GASTROENTEROLOGY 1990;98:1199-1207

Biosynthesis of Alkaline Phosphatase During Differentiation of the Human Colon Cancer Cell Line Caco-2 HISASHI MATSUMOTO, ROGER H. ERICKSON, JAMES R. GUM, MASAHIRO YOSHIOKA, ELIZABETH GUM, and YOUNG S. KIM Gastrointestinal Research Laboratory, Veterans Administration Medical Center, and Department of Medicine, University of California, San Francisco, California

The human colon cancer cell line Caco-2 undergoes spontaneous enterocytic differentiation during growth and expresses a number of brush-border membrane-associated hydrolases typical of a differentiated phenotype. Among these is the enzyme alkaline phosphatase, which is frequently used as a marker of cell differentiation in colon cancer cells. Since the biochemical processes regulating the expression of alkaline phosphatase during cell differentiation are only poorly understood, we examined the biosynthesis and processing of alkaline phosphatases in undifferentiated (O-day confluent) and differentiated (14-day confluent) Caco-2 cells. It was found that both cell phenotypes expressed a single, heat-labile intestinal-like enzyme, which undergoes similar posttranslational processing and glycosylation. Although the rate of enzyme synthesis and alkaline phosphatase messenger ribonucleic acid was 5-6-fold higher in differentiated cells, the degradation rates in both cell types were similar with a half-life of approximately 10 days. These results suggest that the increase in alkaline phosphatase activity during Caco2 cell differentiation is caused by changes in the synthetic rate and that the low turnover rates facilitate accumulation of the enzyme. Furthermore, these studies demonstrate that Caco-2 cells are useful for examining the molecular and biochemical events involved in the differentiation of the small intestinal epithelium.

H

uman alkaline phosphatase is a cell-surface glycoprotein associated with a variety of tissues and organs through all stages of development including fetal, adult, and neoplastic. Current evidence indicates that there are at least four distinct alkaline phosphatase isoenzymes, encoded by four separate genes (1): (a) placental, (b) intestinal, (c) placental-like,

and (d) liver/kidney/bone (or tissue-unspecific) types (1-4). In addition, there may be a fifth gene in humans that encodes a fetal intestinal type of alkaline phosphatase (1). Some of these isoenzymes can be distinguished from each other biochemically by a number of parameters, including thermostability, effect of inhibitors, electrophoretic mobility, molecular weight, and immunologic properties. Previous studies from our laboratory and others (5-10) have shown that a number of human colorectal cancer cell lines express high levels of brush-border membrane-associated hydrolases, including alkaline phosphatase, when grown in the presence of various differentiating agents such as sodium butyrate. In colon cancer cell lines such as HRT-18 (9) and LS174T (7), butyrate causes induction of a placental type of alkaline phosphatase while HT -29 has been reported to produce an intestinal type (10). Butyratemediated induction of alkaline phosphatase in SW620 cells is apparently unique in that the tissue-unspecific (liver/bone/kidney) isozyme is induced (5). In each of these cases, the exact mechanism responsible for the induction of alkaline phosphatase by butyrate and other agents is currently unknown. With colon cancer cells it has been postulated that the increase in brush-border membrane-associated enzyme activities might reflect a more differentiated phenotype. However, microscopic evidence of cellular differentiation is typically not observed in many of the cell lines that have been treated with various "differentiating" agents. In contrast, the cell line Caco-2 will spontaneously

Abbreviations used in this paper: CEA, carcinoembryonic antigen; endo H, endoglycosidase H; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; SDS, sodium dodecyl sulfate; TeA, trichloracetic acid. © 1990 by the American Gastroenterological Association 0016-5085/90/$3.00

1200 MATSUMOTO ET AL.

differentiate in tissue culture to a cell type exhibiting characteristics typical of small intestinal epithelial cells in the absence of any exogenous inducing agents (11). Electron microscopic studies have shown that differentiated Caco-2 cells are highly polarized with a typical brush-border membrane and have tight junctions between individual cells (11). In addition, this cell phenotype is also characterized by high levels of enzymes typically associated with the brush-border membrane and by the formation of domes in culture which are a characteristic marker of polarized epithelial cells (11-14). Thus, Caco-2 cells can serve as a useful model to examine the biochemical basis of cell differentiation and the biosynthesis and expression of brush-border membrane-associated enzymes. Accordingly, we have exploited the unique features of this human colon cancer cell line to study the mechanisms regulating the biosynthesis and expression of membrane-associated alkaline phosphatase during cell differentiation.

Materials and Methods

Cell Culture Caco-2 cells were obtained from the American Type Culture Collection (Rockville, Md.). Caco-2 cells were cultured in 25-cm 2 flasks in Dulbecco modified Eagle's medium supplemented with 20% heat-inactivated fetal calf serum, 1 % nonessential amino acids, penicillin (50 U/ml), and streptomycin (100 ~g/ml) at 37°C in 7% Co/930/0 air atmosphere. The medium was changed daily in all experiments, and the cells were subcultured weekly. The passages of the Caco-2 cell line used in this study ranged from the 30th to the 60th. Enzyme and Protein Assay Alkaline phosphatase activity was assayed at 37°C using p-nitrophenyl phosphate as substrate (9). Enzyme activity was expressed in units; 1 unit is equivalent to the micromoles of substrate hydrolyzed per minute (9). Protein was measured by the modified method of Lowry et al. (15).

Characterization of Alkaline Phosphatase Heat stability. The thermal stability of alkaline phosphatase was examined by heating samples of cell homogenates in 10 mM Tris HCl buffer, pH 7.4, or 650 mM 2-amino-2-methyl-l,3-propandiol-HCl buffer, pH 10.0, at 65°C for 5, 10, or 20 min. A control was incubated at 4°C. Inhibition study. The following inhibitors were used in the inhibition study: L-phenylalanine (5 mM); L-leucine (5 mM); L-homoarginine (10 mM); L-Leu-Gly-Gly (5 mM); and L-Phe-Gly-Gly (5 mM). To approximately 0.01 U of enzyme samples, 0.1 ml of inhibitor and 0.45 ml of substrate/bufferl MgCl 2 were added. After incubation at 37°C, the reaction was stopped with 1 N NaOH and the percentage of inhibition was calculated from the respective controls. Cell ho-

GASTROENTEROLOGY Vol. 98, No.5

mogenates from human placenta and human small intestinal mucosa were included in inhibition studies for comparison. Western Blots af Alkaline Phosphatase Western blots of alkaline phosphatase from Caco-2, LS174T, human small intestine, and human placenta were conducted as described (16), using a 1:100 dilution of antialkaline phosphatase immunoglobulin G (IgG) followed by 106 cpmlml 125 I-labeled protein A.

Preparation of Membrane Fraction The cells were harvested by gentle scraping with a rubber policeman in 6 ml of 2 mM Tris-HCI, 50 mM mannitol, and 40 ~g/ml phenylmethylsulfonyl fluoride (PMSF), pH 7.1. The cell suspension was sonicated twice for 10 s on ice and designated the cell homogenate. The homogenate was centrifuged at 100,000 x g for 1 h to obtain a membrane pellet and soluble (cytoplasmic) fraction.

Labeling With L-rSjMethianine The culture flasks were rinsed two times with phosphate-buffered saline (PBS) and preincubated for 15 or 30 min in methionine-free medium containing 20% dialyzed fetal serum. Thereafter the cells were labeled with 150-300 ~Ci L-[ 35 S]methionine per flask in methionine-free medium for 15 min or 3 h. A chase was performed with medium containing 10 mM unlabeled methionine.

Immunaprecipitatian of Alkaline Phosphatase For immunoprecipitation of alkaline phosphatase, lysates of membrane fractions labeled with [35S]methionine were sonicated 15 s and dissolved in buffer A [1 % Nonidet P-40 (Sigma Chemical Co., St. Louis, Mo.), 0.5 M NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.1 mM PMSF, 1 mg/ml bovine serum albumin, and 10 mM Tris-HCI, pH 8.0) as described (7). A 5-~1 aliquot of normal rabbit serum was added to a final volume of 1 ml, and the mixtures were incubated for 30 min at 30°C followed by 15 min at 4°C. The samples were then treated for 1 h with occasional shaking at 4°C with 100 ~l of a 10% (wt/vol) solution of formalintreated Staphylococcus aureus (protein-A positive) cells (Boehringer Mannheim Biochemicals, Indianapolis, Ind.) and centrifuged for 2 min at 12,000 x g in a microcentrifuge. The supernatant was then treated with 2 ~l of anti-human placental alkaline phosphatase IgG (Dako Corp., Santa Barbara, Calif.) for 30 min at 30°C, 15 min at 4°C, and then 1 h with S. aureus cells as above. The cell pellet was then washed as described (7) and the absorbed antigen-antibody complex was solubilized with electrophoresis sample buffer containing 20/0 (wt/vol) sodium dodecyl sulfate (SDS) and 100 mM dithiothreitol at 100°C for 5 min. Electrophoresis was conducted using 9% polyacrylamide gels in the presence of SDS (17). Proteins were transferred electrophoretically from gels to nitrocellulose membranes (16). The membranes were soaked 1 h in Autofluor (National Diagnostics,

May 1990

Manville, N.}.) and radioautographed at -70°C using Kodak XAR-2 film (Kodak Co., Whittier, Calif.).

Incorporation of rssS]Methionine Into the Total Membrane Fraction Aliquots (lO-JLI) containing the cell membrane fraction were transferred to 1 ml of ice cold 5% trichloracetic acid (TCA) and kept at 4°C for 1 h before filtering with glass fiber filters. The radioactivity (counts per minute) on the filters was counted in a liquid scintillation counter (Beckman Instruments, Fullerton, Calif.) and the incorporation of L-[ 35 Sjmethionine into total membrane protein was determined.

ALKALINE PHOSPHATASE BIOSYNTHESIS

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chased for periods of up to 96 h. Culture medium was changed daily. After the chase, the culture medium was removed and the cell layer was rinsed with ice-cold saline, snap frozen by flotation of the flasks on a bath of liquid nitrogen, and stored at -70°C. Membrane fractions were prepared from each individual flask as described above. The total membrane protein from each flask was solubilized with detergent, and the entire amount was subjected to immunoprecipitation followed by SOS-PAGE and radioautography. A scanning densitometric analysis of the fluorograms was performed to determine the amount and turnover rate of radiolabeled alkaline phosphatase.

Results Incorporation of L-[35 S]Methionine Into Alkaline Phosphatase Alkaline phosphatase was immunoprecipitated from 35S-labeled cell membranes and subjected to SOS-polyacrylamide gel electrophoresis (PAGE) as above. Gels were stained with Coomassie brilliant blue. After destaining, gels were sliced into 2-mm pieces and dissolved in NCS solubilizer (Amersham Corp., Arlington Heights, Ill.) at 50°C for 2 h, and radioactivity was determined. The value obtained with normal rabbit serum was subtracted to correct for any nonspecific precipitation.

Alkaline Phosphatase Activity and Dome Formation During Differentiation of Caco-2 Cells The activity of alkaline phosphatase was measured during differentiation of Caco-2 cells. As shown in Figure 1, the activity increased linearly after confluence and the specific activity (units per milligram

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Northern Analysis of Alkaline Phosphatase Messenger Ribonucleic Acid Total cellular RNA was isolated from cells (0- and 14-day) grown in individual T -150 flasks (3 each) using the guanidium thiocyanate-cesium chloride procedure of Chirgwin et al. (18). Poly(AJ+ RNA was prepared using one cycle of oligo(dT)-cellulose chromatography. Identical amounts of Poly(A)+ RNA isolated from the two cell types were subj ected to electrophoresis and hybridization analysis using the 1.9 kb Kpn I fragment of placental alkaline phosphatase (19) as described (20). Radioautographs were scanned with a densitometer, and the area under the peak was determined to quantify the amount of alkaline phosphatase mRNA.

Endoglycosidase H Treatment Immunoprecipitates were boiled in 50 JLI of 0.1 citric acid-NaOH, pH 5.2, for 3 min in the presence of a cocktail of protease inhibitors containing antipain, benzamidine, pepstatin, aprotinin, and PMSF as described (13). Endoglycosidase H (endo H), 3 JLI (45 JLg/ml) with a fresh aliquot of protease inhibitor cocktail, was added and samples were incubated for 18 h at 37°C, treated with sample buffer for 3 min at 100°C, and subjected to SOS-PAGE. Controls were incubated at 37°C without endo H.

Turnover Rate of Newly Synthesized Alkaline Phosphatase Both 0- and 12-day cells were pulse labeled with 150 JLCi of [35Sjmethionine per culture flask (13) for 3 hand

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DA YS AFTER CONFLUENCE Figure 1. Induction of membrane-associated alkaline phosphatase activity during the differentiation of Caco-2 cells. Cells were grown, harvested, and membrane fraction prepared as described in Materials and Methods. Results are the mean ± SD of 6 separate experiments. Enzyme activity is measured in units per milligram protein.

GASTROENTEROLOGY Vol. 98, No.5

1202 MATSUMOTO ET AL.

protein) in differentiated (14-day) cells was approximately 25 times higher than in undifferentiated (O-day) cells. During this period, the number of cells per T-25 flask approximately doubled from 4.8 ± 1.4 to 10.5 ± 1.9 million cells (mean ± SD, 6 determinations). In addition, domes formed rapidly after confluence, reaching a maximum number on the sixth day (not shown). The number of individual domes then rapidly decreased because of dome fusion and remained constant. After 25-30 days it became difficult to monitor the cells in culture due to detachment of the cell monolayer.

Characterization of Alkaline Phosphatase From Caco-2 Cells Alkaline phosphatase from Caco-2 cells was compared with placental and intestinal types of alkaline phosphatase with regard to its thermo stability and sensitivity to various inhibitors. As shown in Figure 2, human intestinal alkaline phosphatase and the enzyme from Caco-2 cells were unstable when treated at 65°C at pH 7.4 or 10.0. However, the placental type of alkaline phosphatase was comparatively more stable under these conditions. The sensitivity of alkaline phosphatase to various amino acid and peptide inhibitors is shown in Figure 3. Alkaline phosphatase from Caco-2 cells (0- and 14-day) was partially inhibited by phenylalanine and leucine but was insensitive to inhibition by homoarginine. The placental type of alkaline phosphatase was inhibited by Leu-Gly-Gly

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and Phe-Gly-Gly while the enzyme from Caco-2 cells and intestine were much less so.

Immunoprecipitation of Alkaline Phosphatase The immunological properties of alkaline phosphatase in total cell homogenates from 0- and 14-day cells from Caco-2 cells were examined using a commercially available polyclonal anti-human placental alkaline phosphatase antibody. Fixed S. aureus containing protein A was used to precipitate the antigenantibody complex. No significant difference was found in the immunotitration curves obtained from Caco-2 (0- and 14-day cells), human small intestine, and human full-term placenta (not shown). An excess of antibody completely precipitated the alkaline phosphatase from both types of Caco-2 cells and also the small intestinal and term placental types.

Biosynthesis of alkaline phosphatase during differentiation of the human colon cancer cell line Caco-2.

The human colon cancer cell line Caco-2 undergoes spontaneous enterocytic differentiation during growth and expresses a number of brush-border membran...
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