This article was downloaded by: [Aston University]On: 04 September 2014, At: 22:40Publisher: RoutledgeInforma Ltd Registered in England and Wales Registered Number: 1072954 Registered office: MortimerHouse, 37-41 Mortimer Street, London W1T 3JH, UK

Nutrition and CancerPublication details, including instructions for authors and subscription information:http://www.tandfonline.com/loi/hnuc20

RRR‐α‐tocopheryl succinate inhibits el4 thymiclymphoma cell growth by inducing apoptosis andDNA synthesis arrestWeiping Yu a , Bob G. Sanders a & Kimberly Kline ba Genetics Institute , University of Texas , at Austin, Austin, TX, 78712–1097b Div. of Nutritional Sciences/A2703, Genetics Institute , University of Texas , at Austin,Austin, TX, 78712–1097 Phone: 512–471–8911 Fax: 512–471–8911 E-mail:Published online: 04 Aug 2009.

To cite this article: Weiping Yu , Bob G. Sanders & Kimberly Kline (1997) RRR‐α‐tocopheryl succinate inhibits el4thymic lymphoma cell growth by inducing apoptosis and DNA synthesis arrest, Nutrition and Cancer, 27:1, 92-101, DOI:10.1080/01635589709514508

To link to this article: http://dx.doi.org/10.1080/01635589709514508

PLEASE SCROLL DOWN FOR ARTICLE

Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) containedin the publications on our platform. However, Taylor & Francis, our agents, and our licensors make norepresentations or warranties whatsoever as to the accuracy, completeness, or suitability for any purposeof the Content. Any opinions and views expressed in this publication are the opinions and views of theauthors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should notbe relied upon and should be independently verified with primary sources of information. Taylor and Francisshall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, andother liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relationto or arising out of the use of the Content.

This article may be used for research, teaching, and private study purposes. Any substantial or systematicreproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in anyform to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions

NUTRITION AND CANCER, 27(1), 92-101

Copyright © 1997, Lawrence Erlbaum Associates, Inc.

RRR-α-Tocopheryl Succinate Inhibits EL4 Thymic Lymphoma CellGrowth by Inducing Apoptosis and DNA Synthesis Arrest

Weiping Yu, Bob G. Sanders, and Kimberly Kline

Abstract: RRR-α-tocopheryl succinate (vitamin E succinate,VES) treatment ofmurine ElA T lymphoma cells induced thecells to undergo apoptosis. After 48 hours of VES treatmentat 20 μglml, 95% of cells were apoptotic. Evidence for theinduction of apoptosis by VES treatments is based on stain-ing of DNA for detection of chromatin condensation/frag-mentation, two-color flow-cytometric analyses of DNAcontent, and end-labeled DNA and electrophoretic analysesfor detection of DNA ladder formation. VES-treated ElAcells were blocked in the G1 cell cycle phase; however,apoptotic cells came from all cell cycle phases. Analyses ofmRNA expression of genes involved in apoptosis revealeddecreased c-myc and increased bcl-2, c-fos, and c-junmRNAs within three to six hours after treatment. Westernanalyses showed increased c-Jun, c-Fos, and Bcl-2 proteinlevels. Electrophoretic mobility shift assays showed in-creased AP-1 binding at 6, 12, and 24 hours after treatmentand decreased c-Myc binding after 12 and 24 hours of VEStreatment. Treatments of ElA cells with VES + RRR-α-to-copherol reduced apoptosis without effecting DNA synthesisarrest. Treatments of ELA cells with VES + rac-6-hydroxyl-2,5,7,8-tetramethyl-chroman-2-carboxylic acid, butylated hy-droxytoluene, or butylated hydroxyanisole had no effect onapoptosis or DNA synthesis arrest caused by VES treat-ments. Analyses of bcl-2, c-myc, c-jun, and c-fos mRNAlevels in cells receiving VES + RRR-α-tocopherol treatmentsshowed no change from cells receiving VES treatmentsalone, implying that these changes are correlated with VEStreatments but are not causal for apoptosis. However, treat-ments with VES + RRR-α-tocopherol decreased AP-1 bind-ing to consensus DNA oligomer, suggesting AP-1involvement in apoptosis induced by VES treatments.

Introduction

RRR-α-tocopheryl succinate (vitamin E succinate, VES)has been demonstrated to be a potent growth inhibitor ofvarious cancer cell types in vitro and in vivo (1-3; reviewedin Ref. 4). Tumor cells responsive to VES antiproliferative

effects include murine and human neuroblastoma cells (5-7),rat glioma cells (5), murine B-l6 melanoma cells (8), humanprostatic adenocarcinoma cells (2,9,10), avian lymphoidcells (1,11,12), human promyelocytic cells (13), hamsteroral buccal pouch epidermoid carcinoma cells (3), humanbreast cancer cells (2,14,15), human RL B lymphoma cells(15), and murine EL4 T lymphoma cells (16).

Although the mechanism(s) whereby VES inhibits theproliferation of rapidly dividing cells is not well understood,induction of cell cycle blockages, increased secretion andactivation of potent negative growth factors, namely, trans-forming growth factor-Ps (TGF-Ps), (2,12,14-17) and theirtype II cell surface receptors (15,16), and induction of apop-tosis (3,16,18; unpublished data) have been observed inVES-treated cells. Earlier studies using the EL4 cell modelindicated that VES has a dose-dependent biphasic action onEL4 cells, either enhancing cellular proliferation, in part, viaincreased secretion of the cytokine interleukin-2 or inhibit-ing cellular proliferation, in part, via the induction and secre-tion of biologically active TGF-p (17). Because VES is apotential chemopreventive and chemotherapeutic agent, it isof interest to ascertain the manner in which VES-treated cellsbecome growth inhibited and to try to gain a better under-standing of the molecular events involved. This study char-acterizes the ability of VES to induce murine EL4 lymphomacells to undergo apoptosis, addresses the involvement ofcertain apoptotic-related events in this process, and showsthat the VES-induced apoptotic process is different fromVES-induced DNA synthesis arrest.

Materials and Methods

Cell Culture

EL4.IL-2 (H-2b) thymoma cells (19), a dimethylbenzan-thracene-induced thymic lymphoma cell line derived from aC57BL/6 mouse (no. TIB 181, American Type Culture Col-lection, Rockville, MD), were maintained at 37°C in 5%CO2-95% air by culturing in Dulbecco's modified Eagle'smedium with high glucose (with 4.5 g/ml glucose; HyClone,

The authors are affiliated with the Genetics Institute, University of Texas at Austin, Austin, TX 78712-1097. K. Kline is also affiliated with the Divisionof Nutrition, University of Texas at Austin, Austin, TX 78712-1097.

Dow

nloa

ded

by [

Ast

on U

nive

rsity

] at

22:

40 0

4 Se

ptem

ber

2014

Logan, UT) supplemented with penicillin (100 IU/ml; Hy-Clone), streptomycin (100 ug/ml; HyClone), 2 mM L-glu-tamine (Sigma Chemical, St. Louis, MO), and 10% calf serum(HyClone). Cultures were routinely examined to verify ab-sence of mycoplasma contamination, and cell viability wasdetermined by trypan blue dye exclusion analyses. EL4 cellsat 5 x 105 cells/ml in T-75 flasks were used for analyses ofmRNA, protein, DNA binding, and apoptosis; cells at 1 x107ml cultured in 96-well microtiter plates (2 x 104

cells/well) were used for DNA synthesis assays. Cells wereincubated for different time periods in the presence of 1) VES(Sigma Chemical) at 20 ug/ml [VES was dissolved in abso-lute ethanol and diluted in RPMI-1640 containing 5% fetalbovine serum, penicillin (100 IU/ml), streptomycin (100ug/ml), and 2 mM L-glutamine to a final concn of 20 ug/mlVES and 0.2% (vol/vol) ethanol], 2) succinic acid and ethanolequivalents referred to as vehicle (VEH) control, 3) mediacontaining no treatments, referred to as untreated (UT) con-trols, or 4) VES + RRR-cc-tocopherol (200 ug/ml; EastmanKodak, Rochester, NY), rac-6-hydroxyl-2,5,7,8-tetramethyl-chroman-2-carboxylic acid (Trolox, 100 ng/ml; Hoffmann-La Roche, Nutley, NJ), butylated hydroxytoluene (BHT, 10|iM; Sigma Chemical), or butylated hydroxyanisole (BHA,10 uM; Sigma Chemical).

Apoptosis Measured by DAPI

Treated EL4 cells were pelleted and washed once withphosphate-buffered saline (PBS; 137 mM NaCl, 3 mM KC1,5 mM Na2HPO4, and 1 mM KH2PO4, pH 7.4) and stainedwith 2 ug/ml DAPI (4\6-diamidine-2'-phenylindole dihy-drochloride; Boehringer Mannheim, Indianapolis, IN) in100% methanol for 15 minutes at 37°C. Cells were viewedusing a Zeiss ICM 405 microscope with ultraviolet (UV)excitation and a 487701 filter with a x40 objective/xlO eye-piece and photographed using a Zeiss M35 camera and Ko-dak Tri-X pan 400 film. Cells with nuclei that containedclearly condensed chromatic or cells with fragmented nucleiwere scored as apoptotic.

Apoptosis Measured by Flow Cytometry

EL4 cells at 1 x 107 were pelleted and washed twice withPBS without calcium and magnesium (pH 7.4) containing0.1% glucose and fixed with cold 95% ethanol at 4°C forsix hours. Fixed cells were washed once in PBS and stainedwith 15 ng/ml propidium iodide (Sigma Chemical) and 1mg/ml of deoxyribonuclease-free ribonuclease A (SigmaChemical) in PBS (pH 7.4) overnight at 4°C. DNA contentwas determined using a Coulter Epics Elite Flow Cytometer(Coulter Electronics, Hialeah, FL) with an argon laser settingof 488 nm. Cell size was measured simultaneously, and datawere analyzed using the Coulter Multicycle Program. Cellswith a DNA content of <2N were designated as a pre-Glapoptotic peak. Two-color flow-cytometric analyses were

conducted using TUNEL and propidium iodide-labeling pro-cedures, as described elsewhere (20).

Apoptosis Measured by Laddering of DNA

DNA preparations from 1 x 107 EL4 cells, after twodays of treatment, were isolated following the procedures ofNicoletti and co-workers (21). DNA concentrations weredetermined by absorbency at 260 nm with use of a spectro-photometer (Beckman DU-64). DNA (20 ug) was mixed withloading buffer (0.25% bromphenol blue, 0.25% xylene cy-anol FF, 30% glycerol), before it was loaded into the wells ofa 1% agarose gel containing 0.5 fig/ml ethidium bromide.Electrophoresis was carried out in TAE buffer [40 mM tris-(hydroxymethyl)aminomethane(Tris)-acetate, 1 mMEDTA] at60 V for two hours. Gels were photographed under UV lightwith a Polaroid model MP4+ instant camera and Polaroid 667print film (with a 5.6 aperture and a 4 or 8 speed setting).

Cellular RNA Preparation

The expression and size of bcl-2, c-myc, c-fos, and c-junmRNAs were analyzed by conventional Northern blot analy-ses, and mRNA levels were quantitated by scanning theautoradiograms with a densitometer. Total cellular RNAfrom 2 x 107 EL4 cells was isolated using the method ofManiatis and colleagues (22). The RNA pellets were airdried and washed four times in a total volume of 400 ul byfirst suspending the pellet in 100 ul of TE (10 mM Tris, 1mM EDTA, pH 7.4), adding 24 p.] of 4 M NaCl and 34 u.1of TE containing 0.5% sodium dodacyl sulfate (SDS), thenprecipitating the RNA by adding 1 ml of cold absolute etha-nol and incubating at -70°C for one hour. The resultingRNA pellets were washed once with 70% ethanol, and sam-ples were analyzed by agarose gel electrophoresis or storedat -70°C before electrophoretic analysis.

RNA Electrophoresis and Capillary Transfer

RNA was separated by electrophoresis under denaturingconditions on 1% agarose gels [wt/vol; diluent: 500 mM3-(N-morpholino)ethanesulfonic acid, 125 mM sodium ace-tate, 25 mM EDTA, pH 7.0, containing 0.68 M formalde-hyde]. RNA samples (30 u.g/lane) were dissolved in 20 u.1 ofsample buffer, placed in 5 u.1 of loading buffer (50% glycerol,0.1 % bromphenol blue, and 0.5 ug/ml ethidium bromide), andheated at 65°C for 20 minutes. After electrophoresis for 18hours at 40 V, gels were photographed under UV light todocument migration of the 28S and 18S bands. The 28S and18S migration position, as well as RNA ladders (GIBCO,molecular weight standards), were used to calculate messagesizes. Gels were soaked in 50 mM NaOH for 15 minutes andneutralized twice for 10 minutes in lOx SSC (1.5 M sodiumchloride and 0.15 M sodium citrate, pH 7.0); the RNA wastransferred by capillary action onto Nytran filters (0.45 urn;Schleicher & Schuell, Keene, NH) overnight and immobi-

Vol. 27, No. 1 93

Dow

nloa

ded

by [

Ast

on U

nive

rsity

] at

22:

40 0

4 Se

ptem

ber

2014

lized by UV cross-linking following the manufacturer's in-structions (UV Stratalinker 1800, Stratagene, La Jolla, CA).

Northern Blots

The probes for bcl-2, c-myc, c-jun, c-fos, and 18S RNAwere generous gifts from Dr. Stanley Korsmeyer (HowardHughes Medical Institute and Depts. of Medicine and Pathol-ogy, Washington University School of Medicine, St. Louis,MO), Dr. Kenneth Marcu via Dr. Michael Weller (Dept. ofInternal Medicine, Section of Clinical Immunology, Univer-sity of Zurich School of Medicine, Zurich, Switzerland), Dr.Andrew P. Butler (M. D. Anderson-Science Park ResearchDiv., Smithville, TX), Dr. Paul Gottlieb (Dept. of Microbiol-ogy, The University of Texas, Austin, TX), and Dr. FrankRuscetti (Frederick Cancer Center, Frederick, MD), respec-tively. The cDNA for mouse bcl-2 is an 841-bp fragmentinserted in a pBluescript IIKS vector (23). A 700-bp fragmentof this original bcl-2 cDNA was excised with the BspMIrestriction enzyme for use in these studies. The cDNA formurine c-myc is a 460-bp fragment of exon 2 inserted in apT3T7 vector, which is excised with the Pst I restrictionenzyme (24). The cDNA for murine c-jun is a 1.8-kb fragmentligated into a pGEM-3Z vector, which is excised as an 0.89-kbfragment with EcoRI and Smal (25). The cDNA for rat c-fosis a 2.1-kb fragment ligated into a pSP 65 vector, which isexcised with EcoRI (26). The 18S probe, which was used forverification of equal loading of RNA, is a 2.87-kb EcoRIlinearized partial cDNA of eukaryotic origin. A randomprimer DNA labeling kit (DECA primer II, Ambion, Austin,TX) was used to label 50 ng of each purified probe with[<x-32P]-dCTP (NEN/Dupont, Wilmington, DE). RNA blotswere prehybridized in hybridization solution (10% dextransulfate, 1 M NaCl, 1% SDS, 10 ug/ml denatured salmonsperm DNA) at 56°C in a hybridization chamber (model 310,Robbins Scientific, Sunnyvale, CA) for one hour, labeledprobes (cpm « 1 x lOVml) were added to the hybridizationsolution, and the hybridization was allowed to continueovernight. Blots were washed in 2x SSC containing 1% SDSonce at 56°C and twice at 65°C for one hour. Autoradiogramswere produced by exposure of blots to Kodak BioMax MRfilm (Eastman Kodak) at -80°C with intensifying screens.Results were quantitated by densitometric analyses.

Western Immunoblotting

Nuclear extracts were prepared for c-Fos and c-Jun pro-tein analyses, as described by Goldstone and Lavin (27).Protein from whole cell extracts (2 x 107 cells) were preparedfor Bcl-2 analyses by resuspending cell pellets in 400 |xl oflysis buffer (150 mM NaCl, 1% NP-40,0.5% deoxycholate,0.1% SDS, and 50 mM Tris pH 7.4) on ice for 30 minuteswith periodic vortexing. The resulting preparations werecentrifuged for 10 minutes at 4°C, aliquoted, and stored at-80°C until used. Forty micrograms of protein from nuclearextracts and 140 u.g of protein from whole cell extracts weremixed with an equal volume of 2x Laemmli reducing sample

buffer, boiled for five minutes, fractionated by one-dimen-sional SDS-polyacrylamide gel electrophoresis, and trans-ferred onto a nitrocellulose membrane (no. 68390,Schleicher & Schuell) (28). Membranes were then blockedwith 5% dry milk in 25 mM TrisHCl, pH 8.0, containing12.5 mM NaCl and 0.05% Tween-20 (TBST, blocking buff-er), reacted with primary antibody [rabbit polyclonal anti-human Bcl-2, rabbit polyclonal antimouse c-Jun, and rabbitpolyclonal anti-human c-Fos (catalog no. SC-492, SC-45,and SC-52, respectively, Santa Cruz Biotechnology, SantaCruz, CA)] in blocking buffer at 1 ug/ml, respectively, forone hour at room temperature. Membranes were washedfour times, 10 minutes each, in TBST buffer. Horseradishperoxidase-conjugated secondary antibody [goat anti-rabbitimmunoglobulin G (H + L), no. 111-035-003, Jackson Im-munoresearch Laboratory, West Grove, PA] was diluted to1:2,000 for Bcl-2 and 1:1,000 for c-Fos and c-Jun detectionin blocking buffer and allowed to react for one hour at roomtemperature. After four 10-minute washes in TBST, themembranes were overlaid with a 1:1 mixture of enhancedchemiluminesence reagent (Amersham ECL detection kitRPN 2106) following the manufacturer's instructions. Ko-dak BioMax MR film was exposed for various time intervals(30 sees to 10 mins) before film development.

Electrophoretic Mobflity Shift Assays

Electrophoretic mobility shift assays (EMSAs) were con-ducted by incubating 10 u.g of nuclear extracts with theAP-1 consensus sequence (TGACTCA) and mutant se-quence (TGACTTG) and the c-Myc consensus sequence(CACGTG) and mutant sequence (CACGGA) (Santa CruzBiotechnology). The consensus oligomer was end-labeledwith [y-32P]ATP (NEN/Dupont, Wilmington, DE; 3,000-5,000 Ci/mmol) by use of the T4 polynucleotide kinase(Promega, Madison, WI) or used unlabeled to inhibit thereaction as a control for specificity. Unlabeled mutant se-quences also served as controls for determining the speci-ficity of banding patterns. The reaction mixture [4 u.1 bindingbuffer, 1 u.g poly(dI-dC)-poly(dI-dC), 1 ul labeled oligo, 10Hg protein in 20 \i\ volume] was incubated for 20 minutesat room temperature, then loaded onto a one-dimensionalvertical 5% polyacrylamide-0.25x TBE (22.2 mM Tris-bo-rate and 0.25 mM EDTA) gel and electrophoresed for twohours at 150 V. After electrophoresis, gels were dried andautoradiographed using Kodak BioMax MR film.

Results

VES Treatment of Murine EL4 T Lymphoma CellsReduces [3H]thymidine Uptake and Induces Apoptosis

VES treatment of EL4 cells inhibits [3H]thymidine uptakein a dose-dependent manner. Cells treated for 24 hours with10 or 20 ug/ml VES exhibit 51% and 58% reduction, re-spectively, in [3H]thymidine uptake compared with UT or

94 Nutrition and Cancer 1997

Dow

nloa

ded

by [

Ast

on U

nive

rsity

] at

22:

40 0

4 Se

ptem

ber

2014

55% and 61% reduction, respectively, in [3H]thymidine up-take compared with VEH cells (data not shown).

In an effort to address possible outcomes of DNA syn-thesis arrest (senescence or apoptosis) caused by VES treat-ments, cells were examined for apoptotic characteristics.Apoptosis was documented by characterizing morphologicaland structural changes that are characteristic of this form ofprogrammed cell death, including chromatin condensation,chromatin crescent formation/margination, and fragmenta-tion by fluorescence microscopy of DAPI-labeled cells,DNA fragmentation by fluorescence-activated cell sorteranalyses of propidium iodide-labeled cells showing a pre-Glpeak, DNA fragmentation (i.e., DNA ladder) by electropho-resis of DNA on agarose gels, and DNA fragmentation bya modified TUNEL (TdT-mediated deoxyuridine triphos-phate-biotin nick-end labeling) assay.

Nuclear fragmentation was seen in EL4 cells treated withVES (20 ng/ml) for 36 hours when the cells were stained withthe DNA dye DAPI (Figure IB). For comparison, DAPI-stained untreated cells are presented in Figure 1A. Whereascontrol cells show a chromatin staining pattern typical ofuntreated EL4 cells, VES-treated cells show fragmentednuclei. Quantitation of the percentage of apoptotic cells wasdetermined by counting the number of DAPI-labeled cellsexhibiting nuclear fragmentation vs. intact chromatin stainingand showed that VES treatments for 24, 36, and 48 hoursproduced 13%, 75%, and 95% apoptotic cells, respectively.Figure 1C is a photograph of ethidium bromide-stained DNAafter electrophoresis in a 1% agarose gel. The DNA profilesof EL4 cells cultured in the presence of VEH or VES or UTfor 48 hours are depicted. DNA from cells treated with VESexhibit a laddering of 180-bp multimers, a profile typical ofapoptotic cells. In comparison, UT and VEH-treated cellsexhibit high-molecular-weight DNA only.

Figure 2, A-C, shows histograms obtained from theanalyses of EL4 cells treated for 24 hours with VEH or VESor UT, then stained with propidium iodide and analyzed byflow cytometry. UT and VEH control cells exhibit typicalcell cycle profiles for rapidly dividing EL4 cells with ap-proximately 46-55% of cells in Gl, 37-45% in S, and 8-10% in G2/M cell cycle phases. VES-treated cells exhibita pre-Gl apoptotic cell peak, as detected by multicycleanalyses. The cell distribution profile, excluding non-pre-Glcells, showed 59% of cells in Gl, 19% in S, and 22% inG2/M cell cycle phases. Figure 2D is a composite profileobtained by overlaying the three cell treatment profiles forcomparative purposes.

Figure 3, A and C, depicts flow-cytometric scattergramsfor EL4 cells dual labeled with propidium iodide for deter-mination of total DNA content of cells and labeled withFITC for determination of DNA fragmentation with use ofa modified TUNEL procedure (see Materials and Meth-ods). Figure 3, B and D, also depicts flow-cytometric his-tograms of TUNEL fluorescein isothiocyanate (FITC)staining vs. cell number for determination of percentage ofcells showing a high degree of FITC staining, namely, de-

UT VEH VES

Figure 1. Analyses of DNA fragmentation by fluorescent microscopy andelectrophoresis. EL4 cells at 5 x 105/ml were treated with RRR-cc-tocoph-eryl succinate (VES, 20 ng/ml, B) or cultured untreated (A) for 36 hrs.DNA was labeled with fluorescent DNA-binding dye 4',6-diaminidine-2'-phenylindole dihydrochloride, and cells were examined. Photographsrepresent cells viewed using a Zeiss fluorescent microscope (x40 objective,xlO eyepiece) and photographed using a Zeiss M 35 camera and KodakTri-X Pan 400 film. Ethidium bromide staining after electrophoresis ofDNA samples from EL4 cells treated with VES for 48 hrs provides evidenceof DNA fragmentation (DNA laddering). Only high-molecular-weight DNAis seen in cells treated with vehicle (VEH) or untreated (UT) for 48 hrs(C). Data are representative of 3 independently conducted experiments.

termination of percentage of cells that have undergone apop-tosis. These analyses, conducted on cells treated for 24 hourswith VES (Figure 3, C and D) or VEH (Figure 3, A and

Vol. 27, No. 1 95

Dow

nloa

ded

by [

Ast

on U

nive

rsity

] at

22:

40 0

4 Se

ptem

ber

2014

|

<S

1,000B

ApoptoticCells

DNA Content

Figure 2. Histograms of flow-cytometric analyses of propidium iodide-stained EL4 cells treated for 24 hrs with media only (UT, A), vehiclecontrol (VEH, B), or VES (20 |xg/ml, C). D: composite profile obtainedby overlaying 3 cell treatment profiles for comparative purposes. Horizontaland vertical axes, log fluorescence intensity and relative cell numbers,respectively. Subdiploid peak at left of Gl labeled "apoptotic cells" resultsfrom cells containing fragmented DNA. Diploid DNA content is designatedGl, and 4N DNA content is designated G2/M. Data are representative of3 independently conducted experiments.

B), show that apoptotic cells in the VES-treated cell popu-lation come from all three cell cycle phases, with most ofthe cells from the Gl phase of the cell cycle. The VEHcontrol exhibited 7% apoptosis (Figure 3B), whereas 22%of VES-treated cells exhibit FITC-staining characteristic ofapoptotic cells (Figure 3D).

EL4 Cells Treated With VES Exhibit Elevated bcl-2,c-fos, and c-jun mRNA and Reduced c-myc mRNA

Northern blot analyses revealed that EL4 cells exhibit a7.5-kb bcl-2 mRNA transcript and that VES-treated cellsshow higher levels of the bcl-2 mRNA transcript than UTor VEH-treated cells at 6, 12, and 24 hours after treatment(Figure 4A; note: levels of specific mRNAs/lane after ad-justment for load differences by densitometric analyses ofmRNAs for each of the specific mRNAs and 18S RNA aredepicted in bar graphs).

Northern blot analyses show that c-fos is constitutivelyexpressed at low levels by UT and VEH-treated EL4 cellsand that VES treatment increases the level of a 2.3-kbmRNA transcript at 3, 6, 12, and 24 hours after treatment

1,000

DNA Content FITC

1,000! 1,000

DNA Content FITC

Figure 3. Scattergrams of two-color flow cytometry conducted on EL4cells treated for 24 hrs with vehicle (VEH, A) or VES (20 ug/ml, C). Amodified end-labeling TUNEL procedure and propidium iodide labelingwere conducted as described in Materials and Methods. x-Axis, propidiumiodide labeling of DNA content, y-axis, fluorescein isothiocyanate (FITC)labeling of fragmented DNA. VES-treated cells in all phases of cell cycleexhibit fragmented DNA. B and D, percentage of apoptotic cells after VEHand VES treatment, respectively. Data are representative of 3 independentlyconducted experiments.

(Figure 4B). Levels of a 2.5-kb c-jun mRNA transcript wereincreased over UT and VEH controls at 6, 12, and 24 hoursafter VES treatment (Figure 4C). Northern blot analysesshowed that c-myc mRNA was expressed by UT and VEH-treated EL4 cells as a single 2.3-kb transcript (Figure 4D).Levels of c-myc mRNA from VES-treated cells at all timepoints were decreased below control levels (Figure 4D).

Effects of VES Treatments on Levels of c-Jun, c-Fos,and Bcl-2 Proteins

Western analyses of nuclear or cellular extracts fromVES-treated EL4 cells showed elevated levels of c-Jun, c-Fos, and Bcl-2 compared with VEH controls (Figure 5).Densitometric analyses of protein levels in preparations fromVES-treated cells (as percentage of protein levels in VESpreparations compared with protein preparations obtainedfrom VEH-treated cells) for the 6-, 12-, and 24-hour analysesfor c-Jun, c-Fos, and Bcl-2 revealed the following. c-Junprotein levels were elevated 6.1%, 35%, and 127%, respec-tively. c-Fos levels at 6 and 24 hours after treatment wereelevated 4.3% and 39.3%, respectively. The c-Fos levels at12 hours of treatment were elevated 103%; however, thishigh level is a reflection of lane load differences in the VEHcontrol. Bcl-2 protein levels were elevated 14.2%, 9.6%,and 26.1%, respectively. c-Myc protein levels were not de-tectable in the EL4 cells when assayed with antibodies toc-Myc that were obtained from two different commercialsources, even though these reagents were capable of detect-ing c-Myc in human and avian cell lines when assayed inour laboratory.

96 Nutrition and Cancer 1997

Dow

nloa

ded

by [

Ast

on U

nive

rsity

] at

22:

40 0

4 Se

ptem

ber

2014

«t- •»••; -bcl-2(7.5 Kb)

3-,2.5-

2-1.5-

1-0.5

0I

ZM.i 1

12

24

- c-fos' • (2.3 Kb)

124

"IB -c-jun,5 (2.5 Kb)

i12 24

j i * yg : i -c-myc(2.3 Kb)

I I I = U

3 6 12 24

Time (hours)

II = VEH

Effects of VES Treatments on AP-1 and c-Myc DNABinding Activity

EMSAs were conducted to determine whether VES treat-ments of EL4 cells for various time periods modulated DNAbinding to AP-1 or c-myc consensus sequences (Figure 6,A and B). EMSA showed that nuclear extracts from VES-treated cells exhibited increased binding activity for the AP-1 consensus sequence compared with the binding of nuclearextracts from VEH controls that were run in parallel foreach time point for the 6-, 12-, and 24-hour time points.c-Myc binding activity was decreased at 12 and 24 hoursafter VES treatment initiation compared with binding activ-ity exhibited by nuclear extracts from VEH-treated cells.Specificity of consensus oligomer binding was confirmed

6h 12h 24h J2

VEH VES VEH VES VEH VES 5

AP-1

B12h 24h

oVEH VES VEH VES 5

C-Myc

Figure 4. Expression and levels of bcl-2, c-fos, c-jun, and c-myc mRNAs

from total cellular RNA preparations from EL4 cells were determined by

Northern blot analyses. Expression of bcl-2, c-fos, c-jun, and c-myc mRNAs

in UT, VEH, and VES EL4 cells are shown after 3, 6, 12, and 24 hrs of

treatments. Levels, adjusted for lane load differences calculated from

mRNA levels of 18S RNA and bcl-2, c-myc, c-fos, and c-jun mRNAs, are

depicted by bar graphs. Data are representative of 4 independently

conducted experiments.

12

Figure 5. Western immunoblotting analyses of c-Jun, c-Fos, and Bcl-2

protein levels in VES- and VEH-treated EL4 cells after 6, 12, and 24 hrs

of treatments. Data are representative of 3 independently conducted

experiments.

AP-1

COLU>

Figure 6. Electrophoretic mobility shift assays (EMSAs) for AP-1

(Fos/Jun) DNA binding activities (A) and c-Myc binding activities (B) for

nuclear extracts prepared from EL4 cells receiving vehicle (VEH) or 20

Hg/ml VES treatments for various time periods (6, 12, and 24 hrs for AP-1;

12 and 24 hrs for c-Myc). Specificity of binding was determined by

competition assays with use of 100-fold excess of unlabeled mutant AP-1

or c-Myc oligomer probes (mutant) or 100-fold excess of unlabeled

consensus oligomer probes (blocked). C: effects of RRR-α-tocopherol

(daT) alone and in combination with VES on AP-1 binding to consensus

DNA oligomer. Data are representative of 3 independently conducted

experiments.

Vol. 27, No. 1 97

Dow

nloa

ded

by [

Ast

on U

nive

rsity

] at

22:

40 0

4 Se

ptem

ber

2014

by the use of "cold"-nonradiolabeled wild-type (lane labeled"blocked") or mutant oligomers for each of the two consen-sus oligomers. EMSA data showing elevated AP-1 and de-creased c-Myc binding to consensus sequences are inagreement with the mRNA and protein data (AP-1 only)depicted in Figures 4 and 5.

Investigation of Potential Inhibitors of VES-InducedDNA Synthesis Arrest and/or Apoptosis

To determine the events involved in VES-induced DNAsynthesis arrest or apoptosis, a number of agents of knownbiochemical activity were examined for the capacity to inter-fere with the DNA synthesis arrest or the apoptotic processes.Cycloheximide (inhibitor of protein synthesis) and actinomy-cin D (inhibitor of RNA transcription) induced apoptosis inEL4 cells and therefore could not be used to ascertain whetherVES-induced DNA synthesis arrest or apoptosis requiresactive transcription or translation. Treatment of EL4 cellswith VES (20 ng/ml) + RRR-α-tocopherol (200 ug/ml; alipid-soluble antioxidant) reduced the percentage of apoptoticcells from 60% (VES alone) to 17% (VES + RRR-α-tocoph-erol); DNA synthesis was not affected (Table 1). Treatmentof EL4 cells with RRR-α-tocopherol alone did not induceapoptosis and had no effect on DNA synthesis. Trolox, BHT,and BHA administered separately or in combination with

Table 1. Modulation of VES-Induced Apoptosis but NotVES-Suppressed DNA Synthesis by Treatments of EL4Cells With VES + RRR-α-Tocopherol"

Treatments''

UT

VESRRR-α-tocopherolRRR-α-tocopherol + VES

TroloxTrolox + VES

BHT

BHT + VESBHA

BHA + VES

DNA Synthesis*

[3H]TdR uptake,cpm x 103

99.6 + 2.55.4+ 1.1

99.0 ± 2.2

6.8 + 6.2

106.9 + 4.13.2 ± 1.0

93.2 + 9.4

2.0 + 0.387.1 ± 9 . 3

6.8 ± 0.4

Suppression,%

950

93

097

6

9813

93

Apoptosis/%

0

600

170

554

652

67

a: Abbreviations are as follows: VES, RRR-α-tocopheryl succinate(vitamin E succinate); TdR, thymidine; UT, untreated; Trolox,rac-6-hydroxyl-2,5,7,8-tetramethyl-chroman-2-carboxylic acid; BHT,butylated hydroxytoluene; BHA, butylated hydroxyanisole.

b: Values are means ± SD of triplicate replicas. Percent DNA synthesisinhibition was determined by comparing [3H]TdR (cpm) uptakebetween UT and treated groups.

c: Percent apoptosis was determined by counting no. of apoptotic cellson the basis of morphological criteria of 4',6-diaminidine-2'-phenylin-dole dihydrochloride (DAPI)-stained cells vs. nonapoptotic cells forrespective treatment groups. Data represent average of 2 independentexperiments.

d: EL4 cells in 6-well plates (2 x 106 cells/well) were treated with VES(20 ug/ml), RRR-α-tocopherol (100 ug/ml), Trolox (100 ug/ml), BHT(10 uM), or BHA (10 uM) or treated at these levels of each antioxidant+ VES for 36 hrs. UT cells were cultured with media for 36 hrs.

c-jun

1.5V

m m °-fos

1.5-

1-

.5-

0-

1.5-.

1-

.5-

0

bcl-2

m : c-myc

1.5-j

1-

.5-

0

Figure 7. Expression and levels of c-jun, c-fos, bcl-2, and c-myc mRNAsfrom total cellular RNA preparations from EL4 cells treated for 24 hrsdetermined by Northern blot analyses. Lane 1, RRR-α-tocopherol (daT,200 ug/ml) + VES (20 ug/ml); Lane 2, RRR-α-tocopherol (daT; 200ug/ml); Lane 3, VES (20 ug/ml); Lane 4, UT; Lane 5, VES ( 20 ug/ml).Levels of respective mRNAs, adjusted for lane load differences calculatedfrom mRNA levels of specific mRNAs and 18S RNA, are depicted by bargraphs. Data are representative of 3 independently conducted experiments.

VES had little effect on VES-induced apoptosis or VES-in-duced DNA synthesis arrest. Although treatments with VES+ RRR-α-tocopherol reduced the percentage of cells under-going apoptosis compared with VES treatments alone, VES+ RRR-α-tocopherol did not influence the expression ofc-jun, c-fos, bcl-2, or c-myc mRNAs compared with VEStreatments alone (Figure 7). However, transcription factorbinding to AP-1 consensus DNA was reduced after treatmentswith VES + RRR-α-tocopherol (Figure 6C). Treatments with

98 Nutrition and Cancer 1997

Dow

nloa

ded

by [

Ast

on U

nive

rsity

] at

22:

40 0

4 Se

ptem

ber

2014

VES+RRR-α-tocopherol did not affect the binding of c-Mycto consensus DNA oligomers (data not shown).

Discussion

The antiproliferative actions of VES are complex andmay be due, in part, to the ability of VES to induce autocrineacting biologically active TGF-ps (2,12,14,15,17). However,growth-inhibitory actions, characterized by DNA synthesisarrest as well as apoptosis, by VES can be TGF-P inde-pendent, as was demonstrated by VES-induced DNA syn-thesis arrest and apoptosis of TGF-P-nonresponsive humanprostate cells (10; unpublished data). Certain metabolitesand derivatives of the other three fat-soluble vitamins, A,D, and K, are also capable of inhibiting the growth of tumorcells (29-32). Investigations of the mechanisms of actionof VES, along with certain derivatives of vitamins A, D,and K, in the regulation of tumor cell growth represent anongoing research area with potential clinical applications. Itis important to note that analyses of chemopreventive agents(retinoids, carotenoids, and tocopherols) in animal modelshave indicated common mechanisms for growth-inhibitoryeffects. These mechanisms include an increase in programmedcell death (33,34).

This study reports on VES-induced apoptosis in murineT-helper EL-4 cells. The VES-treated EL-4 cells exhibitedclassical apoptotic characteristics, including nuclear DNAcondensation and DNA fragmentation yielding a charac-teristic ladder pattern indicating internucleosomal cleavage.Approximately 95% of cells exhibited apoptotic charac-teristics within two days of treatment initiation. Cell cycleanalyses showed VES-treated EL-4 cells to be blocked inthe Gl cell cycle stage, and cell cycle analyses of propidiumiodide and FITC-labeled cells showed that a Gl block wasnot necessary for VES-induced apoptosis, because apoptoticcells were derived from all phases of the cell cycle.

This study also shows that treatment of EL-4 cells for36 hours with VES + RRR-α-tocopherol reduced the levelsof apoptosis induced by VES treatment alone from 60% to17% but had no effect on VES-induced DNA synthesis ar-rest. This blockage of apoptosis by RRR-α-tocopherol, awell-known Iipid-soluble antioxidant, suggests that theapoptotic-inducing properties of VES treatments may be dueto prooxidant effects. VES is a derivative of natural vitaminE (RRR-α-tocopherol), containing a succinic acid moietyattached via an ester linkage to the chroman head structureof RRR-α-tocopherol, a modification that eliminates the hy-droxyl moiety that mediates RRR-α-tocopheroI's classicalantioxidant properties. Thus the intact VES compound doesnot function as an antioxidant. Only in situations where theesterification is reversed and RRR-α-tocopherol is "freed"will there be Iipid-soluble antioxidant activity associatedwith this compound. For example, studies show that VES,on removal of the succinate moiety by esterase activity, isan extremely effective antioxidant (35). Because many celltypes have active esterases that can convert VES to RRR-a-tocopherol and succinic acid, it has been unclear whether

it is the intact VES compound that is the biologically activeagent. Recent work by Fariss and co-workers (36) directlyestablished the antiproliferative effects of the intact VEScompound. They prepared vitamin E succinate with a non-hydrolyzable ether linkage and showed it to selectively in-hibit leukemia cell proliferation in a manner identical toVES (36). Thus, if VES remains intact and competes withand displaces RRR-α-tocopherol, it may be possible thatVES treatments reduce the cellular content of RRR-α-to-copherol, the major lipid membrane-associated antioxidant,and thereby increase the net generation of reactive oxygenspecies. To test this possibility, EL-4 cells were treated withVES plus three well-known antioxidants (Trolox, BHT, andBHA). The finding that none of these antioxidants modu-lated VES-induced apoptosis raises the possibility that RRR-a-tocopherol may be blocking VES-induced apoptosis viasome other mechanism. Preliminary experiments designedto determine increases in free radicals or free radical-gen-erated DNA damage also do not support the hypothesis thatVES is acting as a prooxidant.

In an effort to address possible components in the VES-initiated apoptotic signaling cascade, the expression of fourapoptosis-associated genes was examined: bcl-2, c-jun, c-fos, and c-myc. These studies were limited to examinationof mRNA-, protein-, and DNA-binding activity; other im-portant gene family members and status of phosphorylatedstates were not addressed. The investigation of apoptoticgenes and their products in VES-treated EL-4 cells permitsseveral conclusions to be reached and provides clues forfuture studies.

Because the bcl-2 gene encodes a protein that is involvedin the prevention of apoptosis (37) and because VES causedapoptosis, despite increased Bcl-2 message and protein ex-pression, the studies reported here suggest that Bcl-2 doesnot mediate VES-induced apoptosis in this cell type. Thesestudies also suggest that VES is not inducing apoptosis viathe induction of biologically active TGF-P, because TGF-P-induced apoptosis in EL4 cells does not affect the steady-state levels of bcl-2 mRNA (38).

c-Myc, a nuclear DNA-binding phosphoprotein, func-tions as a transcriptional regulator that helps control cellproliferation, differentiation, and apoptosis (39). Because adecrease in c-myc mRNA and c-Myc binding to consensusDNA sequence was observed in VES-treated EL4 cells, thereis the possibility that reduced c-Myc levels and activity maybe involved in VES-induced apoptosis. However, the obser-vations that treatments of EL4 cells with VES + RRR-α-to-copheryl, where VES-induced apoptosis is blocked, did notaffect the message levels or DNA binding activity of c-Mycreduce the possibility that c-myc is playing a causal role inVES-induced apoptosis in EL-4 cells. Prasad and co-workers(40) reported a correlation between RRR-α-tocopheryl suc-cinate-induced growth inhibition and the reduced expressionof c-myc-, N-myc-, and H-ras-specific mRNAs in murineneuroblastoma cells (NBP2) in culture. Perhaps reduced c-Myc activity in VES-treated EL-4 cells is playing a role inVES-induced DNA synthesis arrest.

Vol. 27, No. 1 99

Dow

nloa

ded

by [

Ast

on U

nive

rsity

] at

22:

40 0

4 Se

ptem

ber

2014

The c-fos and c-jun genes, the products of which arecomponents of the immediate-early response also called pri-mary response transcription factor, AP-1, play a regulatoryrole in cellular responses to phorbol esters, UV radiation,tumor necrosis factor-a, and serum growth factors (41). TheAP-1-binding site is recognized by Jun family member ho-modimers and Jun family member/Fos family member het-erodimers, and the balance between Jun and Fos is criticalto the outcome (induction vs. repression) of gene expression(42). VES induction of c-fos and c-jun mRNA and proteinexpression, as well as enhanced AP-1 DNA-binding activity,suggests that VES may be inducing apoptosis via modulationof AP-1-binding activities. This possibility is strengthened,in that nuclear proteins from cells receiving treatments ofVES + RIlR-α-tocopherol exhibit reduced apoptosis andreduced AP-1 binding. Further support for AP-1 involve-ment comes from other studies showing marked increasesin c-Jun in other VES-treated cells (18) and reduction ofapoptosis in avian lymphoid cells expressing a dominantnegative mutant of c-Jun (unpublished results). The role ofelevated c-Jun levels in induction of apoptosis is not under-stood but is considered to be a critical event in growthfactor-deprived induced apoptosis of nerve cells (43).

Although the mechanism(s) involved in VES-inducedapoptosis is unknown, the ability of a compound to induce95% apoptosis in a cancer cell population within 48 hoursis noteworthy. It is also noteworthy that VES is selective,inhibiting the growth of cancer cells but not normal cells.

Acknowledgments and Notes

This work was supported by The Foundation for Research (CarsonCity, NV). Address reprint requests to Kimberly Kline, Ph.D., Div. ofNutritional Sciences/A2703, University of Texas at Austin, Austin, TX78712-1097. Phone: 512-471-8911. FAX: 512-471-5844. E-mail:k.kline@mail.utexas.edu.

Submitted 1 March 1996; accepted in final form 27 August 1996.

References

1. Kline, K, and Sanders, BG: "Anti-Tumor Proliferation Properties ofVitamin E." In Vitamins and Minerals in the Prevention and Treatmentof Cancer, M M Jacobs (ed). Boca Raton, FL: CRC, 1991, pp 189-205.

2. Kline, K, Yu, W, Zhao, B, Israel, K, Charpentier, A, et al.: "VitaminE Succinate: Mechanisms of Action as Tumor Cell Growth Inhibitor."In Nutrients in Cancer Prevention and Treatment, KN Prasad, L San-tamaria, and RM Williams (eds). Totowa, NJ: Humana, 1995, pp 39-56.

3. Schwartz, J: "The Clinical Control of Tumor Cell Growth Throughthe Action of Carotenoids, Retinoids and Tocopherols." In AdjuvantNutrition in Cancer Treatment, P Quillin and RM Williams (eds).Arlington Heights, IL: Cancer Treat Found, 1993, pp 173-233.

4. Prasad, KN, and Edwards-Prasad, J: "Vitamin E and Cancer Preven-tion: Recent Advances and Future Potentials." J Am Coll Nutr 11,487-500, 1992.

5. Rama, BN, and Prasad, N: "Study on the Specificity of Alpha To-copheryl (Vitamin E) Acid Succinate Effects on Melanoma, Gliomaand Neuroblastoma Cells in Culture." Proc Soc Exp Biol Med 174,302-307, 1983.

6. Slack, R, and Proulx, P: "Studies on the Effects of Vitamin E onNeuroblastoma NIE 115." Nutr Cancer 12, 75-82, 1989.

7. Helson, L, Verma, M, and Helson, C: "Vitamin E and HumanNeuroblastoma." In Modulation and Mediation of Cancer by Vitamins,FL Meyskens and KN Prasad (eds). Basel: Karger, 1983, pp 258-265.

8. Prasad, KN, and Edwards-Prasad, J: "Effects of Tocopherol (VitaminE) Acid Succinate on Morphological Alterations and Growth Inhibitionin Melanoma Cells in Culture." Cancer Res 42, 550-554, 1982.

9. Ripoll, EAP, Rama, BN, and Webber, M: "Vitamin E Enhances theChemotherapeutic Effects of Adriamycin on Human Prostate Carci-noma Cells In Vitro." J Urol 136, 529-531, 1986.

10. Israel, K, Sanders, BG, and Kline, K: "RRR-α-Tocopheryl SuccinateInhibits the Proliferation of Human Prostatic Tumor Cells With De-fective Cell Cycle/Differentiation Pathways." Nutr Cancer 24, 161—169, 1995.

11. Kline, K, Cochran, GS, and Sanders, BG: "Growth-Inhibitory Effectsof Vitamin E Succinate on Retrovirus-Transformed Tumor Cells InVitro." Nutr Cancer 14, 27-41, 1990.

12. Simmons-Menchaca, M, Qian, M, Yu, W, Sanders, BG, and Kline,K: "RRR-α-Tocopheryl Succinate Inhibits DNA Synthesis and En-hances the Production and Secretion of Biologically Active Transform-ing Growth Factor-Beta (TGF-P) by Avian Retrovirus-TransformedLymphoid Cells." Nutr Cancer 24, 171-185, 1995.

13. Turley, JM, Sanders, BG, and Kline, K: "RRR-α-Tocopheryl SuccinateModulation of Human Promyelocytic Leukemia (HL-60) Cell Prolif-eration and Differentiation." Nutr Cancer 18, 201-213, 1992.

14. Charpentier, A, Groves, S, Simmons-Menchaca, M, Turley, J, Zhao,B, et al.: "RRR-α-Tocopheryl Succinate Inhibits Proliferation and En-hances Secretion of Transforming Growth Factor-P (TGF-P) by HumanBreast Cancer Cells." Nutr Cancer 19, 225-239, 1993.

15. Charpentier, A, Simmons-Menchaca, M, Yu, W, Zhao, B, Qian, M,et al.: "RRR-α-Tocopheryl Succinate Enhances TGF-β1 -β2, and -β3

and TGF-βR-II Expression by Human MDA-MB-435 Breast CancerCells." Nutr Cancer 26, 237-250, 1996.

16. Turley, JM, Funakoshi, S, Ruscetti, FW, Kasper, J, Murphy, WJ, etal.: "Growth Inhibition and Apoptosis of RL Human B LymphomaCells by Vitamin E Succinate and Retinoic Acid: Role for Transform-ing Growth Factor p." Cell Growth Differentiation 6, 655-663, 1995.

17. Yu, W, Sanders, BG, and Kline, K: "Modulation of Murine EL-4Thymic Lymphoma Cell Proliferation and Cytokine Production byVitamin E Succinate." Nutr Cancer 25, 137-149, 1996.

18. Qian, M, Sanders, BG, and Kline, K: "RRR-α-Tocopheryl SuccinateInduces Apoptosis in Avian Retrovirus-Transformed Lymphoid Cells."Nutr Cancer 25, 9-26, 1996.

19. Farrar, JJ, Fulter-Farrar, J, and Simon, PL: "Thyoma Production of TCell Growth Factor (IL-2)." J Immunol 125, 2555-2558, 1980.

20. Delia, D, Aiello, A, Lombardi, L, Pelicci, PG, Grignani, F, Jr, et al.:"N-(4-Hydroxyphenyl) Retinamide Induces Apoptosis of MalignantHemopoietic Cell Lines Including Those Unresponsive to RetinoicAcid." Cancer Res 53, 6036-6041, 1993.

21. Nicoletti, I, Migliorati, G, Pagliacci, MC, Grignani, F, and Riccardi,C: "A Rapid and Simple Method for Measuring Thymocyte Apoptosisby Propidium Iodide Staining and Flow Cytometry." J Immunol Meth-ods 139, 271-279, 1991.

22. Maniatis, T, Fritsch, EF, and Sambrook, J: Molecular Cloning: ALaboratory Manual. Cold Spring Harbor, NY: Cold Spring HarborLaboratory, 1989.

23. Nunez, G, London, L, Hockenbery, D, Alexander, M, McKeam, JP,et al.: "Deregulated bcl-2 Gene Expression Selectively Prolongs Sur-vival of Growth Factor-Deprived Hemopoietic Cell Lines." J Immunol144, 3602-3610, 1990.

24. Stanton, LW, Fahrlander, PD, Tesser, PM, and Marcu, KB: "Nucleo-tide Sequence Comparison of Normal and Translocated Murine c-mycGenes." Nature 310, 423-425, 1984.

25. Lamph, WW, Wamsley, P, Sassone-Corsi, P, and Verma, IM: "Induc-tion of Proto-Oncogene JUN/AP-1 by Serum and TPA." Nature 334,629-631, 1988.

26. Curran, T, Gordon, MB, Rubino, KL, and Sambucetti, LC: "Isolationand Characterization of the c-fos (Rat) cDNA and Analysis of Post-tTranslational Modification In Vitro." Oncogene 2, 79-84, 1987.

100 Nutrition and Cancer 1997

Dow

nloa

ded

by [

Ast

on U

nive

rsity

] at

22:

40 0

4 Se

ptem

ber

2014

27. Goldstone, SD, and Lavin, MF: "Prolonged Expression of c-jun andAssociated Activity of the Transcription Factor AP-1, During Apop-tosis in a Human Leukaemic Cell Line." Oncogene 9, 2305-2311,1994.

28. Laemmli, UK: "Cleavage of Structural Proteins During the Assemblyof the Head of Bacteriophage T4." Nature 227, 680-685, 1970.

29. Lippman, SM, Kessler, JF, and Meyskens, FL, Jr "Retinoids as Pre-ventive and Therapeutic Anticancer Agents." Cancer Treat Rep 71,391-405, 1987.

30. DeLuca, HF: "Vitamin D, Gene Expression, and Cancer." In Nutrientsin Cancer Prevention and Treatment, KN Prasad, L Santamaria, andRM Williams (eds). Totowa, NJ: Humana, 1995, pp 57-69.

31. Wu, FYH, Chang, NT, Chen, WJ, and Juan, CC: "Vitamin K3-InducedCell Cycle Arrest and Apoptotic Cell Death Are Accompanied byAltered Expression of c-fos and c-myc in Nasopharyngeal CarcinomaCells." Oncogene 8, 2237-2244, 1993.

32. Roberts, AB, and Sporn, MB: "Mechanistic Interrelationships BetweenTwo Superfamilies: The Steroid/Retinoid Receptors and TransformingGrowth Factor-P." Cancer Surv 14, 205-220, 1992.

33. Schwartz, JL: "The Dual Roles of Nutrients as Antioxidants andProoxidants: Their Effects on Tumor Cell Growth." J Nutr 126, 1221S-1227S, 1996.

34. Schwartz, JL: "Molecular and Biochemical Control of Tumor GrowthFollowing Treatment With Carotenoids or Tocopherols." In Nutrientsin Cancer Prevention and Treatment, KN Prasad, L Santamaria, andRM Williams (eds). Totowa, NJ: Humana, 1995, pp 287-316.

35. Carini, R, Poli, G, Dianzani, MU, Maddix, SP, Slater, TF, et al.:"Comparative Evaluation of the Antioxidant Activity of α-Tocopherol,α-Tccopherol Polyethylene Glycol 1000 Succinate and α-Tocopherol

Succinate in Isolated Hepatocytes and Liver Microsomal Suspensions."Biochem Pharmacol 39, 1597-1601, 1990.

36. Fariss, MW, Fortuna, MB, Everett, CK, Smith, JD, Trent, DF, et al.:"The Selective Antiproliferative Effects of α-Tocopheryl Hemisucci-nate and Cholesteryl Hemisuccinate on Murine Leukemia Cells ResultFrom the Action of the Intact Compounds." Cancer Res 54, 3346-3351,1994.

37. Hockenbery, DM, Oltvai, ZN, Yin, XM, Milliman, CL, and Kors-meyer, SJ: "Bcl-2 Functions in an Antioxidant Pathway to PreventApoptosis." Cell 75, 241-251, 1993.

38. Weller, M, Constam, DB, Malipiero, U, and Fontana, A: 'Transform-ing Growth Factor-P2 Induces Apoptosis of Murine T Cell ClonesWithout Down-Regulating bcl-2 mRNA Expression." Eur J Immunol2, 1293-1300.

39. Marcu, KB, Bossone, SA, and Patel, AJ: "Myc Function and Regula-tion." Annu Rev Biochem 61, 809-860, 1992.

40. Prasad, KN, Cohrs, RJ, and Sharma, OK: "Decreased Expressions ofc-myc and H-ras Oncogenes in Vitamin E Succinate Induced Mor-phologically Differentiated Murine B-16 Melanoma Cells in Culture."Cell Biol, 1250-1255, 1990.

41. McMahon, SB, and Monroe, JG: "Role of Primary Response Genesin Generating Cellular Responses to Growth Factors." FASEB J 6,2707-2715, 1992.

42. Diamond, MI, Miner, JN, Yoshinaga, SK, and Yamamoto, KR: "Tran-scription Factor Interactions: Selectors of Positive or Negative Regu-lation From a Single DNA Element." Science 249, 1266-1272, 1990.

43. Ham, J, Babij, C, Whitfield, J, Pfarr, CM, Lallemand, D, et al.: "Ac-Jun Dominant Negative Mutant Protects Sympathetic NeuronsAgainst Programmed Cell Death." Neuron 14, 927-937, 1995.

Vol. 27, No. 1 101

Dow

nloa

ded

by [

Ast

on U

nive

rsity

] at

22:

40 0

4 Se

ptem

ber

2014