Isolation and Characterization of Chinese Hamster Ovary Cells ...

Vol. 267, No.

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.

Issue of March 5, pp. 4889-4896,1992 Printed in U.S.A.

Isolation and Characterizationof Chinese Hamster Ovary Cells Defective inthe Intracellular Metabolism of Low Density Lipoprotein-derived Cholesterol* (Received for publication, October 10, 1991)

Neera K. DahlS, Karen L.ReedQ, Michelle A. Daunais, Jerry R. FaustS, and Laura Liscumll From the Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 021 I 1

We have isolated clones of an established cell line apolipoprotein E (3) have been implicated in intracellular which express defects in intracellular cholesterol me- cholesterol metabolism, the precise role of these factors in tabolism. Chinese hamster ovarycells were mutagen- transporting cholesterol and mediating cholesterol’s regulaized, and clones unable to mobilize low density lipoprotory responses is unknown. tein (LDL)-derivedcholesterol to theplasmamemNiemann-Pick disease type C (NPC) is an inborn error of brane were selected.Biochemical analysis of two metabolism that results in defective trafficking of low density mutant clones revealed a phenotype characteristic of lipoprotein (LDL)-derived cholesterol (4-7). In NPC fibrothe lysosomal storagedisease, Niemann-Pick typeC. blasts, LDL is internalized andhydrolyzed normally; however, The mutant cell lines were found to be defective in the LDL-derived cholesterol accumulates in lysosomes and the regulatory responses elicited by LDL-derived cho- moves to other cell membranes and regulatory sites more lesterol. LDL-mediated stimulation of cholesterol es- slowly than normal. Defective transport of LDL-derived choterification was grossly defective, and LDL suppres- lesterol results in impaired cholesterol homeostasis in NPC. sion of 3-hydroxy-3-methylglutaryl-CoAreductase This specific impairment, which is associated with a single was impaired. However, the mutants modulated these gene mutation, indicates that LDL-derived cholesterol transactivities normally in response to 25-hydroxycholes- port from lysosomes is not random or diffuse but is precisely terol or mevalonate.TheLDL-specific defects were mediated. Thus, NPC represents an appropriate cell model predicated by the inabilityof these mutants to mobilizefor identifying a component of the cholesterol transport pathLDL-derivedcholesterolfromlysosomes.Cell fractionation studies showed that LDL-derived, unesteri- way. However, NPC represents only one possible mutation in a complex pathway by which cholesterol moves to various fied cholesterol accumulated in the lysosomes of mutant cells to significantly higher levels than normal, sites and mediates transcriptional regulation, enzyme activacommensuratewith defective movement of cholesterol tion, and protein degradation. These processes may potento other cellular membranes. Characterization of cell tially involve multiple gene products. In addition, the usefulness of NPC fibroblasts in delineating the mechanism of lines defective in intracellular cholesterol transport will facilitate identification of thegene(s) required for intracellular cholesterol transport is limited because primary cultures of human fibroblasts have a limited lifespan and do intracellular cholesterol movement and regulation. not lend themselves to genetic manipulation. We have developed a selection protocol for isolating cell lines that express defects in the intracellular transport of Our goal is to identify cellular factors that mediate intra- LDL-derived cholesterol. Mutants have been selected which cellular cholesterol transport andregulation in uiuo. Although express defects in the transport of LDL-derived cholesterol proteins such as sterol carrier protein 2 (SCP2)’ (1, 2) and from lysosomes to theplasma membrane. Biochemical analysis of two mutant lines has revealed phenotypes that resemble * This work wassupported by the Whitaker Health Sciences Fund NPC. Characterization of multiple, nonallelic CHO cell lines and by a grant-in-aid award from the American Heart Association and Bristol-Myers. The costs of publication of this article were defective in intracellular cholesterol transport will facilitate defrayed in part by the payment of page charges. This article must identification of the gene(s) required for intracellular cholestherefore be hereby marked “advertisement” in accordance with 18 terol movement and regulation. ’

U.S.C. Section 1734 solelyto indicate this fact. $’ Supported by National Institutes of Health Training GrantDK 07542. Present address: Dept. of Animal Sciences, University of Delaware, Newark, DE. 1Established Investigator of the American Heart Association. To whom correspondence should be addressed Dept. of Physiology, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111. Tel. 617-956-6945. The abbreviations used are: SCPZ, sterol carrier protein 2; LDL, low density lipoprotein; NPC, Niemann-Pick type C; CHO, Chinese hamster ovary; [3H]CL-LDL, LDL that has been labeled with [3H] cholesteryl linoleate; r[25-HC oleate]LDL, LDL that has been reconstituted with 25-hydroxycholesteryl oleate; EBSS, Earle’s balanced salt solution; HMG-CoA, 3-hydroxy-3-methylglutarylcoenzyme A; PITP, phosphatidylinositol/phosphatidylcholinetransfer protein; and U18666A, 3-~-[2-(diethylamino)ethoxy]androst-5-en-l7-one; PI/ PC, phosphatidylinositolphosphatidylcholine; HEPES, 4-(2-hydroxyethy1)-l-piperazineethanesulfonicacid; CT, cholesterol transport.

EXPERIMENTALPROCEDURES

Materials [9,10-3H]Oleic acid (10 Ci/mmol), cholesteryl [l-14C]oleate (57 mCi/mmol), ~~-3-hydroxy-3-[~H]methylglutaryl-CoA (10.2 Ci/ mmol), RS-[2-’4C]mevalonolactone(48.6 mCi/mmol), [1,2,6,7-3H] cholesteryl linoleate (82.9 Ci/mmol), and [4-’4C]cholesterol (53.2 mCi/mmol) were purchased from Du Pont-New England Nuclear. Mevinolin was a generous gift of A. Alberts (Merck). 25-Hydroxycholesterol and cholesterol were added to medium from ethanolic solutions; amphotericin B was dissolved in dimethyl sulfoxide. Newborn calf serum and all other reagents were from Sigma or obtained as described previously (8). Preparation of LDL, Lipoprotein-deficient Serum, PHICL-LDL, rl.25-HC oleate]LDL, Media, and Buffers LDL was prepared by ultracentrifugation (9). Lipoprotein-deficient serum was prepared from newborn calf serum omitting the thrombin

4889

Cholesterol Transport-defective CHO

4890

incubation (9). [3H]CL-LDLwas prepared with an average specific activity of41,000 cpm/nmol of total cholesteryl linoleate (10). 25Hydroxycholesteryl oleate and r[25-HC oleate]LDL were prepared as described (8).The r[25-HC oleate]LDL had a sterol/protein ratio of 0.30. The content of total 25-hydroxycholesterol was 112pg/ml. The concentrations of native and r[25-HC oleate]LDL in culture medium are expressed as pg of protein/ml. The following media were prepared medium A (Ham's F-12 medium containing 5% (v/v) newborn calf serum, 2 mM glutamine, 100 units/ml penicillin, 100 pg/ml streptomycin, and 20 mM HEPES, pH 7.1); mediumB (medium A inwhich 5% (v/v) calf serum was replaced with 5% (v/v) lipoprotein-deficient calf serum); medium C (medium B containing 1%instead of 5% (v/v) lipoprotein-deficient calf serum); and medium D (medium B containing 20 p~ mevinolin and 0.5 mM mevalonate). The following buffers were prepared buffer A (150 mM NaCl, 50 mM Tris-chloride, pH 7.4); buffer B (buffer A containing 2 mg/ml bovine serum albumin); and buffer C (250 mM sucrose, 20 mM HEPES, and1 mM EDTA, pH 7.3). Cultured Cells All cells were grown as monolayers in a humidified incubator (5% CO,) a t 37 "C in medium A. On day 0 of each experiment, monolayer stock flasks were trypsinized, and cells were seeded as indicated in the individual experiment. Protein Determination Cell protein was determined by the method of Lowry et al. (11) using bovine serum albumin as a standard. Selection of Cholesterol Transport Mutants CHO cells (8 X lo6cells in three 150-mm dishes) were mutagenized with 400 pg/ml ethyl methanesulfonate in medium A for 18 h. Cells were washed with EBSS, trypsinized, and seeded into 40 100-mm dishes a t 75,000 cells/dish in medium A. The 40 pools of mutagenized cells were kept separate in subsequent selection steps. Selection I-On day 0, cells were washed with EBSS and refed 8 ml of medium B.On day 1, cells were fed 9 ml of medium D containing 4.5 pg/ml LDL. After 24 h, cells were washed with EBSS andtreated with 175 pg/ml amphotericin B in medium C. After 6 h, cells were washed twice with EBSSand refed medium A. Selection I was repeated once with 4.5 pg/ml LDL and thentwice with 9 pg/ml LDL. These selection parameters (LDLand amphotericin B concentrations and incubation times) were optimized in preliminary experiments. Selection ZZ-On day 0, cells surviving selection I were seeded into 100-mm dishes at 50,000 cells/dish in medium A. On day 1, cells were washed with EBSS and refed medium B. On day 2, cells were refed medium B containing 4pg/ml r[25-HC oleate]LDL. After 24 h, cells were treated with amphotericin B as described above. Selection I1 was repeated once after which colonies were picked using cloning cylinders. Selection I1 incubation conditions were determined by comparing amphotericin B sensitivity in parental CHO and LDL receptor-defective met-18b-2 cells. Amphotericin B Killing

On day 0, cells were seeded into 12-well plates, at cell densities as indicated in the figure legends, in 1 ml of medium A. On day 1, monolayers were washedwith 2 ml of EBSS andrefed 1ml of medium B. On day 3, cells were refed as indicated in the individual experiment. After 24 h, cells were washed with EBSS and refed 1 ml of medium C or 175 pg/ml amphotericin B in medium C. After 6 h, cells were washed twice with EBSS and refed medium A. On day 5, cells were washed with buffer A, fixed for 15 min with 100% methanol, then stained for 5 min with 0.2% (w/v) crystal violet in 50% (v/v) ethanol. Plates were rinsed with water and air dried. Incorporation of PHlOleate into Cholesteryl PHIOleate On day 0, cells were seeded into six-well plates (20,000-35,000 cells/35-mm well) in 1.5 ml of medium A. On day 1, all monolayers werewashed with 2 mlof EBSS and refed 2 mlof mediumB. Experiments were initiated on day 3. Following incubations as indicated, monolayers were pulsed with 100 p~ [3H]oleate (15,000 cpm/ nmol) bound to albumin (9). After 1 h, cells were washed, and cholesteryl [3H]oleatewas isolated (8).After the lipids were extracted, the monolayers were dissolved in 0.1 N NaOH and aliquots removed

Cells

for protein determination. Cholesterol esterification is defined as nmol of cholesteryl [3H]oleateformed per h/mg of protein.

3-Hydroxy-3-methylglutaryl (HMG)-CoA Reductase Activity On day 0, cells were seeded in 60-mm dishes (45,000-70,000 cells/ dish) in 3 ml of medium A. On day 1,monolayers were washed once with 4 ml of EBSS and refed 3 mlof medium B. Experimental additions were made on day 3 as described in the figure legends. At time of harvest, cells were washed with buffer A at 4 "C once quickly, once for 7 min, and once quickly. Cells from each dish were scraped separately, pelleted by centrifugation (10,000 X g, 4 min, 4 " C ) and stored a t -20 "C. Cell pellets were thawed and solubilized as described (185 (12).The conversion of ~~-3-hydroxy-3-[~H]methylglutaryl-CoA pM, 6,980 cpm/nmol) to [3H]mevalonate was measured in 10-pl aliquots of the detergent-solubilized extract (12). Protein content of the detergent-solubilized extract was measured after trichloroacetic acid precipitation. Samples dissolved in Lowry reagent (11) were extracted twice with 1ml of ethyl etherprior to theaddition of Folin's phenol reagent. One unit of HMG-CoA reductase activity represents the formation of 1 nmol of [3H]mevalonate/min. Movement of LDL-derived Cholesterol to the Plasma Membrane

On day 0, cells were seeded in six-well plates at 23,000 cells/well in 1.5 ml of medium A. On day 1, cells were washed twice with 1 ml of EBSS andrefed 1 ml of medium B. On day 4, the cells were pulsed for 2 h with 15 pg/ml [3H]CL-LDL in medium D, washed twice with EBSS, and refed 1 mlof medium Dcontaining 25 p1 of small unilamellar vesicles. Small unilamellar vesicles were prepared with a cholesterol/phosphatidylcholine molar ratio of0.7 (7). Media and cells were harvested at staggered times, and cellular and medium [3H] cholesterol were quantitated as described (7). Percoll Gradient Fractionation of PHICL-LDL-labeled Cells On day 0, cells were seeded in 150-mm dishes (180,000-275,000 cells/dish) in 15 ml of medium A. On day 1, monolayers were washed once with 15 ml of EBSS and thenfed 17 ml of medium B. On day 3, cells were refed 17 ml of medium B. Additions of 10 pg/ml [3H]CLLDL were made a t staggered times, and all monolayers were harvested at thesame time. times (7 min, Monolayers were washed once quickly and then three 4 "C) with buffer B, then washed once quickly with buffer A, and finally washed once quickly and once (7 min, 4 "C) with buffer C. Cells were scraped into 5 ml of buffer C, pelleted by centrifugation (2,000 x g, 5 min, 4 "C), and homogenized in 700 pl of buffer C by four strokes in a 2-ml tight-fitting Dounce tissue grinder (Kontes Co., Vineland, NJ). The homogenates were centrifuged (2,000 X g, 5 min, 4 "C)and thepellets resuspended in 500 p1 of buffer C, rehomogenized, and centrifuged again. The resulting postnuclear supernatants were combined (1.05-1.20 ml total), and 800 p1 was layered onto 9 ml of 11%(v/v) Percoll in buffer C. Gradients were subjected to centrifugation (20,000 X g, 40 min, 4 "C with a model Ti-70.1 rotor, Beckman Instruments) and then fractionated into 10 equal portions. A reference gradient was developed simultaneously by loading 800 pl of buffer C onto 9 ml of 11%Percoll and centrifuged with the samples. Cell Fractionation Assays Aliquots (50-500 pl) of postnuclear supernatants, 2,000 X g pellets, and gradient fractions were assayed for [3H]cholesterol content as described (7). [3H]Cholesterol content in gradient fractions is expressed as pmol/fraction. N-Acetyl-P-glucosaminidase activity was measured as described (7). One unit of N-acetyl-P-glucosaminidase activity represents the formation of 1nmol of p-nitrophenol/30 min. Plasma membranes were localized in the gradient fractions using wheat germ agglutinin linked to horseradish peroxidase. One 150-mm dish of CHO cells, not labeled with [3H]CL-LDL,was used to determine plasma membrane localization in the gradient. The monolayer was washed once quickly and then twice (5 min, 4 "C)with buffer A. The monolayer was incubated (30 min, 4 'C) with 5 ml of buffer A containing 10 pg/ml wheat germ agglutinin linked to horseradish peroxidase. This plate was then harvested, homogenized, and fractionated as described above. Aliquots from this gradient were stored at -20 "C. Peroxidase activity was determined following manufacturer's instructions with o-phenylenediamine tablets (Sigma). Assays were performed in 51 mM sodium phosphate, 24.3 mM citric acid, pH 5, containing 0.4 mg/ml o-phenylenediamine dihydrochloride and 0.4

Cholesterol Transport-defective CHO Cells pl/ml of 30% hydrogen peroxide in a final volume of 800 pl. The reaction was incubated a t room temperature for 5-10 min and stopped by the addition of 200 p1 of 3 N HCl. Absorbance was read a t 492 nm. Parallel incubations were performed with aliquots of the reference (no cell extract) gradient fractions, and the absorbance caused by Percoll was subtracted from experimental values. RESULTS

Isolation of LDL-Cholesterol Transport-defective CHO Cells-Amphotericin B is a polyene antibiotic that forms aqueous pores in cholesterol-rich membranes (13, 14). Cells grown under conditions in which the plasma membrane is replete with endogenously synthesized or LDL-derived cholesterol are lysed and killed by amphotericin B (15, 16). However, cells can be made relatively resistant to amphotericin B killing by culturing in medium with lipoprotein-deficient serum plus mevinolin (an inhibitor of endogenous cholesterol synthesis) or 25-hydroxycholestero1(a suppressor of endogenous cholesterol synthesis) (15). We utilized amphotericin B in a two-part selection to isolate CHO cells with defects in thetransport of LDL-derived cholesterol to the plasma membrane. Selection I, which is depicted in Fig. lA, was a modification of the strategy used by Krieger et al. (15) to isolate LDL receptor-defective CHO cells. Ethyl methanesulfonate-mutagenized CHO cells were first preincubated in medium D, which contains lipoproteindeficient serum plus mevinolin. LDL was added, and 24 h later the cells were treated with amphotericin B. Wild-type CHO cells are killed by this treatment. Cells that survived amphotericin B treatment may have defects at one of the three steps (designated in Fig. lA)which alter thedelivery of LDL-derived cholesterol to the plasma membrane: 1) LDL binding andinternalization; 2) lysosomal cholesteryl ester hydrolysis; or 3) mobilization of LDL-derived cholesterol to the plasma membrane. An experiment designed to illustrate Selection I is shown in Fig. 2. In this experiment, amphotericin B selection was performed on parental CHO cells and two of the cholesterol transport mutants selected in this study. Cells were preincubated for 24 h in medium B with the indicated additions of mevinolin and LDL and thenincubated for 6 h in theabsence or presence of amphotericin B. Cells preincubated in medium B without mevinolin were sensitive to amphotericin B; however, mevinolin inhibited endogenous cholesterol synthesis and rendered cells amphotericin B resistant. The addition of 3-5 pg/ml LDL conferred amphotericin B sensitivity to parental CHO cells whereas mutants 2-2 and 4-4 were relatively resistant to amphotericin B treatment after incubation with LDL. From the survivors of Selection I, we wished to isolate only those cells with defects in step 3, the transport of LDLderived cholesterol to the plasma membrane. Therefore Selection I1 was designed to eliminate cells expressing defects in steps 1 and 2 by selecting for cells with normal receptormediated endocytosis of LDL and hydrolysis of LDL-derived steryl esters (Fig. 1B). For this selection we utilized LDL from which the endogenous cholesteryl esters had been removed and replaced with 25-hydroxycholesteryl oleate. Survivors of step 1 were incubated in medium B supplemented with r[25-HC oleate]LDL for 24 h, and thencells were treated with amphotericin B. To survive, cells must be able to internalize the LDL (step 1)and hydrolyze the 25-hydroxycholesteryl oleate (step 2). The unesterified 25-hydroxycholestero1 that is released rescues the cells from amphotericin B killing by suppressing endogenous cholesterol synthesis and lowering plasma membrane cholesterol levels. Fig. 3 illustrates Selection I1 carried out on parental CHO

489 1

Selection I

A.

0

I Acetyl CoA

Cholesterol

Cholesterol

Amphotericin

B.

P B

Selection I1

f

I

25-HC Oleate

\

inhibilion

3

Acetyl CoA

I

Cholesterol Amphotericin B

/

FIG. 1. Depiction of Selection I (panel A ) and Selection I1 (panel B ) for the isolation of cholesterol transport-defective CHO cells. For Selection I (panel A ) , mutagenized CHO cells were incubated in medium D, which contains lipoprotein-deficient serum and mevinolin (to inhibit cholesterol synthesis). LDL was added, and 24 h later the cells were treated with amphotericin B. To survive amphotericin B treatment, cells must have a defect at one of the three steps that alter thedelivery of LDL-derived cholesterol to the plasma membrane: 1 , LDL binding and internalization; 2, lysosomal cholesteryl ester hydrolysis; or 3, mobilization of LDL-derived cholesterol to theplasma membrane. For Selection I1 (panel B ) ,survivors of Selection I were incubated in medium B supplemented with r[25HC oleateJLDL for 24 h followed by amphotericin B treatment. To survive, cells must be able to internalize the LDL (step 1) and hydrolyze the 25-hydroxycholesteryl oleate (step 2). The unesterified 25-hydroxycholestero1that is released rescues the cells from amphotericin B killing by suppressing endogenous cholesterol synthesis, thus lowering cellular cholesterol levels (15). Presumably, cells surviving this dual selection procedure would exhibit normal binding, internalization, and hydrolysis of LDL (steps 1 and 2) but defective transport of LDL-derived cholesterol to the plasma membrane (step 3).

cells, LDL receptor-defective met-18b-2 CHO cells (12), and mutant cell lines 2-2 and 4-4. Cells were preincubated for 24 h in medium B with the indicated additions and then incubated for 6 h in the absence or presence of amphotericin B. The addition of r[25-HC oleate]LDL allowed cells with intact LDL receptor activity to survive amphotericin B treatment. LDL receptor-defective met-18b-2 cells were unable to internalize the r[25-HC oleate]LDL and remained amphotericin B sensitive. The addition of 25-hydroxycholestero1directly to the medium caused down-regulation of endogenous cholesterol synthesis and resistance to amphotericin B in all cell lines. Six cell lines were cloned from each of eight independent stocks of mutagenized and selected CHO cells. From preliminary biochemical analysis, two mutant cell lines from differ-

4892

CHO Cells

Transport-defective Cholesterol cells

date have exhibited impaired LDL-mediated regulation of cellular cholesterol metabolism (4-7,17).Therefore, we tested the regulatory responses elicited by LDL in mutants 2-2 and 4-4.Fig. 4 shows LDL (panel A) and 25-hydroxycholesterol (panel B ) stimulation of cholesterol esterification in parental CHO cells and mutants2-2 and 4-4. Parental CHO cells incubatedin the absence of LDL + 0 showed low rates of incorporation of [3H]oleate into cholesteryl [3H]oleate (0.25 nmol/h/mg). When LDL was added, cholesterol esterification was stimulated in a concentrationdependent manner, such that 30 pg/ml LDL increased cholesterol esterification 6.5-fold. Mutants 2-2 and 4-4also exhibited low rates of cholesterol esterification in the absence of LDL (0.17 and 0.19 nmol/h/mg, respectively); however, LDL failed to stimulate cholesterol esterification appreciably. 25-Hydroxycholesterol maximally stimulated cholesterol es+ + 5 terification equally in all three cell lines, suggesting that acyl CoAcholesterol acyltransferaseis not defective in the mutant + + 7 cells. We also observed that high concentrations of exogenous mevalonate stimulated cholesterol esterification similarly in .all three cell lines (data notshown). - . Suppression of HMG-CoA Reductase Activity by LDL, 25+ + 9 ‘i,- B Hydroxycholesterol, and Meualonate-Since mutants 2-2 and FIG. 2. LDL-dependent amphotericin B killing of parental 4-4exhibit defective LDL stimulation of cholesterol esterifiand cholesterol transport-defective CHO cells. On day 0, cells cation, we sought to determine if other aspects of LDLwere seeded into 12-well plates (50,00O/well) in medium A. On day 1, mediated regulation are also impaired. We tested the effect of monolayers were washed with 1ml of EBSS andrefed 1ml of medium LDL, 25-hydroxycholestero1, and mevalonate in suppressing B. On day 3, monolayers received 1 ml of medium B, and additions HMG-CoA reductase in the threecell lines. of mevinolin (along with 0.5 mM mevalonate) and LDL were made Fig. 5 shows that HMG-CoA reductase activities in parental as indicated. After 24 h, cells received 1 ml of medium C in the absence or presence of amphotericin B as described under “Experi- CHO cells and mutants 2-2 and 4-4cultured in medium B mental Procedures.” On day 5, cells were washed, fixed, and stained were very similar (1.04,1.04,and 1.08 units/mg, respectively). with crystal violet as described under “Experimental Procedures.” In CHO cells, LDL suppressed HMG-CoA reductase activity in a concentration-dependent manner, with maximal suppresCells sion to 36% of control with 10 pg/ml LDL. However, mutants Addition Ampho 2-2 4-4 Met CHO 2-2 and 4-4showed impaired LDL-mediated suppression at B 18h-2 10 pg/ml LDL, and HMG-CoA reductase activities remained at 79 and 93% of control, respectively. None As seen in Table I, mutants 2-2 and 4-4responded normally i to both 25-hydroxycholesterol and mevalonate. This experiment indicated that the defective regulation of HMG-CoA reductase was specific for LDL-derived cholesterol. None + Movement of LDL-derived Cholesterol to the Plasma Membrane-The relative rate of movement of LDL-derived [3H] cholesterol to theplasma membrane was assessed in parental Ampho

Mcvinolin

LDL

CHO

2-2

4-4

f-

A.

2.0

FIG.3. Effect of r[25-HC oleate]LDL and 25-hydroxycholesterol on amphotericin B-mediated cell killing. On day 0, cells were seeded into 12-well plates (15,00O/well) in medium A. On day 1, monolayers were washed with 1ml of EBSS andrefed 1ml of medium B. On day 3, monolayers received 1 ml of medium B, and additions of 4 pg/ml r[25-HC oleate]LDL or 1 pg/ml 25-hydroxycholestero1 were made as indicated. After 24 h, cells received 1 ml of medium C in the absence or presence of amphotericin B as described under “Experimental Procedures.” On day 5, cells were washed, fixed, and stained with crystal violet as described under “Experimental Procedures.”

ent mutagenized stocks were chosen for further analysis. The first digit of the clone designation indicates the pool of mutagenized cells from which the clone was derived; the second digit indicates the clone number. Stimulation of Cholesterol Esterification byLDL, 25-Hydroxycholesterol,und Mevalonate-All LDL-cholesterol transport-defective human fibroblasts and CHO cells examined to

-0.0

-0.0 0

10

20

LDL (ug/ml)

30

0.0

0.5

1.0

1.5

2.0

25-Hydroxycholesterol (uglml)

FIG.4. Stimulation of cholesterol esterification by LDL (punel A ) and 25-hydroxycholestero1 (panel B ) . Parental CHO cells (W), mutant 2-2 (a), and mutant4-4 (A) were grown as described under “Experimental Procedures.” On day 3, each monolayer received 1ml of medium B containing the indicated concentration of LDL or 25-hydroxycholestero1 (plus 10 pg/ml cholesterol). After 7 h of incubation, cells were pulse labeled with [’Hloleate for 1 h. The cellular content of cholesteryl [‘Hloleate was determined as described under “Experimental Procedures.” Each data point represents the average of two wells.

Cholesterol Transport-defective CHO

Cells

4893

I

1.21

CI

0.8 0.6 0.4

CHO

0

1

2

3

4

5

Time (Hr)

LDL (uglml) FIG.5. LDL-mediated suppression of HMG-CoA reductase activity. Parental CHO cells (a),mutant 2-2 (O), and mutant 4-4 (A) were grown as described under “Experimental Procedures.” On day 3, each monolayer received 3 mlof medium B containing the indicated concentration of LDL. After 8.5 h, cells were harvested, and HMG-CoA reductase activity was measured as described under “Experimental Procedures.’’ Each data point represents the average of two dishes.

TABLE I Suppression of HMG-CoA reductase activity by mevalomte and 25-hydroxycholestero1 Cells were grown as described under “Experimental Procedures.’’ On day 3, cells were refed 1 ml of medium D containing either no addition, mevalonate, or 25-hydroxycholesterol plus 10 pg/ml cholesterol. After 12 h, cells were harvested, and HMG-CoA reductase activity was measured as described under “Experimental Procedures.” HMG-CoA reductase activity is expressed as units/mg and represents the average of two dishes.

FIG.6. Movement of LDL-derived cholesterol to the plasma membrane. Parental CHO cells (a),mutant 2-2 (O), and mutant 44 (A) were grown as described under “Experimental Procedures.” On day 4, cells were pulsed with 15 pg/ml [3H]CL-LDLin medium D for 2 h. Monolayers were then washed and refed 1 mlof medium D containing 25 pl of small unilamellar vesicles. The media were removed and cells harvested at staggered times. Cellular [3H]cholesterol and medium [3H]cholesterol were determined as described under “Experimental Procedures.” The LDL-derived medium [3H]cholesterol is expressed as a percentage of the total cellular and medium [3H]cholesterol at each time point. Each data point represents the average of three wells. 1200

HMG-CoA reductase Addition CHO

2-2

4-4

unitslmg

None Mevalonate (30 mM) 25-Hvdroxvcholesterol (0.05 d m l )

1 2 3 4 5 6 7 8 9 1 0

1.73 1.60 1.78 0.26

0.30

0.52

0.85 0.73 0.76

and mutant CHO cells. Cells were pulsed for 2 h with [3H] CL-LDL followedby chase incubations for various times. Movement of [3H]cholesterol to the plasma membrane was determined by quantitating the amount of [3H]cholesterol desorbed into the medium and trapped by small unilamellar vesicles during the chase incubations. We found that all three cell lines internalized and hydrolyzed similar amounts of [3H]CL-LDL during the 2-h pulse. Parental CHO, 2-2, and 4-4 cells contained 11.95, 11.30, and 11.83 nmol/mg, respectively, of LDL-derived [3H]cholesteryl esters and[3H]cholesterol.Throughout the course of the chase incubations, cellular levels of [3H]cholesteryl esters declined, and [3H]cholesterol increased in all three cell lines (data not shown). Despite similar rates of LDL uptake and lysosomal hydrolysis, the amount of [3H]cholesterolthat desorbed from the cells to themedium was strikingly different in the various cell lines (Fig. 6). In CHO cells, there was a time-dependent increase in LDL-derived [3H]cholesterol in the medium such that by 5 h 7.4% of the [3H]cholesterol had desorbed. However, there was only a marginal increase in desorption of [3H] cholesterol from either mutant cell line during the 5-h time course. We conclude that both mutant cell lines are defective in transporting LDL-derived cholesterol to theplasma membrane. Intracellular Movement of LDL-derived Cholesterol in Con-

Fraction

FIG. 7. Distribution of LDL-derived [SH]cholesterolin Percoll gradient fractions from cells continuously labeled for 6 h. Parental CHO cells, mutant 2-2, and mutant 4-4 were grown as given under “Experimental Procedures.” On day 4, cells were pulsed for 5 h with [3H]CL-LDL(10 pg/ml) in medium B. Percoll gradient fractionation of postnuclear supernatants was carried out, and the [3H]cholesterol content of gradient fractions was determined as described under “Experimental Procedures.” Each vertical bar represents thepmol of [3H]cholesterolin one gradient fraction.

tinuously Labeled Cells-The accumulation of LDL-derived [3H]cholesterol in the lysosomes of mutants 2-2 and 4-4 was revealed when cells were labeled continuously with [3H]CLLDL. Postnuclearsupernatants of cellhomogenate9were fractionated on Percoll gradients to separate lysosomes from other subcellular organelles. Ten fractions were obtained from each gradient, and the location of plasma membranes (fractions 2-5) and lysosomes (fractions 9 and 10) was determined by measuring the distribution of marker enzyme activities (data not shown). The total cellular levels of LDL-derived [3H]cholesterol were similar for the three cell lines (data not shown). Fig. 7 shows the contentof LDL-derived [3H]cholesterolin gradient fractions from CHO, 2-2, and 4-4 cells pulsed with [3H]CLLDL for 5 h. Inall cases, the [3H]cholesterol was found primarily in the lysosomes (fractions9and 10) and light membranes (fractions 2-5). In 2-2 and 4-4 cells, the LDLderived [3H]cholesterol had accumulated to very high levels in thelysosomes whereas in the CHO cells the [3H]cholesterol

Cholesterol Transport-defective CHO Cells

4894

was higher in the light membranes. A time course of continuous labeling of the cells shows that mutants 2-2 and 4-4 exhibited intracellular LDL-derived cholesterol transport that was remarkably different from those of the parental CHO cells (Fig. 8). In the parentalCHO cells, LDL-derived [3H]cholesterolaccumulated in lysosomes to 0.3 nmol in 1 h and remained at this level for the remainder of the time course (panel A ) . In contrastto theplateau that was observed in the lysosomal fractions, the appearance of LDLderived cholesterol in the light membrane fractions increased linearly during the time course (panel B ) . This indicates that the appearance of LDL-derived cholesterol in the lysosomes (receptor-mediated uptake and hydrolysis) is balanced by the movement of the sterol fromlysosomes to other cellular membranes. In contrast, mutants 2-2 and 4-4 accumulated much more LDL-derived cholesterol in lysosomal fractions (panel A ) , and the appearance of the sterolin the light membranes fractions was slower (panel B ) . These data indicate that LDL-derived cholesterol transport from lysosomes to other cell membranes is impaired in mutants 2-2 and 4-4. DISCUSSION

The first in uiuo evidence that a gene product facilitates the directed movement of LDL-derived cholesterol emerged from studies with fibroblasts isolated from patients with the autosomal recessive lysosomalstorage disease, Niemann-Pick type C . NPC cells exhibit impaired intracellular transportof LDLderived cholesterol (4-7). The precise gene defect in NPC is unknown. Delineating the defective gene will facilitate our understanding of the mechanism underlying intracellular cholesterol transport and regulation. In this paper, we report a selection protocol that has enabled us to select CHO mutants expressing a NPC phenotype. Studying CHO mutants defective in cholesterol transport rather than human NPC fibroblasts has several advantages. Primaryhuman fibroblasts become senescent after 30-40 population doublings whereas CHO cells represent an immortalized cell line. Multiple alleles regulating intracellular cholesterol transport may be revealed in studies of defective CHO cells whereas the clinical NPC

--

f”J

2.0

2.0

A. Lysosomes

0.5

0.5

-4-4 0.0

00 0

1

2

3

Time (Hr)

4

5

0

1

2

3

4

5

Time (Hr)

FIG. 8. [SH]Cholesterolcontent in lysosomal (panel A ) and light membrane (panel B ) Percoll gradient fractions from continuously labeled cells. Parental CHO cells (M), mutant 2-2 (O), and mutant 4-4(A) were grownas described under “Experimental Procedures.” On day 4,cells from the same experiment as shown in Fig. 7 were pulsed at staggered times with [3H]CL-LDL (10 pg/ml). All dishes were harvested at thesame time. Percoll gradient fractionation of postnuclear supernatants was carried out, and the I3H] cholesterol content of gradient fractions was determined as described under “Experimental Procedures.” The [3H]cholesterol content of gradient fractions with high N-acetyl-P-glucosaminidase activity (fractions 9 and 10) were summed (Lysosomes, panel A ) . The [3H] cholesterol content of gradient fractions with high wheat germ agglutinin-horseradish peroxidase activity (fractions 2-5) were summed (Light Membranes, panel B ) . Each data point represents the sum of [:’H]cholesterol in the designated fractions from one gradient, expressed as nmol/fraction.

phenotype may be exhibited in individuals expressing mutations inonly one gene or alimited number of genes. Exogenous DNA can be introduced into mutant CHO cells readily, thus facilitating the cloning of complementing genes. In addition, genetic manipulation of CHO cells is simplified by virtue of their haploid state for the expression of many genes (18). For ourmutant selection, we utilized the polyene antibiotic amphotericin B. Amphotericin B complexes with sterol-rich membranes forming aqueous pores that result incell lysis (13, 14, 16). Polyene antibiotics, such as amphotericin B and filipin, have proven to be valuable tools for isolating mutants in cholesterol synthesis (19-21), regulation of cholesterol metabolism (22), and LDL receptor activity (15). Our two-step selection for LDL-cholesterol transport mutants was based on the following criteria: the mutants must be able to bind and internalizeLDL normally and tohydrolyze the cholesteryl esters present inthe LDL core but be defective in the movement of LDL-derived cholesterol to the plasma membrane. Selection I isolated mutants defective in any facet of LDL metabolism. Cells capable of binding, internalizing, and hydrolyzing LDL-derived cholesterol esters would survive this initial selection if they were defective in the transportof LDL-derived cholesterol (phenotypically similar to NPC). However, two additional types of nontransport-defective mutants could also survive Selection I: cells defective in binding or internalizing LDL (phenotypically similar to familial hypercholesterolemia) and cells with normal LDL binding and internalization which are unable to hydrolyze LDL-derived cholesterol esters (phenotypically similar to cholesterol ester storage disease). Selection I1 was devised to isolate only cholesterol transport-defective mutants. Cells cultured in medium with lipoprotein-deficient serum are amphotericin B sensitive because endogenously synthesized cholesterol is supplying the plasma membrane. However, 25-hydroxycholestero1does not replace cholesterol in cellular membranes and, by itself, does not cause amphotericin B sensitivity. In fact, when cholesterol synthesis is suppressed, by the addition to the medium of 25hydroxycholesterol, cells are rendered amphotericin B resistant. We incubated survivors of Selection I with LDL that is devoid of cholesterol and reconstituted with 25-hydroxycholesteryl oleate. For 25-hydroxycholestero1to “rescue” the cells from amphotericin B killing, the r[25-HC oleate]LDL must be bound, internalized, and delivered to lysosomes. Once in lysosomes, the 25-hydroxycholesteryI oleate must be hydrolyzed before endogenous cholesterol synthesis can be suppressed. These cells will subsequently survive amphotericin B treatment whereas other types of mutants (familial hypercholesterolemia and cholesterol ester storage disease phenotypes) will continue to synthesize cholesterol and be amphotericin B sensitive. Thus, we have selected for a very specific type of cell mutant, one that metabolizes LDL normally but does not transport the LDL-derived cholesterol to the plasma membrane. Biochemical analysis of two mutants isolated by this selection protocol reveals that we have indeed isolated cells expressing a NPC phenotype. We have found that LDL-mediated regulatory responses are impaired in mutants 2-2 and 4-4. When compared with parental CHO cells, LDL does not stimulate cholesterol esterification, nor does it suppress HMG-CoA reductase activity in themutants. Thesedefective responses appear to be specificfor LDL since 25-hydroxycholesterol and mevalonate elicit normal regulatory responses. Mutants 2-2 and 4-4 survived selection presumably because they were unable to move LDL-derived cholesterol to the plasma membrane, the site of amphotericin B action. Delayed

4895

Cholesterol Transport-defective CHO Cells movement of LDL-derived cholesterol to the plasma membrane was confirmed using [3H]CL-LDL. We observed that LDL-derived [3H]cholesterol movement to plasma membranes and desorption into the medium were impaired. Cell fractionation studies indicated that LDL-derived [3H]cholesterol accumulates to very high levels in lysosomes of 2-2 and 4-4 cells, and its transfer to other cell membranes is delayed as compared with parental CHO cells. Therefore, the defect in LDL-mediated regulatory responses is most likely predicated by the inability of mutants 2-2 and 4-4 to mobilize LDLderived cholesterol from lysosomes. This mutant phenotype is highly reminiscent of NPC, characterized by an accumulation of free cholesterol in lysosomes in response to LDL coincident with sluggish LDL-mediated regulation of cholesterol metabolism. It is well established that NPC is a single gene defect that is transmitted in an autosomal recessive manner (23). However, there are many manifestations of the disease confined to the clinical spectrum NPC (24). It is possible, therefore, that more than one single gene defect can give rise to the clinical entity known asNPC. We are in the process of performing genetic complementation analysis between multiple cell clones that we have selected. This complementation analysis may enumerate the gene defects that lead to the expression of the NPCphenotype. Cadigan and co-workers (17) have also reported the isolation of CHO-derived cholesterol transport (CT) mutantsfrom 25-RA cells. 25-RAcells were derived from mutagenized CHO cells selected for the ability to grow in the presence of 25hydroxycholesterol. Isolation of CT cells was achieved by selecting mutagenized 25-RA cells under cholesterol starvation conditions. Characterization of the CT mutantsrevealed an NPC-like biochemical phenotype, that is, reduced LDLstimulation of cholesterol esterification and an accumulation of unesterified cholesterol as detected by filipin immunofluorescence. Additional LDL-mediated regulatory events, such as suppression of HMG-CoA reductase activity and intracellular transport of LDL-derived cholesterol, were not determined in the CT cells. Our selection protocol was vastly different from that of Cadigan et al. (17),and it willbe interesting to determine if our respective mutants belong to the same complementation groups. The mechanics of intracellular cholesterol transport are incompletely understood. Newly synthesized cholesterol is transported from the endoplasmic reticulum to the plasma membrane in lipid-rich vesicles, a process requiring metabolic energy (25). This transport appears to be independent of Golgi-mediated transport of secretory proteins as it is not blocked by treatment with Brefeldin A (26).Intracellular transport of exogenous cholesterol from lysosomes to the plasma membrane maybe a mediated process, occurring within 2 min in CHO cells (27) and thought to be constitutively active (28). This transport bypasses the endoplasmic reticulum (28) and does not require metabolic energy (29). Intracellular cholesterol transport may occur by either cytosolic sterol carrierproteins, lipid-rich vesicles, passage through contiguous membranes, or monomeric diffusion. Several proteins have been described which mediate sterol and/or phospholipid movement in uitro. However, in uiuo evidence for a role of any of these proteins in directed lipid transfer hasbeen lacking. The human gene for the nonspecific lipid transfer protein, SCP', was cloned (2), and expression studies have indicated that SCPzmay mediate the movement of cholesterol to mitochondria for processing in steroid hormone synthesis. However, a role for SCP, in the bulk transport of cholesterol has yet to be determined. A second lipid

transfer protein, the phosphatidylinositol/phosphatidylcholine (PI/PC)transport protein (PITP) is also undergoing extensive study. The gene for PITP (also known as SEC14) is essential in yeast for secretion from late Golgi compartments (30, 31). Since yeast mutants that bypass the requirement for SEC14p or PITP are unable to convert choline to PC (32), speculation has emerged that PITP is responsible for maintaining the correct PI to PCratio at a specific intracellularsite, which is crucial for late Golgi-mediated secretion and yeast growth. Cleves et al. (33) hypothesize that PITP is not involved in the bulk transfer of PC or PI, as mutants that bypass the SEC14 requirement exhibit no PI or PC transfer yet maintain viability. SCP' may function in a similar manner, modulating cholesterol content at specific sites rather than participating in the bulk transfer between multiple organelles within cells. Wild-type CHO cells display the NPC phenotype when incubated with either the steroid derivative U18666A (8) or the phenothiazine derivative imipramine (34). Both U18666A and imipramine are amphophilic tertiary amines that may inhibit the activity or synthesis of a protein or lipid that facilitates cholesterol movement. It is unknown whether U18666A or imipramine blocks the step in the intracellular cholesterol transport that is defective in NPC or in mutants 2-2 or 4-4. It is possible that intracellular cholesterol transport is a multistep process, and blockage of any one step will give rise to a similar phenotype. Complementation analysis between mutant 2-2, 4-4, andNPC fibroblasts willprovide information in this regard. Among the 42 clones analyzed in this mutant selection, there were several that differed from the classic NPC phenotype.' Further analysis will determine if these mutants are defective in an as yet undefined step in cholesterol transport. With a battery of mutant CHO cells defective in intracellular cholesterol transport, it should be possible to characterize all potential genes involved in this process as well as genes that may secondarily affect this process. Acknowledgments-We thank Eliezar Dawidowicz, Regina Ruggiero, and Monty Krieger for many helpful discussions during the course of these studies. REFERENCES 1. Trzaskos, J. M., and Gaylor, J. L. (1983) Biochim. Biophys. Acta

751,52-65 2. Yamamoto, R., Kallen, C.B., Babalola, G. O., Rennert, H., Billheimer, J. T., and Strauss, J. F. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 463-467 3. Reyland, M. E., Gwynne, J. T., Forgez, P., Prack, M. M., and Williams, D. L. (1991) Proc. Natl. Acad. Sci. U.S. A. 88, 23752379 4. Pentchev, P. G., Comly, M. E., Kruth, H. S., Vanier, M. T., Wenger, D. A., Patel, S., and Brady, R. 0. (1985) Proc. Natl. Acad. Sci. U. S. A. 82,8247-8251 5. Pentchev, P. G., Comly, M. E., Kruth, H. S., Tokoro, T., Butler, J., Sokol, J., Filling-Katz, M., Quirk, J. M., Marshall, D. C., Patel, S., Vanier, M. T., and Brady, R.0. (1987) FASEB J. 1, 40-45 6. Liscum, L., and Faust, J. R. (1987) J. Biol. Chem. 2 6 2 , 1700217008 7. Liscum, L., Ruggiero, R. M., and Faust, J. R. (1989) J. Cell Biol. 108, 1625-1636 8. Liscum, L., and Faust, J. R. (1989) J. Biol. Chem. 264, 1179611806 9. Goldstein, J. L., Basu, S. K., and Brown, M. S. (1983) Methods Enzyrnol. 98, 241-260 10. Faust, J. R., Goldstein, J. L., and Brown, M. S. (1977) J. Biol. Chern. 262,4861-4871

* N. K. Dahl, K.L.Reed, M. A. Daunais, J. R. Faust, and L. Liscum, unpublished results.

4896

Cholesterol Transport-defective CHOCells

11. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.

12. 13. 14. 15.

16. 17. 18. 19. 20. 21. 22. 23.

(1951)J. Biol. Chem. 193,265-275 Faust, J. R., and Krieger, M. (1987) J. Biol. Chem. 262, 19962004 Kinsky, S. C. (1970)Annu. Rev. Phurmucol. 10, 119-142 Norman, A. W., Demel, R. A., De Kruyff, B., and Van Deenen, L. L. M. (1972) J. Biol. Chem. 247, 1918-1929 Krieger, M., Martin, J., Segal, M., and Kingsley, D. (1983) Proc. Natl. Acad. Sci. U. S. A . 80,5607-5611 De Kruijff, B. (1990) Biosci. Rep. 10, 127-130 Cadigan, K. M., Spillane, D. M., and Chang, T.-Y. (1990) J. Cell Biol. 1 1 0 , 295-308 Siminovitch, L. (1985) in Molecular Cell Genetics (Gottesman, M. M., ed) pp. 869-879, Wiley-Interscience Publications, New York Saito, Y., Chou, S. M., and Silbert, D. F. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 3730-3734 Hidaka, K., Endo, H., Akiyama, S., and Kuwano, M. (1978) Cell 1 4 , 415-421 Krieger, M. (1983)Anal. Biochem. 135, 383-391 Chang, T.-Y., and Chang, C. C. Y. (1982) Biochemistry 21,53165323 Spence, M.W., and Callahan, J. W.(1989) in The Metabolic Basis of Inherited Disease (Scriver, C. R., Beaudet, A. L., Sly, W. S., and Valle, D., eds) 6th Ed., pp. 1655-1676, McGraw-Hill

Publications, Minneapolis, MN 24. Fink, J. K.,Filling-Katz, M. R., Sokol, J., Cogan, D. G., Pikus, A., Sonies, B., Soong, B., Pentchev, P. G., Comly, M. E., Brady, R. O., and Barton, N. W. (1989) Neurology 39,1040-1049 25. Kaplan, M. R., and Eimoni, R. D. (1985) J. Cell Biol. 101, 446453 26. Urbani, L., and Simoni, R. D. (1990) J. Biol. Chem. 265, 19191923 27. Brasaemle, D. L., and Attie, A. D. (1990) J. Lipid Res. 31, 103112 28. Johnson, W. J., Chacko, G. K., Phillips, M. C., and Rothblat, G. H. (1990) J. Biol. Chem. 265,5546-5553 29. Liscum, L. (1990) Biochirn. Biophys. Acta 1045,40-48 30. Aitken, J. F., Van Heusden, G. P. H., Temkin, M., and Dowhan, W. (1990) J. Biol. Chem. 265,4711-4717 31. Bankaitis, V.A., Aitken, J. R., Cleves, A. E., and Dowhan, W. (1990) Nature 347, 561-562 32. Cleves, A. E., McGee, T. P., Whitters, E. A., Champion, K. M., Aitken, J. R., Dowhan, W.,Goebl, M., and Bankaitis, V. A. (1991) Cell 64,789-800 33. Cleves, A., McGee, T., and Bankaitis, V. (1991) Trends Cell Biol. 1,30-34 34. Rodriguez-Lafrasse, C., Rousson, R., Bonnet, J., Pentchev, P. G., Louisot, P., and Vanier, M. T. (1990) Biochim. Biophys. Acta 1043,123-128