Induction of apoptosis in HL-60 cells by skimmed milk digested with a proteolytic enzyme from the yeast Saccharomyces cerevisiae

JOURNAL OF BIOSCIENCE Vol. 88, No. 4, 426-432.

AND BIOENGINEERING 1999

Induction of Apoptosis in HL-60 Cells by Skimmed Milk Digested with a Proteolytic Enzyme from the Yeast Saccharomyces cerevisiae MOLAY KUMAR ROY, YASUO WATANABE, AND YOUICHI TAMAI’; Department of Bioresources, Faculty of Agriculture, Ehime University, Matsuyama, Ehime 790, Japan Received 26 May 1999/Accepted I July 1999

Bovine skimmed milk digested with cell-free extract of the yeast Succharomyces cerevisiae was found to exhibit proliferation inhibition activity towards human leukemia (HL-60) cells. The optimum pH for digestion of skimmed milk and production of the proliferation inhibition factor was pH 4.8. Nondigested skimmed milk exhibited little suppressive effect on the proliferation of HL-60 cells. An active enzyme involved in the production of cell proliferation inhibitory materials from skimmed milk was purified from the cell-free extract of 5’. cerevisiae by a series of column chromatographies: DEAE-Sephacel, D-tryptophan methyl esterSepharose 4B, Hiload Superdex G-200 and HPLC Mono Q. The homogeneous purified enzyme and exhibited a molecular mass of 33 kDa in sodium dodeceyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and was identified as protease B by N-terminal amino acid sequence analysis. Bovine skimmed milk digested with purified protease B was found to inhibit proliferation activity of HL-60 cells most strongly when digestion was conducted at pH 4.8. The cell proliferation inhibition activity induced by digested skimmed milk was shown to be due to the induction of apoptosis, demonstrated by the formation of apoptotic bodies and fragmentation of DNA in treated cells. The proliferation inhibition factors produced were recovered in the soluble fraction of 92% ethanol, suggesting that the factors were hydrophilic low molecular mass substances derived from skimmed milk. [Key words: apoptosis, proliferation

inhibition,

skimmed milk, protease B]

Apoptosis, a highly regulated process of cell death, occurs during normal development and is also the cellular response to many physiological and biochemical stimuli (1). The process plays a crucial role in embryonic development, metamorphosis, hormone-dependent atrophy, tumor cell removal, regulation of cell number and elimination of damaged cells (2, 3). There is increasing interest in apoptosis-inducing materials and significant advances have been made in elucidating the molecular mechanisms responsible for the regulation of apoptosis. Recent studies have shown that some food components, which may not have any nutritive role, are biologically active and participate in the regulation of cell growth, differentiation and apoptosis (4, 5). The physiological functions of milk and milk products have been well characterized. Milk contains a wide array of molecules that have diverse biological functions such as antimicrobial, antihypertensive, antithrombotic, and opioid activities. In most cases, the materials that are usually dormant in raw milk can be activated by enzymatic treatment such as that occurring during gastrointestinal digestion or during fermentation. Fermented milk is usually prepared by culturing milk with lactic acid bacteria (e.g., Lactobacillus helveticus, Bl$dobacterium longum, Lactococcus lactis, and L. delbrueckii) but yeast (Saccharomyces cerevisiae) has also been used and the products have been shown to exhibit several physiological functions (6-8). In a previous study, we found that bovine milk fermented with lactic acid bacteria (B. longum) and yeast (S. cerevisiae) has marked proliferation inhibition activity towards HL-60 cells but no activ-

ity was detected using fermented milk prepared only with lactic acid bacteria such as B. longum (9). These observations suggested that yeast may play an important role in the production of proliferation inhibitors from milk. During fermentation, lactic acid bacteria characteristically produce lactic acid from lactose. However, there have been no systematic studies of the activity of S. cerevisiae, i.e., degradation of milk protein or the specificity of proteases toward milk proteins, the cellular location of the active proteases or their catalytic mechanism. Yeast vacuoles, which are lysozome-like organelles, contain several proteases including protease A, protease B, carboxypeptidase Y, carboxypeptidase S, aminopeptidase I, aminopeptidase Co and dipeptidyl aminopeptidase B (for review see 10). During fermentation, the vacuolar proteases of yeasts are released from autolyzed cells and may participate in protein degradation to generate smaller peptide fragments. In this study, we digested skimmed milk with cell-free extract of the yeast S. cerevisiae and the product was found to inhibit proliferation of HL-60 cells. The objective of this study was to identify the main enzyme present in the vacuoles of yeast that could lead to the production of apoptosis-inducing materials from precursor molecules in bovine skimmed milk. MATERIALS

AND METHODS

Materials Azocol (50-100 mesh) was obtained from Calbiochem Inc., D-tryptophan methyl ester and fluorescein isothiocyanated casein from Sigma Chemical Co. and DEAE-Sephacel and activated CH-Sepharose 4B were from Pharmacia Fine Chemicals Inc. The cell titer 96TM aqueous nonradioactive cell proliferation assay system (MTS assay system) was from Promega. RPM1 medium and Dulbecco’s PBS (-) were from Nissui Pharma-

* Corresponding author. Abbreviations: DSM-1, digested skimmed milk with cell-free extracts; DSM-2, digested skimmed milk with purified enzyme (protease W. 426

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ceutical Co. Ltd., Agarose was obtained from FMC Bioproducts and Micro BCA from PIERCE. All other reagents were from Wako Pure Chemical Inc., Osaka unless otherwise stated. Preparation of cell-free extracts of yeast The yeast strain S. cerevisiae X2180 1A used in this study was grown to the stationary phase with shaking at 30°C in YEPG (yeast extracts l%, Polypepton 2%, glucose 2%) medium. Cells were collected from the medium by centrifugation at 3000 x g for 5 min and washed twice with ice-cold water. Extracts were prepared by disrupting the cells with clear glass beads (diameter 0.3 mm) in a Vibrogen homogenizer for 25 min at 0°C and the extract thus obtained was centrifuged at 15,OOOxg for 10min. The clear supernatant was collected and dialyzed against distilled water at 0°C. The dialysate was centrifuged and the resulting clear supernatant was lyophilized and used as the cell-free extract. Enzyme activity assay Enzyme activity for the production of materials with cell proliferation inhibition activity from skimmed milk was assayed as follows: five ml of 1% skimmed milk was mixed with 1 ml of enzyme solution and the mixture was adjusted to pH 4.8 and incubated at 37°C for 3 h with shaking. The resulting mixture was heated at 90°C for 5 min to inactivate the enzymes and the pH of the solution was readjusted to 7.0. After centrifugation at 15,OOOXg for lOmin, the clear supernatant was collected and lyophilized. The lyophilized material was dissolved in 0.5 ml of distilled water and centrifuged again to remove the insoluble materials. Then, cell proliferation inhibition activity of the supernatant was measured by MTS assay. One unit of enzyme activity was expressed as the amount of enzyme yielding one unit of cell proliferation inhibitors per hour during digestion of skimmed milk at 37°C. Proliferation inhibition activity was measured by MTS assay under mentioned conditions. The proteolytic activity of the enzyme fraction was measured according to the method of Saheki and Holzer (ll), using azocol as a substrate and by the Twining method (12). Cell culture and proliferation inhibition activity assay Human acute myeloid leukemia (HL-60) cells were obtained from the Japanese Cancer Research Resources Bank (JCRB) and routinely cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 0.1 mg/ml streptomycin, 100 U/ml penicillin and 1.4 mg/ml NaHC03 at 37°C in a humidified atmosphere containing 5% COZ. Human dermal fibroblasts (Fb) were purchased from Cell System Corporation (USA) and cultured in a fibroblast growth medium system (CS-3FO-250) at 37°C in a humidified atmosphere containing 5% CO*. Cell proliferation activity was measured by the cell titer 96TM aqueous nonradioactive cell proliferation assay (MTS assay) or by counting viable cells. In MTS assay, cells (5 x lo4 cells/ml) suspended in 100 ~1 of medium were added to each wells of 96-well plates, to which 10 ~1 of digested skimmed milk had been previously added, and the plates were then incubated at 37°C in a humidified atmosphere containing 5% COZ. Incubation was continued for 4, 24 or 48 h, after which 22 ~1 of combined MTS/PMS solution was added. After incubation for 4 h, absorbance of the reaction mixture was measured at 490nm using a microplate reader (Bio Rad, Model 450). One unit of cell proliferation inhibition activity was expressed as the potency that exhibits 50% proliferation inhibition after 48 h of incubation under the above men-

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IN HL-60

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tioned conditions. For counting viable cells, HL-60 cells were seeded at an initial concentration of 1 x 105/ml in a mixture of 9 ml of culture medium and 1 ml of digested skimmed milk. The suspension was incubated at 37°C in a humidified atmosphere containing 5% COZ. After 24, 48 or 72 h of incubation, viable cells were counted by the trypan blue exclusion method with a minimum of 100 cells/field. Effects of pH on the digestion of skimmed milk Five ml of 1% skimmed milk was mixed with lyophilized cell-free extract (4.5 mg of protein) and the pH was adjusted to 3.6, 4.0, 4.4, 4.8, 5.2, 5.6, 6.0 or 6.8 (nonadjusted suspension) with acetic acid. Purified enzyme (150 pg) was also mixed with same volume of 1% skimmed milk and the pH was adjusted to 3.8, 4.8, 5.8 or 6.8 (nonadjusted suspension) with acetic acid. Then, samples were prepared by incubating the skimmed milk at 37°C for 3 h with shaking as described above and proliferation inhibition activity towards HL-60 cells was measured by MTS assay. The samples, prepared at different pHs upon digestion with cell-free extract and purified enzyme, were applied similarly to Fb to determine their effects on the proliferation of normal cells. Morphological examination HL-60 cells (1 x 106) suspended in 9ml of RPMI-1640 medium were incubated with 1 ml of digested skimmed milk and photographs were taken under a light microscope after 24 h. DNA fragmentation assay HL-60 cells (2 x 106) suspended in 9 ml of culture medium and 1 ml of skimmed milk digested with either cell free extract (DSM-1) or purified enzyme (DSM-2) were separately incubated for 5, 10, 15 and 20 h. Cells were also cultured similarly without any additives (negative control) or with actinomycin D (10 PM/ml) (positive control). After harvesting, by centrifugation cells were collected and washed in icecold PBS(-). Then, DNA was extracted from the cells using the ApopLadder ExTM kit (Takara Biomedicals, Osaka). Obtained DNA fragments were loaded onto 1.5% agarose gels in TBE buffer (89mM Tris, 89 mM boric acid, 2 mM EDTA) and electrophoresed at a constant voltage (100~). The gels were stained with ethidiurn bromide and photographed using a UV transilluminator . Ethanol fractionation of digested skimmed milk Five ml of 1% skimmed milk was digested with cell-free extract or purified enzyme as described above at 37°C for 3 h. The reaction mixture was heated at 90°C for 5 min and the pH was readjusted to 7.0. The resultant precipitates were removed by centrifugation. Ice-cold ethanol was added to the supernatant to a concentration of 67% and samples were kept overnight at -20°C. Following centrifugation at 15,OOOxg for 10 min, the obtained precipitate (ppt. 1) was lyophilized. The supernatant was made up to 82% ethanol and kept overnight at -20°C after which it was centrifuged. The resulting precipitate (ppt. 2) was collected by centrifugation and then lyophilized, and the supernatant was adjusted to 92% ethanol and treated as above. The resultant precipitate (ppt. 3) was lyophilized and the supernatant was evaporated. Obtained materials (EtOH sup) were lyophilized after dissolving in distilled water. Prior to analysis, each of the above four samples (ppt. 1, ppt. 2, ppt. 3 and EtOH sup) was dissolved in 0.5 ml of distilled water. Then, the cell proliferation inhibition activity towards HL-60 cells was measured by MTS assay. Other general methods The N-terminal amino acid

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sequences were determined using a PSQ-2 amino acid sequencer (Shimadzu). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to the method of Laemmli (13) on 10% polyacrylamide gels. The gels were stained with a silver reagent (Wako silver staining kit). Protein concentration was routinely measured using a micro-BCA protein assay kit (PIERCE).

fication were carried out at 0°C to 4°C unless otherwise stated. Step 1. Ammonium sulfate fractionation Cell-free extracts obtained as described in Materials and Methods were precipitated by adding solid ammonium sulfate up to 50% saturation. The solution was adjusted to pH 5.0 and kept overnight at 0°C. Following centrifugation at 15,000 x g, the precipitate was removed and the remaining supernatant was made up to 90% saturation with ammonium sulfate. Then the precipitate was again collected by centrifugation of the remaining supernatant. The activity in the production of cell proliferation inhibition materials from skimmed milk was detected in the fraction precipitated between 50% to 90% saturated ammonium sulfate. Therefore, the precipitate was dialyzed against buffer A (50 mM Tris-HCI buffer, pH 7.5) after dissolving in the same buffer.

RESULTS Production of proliferation inhibitory materials from skimmed milk The materials formed at different pHs upon digestion of skimmed milk with cell-free extract were examined for their inhibitory activity on the proliferation of HL-60 cells. Bovine skimmed milk digested with cell free-extract of S. cerevisiae was found to inhibit proliferation of HL-60 cells and pH 4.8 was optimum for the production of cell proliferation inhibition materials (Fig. 1A). On the other hand, whole skimmed milk (nondigested skimmed milk) exhibited little suppressive effect on the proliferation of HL-60 cells. The results suggest that the cell proliferation inhibition materials are generated by enzymatic cleavage of milk components. The identification and characterization of a yeast enzyme involved in the production of cell proliferation inhibition materials appeared to be very important for clarifying the degradation mechanism of milk components and production of biologically active substances. Thus, we performed purification of the enzyme from yeast by a series of column chromatographies. Also, protease activity in the fraction was measured after each purification step, because most biologically active substances in milk have been demonstrated to be derived by proteolytic degradation from the major milk proteins. Enzyme purification All procedures of enzyme puri_ ,

(A) HL-60

cells

(El) HL-60

Step 2. DEAE-Sephacel column chromatography The resulting clear enzyme solution was applied to a DEAE-Sephacel column (1.8 x 20 cm) pre-equilibrated with buffer A. The column was washed with the same buffer and eluted first with buffer A containing 0.1 M NaCl and then successively with the same buffer containing 0.2, 0.3, 0.4, 0.5 and 1.O M NaCl. In this and subsequent chromatographic steps, protein concentration was measured at an absorbance of 280 nm. The highest activity in the production of cell proliferation inhibition materials was detected in the 0.1 M NaCl-eluted fraction. The highest protease activity was also detected in the same 0.1 M NaCl-eluted fraction. Step 3. AJinity chromatography Active fractions obtained by DEAE-Sephacel chromatography were concentrated by ultrafiltration (Amicon PM-lo) and dialyzed against 0.1 M potassium phosphate buffer at pH 7.0, containing 0.5 M NaCl (buffer B). The clear solution was applied to a D-tryptophan methyl ester-substituted CHcells

(Cl Fb

1 o-n

10

20

30

Culture time (h)

JO

.x1

0.5! ’

0-1 0

10

20

30

40

Culture time (h)

SO

0

I

I

I

I

IO

20

XI

40

Culture time (h)

FIG. 1. Effects of skimmed milk digested with cell-free extracts (DSM-1) or purified enzyme (DSM-2) on the proliferation of HL-60 or Fb cells. Skimmed milk was digested with cell-free extract or purified enzyme at different pHs as described in Materials and Methods. Proliferation activity was measured by MTS assay. (A) Effect of DSM-1 on HL-60 cells; 0 , in the absence of digested milk; 0, in the presence of nondigested skimmed milk; Q, digested at pH 3.6; + , digested at pH 4.0; A, digested at pH 4.4; 0, digested at pH 4.8; ?, digested at pH 5.2; D, digested at pH 5.6; V, digested at pH 6.0 and x , digested at pH 6.8. (B) Effect of DSM-2 on HL-60 cells; q , in the absence of digested milk; 0, in the presence of nondigested skimmed milk; a, digested at pH 3.8; 0, digested at pH 4.8; A, digested at pH 5.8 and 0, digested at pH 6.8. (C) Effect of DSM-2 on Fb; 0, in the absence of digested milk; 0, in the presence of nondigested skimmed milk; 4, digested at pH 3.8; 0, digested at pH 4.8; A, digested at pH 5.8 and @, digested at pH 6.8.

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Purification

Total activity W) 3850.6 4120.2 1610.3 210.6 155.0 29.2 27.6

(rng)

Cell-free extract Ammonium sulfate fractionation DEAE-Sephacel chromatography Affinity chromatography Superdex G-200 1st Mono Q 2nd Mono Q

2071.5 1620 98.6 5.66 3.5 0.315 0.26

Sepharose 4B (14) column (1 X 10 cm) pre-equilibrated with buffer B. The column was washed first with the same buffer and then with 0.1 M acetate buffer, pH 5.0. Finally, the activity for the production of cell proliferation inhibition materials was found in the fractions eluted with 0.1 M acetate buffer (pH 3.8). The protease activity was also detected in the same fraction eluted with 0.1 M acetate buffer (pH 3.8). The pH of the active fractions was immediately increased to 7.0 by adding solid Tris and the enzyme solution was dialyzed against buffer A containing 0.1 M NaCl. Step 4. Hiload Superdex G-200 chromatography After

dialysis,

the clear

active

enzyme

solution

obtained

by affinity chromatography was applied to a Hiload 161 60 Superdex 2OOpg column (Pharmacia Fine Chemicals) pre-equilibrated with 50 mM Tris-HCl buffer (pH 7.5) containing 0.1 M NaCl. The column was eluted with the same buffer at a flow rate of 1 ml/min at 3”C, and 2 ml fractions were collected. The fraction active in the production of cell proliferation inhibition materials was pooled and concentrated by ultrafiltration (Amicon PM10) following dialysis against buffer A. The protease ac-

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1. Summary of enzyme purification

Total protein

step

OF APOPTOSIS

Specific activity (U/ma) 1.85 2.5 16.3 37.2 44.3 92.7 106.2

Purification

fold

1 1.35 8.8 20.1 23.9 50.1 57.4

tivity was also detected in the same active fraction. Step 5. Mono-Q column chromatography The concentrate was applied to a HPLC Mono-Q column preequilibrated with buffer A. The column was washed for 5 min and then eluted with a linear gradient of 0 to 0.3 M NaCl in the same buffer. The active fraction was collected and dialyzed against buffer A and the concentrated sample was reapplied to the same Mono-Q column for a second round of ion-exchange chromatography. The final fraction with activity for the production of cell proliferation inhibition materials was used as the purified enzyme. The protease activity was also detected in the same active fraction. The results of purification are summarized in Table 1. After purification of the enzyme, we performed digestion of skimmed milk at different pHs. The purified enzyme was found to show the highest activity around pH 4.8 for the production of inhibitors from skimmed milk (Fig. 1B). In contrast, digested skimmed milk showed little effect on the proliferation of normal Fb (Fig. IC), suggesting that sensitivity to the digested milk differed between different cell lines, such as transformed (HL-60) and untransformed cells (Fb). SDS-polyacrylamide gel electrophoresis To examine the molecular mass and homogeneity of the purified enzyme fraction, samples were analyzed by SDS-PAGE, and visualized by silver staining. With SDS-PAGE, the active fraction obtained from the Mono-Q column

-P

45

FIG. 2. SDS-polyacrylamide gel electrophoresis of purified enzyme. Electrophoresis of molecular marker proteins and denatured protein was performed on 10% (w/v) SDS-PAGE gels. Lane 1, Molecular marker proteins including bovine albumin (Mm=66 kDa), egg albumin (45 kDa), glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle (36 kDa), carbonic anhydrogenase from bovine erythrocytes (29 kDa), trypsin inhibitor from soybean (20.1 kDa) and a-lactalbumin from bovine milk (approximate molecular weight 14 kDa). Lanes 2 and 3 show lysozyme from chicken egg white (14.3 kDa), lane 4 is purified enzyme. The gel was stained with a silver staining reagent.

0

20

40

60

-I

80

Culture time (h) FIG. 3. Effects of DSM-1 or DSM-2 on the viability of HL-60 cells. Cells were seeded at an initial concentration of 1 x 10S/ml and incubated with two preparations of digested skimmed milk (DSM-1 and DSM-2) for 24,48 or 72 h as described in Materials and Methods. Cell viability was estimated by counting viable cells: u , in the absence of digested skimmed milk; 0, in the presence of DSM-1; 0, in the presence of DSM-2.

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gave a single band corresponding to a molecular mass of about 33 kDa (Fig. 2). This value was comparable to that of other preparations (32 to 33 kDa) of protease B from yeast (14-16). N-Terminal amino acid sequence of purified enzyme The N-terminal amino acid sequence of this purified enzyme was analyzed and the sequences of the 15 N-terminal residues were NHz-Glu-Phe-Asp-Thr-Gln-Asn-SerAla-Pro-Trp-Giy-Leu-Ala-Arg-Ile. By comparing with the sequence of protease B reported by Charles et al. (16), it was found that the amino acid sequence of the purified enzyme perfectly matched the amino acid sequence of protease B. This and the above results indicated that the purified enzyme was protease B from yeast. Apoptosis-inducing activity of skimmed milk digested with cell-free extract and protease B on HL-60 cells

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FIG. 5. Effects of DSM-I or DSM-2 on DNA fragmentation of HL-60 cells. Time course analysis was performed by incubating DSM-1 or DSM-2 for 5, 10, 15 or 20 h. Lane 1, control cells. Lane 2, cells were treated with actinomycin D. Lanes 3, 5, 7 and 9, cells were treated with DSM-1 for 5, lo,15 or 20 h, respectively. Lanes 4,6,8 and 10, cells were treated with DSM-2 for 5, 10, 15 or 20 h, respectively.

Skimmed milk digested with both cell-free extract and purified enzyme (protease B) exhibited marked cytotoxic effects and apoptosis-inducing activity towards HL-60 cells. Cytotoxicity was estimated by measuring cell proliferation using MTS assay or by counting viable cells. Apoptosis-inducing activity was estimated by morphological examination under a light microscope and by DNA fragmentation assay. Treatment with digested skimmed milk strongly suppressed proliferation of HL60 cells (Fig. 3) as ascertained by counting viable cells. Morphological examination of the treated HL-60 cells under a light microscope (Fig. 4A, B) apparently showed typical apoptotic characteristics such as cell (A)

Culture

FIG. 4. Morphological appearance of HL-60 cells. Cells were incubated in the presence of DSM-1 and DSM-2 for 24 h. (A) Cells incubated in the presence of DSM-1; (B) cells incubated in the presence of DSM-2 and (C) cells incubated in the absence of skimmed milk (control).

W

time (h)

Culture

time (h)

FIG. 6. Effects of ethanol-fractionated-digested skimmed milk (ppt. 1, ppt. 2, ppt. 3, and EtOH sup.) on the proliferation of HL-60 cells measured by MTS assay. Samples were prepared as described in Materials and Methods. (A) Skimmed milk was digested with cell-free extract; q , in the absence of skimmed milk; 0, in the presence of nondigested skimmed milk; A, ppt. 1; V, ppt. 2; rt), ppt. 3; 0, EtOH sup. (B) Skimmed milk was digested with purified enzyme; 0 , in the absence of digested milk; 0, in the presence of nondigested skimmed milk; A, ppt. 1; V, ppt. 2; 8, ppt. 3; C:, EtOH sup.

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shrinkage and membrane blebbing, whereas untreated cells (Fig. 4C) retained their normal morphology during the experimental period. Agarose gel electrophoresis of the DNA extracts of treated cells exhibited a ladder-like pattern of fragmented DNA (Fig. 5), a characteristic feature of apoptosis. Ethanol fractionation and proliferation inhibition activity Proliferation inhibition activities of all the ethanol-fractionated samples (ppt. 1, ppt. 2, ppt. 3 and EtOH sup) towards HL-60 cells were measured by MTS assay; the 92% EtOH soluble fraction was found to be the most active, whereas the precipitated samples showed almost no activity (Fig. 6). DISCUSSION The proteolytic system of S. cerevisiae has been intensively investigated over the past two decades. However, this is the first study to utilize the proteolytic ability of S. cerevisiae for the formation of physiologically active components from bovine milk. In this study we purified an enzyme from the cell-free extract of S. cerevisiae that can release apoptosis-inducing factor from bovine skimmed milk, and the enzyme was identified as protease B from yeast. For the last few years, S. cerevisiae has been used in starter cultures with lactic acid bacteria for the production of fermented milk and these milk products have been found to have several physiological functions (6, 8). The mechanism of production of these functional materials from fermented milk is not yet clear, but some authors have suggested that it is due to the proteolytic activity of the microbes (17). Yeast cells are known to contain various proteolytic enzymes such as protease A, protease B, carboxypeptidases Y, carboxypeptidase S, aminopeptidase I, aminopeptidase Co and dipeptidyl aminopeptidase B (for review see 10). However, the structure of the cleavage sites of the substrates of protease A and protease B has not yet been determined. In this study, we found that protease B from yeast can produce cell proliferation inhibitory materials from bovine skimmed milk. In the first step of chromatography, cell proliferation inhibition activity and proteolytic activity were detected in the 0.1 M NaCl fraction which predominantly contained protease B (16, 18). Little or no activity was detected in the fractions eluted with 0.2M, 0.3 M, 0.4M and 0.5 M, NaCl-containing buffer (data not shown), which were reported to contain other yeast proteases such as protease A and carboxypeptidase Y (19, 20). These observations suggest that neither protease A nor carboxypeptidase Y is the main enzyme involved in the degradation of milk proteins. After gel filtration on a Hiload Superdex G-200 column, the active fractions were further purified by HPLC (Mono-Q column). However, after affinity chromatography, the enzyme was found to be very labile when undergoing the purification process. Due to the possibility of autodigestion, all steps after affinity chromatography were accelerated. During purification, the active enzyme fractions for the production of cell proliferation inhibitory materials were always found to be proteolytically active. This observation suggested that the production of these functional materials from skimmed milk was due to the action of a proteolytic enzyme. Homogeneity of the final enzyme preparation was verified by SDS-PAGE and characterized with respect to molecular mass, optimum pH activity and N-terminal

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amino acid sequence. Skimmed milk digestion with cellfree extract and purified enzyme (protease B) was conducted at different pHs, and the greatest amounts of materials with cell proliferation inhibition activity were formed by digestion at pH 4.8, although pH 7.0 was determined optimum for hydrolyzing azocol by protease B (14). This discrepancy in the pH dependence of digestion might be due to the differences in substrates, i.e., azocol and milk proteins, and digestion at pH 4.8 appeared to be optimum for milk protein. Several authors also demonstrated that low pH treatment (pH 5.0) enhanced the activity of protease B by inactivating its inhibitor complex (16, 18). Some milk proteins such as bovine casein and lactoferrin have been treated with digestive enzymes such as pepsin and trypsin, resulting in the production of several biologically active peptides with antibacterial (21) or antihypertensive properties (22). However, very few experiments have been conducted with respect to proliferation inhibition activity towards animal cells. This study demonstrated that protease B from yeast induced the release of proliferation inhibitory materials from precursors in bovine skimmed milk. These materials were recovered in the 92% ethanol soluble fraction and thus were characterized as low-molecular-mass compounds produced from bovine milk proteins. Inhibition of cell proliferation by digested skimmed milk was inferred to be due to the induction of apoptosis of HL-60 cells, ascertained by changes in cell morphology (Fig. 4A, B) and DNA laddering (Fig. 5). In this study inhibition of proliferation was first observed after 8-10 h of incubation, but DNA laddering was observed within 5 h of incubation, suggesting on earlier collapse of chromatin materials in the presence of digested skimmed milk. After dissociation of the nuclear matrix, morphological changes such as cell shrinkage and membrane blebbing occurred which accounted for the inhibition of cell proliferation revealed by MTS assay. The detailed mechanism by which digested skimmed milk induces apoptosis in HL-60 cells is still unclear. Experiments to elucidate this mechanism in addition to identification of the active fragments are currently underway in our laboratory. REFERENCES

I. WyIIie, A. H.: Apoptosis (the 1992 Frank Rose memorial lecture). Br. J. Cancer, 67, 205-208 (1993). 2. Raff, M. C.: Social controls on cell survival and cell death. Nature, 356, 397-400 (1992). 3. Kerr, J. F., Wyilie, A. H., and Currie, A. R.: Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer, 26, 239-257 (1972). 4. Kuo, M. L., Huang, T. S., and Lin, J. K.: Curcumin, an anti oxidant and anti-tumor promoter, induces apoptosis in human leukemia cells. Biochim. Biophys. Acta, 1317, 95-100 (1996). 5. Sakamoto, K., Lawson, L. D., and Mimer, J. A.: Ally1 sulfides from garlic suppress the in vitro proliferation of human A549 lung tumor cells. Nutr. Cancer, 29, 152-156 (1997). 6. Tamai, Y., Oishi, H., Nakagawa, H., Kuwabara, Y., Yamamoto,

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