Vol. 7, 31 1-318, March 1996 Cell Growth & Differentiation 311

BcI-2 Expression Regulates Sodium Butyrate-inducedApoptosis in Human MCF-7 Breast Cancer Cells1

Mahitosh Mandal and Rakesh Kumar�

Departments of Medicine [M. M., A. K.) and Cellular and MolecularPhysiology ER. K.], Pennsylvania State University College of Medicine,Hershey, Pennsylvania 17033

AbstractSodium butyrate (butyrate) is a potent growth inhibitorand differentiating agent for many cell types, includingbreast cancer cells. Programmed cell death, orapoptosis, is a physiological mechanism of cell deaththat is dependent on both preexisting proteins and denovo protein synthesis. In the studies presented here,we investigated the role of apoptosis in the growthregulation of human MCF-7 breast cancer cells bysodium butyrate. We report that butyrate treatment ofbreast cancer MCF-7 cells causes a nonreversiblegrowth inhibition by inducing apoptosis in a time- anddose-dependent manner. Treatment of MCF-7 cells foras little as 12 h with butyrate caused a 5.6-foldinduction in apoptotic cell death, which continued toincrease up to 27-fold by 48 h treatment The butyrate-induced apoptosis in MCF-7 cells was closely linkedwith the down-regulation of expression of Bcl-2 mRNAand Bcl-2 protein, a gene product known to beinvolved in the regulation of apoptosis in mammaliancells. The observed relationship between the down-regulation of Bcl-2 and induction of apoptosis was notcausal because stable overexpression of BcI-2 resultedin protection of MCF-7 cells from the cytotoxicmorphological changes and growth-inhibitory effects ofbutyrate (15% growth inhibition compared to 60%growth Inhibition in the parental cells). In addition, Bcl-2-overexpressing MCF-7 cells exhibited a significantsuppression in butyrate-induced stimulation ofapoptosis (5-fold increase in apoptosis compared to27-fold in parental MCF-7 cells). These findingsdemonstrate that the levels of Bcl-2 expressionregulate the butyrate-induced apoptosis in breastcancer cells and that butyrate may potentially be usefulin sensitizing the breast cancer cells to chemotherapy-induced apoptosis.

Received 8/30/95; revised 12/1/95; accepted 12/20/95.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to mdi-cate this fact.1 This study was supported in part by the American Institute for CancerResearch Grant 94B93.2 To whom requests for reprints should be addressed at, Department ofMedicine/Oncology Division, Hershey Medical Center, P.O. Box 850, Her-shey, PA 17033.

Introduction

Programmed cell death, or apoptosis, is a physiologicalmechanism of cell death that is dependent on both preex-sting proteins and de novo protein synthesis (1-3). Apopto-sis plays an important role during development, metamor-phosis, organ involution, and in many diseases, includingcancer (1 , 2). Apoptosis is characterized by nuclear conden-sation and fragmentation and degradation of DNA into oh-gonucleosome fragments (1). Regulation of apoptosis is acomplex process and involves a number of cellular genes,including BcI-2 (4, 5) and Bc/-2-related family members such

as BcI-XL, Bc!-X�, and Bax (6-8). The Bc!-2 (B-cell Ieukemia/lymphoma 2) gene was first identified at the breakpoint of achromosomal translocation t(14;18) in B follicular lymphoma(9). The BcI-2 gene encodes a protein of Mr 26,000 thatprotects cells against apoptosis in a variety of experimentalsystems. Overexpression of Bcl-2 has been shown to sup-press the initiation of apoptosis in response to a number ofstimuli, including anticancer drugs (1 0-1 4). Furthermore, in-hibition of Bcl-2 expression by antisense ohigonucleotide (1S,16), dominant-negative inhibitor Bcl-Xs (8), and dexametha-sone (1 7, 1 8) has been shown to promote apoptosis and alsosensitize cells to chemotherapy-induced apoptosis. Thuscancer cells may primarily depend on Bcl-2 or related familymembers to prevent cell death. Recent studies have mdi-

cated that cells from a variety of human cancers includingbreast may have a decreased ability to undergo apoptosis inresponse to some physiological stimuli (2, 19), and thus adefect in apoptosis may be involved in the aberrant survivaland/or development of cancer. Therefore, identification ofagent(s) that can negatively regulate the expression of theBcl-2 pathway in breast cancer cells, and thus trigger apop-tosis, will provide a therapeutic approach to inhibit breastcancer cell growth.

Dietary factors play a vital role both in the development

and prevention of human cancers, including breast cancer(20, 21). Exploration of dietary factors and their active mi-cronutnents with growth-inhibitory properties on human tu-mor cells could provide new tools for prevention and treat-ment of human cancer. One of such dietary micronutrient issodium butyrate, the major short-chain fatty acid producedby fermentation of dietary fiber(22, 23). Sodium butyrate andits more stable derivatives, such as monobut-3 (24, 25), areknown to influence several physiological processes, inhibitthe growth of many cell types, including breast cancer cells(24, 25), and induce markers of differentiation, such as milk-related glycoprotemn and lipid deposition in breast cancercells (26, 27). In addition, studies in recent years have dem-onstrated the ability of sodium butyrate to cause apoptosis inhuman colorectal cancer and lymphoid cells (28-30). Al-though sodium butyrate has been shown to be a potent

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Fig. 1. Sodium butyrate inhibits growth ofbreast cancer cells and induces apoptosis inMCF-7 cells. A, breast cancer cells were treatedwith () or without (D) 3 mM butyrate for 48 h.Cell growth was measured by MU assay, andresults are presented as a percentage of controlfor all three cell lines. The data shown is repre-sentative of four experiments; bars, SEM. B andC, induction of apoptosis in MCF-7 cells treatedwith sodium butyrate. Cells were treated witheither different doses of sodium butyrate for 24h (B) or with 3 mM sodium butyrate for differenttimes (C). Cytoplasmic extracts were preparedand used to quantitate apoptotic cell death byusing an EUSA assay as described in the “Ma-terials and Methods.” Each treatment was per-formed in triplicate, and results shown are rep-resentative of three experiments with similarresults. Results are presented as relative foldincrease in apoptotic cell death as compared tocontrol untreated cells.

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growth inhibitor and inducer of apoptosis in some cancer celltypes, the biochemical mechanism of sodium butyrate-induced apoptosis and its role in human breast cancer re-mains unexplored.

In the studies presented here, we investigated the possiblerole of apoptosis in the growth inhibition of human breastcancer cells by sodium butyrate. We report that the sodiumbutyrate-induced apoptosis in MCF-7 breast cancer cells ismediated by down-regulation of Bcl-2, and overexpressionof Bcl-2 leads to suppression of sodium butyrate-inducedapoptosis and growth inhibition. In addition, treatment withbutyrate sensitizes MGF-7 cells to chemotherapy-inducedapoptosis.

ResultsIn our initial experiments, three human breast cancer celllines were examined for growth inhibition by 48-h treatment

with butyrate. Fig. 1A shows that MGF-7 cells were mostsensitive (75% growth inhibition compared to the growth ofuntreated cells), and ZR-75-R cells were least sensitive (24%growth inhibition compared to the growth of untreated cells)to growth inhibition by sodium butyrate. The growth inhibi-tion of breast cancer cells by sodium butyrate was not re-

hated to the presence or absence of estrogen receptor be-

cause sodium butyrate also inhibited the growth of estrogenreceptor-negative MDA-MB-231 cells. During these experi-ments, we consistently observed that a significant number ofMGF-7 cells started rounding-up and changing their mor-phology between 36-48 h after sodium butyrate treatment.We were intrigued by the possibility of apoptosis in the actionof sodium butyrate in MGF-7 cells. To test this, we exploredthe effect of sodium butyrate on apoptotic cell death using aquantitative ELISA assay that measures cytoplasmic his-tone-bound DNA complexes generated during apoptoticDNA fragmentation (31-33). In the past, classical DNA lad-denng had not been observed in mammary epithehial cells(such as MGF-7) undergoing apoptosis (34, 35), and ELISAassay used here has been a method of choice to quantitateapoptosis in breast cancer cells (32, 33). As shown in Fig. 1,B and C, sodium butyrate treatment of MGF-7 stimulatedapoptosis in a dose- and time-dependent manner. As little as12 h treatment with sodium butyrate caused a 5.6-fold in-

crease in apoptotic cell death, which continued to increaseup to 27-fold by 48 h after treatment. To examine whether theobserved growth inhibition of breast cancer cells by sodiumbutyrate was related to the induction of apoptosis, the quan-titation of apoptotic cell death was also performed in ZR-75-A cells treated with or without sodium butyrate. Results

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Fig. 2. Down-regulation of Bcl-2 expression by sodium butyrate inMGF-7 cells. A, time course of inhibition of Bcl-2 expression in MGF-7cells by sodium butyrate. Cells were treated with or without sodiumbutyrate (3 mM). Cell lysates were made, and equal amounts (20 �g) ofprotein were immunoblotted with Bcl-2 mAb. As an internal control, theupper part of the same blot was blotted with an unrelated HSP-40 Ab.Lane 1, control; Lane 2, 1 .5 h; Lane 3, 3 h; Lane 4, 6 h; Lane 5, 12 h; Lane6, 24 h. B, effect of sodium butyrate on Bcl-2 expression as a function ofdose. Cells were treated with or without sodium butyrate for 1 6 h. Celllysates (20 ,.Lg protein) were immunoblotted with Bcl-2 mAb and HSP-40Ab as described above. Bcl-2 signal was detected by using 1251-labeledprotein A. Lane 1, control; Lane 2, 1 mM; Lane 3, 3 mM; Lane 4, 6 m�. C:Top, inhibition of Bcl-2 mRNA by sodium butyrate. MCF-7 cells weretreated with 3 mM sodium butyrate for 24 h. Total cellular RNA wasisolated. Twenty �.tg RNAS were analyzed by Northern blotting using a32P-labeled cDNA fragment (0.91 kb) specific for human Bcl-2 mANA.Right, position of Bcl-2 mRNA. The Northern blot was rehydndized with anunrelated cDNA probe for actin mRNA. Bottom, ethidium bromide-stainedrRNA bands. These experiments were repeated three times.

Gell Growth & Differentiation 313

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indicated that treatment of ZA-75-A cells with sodium bu-

tyrate (3 mM; 24 h) resulted in only a 3.8-fold increase in

apoptotic cell death, suggesting that the inhibitory effect ofbutyrate in MCF-7 cells was probably associated with its

ability to induce apoptosis.

Because apoptosis in mammalian cells has been shown tobe regulated by Bcl-2 (4) and because of the fact that BcI-2inhibition promoted apoptosis (1 7, 18), we investigatedwhether the sodium butyrate-induced apoptosis in MCF-7

cells was related with the possible modulation in the levels of

BcI-2. To test this possibility, we examined the expression of

Bcl-2 by Western immunoblotting in MCF-7 cells treated with

or without butyrate. As shown in Fig. 2, A and B, it was

interesting to note that treatment of MCF-7 cells with sodiumbutyrate resulted in down-regulation of Bcl-2 expression in a

time-dependent manner starting 3 h after treatment. Treat-

ment with sodium butyrate for 6-1 2 h was sufficient to inhibit

the levels of Bcl-2 to a significant extent (40-60% inhibition

compared to the levels in untreated cells), and this cellulareffect of sodium butyrate was also dose dependent (Fig. 2B).

Results of other experiments demonstrated that treatment

(48 h) with low doses of sodium butyrate (0.5 or 1 mM) also

down-regulated (20-42%, compared to the levels in un-

treated cells) the Bcl-2 expression in MCF-7 cells.3 The ob-

served down-regulation of Bcl-2 expression was a specific

effect of sodium butyrate because there was no influence of

sodium butyrate on the levels of an unrelated HSP-40.4 Blots

in Fig. 2, A and B, were cut into two pieces, and upper

portions were immunoblotted with a HSP-40 Ab (36) as aninternal control within the same blot. To determine whetherthe observed down-regulation of BcI-2 expression in sodiumbutyrate-treated MCF-7 cells was associated with de-creased expression of Bcl-2 mANA, Northern blot analysiswas performed. Fig. 2C shows that 16 h treatment withsodium butyrate inhibited the steady-state levels of Bcl-2mANA by 75% compared to the levels in untreated control

cells. In brief, results from Figs. 1 and 2 indicated that the

observed sodium butyrate-induced down-regulation of Bcl-2expression was an early event that correlated with the period

of induction of apoptosis, suggesting that sodium butyratecan inhibit a gene product known to be involved in apoptosis.

To further examine the possible involvement of Bcl-2 in the

induction of apoptosis and growth inhibition of MCF-7 cells

by butyrate, we examined whether overexpression of Bcl-2

will modulate the sensitivity of MCF-7 cells to the inhibitoryeffect(s) of sodium butyrate. For these studies, MCF-7 cellswere transfected with a Bcl-2 expression vector (1 3). A num-

ber of clonal cell lines stably overexpressing Bcl-2 were

generated. Fig. 3A shows the levels of Bcl-2 protein in two

such clones, MCF-7/BCL2-1 and MCF-7/BCL2-2 cells,

which overexpress 2.7- and 1 .9-fold higher levels of Bcl-2,respectively, compared to the levels in the parental MCF-7

cells. Results in Fig. 3B show the effect of overexpression of

Bcl-2 on the sensitivity of MCF-7 cells to butyrate. As shownin Fig. 3C, both MCF-7/BCL2-1 and MCF-7/BCL2-2 cells

were protected from the growth-inhibitory action of butyrate

to a significant extent compared to growth inhibition inMCF-7 cells. The observed partial protection of cells wasdependent on the duration of treatment, with maximum pro-

tection observed by 48 h after treatment (1 5-25% growth

inhibition in clones compared to 60-75% growth inhibition

MCF-7 cells). The observed suppression of growth inhibition

of MCF-7 cells overexpressing Bcl-2 by sodium butyrate wasnot due to G41 8 selection because MCF-7 cells transfected

with the neo vector alone (MCF-7/neo cells) demonstrated

no change in the levels of Bcl-23 and were also sensitive to

growth inhibition (72%±5) and induction of apoptosis (26-fold ± 7) by 48 h treatment with 3 m�i sodium butyrate.

Since Bcl-2 overexpression reduced the growth-inhibitory

effect of sodium butyrate, we also examined the sensitivity of

BcI-2-overexpressing MCF-7 cells to the cytotoxic effects ofsodium butyrate by the criteria of morphological changes.

Aesults in Fig. 4 show that MCF-7/BCL2-1 cells were dis-tinctively protected from the cytotoxic morphological

changes observed in MCF-7 cells treated with sodium bu-tyrate (3 mM; 48 h treatment). Similar results were also ob-

tamed with MCF-7/BCL2-2 cells.3 The observed protection

3 Unpublished observations.4 The abbreviations used are: HSP, heat shock protein; Ab, antibody;mAb, monoclonal antibody; MiT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-nyltetrazolium bromide.

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McF.7 McF-7IacL2-1 MCF-7/BCL2�2

314 Butyrate-induced Apoptosis in Breast Cancer Cells

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Fig. 3. Effect of sodium butyrate on the growth of MCF-7 cells andMCF-7 cells overexpressing Bcl-2. A, expression of BcI-2 in clones over-expressing Bcl-2. B, kinetics of growth inhibition of MCF-7 cells andclones overexpressing Bcl-2 by sodium butyrate. MCF-7, MCF-7/BCL2-1 , and MCF-7/BCL-2-2 cells (3 x 1 o� cells) were plated into eachwell of a 48-well plate. After 24 h (time 0), some cells were treated inquadruplicate either without (LI) or with 3 m� sodium butyrate for 4 days(U), 3 days (a), 2 days (D), or 1 day (�). On day 4, cell growth wasdetermined by MU assay. Results are presented as a percentage ofcontrols for each cell line. Results shown are representative of four ex-

periments; bars, SEM.

of MCF-7/BCL2-1 cells against the inhibitory effects of so-dium butyrate was also quantitated by the possible revers-ibility of cell growth after 48 h treatment with sodium butyrate(3 mM) over a period of an additional 4 days. Aesults mdi-cated that the removal of sodium butyrate after 48 h treat-ment had no beneficial effect on the survival of MCF-7 cellscompared to MCF-7 cells treated continuously with sodiumbutyrate (95% ± 7%; growth inhibition at day 6). In contrast,

the removal of sodium butyrate after 48 h treatment of MCF-7/BCL2-1 cells resulted in a 27% increase in cell growth(48% ± 3%; growth inhibition at day 6) as compared to

MCF-7/BCI2-1 cells treated continuously with sodium bu-

tyrate (75% ± 5%; growth inhibition at day 6).

Next we examined the effect of Bcl-2 overexpression inMCF-7 cells on the ability of sodium butyrate to induceapoptosis. Aesults in Fig. 5 show that Bcl-2 overexpression

in MCF-7 cells leads to significant suppression in the sodium

butyrate-induced stimulation of apoptosis (5-fold increase inapoptosis compared to 27-fold apoptosis in the parentalMCF-7 cells by 48 h sodium butyrate treatment). Takentogether, results from Figs. 3-5 suggest that the observedprotective effects of Bcl-2 overexpression against inhibitoryeffects of butyrate may be closely linked with the ability of

sodium butyrate to induce apoptosis in MCF-7 cells.

Since overexpression of Bcl-2 in MCF-7 cells resulted insuppression but not complete prevention of sodium bu-

tyrate-induced apoptosis and growth inhibition, we exam-

med the effect of sodium butyrate on BcI-2 expression inMCF-7 clones. Aesults in Fig. 6 show that sodium butyrate (3

mM; 24 h) also down-regulated the levels of Bcl-2 in MCF-7cells overexpressing Bcl-2 (45% inhibition in Bcl-2 expres-sion as compared to the levels in untreated cells; Fig. 6A,Lanes 7 and 8) but to a lesser extent compared to down-

regulation of Bcl-2 in MCF-7 cells (Fig. 6, Lanes 1 and 2) or

MCF-7/Neo cells (Fig. 6, Lanes 3 and 4; up to 80% inhibitionin Bcl-2 expression). In brief, these results suggested that the

levels of Bcl-2 expression play a regulatory role(s) in sodium

butyrate-induced apoptosis in MCF-7 cells; perhaps cellsneed a threshold level of Bcl-2 to protect against apoptosisin response to sodium butyrate treatment in MCF-7 breastcancer cells.

Since Bcl-2 has been shown to protect cells by inhibitingthe induction of apoptosis triggered by several cytotoxicagents (12, 14) and because of the fact that sodium butyratedown-regulated Bcl-2 expression (this study), we explored

the effect of sodium butyrate on the modulation of sensitivityof MCF-7 cells to induce apoptosis in response to doxoru-

bicin, a commonly used chemotherapeutic agent. For theseexperiments, we have selected a dose (0.5 mM) of sodium

butyrate that alone did not induce apoptosis.3 Aesults inTable 1 show that cotreatment of MCF-7 cells with sodium

butyrate, which down-regulated the levels of Bcl-2, in-

creased the sensitivity of cells to the induction of apoptosis.

In the past, 72 h treatment of MCF-7 cells with doxorubicin

has been shown to induce about 50% or less increase in

apoptotic cell death compared to control cells (35).

DiscussionApoptosis is a physiological mechanism of cell death thatplays an important role during embryonic development, or-gan involution, and in diseases like cancer (1-4). Apoptosisis regulated by specific cellular pathways, including Bcl-2 (4,

5). A number of recent studies suggested that the overex-

pression of Bcl-2 protects cells against apoptosis (1 1 , 14,37), and inhibition of Bcl-2 expression promotes apoptosis

(1 7, 1 8) in response to a number of stimuli. Therefore, cancer

cells may depend on BcI-2 or related family members toprevent apoptosis, and a defect in regulation of apoptosis

may be possibly involved in the aberrant survival and/ordevelopment of cancer. The ability of Bcl-2 to inhibit apop-tosis and its relationship with cancer, provided by transgenic

studies involving the BcI-2-Ig locus as a transgene, lendsupport for this model (38). In recent years, approachesaimed to modulate the expression of cellular apoptotic path-ways such as BcI-2 are subject of active investigation in a

effort to control cancer cell growth. The present investigation

was undertaken to explore the role of apoptosis and its

possible modulation by Bcl-2 expression in the growth inhi-

bition of human MCF-7 breast cancer cells by sodium bu-tyrate, a potent growth inhibitor and inducer of differentiation

that is produced by fermentation of dietary fiber (22). Wehave selected the MCF-7 cell system for experimental con-venience as well as to enable us to take advantage of the

wealth of knowledge available about its biology.

The results presented here demonstrated that the treat-

ment of MCF-7 cells with sodium butyrate induced apoptosis

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Cell Growth & Differentiation 315

Fig. 4. Effect of sodium butyrate on the morphol-ogy of MCF-7 (A and B) and MCF-7/BCL2-i (Cand D) cells. Cells were cultured for 48 h in theabsence (Con, A and C) or presence (B and D) of3 mM sodium butyrate (+BA). Phase-contrastphotomicrographs are shown.

Fig. 5. Overexpression of Bcl-2suppresses sodium butyrate-in-duced apoptosis in MCF-7 cells.Equal numbers (2 x i0�) ofMCF-7, MCF-7/BCL2-1 , andMCF-7/BCL2-2 cells weretreated with sodium butyrate (3mM)for48 h (U), 24 h (�), orO h(0). Cytoplasmic extracts weremade to quantitate the apoptoticcell death as described in Fig. 1.All results are presented as foldincrease in apoptotic cell deathcompared to MCF-7 cells as abase value. Note that control un-treated Bcl-2-overexpressingMCF-7 cells were also protectedfrom the spontaneous apoptosiscompared to control MCF-7cells. These experiments wererepeated three times; bars, SEM.

and growth inhibition, and this was closely linked with the

down-regulation of Bcl-2 expression. In addition, overex-

pression of Bcl-2 in MCF-7 cells resulted in significant inhi-bition of sodium butyrate-induced apoptosis and also pro-tected the MCF-7 cells against the inhibitory effects of

sodium butyrate. The observed down-regulation of Bcl-2

expression by sodium butyrate may also provide the bio-

chemical basis of some of the known growth-inhibitory ac-

tions of sodium butyrate in MCF-7 cells (24-27). The finding

that sodium butyrate inhibited the expression of Bcl-2, agene product involved in apoptosis, is important because it

suggests that a dietary micronutrient can modulate the ox-

pression of a specific apoptotic regulatory pathway. In the

past, there was precedence to correlate the levels of Bcl-2

with the induction of apoptosis. In this context, it is important

to note that dexamethasone and interleukin 6 have been

shown recently to down-regulate Bcl-2 expression and in-

duce apoptosis in myeloid leukemic cells (1 7, 1 8). The mech-

anism of observed down-regulation of Bcl-2 by sodium bu-

tyrate remains to be delineated. This may occur at the

transcription level and/or posttranscription level and may

also involve reduced BcI-2 mANA stability, leading to a de-

crease in Bcl-2 expression.The observed temporal relationship between the down-

regulation of Bcl-2 expression and induction of apoptosis in

MCF-7 cells was not causal because stable overexpressionof Bcl-2 in MCF-7 cells resulted in a significant suppression

of sodium butyrate-induced stimulation of apoptosis and

inhibitory effects. These results are consistent with the pro-vious studies (1 1 , 14, 37), and support the notion of a pro-

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sponse to a chemotherapeutic agent(s) and opens an interest-

ing area of investigation to further explore the possible thera-

316 Butyrate-induced Apoptosis in Breast Cancer Cells

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Fig. 6. Effect of sodium butyrate on the levels of Bcl-2 in MCF-7 cellswith or without BCL2 overexpression. Cells were treated with (+) orwithout (-) 3 mM sodium butyrate for 24 h. Cell lysates were made, andequal amounts (20 �.tg) of protein were analyzed by immunoblotting witha Bcl-2 mAb, followed by signal development using 1251-labeled protein A(A and B resulted from exposure of the same blot for 3- and 12-hexposures, respectively). The upper portion of Bcl-2 blot was immunob-lotted with a HSP-40 Ab (C). D, immunoblotting of above samples with apolyclonal actin antibody (Sigma). Lanes 1 and 2, MCF-7 cells; Lanes 3and 4, MCF-7/Neo cells; Lanes 5 and 6, MCF-7/BCL2-2 cells; Lanes 7 and8, MCF-7/BCL2-i cells.

Table 1 Sodium butyrate potentiates doxorubicin-induced apoptosis inMCF-7 cells

TreatmentRelative fold increase in apoptosis#{176}

(over control)

Control 1

Doxorubicin (100 flM) 1.13 � 0.09

Sodium butyrate (500 flM) 1 .28 ± 0.08

Doxorubicin 9100 flM) + 3.0 � 0.36sodium butyrate (500 nM)

a DNA fragmentation was measured by quantitative ELJSA assay after 72

h treatment; SD (n = 4).

tective role(s) of Bcl-2 against apoptosis in breast cancercells. It was curious to note that although MCF-7/BCL2-1cells overexpress only 2.7-fold higher levels of Bcl-2 corn-

pared to the levels in MCF-7 cells, they demonstrated a

significant suppression in sodium butyrate-induced apopto-

sis, implying that a small change in the levels of Bcl-2 overthe presumptive apoptotic threshold levels could possibly

modulate the induction of apoptosis. In addition, it will beinteresting to explore whether sodium butyrate can alsomodulate the expression of other members of genes that

either suppress induction of apoptosis, such as Bcl-XL (6), orpromote apoptosis, such as Bcl-X� and Bax (7, 8), and such

efforts are under way.

Our finding that treatment with butyrate, which down-regu-

lated the Bcl-2 expression, could potentiate the sensitivity ofMCF-7 cells to undergo apoptosis in response to doxorubicin

provides further support to a regulatory role of Bcl-2 in the

modulation of apoptosis in human breast cancer cells in re-

peutic potential of sodium butyrate and/or a more stable

analogue ofsodium butyrate in the development of strategies to

control the growth of human breast cancer cells.

Materials and MethodsCell Lines and Growth Assays. Human breast cancer MCF-7 cells (39)were maintained in DMEM-F12 (1:1) supplemented with 10% FCS. Otherhuman breast cancer cells (ZR-75-R and MDA-MB-231) were obtained

from the American Type Culture Collection and were grown in L-15medium supplemented with 10% FCS. Cell growth was measured by theMiT dye (Sigma Chemical Co.) uptake procedure, as described previ-

ously (40, 41). About 2 x 1 o� cells/0.5 ml culture medium were seededinto each well of a 48-well plate. After 24 h, the medium was changed, andappropriate cultures were supplemented with sodium butyrate. For eachpoint, the medium was removed from triplicate wells, MU dye (5 mg/mI

in PBS) was added, and the plate was wrapped in aluminum foil and

incubated at 37#{176}Cfor 4 h. Dye taken up by living cells was extracted inisopropyl alcohol:1 N HCI (96:4) for the determination of absorbance at a

wavelength of 570 nm, and results are presented as a percentage ofcontrol.

Quantitation of Apoptosis. To measure apoptotic cell death, we useda “cell death” ELISA (Boehringer Mannhein, Indianapolis, IN) that measure

cytoplasmic histone-bound DNA fragments (mono- and oligonucleo-somes) generated during apoptotic DNA fragmentation (31-33) and not

free histone or DNA that could be released during nonapoptotic cell death(42). Results obtained by this ELISA assay has been shown to correlatewith those obtained by other methods (31). MCF-7 cells or MCF-7 clones

overexpressing Bcl-2 (2 x 10�) cells were plated into each well of a 48-well

plate. After desired treatment, cytoplasmic extracts were made from bothfloating and attached cells, according to manufacturer’s protocol. Controland drug-treated cell extracts were equalized on the basis of equal cell

number as well as protein in extracts. Briefly, wells were first coated with

anti-histone antibody, loaded with cytoplasmic extracts, and followed byincubation with anti-DNA second antibody conjugated with peroxidase.ELISA was developed with peroxidase substrate, and the absorbance at

405 nm was measured using Microplate autoreader (Bio-Tek Instruments).Preparation of Cell Extracts. All experiments were performed with

cells in logarithmic phase by controlling the plating density. The viability of

cells was assayed by trypan blue dye exclusion. Cells were treated with orwithout sodium butyrate (Sigma); extracts were prepared as described

(43). Briefly, cells were washed twice with cold PBS on ice and lysed inbuffer (50 mM Tris-HCI (pH 7.5), 120 m�i NaGI, 0.5% NP4O, 100 m�.i NaF,200 �M NaVO5, 1 mM phenylmethylsulfonyl fluoride, and 1 0 �g/ml eu-peptin) for 15 mm on ice. The lysates were centrifuged in an Eppendorfcentrifuge at 4#{176}Cfor 15 mm. The protein concentration in extracts was

determined by using the Bio-Rad kit.Immunoblotting. Cell lysates containing an equal amount of total

protein (40 ig) were resolved on a SDS-PAGE, followed by transfer ontonitrocellulose (41 , 44). Membranes were blocked in 2% BSA in TBS [10

mM Tris-HCI (pH 7.5), 150 mM NaCI], followed by probing with eitheranti-Bcl-2 mAb (Dako or Neomarkers, Inc.) or anti-HSP-40 Ab (36), andimmune complexes were detected by using a secondary antibody-based

alkaline phosphatase color reaction or 1251-labeled protein A. Quantitationof specific protein bands was performed by using a protein databasesscanner (Molecular Dynamics). As a internal control, the blot was alwayscut into two pieces after transfer of proteins. The lower portion of blot was

probed with anti-Bcl-2 mAb and the upper portion with an unrelated

antibody. Low-molecular-mass markers (Amersharn Corp.) were used asstandard.

Northern Analysis. Total cellular RNA was isolated and analyzed by

Northern blot methods as described previously (44). Briefly, 20 �g ANAS

denatured in the presence of formaldehyde were fractionated on a 1 %agarose formaldehyde gel and transferred to a nitrocellulose filter, withhybridization and washing as described previously (44). Loading of theRNA was monitored by intensities of ethidium bromide-stained rRNAbands (28 5 and 18 5). The quantification of the specific signal wasperformed by densitometric scanning of the autorads with necessary

precautions to ensure a linear response between different autorad expo-

sures of the same blot being taken. The specific cDNA clone (clone pB4;Ref. 45) for human Bcl-2 mRNA was from the American Type Culture

Cell Growth & Differentiation 317

Collection, and the 0.91-kb EcoRI fragment was used for hybridization

experiments. As a negative control, the filter was rehybridized with an

unrelated actin cDNA probe (43).Expression of Bcl-2 in MCF-7 Cells. MCF-7 cells at a density of 106

cells/i 00-mm diameter plate were transfected with plasmid DNA contain-ing the full-length human Bcl-2 cDNA and a selectable marker, neomycinphosphotransferase gene (pSSFbcl-2; Ref. 13) by calcium phosphateprecipitation procedures (46). At 24 h aftertransfection, selection medium

containing 1 mg of G418, a neomycin analogue, per ml was added. Sevento 10 days of culturing in this medium killed most of the cells, but a few

colonies of cells, one to five per plate, grew. Well-separated colonies wereharvested by trypsinization in cloning cylinders and were propagated inthe presence of G418. Expression of BcI-2 in individually isolated clones

was determined by immunobotting with BcI-2 mAb. As a control, we have

used MCF-7 cells stably transfected with vector containing only neomy-cm. Once a stable cellline from each clone had been established, the drugwas removed from the culture medium. The clonal lines have been main-

tamed in drug-free medium since then.

AcknowledgmentsWe thank Stanley J. Korsmeyer for providing BcI-2 expression vector, C.Kent Osborne for MCF-7 ciones, Kenzo Ohtsuka for HSP-40 Ab, Nec-markers for anti-BcI-2 mAb, and Neeta Shamia for technical assistance.

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