticulum assembly factors (Vma12p, Vma21p, and Vma22p) partially restored a and B expression. That a4 W520L affects both Vo and V1 subunits is a unique phenotype for any V-ATPase subunit mutation and supports the concerted pathway for V-ATPase assembly in vivo.

Impaired biosynthetic processing of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel, constitutes the most common cause of CF. Recently, we have identified a distinct category of mutation, caused by premature stop codons and frameshift mutations, which manifests in diminished expression of COOH-terminally truncated CFTR at the cell surface. Although the biosynthetic processing and plasma membrane targeting of truncated CFTRs are preserved, the turnover of the complex-glycosylated mutant is sixfold faster than its wild-type (wt) counterpart. Destabilization of the truncated CFTR coincides with its enhanced susceptibility to proteasome-dependent degradation from post-Golgi compartments globally, and the plasma membrane specifically, determined by pulse-chase analysis in conjunction with cell surface biotinylation. Proteolytic cleavage of the full-length complex-glycosylated wt and degradation intermediates derived from both T70 and wt CFTR requires endolysosomal proteases. The enhanced protease sensitivity in vitro and the decreased thermostability of the complex-glycosylated T70 CFTR in vivo suggest that structural destabilization may account for the increased proteasome susceptibility and the short residence time at the cell surface. These in turn are responsible, at least in part, for the phenotypic manifestation of CF. We propose that the proteasome-ubiquitin pathway may be involved in the peripheral quality control of other, partially unfolded membrane proteins as well.

Deletion of phenylalanine 508 (delta F508) in the cystic fibrosis transmembrane-conductance regulator (CFTR) prevents the otherwise functional protein from reaching the plasma membrane and is the leading cause of cystic fibrosis. Indirect evidence suggests that the mutant protein, delta F508 CFTR, is misfolded. We address this issue directly, using comparative limited proteolysis of CFTR at steady steady state and during biosynthesis in the native microsomal environment. Distinct protease susceptibilities suggest that cytosolic domain conformations of wild type and delta F508 CFTR differ, not only near F508, but globally. Moreover, delta F508 CFTR proteolytic cleavage patterns were indistinguishable from those of the early folding intermediate of wild type CFTR. The results suggest that the delta F508 mutation causes the accumulation of a form of the protein that resembles an intermediate in the biogenesis of the wild type CFTR, rather than induces the production of non-native variant.

Although the cystic fibrosis transmembrane conductance regulator (CFTR) is primarily implicated in the regulation of plasma-membrane chloride permeability, immunolocalization and functional studies indicate the presence of CFTR in the endosomal compartment. The mechanism of CFTR delivery from the cell surface to endosomes is not understood. To delineate the internalization pathway, both the rate and extent of CFTR accumulation in endosomes were monitored in stably transfected Chinese hamster ovary (CHO) cells. The role of clathrin-dependent endocytosis was assessed in cells exposed to hypertonic medium, potassium depletion or intracellular acid-load. These treatments inhibited clathrin-dependent endocytosis by >90%, as verified by measurements of 125I-transferrin uptake. Functional association of CFTR with newly formed endosomes was determined by an endosomal pH dissipation protocol [Lukacs, Chang, Kartner, Rotstein, Riordan and Grinstein (1992) J. Biol. Chem. 267, 14568-14572]. As a second approach, endocytosis of CFTR was determined after cell-surface biotinylation with the cleavable sulphosuccinimidyl-2-(biotinamido)ethyl-1,3-dithio- propionate. Both the biochemical and the functional assays indicated that arresting the formation of clathrin-coated vesicles inhibited the retrieval of the CFTR from the plasma membrane to endosomes. An overall arrest of membrane traffic cannot account for the inhibition of CFTR internalization, since the fluid-phase endocytosis was not effected by the treatments used. Thus the efficient, constitutive internalization of surface CFTR (5% per min) occurs, predominantly by clathrin-dependent endocytosis. Stimulation of protein phosphorylation by cAMP-dependent protein kinase A and by protein kinase C decreased the rate of internalization of cell-surface biotinylated CFTR, and contributed to a substantial diminution of the internal CFTR pool compared with that of unstimulated cells. These results suggest that the rate of CFTR internalization may participate in the determination of the CFTR channel density, and consequently, of the cAMP-stimulated chloride conductance of the plasma membrane.

The gene product affected in cystic fibrosis, the cystic fibrosis transmembrane conductance regulator (CFTR), is a chlorideselective ion channel that is regulated by cAMP-dependent protein kinase-mediated phosphorylation, ATP binding and ATP hydrolysis. Mutations in the CFTR gene may result in cystic fibrosis characterized by severe pathology (e.g. recurrent pulmonary infection, male infertility and pancreatic insufficiency) involving organs expressing the CFTR. Interestingly, in the kidney, where expression of the CFTR has been reported, impaired ion transport in patients suffering from cystic fibrosis could not be observed. To understand the role of the CFTR in chloride transport in the kidney, we attempted to identify an epithelial cell line that can serve as a model. We demonstrate that the CFTR is expressed constitutively in Madine-Darby canine kidney (MDCK) type I cells, which are thought to have originated from the distal tubule of the dog nephron. We show expression at the mRNA level, using reverse transcriptase-PCR, and at the protein level, using Western blot analysis with three different monoclonal antibodies. Iodide efflux measurements indicate that CFTR expression confers a plasma membrane anion conductance that is responsive to stimulation by cAMP. The cAMP-stimulated iodide release is sensitive to glybenclamide, diphenylamine carboxylic acid and 5-nitro-2-(3-phenylpropylamino)benzoic acid, but not to 4,4'-di-isothiocyanostilbene-2,2'-disulphonic acid, an inhibitor profile characteristic of the CFTR chloride channel. Finally, the polarized localization of the CFTR to the apical plasma membrane was established by iodide efflux measurements and cell-surface biotinylation on MDCK I monolayers. Interestingly, MDCK type II cells, which are thought to have originated from the proximal tubule of the kidney, lack CFTR protein expression and cAMP-stimulated chloride conductance. In conclusion, we propose that MDCK type I and II cells can serve as convenient model systems to study the physiological role and differential expression of CFTR in the distal and proximal tubule respectively.

Misprocessing and mislocalization of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) has been described for the major CF-causing mutation (delta F508) in heterologous expression systems in vitro. We have generated monoclonal antibodies (mAbs) to CFTR with the aim of localizing the protein and its CF variants in vivo. Of the tissues where CFTR was observed, only the sweat gland is readily available and does not undergo secondary changes due to CF disease pathology. Sweat ducts from CF patients homozygous for delta F508 did not show the typical apical membrane staining seen in control biopsies. This demonstrates that the biosynthetic arrest and intracellular retention of delta F508 CFTR initially observed in vitro does occur in vivo and emphasizes the need to focus efforts on understanding the mislocalization.

Circumstantial evidence has accumulated suggesting that CFTR is a regulated low-conductance Cl- channel. To test this postulate directly, we have purified to homogeneity a recombinant CFTR protein from a high-level baculovirus-infected insect cell line. Evidence of purity included one- and two-dimensional gel electrophoresis, N-terminal peptide sequence, and quantitative amino acid analysis. Reconstitution into proteoliposomes at less than one molecule per vesicle was accomplished by established procedures. Nystatin and ergosterol were included in these vesicles, so that nystatin conductance could serve as a quantitative marker of vesicle fusion with a planar lipid bilayer. Upon incorporation, purified CFTR exhibited regulated chloride channel activity, providing evidence that the protein itself is the channel. This activity exhibited the basic biophysical and regulatory properties of the type of Cl- channel found exclusively in CFTR-expressing cell types and believed to underlie cAMP-evoked secretion in epithelial cells.

Independent lines of Chinese hamster ovary cells resistant to the antineoplastic drug, daunorubicin, were obtained by clonal isolation in increasing drug concentrations. A single daunorubicin-resistant phenotype typified by reduced cellular drug accumulation was observed. These mutants displayed a complex phenotype of resistance to a variety of unrelated drugs. Such properties are similar to those of membrane-altered colchicine-resistant lines (V. Ling and L.H. Thompson, J. Cell. Physiol., 83: 103-116, 1974). Analysis of the plasma membrane components of the daunorubicin-resistant clones by gel electrophoresis revealed a prominent cell surface glycoprotein with a molecular weight of about 170,000. This component was immunologically cross-reactive with the cell surface P-glycoprotein of about the same molecular weight, previously identified in colchicine-resistant cells. Thus, it appears that the mechanism of resistance characterized by P-glycoprotein expression could be the basis of many drug-resistant phenotypes.

Chemotherapy often fails because a tumor develops resistance to an array of different drugs. A single glycoprotein turns out to be responsible: it proliferates in some cells and pumps out the drugs. Now that the protein pump has been identified it may be possible to interfere with its action or to make it the target for drugs that destroy the cancer cell.

Multidrug resistance describes a complex phenotype whose predominant feature is resistance to a wide range of structurally unrelated cytotoxic compounds, many of which are anticancer agents. This phenotype occurs frequently in mammalian cell lines and transplantable tumours selected for resistance to a single drug. Reduced cellular accumulation of the drugs involved appears to account for the resistance. This may be a consequence of reduced drug influx, increased drug efflux, or both. A wide variety of biochemical changes have been identified in multidrug resistant cell lines, the most consistent of which is the increased expression of P-glycoprotein, a conserved, high molecular weight, plasma membrane glycoprotein. The level of P-glycoprotein expression correlates with the degree of drug resistance in a variety of different cell types. In a number of multidrug resistant cell lines, overexpression of P-glycoprotein results from gene amplification. While the function of P-glycoprotein is unknown, independent lines of evidence support the notion that P-glycoprotein is the causative molecule mediating the multidrug resistance phenotype. Significant levels of P-glycoprotein expression have been detected in some biopsy specimens from patients with ovarian and sarcoma tumours. These findings suggest that multidrug resistant tumour cells can occur in human malignancies. The presence of such cells may affect the outcome of chemotherapy.

The plasma membranes of hamster, mouse, and human tumor cell lines that display multiple resistance to drugs were examined by gel electrophoresis and immunoblotting. In every case, increased expression of a 170,000-dalton surface antigen was found to be correlated with multidrug resistance. This membrane component is of identical molecular size and shares some immunogenic homology with the previously characterized P-glycoprotein of colchicine-resistant Chinese hamster ovary cells. This finding may have application to cancer therapy.

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.

Multidrug resistance is a complex pleiotropic phenotype of cross-resistance and collateral sensitivity to unrelated compounds observed in many mammalian cell mutants selected for resistance to single agents. In Chinese hamster ovary cells, colchicine-resistant mutants expressing this phenotype have been characterized extensively. Such mutants arise apparently from a single genetic event, and the basis of this phenotype appears to be localized at the membrane level, resulting in altered drug permeability. Expression of a 170,000-dalton surface glycoprotein (P-glycoprotein) has been identified to correlate with the multidrug-resistance phenotype. Selection of a second mutation in colchicine-resistant mutants, for resistance to phytohemagglutinin, results in an alteration of the carbohydrate moiety in P-glycoprotein and other surface components. This mutation does not noticeably affect the multi-drug-resistance phenotype. The altered permeability of mutant cells to drugs, however, can be modulated by nonionic detergents or metabolic inhibitors. These findings are consistent with a molecular mechanism of multidrug resistance whereby the pleiotropic response of the cell is mediated by an overexpression of a cell-surface protein, the P-glycoprotein.

The multidrug-resistance phenotype expressed in mammalian cell lines is complex. Cells selected with a single agent can acquire cross-resistance to a remarkably wide range of compounds which have no obvious structural or functional similarities. The basis for cross-resistance seems to be a decreased net cellular accumulation of the drug involved, and has been attributed to alterations in the plasma membrane. An over-expressed plasma membrane glycoprotein of relative molecular mass (Mr) 170,000 (P-glycoprotein) is consistently found in different multidrug-resistant human and animal cell lines, and in transplantable tumours. Consequently, it has been postulated that P-glycoprotein directly or indirectly mediates multidrug resistance. Here we report the cloning of a complementary DNA encoding P-glycoprotein. Southern blot analysis of hamster, mouse and human DNA using this cDNA as a probe showed that P-glycoprotein is conserved and is probably encoded by a gene family, and that members of this putative family are amplified in multidrug-resistant cells.

One reason for the failure of chemotherapy in the treatment of advanced cancers may be the outgrowth of multidrug-resistant tumour cells. Multidrug resistance has been modelled in numerous mammalian cell lines in which the phenotype is characterized by a pleiotropic cross-resistance to unrelated drugs. In the study reported here, we have produced monoclonal antibodies whose binding to plasma membranes of different multidrug-resistant mammalian cells correlates with the degree of drug resistance. All these antibodies are specific for P-glycoprotein, a cell surface component of relative molecular mass (Mr) 170,000 (170K) that has been described previously, and are directed against three spatially distinct epitopes which define a conserved cytoplasmic domain in the C-terminal region of the P-glycoprotein polypeptide. The conserved nature of P-glycoprotein and its low-level expression is drug-sensitive cells suggest that it has an important function at the cell surface. The monoclonal antibodies against P-glycoprotein described here might serve as diagnostic reagents for clinically unresponsive tumours.