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RRR-α--Tocopheryl Succinate Induction of DNASynthesis Arrest of Human MDA-MB-435 Cells InvolvesTGF-β--Independent Activation of p21Waf1/Cip1

Weiping Yu , Bob G. Sanders & Kimberly KlinePublished online: 18 Nov 2009.

To cite this article: Weiping Yu , Bob G. Sanders & Kimberly Kline (2002) RRR-α--Tocopheryl Succinate Induction of DNA

Synthesis Arrest of Human MDA-MB-435 Cells Involves TGF-β--Independent Activation of p21Waf1/Cip1 , Nutrition and Cancer,43:2, 227-236, DOI: 10.1207/S15327914NC432_13

To link to this article: http://dx.doi.org/10.1207/S15327914NC432_13

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RRR-a-Tocopheryl Succinate Induction of DNA SynthesisArrest of Human MDA-MB-435 Cells InvolvesTGF-b-Independent Activation of p21Waf1/Cip1

Weiping Yu, Bob G. Sanders, and Kimberly Kline

Abstract: RRR-α-tocopheryl succinate (vitamin E succinate,VES), a derivative of vitamin E, is a potent antitumor agent.Cellular events involved in VES-induced DNA synthesis ar-rest of human MDA-MB-435 breast cancer cells were stud-ied. VES induces a dose- and time-dependent inhibition ofDNA synthesis and a G0/G1 cell cycle arrest. VES inducesexpression of p21Waf1/Cip1 mRNA and protein, and antisenseoligomers to p21 block VES-induced growth arrest. Evi-dence suggesting that VES modulates p21 expression in atransforming growth factor-� (TGF-�)-independent fashionincludes failure of TGF-�-neutralizing antibodies to blockVES-induced DNA synthesis arrest or VES activation of ap21 promoter-regulated reporter gene; VES is not capableof inducing the translocation of green fluorescent protein-Smad2 into the nucleus and is not capable of stimulating aTGF-�-dependent reporter gene, and VES induces growthinhibition and upregulates p21 mRNA levels in TGF-� re-ceptor-defective cells.

Introduction

RRR-α-tocopheryl succinate (vitamin E succinate, VES),a derivative of vitamin E, is a potent antitumor agent for var-ious cancer cell types in vitro and in vivo (1–6). The anti-tumor effects of VES on human breast cancer cells includecell growth inhibition, induced cellular differentiation, andapoptosis (4–17).

VES is a potent inducer of apoptosis in human breastcancer cells but not normal human mammary epithelialcells (11). It appears that at least four signal transductionpathways are involved in VES-induced apoptosis of hu-man breast cancer cells, including restoration and signalingby transforming growth factor-β (TGF-β) and Fas (CD95/APO-1) pathways and activation of extracellular signal-regulated kinase (ERK)- and c-Jun NH2-terminal kinase(JNK)-mitogen-activated protein kinase (MAPK) signalingpathways (9–12,15). Less is known about the events in-volved in VES-induced blockage of DNA synthesis and in-

hibition of cell proliferation. Turley et al. (16) reported thatVES inhibited the proliferation of the estrogen receptor-negative human breast cancer cell line BT-20 in the G1 phaseof the cell cycle through a mechanism that involved cyclinA-negative regulation of E2F-mediated transcription. Inves-tigations into the possible role of TGF-β and TGF-β-regu-lated cyclin-dependent kinase (CDK) inhibitors (CDKIs) inVES-induced blockage of DNA synthesis and inhibition ofcell proliferation are the subject of the studies reported here.

Cell cycle checkpoints are crucial for maintaining cellnumber homeostasis. p21Waf1/Cip1 is an important TGF-β- andp53-dependent or -independent cell cycle mediator, acting tocause cell cycle arrest (18–21). The ability of TGF-β to in-hibit the activity of G1 cyclin-CDK complexes, thereby pre-venting phosphorylation of retinoblastoma protein andrelease of the transcription factor E2F, thus inducing a G1

cell cycle block, involves transcriptional induction of CDKIp21Waf1/Cip1 (22–24).

One pathway in which TGF-β-initiated signaling at thecell surface is transmitted to the nucleus involves a family ofsignal-transducing proteins, the Smad family (reviewed inRef. 25). Basically, receptor Smad proteins (Smad2 orSmad3) undergo serine phosphorylation in response to TGF-β receptor I and II complex activation (26). Smads form acomplex with a common partner, Smad4, and the complextranslocates to the nucleus and exhibits transactivating activ-ity (activating or repressing) in association with DNA-binding transcription factors as well as non-DNA-bindingtranscriptional cofactors (26,27). TGF-β-regulated genes[p15, p21, p27, and plasminogen inhibitor activator-1 (PAI-1)] are controlled through direct or indirect binding of Smadcomplexes with DNA-binding transcription factors and non-DNA-binding transcriptional cofactors (26,27). TGF-β re-ceptor activation by exogenous ligand also transduces sig-nals to the nucleus via members of the MAPK superfamily,including ERKs, stress-activated protein kinases-JNKs, andstress-activated p38-Hog (28–31).

This report characterizes VES-mediated growth inhibi-tion in MDA-MB-435 human breast cancer cells. Data show

NUTRITION AND CANCER, 43(2), 227–236Copyright © 2002, Lawrence Erlbaum Associates, Inc.

W. Yu and B. G. Sanders are affiliated with the School of Biological Sciences and K. Kline with the Division of Nutrition, University of Texas at Austin,Austin, TX 78712.

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that VES induces DNA synthesis arrest in a concentration-and time-dependent manner and produces a G0/G1 cell cycleblock. VES induces expression of p21Waf1/Cip1 mRNA andprotein, and antisense oligomers to p21 block VES-inducedDNA synthesis arrest and growth arrest. Evidence suggest-ing that VES modulates DNA synthesis arrest and growthinhibition in a TGF-β-independent fashion includes failureof TGF-β-neutralizing antibodies to block VES-inducedDNA synthesis arrest, failure of TGF-β-neutralizing anti-bodies to block VES activation of a p21 promoter-regulatedluciferase reporter gene, inability of VES to cause the trans-location of green fluorescent protein (GFP)-Smad2 into thenucleus, inability of VES to activate a TGF-β-dependentSmad-binding element (SBE)-regulated reporter gene, andVES-induced growth inhibition and upregulation of p21mRNA levels in TGF-β receptor-defective cells. In contrastto studies demonstrating TGF-β involvement in VES-in-duced apoptosis, these studies show that TGF-β is not in-volved in VES-induced DNA synthesis arrest. The studiesalso show that although MDA-MB-435 breast cancer cellsare refractive to the antiproliferative effects of exogenousTGF-β, treatment of MDA-MB-435 cells with exogenousTGF-β1 stimulates translocation of GFP-Smad2 from thecytoplasm to the nucleus and causes the activation of anSBE-luciferase reporter construct.

Materials and Methods

Cell Culture and VES and TGF-b1 Treatments

MDA-MB-435 (provided by Dr. Janet E. Price, Dept. ofCell Biology, University of Texas M. D. Anderson CancerCenter, Houston, TX) (32) and MDA-MB-231 (AmericanType Culture Collection, Manassas, VA) are non-estrogen-responsive human breast cancer cell lines. T-47D (AmericanType Culture Collection) and MCF-7 (provided by Dr. Su-zanne Fuqua, Baylor College of Medicine, Houston, TX) areestrogen-responsive human breast cancer cell lines (33).MCF-10A (American Type Culture Collection) is an im-mortalized nontumorigenic human breast cell line. Cellswere maintained as previously described (11). For experi-ments, the percentage of fetal bovine serum (Hyclone Labo-ratories, Logan, UT) was reduced to 2% for MDA-MB-435and 5% for MCF-7, MDA-MB-231, and T47D cells. MCF-10A cells were tested using 2% horse serum (GIBCO BRLLife Technologies, Grand Island, NY). Exponentially grow-ing cells were plated at 5 × 106 cells in T-75 flasks formRNA analysis, 1.6 × 106 cells in T-25 flasks for Westernimmunoblot analysis, 1.5 × 104 cells in 96-well plates forDNA synthesis arrest assay, and 1.5 × 105 cells/well in 12-well plates for cell number counting and apoptosis analysisand allowed to attach overnight. VES treatments were con-ducted at 5, 10, or 20 µg/ml VES in 0.2% ethanol (vol/vol)or vehicle control (VEH), which consisted of an equivalentor the highest amount of sodium succinate in 0.2% ethanol

used in the experiment. VES and sodium succinate were pur-chased from Sigma. Purified human platelet TGF-β1 (R & DSystems, Minneapolis, MN) treatments were conducted at 5,10, and 20 ng/ml TGF-β1 for MDA-MB-435 cells and at1.25, 2.5, and 5 ng/ml TGF-β1 for MCF-10A cells.

DNA Synthesis Arrest Assay ([3H]ThymidineIncorporation) and Cell Growth Inhibition Analysis

The effects of VES or TGF-β1 on DNA synthesis byMDA-MB-435 cells were assessed by [3H]thymidine incor-poration as described previously (7). Cell proliferation wasdetermined by counting actual cell numbers (adherent andfloating cells) with a hemocytometer.

Cell Cycle Analyses

MDA-MB-435 cells were cultured for 1 day in the pres-ence of VES (10 µg/ml) or VEH. After culture, cells werewashed twice with phosphate-buffered saline (PBS) at pH7.4 and fixed in l ml of ice-cold 100% ethanol overnight at4°C. Fixed cells were washed once in PBS and stained byresuspension in 0.5 ml of PBS containing propidium io-dide (20 µg/ml) and ribonuclease A (1 mg/ml; Sigma) andanalyzed for DNA content by flow cytometry using a Bec-ton-Dickinson fluorescence-activated cell sorter. Data wereanalyzed by Mod Fit LT software (Becton-Dickinson Im-munocytometry Systems, San Jose, CA).

Determination of Apoptosis (Morphological Analysis of4′,6-Diamidino-2-Phenylindole-Stained Cells)

Because previous studies (9,15) characterized VES-in-duced apoptosis in human breast cancer cells using detectionof DNA fragmentation by gel electrophoresis and TdT-me-diated dUTP nick end labeling, detection of hypodiploidcells by flow cytometry, and analysis of nuclear morphologyusing the fluorescent DNA dye 4′,6-diamidino-2-phenyl-indole (DAPI), we limited apoptosis assessment in thesestudies to the latter technique. The DAPI technique has beendescribed previously (11). Briefly, cells in which the nucleuscontained clearly condensed chromatin or cells exhibitingfragmented nuclei were scored as apoptotic. Apoptotic dataare reported as percent apoptosis, obtained by determiningthe number of apoptotic cells vs. the total number of cells.For each sample, ≥3 counts involving ≥100–200 cells/countwere scored. Apoptotic data are presented as means ± SD forthree independently performed experiments. Reagents formorphological analysis of apoptosis were purchased fromBoehringer Mannheim (Indianapolis, IN).

Western Blot Analysis

p21 protein was detected in whole cell protein extracts byWestern blot analysis. Whole cell lysates were prepared aspreviously described (9), and 100 µg of protein were loaded

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per lane, separated using sodium dodecyl sulfate-polyacryl-amide gel electrophoresis on a 15% gel under reducing con-ditions, and electroblotted onto a nitrocellulose membrane(Optitran BA-S supported nitrocellulose for 0.2-µm pore;Schleicher & Schuell, Keene, NH). Equal loading was veri-fied using glyceraldehyde-3-phosphate dehydrogenase(GAPDH) antibody (rabbit antibody produced in-house).Immunoblotting was performed using a rabbit antibody top21 and a murine monoclonal antibody to p27 (Santa CruzBiotechnology, Santa Cruz, CA). Membranes were reactedwith horseradish peroxidase-conjugated goat anti-rabbit orgoat anti-mouse secondary antibody (Jackson Immunore-search Laboratory, West Grove, PA). Immune complexeswere visualized using enhanced chemiluminescence detec-tion (Pierce Chemical, Rockford, IL). Fold differences in thelevel of chemiluminescence were determined by densito-metric analysis.

Northern Blot Analysis

Total cellular RNA was isolated and analyzed using es-tablished procedures previously described (34). A total of 20µg of RNA was loaded per lane and separated by 1% aga-rose-formaldehyde gel electrophoresis. RNA was trans-ferred to a nylon membrane (Nytran, Schleicher & Schuell)by capillary transfer. Ultraviolet cross-linking (Stratalinker1800, Stratagene, La Jolla, CA) was performed to enhanceRNA binding to the membrane. Membranes were hybridizedin a mixture of 10% dextran sulfate, l M NaCl, 1% sodiumdodecyl sulfate, and 10 µg/ml denatured salmon sperm DNAin the presence of radiolabeled probes at 65°C. The cDNAprobe for p21 (pCEP-WAF-1) was obtained from Dr. BertVogelstein (Johns Hopkins Oncology Center, Baltimore,MD), and the GAPDH cDNA probe was purchased from theAmerican Type Culture Collection. Probes were radio-labeled using a random primer labeling kit (Deca Prime II,Ambion, Austin, TX).

Antisense Oligonucleotide Transfection

Phosphothiorate-modified antisense and sense oligomersto p21 [CGT TTC CAT CTT CGT AGT CAT (antisense)and ATG ACT GCA AAG ATG GAA ACG (sense)] werepurchased from Operon Technologies (Alameda, CA) andtransfected into MDA-MB-435 cells using LipofectAMINE(Life Technologies, Grand Island, NY) as previously de-scribed (9). Briefly, the cells were transfected with 0.5 ml ofminimum essential medium (MEM)-Option serum-free me-dium (Life Technologies) containing 100 µl of oligomer-LipofectAMINE complex for 12-well plate cultures and 3ml of MEM-Option serum-free medium containing 700 µlof oligomer-LipofectAMINE complex for T-25 flask cul-tures. The oligomer-LipofectAMINE complex was made bymixing 2 µg of oligomers and 50 µl of MEM-Option serum-free medium with 8 µg of LipofectAMINE and 50 µl of

MEM-Option serum-free medium and incubating the mix-ture at room temperature for 45 min.

Blockage of TGF-b Signaling With NeutralizingAntibodies to TGF-b1 Ligand

MDA-MB-435 cells were pretreated with 1 µg/ml of neu-tralizing antibody to TGF-β1 (R & D Systems) or irrelevantantibody (control antibody) for 2 h before they were treatedwith VES at 10 µg/ml for 1 day for analysis of DNA synthe-sis arrest or with VES at 20 µg/ml for 3 days for determina-tion of apoptosis.

Luciferase Activity Assays

Three TGF-β-responsive promoter-luciferase reporterconstructs (SBE, p21, and PAI-1) were used to ascertainTGF-β responsiveness: 1) the pSBE4-luciferase (SBE-Luc)construct, which contains two copies of the SBE (5′-GTCTAGAC-3′) in tandem with the luciferase reporter gene(gift from Drs. Kenneth W. Kinzler and Bert Vogelstein,Johns Hopkins University School of Medicine) (35), 2) thep3TP-Lux promoter-reporter (PAI-1-Luc) construct, whichcontains a TGF-β response element from the PAI-1 gene intandem with the luciferase reporter gene (36), and 3) thep21P (p21-Luc) construct, which contains a TGF-β responseelement from the human p21Waf1 gene in tandem with the lu-ciferase reporter gene (37). The p3TP-Luc and p21P vectorswere gifts from Dr. Michael Brattain (Roswell Park CancerInstitute, Buffalo, NY). Empty vectors were used as con-trols. MDA-MB-435 cells were transfected, using Lipofect-AMINE-Plus, independently with the luciferase reporterconstructs plus a β-galactosidase-expressing plasmid (usedfor normalization purposes), following the manufacturer’sinstructions. Transfected cells were treated with VES (10µg/ml), VEH, or TGF-β1 (5 ng/ml) for 12 h, harvested, andlysed. Light units and optical density were determined forluciferase activity and β-galactosidase expression, respec-tively. Luciferase activities were corrected for transfectionefficiencies using β-galactosidase activities and presented asfold induction, where 1.0-fold is arbitrarily established forcorrected luciferase activities generated by VEH or un-treated transfected control cells.

Visualization of GFP-Smad2 Translocation From theCytoplasm to the Nucleus

MDA-MB-435 cells at 3 ×105 cells/well were plated insix-well plates containing poly-L-lysine (0.01% solution;Sigma)-coated coverslips, incubated overnight in culturemedium, and transiently transfected with GFP-Smad 2(PBJ5-JegSmad2; gift from Drs. Brent Stockwell and StuartL. Schreiber; Harvard University, Cambridge, MA) for 3–4h using LipofectAMINE-Plus as described above. Trans-fected cells were treated with VES at 10 µg/ml or TGF-β1 at5 ng/ml (R & D Systems) or untreated (control) for 6 h in

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medium containing 2% serum. Cells were fixed in methanol,and nuclei were stained with DAPI (2 µg/ml) and examinedby confocal microscopy at 488 nm excitation-530 ± 15 nmemission for GFP-Smad2 and 354 nm excitation-440 ± 20nm emission for DAPI-stained nuclei.

Results

VES Induces Growth Inhibition of MDA-MB-435 Cellsin a Concentration- and Time-Dependent Manner

DNA synthesis, determined by [3H]thymidine uptake,was inhibited in a dose-dependent manner, with treatment ofcells, with VES at 5, 10, and 20 µg/ml producing 30%, 54%,and 66% reductions in DNA synthesis after 1 day and 52%,70%, and 83% reductions in DNA synthesis after 2 days(Fig. 1A). A count of total adherent and floating MDA-MB-435 cells showed that cell proliferation was reduced by 14%,36%, and 54% after treatment with VES at 10 µg/ml and by28%, 48%, and 63% after treatment with VES at 20 µg/mlafter 1, 2, and 3 days, respectively (Fig. 1B). Cell cycle anal-ysis of MDA-MB-435 cells showed that VES (10 µg/ml) in-duced a G0/G1 cell cycle arrest after 24 h of treatment (Fig.2). After VES treatment for 1 day, the G0/G1 cell cycle phaseexhibited a 62% increase in cell numbers and the S and G2/Mphases exhibited 32% and 37% decreases, respectively.

VES Induces Increased Expression of p21 mRNA andProtein

In MDA-MB-435 cells, VES (10 µg/ml) induced ele-vated levels of p21 mRNA compared with VEH, starting 6 hafter treatment and reaching high levels by 24 h (Fig. 3A,top). Quantification by densitometry and normalization onthe basis of GAPDH hybridization signals (Fig. 3A, bottom)showed 1.2-, 1.5-, 2.5-, and 3-fold increases in p21 mRNAlevels in VES-treated cells compared with VEH-treated cells3, 6, 12, and 24 h after treatment. Western immunoblot anal-ysis of p21 protein showed elevated levels at 12 and 24 h af-ter VES treatment (Fig. 3B, top). No p21 protein wasdetected in cells treated with VES for 6 h or VEH for 24 h.GAPDH protein levels were used to normalize lane loads(Fig. 3B, bottom). p27 protein was not detected in MDA-MB-435 cells treated with VEH or VES for 3, 6, 12, and 24 h(Fig. 3C, top). As a positive control, low levels of p27 weredetected in whole cell extracts from MCF-7 human breastcancer cells (Fig. 3C, top). GAPDH protein levels were usedfor lane load controls (Fig. 3C, bottom). To address whetherupregulation of p21 at the mRNA level is a common event inhuman breast cancer cell lines treated with VES, three celllines, MCF-7, MDA-MB-231, and T-47D, in addition toMDA-MB-435 cells, were tested (Fig. 3D). Densitometricanalysis showed 3.7-, 1.7-, 1.4-, and 2.6-fold increased ex-pression of p21 mRNA in MDA-MB-435, MCF-7, MDA-

230 Nutrition and Cancer 2002

Figure 1. RRR-α-tocopheryl succinate (vitamin E succinate, VES) inducescell growth inhibition. A: MDA-MB-435 cells cultured at 1.5 × 104 cells/wellin 96-well plates were treated with VES (5, 10, and 20 µg/ml) for 1 or 2 days.DNA synthesis was monitored by [3H]thymidine uptake. DNA synthesis ar-rest was calculated by comparing [3H]thymidine uptake by cells receivingVES treatment with [3H]thymidine uptake by cells receiving vehicle (VEH)control. B: MDA-MB-435 cells cultured at 1.5 × 105 cells/well in 12-wellplates were treated with VES at 10 and 20 µg/ml (VES 10 and VES 20, re-spectively) or VEH for 1, 2, or 3 days, and total cell numbers (adherent andfloating cells) were counted. Values are means ± SD of 3 experiments.

Figure 2. VES induces a G0/G1 cell cycle block. MDA-MB-435 cells werecultured for 1 day in the presence of VES (10 µg/ml) or VEH. After culture,cell cycle analysis was performed by flow cytometry. Data are from a repre-sentative experiment (n = 2).

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MB-231, and T-47D cells, respectively, after 1 day of treat-ment with VES at 10 µg/ml (Fig. 3D).

Antisense Oligomers to p21 Block VES-InducedGrowth Inhibition

MDA-MB-435 cells transiently transfected with anti-sense oligomers to p21 and treated for 1 day for DNA syn-

thesis or for 2 days for growth inhibition with VES at 10 or20 µg/ml exhibited less DNA synthesis arrest (Fig. 4A) andless growth inhibition than VES-treated cells transfectedwith sense oligomers (Fig. 4B). VES at 10 and 20 µg/ml re-sulted in 42% and 40% blockage of DNA synthesis arrestand 46% and 28% blockage of growth inhibition, respec-tively (Fig. 4, A and B). Densitometric analysis showed a3.1-fold reduction of p21 protein levels in cells transfected

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Figure 3. VES induces p21 mRNA and protein expression. A: Northern blotanalysis of p21 mRNA from MDA-MB-435 cells treated with VES (10 µg/ml) or VEH for 3, 6, 12, or 24 h. Top: p21 mRNA; bottom: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA used for normalization oflane loads. B: Western immunoblot analysis of p21 (top) and GAPDH (bot-tom) protein expression. MDA-MB-435 cells were treated with VES (10µg/ml) for 6, 12, or 24 h or VEH for 24 h. C: Western immunoblot analysisof p27 (top) and GAPDH (bottom) protein expression. MDA-MB-435 cellswere treated with VES (10 µg/ml) for 3, 6, 12, or 24 h or VEH for 24 h. Un-treated MCF-7 cells were used as a positive control for expression of p27.D: Northern blot analysis of p21 mRNA from MDA-MB-435, MCF-7,MDA-MB-231, and T-47D cells treated with VES (10 µg/ml) or VEH for24 h. Top: p21 mRNA; bottom: GAPDH mRNA used for normalization oflane loads. Data are representative of ≥2 independent experiments.

Figure 4. Evidence for involvement of p21 in VES-induced DNA synthe-sis arrest and growth inhibition. MDA-MB-435 cells transiently trans-fected with sense or antisense oligomers to p21 were treated with VES (10or 20 µg/ml) or VEH for 24 h before determination of DNA synthesis by[3H]thymidine uptake (A) or growth inhibition by cell counts (B). Per-centage of DNA synthesis arrest or growth inhibition was calculated bycomparing [3H]thymidine uptake or number of cells in VES-treatedcultures with [3H]thymidine uptake or number of cells in VEH controls,respectively. Values are means ± SD of 3 separate experiments. C: p21protein expression in cells transfected with antisense (A) or sense (S) oli-gomers to p21 was detected by Western immunoblot analysis. GAPDHprotein was used as a control for normalization of lane loads. Data are rep-resentative of 2 experiments.

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with antisense oligomers compared with cells transfectedwith sense oligomers (Fig. 4C, top). GAPDH protein levelswere used as lane load controls (Fig. 4C, bottom).

VES-Induced Growth Inhibition Does Not Depend onTGF-b Signaling

VES-induced DNA synthesis arrest was not changedwhen MDA-MB-435 cells were pretreated with TGF-β1-neutralizing antibody for 2 h before addition of VES at 10µg/ml for 1 day (Fig. 5A). As expected, neutralizing anti-body to TGF-β1 blocked VES-induced apoptosis after 3days by 48% (Fig. 5A).

Further evidence that VES-induced growth inhibition is in-dependent of TGF-β signaling comes from the inability ofneutralizing antibody to TGF-β1 to affect the ability of VESto stimulate the transcription of a transiently transfected p21promoter-regulated luciferase reporter gene (Fig. 5B). Treat-ment with VES and irrelevant or TGF-β1-neutralizing anti-bodies for 12 h had no effect on VES-induced activity of thep21 luciferase construct (both exhibited 1.6-fold activity). Asa positive control, neutralizing antibodies to TGF-β1 were ca-pable of blocking the ability of TGF-β1 (5 ng/ml) to stimulatethe transcription of an SBE-regulated luciferase reporter genefrom 5.0- to 2.7-fold, a 46% blockage (Fig. 5B).

MDA-MB-435 Cells Are Nonresponsive to Growth-Inhibitory Properties of TGF-b1, but NuclearTranslocation of Smad2 Occurs in MDA-MB-435 CellsAfter Treatment With TGF-b1 but Not VES

Addition of exogenous TGF-β1 at 5, 10, and 20 ng/mlhad little to no effect on DNA synthesis arrest as measuredby [3H]thymidine uptake after 1 day of treatment (Fig. 6A)or growth inhibition of MDA-MB-435 cells as determinedby counting actual cell numbers after 2 days of treatment(Fig. 6B). MCF-10A cells, which are growth inhibited (i.e.,exhibit DNA synthesis arrest and reduced cell numbers) bylower doses of exogenous TGF-β1, served as a positive con-trol (Fig. 6).

In an attempt to understand the molecular basis for non-responsiveness of MDA-MB-435 cells to exogenous TGF-β,the effects of TGF-β1 and VES on the translocation of GFP-Smad2 from the cytoplasm to the nucleus were investigated.Cells were transiently transfected with GFP-Smad2 and cul-tured with TGF-β1 (5 ng/ml) or VES (10 µg/ml) for 6 h. Thepercentage of cells exhibiting a shift in GFP-Smad2 fromcytosol to nucleus was 18%, 19%, and 60% after no treat-ment, VES treatment, or treatment with TGF-β1, respec-tively (Fig. 7). These data suggest that MDA-MB-435 cellscan respond to exogenous TGF-β ligand by some degree ofSmad activation as measured by Smad translocation.

Differential Induction of TGF-b1-Responsive LuciferaseReporter Gene Constructs (SBE-Luc, PAI-1-Luc, andp21-Luc) by VES and TGF-b1

Studies were conducted to determine whether exogenousTGF-β1 or VES treatments could induce transcriptional re-sponses associated with TGF-β signaling in MDA-MB-435cells transiently transfected with SBE-Luc, PAI-1-Luc, orp21-Luc. TGF-β receptor type II-defective T-47D cells wereused for verification of VES activation of p21-Luc in a TGF-β-independent manner, and MCF-10A immortalized butnontumorigenic cells, which are responsive to the growth-inhibitory properties of TGF-β1, were used to verify thetranscriptional competence and TGF-β1 responsiveness ofthe reporter constructs. All three cell types were transientlytransfected with SBE-Luc, PAI-1-Luc, or p21-Luc andtreated with TGF-β1 (5 ng/ml) or VES (10 µg/ml) for 12 h.TGF-β1, but not VES, induced a 4.7- and a 6.5-fold increase

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Figure 5. VES induces transforming growth factor-β (TGF-β) -independ-ent DNA synthesis arrest and TGF-β-independent p21 promoter-driven re-porter gene expression. Neutralizing antibody (Ab) to TGF-β1 was used toblock TGF-β signaling. A: MDA-MB-435 cells were pretreated with neu-tralizing antibody to TGF-β1 or irrelevant antibody at 1 µg/ml for 2 h beforetreatment with VES (10 µg/ml) or VEH for 1 day for analysis of DNA syn-thesis arrest and 3 days for analysis of apoptosis. B: MDA-MB-435 cellstransiently transfected with a p21- or an Smad-binding element (SBE) pro-moter-driven luciferase reporter gene were pretreated with neutralizing an-tibody to TGF-β1 or irrelevant antibody (1 µg/ml) + VES (10 µg/ml) orTGF-β1 (5 ng/ml), respectively. After 12 h, cells were analyzed for lucifer-ase activity. Values are means ± SD of 3 experiments.

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in transcriptional activity of SBE-Luc in MDA-MB-435 andMCF-10A cells, respectively (Table 1). SBE-Luc was notactivated in TGF-β-defective T-47D cells (Table 1). TGF-β1 was incapable of activating transcriptional activity ofPAI-1-Luc or p21-Luc in MDA-MB-435 or T-47D cells, butPAI-1-Luc and p21-Luc were induced 6.5- and 2.3-fold, re-spectively, by TGF-β1 in MCF-10A cells (Table 1). VESwas incapable of activating SBE-Luc or PAI-1-Luc in any ofthe three cell types; however, VES induced 1.5-, 1.6-, and1.5-fold increases of p21-Luc in MDA-MB-435, MCF-10A,and T47D cells, respectively (Table 1).

Discussion

MDA-MB-435 human breast cancer cells undergo DNAsynthesis arrest and growth inhibition after VES treatment.

These events are correlated with a G1 cell cycle arrest andupregulation of p21Waf1/Cip1 at the mRNA and protein level.Previous studies have characterized cellular events involvedin VES induction of differentiation and apoptosis (1,12,13).It appears that VES causes breast cancer cells to undergoDNA synthesis arrest and differentiation followed by apo-ptosis. After 3 days of VES treatment, >70% of cells withina given cell population exhibit terminal stages of apoptosis(1). Because previous studies have shown that VES can acti-vate latent TGF-β to its biologically active form as well asincrease TGF-β receptor II expression (8) and have demon-strated that TGF-β signaling is necessary for VES-inducedapoptosis (9) and because TGF-β is a well-recognizedantiproliferative factor for epithelial cells and a known regu-lator of p21 transcription (38), expectations were that TGF-βwould be involved in VES inhibition of DNA synthesis andgrowth. However, evidence suggests that TGF-β is not in-volved in VES-induced DNA synthesis and growth arrest.Evidence suggesting that VES induces DNA synthesis arrestand growth arrest in a TGF-β-independent manner includesthe following: 1) TGF-β–neutralizing antibodies fail toblock VES-induced DNA synthesis arrest or VES activation

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Figure 7. TGF-β1, but not VES, activates Smad pathway as measured bytranslocation of green fluorescent protein (GFP)-Smad2 from cytosol to nu-cleus. A: MDA-MB-435 cells transiently transfected with GFP-Smad2were not treated (untreated) or treated with VES (10 µg/ml) or TGF-β1 (5ng/ml) for 6 h. Nuclei were stained with 4′,6-diamidino-2-phenylindole(DAPI). GFP-Smad2 (top) and nuclear morphology (bottom) were visual-ized by confocal microscopy. Data are representative of multiple experi-ments. B: percentage of transfected cells exhibiting GFP-Smad2translocation to nucleus. Values are means ± SD of 3 experiments.

Figure 6. MDA-MB-435 cells are refractive to TGF-β1 growth inhibition.MDA-MB-435 or MCF-10A cells cultured at 1.5 × 104 cells/well in 96-wellplates were treated with TGF-β1 at 1.25, 2.5, and 5 ng/ml (MCF-10A cells)or at 5, 10, or 20 ng/ml (MDA-MB-435 cells) for 1 day before determina-tion of [3H]thymidine uptake (A) or for 2 days before cell numbers werecounted (B). Values are means ± SD of 3 experiments.

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of a p21-dependent reporter gene; 2) VES is not capable ofinducing the translocation of GFP-Smad2 into the nucleus,and VES is not capable of activating a TGF-β–dependentSBE-regulated reporter gene or a PAI-1 TGF-β–regulatedpromoter-dependent reporter gene; and 3) VES is capable ofinducing growth inhibition and upregulating p21 mRNAlevels in TGF-β receptor-defective T-47D human breastcancer cells.

Thus, although TGF-β signaling is implicated in VES-induced apoptosis (9), TGF-β signaling does not appear tobe involved in VES induction of differentiation (13) or, asdemonstrated by the studies reported here, in VES blockageof DNA synthesis and inhibition of cell proliferation.

Additionally, studies reported here show that althoughMDA-MB-435 human breast cancer cells are refractory toexogenous TGF-β–mediated growth inhibition, they arecapable of responding to exogenous TGF-β stimulation bytranslocating GFP-Smad2 from the cytoplasm to the nu-cleus and by differentially activating TGF-β–regulatablereporter genes, suggesting that metastatic MDA-MB-435tumor cells may serve as a useful model for determininghow altered TGF-β signaling contributes to an aggressivetumor phenotype.

As we reported previously (8), VES is a potent concentra-tion- and time-dependent inhibitor of DNA synthesis, as de-termined by measuring [3H]thymidine uptake. Here weshow that treatment of MDA-MB-435 cells with VES at 5,10, and 20 µg/ml for 24 h results in blockage of DNA syn-thesis by 30%, 54%, and 66%, respectively. Likewise, as wereported previously (8), VES is a potent concentration- andtime-dependent inhibitor of cell proliferation as determinedby counting total cell numbers. Here we show that treatmentof MDA-MB-435 cells with VES at 5, 10, and 20 µg/ml for48 h results in reduction of total cell numbers by 52%, 70%,and 83%, respectively. Because total cell numbers reflectcell proliferation as well as survival and because VES is apotent inducer of apoptosis, it is important to point out thatVES at 5, 10, and 20 µg/ml induces ~5%, 19%, and 30% ofMDA-MB-435 cells to undergo apoptosis after 48 h (datanot shown). Also it is important to note that, in determiningtotal cell numbers, floating and adherent cells were counted.Thus blockage of DNA synthesis leading to reductions in to-tal cell numbers is an earlier event in VES-mediated growthinhibition than reduction in cell numbers due to apoptosis.

Investigations into the possible role of CDKIs in VES-mediated growth arrest led to the discovery that VES mark-edly upregulates p21 at the mRNA and protein level in atime-dependent fashion. Another member of the Cip/Kipfamily of inhibitors (39), p27Kip1, was not detectable inMDA-MB-435 cells before or after VES treatment. Becauseantisense oligomers to p21 resulted in a decrease in VES-mediated DNA synthesis arrest and inhibition of cell prolif-eration, the data suggest that increases in p21 are, at least inpart, responsible for VES-mediated reductions in tumor cellnumber. Although p21 is a transcriptional target of p53 (40),it can respond to signals independently of p53. Because VESinduces p21 activation and growth inhibition in MDA-MB-435, MDA-MB-231, and T-47D cell lines, all of which havebeen characterized as p53 defective (41,42), it appears thatVES modulates p21 in a p53-independent fashion. We donot know what cellular events lead to TGF-β- and p53-independent activation of p21 by VES; however, severalfactors have been reported to initiate TGF-β- and p53-in-dependent activation of p21, including signal transducersand activators of transcription-1 and -3 differentiation in-ducers, growth factors, and cytokines and MyoD-inducedterminal differentiation (43–46). Further studies are neededto investigate the mechanisms whereby VES induces TGF-β- and p53-independent activation of p21. Alternatively,VES may increase p21 mRNA by increasing the stability ofthe message.

TGF-β functions as an autocrine and a paracrine negativegrowth regulator of epithelial cells, a function that is oftenlost in more advanced epithelial tumor cells (38). TGF-β ap-pears to play contrasting roles in the progression of epithe-lial cancers, functioning as a growth inhibitor of early-stageTGF-β–responsive epithelial cancers and as a promoter ofgrowth and metastasis in more advanced tumors (25,26,47,48). A switch to a more aggressive tumor phenotype isfrequently accompanied by changes in tumor cell respon-

234 Nutrition and Cancer 2002

Table 1. Induction of Growth Inhibition and Stimulationof TGF-β-Dependent (SBE-Luc) and TGF-β-Regulated(PAI-1-Luc and p21-Luc) Reporter Constructs by VESand TGF-β Treatment of Human Breast Cancer Cellsa

GrowthInhibitionb

Luciferase Activityc

Treatment SBE-Luc PAI-1-Luc p21-Luc

MDA-MB 435 cells

VES 55 ± 6 1.2 0.8 1.5TGF-β 1 ± 2 4.7 0.6 1.0

MCF-10A cells

VES 35 ± 5 1.0 1.1 1.6TGF-β 45 ± 4 5.6 6.5 2.3

T-47D cells

VES 38 ± 4 0.9 1.1 1.5TGF-β 0 1.1 1.0 1.0

a: Human breast cancer cells, MDA-MB-435, MCF-10A, and T-47D,were treated with RRR-α-tocopheryl succinate (VES) at 10 µg/ml ortransforming growth factor-β1 (TGF-β1) at 5 ng/ml for 2 days beforeanalysis of growth inhibition and 12 h before analysis of activation ofluciferase reporter constructs.

b: Growth inhibition was determined by counting cell numbers. Valuesare means ± SD of 3 separate experiments.

c: Stimulation of TGF-β-dependent and TGF-β-regulated reporter con-structs was assessed in cells transiently cotransfected with Smad-binding element (SBE), plasminogen activator inhibitor-1 (PAI-1),or p21 promoter-regulated luciferase reporter constructs (SBE-Luc,PAI-1, Luc, and p21-Luc, respectively) or empty vector control + a β-galactosidase plasmid (see Materials and Methods). Luciferase activ-ities were corrected for transfection efficiencies using β-galactosidaseactivities and presented as fold induction, where 1.0-fold is arbitrarilyestablished for luciferase activities generated by vector control cells.Data are representative of 2 independent experiments.

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siveness to TGF-β. Conversion of tumor cells from TGF-β–responsive to TGF-β–nonresponsive may occur at severallevels, involving defects in TGF-β signaling pathway com-ponents, transcriptional regulators, and cell cycle regulators(38,47–49). As reported here, MDA-MB-435 cells culturedin low 2% serum lack response to the growth-regulatoryproperties of TGF-β. This is in contrast to a previous studyshowing that MDA-MB-435 cells could respond to an exog-enous source of TGF-β by undergoing growth inhibitionas measured by [3H]thymidine uptake or cell number (50).Possible explanations for the differences in ability of MDA-MB-435 cells to respond to exogenous TGF-β include varia-tions in passage number of cells being studied, presence orabsence of various growth factors in the culture medium,and variations in commercial sources of TGF-β. MDA-MB-231 cells are reported to lose responsiveness to TGF-βafter Passage 35 (51). Likewise, late-passage (>500) MCF-7 cells have been reported to not exhibit growth inhibitionby TGF-β1 or TGF-β2 (52). Thus, as is well documented,TGF-β displays pleiotrophic effects that are dependent oncell type, cell density, and cellular context (53).

The inability of MDA-MB-435 cells to undergo growtharrest in response to exogenous TGF-β is also in contrast tothe ability of these cells to respond to VES by upregulatingautocrine TGF-β signaling by increasing the expression ofbiologically active TGF-β via conversion of latent TGF-β tobiologically active TGF-β and increasing TGF-β receptor IIexpression (7,8). Furthermore, VES-induced apoptosis ofMDA-MB-435 cells is mediated, at least in part, by theTGF-β-MAPK signaling pathways (1,9).

Although TGF-β signaling plays a role in VES-inducedapoptosis (9), data presented here show that VES inhibitionof growth in MDA-MB-435 cells does not involve TGF-βbut, rather, involves activation of the CDKI p21Waf1/Cip1 in aTGF-β-independent manner. Furthermore, and perhapsmore important to the aggressive metastatic phenotype ofthese cells, data presented here show that although MDA-MB-435 cells are nonresponsive to the growth-arrest actionsof TGF-β, they retain some type of responsiveness to exoge-nous TGF-β, in that GFP-Smad2 is signaled to translocatefrom the cytosol to the nucleus, and the artificial SBE-Lucreporter construct is rapidly activated. Taken together, thesedata suggest that MDA-MB-435 breast cancer cells have lostthe ability to respond to TGF-β-mediated growth-arrest sig-nals but have retained certain aspects of TGF-β ligand-activated receptor-mediated Smad signaling. Whether thisaltered TGF-β responsiveness is critical to measures of tu-mor cell aggressiveness, for example, enhanced anchorage-independent growth, growth factor independence, invasion,and motility, remains to be determined.

Although treatment of MDA-MB-435 cells with exoge-nous TGF-β1 activated Smads and a TGF-β-dependent con-struct containing SBEs, two TGF-β-regulated reporterconstructs, PAI-1-Luc and p21-Luc, were not activated aftertreatment with TGF-β1. Additionally, TGF-β1 treatment ofMDA-MB-435 cells did not induce p21 protein expression(data not shown). Taken together, this information suggests

that the cell growth refractivity of MDA-MB-435 cells toTGF-β is not due to a failure of TGF-β ligand-activated re-ceptor-mediated Smad activation events but, rather, to someyet to be defined nuclear signaling event(s).

In summary, these data show that VES induces tumor cellgrowth inhibition of human breast cancer cells via TGF-β-and p53-independent activation of p21Waf1/Cip1. Furthermore,these studies show that although MDA-MB-435 carcinomacells are refractory to exogenous TGF-β-mediated negativegrowth control, they are capable of responding to exogenousTGF-β signals by translocating GFP-Smad2 from the cyto-plasm to the nucleus and by activating an SBE-regulated re-porter gene, suggesting that these cells may serve as a usefulmodel for trying to dissect how altered TGF-β signaling maycontribute to the more aggressive metastatic tumor pheno-type exhibited by MDA-MB-435 cells.

Acknowledgments and Notes

The authors thank Barbara Goettgens (Microscopy Laboratory, Insti-tute for Cellular and Molecular Biology, University of Texas, Austin, TX)for assistance in generating the GFP-Smad2 nuclear translocation data andDr. Kent Claypool (M. D. Anderson Cancer Center, Science Park-Research Division, Smithville, TX) for assistance in generating the FACScell cycle data. This work was supported by National Cancer InstituteGrant CA-59739, National Institute for Environmental Health SciencesGrant ES-07784, and the Foundation for Research. Address correspon-dence to K. Kline, Div. of Nutrition/A2703, University of Texas at Austin,Austin, TX 78712-1097. Phone: (512) 471-8911. FAX: (512) 232-7040.E-mail: k.kline@mail.utexas.edu.

Submitted 10 December 2001; accepted in final form 25 March 2002.

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