Upregulation of caveolin in multidrug resistant cancer cells: functional implications.

Multidrug resistance (MDR) is a multifactorial process that involves elevated expression of drug transporters as well as additional biochemical changes that contribute to the drug resistant phenotype. Here we review recent results indicating the upregulation of constituents of rafts and caveolae, including glucosylceramide, cholesterol and caveolin-1, in MDR cells. Accordingly, the number of plasma membrane caveolae is greatly increased in MDR cells. The relationship between caveolin and MDR may be linked to the function of caveolin-1 in mediating cholesterol efflux, a pathway that we hypothesized to facilitate the delivery of drugs from intracellular compartments to plasma membrane resident drug transporters. An additional link seems to exist between the upregulation of GlcCer synthase and attenuation of ceramide-mediated apoptotic signaling. These adaptations may promote cell survival during chemotherapy and, hence, would be positively selected during cell exposure to cytotoxic drugs. However, the overexpression of caveolin-1, an oncosuppressive protein, may also reverse or attenuate important aspects of the phenotypic transformation of MDR cells. The molecular mechanisms by which caveolin-1 exerts its effects on cell proliferation, cell survival, and multidrug resistance remain to be fully elucidated.

Inhibitory effect of steroidal alkaloids on drug transport and multidrug resistance in human cancer cells.

Intrinsic or acquired resistance of tumor cells to multiple cytotoxic drugs (multidrug resistance MDR) is a major cause of failure of cancer chemotherapy. MDR is often caused by elevated expression of drug transporters such as P-glycoprotein (P-gp) or multidrug resistance protein (MRP). A number of compounds, termed chemosensitizers, have little or no cytotoxic action of their own, but inhibit (P-gp) or MRP-mediated drug export and are capable of sensitizing MDR cells to the cytotoxic effects of chemotherapeutic drugs. Here we examined the ability of steroidal alkaloids of plant origin, namely the Veratrum sp. alkaloid cyclopamine and the Lycopersicon sp. alkaloid tomatidine, to act as potent and effective chemosensitizers in multidrug resistant tumor cells. Drug uptake was determined by measuring accumulation of tetramethylrosamine in multidrug resistant NCI AdrR human adenocarcinoma cells. Resistance to adriamycin and vinblastine was determined by utilizing the MTT cell survival assay. Cyclopamine and tomatidine elevate tetramethylrosamine uptake by NCI AdrR cells and sensitize the cells to the cytotoxic action of adriamycin and vinblastine. In both cases these agents are comparable in patency and efficacy to verapamil, a reversal agent commonly used in MDR research. It is concluded that steroidal alkaloids of plant origin act as inhibitors of P-gp-mediated drug transport and multidrug resistance and therefore may serve as chemosensitizers in combination chemotherapy with conventional cytotoxic drugs for treating multidrug resistant cancer.

Multidrug resistance: a role for cholesterol efflux pathways?

Multidrug resistance (MDR) severely impairs the efficacy of cancer chemotherapy. Several protein transporters that mediate drug export have been identified, but additional adaptations appear to be necessary for full-fledged drug resistance. The cell surface density of caveolae and the expression of the caveolar coat protein caveolin are dramatically increased in MDR cancer cells. Acquisition of MDR might thus be accompanied by upregulation of caveolin-dependent cholesterol efflux pathways, raising the possibility that these same pathways are utilized for delivering drugs from intracellular compartments to the plasma membrane, where drugs can be extruded from the cells by drug efflux ATPases. The upregulation of caveolin mandates a phenotypic change of MDR cells in terms of their cholesterol homeostasis and is accompanied by loss of important features of the transformed phenotype of MDR cancer cells.

Changes in phospholipase D isoform activity and expression in multidrug-resistant human cancer cells.

Multidrug resistance (MDR) is a major cause of failure of cancer chemotherapy and is often associated with elevated expression of drug transporters such as P-glycoprotein (P-gp) in the cancer cells. MDR is, however, accompanied by additional biochemical changes including modifications of membrane composition and properties. We have shown that MDR is associated with a massive up-regulation of caveolin expression and an elevated surface density of caveolae. We report that phospholipase D (PLD), a constituent enzyme of caveolae and detergent-insoluble glycolipid-rich membranes (DIGs), is up-regulated in human MDR cancer cells. Lysates of HT-29-MDR human colon adenocarcinoma cells, MCF-7 AdrR human breast adenocarcinoma cells and the corresponding parental drug-sensitive cells, were fractionated on discontinuous sucrose density gradients. PLD activity was found to be enriched in low density fractions that contain DIGs and caveolar membranes, and the activity in these fractions was 4- to 6-fold higher in the MDR cells compared with the parental drug- sensitive cells. Utilizing specific antibodies to PLD1 and PLD2, the distribution of PLD isoforms along the gradient was determined and the PLD localized in DIGs and caveolar membranes has been identified as PLD2. Northern blot analysis of PLD1 and PLD2 mRNA levels has indicated that PLD2 mRNA is elevated in both HT-29-MDR and MCF-7 AdrR cells. PLD1 mRNA levels were either unchanged or reduced in the MDR cells. Finally, in vivo experiments have confirmed previous results showing that activation of PLD by phorbol esters is markedly potentiated in the MDR cells. We conclude that MDR is accompanied by an increase in PLD2 activity in DIGs and caveolar membranes.

Phospholipase D2: functional interaction with caveolin in low-density membrane microdomains.

Low-density detergent-insoluble membrane domains contain caveolin-1 and are enriched in a phospholipase D activity that is not PLD1. Here we show that caveolin-rich fractions, prepared from HaCaT human keratinocytes by either detergent-based or detergent-free methods, contain PLD2. Caveolar membrane PLD activity is stimulated 2-fold by low concentrations (10-30 microM) of the caveolin-1 and caveolin-2 scaffolding domain peptides, whereas it is inhibited at higher concentrations of the peptides. Immunoisolated HA-tagged PLD1 and PLD2 are not stimulated by the peptides, although both enzymes retain sensitivity to their inhibitory effect. Down-regulation of caveolin-1 expression by treatment of the cells with acetyl-leucyl-leucyl-norleucinal decreased caveolar PLD activity by 50%. Similarly, expression of an active form of the sterol regulatory element-binding protein (SREBP(1-490)) down-regulated caveolin-1 expression by 50% and decreased caveolar PLD activity by 60%. These data identify the PLD activity in caveolin-rich membranes as PLD2 and provide in vivo evidence suggesting that caveolin-1 regulates PLD2 activity.

Phospholipase D: molecular and cell biology of a novel gene family.

Interaction of extracellular-signal molecules with cell-surface receptors often activates a phospholipase D (PLD)-mediated hydrolysis of phosphatidylcholine and other phospholipids, generating phosphatidic acid. The activation of PLD is believed to play an important role in the regulation of cell function and cell fate. Multiple PLD activities were characterized in eukaryotic cells, and, more recently, several PLD genes have been cloned. A PLD gene superfamily, defined by a number of structural domains and sequence motifs, also includes phosphatidyltransferases and certain phosphodiesterases. Among the eukaryotic PLD genes are those from mammals, nematodes, fungi and plants. The present review focuses on the structure, localization, regulation and possible functions of cloned mammalian and yeast PLDs. In addition, an overview of plant PLD genes, and of several distinct PLD activities that have not yet been cloned, is provided. Emerging evidence from recent work employing new molecular tools indicates that different PLD isoforms are localized in distinct cellular organelles, where they are likely to serve diverse functions in signal transduction, membrane vesicle trafficking and cytoskeletal dynamics.

Changes in lipid and protein constituents of rafts and caveolae in multidrug resistant cancer cells and their functional consequences.

The carcinogenic process involves a complex series of genetic and biochemical changes that enables transformed cells to proliferate, migrate to secondary sites and, in some cases, acquire mechanisms that make cancer cells resistant to chemotherapy. This phenomenon in its most common form is known as multidrug resistance (MDR). It is usually mediated by overexpression of P-glycoprotein (P-gp) or other plasma membrane ATPases that export cytotoxic drugs used in chemotherapy, thereby reducing their efficacy. However, additional adaptive changes are likely to be required in order to confer a full MDR phenotype. Recent studies have shown that acquisition of MDR is accompanied by upregulation of lipids and proteins that constitute lipid rafts and caveolar membranes, notably glucosylceramide and caveolin. These changes may be related to the fact that in MDR cells a significant fraction of cellular P-gp is associated with caveolin-rich membrane domains, they may be involved in drug transport and they could have an impact on drug-induced apoptosis and on the phenotypic transformation of MDR cancer cells.

Changes in membrane microdomains and caveolae constituents in multidrug-resistant cancer cells.

Cancer chemotherapy often fails because of the development of tumors which are resistant to most commonly used cytotoxic drugs. This phenomenon, multidrug resistance (MDR), is usually mediated by overexpression of P-glycoprotein (P-gp), an ATPase that pumps out the drugs used in chemotherapy, thereby preventing their accumulation in cancer cells and greatly reducing their cytotoxic efficacy. A large body of work indicates that MDR is associated also with marked changes in membrane lipid composition. Most notably, elevated levels of cholesterol, glycosphingolipids (e.g., glucosylceramide), and sphingomyelin have been reported. These lipids are enriched in caveolae and in membrane microdomains termed detergent-insoluble glycosphingolipid-enriched complexes (DIGs). Recently we demonstrated that in multidrug-resistant tumor cells there is a dramatic increase in the number of caveolae and in the level of caveolin-1, an essential structural constituent of caveolae. Another constituent of membrane microdomains, phospholipase D, is also elevated in MDR cells. These findings may be related to the fact that a significant fraction of cellular P-gp is associated with caveolin-rich membrane domains. The possible role of DIGs and caveolae in the acquisition and/or maintenance of the multidrug resistant phenotype is discussed.

Localization and possible functions of phospholipase D isozymes.

The activation of PLD is believed to play an important role in the regulation of cell function and cell fate by extracellular signal molecules. Multiple PLD activities have been characterized in mammalian cells and, more recently, several PLD genes have been cloned. Current evidence indicates that diverse PLD activities are localized in most, if not all, cellular organelles, where they are likely to subserve different functions in signal transduction, membrane vesicle trafficking and cytoskeletal dynamics.

Localization of phospholipase D in detergent-insoluble, caveolin-rich membrane domains. Modulation by caveolin-1 expression and caveolin-182-101.

The activation of cellular phospholipase D (PLD) is implicated in vesicular trafficking and signal transduction. Two mammalian PLD forms, designated PLD1 and PLD2, have been cloned, but their cellular localization and function are not fully understood. Here, we report that in HaCaT human keratinocytes, as well as other cell lines, PLD activity is highly enriched in low density, Triton X-100-insoluble membrane domains that contain the caveolar marker protein caveolin-1. Similar to other PLDs, the PLD activity in these membrane domains is stimulated by phosphatidylinositol 4, 5-bisphosphate and is inhibited by neomycin. Immunoblot analysis indicated that caveolin-rich membrane domains do not contain the PLD1 isoform. Stable transfection of mouse PLD2 in Chinese hamster ovary cells greatly increased PLD activity in these domains compared with PLD activity in control Chinese hamster ovary cells transfected with vector alone. PLD activity is enriched in low density Triton-insoluble membrane domains also in U937 promonocytes, even though these cells do not express caveolin-1. In U937 cells, also, PLD1 is largely excluded from low density Triton-insoluble membrane domains. Expression of recombinant caveolin-1 in v-Src-transformed NIH-3T3 cells resulted in up-regulation of PLD activity in the caveolin-containing membrane domains. The caveolin scaffolding peptide (caveolin-182-101) modulated the caveolar PLD activity, causing stimulation at concentration of 1-10 microM and inhibition at concentrations >10 microM. We conclude that a PLD activity, which is likely to represent PLD2, is enriched in low density Triton-insoluble membrane domains. The effects of caveolin-1 expression and of the caveolin scaffolding peptide suggest that in cells that express caveolin-1, PLD may be targeted to caveolae. The possible functions of PLD in the dynamics of caveolae and related domains and in signal transduction processes are discussed.

Up-regulation of caveolae and caveolar constituents in multidrug-resistant cancer cells.

Cancer chemotherapy often results in the development of multidrug resistance (MDR), which is commonly associated with overexpression of P-glycoprotein (P-gp), a plasma membrane drug efflux ATPase. It was shown recently that glycosphingolipids are elevated in MDR cells. Sphingolipids are major constituents of caveolae and of detergent-insoluble, glycosphingolipid-rich membrane domains. Here we report that multidrug-resistant HT-29 human colon adenocarcinoma cells exhibit a 12-fold overexpression of caveolin-1, a 21-kDa coat/adaptor protein of caveolae. Similar observations were made in adriamycin-resistant MCF-7 human breast adenocarcinoma cells. Caveolin-2 expression is also up-regulated in MCF-7-AdrR cells, but neither caveolin-1 nor caveolin-2 were detected in MCF-7 cells stably transfected with P-gp. The up-regulation of caveolins is associated with a 5-fold increase in the number of caveolae-like structures observed in plasma membrane profiles of HT-29-MDR cells and with the appearance of a comparable number of caveolae in MCF-7-AdrR cells. A significant fraction (approximately 40%) of cellular P-gp is localized in low density detergent-insoluble membrane fractions derived from either HT-29-MDR or MCF-7-AdrR cells. The distribution of recombinant P-gp in stably transfected MCF-7 cells was similar, even though these cells do not express caveolins and are devoid of caveolae. The possibility that caveolae contribute to the multidrug resistance and thus are co-selected with P-gp during the acquisition of this phenotype is discussed.

Kinetic analysis in mixed micelles of partially purified rat brain phospholipase D activity and its activation by phosphatidylinositol 4,5-bisphosphate.

A partially purified rat brain membrane phospholipase D (PLD) activity was characterized in a mixed micellar system consisting of 1-palmitoyl-2-[6-N-(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)-amino]capr oyl-phosphatidylcholine (NBD-PC) and Triton X-100, under conditions where Triton X-100 has a surface dilution effect on PLD activity and the catalytic rate is dependent on the surface concentration (expressed in terms of molar ratio) of NBD-PC. PLD activity was specifically activated by phosphatidylinositol 4,5-bisphosphate (PIP2), and the curve of activation versus PIP2 molar ratio fitted a Michaelis-Menten equation with a K(act) value between molar ratios of 0.001-0.002. Maximal activation was observed at a PIP2 molar ratio of 0.01. Similar values were obtained when activities of partially purified PLD as well as membrane-bound PLD were determined towards pure NBD-PC micelles. In the mixed micellar system PIP2 was shown to elevate by 6-22 fold the specificity constant of PLD towards NBD-PC (K(A), which is proportional to Vmax/Km). Kinetic analysis of PLD trans-phosphatidylation activity towards ethanol, 1-propanol and 1-butanol revealed a Michaelis-Menten type dependence on alcohol concentration up to 1000, 200 and 80 mM, respectively. While Vmax values were similar towards all three alcohols, enzyme affinity increased as the alcohol was longer, and Km values for ethanol, 1-propanol and 1-butanol were 291, 75 and 16 mM (respectively). PLD specificity constants (K(A)) towards ethanol, 1-propanol and 1-butanol were shown to be respectively 260, 940 and 5,920 times higher than to water, the competing substrate. 1-Propanol and 1-butanol inhibited PLD activity above 400 and 100 mM, respectively. The present results indicate that partially purified PLD obeys surface dilution kinetics with regard to its phospholipid substrate PC and its cofactor PIP2, and that in the presence of alcohols, its transphosphatidylation activity may be analyzed as a competitive reaction to the hydrolysis reaction.

Ras GTPase-activating protein-associated p62 is a major v-Src-SH3-binding protein.

Oncogenic transformation by v-Src is accompanied by marked morphological changes and cytoskeletal reorganization. Yet, the cytoskeleton-associated proteins with which v-Src interacts are largely unknown. We have studied the binding of v-Src-SH3 domain to cellular proteins utilizing a blot overlay procedure with a GST-v-Src-SH3 fusion protein as probe. A major 62-64 kDa v-Src-SH3-binding protein, present in detergent-insoluble cellular fractions, was identified as p21ras-GTPase-activating protein-associated p62 (GAPA62). In non-transformed cells, including NIH 3T3 cells, GAPA62 was present in both the RIP A-soluble and RIP A-insoluble fractions, but only the latter form was tyrosine-phosphorylated. In contrast, in polyoma middle T antigen-transformed 3T3 cells, GAPA62 was present only in the RIP A-insoluble fraction, where it was highly phosphorylated. It is suggested that tyrosine phosphorylation of GAPA62 may be an important determinant of its cellular localization and its possible function as a mediator of v-Src actions.

Identification of a novel Ca2+-dependent, phosphatidylethanolamine-hydrolyzing phospholipase D in yeast bearing a disruption in PLD1.

We have previously reported the identification and partial characterization of a gene encoding a phospholipase D activity (PLD1) in the yeast, Saccharomyces cerevisiae. Here we report the existence of a second phospholipase D activity, designated PLD2, in yeast cells bearing disruption at the PLD1 locus. PLD2 is a Ca2+-dependent enzyme which preferentially utilizes phosphatidylethanolamine over phosphatidylcholine as a substrate. In contrast to PLD1, the activity of PLD2 is insensitive to phosphatidylinositol 4,5-bisphosphate, and the enzyme is incapable of catalyzing the transphosphatidylation reaction with short chain alcohols as acceptors. Subcellular fractionation shows that PLD2 localizes mainly to the cytosol, but could also be detected in the particulate fraction. Thus, the biochemical properties of PLD2 appear to be substantially different from those of PLD1. PLD2 activity is significantly and transiently elevated upon exit of wild type yeast cells from stationary phase, suggesting that it may play a role in the initiation of mitotic cell division in yeast. In view of the significantly different properties of PLD1 and PLD2, and because the yeast genome contains PLD1 as the sole member of the recently defined PLD gene family, it may be concluded that PLD2 is structurally unrelated to PLD1. Thus, the novel PLD2 activity described herein is likely to represent the first identified member of a new PLD gene family.

Phospholipase D: role in signal transduction and membrane traffic.

The activation of phospholipase D (PLD) in response to cell stimulation by extracellular signal molecules is a widespread phenomenon. A variety of extracellular signal molecules cause a rapid and dramatic stimulation of PLD activity. G proteins and protein kinases appear to be involved in the receptor-mediated regulation of PLD. There is indirect evidence for the existence of multiple PLD subtypes, both membrane-associated and cytosolic. Recent studies indicate that PLD activities require a lipid cofactor, phosphatidylinositol 4,5-bisphosphate (PIP2). Addition of PIP2 at physiological concentrations stimulates both membrane-associated and partially purified PLD activity. Other acidic phospholipids have little or no effect. Neomycin, a high affinity ligand of PIP2, inhibits membrane PLD activity, presumably by binding to endogenous PIP2. A monoclonal antibody to phosphatidylinositol 4-kinase inhibits PIP2 synthesis in permeabilized U937 cells and blocks PLD activation by GTP gamma S and TPA. These results indicate that PIP2 synthesis is required for G protein- and protein kinase C-mediated activation of PLD in the cells. Recent evidence has implicated PLD and phosphoinositide kinases in vesicular trafficking. The main lipid mediator produced by PLD, phosphatidic acid, could regulate membrane traffic events by direct regulation of target proteins involved in vesicle targeting, docking and fusion. In addition, under certain circumstances, the formation of phosphatidic acid may lead to changes in lipid bilayer properties that would facilitate vesicle budding and fusion events in the course of intracellular membrane traffic.

Enzymology of mammalian phospholipases D: in vitro studies.

The existence of multiple forms of phopholipase D was clearly established in a large number of biochemical studies that described and characterized the enzymological properties of the different PLD activities. This review summarizes the in vitro evidence showing differential subcellular localization and chromatographic properties of putative PLD isozymes, their phospholipid and alcohol substrate specificities, their modulation by various divalent cations, small G proteins and protein kinase c isozymes, and the role of phosphatidylinositol 4,5-bisphosphate as a cofactor of phospholipase D.

Identification and characterization of a gene encoding phospholipase D activity in yeast.

We have identified an open reading frame on chromosome XI of the yeast, Saccharomyces cerevisiae, as encoding a protein with phospholipase D (PLD) activity. We have named this open reading frame, PLD1, and show that yeast bearing a disruption in this gene are unable to catalyze the hydrolysis of phosphatidylcholine. PLD1 encodes a hypothetical protein of 1683 amino acids and has a predicted molecular mass of 195 kDa. Yeast bearing disruptions at the PLD1 locus are morphologically normal and grow vegetatively like wild-type cells. In contrast, homozygous delta pld1 diploid cells are unable to sporulate and do not produce asci under conditions that induce meiosis and sporulation in wild-type cells. Thus, PLD1 is likely to be essential for the meiotic cycle in yeast cells. This is the first identification of a eukaryotic, nonplant, phosphatidylcholine-hydrolyzing phospholipase D gene. Because the biological role of PLD is not well understood, we expect that delta pld1 yeast will become a useful tool for the characterization of PLD functions as well as for the identification of mammalian PLD homologs.

Release of gelatinase A (matrix metalloproteinase 2) induced by photolysis of caged phosphatidic acid in HT 1080 metastatic fibrosarcoma cells.

Phosphatidic acid (PA) is a putative novel messenger in signal transduction and membrane traffic. We have synthesized a photolyzable derivative of PA, termed caged PA (cPA), which may be utilized as a new tool in studies of PA-mediated cellular events. 1-(2-Nitrophenyl)diazoethane, synthesized from 2-nitroacetophenone, was reacted with dipalmitoyl-PA to yield a 1-(2-nitrophenyl)ethyl ester of PA. Photolysis of the compound by ultraviolet light resulted in the formation of phosphatidic acid. The structure of the compound and of its photolytic products was verified by NMR spectroscopy. The utility of cPA was examined in HT 1080 metastatic fibrosarcoma cells, in which the formation of PA by phospholipase D was implicated in laminin-induced release of gelatinase A (matrix metalloproteinase 2 (MMP-2)). The uptake of cPA by HT 1080 cells reached a plateau after 120 min of incubation. Ultraviolet illumination of cPA-loaded cells for 5 s resulted in photolysis of 1.8% of the cell-incorporated cPA. The photolysis of cPA caused a 2-fold elevation in the release of MMP-2 to the medium, whereas nonphotolyzed cPA caused no change in MMP-2 release. Moreover, the effect of cPA photolysis was significantly higher than that obtained with extracellularly introduced PA. Thus, the effect of laminin on MMP-2 secretion can be mimicked by photolysis of cPA, suggesting a pivotal role for phospholipase D in laminin-induced cancer cell invasiveness and metastasis. These results indicate that cPA could serve as a unique tool for studying the cellular roles of PA.