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(Journal of Nutrition. 1999;129:804-813.)
© 1999 The American Society for Nutritional Sciences

Article

Apoptosis and Cell-Cycle Arrest in Human and Murine Tumor Cells Are Initiated by Isoprenoids1

Huanbiao Mo and Charles E. Elson2

Department of Nutritional Sciences, University of Wisconsin-Madison, Madison, WI 53706

2To whom correspondence should be addressed.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Abstract

Diverse classes of phytochemicals initiate biological responses that effectively lower cancer risk. One class of phytochemicals, broadly defined as pure and mixed isoprenoids, encompasses an estimated 22,000 individual components. A representative mixed isoprenoid, {gamma}-tocotrienol, suppresses the growth of murine B16(F10) melanoma cells, and with greater potency, the growth of human breast adenocarcinoma (MCF-7) and human leukemic (HL-60) cells. ß-Ionone, a pure isoprenoid, suppresses the growth of B16 cells and with greater potency, the growth of MCF-7, HL-60 and human colon adenocarcinoma (Caco-2) cells. Results obtained with diverse cell lines differing in ras and p53 status showed that the isoprenoid-mediated suppression of growth is independent of mutated ras and p53 functions. ß-Ionone suppressed the growth of human colon fibroblasts (CCD-18Co) but only when present at three-fold the concentration required to suppress the growth of Caco-2 cells. The isoprenoids initiated apoptosis and, concomitantly arrested cells in the G1 phase of the cell cycle. Both suppress 3-hydroxy-3-methylglutaryl CoA reductase activity. ß-Ionone and lovastatin interfered with the posttranslational processing of lamin B, an activity essential to assembly of daughter nuclei. This interference, we postulate, renders neosynthesized DNA available to the endonuclease activities leading to apoptotic cell death. Lovastatin-imposed mevalonate starvation suppressed the glycosylation and translocation of growth factor receptors to the cell surface. As a consequence, cells were arrested in the G1 phase of the cell cycle. This rationale may apply to the isoprenoid-mediated G1-phase arrest of tumor cells. The additive and potentially synergistic actions of these isoprenoids in the suppression of tumor cell proliferation and initiation of apoptosis coupled with the mass action of the diverse isoprenoid constituents of plant products may explain, in part, the impact of fruit, vegetable and grain consumption on cancer risk.

KEY WORDS: • isoprenoids • cell cycle arrest • human and murine tumors • apoptosis • lamin B prenylation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The tocotrienols and ionones are found among the estimated 22,000 isoprenoid products of secondary plant metabolism which share a common precursor, mevalonic acid (Bach 1995Citation ). ß-Ionone, an end ring analog of ß-carotenoid, represents a subclass of cyclic isoprenoids. The tocotrienols, the less potent of the vitamin E-active tocols, represent a group of mixed isoprenoids with only a part of the molecule being derived via the isoprenoid pathway.

The tocotrienols suppress the proliferation of B16(F10) melanoma cells (He et al. 1997Citation ), MCF-7, MDA-MB-231 and MDA-MB-435 breast cancer cells (Guthrie et al. 1997Citation , Nesaretnam et al. 1998Citation ), and H69 lung carcinoma, HeLa cervical epitheloid carcinoma, and P388 leukemia cells (Komiyama et al. 1989Citation ). ß-Ionone suppresses the proliferation of B16 melanoma cells (He et al. 1997Citation ) and MCF-7 and MDA-MB-231 breast cancer cells (Elson 1995Citation ).

ß-Ionone and {gamma}-tocotrienol suppress the growth of implanted B16(F10) melanomas, the latter when fed at the dietary equivalent of the tocol content of the AIN-76A diet (He et al. 1997Citation ). The suppression of tumor growth by these and other dietary isoprenoids is attributed to both the suppression of cell division (Elson 1995Citation , 1996Citation and references therein) and the initiation of apoptosis (Elson 1995Citation , He et al. 1997Citation , Mills et al. 1995Citation , Reddy et al. 1997Citation ).

Lovastatin and other competitive inhibitors of 3-hydroxy-3-methylglutaryl CoA (HMG CoA)3 reductase activity, the limiting step in the synthesis of farnesyl pyrophosphate (Goldstein and Brown 1990Citation ), suppress cell division (Doyle and Kandutsche 1988Citation , Fairbanks et al. 1986Citation ), and induce apoptosis (Perez-Sala et al. 1995Citation ) in a wide variety of cells. Farnesyl pyrophosphate is a rate-limiting substrate for the posttranslational modification and membrane association of ras (Dricu et al. 1997Citation ) and the nuclear lamins (Zhang and Casey 1996Citation ) and for the synthesis of dolichol, an endogenous isoprenoid essential for the glycosylation and membrane attachment of growth factor receptors (Dricu et al. 1997Citation ). Tocotrienols (Parker et al. 1993Citation , Qureshi et al. 1986Citation ) and ß-ionone (Elson et al. 1998, Yu et al. 1994Citation ) modulate HMG-CoA reductase activity via posttranscriptional actions and, essential for explaining their in vivo tumor-suppressive action, with greater potency in neoplastic cells.

We now trace the tumor growth suppressive actions of ß-ionone and {gamma}-tocotrienol, isoprenoids, respectively, representing subclasses of pure and mixed isoprenoids, to the initiation of apoptosis and G1-phase arrest. Cell lines derived from human cancer tissues respond with 3–5-fold greater sensitivity than normal cells, specifically colon fibroblasts, to the growth-suppressive action of isoprenoids.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell lines and culture media.

Murine B16(F10) melanoma cells (He et al. 1997Citation ) were grown in monolayer culture (35 x 10 mm flasks) in 3 mL of RPMI 1640 medium (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS, Sigma) and 80 mg/L of gentamycin (Sigma). Cultures, seeded with 3.3 x 107 cells/L, were incubated for 24 h at 37°C in a humidified atmosphere of 5% CO2. The medium was decanted and replaced with fresh medium containing the test agents, and incubations were continued as shown in the results section.

Human MCF-7 breast adenocarcinoma cells (HTB-22, ATCC; American Type Culture Collection, Manassas, VA) were grown in monolayer culture (25 cm2 flasks) in 8 mL of minimum essential medium (MEM; Gibco BRL, Grand Island, NY), supplemented with 1 mmol/L of sodium pyruvate, 10 mg/L of bovine insulin (Gibco BRL), 10% FBS and 2% penicillin/streptomycin (1 x 107 units penicillin and 1 x 107 µg streptomycin/L; Gibco BRL). The cultures, seeded with 1.25 x 107 cells/L, were incubated for 24 h at 37°C in a humidified atmosphere of 5% CO2. The medium was decanted and replaced with fresh medium containing the test agents, and incubations were continued as shown in the results section; the medium was replaced at 48-h intervals.

Human Caco-2 colon adenocarcinoma cells (HTB-37, ATCC) were grown in monolayer culture (25 cm2 flasks) in 8 mL of MEM supplemented with 20% FBS and 2% penicillin/streptomycin. The cultures, seeded with 1.25 x 107 cells/L, were incubated for 24 h at 37°C in a humidified atmosphere of 5% CO2. The medium was decanted and replaced with fresh medium containing the test agents, and incubations were continued as shown in the results section; the medium was replaced at 48-h intervals.

Human CCD-18Co normal colon fibroblast cells (CRL-1459, ATCC) were grown in monolayer culture (25 cm2 flasks) in 8 mL of MEM containing nonessential amino acids with Earle's Salts (Gibco BRL) supplemented with 10% FBS and 2% penicillin/streptomycin. The cultures, seeded with 6.25 x 106 cells/L, were incubated for 24 h at 37°C in a humidified atmosphere of 5% CO2. The medium was decanted and replaced with fresh medium containing the test agents, and incubations were continued as shown in the Results section; the medium was replaced at 48-h intervals.

Human HL-60 acute promyelocytic leukemia cells (CCL-240, ATCC) were grown in suspension culture (25 cm2 flasks) in 8 mL of RPMI 1640 medium with 20% of FBS and 2% of penicillin/streptomycin. Cultures, seeded with 1.25 x 108 cells/L, were incubated with test agents for 24 h at 37°C in a humidified atmosphere of 5% CO2. The cells were pelleted by low-speed centrifugation, the media decanted and cells were resuspended in media containing the test agents.

Chemicals.

{gamma}-Tocotrienol was isolated from rice bran oil as previously described (He et al. 1997Citation ), and ß-ionone (96%) was purchased from Aldrich Chemical Company (Milwaukee, WI). The isoprenoids, dissolved in absolute ethanol, were added to cultures at 24 h ({gamma}-tocotrienol, 5–30 µmol/L and ß-ionone, 5–300 µmol/L); all cultures contained 5 mL of ethanol/L (80 mmol/L). Lovastatin, a gift from Merck Research Laboratories (Rahway, NJ), was dissolved in chloroform and added to cultures as indicated below. Chloroform at concentrations to 60 mmol/L had no impact on cell growth. [2-14C]-Acetic acid, sodium salt, (sp. act. 1.85 GBq/mmol) and R-[2-14C]-mevalonolactone (sp. act. 1.85 GBq/mmol) were purchased from American Radiolabeled Chemicals, Inc. (St. Louis, MO).

Cell harvest.

The medium and detached cells were decanted from cells grown in monolayer culture, the monolayer was washed twice with Hanks' Balanced Salt Solution (HBSS, Sigma) and then incubated with a trypsin-EDTA solution (Sigma) at 37°C for 2 min. Trypsin was inactivated by suspending the cells in medium containing 10% of FBS. The trypsinized cells were pelleted at 250 x g and resuspended in HBSS. Viable cells, [cells that excluded 0.4% of trypan blue (Gibco BRL)], were counted with a hemocytometer; 0-time (24-h) cell counts were deducted from final cell counts to provide an estimate of the net increase in cell number.

HL-60 cells were pelleted at 250 x g and resuspended in HBSS. Viable cells, cells that excluded 0.4% trypan blue, were counted with a hemocytometer; 0-time (seeding) cell counts were deducted from final cell counts to provide an estimate of the net increase in cell number.

IC50 value.

The IC50 value represents the concentration of an isoprenoid required to inhibit the net increase in cell count by 50% at a time point within the linear growth period plotted for control cells. The range of concentrations for each isoprenoid tested for each cell line was determined with screening assays. The cells were then incubated with isoprenoids at concentrations that suppressed the proliferation of cells by 90% or less. Cell proliferation, that is the net increase in cell population, was plotted over concentration; the IC50 value falls at the midpoint of the linear portion of the sigmoidal plot averaged over two or more assays.

Cell cycle distribution.

Flow cytometry, in situ detection of apoptosis with the deoxynucleotide transferase mediated dUTP nick end labeling (TUNEL), and DNA fragmentation assays were applied to evaluations of isoprenoid-mediated time- and concentration-dependent effects on the distribution of tumor cells in G1, S and G2/M phases of the cell cycle and the induction of apoptotic cell death.

Flow cytometry.

Isoprenoid-initiated changes in cell cycle distribution and apoptosis were monitored by flow cytometry. Cell pellets (>1 x 106 cells), harvested as described above, were fixed in 1 mL of 70% ethanol at 4°C for 60 min, washed in 1 mL of PBS and resuspended in 400 µL of PBS containing 0.5 mg of RNAse A (Sigma). After gentle mixing, a 100-µL aliquot of propidium iodide (1 g/L PBS) (Sigma) was added (Nicoletti et al. 1991Citation ). The cells were incubated in the dark at room temperature for 15 min and then held at 4°C in the dark for flow cytometric analysis. For each sample, at least 1 x 104 cells were analyzed for DNA content using an Epics XL flow cytometer (Coulter Corporation, Miami, FL). The distribution of cells in sub-G1, G1, S and G2-M was determined using MultiCycle AV software (Phoenix Flow Systems, San Diego, CA). The sub G1 peak is an indicator of the onset of apoptosis (Hotz et al. 1994Citation ).

In situ detection of apoptosis.

The TUNEL analysis, an in situ measure of apoptosis, was carried out with the In Situ Cell Death Detection kit (Boehringer Mannheim, Indianapolis, IN). Briefly, cell pellets (2 x 106 cells), suspended in 100 µL of PBS, were added to 1.4 mL of fixation solution (4% paraformaldehyde in PBS, pH 7.4) and held on ice for 15 min. Fixed cells were rinsed twice with PBS and then incubated at ice temperature for 2 min in 1 mL of permeabilization solution (0.1% Triton X-100, 0.1% sodium citrate). After pelleting, the cells were rinsed twice with PBS, suspended in 50 µL TUNEL reaction mixture and incubated for 60 min at 37°C. The cells were washed three times with PBS, and aliquots were then taken for evaluation of DNA strand breakage using a fluorescent microscope.

DNA fragmentation.

B16 melanoma and Caco-2 colon adenocarcinoma cells were harvested for analysis of genomic DNA as described by Compton and Cidlowski (1986)Citation and Sambrook et al. (1989)Citation . Aliquots (2.5 x 106 cells) were incubated in 500 µL of buffer (10 mmol/L Tris-HCl, 1 mmol/L EDTA, 0.1 mol/L NaCl, pH 8.0) containing 100 mg proteinase K/L solution (Boehringer Mannheim) and 0.5% SDS for 1 h at 50°C. The lysed samples were extracted sequentially with equal volumes of phenol, phenol/chloroform/isopropanol (25:24:1), and chloroform. The residues, suspended in 0.3 mmol/L of sodium acetate in 67% ethanol, were held on ice for 10 min and then collected by centrifugation. The DNA precipitates, resuspended in 15 µL buffer (10 mmol/L Tris-HCl, 1 mmol/L EDTA, pH 8.0), were incubated with 60 µg of RNase A for 30 min at 37°C. Aliquots of the RNA-free DNA were electrophoresed as described below.

Fragmented DNA was extracted from HL-60 leukemic cells as described by Gong et al (1994)Citation . Cell pellets (0.5–1 x 107 cells), harvested as described above, were fixed with 1 mL of 70% cold ethanol for 1 h. The cells were harvested by centrifugation at 800 x g for 5 min and the ethanol thoroughly removed. Cell pellets were suspended in 40 µL of phosphate-citrate buffer (96% of 0.2 mol/L of Na2HPO4 and 4% of 0.1 mol/L of citric acid, pH 7.8) with mixing for 1 h at room temperature. After centrifugation at 1000 x g for 10 min, the supernatant fraction was concentrated to 10 µL by vacuum in a SpeedVac concentrator (SVC 100; Savant Instruments, Inc., Farmingdale, NY) for 1 h. Detergent (3 µL 0.25% NP-40, Sigma) and 3 µg of RNase A were added in order, the mixture incubated for 30 min at 37°C, then 3 µg of proteinase K was added. After continuing the incubation for 30 min, 3 µL of loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol FF, 30% glycerol, 6 mmol/L EDTA) was added.

Electrophoresis.

DNA extracts adjusted to common cell number and a reference ({phi}X174 RF DNA/Hae III Fragments; Gibco BRL) were loaded on 2% of agarose gel (Gibco BRL). DNA fragments were separated by horizontal electrophoresis (USA Scientific Plastics, Ocala, FL; 60 V for 2–3 h, Tris-Borate-EDTA buffer (TBE, pH 8.0, Gibco BRL). The gels were stained with ethidium bromide solution (Boehringer Mannheim) and viewed under ultraviolet light. Ethidium bromide-stained bands showing DNA fragmentation provide confirmation of apoptotic cell death.

Lamin B processing.

We first determined that cell division proceeded normally in cultures seeded with 3 x 109 HL-60 cells/L. Next, we used 24-h cell counts and cell-cycle profiles determined by FACScan as end points in determining the "no effect" concentrations of ß-ionone and lovastatin. For these experiments, 25 cm2 flasks containing 5 mL of RPMI 1640 medium with 20% FBS and 2% of penicillin/streptomycin were seeded with 3 x 109 cells/L and incubated with "no-effect" levels of the test agents and tracer quantities of radiolabeled substrates (acetic acid, 90 GBq/L; mevalonolactone, 120 and 360 GBq/L) for 24 h at 37°C in a humidified atmosphere of 5% CO2.

The cells were pelleted and resuspended in lysis buffer [100 µL buffer/1 x 106 cells; 50 mmol/L Tris-HCl, pH 7.4, 10 mmol/L of sodium pyrophosphate, 50 mmol/L of sodium fluoride, 0.5% NP-40, 2 mmol/L of benzamidine, 50 mmol/L of ß-glycerophosphate, 25 mg/L of p-nitroguanidinobenzoate (Sigma), 5 mmol/L of EDTA, 1 mg/L of leupeptin (Sigma), 1 mg/L of pepstatin (Sigma), 1 mmol/L of phenylmethylsulfonyl fluoride (Sigma), 100 mg/L of soybean trypsin inhibitor (Sigma), and 1% of SDS]. The lysate was collected by centrifugation and the protein content determined using bicinchoninic acid (Sigma). The lysate was added to an equal volume of immunoprecipitation buffer (80 mmol/L of ß-glycerophosphate, 50 mmol/L of sodium fluoride, 1 mmol/L of ATP, 5 mmol/L of sodium pyrophosphate, 5 mmol/L of EDTA, 1% of Triton X-100, 1% of sodium deoxycholate, 0.1% of SDS) containing 100 g of nonfat dry milk and 50 g of bovine serum albumin/L and incubated with mixing at 4°C for 1 h. A quantity of Protein A-Sepharose (Sigma) equal to the total lysate protein was added and the incubation continued for 1 h. Sepharose beads were removed by centrifugation, and the precleared lysate was incubated with lamin B antibody (13 µg/mg of protein; CALBIOCHEM, San Diego, CA). After an overnight incubation at 4°C, the antigen-antibody complexes were collected with protein A-Sepharose (3 mg/mg protein) and washed extensively with immunoprecipitation buffer twice and finally with immunoprecipitation buffer lacking detergents.

The washed immunoprecipitates were heated to 65°C in 15 µL of loading buffer (50 mmol of Tris-HCl/L, 2% of SDS, 0.1% of bromophenol blue, 10% of glycerol 100 mmol dithiothreitol/L, pH 6.8) for 10 min to dissociate the antigen-antibody complex and then loaded on 0.75 mm of 12% SDS-polyacrylamide minigels which were run at 15 mA for 5–6 h with Tris buffer, pH 8.3). The proteins were transferred (Model TE22 transphor electrophoresis unit; Hoefer, San Francisco, CA) to nitrocellulose membranes (Bio-Rad) at 100 mA overnight. The intensity of the radiolabel on each membranes was determined by exposure to a Packard Cyclone Storage Phosphor Screen (Packard Instrument Company, Inc., Meriden, CT) for 48 h and then scanning the bands with a Cyclone imaging system (Packard). Digital Light Units (DLU) reflect intensity of the radiolabel. The lamin B band was quantitated by Western blotting. The nitrocellulose membranes were incubated in blocking solution (5% of dry milk, 0.05% of Tween-20 in PBS) for 2 h, washed with 0.05% of Tween-20 in PBS, and then incubated with lamin B monoclonal antibody (CALBIOCHEM, 100 mg/L) for 1 h. After washing with Tween-20/PBS, the membranes were incubated with the secondary antibody (horseradish peroxidase diluted 1:1,500, Enhanced Chemiluminescence Kit; Amersham Life Science, Arlington Heights, IL) for 1 h, washed, and then exposed to ECL film for 15–60 s. The relative densities of the lamin B bands (O.D. x mm2) were determined with a pdi Model DNA 35 Imaging Device and Quantity One software (Huntington Station, NY).

Statistical methods.

StatView software (Abacus Concepts, Berkeley, CA) was used for the assessment (unpaired t test) of treatment-mediated effects on the incorporation of 2-14C-mevalonolactone into lamin B and cell proliferation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Tumor cell lines differed substantially in their response to ß-ionone. IC50 values, the concentrations of ß-ionone that caused a 50% reductions in the populations of murine B16 melanoma cells, human HL-60 acute promyelocytic leukemia cells, human MCF-7 breast adenocarcinoma cells, human Caco-2 colon adenocarcinoma cells and CCD-18Co normal human colon fibroblasts, are listed on Table 1.Citation The values estimated for the four cell lines derived from human tumors were 20–40% of the values estimated for normal human fibroblasts and for the murine melanoma. The greater sensitivity of the human tumor cell lines to ß-ionone was confirmed by findings that their growth was blocked when incubated with 150 µmol/L of ß-ionone, the concentration of ß-ionone that suppressed the growth of B16 melanoma cells and normal human fibroblasts by 50 and 57%, respectively. HL-60 and MCF-7 cells (Guthrie et al. 1997Citation , Nesaretnam et al. 1998Citation ) were also 3–5-fold more sensitive than the highly metastatic murine melanoma to the growth-suppressive action of {gamma}-tocotrienol.


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  		Table 1. IC50 values showing the relative sensitivities of human tumor cell lines to ß-ionone and {gamma}-tocotrienol1

 
Growth suppression as determined in these studies reflects the initiation of apoptosis and the arrest of cell division. Representative DNA histograms recorded for control and isoprenoid-treated B16 (20 µmol of {gamma}-tocotrienol/L, 3 h incubation), Caco-2 (150 µmol of ß-ionone/L, 24 h incubation) and HL-60 (100 µmol of ß-ionone/L, 8 h incubation) cells are shown in Figure 1.Citation All DNA histograms of isoprenoid-treated cells revealed a sub-G1 (apoptotic) peak of varying magnitude. The distribution of asynchronous B16 cells in the phases of the cell cycle remained remarkably constant as the population increased from 8.2 x 106 to 28.7 x 106 cells/cultures during a 24-h incubation (Table 2Citation ).The cell cycle analyses of cells harvested after a 3-h incubation in medium containing 20 µmol of {gamma}-tocotrienol/L revealed the initiation of apoptosis, an increase in the proportion of G1-phase cells, a decrease in the proportion of S-phase cells, and an increase in the G1/S ratio, a marker for G1 arrest. Because of these growth-suppressive actions, the 3-h cell population was reduced by 25% (Table 2)Citation .



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  		Figure 1. Cell-cycle analysis following isoprenoid treatment. Murine B16(F10) melanoma cells incubated with 20 µmol/L of {gamma}-tocotrienol for 3 h, human HL-60 acute promyelocytic leukemia cells incubated with 100 µmol/L of ß-ionone for 8 h, and human Caco-2 colon adenocarcinoma cells incubated with 150 µmol/L of ß-ionone for 24 h were harvested and analyzed by flow cytometry. Control cells, harvested from identical cultures, were grown in the absence of the isoprenoid. The distribution of cells in G1, S, and G2-M phases of the cell cycle and proportion of apoptotic cells are shown on the panels.

 

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  		Table 2. Impact of {gamma}-tocotrienol and ß-ionone on the distribution of B16 cells in the cell cycle

 
The sequence of these changes in the cell cycle distribution initiated by ß-ionone (150 µmol/L) is shown in Figure 2Citation A–F.The series of plots drawn from two studies show the time-dependent impact of ß-ionone on the proportions of cells in G1 (Fig. 2A)Citation , S (Fig. 2B)Citation and G2/M (Fig. 2C)Citation phases of the cell cycle through 12 h. The time required for the doubling of the control cell population was about 8 h (Fig. 2FCitation ; see also Table 2Citation ). The initiation of apoptosis (Fig. 2D)Citation preceded the G1-phase arrest (Fig. 2E)Citation . The apparent decrease in the fraction of apoptotic cells (Fig. 2D)Citation , preceding the increase in the G1/S ratio, a marker of G1 phase arrest (Fig. 2E)Citation may be misleading. As apoptosis proceeds, the cells swell, round up and detach from the monolayer (Cotter et al. 1990Citation , Stewart 1994Citation ). As outlined in the procedure, detached cells were not collected for this analysis. Under these conditions the experimental cell population was reduced by 20% at 12 h (Fig. 2F)Citation and by 50% at 48 h (Table 1)Citation .



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  		Figure 2. A representative analysis of the time-dependent impact of ß-ionone (150 µmol/L) on growth and distribution of murine B16 melanoma cells in the cell cycle. B16 cells were incubated with ß-ionone for 0, 1, 2, 3, 8 and 12 h. Cells remaining on the monolayer after washing were harvested, counted and aliquots were analyzed by flow cytometry. Figures 2A–CCitation record the percentages of cells in the G1 (A), S (B) and G2M (C) phases of the cell cycle. Figure 2DCitation records the percentage of apoptotic cells harvested at each time point. The values plotted for control cells (- - -) are the means ± SD (n = 6). Values plotted for experimental cells (—) are means of two determinations at each time point. The G1/S ratios, indicators of G1 arrest, are plotted on Figure 2ECitation . The values plotted on Figure 2FCitation are means ± SD (n = 20) of the populations of control (- - -) and experimental (—) cells.

 
Relatively slow-growing Caco-2 cells responded in similar fashion to the growth-suppressive action of ß-ionone. Under control conditions, the distribution of cells in G1 (57.4 ± 2.9%), S (38.6 ± 9.8%) and G2/M (4.0 ± 6.9%) phases of the cell cycle remained constant during a 24-h incubation. The distribution profile for cells incubated with 150 µmol of ß-ionone/L for 24 h recorded 73% of cells in G1, 15% in S, and 12% in G2/M. The increase in the G1/S ratio (1.5 to 4.9) shows the ß-ionone-mediated G1 arrest. Flow cytometry further showed that 21% of the Caco-2 cells remaining on the monolayer were apoptotic. Apoptotic cells released from monolayers were not harvested for this analysis.

We next evaluated the time- and concentration-dependent impact of ß-ionone on the cell-cycle distribution of rapidly proliferating HL-60 cells. The cell-cycle distribution of control cells at 24 h (39.0 ± 1.8% G1, 50.6 ± 1.5% S, 10.4 ± 0.6% G2/M) differed little with that recorded at 0-time (38.3 ± 0.2% G1, 51.6 ± 0.1% S, 10.2 ± 0.3% G2/M). The series of plots drawn from two studies show the time-dependent impact of ß-ionone on the proportions of HL-60 cells in the G1 (Fig. 3Citation A),S (Fig. 3B)Citation and G2/M (Fig. 3C)Citation phases of the cell cycle. The initiation of apoptosis (Fig. 3D)Citation preceded the G1-phase arrest (Fig. 3E)Citation .



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  		Figure 3. A representative analysis of the time-dependent impact of ß-ionone (100 µmol/L) on the growth and distribution of HL-60 cells in the cell cycle. HL-60 cells were incubated with ß-ionone for 0, 8, and 12 h. Aliquots of cells were analyzed by flow cytometry. Figures 3A–CCitation record the percentages of HL-60 cells in the G1 (A), S (B) and G2M (C) phases of the cell cycle. Figure 3DCitation records the impact of ß-ionone on the percentage of apoptotic cells. Values are means ± SD, n = 2. The G1/S ratios, indicators of G1 arrest, are plotted on Figure 3ECitation . Imposed on the plots are values (means ± SD, n = 6) obtained for control cells at 0 and 24 h.

 
HL-60 cells were also incubated with graded concentrations of ß-ionone for 24 h. As shown in Figures 4Citation A–F,the cell cycle profile parameters and 24-h cell populations remained relatively constant as the ß-ionone concentration was increased to 20 µmol/L. The onsets of apoptosis (Fig. 4D)Citation and G1 arrest, indicated by the increase in the G1/S ratio (Fig. 4E)Citation , were attained at a concentration of ß-ionone falling between 50 and 75 µmol/L. Increasing the ß-ionone concentration to 75 µmol/L amplified both the G1 arrest and apoptosis and concomitantly, the population of cells was reduced by 99% when compared with the population in the 24-h control culture (Fig. 4F)Citation . More than 80% of the HL-60 cells harvested at 24 h from medium containing 100 µmol ß-ionone/L, a concentration double the IC50 value (Table 1)Citation , were apoptotic (Fig. 4D)Citation . HL-60 cells are incubated in suspension culture. It should be noted that the plasma membrane of cells undergoing the early stages of apoptosis remain intact (Cotter et al. 1990Citation , Stewart 1994Citation ). As a result, all HL-60 cells that exclude trypan blue are harvested for cell-cycle analysis. Prior reference was made to the decanting of a portion of apoptotic B16 and Caco-2 cells (those released from the monolayer) prior to cell-cycle analysis.



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  		Figure 4. A representative analysis of the concentration-dependent impact of ß-ionone on the 24-h cell cycle distribution of HL-60 cells. HL-60 cells were incubated with various concentrations of ß-ionone (0–100 µmol/L) for 24 h. Cells were counted and cell cycle distribution analyzed by flow cytometry. Figures 4A–CCitation record the impact of ß-ionone (0–75 µmol/L) on the percentages of cells in the G1 (A), S (B), and G2/M (C) phases of the cell cycle. Figure 4DCitation records the percentage of apoptotic cells. Apoptotic cells accounted for 90% of the cells in cultures incubated with 100 µmol of ß-ionone/L. The G1/S ratios, indicators of G1 arrest, are plotted on Figure 4ECitation . The values plotted on Figure 4FCitation are means ± SD (n = 20) of 24-h cell populations. Control values (0 µmol of ß-ionone/L) are means ± SD, n = 6.

 
The isoprenoid-mediated initiation of apoptosis shown by flow cytometry was confirmed using TUNEL analysis and DNA electrophoresis. Representative fluorescent photomicrographs of isoprenoid-treated cells harvested for the TUNEL assay are shown in Figure 5.Citation The extent of DNA strand breakage present in B16 cells incubated with 20 µmol of {gamma}-tocotrienol/L for 3 h and in HL-60 cells incubated with 100 µmol of ß-ionone/L for 12 h reflected the proportions of apoptotic cells detected by FACScan analysis. DNA strand breakage was recorded in MCF-7 cells which were incubated with 100 µmol of ß-ionone/L for 48 h (Fig. 5)Citation . B16 and MCF-7 cells in advanced stages of apoptosis were not recovered for these analyses. All control cells were essentially free of strand breakage.



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  		Figure 5. Isoprenoid-initiated apoptosis detected by terminal deoxynucleotidyl transferase reaction (TUNEL) analysis of murine B16 melanoma cells after incubation with 20 µmol/L of {gamma}-tocotrienol for 12 h, of human HL-60 acute promyelocytic leukemic cells after incubation with 100 µmol/L of ß-ionone for 12 h, and of human MCF-7 adenocarcinoma breast cells after incubation with 100 µmol/L of ß-ionone for 48 h. Control cells were harvested from cultures grown in the absence of the isoprenoid. Aliquots containing identical numbers of control and treated cells were analyzed. Fluorescence marks apoptotic cells. Objective magnification, 40x.

 
Electrophoresis of DNA extracted from HL-60 cells incubated for 3 h with the DNA topoisomerase I inhibitor camptothecin yielded a laddering pattern consisting of segments differing by ~200 bp (Gong et al. 1994Citation ). The electrophoretic pattern of DNA extracted from HL-60 cells incubated for 12 h with 10 µmol of lovastatin/L (Perez-Sala et al. 1995Citation ) was characterized by a more generalized and diffuse pattern of DNA segments. Figure 6Citation records a similar pattern showing the generalized and diffuse segmenting of the DNA extracted from HL-60 cells following an 8 h incubation with 100 µmol of ß-ionone/L. The electrophoretic pattern for DNA extracted from Caco-2 cells incubated with 100 µmol/L of ß-ionone for 8 h showed a similar smear (Fig. 6)Citation . Smearing characterized also the electrophoresis patterns of the DNA extracted from B16 cells incubated with 250 µmol/L of ß-ionone for 12 h (Fig. 6)Citation .



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  		Figure 6. Isoprenoid-induced DNA fragmentation detected by agarose gel electrophoresis of DNA isolated from human HL-60 acute promyelocytic leukemia cells, human Caco-2 colon adenocarcinoma cells, and murine B16 melanoma cells. HL-60 cells were incubated with 100 µmol of ß-ionone/L for 0–8 h. DNA isolated from an aliquot of 5 x 106 cells was loaded in each lane. The marker shows {phi}{chi}174 RF DNA/Hae III fragments. Caco-2 cells were incubated with 100 µmol of ß-ionone/L for 8 h. DNA isolated from 2.5 x 106 cells was loaded in each lane. B16 cells were incubated with 250 µmol of ß-ionone/L for 12 h. DNA isolated from 2.5 x 106 cells was loaded in each lane.

 
We tested the hypothesis that the isoprenoid-mediated suppression of HMG-CoA reductase activity (and farnesyl pyrophosphate synthesis) suppresses the farnesylation of lamin B. Western blots of lamin B immunoprecipitated from lysates of HL-60 cells incubated for 24 h with 50 or 100 µmol/L of ß-ionone revealed the presence of low molecular weight bands (data not shown), whereas that from cells incubated with 20 µmol of ß-ionone/L showed a single 68 kDa band. Lovastatin served as a positive control for the following assays. We first examined the concentration-dependent impact of lovastatin on the 24-h growth of HL-60 cells (Fig. 7Citation A–F).The proportions of cells in G1 (Fig. 7A)Citation , S (Fig. 7B)Citation , and G2/M (Fig. 7C)Citation remained relatively constant as lovastatin was increased to 0.1 µmol/L. The onset of apoptosis (Fig. 7D)Citation coincided with the decrease in cell population (Fig. 7F)Citation . The IC50 value, estimated to be about 0.075 µmol/L (Fig. 7F)Citation , fell between the concentration of lovastatin that initiated apoptosis (Fig. 7D)Citation and concentration that caused G1 arrest (Fig. 7E)Citation .



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  		Figure 7. A representative analysis of the concentration-dependent impact of lovastatin (0–0.2 µmol/L) on the 24-h cell cycle distribution of HL-60 cells. Cells were counted and cell cycle distribution analyzed by flow cytometry. Figures 7A–CCitation record the impact of lovastatin on the percentages of cells in the G1 (A), S (B) and G2M (C) phases of the cell cycle. Figure 7DCitation records the impact of lovastatin on the percentage of apoptotic cells. The G1/S (see Figs. 7A, 7BCitation ) ratios, indicators of G1 arrest, are plotted on Figure 7ECitation . Control (0 µmol lovastatin/L) values are means ± SD, n = 6. The values plotted on Figure 7FCitation are means ± SD (n = 20) of 24-h cell populations.

 
Assays employing the "no-effect" level of the test agent, ß-ionone (20 µmol/L, Fig. 4Citation ) and that of the positive control, lovastatin (0.05 µmol/L; Fig. 7Citation ) permitted the evaluation of treatment-mediated effects of lamin B processing in the absence of the confounding presence of apoptosis-associated degraded lamin B (Shimizu et al. 1998Citation ). We first confirmed that these "no-effect" treatments had no impact on cell growth. Neither the quantity of protein in the cell lysate nor the quantity of lamin B immunoprecipitated from the lysates varied among treatments (Fig. 8Citation ).Also recorded on Figure 8Citation are the DLU (49 h exposure)/lamin B immunoprecipitated from 700 µg aliquots of the cell lysate protein. Acetate incorporation by ß-ionone-treated cells during a 24 h incubation was 51% of control and that by lovastatin-treated cells, 53% of control (Fig. 8)Citation . The ß-ionone effect was confirmed in a second assay that recorded a 37% suppression of acetate incorporation into lamin B (data not shown).



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  		Figure 8. Impact of ß-ionone and lovastatin on the incorporation of radiolabeled acetic acid and mevalonolactone into nuclear lamin B. HL-60 cells were seeded with 3 x 109 cells/L and incubated with 20 µmol/L of ß-ionone or 0.05 µmol/L of lovastatin and tracer quantities of radiolabeled substrates (acetic acid, 90 GBq/L; mevalonolactone, 120 GBq/L) for 24 h at 37°C in a humidified atmosphere of 5% CO2. Quantitative values (mean ± SD) recorded for lysate protein (mg/culture, n = 4), density of the lamin B band (O.D. x mm2, n = 2), radiolabeled acetate incorporation/lamin B precipitated from 700 µg of lysate protein, (digital light units (DLU)/band, 49 h exposure, n = 1) and radiolabeled mevalonolactone incorporation/lamin B precipitated from 1500 µg of lysate protein (DLU/band, 192 h exposure, n = 1) are presented in the bars; the plots show experimental values as percentage of control values.

 
Inhibition of HMG-CoA reductase with the agents potentiated the incorporation of mevalonolactone into lamin B. Figure 8Citation records the DLU (192 h exposure)/lamin B immunoprecipitated from 1500 µg aliquots of the cell lysate protein. Incorporation of mevalonolactone into lamin B by cells treated with lovastatin was 233% of control and that by cells treated with ß-ionone, 173% of control. In a follow-up study, the medium was supplemented with 198 µmol of mevalonolactone/L, a concentration sufficient to reverse lovastatin-initiated apoptosis in HL-60 cells (Perez-Salas et al. 1995Citation ). Under these conditions, radiolabeled mevalonolactone incorporation into lamin B by ß-ionone-treated cells was 124% of control (P < 0.05, n = 3).

We earlier reported a diet patterned after the AIN-76A formulation but modified only with the substitution of d-{gamma}-tocotrienol for dl-{alpha}-tocopherol significantly suppressed the growth of implanted B16 melanomas (He et al. 1997Citation ). The IC50 values we calculated for the {gamma}-tocotrienol-mediated suppression of tumor cell proliferation, 4–20 µmol/L (Table 1)Citation , fall in the range of the plasma tocol values reported for rodents and as well as humans. On the other hand, the concentration of ß-ionone required to suppress the proliferation of human and murine tumor cells (Table 1)Citation fell beyond a physiological concentration. Crucial findings, we believe, are presented on Figure 9.Citation The growth of B16 melanoma cells incubated for 48 h with 7.5 µmol of {gamma}-tocotrienol/L and with 75 µmol of ß-ionone/L was inhibited by 7% (P = 0.460) and 27% (P < 0.001), respectively. Doubling the concentrations of the respective isoprenoids yielded 23% (P < 0.001) and 56% (P < 0.001) inhibitions of growth. An additive and potentially synergistic growth-suppressive action was suggested by findings that each of the four blends tested suppressed growth to a greater degree than that predicted by the sums of the individual actions (Fig. 9A)Citation ; the observed response was 21 ± 13% greater than the predicted response (paired t test, P = 0.0537). The growth-suppressive potencies of {gamma}-tocotrienol and ß-ionone shown in Figures 9B and Citation 9CCitation fall in the range recorded in Figure 9ACitation . That is, the impact of 10 µmol of {gamma}-tocotrienol/L (Fig. 9B)Citation fell between that of 7.5 and 15 µmol of {gamma}-tocotrienol/L (Fig. 9A)Citation and that of 75 µmol of ß-ionone/L (Fig. 9C)Citation essentially matched that shown on Figure 9ACitation . Lovastatin (1 and 2 µmol/L) suppressed the growth of B16 cells (27%, Fig. 9BCitation , and 49%, Fig. 9CCitation , respectively). Synergy between the growth-suppressive actions of lovastatin and the two isoprenoids is suggested. A combination consisting of 10 µmol of {gamma}-tocotrienol/L (17% growth inhibition) and 1 µmol of lovastatin/L (27% growth inhibition) inhibited growth by 61% (Fig. 9B)Citation . Similar synergy was obtained with the blend of ß-ionone and lovastatin (Fig. 9C)Citation .



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  		Figure 9. The synergistic impact of blends of {gamma}-tocotrienol, ß-ionone and lovastatin on the 48-h growth of murine melanoma B16 cells. Cells remaining on the monolayer after washing were harvested and counted. The 48-h cell counts (means ± SD, n = 20) are plotted on the figures. The inhibition of growth by each individual treatment, [1-(Treatment count/Control count)] x 100, is recorded as Growth, % Inhibition. The predicted inhibition recorded for a blend reflects the sum of the inhibitions imposed by the constituents. Figure 9ACitation shows the synergistic effects of {gamma}-tocotrienol and ß-ionone, Figure 9BCitation , the synergy obtained by combining {gamma}-tocotrienol with lovastatin, and 9C, the synergy obtained by combining ß-ionone with lovastatin.a–f Means not sharing a superscript are different (unpaired t test, P < 0.05). The difference between the values, "Growth, % Inhibition" and "Predicted % Inhibition", (Fig. 9A)Citation approaches significance (paired t test, P = 0.0537).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The human leukemic, breast and colon tumor cell lines selected for this study had a greater sensitivity than that of murine melanoma cells to the growth-suppressive action of the two isoprenoids. Growth, measured as the increase in cell population, reflects rates of cell division (proliferation) and cell death (apoptosis). Isoprenoids trigger actions at both levels; we report the concentration-dependent onset of G1 arrest and apoptosis. Aberrant tumor growth can be traced to mutations of ras and p53, the former by sustaining cell proliferation and the latter, by blocking apoptosis.

The isoprenoid-mediated suppression of cell growth was clearly independent of a mutated ras function. ß-Ionone suppressed the proliferation of B16 and HL-60 cells that respectively express ki-ras (Kris et al. 1985Citation ) and n-ras (Murray et al. 1983Citation ). Our finding that ß-ionone also suppressed the proliferation of Caco-2 and MCF-7 cells which express wild-type ras (Delage et al. 1993Citation , Sukumar et al. 1988Citation ) confirms earlier work (Ruch and Sigler 1994Citation ), showing that the tumor-suppressive action of cyclic isoprenoids, like that of lovastatin (DeClue et al. 1991Citation ), is independent of ras function. ß-Ionone and lovastatin initiated concentration-dependent actions which arrested HL-60 promyelocytic leukemia cells in the G1 phase of the cell cycle. Studies utilizing lovastatin showed that mevalonate synthesis is essential for the dolichol-supported glycosylation and membrane attachment of growth factor receptors (Dricu et al. 1997Citation ). ß-Ionone, we propose, similarly suppresses dolichol synthesis and, concomitantly, the glycosylation and membrane attachment of growth factor receptors.

The isoprenoid-mediated initiation of apoptosis was clearly independent of a mutated p53 function. ß-Ionone and {gamma}-tocotrienol initiated apoptosis in B16 and MCF-7 cells which express wild type p53 (David-Pfeuty et al. 1996Citation , Gudas et al. 1996Citation , Parker et al. 1994Citation ) and in Caco-2 cells which express mutated p53 (Gartel et al. 1996Citation ) as well as in p53-null HL-60 cells (Koeffler et al. 1986Citation , Wolf and Rotter 1985Citation ). The concentration-dependent actions of ß-ionone and lovastatin initiated apoptosis in HL-60 promyelocytic leukemia cells. HL-60 cells express primarily lamin B (Moir et al. 1995Citation ). The structural and functional integrity of the nuclear lamina is strictly related to the covalent modification of nuclear lamin B during the cell cycle. Farnesyl pyrophosphate, the rate-limiting substrate for the posttranslational modification of the carboxyl-terminal cysteine of the CAAX sequence of neosynthesized lamin B (Zhang and Casey 1996Citation ), is required for the reassembly of daughter nuclei during interphase (Bruscalupi et al. 1997Citation ; Hutchison et al. 1994Citation ; McKeon 1991Citation ). The breakdown of lamin B processing subsequent to lovastatin- or isoprenoid-mediated suppression of HMG-CoA reductase activity, we postulate, interferes with the assembly of daughter nuclei and renders DNA available to p53-independent apoptotic endonuclease activities as demonstrated by FACScan, DNA gel electrophoresis and TUNEL assays. At "no effect" concentrations, concentrations shown to have no immediate impact on cell-cycle distribution, lovastatin and ß-ionone equally suppressed the incorporation of radiolabeled acetate, the precursor of mevalonate, into lamin B and both enhanced the incorporation of radiolabeled mevalonate into lamin B.

The multivalent regulation of HMG-CoA reductase, elegantly delineated by Goldstein and Brown (1990)Citation , consists of three entities, the sterol feedback modulation of transcription, the modulation of the efficiency of HMG-CoA reductase mRNA processing, and the degradation of HMG CoA reductase catalyzed by a cytosolic cysteine protease. Farnesol, (Correll et al. 1994Citation ) diverted from the mevalonate pathway, modulates the latter activity. The prenyl pyrophosphate pyrophosphatase which potentially plays a key role in the modulation of HMG-CoA reductase activity by diverting farnesol from the mevalonate pathway (Meigs and Simoni 1997Citation ) might be induced by ß-ionone (Case et al. 1995Citation , Miegs and Simoni 1997Citation ). If so, the potential synergy obtained with ß-ionone and {gamma}-tocotrienol could reflect the dual consequences of the induction of a prenyl pyrophosphate pyrophosphatase activity by the former and the latter's action as a farnesol analogue. This prospect draws support from data published elsewhere. He et al. (1997)Citation evaluated the impact of four blends of ß-ionone (50 and 100 µmol/L) and {gamma}-tocotrienol (7.5 and 15 {gamma}-tocotrienol/L) and Mo et al. (1998)Citation that of blends of ß-ionone (70 µmol/L) with {gamma}-tocotrienol (10 µmol/L) or {delta}-tocotrienol (5 µmol/L) on the proliferation of B16 melanoma cells. The combined data from the three studies record an observed response 19.6 ± 15.7 greater than the predicted response (paired t test, P = 0.0038). Synergy was not attained with binary blends consisting only of cyclic or acyclic isoprenoids. The observed response obtained with a blend of two cyclic isoprenoids, carvacrol and ß-ionone, of about equal potency [IC50 values, 120 and 150 µmol/L, respectively, (He et al. 1997Citation )] was 9.4 ± 3.4% less than the predicted response (n = 4, P < 0.015). Finally, the observed response obtained with blends of two acyclic isoprenoids, one a potent farnesyl analog and the second, a geranyl analog with lower tumor-suppressive potency [{gamma}-tocotrienol and geranyl tiglate, IC50 values, 20 and 38 µmol/L, respectively, (Elson and Mo, unpublished results)], was 20.0 ± 11.5% less than the predicted response (n = 4, P < 0.041). These preliminary findings suggest that the less-potent member of each binary blend consisting of either cyclic or acyclic isoprenoids attenuates the tumor-suppressive action of the more potent member. If farnesol proves to be the dominant posttranscriptional modulator of isoprenoid synthesis (Correll et al. 1994Citation ), the synergy realized with a blend of cyclic and acyclic isoprenoids can be traced, respectively, to the induction of a prenyl pyrophosphate pyrophosphatase activity (Case et al. 1995Citation , Meigs and Simoni 1997Citation ) and to the induction of cytosolic cysteine protease with specificity for HMG CoA reductase.

ß-Ionone (Yu et al. 1994Citation ) and {gamma}-tocotrienol (Parker et al. 1993Citation ) modestly lower serum cholesterol levels of animals fed a cholesterol-free diet. This stands in contrast to the marked impact these isoprenoids have on the growth of the B16 melanoma, a rigorous model for assessing the potency of pharmacological agents (Kuwashima et al. 1990Citation , Tsukamoto et al. 1991Citation ), when implanted in the flanks of mice (He et al. 1997Citation ). Comparisons of the relative sensitivities of normal and tumor cells (primary hepatocytes vs. HepG2 cells, Parker et al. 1993Citation ; CCD-18Co normal colon fibroblasts vs. Caco-2 colon adenocarcinoma cells,Table 1Citation ) show normal cells to be 3- to 40-fold less sensitive than tumor cells to isoprenoid-mediated effects on HMG-CoA reductase activity and growth. While resistant to sterol-feedback inhibition of transcription, the primary regulatory action controlling sterol synthesis, the elevated HMG-CoA reductase activity characteristic of tumors retains sensitivity to the secondary regulatory action of nonsterol factors that modulate translational efficiency and reductase degradation (Elson 1995Citation , 1996Citation , Mo et al. 1998Citation , and references therein).

Epidemiologic studies reveal a strong inverse association between frequency of intake of plant-derived foods and cancer incidence (Block et al. 1992Citation , Willett 1994Citation ). ß-Ionone, widely distributed in fruits and vegetables, and {gamma}-tocotrienol, widely present in cereals and vegetable oils of monocot origin, as well as many other phytochemicals exert broad and potent anticarcinogenic and antitumor activities when fed at pharmacological levels. Our studies with the murine B16 melanoma, a cell line relatively resistant when compared to human tumor cell lines to the isoprenoid actions, reveal additive and potentially synergistic growth-suppressive actions of ß-ionone, {gamma}-tocotrienol and other isoprenoids (He et al. 1997Citation , Mo et al. 1998Citation ). These findings support the concept that diet relevancy is found in the mass action of assorted isoprenoids (Bach 1995Citation ) and those of the other phytochemicals broadly distributed in plant products rather than in the singular action of a food or a phytochemical.


   FOOTNOTES
 
1 Supported by Public Health Service grant CA 73418 and the College of Agricultural and Life Sciences, University of Wisconsin. Back

2 Abbreviations used: ATCC, American Type Culture Collection; FBS, fetal bovine serum; HBSS, Hanks' Balanced Salt Solution; HMG-CoA, 3-hydroxy-3-methylglutaryl CoA; IC50, the concentration required to suppress the increase in the population of cells by 50%; MEM, minimum essential medium; TUNEL, deoxynucleotide transferase mediated dUTP nick end labeling. Back

Manuscript received July 16, 1998. Initial review completed August 19, 1998. Revision accepted December 21, 1998.


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 DISCUSSION
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