Cholesterol metabolism is particularly active in malignant, proliferative cells, whereas cholesterol starvation has been shown to inhibit cell proliferation. Inhibition of enzymes involved in cholesterol biosynthesis at steps before the formation of 7-dehydrocholesterol has been shown to selectively affect cell cycle progression from G2 phase in human promyelocytic HL-60 cells. In the present work, we explored whether cholesterol starvation by culture in cholesterol-free medium and treatment with different distal cholesterol biosynthesis inhibitors induces differentiation of HL-60 cells. Treatment with SKF 104976, an inhibitor of lanosterol 14-α demethylase, or with zaragozic acid, which inhibits squalene synthase, caused morphologic changes alongside respiratory burst activity and expression of cluster of differentiation antigen 11c (CD11c) but not cluster of differentiation antigen 14. These effects were comparable to those produced by all-trans retinoic acid, which induces HL-60 cells to differentiate following a granulocyte lineage. In contrast, they differed from those produced by vitamin D3, which promotes monocyte differentiation. The specificity of the response was confirmed by addition of cholesterol to the culture medium. Treatment with PD 98059, an inhibitor of extracellular signal–regulated kinase, abolished both the activation of NADPH oxidase and the expression of the CD11c marker. In sharp contrast, BM 15766, which inhibits sterol Δ7-reductase, failed to induce differentiation or arrest cell proliferation. These results show that changes in the sterol composition may trigger a differentiation response and highlight the potential of cholesterol pathway inhibition as a possible tool for use in cancer therapy. [Cancer Res 2007;67(7):3379–86]

Cholesterol homeostasis is abnormal in malignant cells. Freshly isolated acute myeloid leukemia (AML) and chronic myelogenous leukemia cells display much higher specific low-density lipoprotein uptake and degradation (13) and hydroxymethylglutaryl (HMG)-CoA reductase activity (2) than leukocytes from healthy subjects. A recent study by Banker et al. (4) reported that the levels of mRNAs for the low-density lipoprotein receptor and 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA reductase) are increased by daunorubicin or cytarabine treatment in almost all cultured AML samples, an effect which may increase leukemia cell survival and impart relative resistance to therapy.

Statins, which block cholesterol biosynthesis through competitive inhibition of HMG-CoA reductase, the rate-limiting enzyme in the pathway, have been shown to inhibit cell proliferation and to have several effects that are of interest in relation to cancer prevention, including effects on cell differentiation (5, 6). However, the effects of inhibiting cholesterol biosynthesis are dependent on the model used. In both neuroblastoma cells (7) and AML cells (8), lovastatin induces widespread differentiation followed by extensive apoptosis. In human malignant glioma cell lines and untransformed rat astrocytes, lovastatin also causes effective apoptosis but does not induce differentiation (9). Treatment with statins has been also shown to sensitize AML cells to radiochemotherapy, suggesting that cellular cholesterol is critical for cell survival (10). Acute lymphoid leukemias, however, seem to be resistant to statins (11). Phenylacetate, an inhibitor of mevalonate-5-pyrophosphate decarboxylase, has also been reported to inhibit cell growth in a variety of cell lines but data on its effects on cell differentiation are inconclusive (9, 12).

Administration of relatively high doses of statins to rodents has been shown to reduce the growth of various types of tumor (13, 14). Some retrospective studies of patients receiving statins as lipid-lowering drugs for the prevention of cardiac events showed a slight reduction in the incidence of breast cancer (15) and colorectal cancer (16). However, some other clinical studies (1719) and two recent meta-analyses (20, 21) found no evidence that the use of statins reduces the risk of cancer. A recent phase II trial also failed to reveal any relevant activity of phenylacetate against malignant glioma (22).

Both statins and phenylacetate act very early in the cholesterol pathway, inhibiting the synthesis of both sterols and nonsterol mevalonate derivatives. These different mevalonate derivatives exert many distinct biological actions, with important physiologic implications. Cholesterol is used for membrane formation and is a principal component of lipid rafts and caveolae, in which many receptors for extracellular signals reside (23). Nonsterol isoprenoids are precursors of ubiquinone and heme A and are used for protein prenylation, which allows certain proteins to localize to membranes where they exert specific functions (24). Thus, it is not surprising that blockade of their synthesis results in the alteration of multiple processes. Furthermore, differences in the sensitivity to these inhibitors and/or the demand for nonsterol isoprenoid derivatives among cell types may explain the variability of the results obtained. The profound consequences of these various effects are highlighted in the results of previous studies from our laboratory showing that different inhibitors of cholesterol biosynthesis, acting on distinct enzymes, have different effects on cell cycle progression (2528).

Promyelocytic HL-60 cells represent a useful model in which to study myeloid cell differentiation. Treatment with all-trans retinoic acid (RA) or DMSO has been shown to cause HL-60 to differentiate into granulocytes (29). In contrast, treatment with vitamin D3 induces differentiation into monocytes (30).

Exposure of HL-60 cells to agents that cause granulocyte differentiation is accompanied by a rapid decrease in sterol synthesis, whereas this effect is not observed with factors that cause monocyte or macrophage differentiation (31, 32). This is consistent with the observation that peripheral blood granulocytes almost entirely lack the ability to synthesize post-squalene products, whereas monocytes posses an active cholesterol biosynthesis pathway (32). However, it is unclear whether cholesterol biosynthesis is directly involved in myeloid differentiation.

In this study, we examined the effects of cholesterol starvation on differentiation of HL-60 cells. We found that distal inhibition of cholesterol biosynthesis by treatment with either SKF 104976, an inhibitor of lanosterol 14-α demethylase, or zaragozic acid, an inhibitor of squalene synthase, specifically induces respiratory burst activity and the expression of cluster of differentiation antigen 11c (CD11c) but not cluster of differentiation antigen 14 (CD14). These effects were mediated by the mitogen-activated protein kinase (MAPK) pathway. In contrast, BM 15766, which inhibits sterol Δ7-reductase, failed to induce differentiation. Our results show that changes in sterol composition may trigger a differentiation response and highlight the potential of cholesterol pathway inhibition as a possible tool in cancer therapy.

Materials. 2-[2-Amino-3methoxyphenyl]-4H-1-benzopyran-4-one (PD 98059), phorbol 12-myristate 13-acetate (PMA), and all-trans RA were purchased from Sigma Chemical (St. Louis, MO) and 5-(and-6)-chloromethyl-2′,7′- dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) was from Molecular Probes (Eugene, OR). Cholesterol was from Steraloids, Inc. (Newport, RI) and 1α,25-dihydroxyvitamin D3 (D3) was from Fluka (Buchs, Aargau, Sweden). Cholesterol biosynthesis inhibitors were gifts from the following laboratories: SKF 104976 from SmithKline Beecham Pharmaceuticals (Harlow, Essex CM19 5AW, United Kingdom), zaragozic acid from Merck Research Laboratories (Rahway, NJ), and BM 15766 from Boehringer Mannheim (Barcelona, Spain). Antibiotics were from Life Technologies, Inc. (Barcelona, Spain). Phycoerythrin-labeled CD14 antibody and allophycocyanin-labeled CD11c were obtained from BD Biosciences (San Jose, CA). Rabbit polyclonal anti–extracellular signal–regulated kinase (ERK) antibody (M-5670) and mouse monoclonal anti–diphospho-ERK (M-8159) were purchased from Sigma. Mouse monoclonal anti-p67phox antibody (610913) and mouse monoclonal anti-p47phox (610355) were from BD Transduction Laboratories, BD Biosciences (San Jose, CA). Rabbit polyclonal anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (sc-25778) was purchased to Santa Cruz Biotechnology (Santa Cruz, CA). Peroxidase-conjugated donkey anti-rabbit immunoglobulin G (IgG) (H + L) and peroxidase-conjugated donkey anti-mouse IgG (H+L) were obtained from Jackson ImmunoResearch Laboratories, Inc. (Suffolk, United Kingdom). [14C]Acetate (53 mCi/mol) was provided by Amersham Biosciences (Buckinghamshire, United Kingdom) and 3H-cholesterol (51 Ci/mmol) by New England Nuclear (Zaventem, Belgium). All other chemical products were of analytic grade.

PD 98059, PMA, D3, CM-H2DCFDA, zaragozic acid, SKF 104976, and BM 15766 were dissolved in DMSO. All-trans RA and cholesterol were dissolved in ethanol. Stock solutions were stored at −20°C.

Cells and cell culture. HL-60 (ECACC 98070106) cells were obtained from the European Collection of Cell Cultures (Salisbury, United Kingdom) and cultured at 37°C in a humidified atmosphere containing 5% CO2. Cells were cultured in cholesterol-free medium containing insulin, transferrin, and selenium supplements: RPMI 1640 (Life Technologies) supplemented with 625 μg/mL transferrin (Roche, Basel, Switzerland), 625 μg/mL insulin, 535 μg/mL linoleic acid-bovine serum albumin, 625 ng/mL sodium selenite (Sigma), 125 mg/mL human serum albumin (Grifols 20%, Barcelona, Spain), and antibiotics (100 units/mL penicillin, 100 μg/mL streptomycin, and 10 μg/mL gentamicin). Cells were subcultured two to three times weekly to maintain logarithmic growth. Viable cells, characterized by trypan blue exclusion, were counted with a Neubauer hemocytometer.

Cells (2 × 105/mL) in exponential growth phase were suspended in fresh medium and treated with the different compounds dissolved as indicated above. The final concentration of DMSO and ethanol in the medium was 0.044% and 0.44% (v/v), respectively, in both control and experimental conditions. All experiments were repeated at least thrice.

Morphologic studies. Cell morphology was examined by microscopy using the May Grünwalds-Giemsa stain according to manufacturer's instructions (Merck Diagnostica, Darmstadt, Germany). Cells were centrifuged onto glass slides in a Shandon Cytospin 3 at 750 rpm for 3 min. The slides were then dried, stained, and examined at ×400 magnification using an Olympus BX51 light microscope fitted with a JVC 3-CCD digital color video camera.

NADPH oxidase activity. The respiratory burst was detected by oxidation of the fluorescent probe CM-H2DCFDA, as described elsewhere (33). Cells (5 × 105) were harvested by centrifugation at 1,500 rpm for 5 min, washed with PBS, and resuspended in 100 μL of fresh culture medium. Then, the cells were incubated for 2 h with 10 μmol/L CM-H2DCFDA and 100 ng/mL PMA (activated) or PBS (resting) at 37°C in the dark. Fresh culture medium (400 μL) was added before acquisition with a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA). Data were analyzed with the WinMDI 2.8 software (Build #13 01-19-2000, Copyright Joseph Trotter).

CD marker expression. To detect cell-surface markers by flow cytometry, 5 × 105 cells were harvested by centrifugation at 1,500 rpm for 5 min, washed with PBS, and resuspended in 100 μL of fresh culture medium. Then, cells were incubated for 15 min in the dark at room temperature with 10 μL of phycoerythrin-labeled CD14 and 5 μL of allophycocyanin-labeled CD11c antibodies. The cells were washed twice with PBS and resuspended in 0.5 mL of 1% paraformaldehyde, then analyzed by flow cytometry. For each sample, separate aliquots of cells were treated with PBS instead of antibodies to determine the fluorescence relative to unlabeled controls.

Western blotting. For Western blot analysis, 4 × 106 to 8 × 106 cells were harvested and washed twice with ice-cold PBS, and whole-cell extracts were prepared by mixing the cell pellets with an extraction buffer [50 mmol/L Tris-HCl (pH 7.5), 125 mmol/L NaCl, 1% NP40, 0.225 mg/mL NaF, 0.626 mg/mL Na4P2O7, 1 mmol/L Na3VO4, 10% glycerol, 1 mmol/L Na2EDTA, and 5 μL/mL Calbiochem Proteases Inhibitor Cocktail Set III]. Equal amounts of extracts (40 μg of protein) were separated by SDS-PAGE (12% polyacrylamide gel containing SDS) and transferred to polyvinylidene difluoride membranes (Hybond-P, Amersham Biosciences). The membranes were blocked with 5% nonfat dried milk in TBS containing 0.05% Tween 20 (TBS-T) and then blotted overnight at 4°C with mouse monoclonal anti–diphospho-ERK (1:2,000 dilution), mouse monoclonal anti-p47phox antibody (1:500 dilution), and mouse anti-p67phox antibody (1:500 dilution) in TBS-T and 1% nonfat dried milk. Then, the membranes were washed thrice with TBS-T and incubated for 1 h with a peroxidase-conjugated donkey anti-mouse antibody (1:2,000 dilution) in TBS-T and 1% nonfat dried milk. The membranes were washed and the protein bands were detected with a chemiluminescence assay system (ECL Western Blotting Analysis system, Amersham Biosciences) and visualized with the VersaDoc Model 4000 Imaging System (Bio-Rad, Hercules, CA) using Quantity One 4.5.2 1D Imaging Software. The blots were stripped for 30 min at 50°C with a stripping buffer [100 mmol/L 2-mercaptoethanol, 2% SDS, 62.5 mmol/L Tris-HCl (pH 6.7)] and reprobed with rabbit polyclonal anti-ERK antibody (1:5,000 dilution) or finally with a rabbit polyclonal anti-GAPDH antibody (1:5,000 dilution).

Analysis of cell sterol content. HL-60 cells (7.5 × 106) were incubated in the absence (control) or presence of the different inhibitors for 72 h and then washed with ice-cold PBS and resuspended in 0.5 mL of 10% (w/v) KOH. 3H-Cholesterol was added as an internal standard. The samples were treated sequentially with chloroform-methanol (2:1, v/v) and distilled water to obtain the lipid and water-soluble fractions. Then, the nonsaponifiable fractions were obtained and used for sterol separation by reverse-phase high-performance liquid chromatography as described elsewhere (25, 34).

Statistical analysis. All experiments were done at least thrice. Data are shown as mean ± SE. Effects of the different treatments were analyzed by one-way ANOVA and post hoc multiple comparisons were done with Tukey's test. Calculations were done using Statgraphics Plus v5.0 software (Statistical Graphics, Herndon, VA).

Induction of oxidative burst via inhibition of lanosterol demethylase. To reduce the cell cholesterol content, HL-60 cells were incubated in a defined medium lacking cholesterol and were treated with SKF 104976, a specific inhibitor of lanosterol 14-α demethylase. This treatment led to a marked decrease in cholesterol content (1.18 ± 0.1 versus 6.01 ± 0.4 μg/mg of cell protein in treated and control cells, respectively). NADPH oxidase activity was analyzed as a functional marker of differentiation in human myeloid cells lines. Cells were stimulated with PMA to activate NADPH oxidase and incubated in the presence of CM-H2DCFDA. CM-H2DCFDA passively diffuses into cells and, following cleavage of acetate groups by intracellular esterases, oxidation by superoxide anion/hydrogen peroxide yields a fluorescent adduct that is trapped inside the cell, facilitating its detection by flow cytometry (33, 35). Cells treated for 5 days with 1 μmol/L RA or 1 μmol/L D3, which cause differentiation of HL-60 cells to granulocytes and monocytes, respectively, were used as positive controls. Differentiation induced by any of the agents resulted in intense CM-H2DCFDA oxidation when compared with undifferentiated cells (Fig. 1A). Treatment with SKF 104976 also increased respiratory burst activity in a time- and dose-dependent manner. The maximum effect was achieved with 1.5 μmol/L SKF 104976 at 4 days; under those conditions, the mean fluorescence was even higher than with either RA or D3 (Fig. 1B and C).

Figure 1.

NADPH oxidase activity in differentiated HL-60 cells. Flow cytometry histograms showing CM-H2DCFDA (DCF) fluorescence from resting (open) and PMA-stimulated (shaded) HL-60 cells. Numbers indicate the geometric mean fluorescence of a representative experiment. A, effect of treatment with 1 μmol/L all-trans RA (RA) or 1 μmol/L D3 for 5 d. Untreated cells were used as control. B, effect of incubation time in cells treated with 1.5 μmol/L SKF 104976. C, effect of the indicated doses of SKF 104976 (SKF) in cells treated for 3 d. D, Western blot analysis of whole-cell extracts from cells treated with 1.5 μmol/L SKF 104976 for 3 d. p47phox and p67phox expression is shown in the same immunoblot (top) and GAPDH was visualized as a loading control (bottom).

Figure 1.

NADPH oxidase activity in differentiated HL-60 cells. Flow cytometry histograms showing CM-H2DCFDA (DCF) fluorescence from resting (open) and PMA-stimulated (shaded) HL-60 cells. Numbers indicate the geometric mean fluorescence of a representative experiment. A, effect of treatment with 1 μmol/L all-trans RA (RA) or 1 μmol/L D3 for 5 d. Untreated cells were used as control. B, effect of incubation time in cells treated with 1.5 μmol/L SKF 104976. C, effect of the indicated doses of SKF 104976 (SKF) in cells treated for 3 d. D, Western blot analysis of whole-cell extracts from cells treated with 1.5 μmol/L SKF 104976 for 3 d. p47phox and p67phox expression is shown in the same immunoblot (top) and GAPDH was visualized as a loading control (bottom).

Close modal

Expression of the regulatory subunits of NADPH oxidase, p47phox and p67phox, was analyzed by Western blot. As shown in Fig. 1D, the proteins were barely detected in undifferentiated HL-60 and their expression rapidly increased as a result of treatment with 1.5 μmol/L SKF 104976.

Morphology and CD marker expression in HL-60 cells treated with SKF 104976. The morphology of HL-60 cells treated with SKF 104976 compared with control cells and cells induced to differentiate with D3 or RA is shown in Fig. 2A. Undifferentiated HL-60 cells were round and had unsegmented nuclei that occupied >75% of the cross-sectional area of the cell. RA-treated cells had bilobed and segmented nuclei, comparable to those found in metamyelocytes and banded neutrophils, and were relatively regular in shape. D3-treated cells had a reduced nucleus-to-cytoplasm ratio. Moreover, the nuclei assumed the reniform appearance typical of monocytes. Preparations of cells treated with 1.5 μmol/L SKF 104976 for 3 or 5 days were more heterogeneous. A subset of cells were binucleated whereas those cells that were mononucleated were smaller than the undifferentiated, control cells. Moreover, some cells were multinucleated and mitotic aberrations were observed.

Figure 2.

Morphology and cell-surface protein expression in differentiated HL-60 cells. A, May Grünwalds stain in HL-60 cells treated with vehicle (Control), 1 μmol/L all-trans RA, 1 μmol/L D3, or 1.5 μmol/L SKF 104976 for the indicated days. Photographs were taken at ×400 magnification. B, fluorescence-activated cell sorting (FACS) analysis of CD11c and CD14 expression on HL-60 cells treated with RA or D3 for 5 d. Percentages of positive cells are indicated. C, effect of incubation time in cells treated with 1.5 μmol/L SKF 104976. D, effect of the indicated doses of SKF 104976 in cells treated for 3 d.

Figure 2.

Morphology and cell-surface protein expression in differentiated HL-60 cells. A, May Grünwalds stain in HL-60 cells treated with vehicle (Control), 1 μmol/L all-trans RA, 1 μmol/L D3, or 1.5 μmol/L SKF 104976 for the indicated days. Photographs were taken at ×400 magnification. B, fluorescence-activated cell sorting (FACS) analysis of CD11c and CD14 expression on HL-60 cells treated with RA or D3 for 5 d. Percentages of positive cells are indicated. C, effect of incubation time in cells treated with 1.5 μmol/L SKF 104976. D, effect of the indicated doses of SKF 104976 in cells treated for 3 d.

Close modal

Expression of CD11c and CD14 on the cell surface is also shown in Fig. 1. As expected, most of the undifferentiated HL-60 cells did not express any of these antigens. In contrast, most of the cells induced to differentiate to granulocytes with RA expressed CD11c, whereas those that differentiated to monocytes using D3 expressed both CD11c and, characteristically, CD14 (Fig. 2B). These results are consistent with those of previous reports (36, 37). Treatment with 1.5 μmol/L SKF 104976 was accompanied by a progressive increase in the proportion of cells expressing CD11c, which accounted for ∼60% of the total at day 4 of treatment (Fig. 2C). This effect of SKF 104976 on CD11c expression was dose dependent (Fig. 2D). In contrast, treatment with SKF 104976 did not produce any detectable change in CD14 expression (Fig. 2C and D).

Prevention of SKF 104976–induced cell differentiation by cholesterol. Having shown that cholesterol starvation induced HL-60 cell differentiation, we analyzed the specificity of this effect by supplementing the medium with free cholesterol (Table 1). In control cells, supplementing the medium with 90 μg/mL cholesterol had no apparent effect on respiratory burst activity or cell-surface CD expression. Simultaneous addition of cholesterol and 1.5 μmol/L SKF 104976 for 3 days practically abrogated the effects of the cholesterol biosynthesis inhibitor alone on both cell-surface expression of CD11c and respiratory burst activity. These results show that cholesterol deficiency specifically induces differentiation of human promyelocytic HL-60 cells.

Table 1.

Abrogation of SKF 104976–induced HL-60 cell differentiation by cholesterol

ΔDCF (n = 4)%CD11c (n = 3)
Control 25.0 ± 12.4a 8.44 ± 0.90a 
Cholesterol 6.8 ± 7.6a 6.33 ± 0.96a 
SKF 104976 172.5 ± 31.7b 59.9 ± 5.86b 
SKF 104976 + cholesterol 36.6 ± 2.9a 12.7 ± 0.60a 
P (ANOVA) <0.001 <0.001 
ΔDCF (n = 4)%CD11c (n = 3)
Control 25.0 ± 12.4a 8.44 ± 0.90a 
Cholesterol 6.8 ± 7.6a 6.33 ± 0.96a 
SKF 104976 172.5 ± 31.7b 59.9 ± 5.86b 
SKF 104976 + cholesterol 36.6 ± 2.9a 12.7 ± 0.60a 
P (ANOVA) <0.001 <0.001 

NOTE: Data are shown as mean ± SE. HL-60 cells were treated with vehicle (control), 1.5 μmol/L SKF 104976, or 90 μg/mL cholesterol for 3 d. Effects of the different treatments were analyzed by one-way ANOVA and post hoc multiple comparisons were done with Tukey's test. Groups in the same column that do not share a superscript letter are statistically different (P < 0.05). ΔDCF, difference of the medians of CM-H2DCFDA fluorescence between PMA-stimulated and resting cells. %CD11c, percentage of cells expressing the CD11c antigen.

Effects of other inhibitors of cholesterol biosynthesis on differentiation of HL-60 cells. Zaragozic acid is a potent inhibitor of squalene synthase (38). Treatment of HL-60 cells with 60 μmol/L zaragozic acid for 3 days resulted in a notable reduction of the cell cholesterol content (1.46 ± 0.2 versus 6.01 ± 0.4 μg/mg of cell protein in treated and control cells, respectively). As recently shown in our laboratory, this treatment leads to inhibition of cell proliferation and cell cycle arrest at G2-M phase, similarly to SKF 104976 (27). We therefore assessed whether cell deprivation of any sterol is a stimulus for HL-60 cell differentiation.

Treatment of HL-60 cells with zaragozic acid in medium lacking cholesterol resulted in stimulation of respiratory burst in a time- and dose-dependent fashion (Fig. 3A and B, respectively) and induced cell-surface expression of CD11c but not CD14 (Fig. 3C). A gradual induction of p47phox and p67phox proteins was observed in cells treated with zaragozic acid (Fig. 3D). The effects closely resembled those of SKF 104976. Moreover, the effects of zaragozic acid on both oxidative activity (Fig. 4A) and expression of CD11c (Fig. 4B) were prevented by simultaneous addition of free cholesterol to the incubation medium. These results indicate that the effects of zaragozic acid are due to the cholesterol deficiency it produces.

Figure 3.

Zaragozic acid–induced differentiation in HL-60 cells. A, NADPH oxidase activity in HL-60 cells treated with 60 μmol/L zaragozic acid for the indicated time. Cells treated with vehicle were used as controls. CM-H2DCFDA fluorescence histograms of resting (open) and PMA-stimulated (shaded) HL-60 cells. Numbers indicate the geometric mean fluorescence of a representative experiment. B, dose effect of zaragozic acid (ZA) in cells treated for 3 d. C, FACS analysis of CD11c and CD14 expression on HL-60 cells treated with 60 μmol/L zaragozic acid for 3 d compared with untreated cells (control). D, Western blot analysis of whole-cell extracts of cells treated with the indicated doses of zaragozic acid for 3 d. p47phox and p67phox expression is shown in the same immunoblot (top) and GAPDH was visualized as a loading control (bottom).

Figure 3.

Zaragozic acid–induced differentiation in HL-60 cells. A, NADPH oxidase activity in HL-60 cells treated with 60 μmol/L zaragozic acid for the indicated time. Cells treated with vehicle were used as controls. CM-H2DCFDA fluorescence histograms of resting (open) and PMA-stimulated (shaded) HL-60 cells. Numbers indicate the geometric mean fluorescence of a representative experiment. B, dose effect of zaragozic acid (ZA) in cells treated for 3 d. C, FACS analysis of CD11c and CD14 expression on HL-60 cells treated with 60 μmol/L zaragozic acid for 3 d compared with untreated cells (control). D, Western blot analysis of whole-cell extracts of cells treated with the indicated doses of zaragozic acid for 3 d. p47phox and p67phox expression is shown in the same immunoblot (top) and GAPDH was visualized as a loading control (bottom).

Close modal
Figure 4.

Abrogation of zaragozic acid–induced differentiation of HL-60 cells by cholesterol. A, NADPH oxidase activity in HL-60 cells treated with 60 μmol/L zaragozic acid and/or 90 μg/mL cholesterol (Chol) for 3 d. Untreated cells were used as controls. CM-H2DCFDA fluorescence histograms of resting (open) and PMA-stimulated (shaded) HL-60 cells. Numbers indicate the geometric mean fluorescence of a representative experiment. B, FACS analysis of CD11c expression on HL-60 cells treated as indicated above. Numbers indicate the percentages of positive cells in a representative experiment.

Figure 4.

Abrogation of zaragozic acid–induced differentiation of HL-60 cells by cholesterol. A, NADPH oxidase activity in HL-60 cells treated with 60 μmol/L zaragozic acid and/or 90 μg/mL cholesterol (Chol) for 3 d. Untreated cells were used as controls. CM-H2DCFDA fluorescence histograms of resting (open) and PMA-stimulated (shaded) HL-60 cells. Numbers indicate the geometric mean fluorescence of a representative experiment. B, FACS analysis of CD11c expression on HL-60 cells treated as indicated above. Numbers indicate the percentages of positive cells in a representative experiment.

Close modal

We then analyzed the effects of other more distal inhibitors of the cholesterol biosynthesis pathway. BM 15766 is a competitive inhibitor of sterol Δ7-reductase, and treatment of HL-60 cells with 25 μmol/L BM 15766 has been shown to block [14C]acetate incorporation into cholesterol, with a reduction in cell cholesterol content but an accumulation of 7-dehydrocholesterol (27). In the present study, treatment of HL-60 cells with BM 15766 for up to 5 days did not result in any appreciable increase in respiratory burst activity or the expression of CD markers (data not shown).

The ERK pathway is required for differentiation of HL-60 cells induced by cholesterol starvation. The ERK pathway is known to be involved in myeloid cell differentiation (39). Analysis of ERK protein expression in HL-60 cells revealed that treatment with SKF 104976 was accompanied by a rapid and marked increase in the cell levels of phosphorylated ERK without apparent changes in total ERK (Fig. 5A). To ascertain whether the ERK pathway is involved in cell differentiation induced by cholesterol starvation, HL-60 cells were treated with increasing concentrations of SKF 104976 and 30 μmol/L PD 98059, a specific inhibitor of MAPK/ERK kinase (MEK). As shown in Fig. 5B, PD 98059 totally prevented the expression of CD11c. This finding indicates that active ERK is required for differentiation induced by SKF 104976.

Figure 5.

Role of ERK in SKF 104976–induced HL-60 cell differentiation. A, Western blot analysis of whole-cell extracts of cells treated with 1.5 μmol/L SKF 104976 for 3 d. Membranes were blotted with anti–diphospho-ERK antibody and then reprobed with anti-ERK antibody. GAPDH was visualized as a loading control. B, FACS analysis of CD11c expression on HL-60 cells treated with increasing concentrations of SKF 104976 for 3 d in the absence (top) or presence (bottom) of 30 μmol/L PD 98059, a specific inhibitor of MEK. Numbers indicate the percentages of positive cells in a representative experiment.

Figure 5.

Role of ERK in SKF 104976–induced HL-60 cell differentiation. A, Western blot analysis of whole-cell extracts of cells treated with 1.5 μmol/L SKF 104976 for 3 d. Membranes were blotted with anti–diphospho-ERK antibody and then reprobed with anti-ERK antibody. GAPDH was visualized as a loading control. B, FACS analysis of CD11c expression on HL-60 cells treated with increasing concentrations of SKF 104976 for 3 d in the absence (top) or presence (bottom) of 30 μmol/L PD 98059, a specific inhibitor of MEK. Numbers indicate the percentages of positive cells in a representative experiment.

Close modal

Induction of a differentiation response in malignant cells can have positive clinical implications, such as the loss of proliferative potential and the induction of apoptosis (8, 11). In previous studies, we showed that inhibition of cholesterol biosynthesis in HL-60 cells resulted in the arrest of cell cycle progression (2528). In this study, we have shown that inhibition of cholesterol biosynthesis induces differentiation in promyelocytic HL-60 cells, as indicated by morphologic changes, gradual induction of p47phox and p67phox protein expression, increased NADPH oxidase activity, and the expression of specific cell-surface markers.

To deprive cells of cholesterol, they were incubated in a cholesterol-free medium and treated with zaragozic acid, SKF 104976, or BM 15766, which inhibit squalene synthase, lanosterol 14-α-demethylase, and sterol Δ7-reductase, respectively. These inhibitors caused a reduction in cell cholesterol content that was accompanied by accumulation of the substrates of the respective target enzymes: lanosterol and dihydrolanosterol in cells treated with SKF 104976 and 7-dehydrocholesterol in cells treated with BM 15766; no intermediate sterol accumulated in cells treated with zaragozic acid (27). Treatment with SKF 104976 or zaragozic acid induced extensive differentiation of HL-60 cells. In both cases, this effect was specific because it was prevented by adding cholesterol to the incubation medium. In sharp contrast, cells treated with BM 15766 did not show any signs of differentiation. These findings correlated with the differential action of these cholesterol biosynthesis inhibitors on cell growth: SKF 104976 and zaragozic acid have been reported to arrest cell cycle progression and cell growth, whereas BM 15766 was ineffective (27). In light of the requirement for cholesterol during cytokinesis (40, 41), these results indicate that 7-dehydrocholesterol, which accumulates in BM 15766-treated cells, may substitute cholesterol for cell division, whereas lanosterol and dihydrolanosterol, which accumulate in cells treated with SKF 104976, may not (27). The similarity between the effects of cholesterol biosynthesis inhibition on cell proliferation and cell differentiation is consistent with the suggestion that these two processes are regulated simultaneously (42, 43).

The differentiation program induced by cholesterol starvation differs from that induced by either RA or D3. Cells treated with SKF 104976 were morphologically heterogeneous; binucleated cells were abundant and many of them showed evidence of abnormal mitosis. In addition, the mononucleated cells present in SKF 104976–treated preparations were smaller than control, undifferentiated cells. This complex phenotype contrasted with those achieved with RA (bilobed and segmented nuclei characteristic of neutrophils) or D3 (reniform nuclei typical of monocytes). Cells treated with SKF 104976 or zaragozic acid showed increased expression of the CD11c antigen, an α-integrin chain known to be strongly up-regulated during myeloid differentiation (36, 37). The lipopolysaccharide receptor CD14, however, was not expressed in cholesterol-deprived cells, indicating that monocyte differentiation did not occur.

Analysis of the effects of inhibiting the mevalonate pathway on differentiation has shown that treatment with statins induces a granulocyte-type differentiation in AML but not ALL cells, as indicated by morphologic changes and increased expression of CD11b or cluster of differentiation antigen 18 (CD18; ref. 8). In keratinocytes, cholesterol depletion by treatment with a combination of lovastatin and methyl-β-cyclodextrin produced a strong up-regulation of mRNA for involucrin, a marker of epidermal differentiation (44). On the other hand, patients with acute leukemia, especially those with monocytic or myelomonocytic leukemia, frequently display hypocholesterolemia in association with increased low-density lipoprotein receptor activity in malignant cells (45). This effect is presumably due to the increased demand for cholesterol associated with cell proliferation (46). Taken together, those results suggest that blocking cholesterol provision by inhibition of cholesterol biosynthesis may be of interest as a cancer therapy due to its effects on both cell proliferation and differentiation.

The MEK/ERK/MAPK signaling pathway has been shown to play a critical role during both monocyte and granulocyte differentiation of HL-60 cells stimulated with PMA and RA, respectively (39). In this study, we have shown that treatment of HL-60 cells with SKF 104976 or zaragozic acid produces sustained activation of ERK1/2 and that coincubation with PD 98059 abrogated differentiation. This pathway is also known to be required for neuronal differentiation in rat PC12 cells (47), maturation of thymocytes from CD4CD8 to CD4+CD8+ cells (48), and adipogenesis (49). Taken together, these results firmly indicate the universal role of the MEK/ERK/MAPK signaling pathway in cell differentiation.

In summary, the results of this study indicate that cholesterol starvation may lead to myeloid differentiation. Furthermore, our observation of distinct responses to different cholesterol biosynthesis inhibitors that reduce the cell cholesterol content to a similar extent suggests that differentiation is triggered by specific changes in the sterol composition of the cell. Thus, modulation of the expression of the different enzymes involved in cholesterol biosynthesis may have a role in the differentiation process.

Note: Present address for C. Fernández-Hernando: Department of Pharmacology, Boyer Center for Molecular Medicine, School of Medicine, Yale University, New Haven, CT 06536-0812.

Grant support: Ministerio de Educación y Ciencia, Spain, grants AGL2004-07075-C02-01 and SAF2005-7308 (M.A. Lasunción); Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Spain, CIBER CB06/0/0021 (M.A. Lasunción).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Beatriz Ledo for her assistance in the laboratory and K. Patten for editorial advice.

1
Ho YK, Smith RG, Brown MS, Goldstein JL. Low-density lipoprotein (LDL) receptor activity in human acute myelogenous leukemia cells.
Blood
1978
;
52
:
1099
–114.
2
Vitols S, Norgren S, Juliusson G, Tatidis L, Luthman H. Multilevel regulation of low-density lipoprotein receptor and 3-hydroxy-3-methylglutaryl coenzyme A reductase gene expression in normal and leukemic cells.
Blood
1994
;
84
:
2689
–98.
3
Rudling M, Gafvels M, Parini P, Gahrton G, Angelin B. Lipoprotein receptors in acute myelogenous leukemia: failure to detect increased low-density lipoprotein (LDL) receptor numbers in cell membranes despite increased cellular LDL degradation.
Am J Pathol
1998
;
153
:
1923
–35.
4
Banker DE, Mayer SJ, Li HY, Willman CL, Appelbaum FR, Zager RA. Cholesterol synthesis and import contribute to protective cholesterol increments in acute myeloid leukemia cells.
Blood
2004
;
104
:
1816
–24.
5
Jakobisiak M, Golab J. Potential antitumor effects of statins (Review).
Int J Oncol
2003
;
23
:
1055
–69.
6
Demierre MF, Higgins PD, Gruber SB, Hawk E, Lippman SM. Statins and cancer prevention.
Nat Rev Cancer
2005
;
5
:
930
–42.
7
Dimitroulakos J, Yeger H. HMG-CoA reductase mediates the biological effects of retinoic acid on human neuroblastoma cells: lovastatin specifically targets P-glycoprotein-expressing cells.
Nat Med
1996
;
2
:
326
–33.
8
Dimitroulakos J, Thai S, Wasfy GH, Hedley DW, Minden MD, Penn LZ. Lovastatin induces a pronounced differentiation response in acute myeloid leukemias.
Leuk Lymphoma
2000
;
40
:
167
–78.
9
Schmidt F, Groscurth P, Kermer M, Dichgans J, Weller M. Lovastatin and phenylacetate induce apoptosis, but not differentiation, in human malignant glioma cells.
Acta Neuropathol (Berl)
2001
;
101
:
217
–24.
10
Li HY, Appelbaum FR, Willman CL, Zager RA, Banker DE. Cholesterol-modulating agents kill acute myeloid leukemia cells and sensitize them to therapeutics by blocking adaptive cholesterol responses.
Blood
2003
;
101
:
3628
–34.
11
Dimitroulakos J, Nohynek D, Backway KL, et al. Increased sensitivity of acute myeloid leukemias to lovastatin-induced apoptosis: a potential therapeutic approach.
Blood
1999
;
93
:
1308
–18.
12
Samid D, Shack S, Sherman LT. Phenylacetate: a novel nontoxic inducer of tumor cell differentiation.
Cancer Res
1992
;
52
:
1988
–92.
13
Alonso DF, Farina HG, Skilton G, Gabri MR, De Lorenzo MS, Gomez DE. Reduction of mouse mammary tumor formation and metastasis by lovastatin, an inhibitor of the mevalonate pathway of cholesterol synthesis.
Breast Cancer Res Treat
1998
;
50
:
83
–93.
14
Denoyelle C, Vasse M, Korner M, et al. Cerivastatin, an inhibitor of HMG-CoA reductase, inhibits the signaling pathways involved in the invasiveness and metastatic properties of highly invasive breast cancer cell lines: an in vitro study.
Carcinogenesis
2001
;
22
:
1139
–48.
15
Boudreau DM, Gardner JS, Malone KE, Heckbert SR, Blough DK, Daling JR. The association between 3-hydroxy-3-methylglutaryl conenzyme A inhibitor use and breast carcinoma risk among postmenopausal women: a case-control study.
Cancer
2004
;
100
:
2308
–16.
16
Poynter JN, Gruber SB, Higgins PD, et al. Statins and the risk of colorectal cancer.
N Engl J Med
2005
;
352
:
2184
–92.
17
Group HPSC. The effects of cholesterol lowering with simvastatin on cause-specific mortality and on cancer incidence in 20,536 high-risk people: a randomised placebo-controlled trial [ISRCTN48489393].
BMC Med
2005
;
3
:
6
.
18
Baigent C, Keech A, Kearney PM, et al. Efficacy and safety of cholesterol-lowering treatment: prospective meta-analysis of data from 90,056 participants in 14 randomised trials of statins.
Lancet
2005
;
366
:
1267
–78.
19
Boudreau DM, Rutter CM, Buist DS. The influence of statin use on breast density.
Cancer Epidemiol Biomarkers Prev
2006
;
15
:
1026
–9.
20
Bonovas S, Filioussi K, Tsavaris N, Sitaras NM. Use of statins and breast cancer: a meta-analysis of seven randomized clinical trials and nine observational studies.
J Clin Oncol
2005
;
23
:
8606
–12.
21
Dale KM, Coleman CI, Henyan NN, Kluger J, White CM. Statins and cancer risk: a meta-analysis.
JAMA
2006
;
295
:
74
–80.
22
Chang SM, Kuhn JG, Robins HI, et al. Phase II study of phenylacetate in patients with recurrent malignant glioma: a North American Brain Tumor Consortium report.
J Clin Oncol
1999
;
17
:
984
–90.
23
Schlegel A, Pestell RG, Lisanti MP. Caveolins in cholesterol trafficking and signal transduction: implications for human disease.
Front Biosci
2000
;
5
:
D929
–37.
24
McTaggart SJ. Isoprenylated proteins.
Cell Mol Life Sci
2006
;
63
:
255
–67.
25
Martínez-Botas J, Suárez Y, Ferruelo AJ, Gómez-Coronado D, Lasunción MA. Cholesterol starvation decreases p34(cdc2) kinase activity and arrests the cell cycle at G2.
FASEB J
1999
;
13
:
1359
–70.
26
Martínez-Botas J, Ferruelo AJ, Suárez Y, Fernández C, Gómez-Coronado D, Lasunción MA. Dose-dependent effects of lovastatin on cell cycle progression. Distinct requirement of cholesterol and non-sterol mevalonate derivatives.
Biochim Biophys Acta
2001
;
1532
:
185
–94.
27
Fernández C, Martín M, Gómez-Coronado D, Lasunción MA. Effects of distal cholesterol biosynthesis inhibitors on cell proliferation and cell cycle progression.
J Lipid Res
2005
;
46
:
920
–9.
28
Suárez Y, Fernández C, Ledo B, Martín M, Gómez-Coronado D, Lasunción MA. Sterol stringency of proliferation and cell cycle progression in human cells.
Biochim Biophys Acta
2005
;
1734
:
203
–13.
29
Collins SJ. The HL-60 promyelocytic leukemia cell line: proliferation, differentiation, and cellular oncogene expression.
Blood
1987
;
70
:
1233
–44.
30
Tanaka H, Abe E, Miyaura C, et al. 1α,25-Dihydroxycholecalciferol and a human myeloid leukaemia cell line (HL-60).
Biochem J
1982
;
204
:
713
–9.
31
Cooper RA, Ip SH, Cassileth PA, Kuo AL. Inhibition of sterol and phospholipid synthesis in HL-60 promyelocytic leukemia cells by inducers of myeloid differentiation.
Cancer Res
1981
;
41
:
1847
–52.
32
Yachnin S, Toub DB, Mannickarottu V. Divergence in cholesterol biosynthetic rates and 3-hydroxy-3-methylglutaryl-CoA reductase activity as a consequence of granulocyte versus monocyte-macrophage differentiation in HL-60 cells.
Proc Natl Acad Sci U S A
1984
;
81
:
894
–7.
33
Trayner ID, Rayner AP, Freeman GE, Farzaneh F. Quantitative multiwell myeloid differentiation assay using dichlorodihydrofluorescein diacetate (H2DCF-DA) or dihydrorhodamine 123 (H2R123).
J Immunol Methods
1995
;
186
:
275
–84.
34
Fernández C, Suárez Y, Ferruelo AJ, Gómez-Coronado D, Lasunción MA. Inhibition of cholesterol biosynthesis by Δ22-unsaturated phytosterols via competitive inhibition of sterol Δ24-reductase in mammalian cells.
Biochem J
2002
;
366
:
109
–19.
35
Rothe G, Valet G. Flow cytometric assays of oxidative burst activity in phagocytes.
Methods Enzymol
1994
;
233
:
539
–48.
36
Scatena R, Nocca G, Sole PD, et al. Bezafibrate as differentiating factor of human myeloid leukemia cells.
Cell Death Differ
1999
;
6
:
781
–7.
37
White SL, Belov L, Barber N, Hodgkin PD, Christopherson RI. Immunophenotypic changes induced on human HL60 leukaemia cells by 1α,25-dihydroxyvitamin D3 and 12-O-tetradecanoyl phorbol-13-acetate.
Leuk Res
2005
;
29
:
1141
–51.
38
Bergstrom JD, Kurtz MM, Rew DJ, et al. Zaragozic acids: a family of fungal metabolites that are picomolar competitive inhibitors of squalene synthase.
Proc Natl Acad Sci U S A
1993
;
90
:
80
–4.
39
Miranda MB, McGuire TF, Johnson DE. Importance of MEK-1/-2 signaling in monocytic and granulocytic differentiation of myeloid cell lines.
Leukemia
2002
;
16
:
683
–92.
40
Fernández C, Lobo MV, Gómez-Coronado D, Lasunción MA. Cholesterol is essential for mitosis progression and its deficiency induces polyploid cell formation.
Exp Cell Res
2004
;
300
:
109
–20.
41
Ng MM, Chang F, Burgess DR. Movement of membrane domains and requirement of membrane signaling molecules for cytokinesis.
Dev Cell
2005
;
9
:
781
–90.
42
Drayson MT, Michell RH, Durham J, Brown G. Cell proliferation and CD11b expression are controlled independently during HL60 cell differentiation initiated by 1,25α-dihydroxyvitamin D(3) or all-trans-retinoic acid.
Exp Cell Res
2001
;
266
:
126
–34.
43
Brown G, Hughes PJ, Michell RH. Cell differentiation and proliferation-simultaneous but independent?
Exp Cell Res
2003
;
291
:
282
–8.
44
Jans R, Atanasova G, Jadot M, Poumay Y. Cholesterol depletion up-regulates involucrin expression in epidermal keratinocytes through activation of p38.
J Invest Dermatol
2004
;
123
:
564
–73.
45
Vitols S, Gahrton G, Bjorkholm M, Peterson C. Hypocholesterolaemia in malignancy due to elevated low-density-lipoprotein-receptor activity in tumour cells: evidence from studies in patients with leukaemia.
Lancet
1985
;
2
:
1150
–4.
46
Kruth HS, Avigan J, Gamble W, Vaughan M. Effect of cell density on binding and uptake of low density lipoprotein by human fibroblasts.
J Cell Biol
1979
;
83
:
588
–94.
47
Qui MS, Green SH. PC12 cell neuronal differentiation is associated with prolonged p21ras activity and consequent prolonged ERK activity.
Neuron
1992
;
9
:
705
–17.
48
Crompton T, Gilmour KC, Owen MJ. The MAP kinase pathway controls differentiation from double-negative to double-positive thymocyte.
Cell
1996
;
86
:
243
–51.
49
Prusty D, Park BH, Davis KE, Farmer SR. Activation of MEK/ERK signaling promotes adipogenesis by enhancing peroxisome proliferator-activated receptor γ (PPARγ) and C/EBPα gene expression during the differentiation of 3T3-1 preadipocytes.
J Biol Chem
2002
;
277
:
46226
–32.