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

Transcription

1 Vol. 7, , Mrch 1996 ell Growth & Differentition 311 BcI-2 Expression Regultes Sodium Butyrte-induced Apoptosis in Humn MF-7 Brest ncer ells1 Mhitosh Mndl nd Rkesh Kumr Deprtments of Medicine [M. M., A. K.) nd ellulr nd Moleculr Physiology ER. K.], Pennsylvni Stte University ollege of Medicine, Hershey, Pennsylvni 1733 Abstrct Sodium butyrte (butyrte) is potent growth inhibitor nd differentiting gent for mny cell types, including brest cncer cells. Progrmmed cell deth, or poptosis, is physiologicl mechnism of cell deth tht is dependent on both preexisting proteins nd de novo protein synthesis. In the studies presented here, we investigted the role of poptosis in the growth regultion of humn MF-7 brest cncer cells by sodium butyrte. We report tht butyrte tretment of brest cncer MF-7 cells cuses nonreversible growth inhibition by inducing poptosis in time- nd dose-dependent mnner. Tretment of MF-7 cells for s little s 12 h with butyrte cused 5.6-fold induction in poptotic cell deth, which continued to increse up to 27-fold by 48 h tretment The butyrteinduced poptosis in MF-7 cells ws closely linked with the down-regultion of expression of Bcl-2 mrna nd Bcl-2 protein, gene product known to be involved in the regultion of poptosis in mmmlin cells. The observed reltionship between the downregultion of Bcl-2 nd induction of poptosis ws not cusl becuse stble overexpression of BcI-2 resulted in protection of MF-7 cells from the cytotoxic morphologicl chnges nd growth-inhibitory effects of butyrte (15% growth inhibition compred to 6% growth Inhibition in the prentl cells). In ddition, Bcl- 2-overexpressing MF-7 cells exhibited significnt suppression in butyrte-induced stimultion of poptosis (5-fold increse in poptosis compred to 27-fold in prentl MF-7 cells). These findings demonstrte tht the levels of Bcl-2 expression regulte the butyrte-induced poptosis in brest cncer cells nd tht butyrte my potentilly be useful in sensitizing the brest cncer cells to chemotherpyinduced poptosis. Received 8/3/95; revised 12/1/95; ccepted 12/2/95. The costs of publiction of this rticle were defryed in prt by the pyment of pge chrges. This rticle must therefore be hereby mrked dvertisement in ccordnce with 18 U.S.. Section 1734 solely to mdicte this fct. 1 This study ws supported in prt by the Americn Institute for ncer Reserch Grnt 94B93. 2 To whom requests for reprints should be ddressed t, Deprtment of Medicine/Oncology Division, Hershey Medicl enter, P.O. Box 85, Hershey, PA Introduction Progrmmed cell deth, or poptosis, is physiologicl mechnism of cell deth tht is dependent on both preexsting proteins nd de novo protein synthesis (1-3). Apoptosis plys n importnt role during development, metmorphosis, orgn involution, nd in mny diseses, including cncer (1, 2). Apoptosis is chrcterized by nucler condenstion nd frgmenttion nd degrdtion of DNA into ohgonucleosome frgments (1). Regultion of poptosis is complex process nd involves number of cellulr genes, including BcI-2 (4, 5) nd Bc/-2-relted fmily members such s BcI-XL, Bc!-X, nd Bx (6-8). The Bc!-2 (B-cell Ieukemi/ lymphom 2) gene ws first identified t the brekpoint of chromosoml trnsloction t(14;18) in B folliculr lymphom (9). The BcI-2 gene encodes protein of Mr 26, tht protects cells ginst poptosis in vriety of experimentl systems. Overexpression of Bcl-2 hs been shown to suppress the initition of poptosis in response to number of stimuli, including nticncer drugs (1-1 4). Furthermore, inhibition of Bcl-2 expression by ntisense ohigonucleotide (1S, 16), dominnt-negtive inhibitor Bcl-Xs (8), nd dexmethsone (1 7, 18) hs been shown to promote poptosis nd lso sensitize cells to chemotherpy-induced poptosis. Thus cncer cells my primrily depend on Bcl-2 or relted fmily members to prevent cell deth. Recent studies hve mdicted tht cells from vriety of humn cncers including brest my hve decresed bility to undergo poptosis in response to some physiologicl stimuli (2, 19), nd thus defect in poptosis my be involved in the berrnt survivl nd/or development of cncer. Therefore, identifiction of gent(s) tht cn negtively regulte the expression of the Bcl-2 pthwy in brest cncer cells, nd thus trigger poptosis, will provide therpeutic pproch to inhibit brest cncer cell growth. Dietry fctors ply vitl role both in the development nd prevention of humn cncers, including brest cncer (2, 21). Explortion of dietry fctors nd their ctive micronutnents with growth-inhibitory properties on humn tumor cells could provide new tools for prevention nd tretment of humn cncer. One of such dietry micronutrient is sodium butyrte, the mjor short-chin ftty cid produced by fermenttion of dietry fiber(22, 23). Sodium butyrte nd its more stble derivtives, such s monobut-3 (24, 25), re known to influence severl physiologicl processes, inhibit the growth of mny cell types, including brest cncer cells (24, 25), nd induce mrkers of differentition, such s milkrelted glycoprotemn nd lipid deposition in brest cncer cells (26, 27). In ddition, studies in recent yers hve demonstrted the bility of sodium butyrte to cuse poptosis in humn colorectl cncer nd lymphoid cells (28-3). Although sodium butyrte hs been shown to be potent

2 312 Butyrte-induced Apoptosis in Brest ncer ells A. -.ioe#{149}.) B U 5 U...,.) U S U. y Fig. 1. Sodium butyrte inhibits growth of brest cncer cells nd induces poptosis in MF-7 cells. A, brest cncer cells were treted with () or without (D) 3 mm butyrte for 48 h. ell growth ws mesured by MU ssy, nd results re presented s percentge of control for ll three cell lines. The dt shown is representtive of four experiments; brs, SEM. B nd, induction of poptosis in MF-7 cells treted with sodium butyrte. ells were treted with either different doses of sodium butyrte for 24 h (B) or with 3 mm sodium butyrte for different times (). ytoplsmic extrcts were prepred nd used to quntitte poptotic cell deth by using n EUSA ssy s described in the Mterils nd Methods. Ech tretment ws performed in triplicte, nd results shown re representtive of three experiments with similr results. Results re presented s reltive fold increse in poptotic cell deth s compred to control untreted cells I I S 2I 25 SodIum Butyrte (mm) Sodium Butyrte (h) growth inhibitor nd inducer of poptosis in some cncer cell types, the biochemicl mechnism of sodium butyrteinduced poptosis nd its role in humn brest cncer remins unexplored. In the studies presented here, we investigted the possible role of poptosis in the growth inhibition of humn brest cncer cells by sodium butyrte. We report tht the sodium butyrte-induced poptosis in MF-7 brest cncer cells is medited by down-regultion of Bcl-2, nd overexpression of Bcl-2 leds to suppression of sodium butyrte-induced poptosis nd growth inhibition. In ddition, tretment with butyrte sensitizes MGF-7 cells to chemotherpy-induced poptosis. Results In our initil experiments, three humn brest cncer cell lines were exmined for growth inhibition by 48-h tretment with butyrte. Fig. 1A shows tht MGF-7 cells were most sensitive (75% growth inhibition compred to the growth of untreted cells), nd ZR-75-R cells were lest sensitive (24% growth inhibition compred to the growth of untreted cells) to growth inhibition by sodium butyrte. The growth inhibition of brest cncer cells by sodium butyrte ws not rehted to the presence or bsence of estrogen receptor becuse sodium butyrte lso inhibited the growth of estrogen receptor-negtive MDA-MB-231 cells. During these experiments, we consistently observed tht significnt number of MGF-7 cells strted rounding-up nd chnging their morphology between h fter sodium butyrte tretment. We were intrigued by the possibility of poptosis in the ction of sodium butyrte in MGF-7 cells. To test this, we explored the effect of sodium butyrte on poptotic cell deth using quntittive ELISA ssy tht mesures cytoplsmic histone-bound DNA complexes generted during poptotic DNA frgmenttion (31-33). In the pst, clssicl DNA lddenng hd not been observed in mmmry epithehil cells (such s MGF-7) undergoing poptosis (34, 35), nd ELISA ssy used here hs been method of choice to quntitte poptosis in brest cncer cells (32, 33). As shown in Fig. 1, B nd, sodium butyrte tretment of MGF-7 stimulted poptosis in dose- nd time-dependent mnner. As little s 1 2 h tretment with sodium butyrte cused 5.6-fold increse in poptotic cell deth, which continued to increse up to 27-fold by 48 h fter tretment. To exmine whether the observed growth inhibition of brest cncer cells by sodium butyrte ws relted to the induction of poptosis, the quntittion of poptotic cell deth ws lso performed in ZR- 75-A cells treted with or without sodium butyrte. Results

3 Gell Growth & Differentition 313 A #{176}-BcI2. IBc12 B. 12 I *Bc Fig. 2. Down-regultion of Bcl-2 expression by sodium butyrte in MGF-7 cells. A, time course of inhibition of Bcl-2 expression in MGF-7 cells by sodium butyrte. ells were treted with or without sodium butyrte (3 mm). ell lystes were mde, nd equl mounts (2 g) of protein were immunoblotted with Bcl-2 mab. As n internl control, the upper prt of the sme blot ws blotted with n unrelted HSP-4 Ab. Lne 1, control; Lne 2, 1.5 h; Lne 3, 3 h; Lne 4, 6 h; Lne 5, 12 h; Lne 6, 24 h. B, effect of sodium butyrte on Bcl-2 expression s function of dose. ells were treted with or without sodium butyrte for 1 6 h. ell lystes (2,.Lg protein) were immunoblotted with Bcl-2 mab nd HSP-4 Ab s described bove. Bcl-2 signl ws detected by using 1251-lbeled protein A. Lne 1, control; Lne 2, 1 mm; Lne 3, 3 mm; Lne 4, 6 m. : Top, inhibition of Bcl-2 mrna by sodium butyrte. MF-7 cells were treted with 3 mm sodium butyrte for 24 h. Totl cellulr RNA ws isolted. Twenty.tg RNAS were nlyzed by Northern blotting using 32P-lbeled cdna frgment (.91 kb) specific for humn Bcl-2 mana. Right, position of Bcl-2 mrna. The Northern blot ws rehydndized with n unrelted cdna probe for ctin mrna. Bottom, ethidium bromide-stined rrna bnds. These experiments were repeted three times. indicted tht tretment of ZA-75-A cells with sodium butyrte (3 mm; 24 h) resulted in only 3.8-fold increse in poptotic cell deth, suggesting tht the inhibitory effect of butyrte in MF-7 cells ws probbly ssocited with its bility to induce poptosis. Becuse poptosis in mmmlin cells hs been shown to be regulted by Bcl-2 (4) nd becuse of the fct tht BcI-2 inhibition promoted poptosis (1 7, 18), we investigted whether the sodium butyrte-induced poptosis in MF-7 cells ws relted with the possible modultion in the levels of BcI-2. To test this possibility, we exmined the expression of Bcl-2 by Western immunoblotting in MF-7 cells treted with or without butyrte. As shown in Fig. 2, A nd B, it ws interesting to note tht tretment of MF-7 cells with sodium butyrte resulted in down-regultion of Bcl-2 expression in time-dependent mnner strting 3 h fter tretment. Tretment with sodium butyrte for h ws sufficient to inhibit the levels of Bcl-2 to significnt extent (4-6% inhibition compred to the levels in untreted cells), nd this cellulr effect of sodium butyrte ws lso dose dependent (Fig. 2B). Results of other experiments demonstrted tht tretment (48 h) with low doses of sodium butyrte (.5 or 1 mm) lso down-regulted (2-42%, compred to the levels in untreted cells) the Bcl-2 expression in MF-7 cells.3 The observed down-regultion of Bcl-2 expression ws specific effect of sodium butyrte becuse there ws no influence of sodium butyrte on the levels of n unrelted HSP-4.4 Blots in Fig. 2, A nd B, were cut into two pieces, nd upper portions were immunoblotted with HSP-4 Ab (36) s n internl control within the sme blot. To determine whether the observed down-regultion of BcI-2 expression in sodium butyrte-treted MF-7 cells ws ssocited with decresed expression of Bcl-2 mana, Northern blot nlysis ws performed. Fig. 2 shows tht 16 h tretment with sodium butyrte inhibited the stedy-stte levels of Bcl-2 mana by 75% compred to the levels in untreted control cells. In brief, results from Figs. 1 nd 2 indicted tht the observed sodium butyrte-induced down-regultion of Bcl-2 expression ws n erly event tht correlted with the period of induction of poptosis, suggesting tht sodium butyrte cn inhibit gene product known to be involved in poptosis. To further exmine the possible involvement of Bcl-2 in the induction of poptosis nd growth inhibition of MF-7 cells by butyrte, we exmined whether overexpression of Bcl-2 will modulte the sensitivity of MF-7 cells to the inhibitory effect(s) of sodium butyrte. For these studies, MF-7 cells were trnsfected with Bcl-2 expression vector (1 3). A number of clonl cell lines stbly overexpressing Bcl-2 were generted. Fig. 3A shows the levels of Bcl-2 protein in two such clones, MF-7/BL2-1 nd MF-7/BL2-2 cells, which overexpress 2.7- nd 1.9-fold higher levels of Bcl-2, respectively, compred to the levels in the prentl MF-7 cells. Results in Fig. 3B show the effect of overexpression of Bcl-2 on the sensitivity of MF-7 cells to butyrte. As shown in Fig. 3, both MF-7/BL2-1 nd MF-7/BL2-2 cells were protected from the growth-inhibitory ction of butyrte to significnt extent compred to growth inhibition in MF-7 cells. The observed prtil protection of cells ws dependent on the durtion of tretment, with mximum protection observed by 48 h fter tretment (1 5-25% growth inhibition in clones compred to 6-75% growth inhibition MF-7 cells). The observed suppression of growth inhibition of MF-7 cells overexpressing Bcl-2 by sodium butyrte ws not due to G41 8 selection becuse MF-7 cells trnsfected with the neo vector lone (MF-7/neo cells) demonstrted no chnge in the levels of Bcl-23 nd were lso sensitive to growth inhibition (72%±5) nd induction of poptosis (26- fold ± 7) by 48 h tretment with 3 mi sodium butyrte. Since Bcl-2 overexpression reduced the growth-inhibitory effect of sodium butyrte, we lso exmined the sensitivity of BcI-2-overexpressing MF-7 cells to the cytotoxic effects of sodium butyrte by the criteri of morphologicl chnges. Aesults in Fig. 4 show tht MF-7/BL2-1 cells were distinctively protected from the cytotoxic morphologicl chnges observed in MF-7 cells treted with sodium butyrte (3 mm; 48 h tretment). Similr results were lso obtmed with MF-7/BL2-2 cells.3 The observed protection 3 Unpublished observtions. 4 The bbrevitions used re: HSP, het shock protein; Ab, ntibody; mab, monoclonl ntibody; MiT, 3-(4,5-dimethylthizol-2-yl)-2,5-diphenyltetrzolium bromide.

4 314 Butyrte-induced Apoptosis in Brest ncer ells B. A. 123 McF.7 McF-7IcL2-1 MF-7/BL22 Fig. 3. Effect of sodium butyrte on the growth of MF-7 cells nd MF-7 cells overexpressing Bcl-2. A, expression of BcI-2 in clones overexpressing Bcl-2. B, kinetics of growth inhibition of MF-7 cells nd clones overexpressing Bcl-2 by sodium butyrte. MF-7, MF-7/ BL2-1, nd MF-7/BL-2-2 cells (3 x 1 o cells) were plted into ech well of 48-well plte. After 24 h (time ), some cells were treted in qudruplicte either without (LI) or with 3 m sodium butyrte for 4 dys (U), 3 dys (), 2 dys (D), or 1 dy (). On dy 4, cell growth ws determined by MU ssy. Results re presented s percentge of controls for ech cell line. Results shown re representtive of four experiments; brs, SEM. of MF-7/BL2-1 cells ginst the inhibitory effects of sodium butyrte ws lso quntitted by the possible reversibility of cell growth fter 48 h tretment with sodium butyrte (3 mm) over period of n dditionl 4 dys. Aesults mdicted tht the removl of sodium butyrte fter 48 h tretment hd no beneficil effect on the survivl of MF-7 cells compred to MF-7 cells treted continuously with sodium butyrte (95% ± 7%; growth inhibition t dy 6). In contrst, the removl of sodium butyrte fter 48 h tretment of MF- 7/BL2-1 cells resulted in 27% increse in cell growth (48% ± 3%; growth inhibition t dy 6) s compred to MF-7/BI2-1 cells treted continuously with sodium butyrte (75% ± 5%; growth inhibition t dy 6). Next we exmined the effect of Bcl-2 overexpression in MF-7 cells on the bility of sodium butyrte to induce poptosis. Aesults in Fig. 5 show tht Bcl-2 overexpression in MF-7 cells leds to significnt suppression in the sodium butyrte-induced stimultion of poptosis (5-fold increse in poptosis compred to 27-fold poptosis in the prentl MF-7 cells by 48 h sodium butyrte tretment). Tken together, results from Figs. 3-5 suggest tht the observed protective effects of Bcl-2 overexpression ginst inhibitory effects of butyrte my be closely linked with the bility of sodium butyrte to induce poptosis in MF-7 cells. Since overexpression of Bcl-2 in MF-7 cells resulted in suppression but not complete prevention of sodium butyrte-induced poptosis nd growth inhibition, we exmmed the effect of sodium butyrte on BcI-2 expression in MF-7 clones. Aesults in Fig. 6 show tht sodium butyrte (3 mm; 24 h) lso down-regulted the levels of Bcl-2 in MF-7 cells overexpressing Bcl-2 (45% inhibition in Bcl-2 expression s compred to the levels in untreted cells; Fig. 6A, Lnes 7 nd 8) but to lesser extent compred to downregultion of Bcl-2 in MF-7 cells (Fig. 6, Lnes 1 nd 2) or MF-7/Neo cells (Fig. 6, Lnes 3 nd 4; up to 8% inhibition in Bcl-2 expression). In brief, these results suggested tht the levels of Bcl-2 expression ply regultory role(s) in sodium butyrte-induced poptosis in MF-7 cells; perhps cells need threshold level of Bcl-2 to protect ginst poptosis in response to sodium butyrte tretment in MF-7 brest cncer cells. Since Bcl-2 hs been shown to protect cells by inhibiting the induction of poptosis triggered by severl cytotoxic gents (12, 14) nd becuse of the fct tht sodium butyrte down-regulted Bcl-2 expression (this study), we explored the effect of sodium butyrte on the modultion of sensitivity of MF-7 cells to induce poptosis in response to doxorubicin, commonly used chemotherpeutic gent. For these experiments, we hve selected dose (.5 mm) of sodium butyrte tht lone did not induce poptosis.3 Aesults in Tble 1 show tht cotretment of MF-7 cells with sodium butyrte, which down-regulted the levels of Bcl-2, incresed the sensitivity of cells to the induction of poptosis. In the pst, 72 h tretment of MF-7 cells with doxorubicin hs been shown to induce bout 5% or less increse in poptotic cell deth compred to control cells (35). Discussion Apoptosis is physiologicl mechnism of cell deth tht plys n importnt role during embryonic development, orgn involution, nd in diseses like cncer (1-4). Apoptosis is regulted by specific cellulr pthwys, including Bcl-2 (4, 5). A number of recent studies suggested tht the overexpression of Bcl-2 protects cells ginst poptosis (1 1, 14, 37), nd inhibition of Bcl-2 expression promotes poptosis (1 7, 1 8) in response to number of stimuli. Therefore, cncer cells my depend on BcI-2 or relted fmily members to prevent poptosis, nd defect in regultion of poptosis my be possibly involved in the berrnt survivl nd/or development of cncer. The bility of Bcl-2 to inhibit poptosis nd its reltionship with cncer, provided by trnsgenic studies involving the BcI-2-Ig locus s trnsgene, lend support for this model (38). In recent yers, pproches imed to modulte the expression of cellulr poptotic pthwys such s BcI-2 re subject of ctive investigtion in effort to control cncer cell growth. The present investigtion ws undertken to explore the role of poptosis nd its possible modultion by Bcl-2 expression in the growth inhibition of humn MF-7 brest cncer cells by sodium butyrte, potent growth inhibitor nd inducer of differentition tht is produced by fermenttion of dietry fiber (22). We hve selected the MF-7 cell system for experimentl convenience s well s to enble us to tke dvntge of the welth of knowledge vilble bout its biology. The results presented here demonstrted tht the tretment of MF-7 cells with sodium butyrte induced poptosis

5 ell Growth & Differentition 315 MF-7 I + BA r _. - f, : : Fig. 4. Effect of sodium butyrte on the morphology of MF-7 (A nd B) nd MF-7/BL2-i ( nd D) cells. ells were cultured for 48 h in the bsence (on, A nd ) or presence (B nd D) of 3 mm sodium butyrte (+BA). Phse-contrst photomicrogrphs re shown. A MF-7IBL 2-I := Fig. 5. Overexpression of Bcl-2 suppresses sodium butyrte-induced poptosis in MF-7 cells. Equl numbers (2 x i) of MF-7, MF-7/BL2-1, nd MF-7/BL2-2 cells were treted with sodium butyrte (3 mm)for48 h (U), 24 h (), oro h (). ytoplsmic extrcts were mde to quntitte the poptotic cell deth s described in Fig. 1. All results re presented s fold increse in poptotic cell deth compred to MF-7 cells s bse vlue. Note tht control untreted Bcl-2-overexpressing MF-7 cells were lso protected from the spontneous poptosis compred to control MF-7 cells. These experiments were repeted three times; brs, SEM. (I) (I,.. 4 l) D D I-.) V U- MF-7 MF-7IBL2-1 MF-7/BL2-2 nd growth inhibition, nd this ws closely linked with the down-regultion of Bcl-2 expression. In ddition, overexpression of Bcl-2 in MF-7 cells resulted in significnt inhibition of sodium butyrte-induced poptosis nd lso protected the MF-7 cells ginst the inhibitory effects of sodium butyrte. The observed down-regultion of Bcl-2 expression by sodium butyrte my lso provide the biochemicl bsis of some of the known growth-inhibitory ctions of sodium butyrte in MF-7 cells (24-27). The finding tht sodium butyrte inhibited the expression of Bcl-2, gene product involved in poptosis, is importnt becuse it suggests tht dietry micronutrient cn modulte the oxpression of specific poptotic regultory pthwy. In the pst, there ws precedence to correlte the levels of Bcl-2 with the induction of poptosis. In this context, it is importnt to note tht dexmethsone nd interleukin 6 hve been shown recently to down-regulte Bcl-2 expression nd induce poptosis in myeloid leukemic cells (1 7, 1 8). The mechnism of observed down-regultion of Bcl-2 by sodium butyrte remins to be delineted. This my occur t the trnscription level nd/or posttrnscription level nd my lso involve reduced BcI-2 mana stbility, leding to decrese in Bcl-2 expression. The observed temporl reltionship between the downregultion of Bcl-2 expression nd induction of poptosis in MF-7 cells ws not cusl becuse stble overexpression of Bcl-2 in MF-7 cells resulted in significnt suppression of sodium butyrte-induced stimultion of poptosis nd inhibitory effects. These results re consistent with the provious studies (1 1, 14, 37), nd support the notion of pro-

6 316 Butyrte-induced Apoptosis in Brest ncer ells A. B. D #{176}-BcI Hsp4O sponse to chemotherpeutic gent(s) nd opens n interesting re of investigtion to further explore the possible ther- I 1#{176}-Actin Fig. 6. Effect of sodium butyrte on the levels of Bcl-2 in MF-7 cells with or without BL2 overexpression. ells were treted with (+) or without (-) 3 mm sodium butyrte for 24 h. ell lystes were mde, nd equl mounts (2.tg) of protein were nlyzed by immunoblotting with Bcl-2 mab, followed by signl development using 1251-lbeled protein A (A nd B resulted from exposure of the sme blot for 3- nd 12-h exposures, respectively). The upper portion of Bcl-2 blot ws immunoblotted with HSP-4 Ab (). D, immunoblotting of bove smples with polyclonl ctin ntibody (Sigm). Lnes 1 nd 2, MF-7 cells; Lnes 3 nd 4, MF-7/Neo cells; Lnes 5 nd 6, MF-7/BL2-2 cells; Lnes 7 nd 8, MF-7/BL2-i cells. Tble 1 Sodium butyrte potentites doxorubicin-induced poptosis in MF-7 cells Tretment ontrol 1 Reltive fold increse in poptosis#{176} (over control) Doxorubicin (1 flm) Sodium butyrte (5 flm) 1.28 ±.8 Doxorubicin 91 flm) sodium butyrte (5 nm) DNA frgmenttion ws mesured by quntittive ELJSA ssy fter 72 h tretment; SD (n = 4). tective role(s) of Bcl-2 ginst poptosis in brest cncer cells. It ws curious to note tht lthough MF-7/BL2-1 cells overexpress only 2.7-fold higher levels of Bcl-2 cornpred to the levels in MF-7 cells, they demonstrted significnt suppression in sodium butyrte-induced poptosis, implying tht smll chnge in the levels of Bcl-2 over the presumptive poptotic threshold levels could possibly modulte the induction of poptosis. In ddition, it will be interesting to explore whether sodium butyrte cn lso modulte the expression of other members of genes tht either suppress induction of poptosis, such s Bcl-XL (6), or promote poptosis, such s Bcl-X nd Bx (7, 8), nd such efforts re under wy. Our finding tht tretment with butyrte, which down-regulted the Bcl-2 expression, could potentite the sensitivity of MF-7 cells to undergo poptosis in response to doxorubicin provides further support to regultory role of Bcl-2 in the modultion of poptosis in humn brest cncer cells in repeutic potentil of sodium butyrte nd/or more stble nlogue ofsodium butyrte in the development of strtegies to control the growth of humn brest cncer cells. Mterils nd Methods ell Lines nd Growth Assys. Humn brest cncer MF-7 cells (39) were mintined in DMEM-F12 (1:1) supplemented with 1% FS. Other humn brest cncer cells (ZR-75-R nd MDA-MB-231) were obtined from the Americn Type ulture ollection nd were grown in L-15 medium supplemented with 1% FS. ell growth ws mesured by the MiT dye (Sigm hemicl o.) uptke procedure, s described previously (4, 41). About 2 x 1 o cells/.5 ml culture medium were seeded into ech well of 48-well plte. After 24 h, the medium ws chnged, nd pproprite cultures were supplemented with sodium butyrte. For ech point, the medium ws removed from triplicte wells, MU dye (5 mg/mi in PBS) ws dded, nd the plte ws wrpped in luminum foil nd incubted t 37#{176} for 4 h. Dye tken up by living cells ws extrcted in isopropyl lcohol:1 N HI (96:4) for the determintion of bsorbnce t wvelength of 57 nm, nd results re presented s percentge of control. Quntittion of Apoptosis. To mesure poptotic cell deth, we used cell deth ELISA (Boehringer Mnnhein, Indinpolis, IN) tht mesure cytoplsmic histone-bound DNA frgments (mono- nd oligonucleosomes) generted during poptotic DNA frgmenttion (31-33) nd not free histone or DNA tht could be relesed during nonpoptotic cell deth (42). Results obtined by this ELISA ssy hs been shown to correlte with those obtined by other methods (31). MF-7 cells or MF-7 clones overexpressing Bcl-2 (2 x 1) cells were plted into ech well of 48-well plte. After desired tretment, cytoplsmic extrcts were mde from both floting nd ttched cells, ccording to mnufcturer s protocol. ontrol nd drug-treted cell extrcts were equlized on the bsis of equl cell number s well s protein in extrcts. Briefly, wells were first coted with nti-histone ntibody, loded with cytoplsmic extrcts, nd followed by incubtion with nti-dna second ntibody conjugted with peroxidse. ELISA ws developed with peroxidse substrte, nd the bsorbnce t 45 nm ws mesured using Microplte utoreder (Bio-Tek Instruments). Preprtion of ell Extrcts. All experiments were performed with cells in logrithmic phse by controlling the plting density. The vibility of cells ws ssyed by trypn blue dye exclusion. ells were treted with or without sodium butyrte (Sigm); extrcts were prepred s described (43). Briefly, cells were wshed twice with cold PBS on ice nd lysed in buffer (5 mm Tris-HI (ph 7.5), 12 mi NGI,.5% NP4O, 1 m.i NF, 2 M NVO5, 1 mm phenylmethylsulfonyl fluoride, nd 1 g/ml eupeptin) for 15 mm on ice. The lystes were centrifuged in n Eppendorf centrifuge t 4#{176} for 15 mm. The protein concentrtion in extrcts ws determined by using the Bio-Rd kit. Immunoblotting. ell lystes contining n equl mount of totl protein (4 ig) were resolved on SDS-PAGE, followed by trnsfer onto nitrocellulose (41, 44). Membrnes were blocked in 2% BSA in TBS [1 mm Tris-HI (ph 7.5), 15 mm NI], followed by probing with either nti-bcl-2 mab (Dko or Neomrkers, Inc.) or nti-hsp-4 Ab (36), nd immune complexes were detected by using secondry ntibody-bsed lkline phosphtse color rection or 1251-lbeled protein A. Quntittion of specific protein bnds ws performed by using protein dtbses scnner (Moleculr Dynmics). As internl control, the blot ws lwys cut into two pieces fter trnsfer of proteins. The lower portion of blot ws probed with nti-bcl-2 mab nd the upper portion with n unrelted ntibody. Low-moleculr-mss mrkers (Amershrn orp.) were used s stndrd. Northern Anlysis. Totl cellulr RNA ws isolted nd nlyzed by Northern blot methods s described previously (44). Briefly, 2 g ANAS dentured in the presence of formldehyde were frctionted on 1 % grose formldehyde gel nd trnsferred to nitrocellulose filter, with hybridiztion nd wshing s described previously (44). Loding of the RNA ws monitored by intensities of ethidium bromide-stined rrna bnds (28 5 nd 18 5). The quntifiction of the specific signl ws performed by densitometric scnning of the utords with necessry precutions to ensure liner response between different utord exposures of the sme blot being tken. The specific cdna clone (clone pb4; Ref. 45) for humn Bcl-2 mrna ws from the Americn Type ulture

7 ell Growth & Differentition 317 ollection, nd the.91-kb EcoRI frgment ws used for hybridiztion experiments. As negtive control, the filter ws rehybridized with n unrelted ctin cdna probe (43). Expression of Bcl-2 in MF-7 ells. MF-7 cells t density of 16 cells/i -mm dimeter plte were trnsfected with plsmid DNA contining the full-length humn Bcl-2 cdna nd selectble mrker, neomycin phosphotrnsferse gene (pssfbcl-2; Ref. 13) by clcium phosphte precipittion procedures (46). At 24 h ftertrnsfection, selection medium contining 1 mg of G418, neomycin nlogue, per ml ws dded. Seven to 1 dys of culturing in this medium killed most of the cells, but few colonies of cells, one to five per plte, grew. Well-seprted colonies were hrvested by trypsiniztion in cloning cylinders nd were propgted in the presence of G418. Expression of BcI-2 in individully isolted clones ws determined by immunobotting with BcI-2 mab. As control, we hve used MF-7 cells stbly trnsfected with vector contining only neomycm. Once stble cellline from ech clone hd been estblished, the drug ws removed from the culture medium. The clonl lines hve been mintmed in drug-free medium since then. Acknowledgments We thnk Stnley J. Korsmeyer for providing BcI-2 expression vector,. Kent Osborne for MF-7 ciones, Kenzo Ohtsuk for HSP-4 Ab, Necmrkers for nti-bci-2 mab, nd Neet Shmi for technicl ssistnce. References 1. Steller, H. Mechnisms nd genes of cellulr suicide. Science (Wshington D), 267: , Thompson,. B. Apoptosis in the pthogenesis nd tretment of disese. Science (Wshington D), 267: , Willims, G. T., nd Smith,. A. Moleculr regultion of poptosis: genetic controls on cell deth. ell, 74: , Korsmeyer, S. J. BcI-2 initites new ctegory of oncogenes: regultor of cell deth. Blood, 8: , Hockenbery,. M., Zither, M., Hickey, W., Nhm, M., nd Korsmeyer, S. J. BcI-2 protein is topogrphiclly restricted in tissues chrcterized by poptotic cell deth. Proc. Nti. Acd. Sci. USA, 88: , Boise, L H., Gonzlez-Grci, M., Postem,. E., Ding, L, Undsten, T., Turks, L A., Mo, X., Nunez, G., nd Thompson,. B. Bci-x, bcl-2-relted gene tht functions s dominnt regultor of poptotic cell deth. ell, 74: , Gottschlk, A. R., Boise, L H., Thompson,. B., nd Quintns, J. Identifiction of immunosuppressnt-induced poptosis in murine B-cell line nd its prevention by bcl-x but not bcl-2. Proc. NtI. Acd. Sci. USA, 91: , Oltvi, Z., Millimn,. L, nd Korsmeyer, S. J. BcI-2 heterodimerizes in vivo with conserved homolog, Bx, tht ccelertes progrmmed cell deth. ell, 74: , Tsujimoto, V., Gorhm, J., ossmn, J., Jffe, E., nd roce,. M. The t(1 4;1 8) chromosome trnsloction involved in B-cell neoplsms results from mistke in VDJ joining. Science (Wshington D), 299: , Hockenbery, D. M., Nunez, G., Millimn,., Schreiber, A. D., nd Korsmeyer, S. J. BcI-2 is n inner mitochondril membrne protein tht blocks progrmmed cell deth. Nture (Lond.), 348: , Sentrnn,. L, Shutter, J. A., Hockenbery, D., Kngw,. nd Korsmeyer, S. J. 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Androgens induce relstnce to bcl-2-medited poptosis in LNP prostte cncer cells. ncer Res., 5:73-738, Lotem, J., nd Schs, L ontrol of sensitivity to induction of poptosis in myeloid leukemi cells by differentition nd bcl-2 dependent nd independent pthwys. ell Growth & Differ., 5: , Lotem, J., nd Schs, L Regultion of bcl-2, bi-xl nd bx in the control of poptosis by hemtopoietic cytokines nd dexmethsone. ell Growth & Differ., 6: , rson, D. A., nd Ribeiro, J. M. Apoptosis nd disese. Lncet, 341: , Doll, A., nd Peto. A. The cuses of cncer: quntittive estimtes of voidble risks of cncer in the United Sttes. Oxford: Oxford University Press, Snyderwmne, E. Some perspectives on the nutritionl spects of brest cncer reserch. ncer (Phil.), 74: , ummings, J. H. Short-chin ftty cids in the humn colon. Gut, 22: , Kwh, J. Effects of sodium butyrte, new phrmcologicl gent, on cells in culture. Mol. ell. 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Sodium butyrte induces poptosis in humn co- Ionic tumor cell lines in p53-independent pthwy: implictions for the possible role of dietry fibre in the prevention of lrge-bowel cncer. mt. J. ncer, 55: , Hgue, A., Elder, D. J. E., Hicks, D. J., nd Prskev,. Apoptosis in colorectl tumors cells: induction bythe short chin ftty cids butyrte, propionte, nd cette nd by the bile slt deoxycholte. Int. J. ncer, 6: 4-46, Filippovich, I., Sorokin, N., Khnn, K. K., nd Lvin, M. F. Butyrte induced poptosis in lymphoid cells preceded by trnsient overexpression of HSP7O mrna. Biochem. Biophysicl. Res. ommun., 198: , Leist, M., Grtner, F., Bohlinger, I., Tiegs, G., nd Wendel, A. Appliction of the cell deth ELISA for the detection of tumor necrosis fctor induced DNA frgmenttion in munne models of inflmmtory orgn filure. Biochemic, 11: 2-22, Sumntrn, V. N., Eloveg, M. W., Nunez, G., lrke, M. F., nd Wich, M. S. 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8 318 Butyrte-induced Apoptosis in Brest ncer ells A. Induction of differentition nd growth rrest is ssocited with nscent DNA frgmenttion nd reduced c-myc expression in MF-7 humn brest cncer cells fter continuous exposure to sublethl concentrtions of doxorubicin. ell Growth & Differ., 5: , Httori, H., Uu, V-., Tohnl, I., Ued, M., Kned, T., Kobyshi, T., Tnbe, K, nd Ohtsuk, K Intrcellulr locliztion nd prtil mino cid sequence of stress-inducible 4 kd protein in Hel cells. ell Struct. & Funct., 17: 77-86, Dole, M., Nunez, G., Merchnt, A. K., Mybum, J., Rode,. K., Bloch,. A., nd stle, V. P. Bcl-2 inhibits chemotherpy-induced poptosis in neuroblstom. ncer Res., 54: , McDonelI, T. J., Dene, N., Pltt, F. M., Nunez, G., McKern, J. P., nd Korsmeyer, S. J. Bcl-2 immunoglobulin trnsgenic mice demonstrte extended B cell survivl nd folliculrlymphoprolifertion. ell, 57:79-88, Benz,.., Scott, G. K., Srup, J.., Johnson, A. M., Tripthy,., orondo, E., Sheprd, M., nd Osborne,. K Estrogen-dependent, tmoxifen-resistnt tumongenic growth of MF-7 cells trnsfected with HER2/neu. Brest ncer Res. Tret., 24: 85-95, Edmondson, J. M., Armstrong, L S., nd Mrtinez, A.. A rpid nd simple MiT-bsed spectrophotometric ssy for determining drug snsitivity in monolyer cultures. J. Tissue ulture Methods, 11: 5-17, Kumr, A., nd Mendelsohn, J. Growth regultion of A431 cells: modultion of expression of trnsforming growth fctor-mrna nd 2,5 -oligodenylte synthetse ctivity. J. Biol. hem., 265: , Del-Biobo, G., Skierski, J.., nd Drzynkiewicz, Z. The concentrtion dependent diversity of effects of DNA topoisomerse I nd II inhibitors on the cell cycle of HL-6 cells. Exp. ell Res., 195: , Kumr, A., nd Atls, I. Interferon- induces the expression of retinoblstom gene product in humn Burkitt s lymphom Dudi cells: role in growth regultion. Proc. NtI. Acd. Sci. USA, 89: , Kumr, R., Korutl, L, Zhng, K. ell cycle-dependent modultion of interferon inducible genes expression nd ctivtion of signling components in Dudi cells. J. Biol. hem., 269: , Tsujimoto, V., nd roce,. A. Anlysis of the structure, trnscripts, nd protein products of bci-2: the gene involved in humn folliculr lymphom. Proc. NtI. Acd. Sci. USA, 83: , Kumr, A., Tiwn, A., Kusn, J., nd Sn, G. lonl derivtives of RD-i 14 cells respond differently in their ntivirl nd gene inducing responses to interferons. J. Virol., 61: , 1987.