Journal of Dental Research

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Butyric-acid-induced Apoptosis in Murine Thymocytes and Splenic T- and B-cells Occurs in the Absence of p53 T. Kurita-Ochiai, K. Ochiai and K. Fukushima J DENT RES 2000 79: 1948 DOI: 10.1177/00220345000790120501 The online version of this article can be found at: http://jdr.sagepub.com/content/79/12/1948

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J Dent Res 79(1 2):1948-1954, 2000

Butyric-acid-induced Apoptosis in Murine TIymocytes and Splenic T- and B-cells Occurs in the Absence of p53

ABSTRACT

INTRODUCTION

T. Kurita-Ochiail*, K. Ochiai2, and K. Fukushimal 'Department of Microbiology, Nihon University School of Dentistry at Matsudo, Matsudo-shi, Chiba 271-8587, Japan; and 2Department of Microbiology, Meikai University School of Dentistry, Sakado, Saitama 350-0283, Japan; *corresponding author, [email protected]

Butyric acid, an extracellular metabolite from periodontopathic bacteria, induces apoptosis in murine thymocytes, splenic T-cells, and human Jurkat T-cells. The present study examines the contributions of apoptosis-related proteins (Bcl-2, Bcl-XL, Bax, and p2lWAFI/CIPI) in the regulation of T-cell death induced by butyric acid, using p53 knock-out (p53-/-) and wild-type (p53k +) mice. The results of a DNA fragmentation assay indicated that thymocytes, splenic T-cells, and Bcells from p53-/- mice were susceptible to butyricacid-induced apoptosis to a degree similar to those from p53` mice. Moreover, butyric acid significantly induced apoptosis in lymphocytes from both p53+/+ and p53-/- mice in a concentration- and time-dependent fashion. Experiments with fractionated subpopulations of splenic T-cells revealed that DNA fragmentation was equally observed in CD4+ and CD8+ splenic T-cells from both p53 ++ and p53-/- lymphocytes. Activation of caspase-3, caspase-6, and caspase-8, but not of caspase-1, in butyric-acid-induced Tcell apoptosis occurred regardless of the presence of p53. Western blotting analysis of splenic Tcells showed that butyric acid treatment decreased Bcl-2 and Bcl-XL expressions in p53 ++ and p53/cells. Splenic T-cells had barely detectable Bax and p2lWAFi CIPi, regardless of whether butyric acid and/or p53 was present. These results suggest that butyric-acid-mediated apoptosis of murine Tcells takes place via a pathway that is independent of p53, and is followed by the p53-regulated proteins Bax and p21WAFI/CIPI, which lower the levels of the apoptosis antagonists Bcl-2 and BclXL in cells.

KEY WORDS: butyric acid, apoptosis, p53, knockout mice.

Received February 18, 2000; Last revision August 10, 2000; Accepted September 5, 2000

1948

A dult periodontitis is a chronic destructive disease characterized by

/Ninteractions between Gram-negative bacteria and host inflammatory

responses. A recent study indicated that severe destructive adult periodontitis is caused by a multi-bacterial infection and that certain combinations of periodontopathogens namely, Porphyromonas, Prevotella, and Fusobacterium spp. seem to be important in the pathogenesis of the disease (Soder et al., 1993). These bacteria produce an elaborate variety of virulence factors such as proteases, lipopolysaccharides (LPS), fimbriae, and short-chain fatty acids. Butyric acid, one of the short-chain fatty acids, has been shown to suppress proliferation of a variety of cancer cell lines in vitro (Langdon et al., 1988; Tsutsumi et al., 1994; Hodin et al., 1996). Our previous study (Kurita-Ochiai et al., 1995) also demonstrated that short-chain fatty acidsespecially volatile fatty acids present in the culture filtrates of Porphyromonas gingivalis, Prevotella loescheii, and Fusobacterium nucleatum markedly inhibited murine T- and B-cell proliferation and cytokine production. Furthermore, we found that a representative volatile fatty acid, butyric acid, induced cytotoxicity and apoptosis in murine- and human T- and B-cells (Kurita-Ochiai et al., 1997, 1998). Butyric acid inhibits deacetylation of histones, leading to alteration in chromosomal structure and gene expression (Tsutsumi et al., 1994; Hodin et al., 1996). However, the precise mechanisms of butyric-acid-induced apoptosis have not been elucidated. p53 is known to play a critical role in regulating apoptosis. DNA damage caused by ultraviolet (UV), gamma-rays, or chemotherapeutic agents (elDeiry et al., 1994) induces a wild-type p53 expression which in tum induces cell death through apoptosis (Miyashita et al., 1995). p53 is also a transcription factor (Vogelstein and Kinzler, 1992), and functions to activate target genes such as p2]WAFJ CIP] and Bax, which encode proteins that govern cell cycle progression and apoptosis. Emerging evidence indicates that bacterium-modulated apoptosis is an important phenomenon in the pathogenesis of infectious disease (Chen and Zychlinsky, 1994). Specific pathogens or their exocellular products may directly induce apoptosis of host cells (Zychlinsky et al., 1992). A recent report indicated that apoptosisrelated morphological changes with aging may be related to susceptibility to periodontal disease (Sakai et al., 1999), while another report indicated that apoptosis-associated DNA damage and expression of the p53 and Bcl-2 apoptosis-regulating genes were prevalent phenomena at chronic bacterially induced inflammation sites in human gingiva (Tonetti et al., 1998). The aim of the present investigation was to examine whether the p53pathway is activated during butyric-acid-induced T-cell apoptosis, and whether induction of the cell-cycle- and cell-death-regulatory genes p21WAFI/CIPI Bcl-2, Bcl-XL, and Bax plays a role in butyric-acid-mediated cell death. In this study, we found that the p53 tumor suppressor molecule is not required for butyric-acid-induced apoptosis of murine T-cells. Furthermore, we found evidence that the activation of caspase-3, caspase-6, and caspase-8, as well as suppression of the expression of the cell-deathregulatory proteins Bcl-2 and Bcl-XL, but not those of Bax or p21WAFI1CIP1 lead to butyric-acid-mediated cell death.


Butyric-acid-induced Apoptosis in p53-deficient Mice

J Dent Res 79(12) 2000

1949

MATERIALS & METHODS Reagents Highly purified butyric acid was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Solutions of butyric acid ranging in concentrations from 0.31 to 5 mM were diluted in RPMI 1640 (Gibco Laboratories, Grand Island, NY, USA) medium and adjusted to pH 7.2 with sodium hydroxide.

Mice p53-deficient in-bred mice (p53-/CBA/OYC, Homozygous,SPF) (Ishizaki et al., 1994) and p53 wild- Figure 1. Butyric acid dose-response curves for apoptosis. (A) Thymocytes, (B) splenic T-cells, and (C) of butyric acid type mice (p53k +CBA/OYC, Wild splenic B-cells from p53+/+ or p53+1- mice were cultured with the indicated concentrations for 21 hrs. Harvested cells were assayed by the DPA assay. The results are expressed as the mean E SE of type, SPF) were purchased from three different experiments with triplicate cultures. Values significantly different from the corresponding Oriental Ferment Company negative controls without butyric acid at p < 0.01 are indicated by asterisks. (Saitama, Japan). The mice were maintained in the Animal Facility of 30 min at 37°C. This purified B-cell preparation contained less Nihon University School of Dentistry at Matsudo under standard than 2% Thy-l+ cells, as determined by immunofluorescence with care, and given food and water ad libitum. All mice were from nine a FACScan fluorescence-activated cell sorter. These cells were to ten weeks of age and were matched by age within each cultured at 37°C in a moist atmosphere of 5% CO2 in complete experiment. The animal use protocol was reviewed and approved medium consisting of RPMI 1640 supplemented with 10% heatby the institutional review board. inactivated fetal calf serum, 2 mM L-glutamine, 100 U of T-cell Preparation penicillin/mL, 100 ptL of streptomycin/mL, and 0.05 mM 2mercaptoethanol. Spleens and thymocytes were aseptically removed, and singlecell suspensions were prepared by gentle teasing of the cells Cell Culture for Apoptosis through sterile stainless steel screens. The thymocyte cell mouse from T-cells of Preparations directly. used was suspension The cells (4 x 106/well) were cultured in 1 mL of complete spleens were obtained as described previously (Kurita-Ochiai et medium in 24-well tissue culture plates (Falcon; Becton Dickinson al., 1994). Briefly, we obtained T-cell-enriched fractions by Labware, Lincoln Park, NJ, USA) with various concentrations of passing spleen cells through a Sephadex G-10 column butyric acid. At the times indicated in the Figs., the cells were (Pharmacia, Piscataway, NJ, USA), followed by panning harvested, centrifuged at 400 x g for 5 min, and then washed twice (Wysocki et al., 1978) the cells on plastic Petri dishes (15 x 100 with ice-cold phosphate-buffered saline. The cells were remm) coated with rabbit anti-mouse F(ab')2 immunoglobulin G suspended in 400 1tL of hypotonic lysis buffer (0.2% Triton X-100, (Organon Tekunika Co., West Chester, PA, USA). After 10 mM Tris, 1 mM EDTA, pH 8.0) and centrifuged for 15 min at incubation for 90 min at 4°C, the non-adherent T-cell-enriched 13,800 x g (Newell et al., 1990). Half of the supematant containing population was recovered, which usually consisted of greater small DNA fragments, as well as the pellet containing large pieces than 95% Thy 1.2+ and less than 5% Ig+ cells, as determined by of DNA and cell debris, were used for the diphenylamine (DPA) immunofluorescence with a FACScan fluorescence-activated cell assay (see below). sorter (Becton Dickinson Co., Sunnyvale, CA, USA). Splenic Tcells were further incubated with anti-CD4- and anti-CD8-coated DNA Fragmentafion Assay magnetic beads (PerSeptive Diagnostics, Inc., Cambridge, MA, The DPA reaction was performed as described previously USA), and passed over a magnet for several cycles. Recovered in both (Peradones et al., 1993). Perchloric acid (0.5 M) was added to the resulted selection cells obtained by negative magnetic pellets containing uncut DNA (re-suspended in 200 gL of enriched CD4+ (> 95%) and CD8+ (> 95%) populations by hypotonic lysis buffer) and to the other half of the supematant fluorescence-activated cell-sorter analysis. containing DNA fragments. Then, 2 volumes of a solution B-cell Preparation containing 0.088 M DPA, 98% (vol/vol) glacial acetic acid, 1.5% (vol/vol) sulfuric acid, and 0.5% (vol/vol) of a 1.6% acetaldehyde described as occurred mouse from spleens B-cells of Preparation solution were added. The samples were stored at 4°C for 48 hrs. previously (Kurita-Ochiai et al., 1995). Briefly, splenic cell The colorimetric reaction was quantified spectrophotometrically suspensions were treated with a cocktail of monoclonal antibodies for at 575 nm with a model UV-160A UV spectrophotometer antibodies) and anti-L3T4 (rat anti-mouse Thy 1.2, anti-Lyt 2, (Shimazu Co., Ltd., Tokyo, Japan). The percentage of 30 min at 4°C, followed by incubation with rabbit anti-rat fragmentation was calculated as the ratio of DNA in the immunoglobulin G and complement (Low Tox rabbit for supematant to total DNA. complement, Cedarlane Laboratories Ltd., Ontario, Canada) Downloaded from jdr.sagepub.com by guest on July 12, 2011 For personal use only. No other uses without permission.

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J Dent Res 79(12) 2000

Bonferroni-Dunn test. Where appropriate, Student's t test was used to compare two groups.

RESULTS Butyric Acid Induces Apoptosis in the Absence of p53

0

I

Figure 2. Butyric-acid-induced apoptosis in CD4+ and CD8+ T-cells. Splenic T-cells were fractionated into (A) CD4+ and (B) CD8+ T-cells by negative magnetic selection. The cells were then cultured with the indicated concentrations of butyric acid for 21 hrs. Harvested cells were assayed by the DPA assay. The results are expressed as the mean ± SE of three different experiments with triplicate cultures. Values significantly different from the corresponding negative controls without butyric acid at p < 0.01 are indicatedby asterisks.

Measurement of Caspase Protease Activity After incubation of cells (16 x 106) in 24-well tissue culture plates for the indicated times with or without 5 mM butyric acid, all cells were collected, washed as described above, and suspended in 50 mM Tris-HCl (pH 7.4)-l mM EDTA-10 mM EGTA. After the addition of 10 mM digitonin, the cells were incubated at 37°C for 10 min. The lysates were clarified by centrifugation at 18,360 x g for 3 min, and cleared lysates containing 50 mg of protein were incubated with 50 gM each of Ac-YVAD-MCA (caspase-1 substrate), Ac-DEVD-MCA (caspase-3 substrate), Ac-VEID-MCA (caspase-6 substrate), and Ac-IETD-MCA (caspase-8 substrate) at 37°C for 1 hr. Levels of released 7-amino-4-methylcoumarin were measured with an MT32 spectrofluorometer (Corona Electric Co., Ibaraki, Japan) with excitation at 380 nm and emission at 460 nm. One unit was defined as the amount of enzyme required to release 0.22 nmol of 7-amino-4-methylcoumarin/min at 37°C.

Western Blotting Cells underwent lysis in lysis buffer [10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonident P-40, 1 mM EDTA, 1 mM EGTA, 0.1 mM PMSF, 8 gg/mL aprotinin, and 2 [tg/mL leupeptin], and were centrifuged at 14,000 rpm for 10 min at 4°C. The supematant was collected, and the amount of protein was measured by means of a BioRad protein assay (Bio-Rad, Hercules, CA, USA). Equal amounts (25 gg) of protein from each sample were separated in 12.5% SDSPAGE, and transferred to a polyvinylfluoride membrane (PVDF; Millipore, Bedford, MA, USA). Western blotting was performed with monoclonal antibodies against either anti-mouse Bcl-2 (clone N-19, Santa Cruz Biotechnology, CA), Bcl-XL (clone H-5, Santa Cruz), Bax (clone N-20, Santa Cruz), or p2lwAFl/cIPl (clone F-5, Santa Cruz). Primary antibodies were detected with either a goat anti-mouse or goat anti-rabbit horseradish-peroxidase-conjugated secondary antibody (Amersham, Little Chalfont, UK). Detection of chemiluminescence was performned with an ECL Western blot detection kit (Amersham), according to the supplier's recommendations.

Statistics Multiple-group comparisons were made by one-way analysis of variance, followed by post hoc intergroup comparison by the

We examined the effects of various concentrations of butyric acid on the DNA fragmentation of thymocytes, splenic T-cells, and splenic B-cells derived from homozygous p53'-1 and wild-type p53+'1 mice. When the three types of cells were cultured in the presence of 0.625 to 5.0 mM butyric acid for 21 hrs and quantitated by the DNA fragmentation assay, a dose-dependent increase in DNA fragmentation was seen in both p53-'- and p53+'1 cells (Fig. 1). Butyric acid induced a substantial and nearmaximal increase (75.8% and 69.5%, respectively, with 5 mM butyric acid) in DNA fragmentation (68.2% and 59.8%, respectively) for both p53+'1 and p53-'- thymocytes at 0.625 mM (p < 0.01). The relative amount of butyric-acid-induced apoptosis was slightly lower in the p53'-1 thymocyte population (-8.4% for thymocytes with 5 mM butyric acid) as compared with the otherwise isogenic p53+1+ thymocytes; however, statistically significant differences were not observed. For splenic T-cells and splenic B-cells from p53+'1 and p53-'- mice, 2.5 mM butyric acid significantly increased the amount of DNA fragmentation (57.2% and 54.0% for p53+'1 and p53-'- splenic T-cells, respectively; 66.3% and 56.1% for p53+'+ and p53'1- splenic B-cells, respectively). With 1.25 mM butyric acid exposure, butyric-acidinduced apoptosis was slightly enhanced in p53+'1 cells as compared with the p53-'- cells, though statistically significant differences were not observed. No differences in sensitivity or butyric-acid-induced apoptosis were observed between p53 heterozygous (+/-) and p53 homozygous wild-type (+/+) thymocytes, or splenic T- and B-cells (data not shown). In similar experiments, cells were cultured with 5 mM butyric acid, which induced a maximal increase in DNA fragmentation after 21 hrs of culture, and were then examined for DNA fragmentation at various times over a 21-hour time period. Treatment with butyric acid for 6 hrs resulted in a marked increase in DNA fragmentation of p53+1+ and p53-/- thymocytes (data not shown). For splenic Tand B-cells from p53+1+ and p53-'- mice, butyric acid rapidly induced apoptosis up to 16 hrs after treatment. However, statistically significant differences among DNA fragmentation of p53+/' and p53-'- mice were not observed in a time-dependent fashion in cells from any of the sources. Next, we fractionated splenic T-cells into CD4+ and CD8+ Tcells by negative magnetic selection. Experiments with these fractionated subpopulations of splenic T-cells revealed that DNA fragmentation was similarly observed in the CD4+- and CD8+T-cell populations from both p53+1+ and p53-/- lymphocytes (Fig. 2). Although the relative amount of butyric-acid-induced DNA fragmentation in CD4+- and CD8+- T-cells was slightly lower in the p53-/- lymphocytes than in the p53+/+ lymphocytes, statistically significant differences were not observed.

Activation of Caspase in Butyric-acid-induced Apoptosis Occurs Regardless of p53 The requirements of caspase- 1, caspase-3, caspase-6, and caspase8 in butyric-acid-induced apoptosis were determined by their capacity to cleave caspase- 1 substrate Ac-YVAD-MCA, caspase-3 substrate Ac-DEVD-MCA, caspase-6 substrate Ac-VEID-MCA,



J Dent Res 79(12) 2000 and caspase-8 substrate Ac-IETDMCA, respectively. Analysis of protease activation during cell death induced by treatment of splenic Tcells with butyric acid revealed similar results in increased levels of caspase-3, caspase-6, and caspase-8 protease activity in both p53+'1 and p53¾1- lymphocytes, but not of caspase-1 (Fig. 3). Notably, the increases in caspase-3 and caspase-6 protease activity began about 8 hrs after the addition of butyric acid and peaked after 16 hrs, reaching levels more than 12 to 14 times and 16 to 18 times, respectively, greater than those of control populations (Figs. 3B, 3C). In contrast, the increase in caspase-8 protease activity began immediately after the addition of butyric acid, peaked after 4 to 8 hrs, and then declined to the basal level after 16 hrs of culture (Fig. 3D). Clear time-lags in peak levels among caspase-8, caspase-3, and caspase-6 protease activities were recognized. However, no differences in the rate of increase among caspase-3, caspase-6, and caspase-8 protease activities induced by butyric acid were observed in p53"'+ or p53-splenic T-cells. Enhancement of caspase-3, caspase-6, and caspase-8 proteolytic activity induced by treatment of splenic T-cells with butyric acid was inhibited in a dosedependent manner by treatment with the caspase-3 inhibitor DEVD-CHO, caspase-6 inhibitor VEID-CHO, and caspase-8 inhibitor IETD-CHO, indicating that DEVD-CHO, VEIDCHO, and IETD-CHO inhibit the activation of caspase-3, caspase-6, and caspase-8 proteases, respectively, induced by butyric acid (data not shown).

Effect of Butyric Acid on Apoptosis-related Protein Expression in T-cells

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Splenic TFigure 3. Caspase activities in butyric-acid-treated splenic T-cells from p53+1+ or p53-/- mice. times. Cell cells were cultured in the presence or absence of 5 mM butyric acid (BA) for the indicated activities extracts were prepared, and (A) caspase-1, (B) caspase-3, (C) caspase-6, and (D) caspose-8 were measured by incubation with each respective caspase substrate. The results are expressed as the mean ± SE of three different experiments with triplicate cultures. Values significantly different from the corresponding negative controls without butyric acid at p < 0.01 are indicated by asterisks.

To determine whether the regulation of Bcl-2, Bcl-XL, Bax, and p2 1WAFI/CIPI protein expressions by butyric-acid stimulation was mediated by p53, we tested these protein expressions using Western blot analysis (Fig. 4). Splenic Tcell lysates from p53+/' and p53-/- mice showed strong signals for Bcl-2 and Bcl-XL expressions, and weak signals for Bax and p21WAF1/CIP1 expression. Bcl-2 and Bcl-XL expressions were similarly detected in splenic T-cells from p53-/- mice as well as from p53+/' mice. However, Bax and p21WAFI/CIPI expressions were not evident in p53-/- mice, inin contrast to p53+/' mice. With butyric-acid treatment

p53+'+ and p53-'- mice, Bcl-2 and Bcl-XL expressions

were

decreased, whereas Bax and p21WAFI'CIP1 levels remained constant. Thus, in splenic T-cells from p53 ++ and p53-1' mice, butyric-acid-induced apoptosis occurred independently of the p53-regulated proteins Bax and p21WAFI/cIPI and was accompanied by decreases in Bcl-2 and Bcl-XL expressions.

DISCUSSION The use of butyric-acid-sensitive T-cell cultures from p53-/mice allowed us to establish that p53 is not required for butyric-acid-induced T-cell apoptosis. The results of the DNA


Kurito-Ochlai eta/.

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Figure 4. Western blot analysis of mouse Bcl-2, Bcl-XL, Bax and p2]WAFI/CPi in splenic T-cells from p53+/+ or p53 mice. Splenic Tcells were cultured in the presence or absence of 5 mM butyric acid (BA) for 16 hrs. Extracts of the harvested cells were analyzed for Bc-2, BclXL, Bax, and p21WAFI/CIPI protein levels by immunoblot analysis with rabbit anti-mouse Bcl-2, Bcl-XL, Bax, and p2] WAH /clcp by means of an ECL detection technique.

fragmientation assay indicated that thymilocytes and spicnic Taiid B-cclIs fi-om p53 micc WCI C susceptible to butyric-acidinduccd apoptosis to a slimlar degrce as those firom p53 imice. Howxever, in all cell pOpUlation's and butyri c acid concentrations tested, the relative amoulnt of butyric-acidinduced apoptosis was slightly loweie in p53 micc than in p53 lnliCe, thougIl the dif'fcircices wcrc not statistically sigifiiicant. Therefore, it may bc possible that p53 contributes only slightly to butylic-acid-induced apoptosis. In contrast, it has becn reported that butylic acid decreased p53 expression in humlian hepatoma cell Iies havinig the wild-type p53 gene (Saito it al., 1 998). Analysis of oUr Western blotting data also indicatcd that p53 expression was decrcased slightly in butyi ic-acid-treated mrlilnc splcnic T-cels (data not shown). ButyriC acid significantly induced apoptosis in lympilocytes f'romi p53 and p53- micc in a concentrationi- and timedepencdlenit fashion. I hcsc results togethel suggest that p53 is not appicciably iinvolved in the decrcase of butyric-acidinduced apoptosis. In spleinic T-cells, bLItyric acid induced both C 1)4 - and ('[)8 -T cell apoptosis equally. Our previous study (KKulita-()ciiai ti a!., 1999) also indicated that butyric acid simila -ly induced both C[D4 - and C D8 - T-ccil apoptosis in PBM('. 1Ihsc rcsults indicate that there is no difference in the scnsitivity to butyi-ic acid betwexie CD4 - and CD8 spleinic T-cells tiom both pS 3 and p53 miice. It has also becn showin that C(1)4 aid CD[)8 cels fiom Ii IV-infectcd patients wvic c qually susccptible to apoptosis wheln induced by exposuic to the F as ligand oi Fas triggering antibody ( staq(iCi ci a!., 1 996). BUtylic-acid-illnlucCd D)NA fiagimcritation of splcnic T-cclls w as accomipanied by an incicase in caspase-3. caspase-6, and

J Dent Res 79(1 2) 2000

caspase-8 activities. but not in caspase- I activity, regaidless of presenice of p53. Time-coursc studies showed that easpase8, caspase-3, and caspase-6 are activated in a stepwise fashiioni, suggesting that they are organized into onle or imlltiple protease cascades. This suggests that caspase-3, caspase-6, and caspase8 each plays a required role in butyric-acid-induced T-cell death. C'aspases, a familily of cysteine proteases, cleave to other meimibers of the caspase family to activate their proteolytic activity in a cascade-like fashion, and the final taiget proteins prosecute apoptosis. It has been proposed that "initiator" caspases with long prodomains, suchi as caspase-8 (MACH/FLICE/MchS), either directly or indirectly activate "effector" caspases, sucil as caspase-3, -6. and -7 (Fraser and Evan, 1996; Srinivasula et al., 1996: Coheln, 1997). Procaspase-9 has also becn proposed as an initiator caspase, SiincC, in the preseince of dATP and cytochi omie C. its long Ntei-iniinal domaini interacts with Apaf 1, resultilng in thc activation of caspase-9 (Li et a!., 1997; Zou el al., 1997). Active caspase-9 can theln activate the effectors caspase-3, -6. and -7 (Zou Ct W! . 1997; Srinivasula el a!., 1998). Thus, multipie species of caspase-3 and caspase-6 constitute the mnaljor pool of activated caspases regardless of the initial apoptotic stimulus (Faleiro et al., 1 997). Western1 blotting analysis of splenic T-cells showed that butyric acid treatmieint decreased 13c1-2 and Bcl-XL expressioni, whereas Bax and p2l WAX ( I'P levels reimained constanit, regardless of the addition of butyric acid, in p53 and p53 cells. The mamminalian Bcl-2 family of apoptosis-associated pliotciiis consists of ieinibers that iilnibit apoptosis (Bcl-2, BclXL, Mcl-I, Al, etc.) and othcis that induce apoptosis (Bax, Bak, Bad, Bcl-XS, Bik, etc.), and the balance betweeni these pro-apoptotic and anti-apoptotic meimibers deteriniies the fate of cells in many systems (Oltvai and Korsimieyer, 1994; Kroemer, 1997). The Bcl-2 family regulates apoptosis by controllinig the activity of caspases, the executioners of apoptosis, via the rlcease of cytochrome C from mitoclionidria (Kroemei etal., 1998). The up-legulation of anti-apoptotic Bcl2 or its close hoinologue Bel-XL is known to inhibit apoptosis (Vaux et cl., 1988; Boise ct a!., 1993), whereas downregulation of Bcl-2 or its antagonizatioii by diinei'izatioil with Bax- x promotes programmed cell death (Oltvai et al., 1993). In studies of rat intestinal epithelial cells transfected with cyclooxygenase 2, elevated Bcl-2 expression was correlated with resistance to butyrate-iniduced apoptosis (Tsujii and Dubois, 1995). This observation, combined with our results, suggests that Bcl-2 may protect against the apoptosis induced by butyric acid. The corielation between butyric-acid-induced down-rcgulatioll of the expressioni of the apoptosis antagoniists. 13c1-2 and Bcl-XL, and the increased apoptosis induced by butyric acid suggests that butylic acid may act by lowerinig the levels of Bc1-2 and Bcl-XL, followed by an activatioii of caspases. Some studies have showni that Bcl-2 and Bcl-XL are equally effective and interchangeablc in their protectioni against the induction of apoptosis (Hluang el al., 1997). Since the great majority of tumoi cells shiow an increased expressioin of BclXL (Dole el al., 1995; Thompsoni, 1995), and because higi cellular levels of Bcl-XL correlate to rcsistance to the induction of apoptosis (Dole et il., 1995; Stiasser el al., 1995), inhibitioni of the expressioni of these apoptosis antagonists may repiesenit a major target for killiig mechanisms. the


J Dent Res 79(12) 2000


In this study, the expression of the p53-related proteins, Bax and p2 1WAFI/CIP1, was not influenced by butyric acid treatment in either p53-'- or p53+'1 mice. It has been shown that butyrate activates the p21 WAFI/CIPI gene promoter through Spl sites in a p53-negative human colon cancer cell line WiDr, which means that the butyrate-induced growth arrest in WiDr cells is due to a p53-independent activation of the p2 1WAF1/CIPI promoter (Nakano et al., 1997). Butyric acid was also found to induce cellular differentiation, as well as suppress the growth and tumorigenicity of SW620 hepatocellular carcinoma cells by a mechanism independent of p53 but possibly dependent on p2iWAFI/CIPI (Yamamoto et al., 1998). These results indicate that butyrate activates p2 1WAF/CIP1 independently of p53 in carcinoma cell growth arrest. However, our preliminary study showed that these WiDr and SW620 cells did not cause apoptosis, though they increased p2iWAFI/CIPI expressions by butyric acid (data not shown). This indicates that the sensitivity for growth arrest and apoptosis induced by butyric acid may differ among such cell types as carcinoma cell lines and normal systemic cells. Moreover, it suggests that there is no correlation between butyric-acid-induced p21 WAFI/CIP1 expression and butyric-acid-induced apoptosis in normal systemic cells. In conclusion, analysis of our data indicates that the p53 tumor suppressor molecule is not necessary for butyric-acidinduced apoptosis of murine T-cells. Furthermore, we found that butyric acid did not affect the expression of the p53regulated proteins, Bax and p2 1wAF/CIP1, during butyric-acidinduced apoptosis. Our study suggests that, by lowering the levels of the apoptosis antagonists Bcl-2 and Bcl-XL in cells, butyric acid can act to induce apoptosis in murine T-cells.

ACKNOWLEDGMENTS This work was supported in part by a research grant for frontier science and a grant-in-aid (11671818) for scientific research from the Ministry of Education, Science, and Culture of Japan, and by a Suzuki memorial grant (98-1003) from Nihon University School of Dentistry at Matsudo.

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