PtK1 cells contain a nondiffusible, dominant factor that makes the Golgi apparatus resistant to brefeldin A

Brefeldin A (BFA) was shown in earlier studies of numerous cell types to inhibit secretion, induce enzymes of the Golgi stacks to redistribute into the ER, and to cause the Golgi cisternae to disappear. Here, we demonstrate that the PtK1 line of rat kangaroo kidney cells is resistant to BFA. The drug did not disrupt the morphology of the Golgi complex in PtK1 cells, as judged by immunofluorescence using antibodies to 58- (58K) and 110-kD (beta-COP) Golgi proteins, and by fluorescence microscopy of live cells labeled with C6-NBD-ceramide. In addition, BFA did not inhibit protein secretion, not alter the kinetics or extent of glycosylation of the vesicular stomatitis virus (VSV) glycoprotein (G-protein) in VSV-infected PtK1 cells. To explore the mechanism of resistance to BFA, PtK1 cells were fused with BFA-sensitive CV-1 cells that had been infected with a recombinant SV-40 strain containing the gene for VSV G-protein and, at various times following fusion, the cultures were exposed to BFA. Shortly after cell fusion, heterokaryons contained one Golgi complex associated with each nucleus. Golgi membranes derived from CV-1 cells were sensitive to BFA, whereas those of PtK1 origin were BFA resistant. A few hours after fusion, most heterokaryons contained a single, large Golgi apparatus that was resistant to BFA and contained CV-1 galactosyltransferase. In unfused cells that had been perforated using nitrocellulose filters, retention of beta-COP on the Golgi was optimal in the presence of cytosol, ATP, and GTP. In perforated cell models of the BFA-sensitive MA104 line, BFA caused beta-COP to be released from the Golgi complex in the presence of nucleotides, and either MA104 or PtK1 cytosol. In contrast, when perforated PtK1 cells were incubated with BFA, nucleotides, and cytosol from either cell type, beta-COP remained bound to the Golgi complex. We conclude that PtK1 cells contain a nondiffusible factor, which is located on or very close to the Golgi complex, and confers a dominant resistance to BFA. It is possible that this factor is homologous to the target of BFA in cells that are sensitive to the drug.     

Brefeldin A's effects on endosomes, lysosomes, and the TGN suggest a general mechanism for regulating organelle structure and membrane traffic.

Addition of brefeldin A (BFA) to most cells results in both the formation of extensive, uncoated membrane tubules through which Golgi components redistribute into the ER and the failure to transport molecules out of this mixed ER/Golgi system. In this study we provide evidence that suggests BFA's effects are not limited to the Golgi apparatus but are reiterated throughout the central vacuolar system. Addition of BFA to cells resulted in the tubulation of the endosomal system, the trans-Golgi network (TGN), and lysosomes. Tubule formation of these organelles was specific to BFA, shared near identical pharmacologic characteristics as Golgi tubules and resulted in targeted membrane fusion. Analogous to the mixing of the Golgi with the ER during BFA treatment, the TGN mixed with the recycling endosomal system. This mixed system remained functional with normal cycling between plasma membrane and endosomes, but traffic between endosomes and lysosomes was impaired.     

MDR1 P-glycoprotein reduces influx of substrates without affecting membrane potential

MDR1 (multidrug resistance) P-glycoprotein (Pgp; ABCB1) decreases intracellular concentrations of structurally diverse drugs. Although Pgp is generally thought to be an efflux transporter, the mechanism of action remains elusive. To determine whether Pgp confers drug resistance through changes in transmembrane potential (E(m)) or ion conductance, we studied electrical currents and drug transport in Pgp-negative MCF-7 cells and MCF-7/MDR1 stable transfectants that were established and maintained without chemotherapeutic drugs. Although E(m) and total membrane conductance did not differ between MCF-7 and MCF-7/MDR1 cells, Pgp reduced unidirectional influx and steady-state cellular content of Tc-Sestamibi, a substrate for MDR1 Pgp, without affecting unidirectional efflux of substrate from cells. Depolarization of membrane potentials with various concentrations of extracellular K(+) in the presence of valinomycin did not inhibit the ability of Pgp to reduce intracellular concentration of Tc-Sestamibi, strongly suggesting that the drug transport activity of MDR1 Pgp is independent of changes in E(m) or total ion conductance. Tetraphenyl borate, a lipophilic anion, enhanced unidirectional influx of Tc-Sestamibi to a greater extent in MCF-7/MDR1 cells than in control cells, suggesting that Pgp may, directly or indirectly, increase the positive dipole potential within the plasma membrane bilayer. Overall, these data demonstrate that changes in E(m) or macroscopic conductance are not coupled with function of Pgp in multidrug resistance. The dominant effect of MDR1 Pgp in this system is reduction of drug influx, possibly through an increase in intramembranous dipole potential.     

Co-translational effects of temperature on membrane insertion and orientation of P-glycoprotein sequences

P-glycoprotein (pgp) is a membrane transport protein that causes multidrug resistance (MDR) by actively extruding a wide variety of cytotoxic agents out of cells. It may also function as a peptide transporter, a volume-regulated chloride channel, and an ATP channel. Previously, it has been shown that hamster pgp1 Pgp is expressed in more than one topological form and that the generation of these structures is modulated by charged amino acids flanking the predicted transmembrane (TM) segments 3 and 4 and by soluble cytoplasmic factors. Different topological structures of Pgp may be related to its different functions. In this study, we examined the effects of translation temperature on the membrane insertion process and the topologies of Pgp. Using the rabbit reticulocyte lysate expression system, we showed that translation at different temperatures affects the membrane insertion and orientation of the putative TM3 and TM4 of hamster pgp1 Pgp in a co-translational manner. This observation suggests that the membrane insertion process of TM3 and TM4 of Pgp molecules may involve a protein conducting channel and/or the interaction between TM3 and TM4, which act in a temperature sensitive manner. We speculate that manipulating temperature may provide a way to understand the structure-function relationship of Pgp and help overcome Pgp-related multidrug resistance of cancer cells.Abbreviations Pgp P-glycoprotein - MDR multidrug resistance - ABC ATP-binding cassette - RRL rabbit reticulocyte lysate - TM transmembrane - RM rough microsomes - ER endoplasmic reticulum     

Disruption of endoplasmic reticulum to Golgi transport leads to the accumulation of large aggregates containing beta-COP in pancreatic acinar cells.

When transport between the rough endoplasmic reticulum (ER) and Golgi complex is blocked by Brefeldin A (BFA) treatment or ATP depletion, the Golgi apparatus and associated transport vesicles undergo a dramatic reorganization. Because recent studies suggest that coat proteins such as beta-COP play an important role in the maintenance of the Golgi complex, we have used immunocytochemistry to determine the distribution of beta-COP in pancreatic acinar cells (PAC) in which ER to Golgi transport was blocked by BFA treatment or ATP depletion. In controls, beta-COP was associated with Golgi cisternae and transport vesicles as expected. Upon BFA treatment, PAC Golgi cisternae are dismantled and replaced by clusters of remnant vesicles surrounded by typical ER transitional elements that are generally assumed to represent the exit site of vesicular carriers for ER to Golgi transport. In BFA-treated PAC, beta-COP was concentrated in large (0.5-1.0 micron) aggregates closely associated with remnant Golgi membranes. In addition to typical ER transitional elements, we detected a new type of transitional element that consists of specialized regions of rough ER (RER) with ribosome-free ends that touched or extended into the beta-COP containing aggregates. In ATP-depleted PAC, beta-COP was not detected on Golgi membranes but was concentrated in similar large aggregates found on the cis side of the Golgi stacks. The data indicate that upon arrest of ER to Golgi transport by either BFA treatment or energy depletion, beta-COP dissociates from PAC Golgi membranes and accumulates as large aggregates closely associated with specialized ER elements. The latter may correspond to either the site of entry or exit for vesicles recycling between the Golgi and the RER.     

Multidrug resistance (MDR1) P-glycoprotein enhances esterification of plasma membrane cholesterol

Class I P-glycoproteins (Pgp) confer multidrug resistance in tumors, but the physiologic function of Pgp in normal tissues remains uncertain. In cells derived from tissues that normally express Pgp, recent data suggest a possible role for Pgp in cholesterol trafficking from the plasma membrane to the endoplasmic reticulum. We investigated the esterification of plasma membrane cholesterol under basal conditions and in response to sphingomyelinase treatment in transfected and drug-selected cell lines expressing differing amounts of functional class I Pgp. Compared with parental NIH 3T3 fibroblasts, cells transfected with human multidrug resistance (MDR1) Pgp esterified more cholesterol both without and with sphingomyelinase. Esterification also was greater in drug-selected Dox 6 myeloma cells than parental 8226 cells, which express low and non-immunodetectable amounts of Pgp, respectively. However, no differences in total plasma membrane cholesterol were detected. Transfection of fibroblasts with the multidrug resistance-associated protein (MRP) did not alter esterification, showing that cholesterol trafficking was not generally affected by ATP-binding cassette transporters. Steroidal (progesterone, dehydroepiandrosterone) and non-steroidal antagonists (verapamil, PSC 833, LY335979, and GF120918) were evaluated for effects on both cholesterol trafficking and the net content of 99mTc-Sestamibi, a reporter of drug transport activity mediated by Pgp. In Pgp-expressing cells treated with nonselective and selective inhibitors, both the kinetics and efficacy of inhibition of cholesterol esterification differed from the antagonism of drug transport mediated by Pgp. Thus, although the data show that greater expression of class I Pgp within a given cell type is associated with enhanced esterification of plasma membrane cholesterol in support of a physiologic function for Pgp in facilitating cholesterol trafficking, the molecular mechanism is dissociated from the conventional drug transport activity of Pgp.     

Pre- and post-Golgi vacuoles operate in the transport of Semliki Forest virus membrane glycoproteins to the cell surface

The effect of reduced temperature on synchronized transport of SFV membrane proteins from the ER via the Golgi complex to the surface of BHK-21 cells revealed two membrane compartments where transport could be arrested. At 15 degrees C the proteins could leave the ER but failed to enter the Golgi cisternae and accumulated in pre-Golgi vacuolar elements. At 20 degrees C the proteins passed through Golgi stacks but accumulated in trans-Golgi cisternae, vacuoles, and vesicular elements because of a block affecting a distal stage in transport. Both blocks were reversible, allowing study of the synchronous passage of viral membrane proteins through the Golgi complex at high resolution by immunolabeling in electron microscopy. We propose that membrane proteins enter the Golgi stack via tubular extensions of the pre-Golgi vacuolar elements which generate the Golgi cisternae. The proteins pass across the Golgi apparatus following cisternal progression and enter the post-Golgi vacuolar elements to be routed to the cell surface.     

Tumor necrosis factor alpha induces stronger cytotoxicity in ABCG2-overexpressing resistant breast cancer cells compared with their drug-sensitive parental line

Tumor necrosis factor alpha (TNF-α) has been reported to modulate the multidrug resistance (MDR) phenotype in vitro and in vivo. Multidrug-resistant cells overexpressing the ABCB1 transporter are more susceptible to inhibition of proliferation and induction of apoptosis by TNF-α than their drug-sensitive counterparts. This study was aimed to investigate TNF-α modulatory and antiproliferative effects on drug-resistant cells overexpressing ABCG2. The effects of TNF-α on viability and proliferation rate of MCF-7 breast cancer cells and their ABCG2-overexpressing sublines MCF-7/mitoxantrone (MX) cells were studied using dye exclusion assay, dimethylthiazolyl-2,5-diphenyl tetrazolium bromide technique, and flow cytometric analysis of cell cycle. TNF-α influence on MX accumulation was investigated by flow cytometry. ABCG2-overexpressing cells were more susceptible to antiproliferative and cytotoxic effects of TNF-α than their parental cells. TNF-α increased accumulation of MX in both parental and resistant cells. Higher sensitivity of MDR cells to TNF-α cytotoxicity would help in characterization of its complex modulatory effects on cancer cells and benefit us in designing new approaches to overcome MDR.     

Fast kinetic analysis of drug transport in multidrug resistant cells using a pulsed quench-flow apparatus

One major feature of multidrug resistance is the reduced cellular level of drugs maintained by MDR cells. Although there is now strong evidence that drugs are actively pumped out of MDR cells, transport experiments have indicated decreased initial rates of influx at the earliest times at which measurements could be made. We have used a pulsed quench-flow apparatus to study transport characteristics of colchicine resistant MDR cells on a very fast time scale. A rapid association of daunomycin with drug sensitive cells occurred within 0.11 sec. This association is virtually absent in MDR cells. In efflux experiments performed on the same rapid time scale, greater than 50% of daunomycin efflux occurred within 0.1 sec. No substantial efflux from B1, drug sensitive cells was observed. On the other hand, vinblastine accumulation by both cell types was similar for approx. 10 seconds. Thus, kinetically, not all drugs are handled in a similar fashion by MDR cells. The pulsed quench-flow apparatus was useful in making fast time measurements of drug influx and efflux and in demonstrating the differences between drug recognition patterns by MDR cells.     

PDMP Blocks Brefeldin A–induced Retrograde Membrane Transport from Golgi to ER: Evidence for Involvement of Calcium Homeostasis and Dissociation from Sphingolipid Metabolism

In this study, we show that an inhibitor of sphingolipid biosynthesis, d,l-threo-1-phenyl-2- decanoylamino-3-morpholino-1-propanol (PDMP), inhibits brefeldin A (BFA)-induced retrograde membrane transport from Golgi to endoplasmic reticulum (ER). If BFA treatment was combined with or preceded by PDMP administration to cells, disappearance of discrete Golgi structures did not occur. However, when BFA was allowed to exert its effect before PDMP addition, PDMP could not “rescue” the Golgi compartment.Evidence is presented showing that this action of PDMP is indirect, which means that the direct target is not sphingolipid metabolism at the Golgi apparatus. A fluorescent analogue of PDMP, 6-(N-[7-nitro-2,1,3-benzoxadiazol-4-yl]amino)hexanoyl-PDMP (C₆-NBD-PDMP), did not localize in the Golgi apparatus. Moreover, the effect of PDMP on membrane flow did not correlate with impaired C₆-NBD-sphingomyelin biosynthesis and was not mimicked by exogenous C₆-ceramide addition or counteracted by exogenous C₆-glucosylceramide addition. On the other hand, the PDMP effect was mimicked by the multidrug resistance protein inhibitor MK571.The effect of PDMP on membrane transport correlated with modulation of calcium homeostasis, which occurred in a similar concentration range. PDMP released calcium from at least two independent calcium stores and blocked calcium influx induced by either extracellular ATP or thapsigargin. Thus, the biological effects of PDMP revealed a relation between three important physiological processes of multidrug resistance, calcium homeostasis, and membrane flow in the ER/ Golgi system.     

Structural differences between sensitive and resistant L1210 cells

The main structural differences between sensitive L1210 mouse leukaemic cells and their multidrug resistant counterpart, obtained by adaptation of the parental cell line to vincristine (VCR), concern the size and shape of the cells, their surface properties and changes in organelles involved in proteosynthesis and transport of substances. The resistant cells are larger with higher density of microvilli. In light and electron micrographs containing a group of cells, cells were found to be closer to each other in L1210/VCR cells than in L1210 cells. This difference in cell aggregation suggests different surface properties which could be visualised by decreased staining of L1210/VCR cell surface coat (glycocalyx) with a polycationic dye ruthenium red. A decrease in surface to volume ratio as a consequence of increased cell size in resistant cells is compensated by proliferation of villi and cytoplasmic protrusions of the cell surface. L1210/VCR cells were further distinguished by higher amount of euchromatin, increase in density of rough endoplasmic reticulum, more developed Golgi apparatus and aggregation of free ribosomes into tetrameric and pentameric polyribosomes. These structural changes may be interpreted as a sign of increase in proteosynthesis and transport of substances.     

A Golgi-related structure remains after the brefeldin A-induced formation of an ER-Golgi hybrid compartment.

Brefeldin A (BFA) has previously been shown to block protein transport from the endoplasmic reticulum (ER), to cause the redistribution of Golgi components to the ER, and to change profoundly the morphology of the Golgi apparatus. In order to quantitate the effects of this drug on the morphology of the ER and the Golgi apparatus in HeLa cells, the numerical, surface and volume densities of these organelles were determined by stereological means. We found that in cells treated with BFA (5 micrograms/ml) clusters of vesicles and tubules, often located near transitional elements of the ER, replaced the Golgi apparatus. The numerical density of these clusters in cells treated with BFA for 30 min or 4.5 h is similar to that of Golgi complexes and Golgi-related clusters in control cells. The surface density of the vesicles and tubules contained in these clusters is about 50% of that represented by Golgi elements in control cells. Concomitantly, a corresponding increase in the surface density of the ER-Golgi hybrid compartment was observed. This hybrid compartment contained Golgi-specific enzymes effecting modifications of N-linked oligosaccharides and the transfer of O-linked sugars. Antibodies recognizing different subcompartments of the Golgi apparatus or the intermediate compartment, labeled vesicles and tubules of the Golgi-related clusters. Applying low doses of BFA allowed for the dissection of the disassembly of the Golgi apparatus into at least two phases. At very low doses (10-20 ng/ml) the numerical density of vesicles in the clusters increased up to 4-fold above control, while the surface density did not markedly change, suggesting that vesiculation of the Golgi cisternae had occurred. Fusion of Golgi elements with the ER seemed to occur only at doses of BFA higher than 20 ng/ml. Contrary to observations on other cell types, removal of BFA from HeLa cell cultures resulted in a rather slow reformation (1-2 h) of the Golgi complex, which allowed us to observe several intermediate stages in this process. During this time period an ER was restored which no longer contained Golgi-specific O-glycosylation functions. Our results demonstrate that BFA does not simply cause the disappearance of the Golgi apparatus by fusion with the ER, but instead clusters of vesicles and tubules remain that contain Golgi-specific markers.     

O-glycosylation of a precursor to a sweet potato vacuolar protein, sporamin, expressed in tobacco cells

Sporamin, a vacuolar protein of the sweet potato, is synthesized as a precursor that contains signal peptide and an N-terminal propeptide that functions as a vacuolar targeting determinant. Sporamin, when expressed in tobacco cells, migrated as smeared bands on an SDS-polyacrylamide gel. The smearing was due to O-glycosylation of the precursor to sporamin. The smeared bands were stained by a glycan-specific stain but no N-glycosylation site was found in the amino acid sequence of the precursor to sporamin. The glycan attached to sporamin contained galactose and arabinose as major sugar components. Mutations that altered the Pro³⁶ or Ser³⁹ residue of the precursor to sporamin prevented glycosylation of the protein, and analysis by semiquantitative Edman degradation suggested that a glycan moiety was attached to Pro³⁶ and, possibly, to Ser³⁹. Pulse-labeling and cell-fractionation experiments revealed that the O-glycosylation of the precursor to sporamin occurred in the Golgi apparatus. Thus, this modification serves as a good marker of the transport from the endoplasmic reticulum (ER) to the Golgi apparatus of the precursor to sporamin. Treatment of transformed tobacco cells with brefeldin A (BFA) caused the intracellular accumulation of prosporamin that did not migrate as smeared bands. Thus, it appeared that BFA inhibited the transport of the precursor to sporamin to the Golgi apparatus. This result provides the first biochemical evidence that BFA inhibits transport from the ER to the Golgi apparatus in plant cells.     

Pulmonary surfactant phosphatidylcholine transport bypasses the brefeldin A sensitive compartment of alveolar type II cells

Brefeldin A (BFA) causes disassembly of the Golgi apparatus and blocks protein transport to this organelle from the endoplasmic reticulum. However, there still remains considerable ambiguity regarding the involvement of the Golgi apparatus in glycerolipid transport pathways. We examined the effects of BFA upon the intracellular translocation of phosphatidylcholine in alveolar type II cells, that synthesize, transport, store and secrete large amounts of phospholipid for regulated exocytosis. BFA at concentrations as high as 10 microg/ml failed to alter the assembly of phosphatidylcholine into lamellar bodies, the specialized storage organelles for pulmonary surfactant. The same concentration of BFA was also ineffective at altering the secretion of newly synthesized phosphatidylcholine from alveolar type II cells. In contrast, concentrations of the drug of 2.5 microg/ml completely arrested newly synthesized lysozyme secretion from the same cells, indicating that BFA readily blocked protein transport processes in alveolar type II cells. The disassembly of the Golgi apparatus in alveolar type II cells following BFA treatment was also demonstrated by showing the redistribution of the resident Golgi protein MG-160 to the endoplasmic reticulum. These results indicate that intracellular transport of phosphatidylcholine along the secretory pathway in alveolar type II cells proceeds via a BFA insensitive route and does not require a functional Golgi apparatus.     

Intracellular distribution of anthracyclines in drug resistant cells

The unresponsiveness of multidrug resistant tumor cells to antineoplastic chemotherapy is often associated with reduced cellular drug accumulation accomplished by overexpressed transport molecules. Moreover, intracellular drug distribution in resistant cells appears to be remarkably different when compared to their wild type counterparts. In the present paper, we report observations on the intracellular accumulation and distribution of doxorubicin, an antitumoral agent widely employed in chemotherapy, in sensitive and resistant cultured tumor cells. The inherent fluorescence of doxorubicin allowed us to follow its fate in living cells by laser scanning confocal microscopy. This study included flow cytometric analysis of drug uptake and efflux and analysis of the presence of the well known drug transporter P-glycoprotein. Morphological, immunocytochemical and functional data evidentiated the Golgi apparatus as the preferential intracytoplasmic site of drug accumulation in resistant cells, capable of sequestering doxorubicin away from the nuclear target. Moreover, P-glycoprotein has been found located in the Golgi apparatus in drug induced resistant cells and in intrinsic resistant cells, such as melanoma cells. Thus, this organelle seems to play a pivotal role in the intracellular distribution of doxorubicin. This revised version was published online in August 2006 with corrections to the Cover Date.     

Brefeldin A-regulated retrograde transport into the endoplasmic reticulum of internalised wheat germ agglutinin

The effects of the fungal metabolite brefeldin A (BFA) on the endocytic routes of internalised wheat germ agglutinin (WGA) were studied in human HepG2 hepatoma cells, drawing particular attention to the application times in relation to the membrane dynamics occurring at the trans Golgi face during endocytosis. As shown in previous studies, transport of internalised WGA into the Golgi apparatus can be classified in three stages being characterised by predominance of vesicular endosomes (stage I), formation of an extended endocytic trans Golgi network (stage II) and uptake of WGA into the stacked Golgi cisternae (stage III). BFA treatment of the cells led to rapid tubular-reticular transformations of the Golgi stacks. Retrograde transport and further destinations of internalised WGA depended on the time of BFA application. When BFA was administered during stages I or II, WGA was localised within the BFA-induced tubules and networks, but never was found within the endoplasmic reticulum. By contrast, BFA treatment during stage III led to a redistribution of internalised WGA into cisternae of the endoplasmic reticulum. These results show that BFA administered according to a precise time schedule can be used as a regulatory agent that allows to control retrograde traffic of internalised molecules into the endoplasmic reticulum.     

Analysis of the tangled relationships between P-glycoprotein-mediated multidrug resistance and the lipid phase of the cell membrane.

P-glycoprotein (Pgp), the so-called multidrug transporter, is a plasma membrane glycoprotein often involved in the resistance of cancer cells towards multiple anticancer agents in the multidrug-resistant (MDR) phenotype. It has long been recognized that the lipid phase of the plasma membrane plays an important role with respect to multidrug resistance and Pgp because: the compounds involved in the MDR phenotype are hydrophobic and diffuse passively through the membrane; Pgp domains involved in drug binding are located within the putative transmembrane segments; Pgp activity is highly sensitive to its lipid environment; and Pgp may be involved in lipid trafficking and metabolism. Unraveling the different roles played by the membrane lipid phase in MDR is relevant, not only to the evaluation of the precise role of Pgp, but also to the understanding of the mechanism of action and function of Pgp. With this aim, I review the data from different fields (cancer research, medicinal chemistry, membrane biophysics, pharmaceutical research) concerning drug-membrane, as well as Pgp-membrane, interactions. It is emphasized that the lipid phase of the membrane cannot be overlooked while investigating the MDR phenotype. Taking into account these aspects should be useful in the search of ways to obviate MDR and could also be relevant to the study of other multidrug transporters.     

The relationship between 1H-NMR mobile lipid intensity and cholesterol in two human tumor multidrug resistant cell lines (MCF-7 and LoVo)

The high resolution proton nuclear magnetic resonance (1H-NMR) spectra of two different cell lines exhibiting multidrug resistance (MDR) as demonstrated by the expression of the well-known energy-driven, membrane-bound 170 kDa P-glycoprotein pump known as Pgp were investigated. In particular, the mobile lipid (ML) profile, and the growth and biochemical characteristics of MCF-7 (human mammary carcinoma) and LoVo (human colon adenocarcinoma) sensitive and resistant tumor cells were compared. The results indicate that both MCF-7 and LoVo resistant cells have a higher ML intensity than their respective sensitive counterparts. However, since sensitive and resistant cells of each pair grow in the same manner, variations in growth characteristics do not appear to be the cause of the ML changes as has been suggested by other authors in non-resistant tumor cells. In order to investigate further the origin of the ML changes, lipid analyses were conducted in sensitive and resistant cell types. The results of these experiments show that resistant cells of both cell types have a greater amount of esterified cholesterol and saturated cholesteryl ester and triglyceride fatty acid than their sensitive counterparts. From a thorough analysis of the data obtained in this paper utilizing numerous techniques including biological, biophysical and biochemical ones, it is hypothesized that cholesterol and triglyceride play a pivotal role in inducing changes in NMR ML signals. The importance of these lipid variations in MDR is discussed in view of the controversy regarding the origin of ML signals and the paramount role played by the Pgp pump in resistance.     

Staphylococcal ADP-ribosyltransferase-sensitive small G protein is involved in brefeldin A action.

An early event in the action of brefeldin A (BFA) is the dissociation of beta-coat protein (beta-COP) from the Golgi membrane. We have recently shown that staphylococcal ADP-ribosyltransferase (epidermal cell differentiation inhibitor (EDIN)), which specifically modifies a small G protein, rho, mimics the action of BFA and disassembles the Golgi apparatus in Vero cells (Sugai, M., Chen, C-h., and Wu, H. C. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 8903-8907). Three independent BFA-resistant cell lines (BER-40 from Vero cells, PtK1, and MDCK) showed cross-resistance to EDIN regarding the release of the beta-COP from the Golgi membrane by EDIN or BFA. BFA as well as EDIN induced disassembly of the actin microfilaments in Vero cells, and they both failed to induce the disassembly of actin microfilaments in BER-40, PtK1, and MDCK cells. BFA inhibited protein secretion in Vero cells but not in BFA-resistant cell lines, whereas EDIN did not inhibit protein secretion in either Vero or other cell lines. AlF-4 inhibited the effect of EDIN as well as that of BFA on the distribution of the beta-COP. These results suggest that an EDIN-sensitive rho protein together with trimeric and other small G protein(s) is involved in the regulation of the assembly of coated vesicles and vesicular transport in the Golgi apparatus.