Preferential alkaline phosphatase isoenzyme induction by sodium butyrate

Biochimica et BiophysicaActa, 762 (1983) 289-294

289

Elsevier Biomedical Press BBA 11145

PREFERENTIAL ALKALINE PHOSPHATASE ISOENZYME INDUCTION BY SODIUM BUTYRATE FRITZ HERZ and MURRAY HALWER

Department of Pathology, Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, N Y 10467 (U.S.A.) (Received October 20th, 1982)

Key words: Alkaline phosphatase isozyme; Sodium butyrate; Enzyme induction; (S W-620 cell)

SW-620, a continuous cell line derived from a poorly differentiated human colon carcinoma, produces two alkaline phosphatases. Under basal conditions the heat-stable, term-placental is the major isoenzyme and the heat-labile, liver/bone/kidney form represents a minor component. Exposing SW-620 cells to sodium butyrate causes induction of increased levels of activity accompanied by a striking shift in isoenzyme distribution not observed heretofore. The activity increase is accounted for entirely by augmentation of the liver/bone/kidney isoenzyme, with the term-placental form not being affected. Two other known alkaline phosphatase inducers, prednisolone and hyperosmolality, do not influence specific activity and isoenzyme distribution. The preferential induction of the liver/bone/kidney form of alkaline phosphatase in SW-620 cells may reflect a butyrate-elicited expression of a more differentiated state.

Introduction Sodium butyrate has a variety of effects on animal cells which mimic differentiation [1]. These include hemoglobin synthesis [2], interferon production [3], induction of glycoprotein and steroid hormones [4-6], enzyme induction [1]. It has been suggested that these effects may be related to histone hyperacetylation [7] due to butyrate inhibition of histone deacetylases [8]. Transcriptional activity of chromatin [9] and control of gene expression [10] have been correlated with histone acetylation. Alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum) EC 3.1.3.1), a membrane-bound glycoprotein, is among the enzymes induced by butyrate [11]. Three easily distinguishable alkaline phosphatase isoenzymes, coded by three separate structural gene loci, have been identified in humans. They are the heat-stable, term-placental and the heat-labile, intestinal and liver/bone/kidney 0167-4889/83/0000-0000/$03.00 © 1983 Elsevier Science Publishers

forms [12]. The term 'tissue-unspecific' has been introduced to describe the latter enzyme form [13]. Induction by butyrate has been documented with continuous tumor cells producing the term-placental [14,15] and the intestinal isoenzymes [16]. This report concerns the alkaline phosphatase of SW620, a cell line derived from a poorly differentiated human colon carcinoma [17]. We will show that this line produces two alkaline phosphatases of which, under basal conditions, the term-placental is the predominant isoenzyme, with the liver/ bone/kidney form representing a minor component. It has been shown that when a cell line synthesizes two different alkaline phosphatases, only the term-placental [18-21], and rarely both [22], are inducible. We demonstrate here that the butyrate-mediated enzyme induction in SW-620 cells [23] is unique: the fatty acid induces only the liver/bone/kidney isoenzyme, without affecting the oncofetal, term-placental isoenzyme. In addition we will show that in SW-620 cells, both isoenzymes are refractory to prednisolone and to

290 hyperosmolality, known inducing stimuli of the term-placental isoenzyme in many tumor cells [18,20,24]. The novel observation of a preferential amplification of gene expression may reflect a more differentiated state as compared to that of untreated control SW-620 cells and be related to the butyrate-mediated suppression of in vitro tumorigenicity of this line [23]. Materials and Methods

Cell culture SW-620 cells, provided at passage 109 by Dr. W.B. McCombs, III, were grown in plastic T75 flasks using Eagle's minimum essential medium containing 11% fetal bovine serum, penicillin (100 U/ml), streptomycin (100 /~g/ml) and amphotericin B (0.25 /~g/ml). Medium was routinely changed three times a week. After growth for 1 week in a humidified atmosphere of 5% CO z in air, the cells were subcultured using 0.05% trypsin/ 0.02% EDTA [16]. To study the response of SW-620 to alkaline phosphatase inducers, the stimuli were added 24 h after cell transfer. The effect of hyperosmolality was tested by increasing the osmolality of the standard medium from 284 mosmol/kg to 384 mosmol/kg by adding 50 mM NaC1 [20]. The effects of prednisolone and sodium butyrate were tested at final concentrations of 1.4/sM [20] and 2 mM [16], respectively. In some experiments the cultures were supplemented with various combinations of NaC1, prednisolone and sodium butyrate. Cells growing in regular medium served as controls. All experiments were done in duplicate. Cultures for enzyme assays were harvested 48 h later by lysis with 0.5 ml of 0.25% sodium deoxycholate [IlL Enzyme assays Alkaline phosphatase activity was measured in duplicate by the hydrolysis of p-nitrophenyl phosphate [25] using 2-amino-2-methyl-1-propanol-HCl buffer (pH 10.6). Specific activity was expressed in ~mol of p-nitrophenol liberated in 1 min at 37°C per mg protein, the latter determined according to Lowry et al. [26]. Thermostability of alkaline phosphatase was tested by preincubating triplicate 0.05-ml aliquots (containing 100-200 /~g protein) with 0.1 ml of 0.67 M 2-amino-2-methyl-l-pro-

panol-HC1 buffer (pH 10.6) at 56°C [20,25]. The percentage of residual activity was computed from controls kept with buffer at 4°C. Inhibition studies were done on partially purified, n-butanol-extracted enzyme solutions [16]. Extracts from tumor cells of cervical [27], colonic [16] and intracranial [28] origin that synthesize term-placental, intestinal and liver/bone/kidney alkaline phosphatases, respectively, were included for comparison. The following specific inhibitors were tested (range of final concentrations in parenthesis): L-phenylalanine (1-20 mM); L-phenylalanylglycylglycine (0.25-20 mM); L-homoarginine (1-20 mM); levamisole (0.02-5 raM); and 1-bromotetramisole (0.005-2 mM). To triplicate 0.05 ml enzyme samples, 0.1 ml of inhibitor and 0.2 ml of substrate/buffer/ MgC1 z were added. 0.1 ml of inactive isomers was added to each control. After incubation at 37°C the reaction was stopped with 0.25 M NaOH and the percentage of inhibition was computed from the respective controls. The results were expressed according to Goldstein and Harris [29] as that concentration of inhibitor required to produce 50% inhibition ([I]50 ). Results and Discussion

SW-620 cells have relative low baseline alkaline phosphatase activity (Table I). Treatment with prednisolone or growth in hyperosmolar medium did not induce increased enzyme levels. However, exposure to sodium butyrate for 48 h resulted in a significant increase. Specific activity of the experimental cultures was 3.5-times higher than in controis. In contrast to the enhancing effect seen when HeLa $3 [30] or HT-29 [16] cells are exposed to sodium butyrate in combination with glucocorticoids or hyperosmolality, respectively, these mixtures failed to increase activity above the levels seen with the fatty acid alone (Table I). Thermostability, a very sensitive parameter for distinguishing alkaline phosphatases [20,25], was used for the initial characterization of the activity of SW-620. As can be seen in Fig. 1, preincubating preparations from untreated SW-620 cells at 56°C revealed the presence of one major heat-stable enzyme component and one minor heat-labile component. By extrapolation to zero time of preincubation it can be estimated that the former

291 TABLE I EFFECT OF VARIOUS AGENTS ON A L K A L I N E PHOSPHATASE ACTIVITY OF SW-620 CELLS Cells were inoculated into regular medium. Additions were made to duplicate cultures after 24 h. Cultures were harvested 48 h later. Specific activity, determinated in duplicate on replicate cultures, is expressed as ~mol of p-nitrophenol liberated/min per mg protein. Heat-stable and heat-labile activity was computed from percentages of activity remaining after preincubation (in triplicate) at 56°C for 20 min with 0.67 M 2-amino-2-methyl- 1-propanol-HC1 buffer (pH 10.6). Addition to culture medium

Total specific activity (xl03)

Heatstable activity (x103)

Heatlabile activity (>(103 )

None Prednisolone (0.5/tg/ml) NaCI (50 mM) Sodium butyrate (2 mM) Prednisolone + NaC1 Prednisolone + sodium butyrate NaC1 + sodium butyrate Prednisolone + NaCI + sodium butyrate

3.1 4.1 3.6 11.1 3.0

2.1 2.8 2.6 1.6 2.0

1.0 1.3 1.0 9.5 1.0

9.6 13.4

1.9 1.5

7.7 11.9

10.8

1.5

9.3

iO0~_

80. 60-

b.I > ~

40-

~

20-

o

Io 0

5

I0

15

20

TIME AT 56*C(rnin)

Fig. 1. Time-course of heat inactivation of alkaline phosphatase activity of control (e e) and sodium butyrate-treated (O O) SW-620 cells. Specific activity of control cells was 3.2.10 -3 and that of butyrate-treated SW-620 was 11.5. 10 -3. Triplicate 0.05-ml aliquots (containing 100-200 #g protein) of enzyme preparations were incubated with 0.1 ml of 0.67 M 2-amino-2-methyl-l-propanol-HC1 buffer (pH 10.6) at 56°C for time indicated and then stored at 4°C. Residual enzyme activity was related to aliquots kept with buffer at 4°C.

represented over 75% of the activity. Upon exposure of SW-620 to sodium butyrate this proportion was dramatically altered. The heat-stable component comprised now less than 20% of the total activity. This change was not due to an effect of the fatty acid on the heat-stable enzyme; the term-placental alkaline phosphatase of HeLa $3 or of C4I cells [16] treated with butyrate had the same thermostability as that of untreated cells and exposing purified term-placental enzyme (Type XXIV, Sigma) to butyrate did not change its thermostability. In addition, thermal inactivation studies on enzyme mixtures ruled out the presence of extrinsic stabilizing or labilizing factors in control and butyrate-treated SW-620 cells, respectively. From the thermostability tests it is evident that sodium butyrate caused induction of the heat-labile activity without influencing the thermostable (term-placental) enzyme (Table I). Heat stability also was tested on prednisolone- a n d / o r hyperosmolality-treated cells, since there are precedents that these effectors, while not augmenting total activity, can induce increased levels of only one isoenzyme [18,20]. However, neither factor affected the proportion of heat-stable or heat-labile activity, thus providing additional evidence that the alkaline phosphatase of SW-620 was refractory to these inducers. The enzyme was further characterized with specific inhibitors that readily distinguish the various isoenzymes [16,29]. Using multiple inhibitor concentrations we determined the concentration required to produce 50% inhibition, [1150, on heated and unheated preparations of control and sodium butyrate-treated SW-620 cells. As shown in Table II, the inhibition profiles of unheated and heated extracts from untreated cells were similar, reflecting the predominance of the term-placental isoenzyme. By contrast, the profiles of extracts from sodium butyrate-treated cells were strikingly dissimilar. The [I]50 for L-phenylalanine and LPheGly-Gly were 4- and 18-times higher for unheated as compared to heated preparations. On the other hand, the [1150 for L-homoarginine was significantly smaller for the unheated than for the heated extracts and those for levamisole and 1-bromotetramisole in unheated extracts were very similar to those of the liver/bone/kidney isoenzyme. These results indicate that the heat-labile enzyme

292 TABLE 1I CONCENTRATIONS OF INHIBITORS P R O D U C I N G 50% INHIBITION OF ALKALINE PHOSPHATASE ACTIVITY Assays were done in triplicate on n-butanol-extracted enzyme solutions using p-nitrophenol phosphate as substrate in 2-amino-2methyl-l-propanol-HC1 buffer (pH 10.6). At least five concentrations of each inhibitor were used. Estimates of [I]50 were obtained from graphs depicting 100/percentage activity remaining vs. concentration of inhibitor, The inhibitor concentration required to produce 50% inhibition was obtained by putting (100/percentage activity remaining)= 2. Graphical representation and [I]5 o estimation according to Goldstein and Harris [29]. 'Unheated' and 'heated' refer to enzyme preparations that were preincubated prior to the inhibition tests with 0.67 M 2-amino-2-methyl-l-propanol-HCl buffer (pH 10.6) for 20 min at 4°C and 56°C, respectively. All values are of [I]50 expressed in mM. Inhibitor

SW-620 cells Untreated cells Unheated

L-Phenylalanine LPhe-Gly-Gly L-Homoarginine Levamisole 1-Bromotetramisole

4.0 0.6 >> 50.0 1.3 0.6

Term-placental enzyme

Intestinal enzyme

Liver/bone/ kidney enzyme

4.5 0.5 >>50.0 2.0 0.6

3.0 14.0 >>50.0 11.0 3.0

>>50.0 >>50.0 3.7 0.09 0.01

Treated with 2 mM sodium butyrate

Heated

3.5 0.4 >> 50.0 1.2 0.5

Unheated

Heated

12,5 9.0 10.0 0.1 0.02

3.0 0.5 >>50.0 1.0 0.5

preferentially induced by sodium butyrate corresponds to the liver/bone/kidney form of alkaline phosphatase. Heterogeneity of continuous cell lines with respect to alkaline phosphatase is well known [31]. Thus, the possibility that SW-620 consists of two types of cell, one producing term-placental and the other, liver/bone/kidney isoenzymes, had to be considered. Using consecutive single-cell platings

[31] clonal populations were isolated. Upon testing these cells for alkaline phosphatase, as expected [25,31], some variability in specific activity was observed. However, the results of thermostability and inducibility studies were similar to those obtained with the parental line: only the heat-labile activity was induced by butyrate (Table III). Since specific glucocorticoid receptors have been identified in responsive cells [32], the lack of re-

TABLE III ALKALINE PHOSPHATASE ACTIVITY OF CLONES DERIVED FROM SW-620 CELLS Cells of clonal populations were inoculated into regular medium. Sodium butyrate (2 mM) was added to duplicate cultures after 24 h. Cultures were harvested 48 h later. Specific activity, determined in duplicate on replicate cultures was expressed as ~tmol of p-nitrophenol liberated/min per mg protein. Heat-stable and heat-labile activity was computed from percentages of activity remaining after preincubation (in triplicate) at 56°C for 20 min with 0.67 M 2-amino-2-methyl-l-propanol-HC1 buffer (pH 10.6). Clone

G-1 H-4 BC-220 BC-29

Sodium butyrate-containing medium

Regular medium Total specific activity (X103 )

Heat-stable activity (X103 )

Heat-labile activity (X103 )

Total specific activity (X103 )

Heat-stable activity (X103 )

Heat-labile activity (X103 )

0.7 1.6 3.6 16.8

0.5 1.3 2.4 12.8

0.2 0.3 1.2 4.0

2.1 4.3 9.2 36.8

0.3 0.8 2.6 6.9

1.8 3.5 6.6 29.9

293

sponse of SW-620 to prednisolone may be due to an absence or to low levels of such receptors. Whereas the failure of hyperosmolality to influence the l i v e r / b o n e / k i d n e y isoenzyme is in keeping with results obtained on cells producing this isoenzyme monophenotypically [18,28], the lack of response of the term-placental isoenzyme of SW-620 is unexpected: in no cell line synthesizing this enzyme form [18,20,27] has this factor failed to act as an inducer. Induction of termplacental alkaline phosphatase by butyrate has been shown repeatedly with cells producing this enzyme form [11,14,15]. However, in similarity to the lack of response of this isoenzyme in SW-620, there are two cervical cancer lines (C4I and DoT) in which for unknown reasons butyrate does not induce [16,33]. It is possible that the failure of the heat-stable alkaline phosphatase of SW-620 to respond to the fatty acid may be related to the tissue of origin of this continuous cell line. With respect to cells producing the l i v e r / b o n e / k i d n e y isoenzyme monophenotypically, butyrate has not been tested on continuous lines, but acts as an inducer in short-term cultures of pituitary adenoma and hemangioblastoma cells [28]. As with its other effects on animal cells [1], the mode of action of butyrate in modulating alkaline phosphatase is not known. Nevertheless, it is tempting to postulate that the preferential induction of the l i v e r / b o n e / kidney isoenzyme may be related to the in vitro suppression of malignancy elicited by butyrate [23] and be the expression of a more differentiated state as compared to that of untreated SW-620 cells. However, these possibilities have to be considered with caution. It has now been shown that whereas the alkaline phosphatase of normal colonic mucosa is of the intestinal type [34], the enzyme of four human colon cancer tissues was heat-labile, insensitive to L-phenylalanine and inhibited by L-homoarginine [34], thus resembling the enzyme found in very early placenta [35,36] and described in a variety of other malignancies

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Acknowledgements We thank Alexander Schermer for technical assistance and Pearl Parsowith for secretarial help.

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