- Methodology article
- Open Access
Model system for the analysis of cell surface expression of human ABCA1
- Ildikó Kasza†1,
- Zoltán Hegyi†1,
- Katalin Szabó2,
- Hajnalka Andrikovics3,
- Katalin Német3,
- András Váradi2,
- Balázs Sarkadi1, 3 and
- László Homolya1Email author
© Kasza et al; licensee BioMed Central Ltd. 2009
- Received: 16 June 2009
- Accepted: 21 December 2009
- Published: 21 December 2009
The ABCA1 protein plays a pivotal role in reverse cholesterol transport, by mediating the generation of HDL particles and removing cellular cholesterol. Both the proper expression of ABCA1 in the plasma membrane and the internalization along with apoA-I are required for function. Therefore, we developed a model system to investigate the effect of clinically relevant drugs on the cell surface appearance of ABCA1.
By retroviral transduction system, we established stable mammalian cell lines expressing functional and non-functional ABCA1 variants, tagged with an extracellular hemagglutinin epitope. After characterization of the expression, proper localization and function of different ABCA1 variants, we followed quantitatively their cell surface expression by immunofluorescent staining, using flow cytometry. As expected, we found increased cell surface expression of ABCA1 after treatment with a calpain inhibitor, and observed a strong decrease in plasma membrane ABCA1 expression upon treatment with a trans-Golgi transport inhibitor, Brefeldin A. We tested cholesterol level lowering drugs and other potential inhibitors of ABCA1. Here we demonstrate that ezetimibe affects ABCA1 cell surface expression only in the case of a functional ABCA1.
Our model system allows a quantitative detection of cell surface expression of ABCA1, screening of substrates or specific inhibitors, and investigating transport regulation.
- Cell Surface Expression
- Cholesterol Efflux
- Reverse Cholesterol Transport
- Calpain Inhibitor
Elevated low-density lipoprotein (LDL) cholesterol, reduced high-density lipoprotein (HDL) cholesterol, and increased triglyceride levels significantly contribute to the accumulation of lipids in atherosclerotic lesions. Reverse cholesterol transport is believed to be crucial for preventing atherogenesis and hence the development of cardiovascular diseases [1, 2]. It is commonly accepted that the ATP-binding cassette protein, ABCA1 plays a pivotal role in the initial steps of the reverse cholesterol transport pathway by mediating the interactions of amphiphilic apolipoproteins (e.g., apoA-I) with cellular lipids to generate nascent HDL particles thus removing excess cellular cholesterol [3, 4]. Mutations in ABCA1 cause Tangier disease, a disorder characterized by very low HDL levels, cholesterol deposition in macrophages, and premature atherosclerosis [5–7]. There is increasing evidence suggesting a role for ABCA1 not only in the hepatic but also in the intestinal HDL-production .
Therapeutic efforts to raise HDL levels with different drugs have been promising. Clinical trials tested various statins, niacin, and ezetimibe alone and in various combinations to raise plasma HDL- and to reduce the plasma LDL-cholesterol levels [9, 10]. Niacin and ezetimibe together with statins were proven to be the most effective combination in vivo . However, conflicting results were reported about the effects of statins on lipid efflux and the modulation of ABCA1 expression in in vitro experiments [12–16]. Recently, certain types of calcium channel blockers (CCBs), e.g., verapamil, nifedipine, have been found to be anti-atherogenic in clinical trials [17, 18]. When their effects on ABCA1 expression were investigated, contradictory results were obtained. These agents either increased ABCA1 mRNA levels or elevated the protein expression without affecting the mRNA level, depending on the cellular test system used [19, 20].
Similar to other plasma membrane proteins, the cell surface expression of ABCA1 is modulated by a complex process, which includes transcriptional and post-transcriptional regulation, as well as internalization, degradation, and recycling [21, 22]. There is evidence that not only sufficient plasma membrane expression but internalization along with apoA-I is required for proper function of ABCA1 [23, 24]. Thus, in the present study we aimed to develop a quantitative in vitro test system, which is suitable for monitoring the plasma membrane level of ABCA1, independently from direct transcriptional regulation. In order to generate such an experimental tool, we introduced a hemagglutinin (HA) epitope into the first extracellular loop of ABCA1, and stably expressed this tagged ABCA1 in various mammalian cell lines, using a constitutive promoter. In addition, to make our method suitable for studying trafficking processes of ABCA1 in conjunction with its function, we generated different loss-of-function mutant variants of the HA-tagged transporter. In the present study we demonstrate the applicability and reliability of the developed cellular test system, and report the effects of several pharmaceuticals, which are known to have cholesterol-lowering effects in vivo. When studying whether they act through modifying the cell surface expression of functional ABCA1, we found that among several drugs, ezetimibe lowers the plasma membrane level of a functional ABCA1.
Stable expression of HA-tagged ABCA1 variants in mammalian cells
Cellular localization and functional characterization of HA-tagged ABCA1 variants
Similar to apoA-I-dependent cholesterol efflux, apoA-I binding was found to be elevated in cells expressing either the untagged or HA-tagged ABCA1, whereas it was unaffected in cells expressing the mutant form of HA-ABCA1 (Figure 6B). Since the calpain inhibitor Acetyl-L-Leucyl-L-Leucyl-L-Norleucinal (ALLN) and cylcosporin A (CsA) were used in further experiments, we investigated the effect of these drugs on the apoA-I-binding in the established cell lines. We found that ALLN significantly increased apoA-I-binding in cells expressing either the untagged or HA-tagged wt ABCA1-expressing, whereas CsA pretreatment slightly reduced the apoA-I-binding in these cells. This observation is in good accordance with previous studies performed with untagged ABCA1 [35–38], and clearly indicates that HA-tagging did not alter the function of the protein. ALLN and CsA had no significant effect on apoAI-binding in the parental cell line and in cell expressing the mutant form of HA-ABCA1.
In summary, we demonstrated reasonable and stable expression, correct subcellular localization, as well as proper function for the HA-tagged ABCA1. Since all three HA-tagged ABCA1 mutants were proven to be non-functional with normal expression and localization, only the wild type HA-tagged and the double mutant variant (MM) were used in further experiments.
Effect of calpain inhibitor, BFA, and apoA-I on cell surface expression of ABCA1 variants
ApoA-I has also been demonstrated to increase ABCA1 expression by binding to the transporter and preventing it from degradation [36, 37]. As expected, apoA-I significantly increased the cell surface expression of wt HA-tagged ABCA1 (Figure 7C). The MM mutant ABCA1, which was shown to be unable to bind apoA-I , exhibited no alteration of the cell surface expression levels when incubated with apoA-I (Figure 7D). These experiments clearly demonstrate that our model system is suitable to detect the effects of various substances on the cell surface expression of either the functional or non-functional variant of ABCA1.
In similar experiments, HA-ABCA1-expressing MDCKII cells were treated with ALLN or BFA. Even though a wide range of drug concentrations was tested, no marked changes in the ABCA1 cell surface expression were seen in these cells. Therefore, in further experiments HEK293H cells expressing the HA-tagged ABCA1 variants were used.
Effect of different drugs on cell surface expression of ABCA1 variants
Effect of various substances on the cell surface expression of wild type (HA-WT) and mutant (HA-MM) HA-ABCA1 variants
ABCA1 cell surface expression
⇧ (+ 60%)
⇧ (+ 30%)
⇧ (+ 30%)
cholesterol level lowering
trans-Golgi transport inhibitor
⇩ (- 60%)
⇩ (- 40%)
protein synthesis inhibitor
⇩ (- 30%) (viability ⇩)
⇩(- 30%) (viability ⇩)
⇧ (+ 60%)
⇧ (+ 60%)
inhibitor of intestinal cholesterol absorption
⇩ (- 20%)
cholesterol level lowering
no effect (viability ⇩)
no effect (viability ⇩)
no effect (viability ⇧)
no effect (viability ⇧)
We found that atorvastatin (10-50 μM), niacin (0.1-1 mM), verapamil (10-50 μM) and nifedipine (30-100 μM) had no significant effect on the plasma membrane level of ABCA1 even in a wide concentration range. However, CsA (10 μM) substantially elevated the cell surface expression of wt HA-ABCA1 (Figure 7C) in accordance with previous results indicating ABCA1 sequestration in the plasma membrane by CsA . It has not been shown yet whether this effect of CsA is associated with the protein function, therefore we performed similar experiments with the non-functional mutant (MM) variant of ABCA1. We found that CsA equally increased the ABCA1 cell surface expression in wt and MM HA-ABCA1-expressing cells (Figure 7C-D), thus the trapping effect of CsA is independent from ABCA1 function. Unexpectedly, ezetimibe (EZ), a blocker of the intestinal cholesterol absorption, significantly (by 20%) reduced the cell surface expression of wt HA-ABCA1 (Figure 7C). Interestingly, this effect of EZ was found to be associated with ABCA1 function, since no reduction in the cell surface expression of the MM HA-ABCA1 was seen in response to EZ treatment (Figure 7D). Glyburide, which has previously been reported to inhibit ABCA1-mediated cholesterol efflux and apoA-I binding without altering the cell surface levels or total protein levels of ABCA1 [29, 46], had no effect of the plasma membrane level of ABCA1 (see Table 1). However, at higher concentrations a slight reduction in the ABCA1 cell surface expression was observed in parallel with a substantial decline in the cell viability in both wt and MM HA-ABCA1-expressing cells. Treatment with 200-500 μM glyburide for 4 h resulted in 50% cell death in both cell types.
We also investigated the effect of plasma membrane cholesterol levels on the cell surface expression of ABCA1 variants. Treatment with 0.1-2.5 mM methyl-β-cyclodextrin (CD), which results in membrane cholesterol depletion , had no effect on the plasma membrane level of either wild type or MM variant of HA-ABCA1 even in a wide range of incubation time (20-240 min), although a substantial reduction in the cell viability was observed. Similarly, cholesterol overload by cholesterol-containing CD treatment (0.1-2.5 mM, 20-240 min) did not alter the cell surface expression of ABCA1 in either cell type.
ABCA1 protein is involved in lipid metabolism in several regards. This protein promotes reverse cholesterol transport from the peripheral tissues. ABCA1, being located on the basolateral surface of hepatocytes and intestinal cells, has been shown to play a crucial role in the hepatic and intestinal secretion of HDL . In the present study we generated and characterized a model system for the quantitative assessment of the plasma membrane level of ABCA1. Inclusion of an extracellular hemagglutinin epitope into ABCA1 enabled monitoring of its cell surface expression by flow cytometry and confocal microscopy. By using retroviral transduction we established mammalian cell lines stably expressing functional and non-functional HA-tagged ABCA1 variants. This experimental tool allowed us to investigate the effect of different drugs on cell surface expression of ABCA1.
In previous studies, the protein expression level and subcellular localization of wild type ABCA1 and non-functional mutant forms were found to be identical [27, 31]. Similarly, in our model system, both the overall and the plasma membrane expression levels of each HA-tagged ABCA1 variant were comparable, as determined by Western blotting and cell surface labeling (Figures 2B, 3). ABCA1 has been reported to be localized to the basolateral membrane in polarized cells . We found proper subcellular localization for each HA-tagged ABCA1 variant (Figures 4, 5) in accordance with previous reports on the untagged versions of the transporter .
Since the applicability and reproducibility of a cellular model system requires stable protein expression levels, we investigated the persistence of our transgene in various cell lines. We found that ABCA1 expression remains stable in MDCKII and HEK293H cells up to 50 and 20 passages, respectively. Thus, our experimental test system substantially surpasses previous attempts to generate cellular models with ABCA1, since in those cases protein expression rapidly declined after a few passages . We demonstrated the applicability of our system to assess the cell surface appearance of ABCA1 by using BFA and ALLN, which have been reported to influence ABCA1 trafficking and degradation (Figure 7A, B).
In agreement with previous reports [35, 36], we demonstrated the stabilizing effect of apoA-I on the HA-tagged ABCA1 at the cell surface (Figure 7C). Furthermore, we found that the cell surface levels of the Walker motif mutant ABCA1 (MM), which fails to bind apoA-I (Figure 6B) [28, 29], was unaffected by apoA-I (Figure 7D). This observation is in line with previous findings demonstrating that binding of apoA-I is required for the apoA-I-mediated increase of ABCA1 expression [29, 37].
In clinical trials, statins were highly effective in reducing LDL-cholesterol, but only moderate effects were seen on the HDL levels. Among a number of different statins, atorvastatin was the most effective and induced redistribution in HDL subpopulations by increasing the fraction of larger HDL particles . Conflicting results were published on the effect of statins on ABCA1 expression and lipid efflux in various cellular systems. Wong et al. demonstrated down-regulation of ABCA1 mRNA and protein levels as well as reduction in the apoA-I-mediated cholesterol efflux in response to statin treatment of PMA-induced Thp-1 cells [14, 15]. In contrast, another study found that atorvastatin increased mRNA expression of ABCA1 and promoted cholesterol efflux in the same cell type . In hepatoma cells, statins induced ABCA1 expression resulting in cholesterol and phospholipid efflux to apoA-I . These observations leave the question open, whether statins influence apoA-I-dependent cholesterol efflux solely by changing ABCA1 expression levels or by modifying its subcellular trafficking and cell surface appearance. Since our model system excludes transcriptional regulation, our results revealed that atorvastatin has no effect on the plasma membrane expression of ABCA1.
Niacin has been reported to be one of the most potent agents increasing HDL cholesterol levels , and to stimulate ABCA1 expression and function in monocytic cell lines via influencing the cAMP/PKA pathway . By investigating the effect of niacin in our system, we observed no direct effect on cell surface appearance of ABCA1 in accordance with previous observations on the regulatory mechanism of niacin.
Calcium channel blockers have also been reported to influence ABCA1 expression. Hasegawa et al. demonstrated the role of different signaling pathways including PKA, tyrosine kinase, and Janus kinase in ABCA1 expression modulation in response to various CCBs. They found that some of the CCBs, e.g., aranidipine and efonidipine, increase ABCA1 protein expression without altering the mRNA level . In contrast, Suzuki et al. demonstrated a dose-dependent stimulatory effect of other CCBs, such as verapamil, nifedipine, and nicardipine, on both the mRNA and protein expression levels of ABCA1, resulting in elevated cellular lipid release . Our results do not support these findings, since we observed no significant effect on the plasma membrane level of ABCA1 in a wide concentration range of verapamil and nifedipine.
Membrane cholesterol has been implicated to regulate many membrane proteins, including several ABC transporters, e.g. ABCB1 and ABCG2 [47, 52, 53]. Membrane microdomains with high cholesterol content have been suggested to play a role in such regulation . However, neither cholesterol overload nor depletion influenced ABCA1 cell surface expression in our model system. It is noteworthy that treatment with methyl-β-cyclodextrin (CD) reduced, whereas cholesterol-loaded CD significantly increased cell viability. The most plausible explanation for this observation is that CD treatment makes the cell membrane fragile and more sensitive to mechanical stress. This notion should be taken in account in future studies on ABCA1-expressing cells treated with CD.
We studied the effect of glyburide and CsA, which have previously been reported to influence the plasma membrane level or function of ABCA1 [29, 38]. In accordance with previous observations, glyburide had no effect on ABCA1 plasma membrane appearance in our model system , whereas CsA greatly increased the cell surface level of ABCA1 (Figure 7C). ALLN elevated both ABCA1 cell surface expression and apoA-I binding, whereas CsA reduced apoA-I binding despite the increased plasma membrane level of ABCA1. This observation is line with a previous report suggesting that CsA sequesters ABCA1 in the plasma membrane . The observation that cell surface expression of the non-functional ABCA1 mutant (MM) is also increased by CsA pretreatment clearly indicates that the trapping effect of this drug is independent from ABCA1 function.
Ezetimibe, a drug which is known to block the intestinal cholesterol absorption, has not only an inhibitory effect on several brush border membrane proteins, including Niemann-Pick C1-like 1, scavenger receptor B-I and CD36 , but has also been reported to lower the mRNA level of several other genes involved in lipid metabolism. The transcription of ABCA1 is also reduced by EZ , but no study has yet been reported on its effect on the cell surface expression of ABCA1. Our model system allows us to investigate the plasma membrane expression of ABCA1 independently from its transcriptional regulation. Strikingly, we found that EZ significantly lowers the cell surface expression of wt HA-ABCA1 (Figure 7C). Since no reduction in the cell surface expression of the MM HA-ABCA1 was seen in response to EZ treatment (Figure 7D), the effect of EZ occurs only with a functional ABCA1, thus, its effect is likely connected to a functional form of the protein.
There are several possibilities to explain the lack of effect of EZ on cell surface expression of the non-functional MM-ABCA1 variant. It is theoretically possible but very unlikely that EZ inhibits the trafficking of only the de novo synthesized functional proteins. It is more plausible that EZ influences the protein internalization and recycling to the plasma membrane, since ABCA1 has been shown to undergo a relatively rapid internalization and recycling to the plasma membrane, which process is widely accepted to require protein function [39, 56]. Therefore, if EZ either accelerates protein internalization or inhibits recycling, and in the meantime MM-ABCA1 has a lower internalization and recycling turnover compared to wild type, EZ causes a smaller effect on the cell surface appearance of the mutant transporter. This hypothesis is supported by the observation that the effects of ALLN were also smaller in HA-MM-ABCA1-expressing cells, as compared to wild type HA-ABCA1. It is also possible that EZ accelerates ABCA1 degradation, since the internalization itself is dependent on the transporter function. This idea is not supported by our recent observations using confocal microscopy, when we found that following EZ treatment the wt HA-ABCA1, but not the MM variant, accumulated intracellularly in vesicles but not in lysosomes (data not shown). Nevertheless, further studies are needed to reveal the mechanism of EZ effect on the cell surface expression of ABCA1.
In the current study, we have provided a model system suitable for the quantitative assessment of the plasma membrane level of ABCA1. We have shown that this test system can be reliably applied for studying the effect of various pharmaceuticals on the cell surface expression of the transporter. Therefore, our model system provides a new tool for acquiring more information on the post-translational regulation, internalization, degradation and recycling of the ABCA1 protein.
For the generation of SsA1neoS vector, the full length wild type human ABCA1 cDNA  was cloned into a bicistronic retroviral SPsLdS vector , which was modified to contain the neomycin resistance cassette (neo) as previously described . A cassette containing the coding sequence of an HA epitope (YPYDVPDY) was inserted into the full-length human ABCA1 cDNA, between residues 207 and 208 . For control experiments, a vector containing only the neo cDNA was generated. The HA-ABCA1 K939 M, K1952 M mutants were constructed in the pBlscrSK- vector containing XhoI/NotI cassette from the SsHAA1neoS vector. HA-ABCA1 K939 M was constructed by the overlap extension PCR technique using the internal 5'-TGGCTGTGATCATCAAGGGC-3' and 5'-CCAGGACGTCCGCTTCATCCAT-3' and external 5'-ATTGACATGGTGGTCGTCA TCC-3' and 5'-GGAT GACGACCACCATGTCAAT-3' oligonucleotides. The external primer pair includes AAG (lysine) to ATG (methionine) single mutation and a new FokI restriction site (in bold). The HA-ABCA1 K1952 M mutant was generated by using 5'-AGCCAAAAGCTTAAAGAACAAGATCTGGGTG-3' and 5'-CTCCTGTTAACATCTTGAAAGTTGATGACA TC CCAGCC-3' oligonucleotides, which results in a lysine to methionine mutation (in bold) and contains a GC substitution (underlined) to create a new FokI restriction site. The mutant constructs were verified by FokI digestion. HA-ABCA1 variant containing double methionine substitution was created by cassette exchange in pBlscrSK- vector by using HindIII/HpaI digestion. After partial digestion of each mutant construct in pBlscrSK- vector by XhoI/BamHI enzymes the fragments were transferred into SsHAA1neoS vector and verified by sequencing.
Phoenix-eco  was a gift from G. Nolan (Department of Pharmacology, Stanford University, Stanford, CA); PG13 retrovirus producing cells , HepG2, Thp-1, and MDCKII cells were obtained form the ATCC (Manassas, VA). HEK293H cells were purchased from Invitrogen. Most cell lines were grown in DMEM containing 10% FCS, whereas HepG2 were cultured in DMEM and F12 (1:1) + 10% FCS medium. Thp-1 cells were maintained in RPMI + 10% FCS medium, and pretreated with 2 nM PMA for 24 h. Human monocytes were isolated from peripheral blood of healthy subjects, as previously described , and cultured in DMEM containing 10% FCS for 5 days. ABCA1 expression was induced in both PMA-pretreated Thp-1 cells and monocyte-derived macrophages by the addition of a synthetic LXR agonist, T0901317 (1 μM, Alexis Biochemicals) 8 h prior to studies.
Generation of cell lines stably expressing ABCA1 variants
Retroviral particles and cell lines stably expressing the ABCA1 variants were generated as described previously [25, 30] with some modifications. Briefly, the Phoenix-eco packaging cell line was transfected by calcium phosphate coprecipitation (Invitrogen). The cell-free viral supernatant was collected 24 and 48 hours after transfection and immediately used to transduce PG13 cells. The transduction of PG13 cells was repeated on the following day. HEK293H and MDCKII cells were transduced with viral supernatant collected from PG13 packaging cells 24 and 48 hours after transduction. The target cells were then selected with 0.5 mg/ml and 0.8 mg/ml G418 for HEK293H and MDCKII cells, respectively. Single-cell clones of transduced MDCKII cells were isolated by limiting dilutions on 96-well plates under continuous G418 selection. In contrast, HEK293H cells were labeled with direct anti-HA antibody and sorted by a FACSAria (Becton Dickinson) cell sorter. To reduce the heterogeneity, a subsequent cell sorting was performed.
RNA isolation and real time RT-PCR
Total RNA was isolated by a single-step method using TriZol (Invitrogen). The purity of the RNA preparation was checked by measuring the absorbance ratio at 260/280 nm. For each reaction 1 μg total RNA was reverse-transcribed into cDNA according to the manufacturer's protocol (Promega) using random hexamer oligonucleotides. The resulting cDNA was then subjected to real-time quantitative PCR (Light Cycler 480 II, Roche), 45 cycles of 95°C for 30 s, 58°C for 1 s and 72°C for 1 s by using specific primers to detect human ABCA1 cDNA, but not the genomic ABCA1 sequence. Oligonucleotides designed for PCR analysis of ABCA1 or housekeeping Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNAs were: 5-ACAAGATGCTGAGGGCTGAT-3 and 5-CCCAAGACTATGCAGCAATG-3 or 5-GAAGGTGAAGGTCGGAGTCA-3 and 5-GACAAGCTTCCCGTTCTCAG-3, respectively. PCRs contained 3 mM MgCl2, 0.25 μM from ABCA1 and 0.5 μM from GAPDH forward and reverse primers, and 5 μl LightCycler DNA Master SYBR Green I (2×, Roche). Before amplification, a preincubation step (60 seconds at 95°C) was performed to activate FastStart DNA polymer and to ensure complete denaturation of the cDNA. Following the last cycle, melting curve analysis was carried out to specify the integrity and purity of the amplicons. Amplification profiles were analyzed using the Fit Points and 2nd Derivative Maximum calculation described in the LightCycler Relative Quantification Software. The comparative Ct method was used to quantify transcript levels and to normalize for GAPDH expression.
Western blot analysis
Preparation of whole cell lysates and immunoblotting were performed as described previously  with some modifications. Cell were suspended in 2× Laemmli buffer and sonicated for 5 seconds at 4°C. 15 μg and 40 μg of protein per well from HEK293H cells and MDCKII cells, respectively, were loaded onto a Laemmli-type 6% SDS-polyacrylamide gel, separated, and blotted onto PVDF membranes. Immunodetection was performed by using an anti-HA monoclonal (HA.11, BabCo, 1:3000) or rabbit polyclonal anti-ABCA1 (Abcam, 1:250) antibodies. A HRP-conjugated goat anti-mouse or anti-rabbit IgG secondary antibody (Jackson Immunoresearch, 1:10.000), and enhanced chemiluminescence (ECL) technique was applied. For loading control, a mouse anti-Na+/K+ ATPase antibody (BioMol, 1:1000) was used.
For immunolocalization studies, HEK293H cells were seeded onto eight-well Lab-Tek II Chambered Coverglass (Nalge Nunc) at 5 × 104 per well cell density, and grown for 2 days. For plasma membrane staining, the cells were gently fixed with 1% paraformaldehyde (PFA) for 5 min at room temperature, and incubated with 5 μg/ml AlexaFluor633-conjugated wheat germ agglutinin (WGA, Invitrogen) for 10 min at room temperature. For lysosome staining, the living cells were subjected to LysoTracker Red (Invitrogen) in culturing medium for 30 min at 37°C. After marker staining the cells were fixed and permeabilized with 8% PFA for 15 min at room temperature, and labeled with anti-HA (HA.11, MMS-101R BabCo, 1:500) antibody. To label the Golgi-apparatus and endosomes, double-labeling was performed using rabbit anti-Giantin (Abcam, 1:500) and anti-EEA1 antibodies (1:250), respectively, as described previously . For secondary antibodies AlexaFluor-488-conjugated anti-mouse IgG and AlexaFluor-594-conjugated anti-rabbit IgG antibodies (Invitrogen, 1:250) were used. After immunolabeling the samples were counterstained with 1 μM DAPI for 10 min at room temperature.
For subcellular localization studies in polarized cells, the MDCKII cells were seeded at 5 × 105 cells/cm2 density on 6.5-mm Transwell Col filters (pore diameter 0.4, Corning Costar) previously coated with 0.03 mg/ml Vitrogen (Cohesion Technologies). The cultures were regularly washed with culturing medium to remove cell debris. Studies were carried out with confluent cultures 10 days after seeding. The cultures were permeabilized and labeled with anti-HA (HA.11, MMS-101R BabCo, 1:500) and anti-Na+/K+ ATPase (Abcam, 1:250) antibodies, as previously described . AlexaFluor-488-conjugated anti-mouse IgG and AlexaFluor-647-conjugated anti-chicken IgG secondary antibodies (Invitrogen, 1:250) were used.
The blue, green, red and far red fluorescence of stained samples was studied by an Olympus FV500-IX confocal laser scanning microscope using a PLAPO 60× (1.4) oil immersion objective (Olympus) at 405, 488, 543, and 633 nm excitations.
Cholesterol efflux measurement
Cholesterol efflux measurement was performed as described previously . Briefly, near confluent HEK293H and non-polarized MDCKII cultures grown on 6 well plates were labeled with 100 nM (4 μCi/ml) 3H-cholesterol (PerkinElmer) at 37°C for 24 hours in serum-free DMEM containing 2 mg/ml fatty acid free BSA. The media were then replaced with fresh DMEM/BSA in the presence or absence of 15 μg/ml of apoA-I and incubated at 37°C for 24 hours. Supernatants were collected and spun down to remove cellular material. The pellets and the cell layers were lysed in 2% SDS and 1 M NaOH. The radioactivity of both samples was measured by liquid scintillation; the cholesterol efflux was expressed as the percentage of total (extruded plus cellular) 3H-cholesterol. The apoA-I-dependent efflux was obtained from the difference of the effluxes measured in the presence and absence of apoA-I.
Cell surface expression studies
For cell surface expression studies, the cells were detached by trypsinization and after washing steps blocked for 10 min at room temperature in phosphate-buffered saline containing 2 mg/ml bovine serum albumin (BSA/PBS). The samples were then incubated for 40 min at room temperature with anti-HA antibody (HA.11, MMS-101R BabCo, 1:250 in BSA/PBS). After subsequent washing steps AlexaFluor-488-conjugated anti-mouse IgG secondary antibody (Invitrogen, 1:500 in BSA/PBS) was applied for 40 min at room temperature. As an isotype control a mouse IgG1 (1:10) from Sigma-Aldrich was used. The cell surface expression of the HA-ABCA1 variants was detected by flow cytometry (Becton Dickinson FACS Calibur).
When the modifying effect of various drugs was studied, the cells in the culture dish were pretreated for 4 hours with the following substances: 50 μM ALLN, 10 μg/ml apoA-I (Calbiochem), 10-50 μM atorvastatin (Pfizer), 5 μg/ml BFA, 10-50 μM ezetimibe (Schering-Plough), 0.1-1 mM niacin, 10-50 μM verapamil, 30-100 μM nifedipine, 50-500 μM glyburide, 10-50 μM cyclosporin A, 100 μg/ml cycloheximide, 0.1-2.5 mM random methylated beta cyclodextrin (CD, CycloLab), or 0.1-2.5 mM CD preloaded with 4.4% cholesterol (CycloLab). The materials were obtained from Sigma-Aldrich if not indicated otherwise. After treatment, cells were trypsinized, pre-stained for 2 min with 10 mg/ml propidium-iodide, and gently fixed with PFA for 10 min. Immunofluorescent staining of drug-treated cells was performed at 4 C. Relative expression levels were expressed as the ratio of geometric mean fluorescence of drug-treated and the vehicle-treated samples. For statistical analysis Student's t test was used.
ApoA-I conjugation and binding-experiments were performed as described previously  with minor modifications. Briefly, apoA-I was conjugated to the fluorochrome Cy5 (PA25001, Amersham Pharmacia Biotech) according to the recommendation of the manufacturer. For all experiments the labeled apoA-I (apoA-I-Cy5) was diluted in binding buffer (1.8 mM CaCl2, 1 mM MgCl2, 5 mM KCl, 150 mM NaCl, 10 mM HEPES, pH 7.4), and aggregates were removed by ultracentrifugation for 30 min at 100,000 g. Binding was performed in the presence of 20 μg/ml of apoA-I-Cy5 for 1 h at 4°C on 5 × 105 cells detached by trypsinization. After the incubation, cells were rapidly washed and not fixed with 1% PFA. For studying the effect of ALLN and CsA, the cells were pretreated with same concentrations of drugs as in the cell surface expression studies. After incubation for 4 hours cells were washed and apoA-I binding experiment was performed. The apoA-I binding was detected by flow cytometry (Becton Dickinson FACS Calibur) and analyzed by CellQuest Pro software. Binding data are calculated from the median of apoA-I-Cy5 fluorescence intensity.
The authors thank Dr. Olga Újhellyi, Dr. Attila Iliás, Dr János Éles, and Dr. György Váradi for their support and advices; Mónika Bátkai, Gyöngyi Bézsenyi, Judit Kiss, Zsuzsanna Andrási and Györgyi Vermes for the perfect technical assistance. This work was supported by research grants from the OTKA (K68936, T38337).
- Boden WE: High-density lipoprotein cholesterol as an independent risk factor in cardiovascular disease: assessing the data from Framingham to the Veterans Affairs High--Density Lipoprotein Intervention Trial. Am J Cardiol. 2000, 86: 19L-22L. 10.1016/S0002-9149(00)01464-8.View ArticlePubMedGoogle Scholar
- Fielding CJ, Fielding PE: Molecular physiology of reverse cholesterol transport. J Lipid Res. 1995, 36: 211-228.PubMedGoogle Scholar
- Oram JF, Lawn RM: ABCA1. The gatekeeper for eliminating excess tissue cholesterol. J Lipid Res. 2001, 42: 1173-1179.PubMedGoogle Scholar
- Srivastava N: ATP binding cassette transporter A1--key roles in cellular lipid transport and atherosclerosis. Mol Cell Biochem. 2002, 237: 155-164. 10.1023/A:1016506221047.View ArticlePubMedGoogle Scholar
- Oram JF: ATP-binding cassette transporter A1 and cholesterol trafficking. Curr Opin Lipidol. 2002, 13: 373-381. 10.1097/00041433-200208000-00004.View ArticlePubMedGoogle Scholar
- Oram JF: Molecular basis of cholesterol homeostasis: lessons from Tangier disease and ABCA1. Trends Mol Med. 2002, 8: 168-173. 10.1016/S1471-4914(02)02289-X.View ArticlePubMedGoogle Scholar
- Schmitz G, Langmann T: Structure, function and regulation of the ABC1 gene product. Curr Opin Lipidol. 2001, 12: 129-140. 10.1097/00041433-200104000-00006.View ArticlePubMedGoogle Scholar
- Brunham LR, Kruit JK, Iqbal J, Fievet C, Timmins JM, Pape TD, Coburn BA, Bissada N, Staels B, Groen AK, Hussain MM, Parks JS, Kuipers F, Hayden MR: Intestinal ABCA1 directly contributes to HDL biogenesis in vivo. J Clin Invest. 2006, 116: 1052-1062. 10.1172/JCI27352.PubMed CentralView ArticlePubMedGoogle Scholar
- Schachter M: Strategies for modifying high-density lipoprotein cholesterol: a role for nicotinic acid. Cardiovasc Drugs Ther. 2005, 19: 415-422. 10.1007/s10557-005-5685-0.View ArticlePubMedGoogle Scholar
- Executive Summary of The Third Report of The National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, And Treatment of High Blood Cholesterol In Adults (Adult Treatment Panel III). Jama. 2001, 285: 2486-2497. 10.1001/jama.285.19.2486.Google Scholar
- Bays HE, Neff D, Tomassini JE, Tershakovec AM: Ezetimibe: cholesterol lowering and beyond. Expert Rev Cardiovasc Ther. 2008, 6: 447-470. 10.1586/14779072.6.4.447.View ArticlePubMedGoogle Scholar
- Argmann CA, Edwards JY, Sawyez CG, O'Neil CH, Hegele RA, Pickering JG, Huff MW: Regulation of macrophage cholesterol efflux through hydroxymethylglutaryl-CoA reductase inhibition: a role for RhoA in ABCA1-mediated cholesterol efflux. J Biol Chem. 2005, 280: 22212-22221. 10.1074/jbc.M502761200.View ArticlePubMedGoogle Scholar
- Sone H, Shimano H, Shu M, Nakakuki M, Takahashi A, Sakai M, Sakamoto Y, Yokoo T, Matsuzaka K, Okazaki H, Nakagawa Y, Iida KT, Suzuki H, Toyoshima H, Horiuchi S, Yamada N: Statins downregulate ATP-binding-cassette transporter A1 gene expression in macrophages. Biochem Biophys Res Commun. 2004, 316: 790-794. 10.1016/j.bbrc.2004.02.121.View ArticlePubMedGoogle Scholar
- Wong J, Quinn CM, Brown AJ: Statins inhibit synthesis of an oxysterol ligand for the liver x receptor in human macrophages with consequences for cholesterol flux. Arterioscler Thromb Vasc Biol. 2004, 24: 2365-2371. 10.1161/01.ATV.0000148707.93054.7d.View ArticlePubMedGoogle Scholar
- Wong J, Quinn CM, Gelissen IC, Jessup W, Brown AJ: The effect of statins on ABCA1 and ABCG1 expression in human macrophages is influenced by cellular cholesterol levels and extent of differentiation. Atherosclerosis. 2008, 196: 180-189. 10.1016/j.atherosclerosis.2007.03.030.View ArticlePubMedGoogle Scholar
- Martin G, Duez H, Blanquart C, Berezowski V, Poulain P, Fruchart JC, Najib-Fruchart J, Glineur C, Staels B: Statin-induced inhibition of the Rho-signaling pathway activates PPARalpha and induces HDL apoA-I. J Clin Invest. 2001, 107: 1423-1432. 10.1172/JCI10852.PubMed CentralView ArticlePubMedGoogle Scholar
- Mancini GB: Antiatherosclerotic effects of calcium channel blockers. Prog Cardiovasc Dis. 2002, 45: 1-20. 10.1053/pcad.2002.122694.View ArticlePubMedGoogle Scholar
- Waters D, Lesperance J: Calcium channel blockers and coronary atherosclerosis: from the rabbit to the real world. Am Heart J. 1994, 128: 1309-1316. 10.1016/0002-8703(94)90253-4.View ArticlePubMedGoogle Scholar
- Suzuki S, Nishimaki-Mogami T, Tamehiro N, Inoue K, Arakawa R, Abe-Dohmae S, Tanaka AR, Ueda K, Yokoyama S: Verapamil increases the apolipoprotein-mediated release of cellular cholesterol by induction of ABCA1 expression via Liver X receptor-independent mechanism. Arterioscler Thromb Vasc Biol. 2004, 24: 519-525. 10.1161/01.ATV.0000117178.94087.ba.View ArticlePubMedGoogle Scholar
- Hasegawa K, Wakino S, Kanda T, Yoshioka K, Tatematsu S, Homma K, Takamatsu I, Sugano N, Hayashi K: Divergent action of calcium channel blockers on ATP-binding cassette protein expression. J Cardiovasc Pharmacol. 2005, 46: 787-793. 10.1097/01.fjc.0000187976.60262.bf.View ArticlePubMedGoogle Scholar
- Kaminski WE, Piehler A, Wenzel JJ: ABC A-subfamily transporters: structure, function and disease. Biochim Biophys Acta. 2006, 1762: 510-524.View ArticlePubMedGoogle Scholar
- Schmitz G, Liebisch G, Langmann T: Lipidomic strategies to study structural and functional defects of ABC-transporters in cellular lipid trafficking. FEBS Lett. 2006, 580: 5597-5610. 10.1016/j.febslet.2006.08.014.View ArticlePubMedGoogle Scholar
- Lorenzi I, von Eckardstein A, Cavelier C, Radosavljevic S, Rohrer L: Apolipoprotein A-I but not high-density lipoproteins are internalised by RAW macrophages: roles of ATP-binding cassette transporter A1 and scavenger receptor BI. J Mol Med. 2008, 86: 171-183. 10.1007/s00109-007-0267-1.View ArticlePubMedGoogle Scholar
- Smith JD, Le Goff W, Settle M, Brubaker G, Waelde C, Horwitz A, Oda MN: ABCA1 mediates concurrent cholesterol and phospholipid efflux to apolipoprotein A-I. J Lipid Res. 2004, 45: 635-644. 10.1194/jlr.M300336-JLR200.View ArticlePubMedGoogle Scholar
- Albrecht C, McVey JH, Elliott JI, Sardini A, Kasza I, Mumford AD, Naoumova RP, Tuddenham EG, Szabo K, Higgins CF: A novel missense mutation in ABCA1 results in altered protein trafficking and reduced phosphatidylserine translocation in a patient with Scott syndrome. Blood. 2005, 106: 542-549. 10.1182/blood-2004-05-2056.View ArticlePubMedGoogle Scholar
- Tanaka AR, Ikeda Y, Abe-Dohmae S, Arakawa R, Sadanami K, Kidera A, Nakagawa S, Nagase T, Aoki R, Kioka N, Amachi T, Yokoyama S, Ueda K: Human ABCA1 contains a large amino-terminal extracellular domain homologous to an epitope of Sjogren's Syndrome. Biochem Biophys Res Commun. 2001, 283: 1019-1025. 10.1006/bbrc.2001.4891.View ArticlePubMedGoogle Scholar
- Hamon Y, Broccardo C, Chambenoit O, Luciani MF, Toti F, Chaslin S, Freyssinet JM, Devaux PF, McNeish J, Marguet D, Chimini G: ABC1 promotes engulfment of apoptotic cells and transbilayer redistribution of phosphatidylserine. Nat Cell Biol. 2000, 2: 399-406. 10.1038/35017029.View ArticlePubMedGoogle Scholar
- Chambenoit O, Hamon Y, Marguet D, Rigneault H, Rosseneu M, Chimini G: Specific docking of apolipoprotein A-I at the cell surface requires a functional ABCA1 transporter. J Biol Chem. 2001, 276: 9955-9960. 10.1074/jbc.M010265200.View ArticlePubMedGoogle Scholar
- Wang N, Silver DL, Thiele C, Tall AR: ATP-binding cassette transporter A1 (ABCA1) functions as a cholesterol efflux regulatory protein. J Biol Chem. 2001, 276: 23742-23747. 10.1074/jbc.M102348200.View ArticlePubMedGoogle Scholar
- Ujhelly O, Ozvegy C, Varady G, Cervenak J, Homolya L, Grez M, Scheffer G, Roos D, Bates SE, Varadi A, Sarkadi B, Nemet K: Application of a human multidrug transporter (ABCG2) variant as selectable marker in gene transfer to progenitor cells. Hum Gene Ther. 2003, 14: 403-412. 10.1089/104303403321209005.View ArticlePubMedGoogle Scholar
- Alder-Baerens N, Muller P, Pohl A, Korte T, Hamon Y, Chimini G, Pomorski T, Herrmann A: Headgroup-specific exposure of phospholipids in ABCA1-expressing cells. J Biol Chem. 2005, 280: 26321-26329. 10.1074/jbc.M413993200.View ArticlePubMedGoogle Scholar
- Ohama T, Hirano K, Zhang Z, Aoki R, Tsujii K, Nakagawa-Toyama Y, Tsukamoto K, Ikegami C, Matsuyama A, Ishigami M, Sakai N, Hiraoka H, Ueda K, Yamashita S, Matsuzawa Y: Dominant expression of ATP-binding cassette transporter-1 on basolateral surface of Caco-2 cells stimulated by LXR/RXR ligands. Biochem Biophys Res Commun. 2002, 296: 625-630. 10.1016/S0006-291X(02)00853-7.View ArticlePubMedGoogle Scholar
- Bortnick AE, Rothblat GH, Stoudt G, Hoppe KL, Royer LJ, McNeish J, Francone OL: The correlation of ATP-binding cassette 1 mRNA levels with cholesterol efflux from various cell lines. J Biol Chem. 2000, 275: 28634-28640. 10.1074/jbc.M003407200.View ArticlePubMedGoogle Scholar
- Wang N, Silver DL, Costet P, Tall AR: Specific binding of ApoA-I, enhanced cholesterol efflux, and altered plasma membrane morphology in cells expressing ABC1. J Biol Chem. 2000, 275: 33053-33058. 10.1074/jbc.M005438200.View ArticlePubMedGoogle Scholar
- Martinez LO, Agerholm-Larsen B, Wang N, Chen W, Tall AR: Phosphorylation of a pest sequence in ABCA1 promotes calpain degradation and is reversed by ApoA-I. J Biol Chem. 2003, 278: 37368-37374. 10.1074/jbc.M307161200.View ArticlePubMedGoogle Scholar
- Arakawa R, Yokoyama S: Helical apolipoproteins stabilize ATP-binding cassette transporter A1 by protecting it from thiol protease-mediated degradation. J Biol Chem. 2002, 277: 22426-22429. 10.1074/jbc.M202996200.View ArticlePubMedGoogle Scholar
- Wang N, Chen W, Linsel-Nitschke P, Martinez LO, Agerholm-Larsen B, Silver DL, Tall AR: A PEST sequence in ABCA1 regulates degradation by calpain protease and stabilization of ABCA1 by apoA-I. J Clin Invest. 2003, 111: 99-107.PubMed CentralView ArticlePubMedGoogle Scholar
- Le Goff W, Peng DQ, Settle M, Brubaker G, Morton RE, Smith JD: Cyclosporin A traps ABCA1 at the plasma membrane and inhibits ABCA1-mediated lipid efflux to apolipoprotein A-I. Arterioscler Thromb Vasc Biol. 2004, 24: 2155-2161. 10.1161/01.ATV.0000144811.94581.52.View ArticlePubMedGoogle Scholar
- Neufeld EB, Remaley AT, Demosky SJ, Stonik JA, Cooney AM, Comly M, Dwyer NK, Zhang M, Blanchette-Mackie J, Santamarina-Fojo S, Brewer HB: Cellular localization and trafficking of the human ABCA1 transporter. J Biol Chem. 2001, 276: 27584-27590. 10.1074/jbc.M103264200.View ArticlePubMedGoogle Scholar
- Schwartz GG, Olsson AG, Ezekowitz MD, Ganz P, Oliver MF, Waters D, Zeiher A, Chaitman BR, Leslie S, Stern T: Effects of atorvastatin on early recurrent ischemic events in acute coronary syndromes: the MIRACL study: a randomized controlled trial. Jama. 2001, 285: 1711-1718. 10.1001/jama.285.13.1711.View ArticlePubMedGoogle Scholar
- Pedersen TR, Faergeman O, Kastelein JJ, Olsson AG, Tikkanen MJ, Holme I, Larsen ML, Bendiksen FS, Lindahl C, Szarek M, Tsai J: High-dose atorvastatin vs usual-dose simvastatin for secondary prevention after myocardial infarction: the IDEAL study: a randomized controlled trial. Jama. 2005, 294: 2437-2445. 10.1001/jama.294.19.2437.View ArticlePubMedGoogle Scholar
- Davidson MH, McGarry T, Bettis R, Melani L, Lipka LJ, LeBeaut AP, Suresh R, Sun S, Veltri EP: Ezetimibe coadministered with simvastatin in patients with primary hypercholesterolemia. J Am Coll Cardiol. 2002, 40: 2125-2134. 10.1016/S0735-1097(02)02610-4.View ArticlePubMedGoogle Scholar
- Guyton JR, Brown BG, Fazio S, Polis A, Tomassini JE, Tershakovec AM: Lipid-altering efficacy and safety of ezetimibe/simvastatin coadministered with extended-release niacin in patients with type IIa or type IIb hyperlipidemia. J Am Coll Cardiol. 2008, 51: 1564-1572. 10.1016/j.jacc.2008.03.003.View ArticlePubMedGoogle Scholar
- Kerzner B, Corbelli J, Sharp S, Lipka LJ, Melani L, LeBeaut A, Suresh R, Mukhopadhyay P, Veltri EP: Efficacy and safety of ezetimibe coadministered with lovastatin in primary hypercholesterolemia. Am J Cardiol. 2003, 91: 418-424. 10.1016/S0002-9149(02)03236-8.View ArticlePubMedGoogle Scholar
- Sudhop T, Lutjohann D, Kodal A, Igel M, Tribble DL, Shah S, Perevozskaya I, von Bergmann K: Inhibition of intestinal cholesterol absorption by ezetimibe in humans. Circulation. 2002, 106: 1943-1948. 10.1161/01.CIR.0000034044.95911.DC.View ArticlePubMedGoogle Scholar
- Nieland TJ, Chroni A, Fitzgerald ML, Maliga Z, Zannis VI, Kirchhausen T, Krieger M: Cross-inhibition of SR-BI- and ABCA1-mediated cholesterol transport by the small molecules BLT-4 and glyburide. J Lipid Res. 2004, 45: 1256-1265. 10.1194/jlr.M300358-JLR200.View ArticlePubMedGoogle Scholar
- Telbisz A, Muller M, Ozvegy-Laczka C, Homolya L, Szente L, Varadi A, Sarkadi B: Membrane cholesterol selectively modulates the activity of the human ABCG2 multidrug transporter. Biochim Biophys Acta. 2007, 1768: 2698-2713. 10.1016/j.bbamem.2007.06.026.View ArticlePubMedGoogle Scholar
- Attie AD: ABCA1: at the nexus of cholesterol, HDL and atherosclerosis. Trends Biochem Sci. 2007, 32: 172-179. 10.1016/j.tibs.2007.02.001.View ArticlePubMedGoogle Scholar
- Asztalos BF, Horvath KV, McNamara JR, Roheim PS, Rubinstein JJ, Schaefer EJ: Comparing the effects of five different statins on the HDL subpopulation profiles of coronary heart disease patients. Atherosclerosis. 2002, 164: 361-369. 10.1016/S0021-9150(02)00149-1.View ArticlePubMedGoogle Scholar
- Zanotti I, Favari E, Sposito AC, Rothblat GH, Bernini F: Pitavastatin increases ABCA1-mediated lipid efflux from Fu5AH rat hepatoma cells. Biochem Biophys Res Commun. 2004, 321: 670-674. 10.1016/j.bbrc.2004.07.020.View ArticlePubMedGoogle Scholar
- Rubic T, Trottmann M, Lorenz RL: Stimulation of CD36 and the key effector of reverse cholesterol transport ATP-binding cassette A1 in monocytoid cells by niacin. Biochem Pharmacol. 2004, 67: 411-419. 10.1016/j.bcp.2003.09.014.View ArticlePubMedGoogle Scholar
- Bacso Z, Nagy H, Goda K, Bene L, Fenyvesi F, Matko J, Szabo G: Raft and cytoskeleton associations of an ABC transporter: P-glycoprotein. Cytometry A. 2004, 61: 105-116. 10.1002/cyto.a.20081.View ArticlePubMedGoogle Scholar
- Orlowski S, Martin S, Escargueil A: P-glycoprotein and 'lipid rafts': some ambiguous mutual relationships (floating on them, building them or meeting them by chance?). Cell Mol Life Sci. 2006, 63: 1038-1059. 10.1007/s00018-005-5554-9.View ArticlePubMedGoogle Scholar
- Hui DY, Labonte ED, Howles PN: Development and physiological regulation of intestinal lipid absorption. III. Intestinal transporters and cholesterol absorption. Am J Physiol Gastrointest Liver Physiol. 2008, 294: G839-843. 10.1152/ajpgi.00061.2008.View ArticlePubMedGoogle Scholar
- During A, Dawson HD, Harrison EH: Carotenoid transport is decreased and expression of the lipid transporters SR-BI, NPC1L1, and ABCA1 is downregulated in Caco-2 cells treated with ezetimibe. J Nutr. 2005, 135: 2305-2312.PubMedGoogle Scholar
- Neufeld EB, Stonik JA, Demosky SJ, Knapper CL, Combs CA, Cooney A, Comly M, Dwyer N, Blanchette-Mackie J, Remaley AT, Santamarina-Fojo S, Brewer HB: The ABCA1 transporter modulates late endocytic trafficking: insights from the correction of the genetic defect in Tangier disease. J Biol Chem. 2004, 279: 15571-15578. 10.1074/jbc.M314160200.View ArticlePubMedGoogle Scholar
- Szakacs G, Langmann T, Ozvegy C, Orso E, Schmitz G, Varadi A, Sarkadi B: Characterization of the ATPase cycle of human ABCA1: implications for its function as a regulator rather than an active transporter. Biochem Biophys Res Commun. 2001, 288: 1258-1264. 10.1006/bbrc.2001.5905.View ArticlePubMedGoogle Scholar
- Becker S, Wasser S, Hauses M, Hossle JP, Ott MG, Dinauer MC, Ganser A, Hoelzer D, Seger R, Grez M: Correction of respiratory burst activity in X-linked chronic granulomatous cells to therapeutically relevant levels after gene transfer into bone marrow CD34+ cells. Hum Gene Ther. 1998, 9: 1561-1570. 10.1089/hum.1998.9.11-1561.View ArticlePubMedGoogle Scholar
- Pear WS, Nolan GP, Scott ML, Baltimore D: Production of high-titer helper-free retroviruses by transient transfection. Proc Natl Acad Sci USA. 1993, 90: 8392-8396. 10.1073/pnas.90.18.8392.PubMed CentralView ArticlePubMedGoogle Scholar
- Miller AD, Garcia JV, von Suhr N, Lynch CM, Wilson C, Eiden MV: Construction and properties of retrovirus packaging cells based on gibbon ape leukemia virus. J Virol. 1991, 65: 2220-2224.PubMed CentralPubMedGoogle Scholar
- Seres L, Cserepes J, Elkind NB, Torocsik D, Nagy L, Sarkadi B, Homolya L: Functional ABCG1 expression induces apoptosis in macrophages and other cell types. Biochim Biophys Acta. 2008, 1778: 2378-2387. 10.1016/j.bbamem.2008.06.010.View ArticlePubMedGoogle Scholar
- Sarkadi B, Price EM, Boucher RC, Germann UA, Scarborough GA: Expression of the human multidrug resistance cDNA in insect cells generates a high activity drug-stimulated membrane ATPase. J Biol Chem. 1992, 267: 4854-4858.PubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.