Effects of retinoids on differentiation, lipid metabolism, epidermal growth factor, and low-density lipoprotein binding in squamous carcinoma cells

The relationship among keratinocyte differentiation capacity, lipid synthesis, low-density lipoprotein (LDL) metabolism, plasma membrane composition, and epidermal growth factor (EGF) binding has been studied in SCC-12F2 cells. The differentiation capacity of the cells, i.e., ionophore-induced cornified envelope formation, was inhibited by various retinoids and stimulated by hydrocortisone. Retinoids that caused a significant reduction of cornified envelope formation, i.e., retinoic acid and 13-cis-retinoic acid, caused only minor changes in lipid synthesis and plasma membrane composition. Arotinoid ethylsulfone, having a minor effect on cornified envelope formation, caused a drastic inhibition of cholesterol synthesis, resulting in changes in the plasma membrane composition. Hydrocortisone stimulated cornified envelope formation but had only minor effects on lipid synthesis and plasma membrane composition. Of all retinoids tested, only arotinoid ethylsulfone caused a drastic increase in EGF binding, while hydrocortisone had no effect. Retinoic acid, arotinoid ethylsulfone, and hydrocortisone had no effects on LDL binding and only minor effects on LDL degradation. These results clearly demonstrate that the plasma membrane composition is not related to keratinocyte differentiation capacity, but most likely does determine EGF binding. Furthermore, EGF binding does not determine keratinocyte differentiation capacity.     

25-Hydroxycholesterol-induced elevations in 45Ca uptake: permeability changes in P815 cells

Certain oxysterols are capable of suppressing the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase. We have previously demonstrated that treatment of P815 cells with 1 microgram 25-hydroxycholesterol/ml culture results in a rapid influx of 45Ca, and supplemental cholesterol prevents this from occurring. In this paper, we report on investigations into the means whereby this influx of calcium takes place. Through the use of respiratory inhibitors which prevent mitochondrial retention of calcium it was determined that the large increase in slow phase (intracellular) calcium uptake caused by 25-hydroxycholesterol treatment was related to mitochondrial uptake. The effects of various inhibitors of calcium uptake into cells, including verapamil, diltiazem, quinidine, ruthenium red, Co++, Mn++, were tested. Of these only Co++ and ruthenium red had any effect on 45Ca uptake. 25-Hydroxycholesterol has been shown to be capable of membrane insertion and this could result in plasma membrane permeability changes. To test this hypothesis P815 cells were treated with 1 microgram 25-hydroxycholesterol/ml or 5 micrograms mevinolin/ml culture. Mevinolin, being a water soluble competitive inhibitor of HMG-CoA reductase, should be unable to disrupt membrane architecture in a manner analogous to 25-hydroxycholesterol. While both inhibitors rapidly suppressed the synthesis of digitonin-precipitable sterols, only 25-hydroxycholesterol was able to increase 45Ca influx. The implications of these findings are discussed.     

The influence of cholesterol and lipid metabolism on host cell structure and hepatitis C virus replication.

The hepatitis C virus (HCV) replicates on a membrane protein complex composed of viral proteins, replicating RNA, and altered cellular membranes. Small-molecule inhibitors of cellular lipid-cholesterol metabolism such as 25-hydroxycholesterol, cerulenin, lovastatin, and GGTI-286 all show a negative effect on HCV replication. Perturbation of host cell lipid and cholesterol metabolism can disrupt replication complexes by altering membranous structures where replication occurs. Changes in cholesterol and (or) lipid composition can have a general effect on membrane structure. Alternatively, metabolic changes can exert a more subtle influence over replication complexes by altering localization of host proteins through alterations in lipid anchoring. Here, we use Huh-7 cells harboring subgenomic HCV replicons to demonstrate that 25-hydroxycholesterol, cerulenin, lovastatin, and GGTI-286 do not disrupt the membranous web where replication occurs, whereas cholesterol-depleting agents such as beta-cyclodextrin do. Cellular imaging suggests that the HCV RNA can remain associated with subcellular compartments connected with replication complexes in the presence of metabolic inhibitors. Therefore, at least 2 different molecular mechanisms are possible for the inhibition of HCV replication through the modulation of cellular lipid and cholesterol metabolism.     

Human crypt intestinal epithelial cells are capable of lipid production, apolipoprotein synthesis, and lipoprotein assembly

The recent availability of spontaneously proliferating, non-transformed human crypt intestinal epithelial cells (HIEC) affords an opportunity to investigate lipid metabolism in undifferentiated enterocytes. The major purpose of this study was to explore the capability of undifferentiated crypt cells to synthesize, assemble, and secrete lipids and apolipoproteins. HIEC were cultured in medium with 5% fetal bovine serum for 5 to 21 d. The cells were clearly able to incorporate [(14)C]oleic acid (dpm/mg protein) into triglycerides (128,279 +/- 16,988), phospholipids (30, 278 +/- 2,107), and cholesteryl esters (2,180 +/- 207). Although improvement in lipid secretion was noted with prolongation of cell culture periods, low efficiency of lipid export (10.3 +/- 2.2% of intracellular content) characterized the HIEC. All phospholipid classes were elaborated, with phosphatidylcholine accounting for 79. 3 +/- 1.3% of cellular phospholipids. Chylomicrons were the dominant (46.4%) lipoproteins secreted, followed by high, low, and very low density lipoproteins (HDL, LDL, and VLDL) comprising 22.5, 20.2, and 10.8% of the total, respectively. HIEC elaborated most of the major apolipoprotein (apo) classes (A-I, A-IV, B-100, C, and E), but were less efficient in producing apoB-48. In contrast to the production of apoA-I and C as early as 5 days after confluence, apoA-I and A-IV were maximally expressed at 11 d. Culture media accumulated much more apoB-100 than apoB-48 (B-48/B-100 ratio 0.21 +/- 0.03), reflecting limited apoB mRNA editing. HIEC demonstrated both endogenous cholesterol synthesis and LDL receptor expression. Cholesterol synthesis was sensitive to 25-hydroxycholesterol and mevinolin, but unresponsive to LDL treatment, suggesting independent regulation pathways. In contrast, LDL inhibited receptor activity. The present findings provide the first solid evidence that immature HIEC are capable of key fat absorptive functions of well-differentiated enterocytes. The intracellular mechanisms required for lipid and apolipoprotein synthesis as well as for lipoprotein assembly are already present in intestinal crypt cells. These cells also retain the capacity for sterol enzyme and receptor expression. However, certain limitations, especially apoB-48 production and lipoprotein secretion as well as unresponsiveness of cholesterol synthesis to LDL, may be ascribed to the lack of differentiation.     

The ATP-binding cassette transporter-2 (ABCA2) regulates cholesterol homeostasis and low-density lipoprotein receptor metabolism in N2a neuroblastoma cells

The ATP-binding cassette transporter-2 (ABCA2) has been identified as a possible regulator of lipid metabolism. ABCA2 is most highly expressed in the brain but its effects on cholesterol homeostasis in neuronal-type cells have not been characterized. It is important to study the role of ABCA2 in regulating cholesterol homeostasis in neuronal-type cells because ABCA2 has been identified as a possible genetic risk factor for Alzheimer's disease. In this study, the effects of ABCA2 expression on cholesterol homeostasis were examined in mouse N2a neuroblastoma cells. ABCA2 reduced total, free- and esterified cholesterol levels as well as membrane cholesterol but did not perturb cholesterol distribution in organelle or lipid raft compartments. ABCA2 did not modulate de novo cholesterol biosynthesis from acetate. Cholesterol trafficking to the plasma membrane was not affected by ABCA2 but efflux to the physiological acceptor ApoE3 and mobilization of plasma membrane cholesterol to the endoplasmic reticulum for esterification were reduced by ABCA2. ABCA2 reduced esterification of serum and low-density lipoprotein-derived cholesterol but not 25-hydroxycholesterol. ABCA2 decreased low-density lipoprotein receptor (LDLR) mRNA and protein levels and increased its turnover rate. The surface expression of LDLR as well as the uptake of fluroresecent DiI-LDL was also reduced by ABCA2. Reduction of endogenous ABCA2 expression by RNAi treatment of N2a cells and rat primary cortical neurons produced the opposite effects of over-expression of ABCA2, increasing LDLR protein levels. This report identifies ABCA2 as a key regulator of cholesterol homeostasis and LDLR metabolism in neuronal cells.     

Increased rates of lipid exchange between Mycoplasma capricolum membranes and vesicles in relation to the propensity of forming nonbilayer lipid structures

We have studied the effects of modification of the endogenous phosphatidylglycerol (PG) and diphosphatidylglycerol (DPG) content of the plasma membrane of Mycoplasma capricolum on the kinetics of spontaneous [14C]cholesterol and 14C-labeled phospholipid exchange between M. capricolum membranes and lipid vesicles. The PG/DPG molar ratio of M. capricolum membranes changed when cells were grown in media supplemented with 0.5 mM CaCl2 and/or egg phosphatidylcholine (PC) (10-20 micrograms/ml), increasing from 3.9 to 6.3 on supplementation with Ca2+; this ratio decreased to 1.1 in media supplemented with PC and to 1.8 in media containing both PC and Ca2+. The ratio of palmitate to oleate in both PG and DPG decreased when cells were grown with PC or with PC and Ca2+. Bilayer disruptions were seen in freeze-fracture electron micrographs of trypsin-treated M. capricolum membranes from cells grown with both Ca2+ and PC, and numerous lipidic particles and other bilayer disruptions were observed in trypsin-treated M. capricolum membranes and their lipid extracts. The rates of spontaneous exchange of 14C-labeled cholesterol and PC from membranes isolated from cells grown with PC and Ca2+ to acceptor lipid vesicles were exchanged by approximately 30%, and the rate of the rapidly exchangeable cholesterol pool in intact cells was enhanced by 64%. The enhancements in cholesterol and PC exchange rates are considered to result from structural defects expected in the M. capricolum membranes obtained from cells grown with Ca2+ supplementation. Our findings parallel previous examples of functional modifications of membranes induced by bilayer instability arising from a pretransitional state leading to the onset of a nonlamellar phase.     

The effect of 25-hydroxycholesterol on accumulation of intracellular calcium.

Liposomes prepared with 25-hydroxycholesterol and egg phosphatidylcholine (PC) were incubated with bovine arterial smooth muscle cells for 8 h at 37 degrees C. Cells incubated in the absence of liposomes or with liposomes containing cholesterol and PC were used as controls. The results indicated that calcium accumulated in the smooth muscle cells incubated in the presence of 25-hydroxycholesterol containing liposomes in an amount proportional to the time of incubation. The calcium accumulation, as indicated by kinetic analysis, resulted from an increased compartment size. (Ca(2+)+Mg2+)-ATPase exhibited decreased activity after pretreatment with 25-hydroxycholesterol containing liposomes and the increased intracellular calcium content was directly proportional to the decreased (Ca(2+) + Mg2+)-ATPase activity. When lipids in the cell membrane were examined, a failure to change the cholesterol/phospholipids ratio in the membrane was noted. The 25-hydroxycholesterol content in the membrane determined by HPLC did not increase. An increase in sphingomyelin and a decrease in phosphatidylethanolamine and acidic phospholipids in the membrane was noted. We suggest that the accumulation of intracellular calcium comes from both an increase of calcium influx and a decrease of (Ca(2+) + Mg2+)-ATPase activity, which may be the consequence of changes in membrane phospholipid composition.     

Effects of cholesterol sulfate on lipid metabolism in cultured human keratinocytes and fibroblasts

Effects of cholesterol sulfate on acetate incorporation into lipid fractions were examined in normal human fibroblast and keratinocyte cultures. Inhibition of sterologenesis in normal fibroblast cultures by cholesterol sulfate was less profound than that produced by either lipoprotein-containing serum or 25-hydroxycholesterol. Cholesterol sulfate also inhibited sterologenesis in low density lipoprotein receptor-deficient fibroblasts and inhibited both sterologenesis and 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in keratinocytes. Cholesterol sulfate increased incorporation of acetate into fatty acid-containing lipids in preconfluent cultures of both cell types in lipoprotein-depleted media. Similar effects were not observed either in response to lipoprotein-containing serum or 25-hydroxycholesterol. Cholesterol sulfate had no effect on oleic acid incorporation into diglycerides, triglycerides, or phospholipid fractions; neither did it inhibit acid lipase activity; nor did it inhibit fatty acid oxidation, indicating that cholesterol sulfate does not inhibit catabolism of acyl lipids. Because cholesterol sulfate had similar effects on fatty acid metabolism in steroid sulfatase-deficient fibroblasts lines, desulfation to cholesterol is not a prerequisite. Cholesterol sulfate did not significantly affect incorporation of oleic acid into sterol esters in fibroblast cultures, but in contrast, inhibited sterol esterification in keratinocyte cultures. These data suggest a novel role for cholesterol sulfate as a modulator of cellular lipid biosynthesis.     

Inhibition of cholesterol biosynthesis disrupts lipid raft/caveolae and affects insulin receptor activation in 3T3-L1 preadipocytes

Lipid rafts are plasma membrane microdomains that are highly enriched with cholesterol and sphingolipids and in which various receptors and other proteins involved in signal transduction reside. In the present work, we analyzed the effect of cholesterol biosynthesis inhibition on lipid raft/caveolae composition and functionality and assessed whether sterol precursors of cholesterol could substitute for cholesterol in lipid rafts/caveolae. 3T3-L1 preadipocytes were treated with distal inhibitors of cholesterol biosynthesis or vehicle (control) and then membrane rafts were isolated by sucrose density gradient centrifugation. Inhibition of cholesterol biosynthesis with either SKF 104976, AY 9944, 5,22-cholestadien-3β-ol or triparanol, which inhibit different enzymes on the pathway, led to a marked reduction in cholesterol content and accumulation of different sterol intermediates in both lipid rafts and non-raft domains. These changes in sterol composition were accompanied by disruption of lipid rafts, with redistribution of caveolin-1 and Fyn, impairment of insulin-Akt signaling and the inhibition of insulin-stimulated glucose transport. Cholesterol repletion abrogated the effects of cholesterol biosynthesis inhibitors, reflecting they were specific. Our results show that cholesterol is required for functional raft-dependent insulin signaling.     

Disorders in lipid composition and properties of plasma membrane in epithelial cells of the placental villous chorion in chlamydial infection

Lipid composition of plasma membranes of placental epithelial cells of villous chorion of healthy women and those with chlamydiosis has been studied. Lipid composition of plasma membranes of ill women differs from that of healthy women by reduction of total phospholipids quantity, by the increase of the level of free cholesterol and free fatty acids. A disturbance in the ratio between certain lipid fractions and increasing quantity of lysophospholipids is observed. Two-fold oppression of plasma membranes Na+, K+ -ATPase activity of placental epithelial cells of villous chorion in ill women has been detected but Mg2+, Ca2+ -ATPase activity has not changed. Thus chlamydial infection causes significant disorders in lipid composition and functioning of epithelial cells membranes of chorion.     

Sodium-calcium exchanger and lipid rafts in pig coronary artery smooth muscle

Pig coronary artery smooth muscle expresses, among many other proteins, Na+-Ca2+-exchanger NCX1 and sarcoplasmic reticulum Ca2+ pump SERCA2. NCX1 has been proposed to play a role in refilling the sarcoplasmic reticulum Ca2+ pool suggesting a functional linkage between the two proteins. We hypothesized that this functional linkage may require close apposition of SERCA2 and NCX1 involving regions of plasma membrane like lipid rafts. Lipid rafts are specialized membrane microdomains that appear as platforms to co-localize proteins. To determine the distribution of NCX1, SERCA2 and lipid rafts, we isolated microsomes from the smooth muscle tissue, treated them with non-ionic detergent and obtained fractions of different densities by sucrose density gradient centrifugal flotation. We examined the distribution of NCX1; SERCA2; non-lipid raft plasma membrane marker transferrin receptor protein; lipid raft markers caveolin-1, flotillin-2, prion protein, GM1-gangliosides and cholesterol; and cytoskeletal markers clathrin, actin and myosin. Distribution of markers identified two subsets of lipid rafts that differ in their components. One subset is rich in caveolin-1 and flotillin-2 and the other in GM1-gangliosides, prion protein and cholesterol. NCX1 distribution correlated strongly with SERCA2, caveolin-1 and flotillin-2, less strongly with the other membrane markers and negatively with the cytoskeletal markers. These experiments were repeated with a non-detergent method of treating microsomes with sonication at high pH and similar results were obtained. These observations are consistent with the observed functional linkage between NCX1 and SERCA2 and suggest a role for NCX1 in supplying Ca2+ for refilling the sarcoplasmic reticulum.     

The plasma membrane lipid composition affects fusion between cells and model membranes

Investigations were carried out on the effect of plasma membrane lipid modifications on the fusogenic capacity of control and ras-transformed fibroblasts. The plasma membrane lipid composition was modified by treatment of cells with exogenous phospholipases C and D, sphingomyelinase and cyclodextrin. The used enzymes hydrolyzed definite membrane lipids thus inducing specific modifications of the lipid composition while cyclodextrin treatment reduced significantly the level of cholesterol. The cells with modified membranes were used for assessment of their fusogenic capacity with model membranes with a constant lipid composition. Treatment with phospholipases C and D stimulated the fusogenic potential of both cell lines whereas the specific reduction of either sphingomyelin or cholesterol induced the opposite effect. The results showed that all modifications of the plasma membrane lipid composition affected the fusogenic capacity irrespective of the initial differences in the membrane lipid composition of the two cell lines. These results support the notion that the lipid composition plays a significant role in the processes of membrane-membrane fusion. This role could be either direct or through modulation of the activity of specific proteins which regulate membrane fusion.     

Human StarD5, a cytosolic StAR-related lipid binding protein

Recently identified StarD5 belongs to the StarD4 subfamily, a subfamily of steroidogenic acute regulatory related lipid transfer (START) domain proteins that includes StarD4 and StarD6, proteins whose functions remain unknown. The objective of this study was to confirm StarD5's protein localization and sterol binding capabilities as measures to pursue function. Using rabbit polyclonal antibody against newly purified human histidine-tagged/StarD5 protein, StarD5 was detected in human liver. In parallel studies, increased expression of StarD5 in primary hepatocytes led to a marked increase in microsomal free cholesterol. Cell fractionation studies demonstrated StarD5 protein in liver cytosolic fractions only, suggesting StarD5 as a directional cytosolic sterol carrier. Supportive in vitro binding assays demonstrated a concentration-dependent binding of cholesterol by StarD5 similar to that of the cholesterol binding START domain protein StarD1. In contrast to selective cholesterol binding by StarD1, StarD5 bound the potent regulatory oxysterol, 25-hydroxycholesterol, in a concentration-dependent manner. StarD5 binding appeared selective for cholesterol and 25-hydroxycholesterol, as no binding was observed for other tested sterols. The ability of StarD5 to bind not only cholesterol but also 25-hydroxycholesterol, a potent inflammatory mediator and regulatory oxysterol, raises basic fundamental questions about StarD5's role in the maintenance of cellular cholesterol homeostasis.     

Coxiella burnetii inhabits a cholesterol-rich vacuole and influences cellular cholesterol metabolism

Coxiella burnetii directs the synthesis of a large parasitophorous vacuole (PV) required for replication. While some lysosomal characteristics of the PV have been described, the origin and composition of the PV membrane is largely undefined. Cholesterol is an essential component of mammalian cell membranes where it plays important regulatory and structural roles. Here we investigated the role of host cholesterol in biogenesis and maintenance of the C. burnetii PV in Vero cells. The C. burnetii PV membrane stained with filipin and was positive for the lipid raft protein flotillin-1, suggesting PV membranes are enriched in cholesterol and contain lipid raft microdomains. C. burnetii infection increased host cell cholesterol content by 1.75-fold with a coincident upregulation of host genes involved in cholesterol metabolism. Treatment with U18666A, lovastatin, or 25-hydroxycholesterol, pharmacological agents that inhibit cholesterol uptake and/or biosynthesis, altered PV morphology and partially inhibited C. burnetii replication. Complete inhibition of C. burnetii PV development and replication was observed when infected cells were treated with imipramine or ketoconazole, inhibitors of cholesterol uptake and biosynthesis respectively. We conclude that C. burnetii infection perturbs host cell cholesterol metabolism and that free access to host cholesterol stores is required for optimal C. burnetii replication.     

Epidermal growth factor receptors are localized to lipid rafts that contain a balance of inner and outer leaflet lipids: a shotgun lipidomics study

The epidermal growth factor (EGF) receptor partitions into lipid rafts made using a detergent-free method, but is extracted from low density fractions by Triton X-100. By screening several detergents, we identified Brij 98 as a detergent in which the EGF receptor is retained in detergent-resistant membrane fractions. To identify the difference in lipid composition between those rafts that harbored the EGF receptor (detergent-free and Brij 98-resistant) and those that did not (Triton X-100-resistant), we used multidimensional electrospray ionization mass spectrometry to perform a lipidomics study on these three raft preparations. Although all three raft preparations were similarly enriched in cholesterol, the EGF receptor-containing rafts contained more ethanolamine glycerophospholipids and less sphingomyelin than did the non-EGF receptor-containing Triton X-100 rafts. As a result, the detergent-free and Brij 98-resistant rafts exhibited a balance of inner and outer leaflet lipids, whereas the Triton X-100 rafts contained a preponderance of outer leaflet lipids. Furthermore, in all raft preparations, the outer leaflet phospholipid species were significantly different from those in the bulk membrane, whereas the inner leaflet lipids were quite similar to those found in the bulk membrane. These findings indicate that the EGF receptor is retained only in rafts that exhibit a lipid distribution compatible with a bilayer structure and that the selection of phospholipids for inclusion into rafts occurs mainly on the outer leaflet lipids.     

Proteolytic cleavage of epidermal growth factor receptor. A Ca2+-dependent, sulfhydryl-sensitive proteolytic system in A431 cells

The Mr = 160,000 epidermal growth factor (EGF) receptor in A431 cells is partially cleaved during membrane isolation to a Mr = 145,000 polypeptide containing both EGF binding and phosphate acceptor sites. We show that the proteolytic degradation of the EGF receptor depends upon the presence of Ca2+ in the medium used to scrape the cells from the substratum. Only the high molecular weight form of the receptor is detected in membranes prepared in the absence of Ca2+. Ca2+-dependent proteolysis occurs rapidly (t1/2 approximately 5 min) following cell scraping. Proteolysis results in a decrease in EGF-dependent phosphorylation of the receptor while retaining EGF binding capacity. In addition, membranes containing the uncleaved form of the receptor reveal a substantial increase in EGF-dependent phosphorylation of proteins with Mr approximately 80, 89, and 185 X 10(3). In the presence of Ca2+, addition of iodoacetic acid to the scraping medium strongly inhibits receptor fragmentation, whereas other inhibitors (phenylmethylsulfonyl fluoride, leupeptin, and pepstatin) have no effect. The results implicate a role for a Ca2+-dependent, SH-sensitive protease in EGF receptor degradation. Prevention of proteolysis yields membrane preparations with highly active EGF-dependent kinase system.     

Sterol carrier protein-2 expression alters plasma membrane lipid distribution and cholesterol dynamics

Although sterol carrier protein-2 (SCP-2) binds, transfers, and/or enhances the metabolism of many membrane lipid species (fatty acids, cholesterol, phospholipids), it is not known if SCP-2 expression actually alters the membrane distribution of lipids in living cells or tissues. As shown herein for the first time, expression of SCP-2 in transfected L-cell fibroblasts reduced the plasma membrane levels of lipid species known to traffic through the HDL-receptor-mediated efflux pathway: cholesterol, cholesteryl esters, and phospholipids. While the ratio of cholesterol/phospholipid in plasma membranes of intact cells was not changed by SCP-2 expression, phosphatidylinositol, a molecule important to intracellular signaling and vesicular trafficking, and anionic phospholipids were selectively retained. Only modest alterations in plasma membrane phospholipid percent fatty acid composition but no overall change in the proportion of saturated, unsaturated, monounsaturated, or polyunsaturated fatty acids were observed. The reduced plasma membrane content of cholesterol was not due to SCP-2 inhibition of sterol transfer from the lysosomes to the plasma membranes. SCP-2 dramatically enhanced sterol transfer from isolated lysosomal membranes to plasma membranes by eliciting detectable sterol transfer within 30 s, decreasing the t(1/2) for sterol transfer 364-fold from >4 days to 7-15 min, and inducing formation of rapidly transferable sterol domains. In summary, data obtained with intact transfected cells and in vitro sterol transfer assays showed that SCP-2 expression (i) selectively modulated plasma membrane lipid composition and (ii) decreased the plasma membrane content cholesterol, an effect potentially due to more rapid SCP-2-mediated cholesterol transfer from versus to the plasma membrane.     

Compactin (ML-236B) reduces the content of filipin-cholesterol complexes in the plasma membrane of chronic lymphocytic leukemia cells

The polyene antibiotic filipin was used to visualize the presence and distribution of cholesterol in the plasma membrane of glutaraldehyde-fixed human chronic lymphocytic leukemia (CLL) cells. Both compactin (ML-236B), a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, and 25-hydroxycholesterol reduced the content of filipin-cholesterol complexes in the plasma membrane of CLL cells grown in media supplemented with either 15% delipidized horse serum or 15% normal (whole) horse serum. The reduction due to compactin was reversed by the concomitant addition of mevalonolactone. The ability of compactin to reduce the relative cholesterol content (as judged by filipin labeling) in CLL cells grown in lipoprotein-containing (normal) serum suggest that either CLL cells are different from other cells in that they predominantly utilize endogenously synthesized cholesterol for incorporation into the plasma membrane, or that a separate pool of endogenously synthesized cholesterol provides cholesterol for the plasma membrane.