Biosynthesis and Processing of Prostatic and Lysosomal Acid Phosphatases in a Prostate Carcinoma Cell Line PC-3SF121

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[CANCER RESEARCH 46, 4796-4803 September 1986] Biosynthesis and Processing of Prostatic and Lysosomal Acid Phosphatases in a Prostate Carcinoma Cell Line PC-3SF121

Abdul Waheed2 and Robert L. Van Etten

Department of Chemistry, Purdue University, West Lafayette, Indiana 47907

ABSTRACT showed a positive correlation with testosterone levels. A similar correlation was observed between the concentration of prostatic The biosynthesis of distinct prostatic and lysosomal acid phosphatases is demonstrated using a human prostatic carcinoma cell line, PC-3SF12. acid phosphatase and testosterone in isolated epithelium. These The biosynthesis and maturation of the acid phosphatases was studied results imply that the synthesis of prostatic acid phosphatase by metabolic labeling with radioactive leucine, specific immunoprecipi- in human BPH tissue is androgen dependent (10). In contrast, tation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and whole-animal experiments have shown that deprivation of an fluorography. Of the tartrate-inhibitable acid phosphatase activity in PC- drogen results in an increase in the total lysosomal-type acid 3SF12 cells, 60% is lysosomal and 10% is prostatic. The lysosomal-type phosphatase activity in rat ventral prostate (11). Prostatic and acid phosphatase is synthesized as precursor with a molecular weight of lysosomal acid phosphatases are both glycoproteins with mo 68,000, some of which is converted to higher-molecular-weight precursor lecular weights of 100,000. In each case, the total molecular polypeptides (M, 71,000 and 77,000). The multiple forms of the precur weight is due to the presence of two identical subunits with sors are due to differences in the carbohydrate chains on the enzyme molecular weights of 48,000-53,000 each (12-14). Both en because biosynthesis in the presence of tunicamycin eliminates the pre zymes are inhibited by tartrate and show similar, but not cursor multiplicity. The initial precursor (M, 68,000) is processed to a identical, kinetic properties. However, their amino acid com mature polypeptide (M, 49,000), via intermediates with molecular weights of 62,000 and 59,000. The mature polypeptide is degraded to positions are somewhat different (12, 13) and they are Â¡ninni smaller polypeptides with molecular weights of 30,000, 28,000, and nologically distinct (4, 15, 16). Much information is now avail 25,000. Precursor polypeptides of the lysosomal-type enzyme are se able about the pathways for biosynthesis and processing of each creted in the medium. Prostatic acid phosphatase is synthesized as a of these enzymes in normal fetal lung cells (17, 18) and I-cell precursor with a molecular weight of 110,000, which is processed via (19) fibroblasts, but detailed information on the biosynthesis of several intermediates (M, 99,000-93,000, 77,000, and 55,000) to a these enzymes in prostate-derived cell lines has not been pre mature polypeptide with a molecular weight of 49,000. Particularly during sented. Ultrastructural and cytochemical studies suggest that cell homogenization, or lysis, the mature polypeptide is rapidly degraded both acid phosphatases are present in prostatic epithelial cells to an immunoprecipitable polypeptide with a molecular weight of 20,000. (20, 21 ). Two cell lines derived from prostatic carcinoma, PC- None of these polypeptides is secreted in detectable amounts into the 3 and DU-145, have also been reported to contain low but medium. Precursors and mature and smaller polypeptides are present in human prostate extract and seminal fluid. Proteolytic degradation of detectable amounts of prostatic and lysosomal acid phospha prostatic acid phosphatases in cells and tissues is probably catalyzed by tases (22). Because of the clinical significance of acid phospha a plasmin-like or related trypsin-like enzyme because degradation of the tases in prostatic cancer and other diseases, the biosynthesis mature prostatic phosphatase polypeptide is completely prevented by and secretion of the enzymes was studied in the prostatic addition of the plasmin inhibitor bovine pancreatic trypsin inhibitor. carcinoma cell line PC-3SF12, using antibodies obtained Prostatic- and lysosomal-type acid phosphatases are eventually stored at against homogeneous human prostatic and human liver (lyso least in part in two different types of cell organelles. Testosterone does somal) acid phosphatases. Androgen and tunicamycin effects not influence the biosynthesis and secretion of either acid phosphatase on the biosynthesis and secretion of both acid phosphatases in this cell line. was also studied.

INTRODUCTION MATERIALS AND METHODS Acid phosphatases (EC 3.1.3.2) are present in human tissues in a number of distinct molecular forms and locations (1-5). Human liver and prostatic acid phosphatase were purified according The acid phosphatase activity of serum includes contributions to literature procedures (14, 23). Homogeneous human prostatic acid from lysosomal (1) and other acid phosphatases that are re phosphatase was labeled with tritium by the reductive methylation of amino groups with sodium [3H]borohydride (Amersham) according to leased from different cells and tissues (6). A variety of patho logical disorders are known in which elevated acid phosphatase the procedure of Kurnarasamy and Symons (24). Antisera against human seminal fluid and human liver acid phosphatases (4) were raised levels are observed (6, 7). Prostatic acid phosphatase levels are in rabbits. Goat antihuman prostatic acid phosphatase was a gift from often elevated in the serum of patients with cancer of prostate Clinical Assays, Inc., Cambridge, MA. Based on the results of Ouch- gland, although the enzyme levels that are actually present in terlony double-diffusion tests, all antisera were monospecific (4). A the prostatic tissue itself may be reduced (8). Acid phosphatase human prostatic carcinoma cell line, PC-3SF12 was obtained from Dr. levels are increased during the development of several other David Kirk of the Prostate Research Program, Huntington Medical disease states besides cancer of the prostate (9). In human Research Institute (Pasadena, CA). The cells were maintained at 37Â°C, BPH3, tissue concentrations of prostatic acid phosphatase under 5% CO2, in the serum-free medium PFMR-4 (GIBCO Labora tories), supplemented with insulin and antibiotic (25). This cell line was Received 1/17/86; revised 5/8/86; accepted 5/30/86. derived from PC-3 cells by continuous growth in the serum-free medium The costs of publication of this article were defrayed in part by the payment PFMR-4. In turn, PC-3 cells were obtained from a bone metastasis and of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. do not respond to androgens in the growth medium. (For detailed 1This work was supported by Department of Health and Human Services NIH characteristics see Ref. 25.) Grant'To CM whom 27003. requests for reprints should be addressed, at Department of Quantitative Immunoprecipitation. The percentage of immunoprecip itable, tartrate-inhibitable phosphatase activity was determined from Chemistry, Purdue University, West Lafayette, IN 47907. 3Abbreviations used: BPH, benign prostatic hyperplasia; SDS, sodium dodecyl the difference between the activity in the supernatants of antiserum- sulfate; BPTI, bovine pancreatic trypsin inhibitor. and control serum-treated samples (4). The cell extract, containing 4796

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1986 American Association for Cancer Research. ACID PHOSPHATASES IN PROSTATE CARCINOMA CELLS about 1SO /IKof protein in a volume of 20 //I. was mixed with 3 /;! of rpm for 10 min in a Beckman JA-20 rotor. The resulting supernatant goat anti-human prostatic acid phosphatase, with 6 Â¿/Iofrabbit anti- was mixed with 100 //I of rabbit anti-human liver acid phosphatase human liver acid phosphatase or with 3-6 /proteins from New England Nuclear: myosin, M, 200,000; 1.5-1.8 mg of protein per flask. Prior to labeling, the cells were washed phosphorylase B, M, 92,500; ovalbumin, M, 46,000; carbonic anhy- twice with 2 ml of PFMR-4 medium without leucine and then starved drase, M, 30,000; lactoalbumin A, M, 18,400. Protein standards for for 90 min in 3 ml of the same medium in order to deplete the Coomassie staining were from Bethesda Research Laboratories: intracellular leucine concentration. The cells were then labeled for 6 h myosin; phosphorylase A, M, 92,500; bovine serum albumin, M, in 3 ml of leucine-free PFMR-4 medium with 180 ^Ci of L-[4,5-3Hj- 68,000; ovalbumin; 0-chymotrypsinogen, M, 25,700; /Mactoalbumin, leucine (Amersham). In order to check the effect of testosterone and M, 18,400; cytochrome C, M, 12,300. Protein concentrations were tunicamycin, cells were labeled in the presence of 20 nM testosterone determined according to Lowry et al. (29). and tunicamycin, 10 Mg/ml. The cells were also pretreated with these Subcellular Localization of Prostatic and Lysosomal Acid Phospha chemicals for 6 h before labeling experiments. For pulse-chase experi tase. Two 75-cm2 flasks of the cells were labeled with [3H]leucine for ments, labeling was quenched by adding 0.1 ml of cold 5-mg/ml sterile 16 h. After labeling, the cells were harvested and homogenized and the leucine solution to the radioactive medium. At the end of the labeling postnuclear supernatant was prepared according to a previous study period, the medium was collected from the flask and mixed with 150 (30). A Percoli gradient mixture was prepared by mixing 25.7 ml of n\ of fetal calf serum, and 1.5 g of ammonium sulfate was added. This Percoli (Pharmacia Fine Chemicals), 10.0 ml of 2.5 Msucrose and 64.3 mixture was placed on ice for at least 3 h. The protein precipitate was ml of deionized distilled water. The Percoli gradient was set up in a 35- recovered by centrifugation at 20,000 rpm in a Beckman JA-20 rotor at 4Â°Cfor 15 min. The precipitate was solubilized in 100 Â¡i\of H2O ml centrifuge tube using 25 ml of the gradient mixture over a cushion of 3 ml of 2.5 M sucrose. Postnuclear supernatant (3 ml) was applied and dialyzed against 10 mM sodium phosphate, pH 7.0, + 150 mM on top of the gradient mixture and centrifuged at 45,000 x g for 60 NaCl buffer for 6 h at 4Â°C.The dialyzed sample was removed and min at 4Â°C.Percoli gradient-colored density marker beads were centri mixed with 0.07% Triton X-100 + 1 mM EDTA + 4 mM phenylmethanesulfonyl fluoride. Samples were then frozen at â€”20"C. fuged in a separate tube. Fractions of 5 ml were collected from the top of the gradient. Density, radioactivity, and acid phosphatase activity Radioactively labeled cells were harvested from a flask by scraping (3) were determined for each fraction. The membranes were recovered into 2 ml of cold saline with a rubber policeman; additional cold saline by centrifugation of each fraction at 105,000 x g for 4 h. The membrane (2 ml) was used to wash the flask, and the saline suspensions were pellets were suspended in 0.7 ml of 10 mM Tris-HCl, pH 7.5, + 0.1% pooled. After centrifugation at 5000 rpm in a table-top centrifuge the Triton X-100 + 1 mM phenylmethanesulfonyl fluoride and frozen at cells were transferred to an Eppendorf tube with 250 n\ of 50 mM Tris- â€”20Â°Covernight.The membrane suspensions were thawed at room HC1, pH 7.5, containing either 4 mM phenylmethanesulfonyl fluoride temperature and sonicated in an ice-cold water bath. Fifty microliters plus 1 mM EDTA or 50 jig of bovine pancreatic trypsin inhibitor (Sigma Chemical Co.), and frozen at -20Â°C. of protein A-bacterial adsorbent (10%, w/v) was added to each fraction. Frozen cell suspensions and media were thawed at room temperature. The mixture was allowed to stand on ice for 60 min before centrifuga Cell suspensions were subjected to ultrasonication for 30 s in an ice- tion at 18,000 rpm for 1 h in a Beckman JA-20 rotor. The supernatant water bath and mixed with 1 /Â»Iof20% Triton X-100 and 2 n\ of 3% was used for immunoprecipitation of the enzymes as described above. Immunoblotting. The enzyme samples were first subjected to SDS- protamine sulfate. Media and cell sonicate samples were each placed on ice for 15 min and then centrifuged at 18,000 rpm using a Beckman polyacrylamide gel electrophoresis (27). The polypeptides from the gel JA-20 rotor for 60 min at 4Â°C.The clear supernatants thus obtained were transferred electrophoretically to nitrocellulose paper BA 85 were carefully collected and are designated as medium extracts and cell (Schleicher and Schuell, Keene, NH) according to Burnette (31). The acid phosphatase polypeptides were visualized using 100-times diluted extracts, respectively. rabbit anti-human prostatic acid phosphatase as a primary antibody Immunoprecipitation of Radioactively Labeled Proteins. The cell and and peroxidase-conjugated goat antirabbit immunoglobulin G (Miles medium extracts derived from one flask were separately mixed with 15 ul of goat anti-human prostatic acid phosphatase, 1 Mg oÃcarrier Laboratories) as a second antibody. In order to increase the sensitivity enzyme, and a solution of detergent buffer (4% Triton X-100 and 1.6 of the visualization procedure, peroxidase-antiperoxidase from rabbit M KC1 in 0.4 M Tris-HCl, pH 7.5) equal in volume to one quarter of (Miles Laboratories) was also used. Prestained protein standards (Be the original volume of the cell or medium extract. The mixture was left thesda Research Laboratories): myosin, M, 200,000; phosphorylase A, at room temperature for 30 min and then at 4Â°Cfor 20 h. The M, 92,500; bovine serum albumin, M, 68,000; ovalbumin, M, 46,000; immunoprecipitates were collected by centrifugation at 12,000 rpm in a-chymotrypsinogen, M, 25,700; /i-lactoalbumin, M, 18,400; cyto a Beckman JA-20 rotor for 5 min and washed as described (26); the chrome C, M, 12,300 were used to determine the apparent molecular supernatants were saved for immunoprecipitation of lysosomal acid weight of the polypeptides. phosphatase. After washing with 700 /Â¿Iofacetone, the immunoprecip Proteolytic Digestion of Prostatic Acid Phosphatase by Human Plas itates were solubilized in 50 /il of solubilizer (27) by heating the samples min. Homogeneous human seminal fluid acid phosphatase (100 Mg)in at 98Â°Cfor 5 min in preparation for SDS-polyacrylamide gel electro- 50 mM Tris-HCl, pH 7.5, containing 0.05% Triton X-100 was incubated with 30 Â¿igofplasmin (Sigma) in a reaction volume of 200 M'at 37Â°C. phoresis. Lysosomal acid phosphatase was immunoprecipitated from the su At different time intervals 1 /

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1986 American Association for Cancer Research. ACID PHOSPHATASES IN PROSTATE CARCINOMA CELLS casein and TV-a-benzyloxycarbonyl-L-lysine-p-nitrophenyl ester, respec either the cell extracts or the medium extracts when preimmune tively. serum was used. In order to characterize the mature polypep tides of the prostatic enzyme in PC-3SF12 cells, homogeneous RESULTS prostatic acid phosphatase, identical to that which was used to immunize the animal, was labeled with tritium and was found In the PC-3SF12 cell line, 60% of the total acid phosphatase to migrate as a polypeptide with a molecular weight of 53,000. activity was due to tartrate-inhibitable enzyme. Only 10% of The elevated molecular weight (A/r 53,000 versus 49,000) of the the total tartrate-inhibitable enzyme was precipitated by the 3H-labeled marker enzyme is a consequence of the reductive antiserum against prostatic acid phosphatase, while 60% was methylation of the lysine residues in the enzyme molecule, since precipitated by the antiserum against lysosomal (human liver) protein labeled by reductive methylation commonly shows a acid phosphatase. About 30% of the tartrate-inhibitable enzyme higher molecular weight on SDS-polyacrylamide gel electro was not precipitated by either antiserum. phoresis (24). In order to understand the relationship among the high- Biosynthesis of Acid Phosphatases molecular-weight polypeptides, mature polypeptides, and the low-molecular-weight (M, 20,000) polypeptide in the cell ex Prostatic Acid Phosphatase tract, pulse-chase experiments were performed. Typical results Typical results obtained in the studies of the biosynthesis and are shown in Fig. 1 (last three lanes). When cells were chased secretion of prostatic acid phosphatase are shown in Fig. 1. with cold leucine for 12-48 h after a 6-h pulse, the intensities When the prostatic enzyme was immunoprecipitated from cell of several of the high-molecular-weight and mature polypep extracts after 1-h labeling, the majority of the radioactivity was tides were decreased, while the intensity of the polypeptide with associated with a polypeptide with a molecular weight of a molecular weight of 20,000 was increased. In many experi 49,000, which is identical in size to the mature tissue enzyme ments in which cells were labeled for longer periods, the poly polypeptide (12-14). However, several polypeptides of high peptide with a molecular weight of 20,000 accumulated to a molecular weight (Mr 110,000, 93,000-90,000, 77,000, and substantial extent. Thus, in contrast to earlier results with WI- 55,000) and one polypeptide of small molecular weight (M, 38, a normal fetal lung fibroblast cell line (17), the present 20,000) were observed. With longer (6 h) labeling periods, there results with the transformed cell line PC-3SF12 indicate the was no significant change in the polypeptide patterns except presence of substantial amounts of the polypeptide with a that the relative intensity of the high-molecular-weight poly molecular weight of 20,000 in long-term labeling and pulse- peptide bands decreased and the intensity of the polypeptide chase experiments (Fig. 1). No immunoprecipitable polypep band at a molecular weight of 20,000 increased. None of these tides were seen in the medium extracts from 12-48-h chase polypeptides was immunoprecipitated from the medium ex experiments. tracts. The specificity of the immunoprecipitation was estab Proteolytic Cleavage of Prostatic Acid Phosphatase by Human lished by using preimmune serum and by competition using Plasmin. During the present studies on the synthesis of prostatic cold homogeneous prostatic acid phosphatase. None of the acid phosphatase in the prostatic carcinoma cell line PC-3SF12, labeled polypeptides normally immunoprecipitated by the anti- we observed that the prostatic enzyme is quickly degraded into serum were precipitated when an excess of cold prostatic acid low-molecular-weight polypeptides (Fig. 1). It seemed possible phosphatase was present in the reaction mixture. Similarly, no that the increased degradation of mature polypeptides in the immunoprecipitable polypeptide bands were observed from PC-3SF12 cell line was due to the presence of higher amounts

Fig. 1. Prostatic acid phosphatase in pulse/ CELLS MEDIUM CELLS chase-labeled PC-3SF12 cells. The cells were ( CHASE (h)| labeled with [3H]leucine for different times as PULSE(h)| |pULSE(h)| indicated. Prostatic acid phosphatase was im munoprecipitated from cells and medium ex 16 16 122448 tracts using an antiserum against homogene --â€¢â€”no ous prostatic enzyme. Cell extracts from 6-h 110 labeling experiments were used in the immu 90-93 K-C |â€”3-90-93 K noprecipitation of the enzyme with preimmune â€” 77 K (control) serum or with antiserum in the pres 77 K â€”â€”75 K PRECURSOR ence of cold carrier enzyme (prostate AP). Three culture flasks were labeled for 6 h and 55 K- â€” 55 K then cells were chased for the indicated times â€”49 K with cold leucine. The immunoprecipitates 53K 49K- MATURE were solubilized under reducing conditions and subjected to electrophoresis and fluorography. The positions of the polypeptides are indicated by their apparent molecular weight. Radioac tive molecular weight standards are described in "Materials and Methods." Radioactive ho mogeneous prostatic acid phosphatase (| '111â€¢ prostate AP) was used as a marker. A. molec ular weight in thousands. 20 K 20 K'

4798

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1986 American Association for Cancer Research. ACID PHOSPHATASES IN PROSTATE CARCINOMA CELLS of specific proteases, since increased proteolytic enzyme activity I 2 and plasminoceli activator levels have been reported in malig nant cells and tumor tissues (34, 35). We determined the total proteolytic activity against casein and the plasmin activity Vf59 against Ar-Â«-benzyloxycarbonyl-lysine-/>-nitrophenyl ester that was present in the cell extracts from the PC-3SF12 and WI-38 PRECURSORMATI K cell lines. The total proteolytic and plasmin-specific protease 55 K activities were found to be 5 times higher in PC-3SF12 than in IDC90-93K-(="77 WI-38 cells. Therefore, we studied the sensitivity of homoge neous human prostatic acid phosphatase toward human plas min. Typical results are shown in Fig. 2. Within 15 min, the native polypeptide with a molecular weight of 49,000 was cleaved into three different polypeptides (M, 32,000, 26,000, and 16,000) but with little net loss of enzymatic activity. Con 20 Kâ€” tinued incubation resulted in further proteolysis of the native polypeptide. Lower-molecular-weight polypeptides were not ac Fig. 3. Inhibition of proteolytic degradation of mature prostatic acid phospha cumulated to a detectable extent. Significantly, the enzyme tase. PC-3SF12 cells were labeled with [3H]leucine for 16 h. Cells were harvested activity was not reduced up to 30 min, although longer incu in the absence (lane I) or presence (lane 2) of 50 /ig of bovine pancreatic trypsin inhibitor. The prostatic acid phosphatase was immunoprecipitated from the cell bation times resulted in a complete loss in the enzyme activity. extracts and subjected to electrophoresis and fluorography. The absence of the Protection from Proteolytic Degradation of Prostatic Acid M, 20,000 fragment in Lane 2 suggests that much of the proteolytic degradation Phosphatase by Bovine Pancreatic Trypsin Inhibitor. In view of of the mature peptide may occur upon cell homogenization or lysis. K, molecular weight in thousands. the above results we sought to reduce the cleavage of prostatic acid phosphatase in PC-3SF12 by adding the well-established plasmin inhibitor BPTI (36). Two 25-cm2 flasks of PC-3SF12 identical to that obtained in earlier experiments (Fig. 1). How were labeled with [3H]leucine. The cells from one flask were ever, the presence of BPTI (second lane) prevented the degra dation of mature phosphatase polypeptides to the polypeptide harvested and extracted in the presence of BPTI and the other with a molecular weight of 20,000 in the 16-h labeling experi flask was processed as usual. The prostatic acid phosphatase ment. As a result, the presence of BPTI causes the polypeptide was immunoprecipitated from both cell extracts and subjected pattern derived from prostatic acid phosphatase in PC-3SF12 to electrophoresis and fluorography (Fig. 3). The first lane, to more nearly resemble the ones observed earlier with WI-38 where BPTI was not present, showed a polypeptide pattern cells (17).

% OF ORIGINAL ACTIVITY Lysosomal Acid Phosphatase IOO 99 94 Typical gels demonstrating the biosynthesis, maturation, and secretion of lysosomal acid phosphatase by the prostatic carci noma cell line PC-3SF12 are shown in Fig. 4. After labeling for 1 h, one major polypeptide of molecular weight 68,000 and four minor polypeptides of molecular weights 77,000, 71,000, 49K 62,000, and 40,000, respectively, were immunoprecipitated 44K from the cell extract using antiserum against human liver (lysosomal) acid phosphatase. Increases in the labeling time up 32 K to 6 h resulted in increased intensities of these polypeptides. During 1 h of labeling two major polypeptides (M, 68,000 and 26K 62,000) were secreted from the cells. After 6 h of pulse labeling, 45% of the immunoprecipitable radioactivity was secreted into the medium. The polypeptides observed had molecular weights of 71,000, 68,000, and 62,000. When preimmune serum was used, none of these polypeptides was immunoprecipitated either from the cell extract or from the medium extract. In contrast I6K to results recently reported for the biosynthesis of lysosomal acid phosphatases in human skin fibroblasts (19), no polypep 15 30 60 120 tide precursor with a molecular weight of 110,000 was observed in PC-3SF12 cell or medium extracts. The maturation of lysosomal acid phosphatase was followed TIME OF DIGESTION (min) by pulse-chase experiments. Typical results are shown in Fig. 4 Fig. 2. Kinetics of proteolytic degradation of prostatic acid phosphatase by (last three lanes). After a 6-h pulse and a 12-h chase with cold human plasmin. Homogeneous enzyme from seminal fluid (100 *tg)was incubated with 30 Mgof plasmin in 200 Â¡Aof50 mM Tris-HCl, pH 7.5, + 0.05% Triton X- leucine, the major radioactivity associated with the M, 68,000 100 buffer at 37*C. At different time intervals 25-jil aliquots were removed and denatured in Laemmli's (27) solubilizer for electrophoresis and 1-jil aliquots were polypeptide decreased and several low-molecular-weight poly removed at the same time for measurement of enzyme activity. After electropho peptides (M, 59,000, 49,000, 30,000, 28,000, and 25,000) were resis, the polypeptides were electrophoretically transferred to nitrocellulose and observed. The intensity of the polypeptide with a molecular visualized by immunoblotting using peroxidase-conjugated antibody and penivi dase-antiperoxidase complex. Numbers at the top, percentage of the original weight of 68,000 was further decreased after a 48-h chase, while enzyme activity; numbers at the bottom, time of incubation with plasmin; K, the radioactivity in the low-molecular-weight polypeptides was molecular weight in thousands. enhanced, the intensity of the polypeptides with molecular 4799

_ CELLS K g MEDIUM CELLS |PULSEN|Z z|pULSE(h>| | CHASE (h)| I 6 8 S l 6 12 2448 Fig. 4. Lysosomal acid phosphatase in pulse/chase-labeled PC-3SF12 cells. The cells were labeled with (3H|leucine for different 77K- = s â€”77 K y, K times as indicated. Three culture flasks were 7l K labeled for 6 h and then chased for the indi 68 K 62 K- S 62K 68 K PRECURSOR cated times with cold leucine. Lysosomal acid .â€¢ '- 59 K. phosphatase was immunoprecipitated from the -49 K" cell and medium extracts using an antiserum IMATURE against human liver acid phosphatase (14). The 40 K- â€¢: i cell and medium extracts obtained after label ing for 6 h were also used in the immunoprecipitation of the enzyme with preimmune â€¢â€¢â€”30K serum (control serum). The immunoprecipitates were solubilized under reducing condi M 28 K tions and subjected to electrophoresis and fluo- â€¢ 25 K rography. K, molecular weight in thousands.

weights of 77,000 and 62,000 was not changed during the 12- coli gradients in order to separate light membranes from heavy 48-h chase, however a diffuse M, 11,000 polypeptide observed membranes (30, 37). The results of the density-gradient cen- in the I - li chase became more sharp and intense after a 48-h trifugations are shown in Fig. 6. The majority of the radioactiv chase. During the 12-48-h chase polypeptides with molecular ity was located in the low-density (1.036-1.056 g/ml) mem weights of 71,000, 68,000, and 62,000 were also secreted into brane fraction. A small amount of radioactivity was present in the medium (results not shown). the high-density (1.066-1.112 g/ml) membrane fraction. The acid phosphatase activity was present in low- and high-density Effect of Testosterone and I unit aim dn on the Synthesis and membrane fractions, showing a bimodal pattern. Secretion of Acid Phosphatases The prostatic and lysosomal acid phosphatases were immu Prostatic Acid Phosphatase noprecipitated from each membrane fraction and then subjected to SDS-polyacrylamide gel electrophoresis and fluorography. When PC-3SF12 cells were labeled in the presence of 20 /Â¿M Most of the prostatic acid phosphatase was located in fractions testosterone or tunicamycin, 10 tig/m\, there were few differ 1-3 having a density range of 1.036-1.054 g/ml, although trace ences in the polypeptide pattern (Fig. 5). The polypeptide with amounts of prostatic enzyme were present in fractions 4 and 5. a molecular weight of 93,000 appeared absent in testosterone- There was no prostatic acid phosphatase in fractions 6 and 7, treated cells, and the intensities of the mature polypeptides which contained the high-density membrane portions (1.112 were reduced by the presence of tunicamycin. Secretion of the and 1.114 g/ml). enzyme was not induced by either of these chemicals. The lysosomal acid phosphatase was also located in fractions of low density, 1.036-1.066 g/ml. In contrast to the prostatic Lysosomal Acid Phosphatase enzyme, the lysosomal enzyme was also found in the high- In the presence of 20 MMtestosterone, there was no change density membrane fraction (1.114 g/ml), which is rich in sec in the polypeptide pattern of newly synthesized molecules in ondary lysosomes (37). the cell and medium extracts (Fig. 5). However, the presence Presence of High-molecular-weight Polypeptides in Human of tunicamycin, which was used to probe the role of carbohy drate in the synthesis of several lysosomal enzyme precursors, Seminal Fluid and Prostatic Tissue Extracts resulted in a reduction of the number and sizes of immunoprecipitable polypeptides from the cell and the medium extracts. Total seminal fluid and prostatic extract were subjected to SDS-polyacrylamide gel electrophoresis, and the polypeptides Two polypeptides with molecular weights of 71,000 and 61,000 from the cell and a single polypeptide with a molecular weight were characterized by an improved immunoblotting technique using rabbit anti-human prostate acid phosphatase. As ex of 61,000 from the medium were immunoprecipitated. Tuni camycin did not enhance the secretion of lysosomal acid phos pected, the mature enzyme polypeptide with a molecular weight phatase. of 48,000 was present in a large amount. In addition, several high-molecular-weight polypeptides with molecular weights of Subcellular Location of Prostatic and Lysosomal Acid 120,000, 91,000, and 77,000 were observed to cross-react with Phosphatase the antiserum. Small amounts of polypeptides with molecular PC-3SF12 cells were labeled for 16 h with [3H]leucine and weights of 55,000,43,000, and 26,000 were also seen in seminal the postnuclear supernatant was applied to self-generating Per fluid and prostatic extracts. 4800

PROSTATIC LYSOSOMAL CELLS MEDIUM CELLS MEDIUM

Fig. 5. Effect of testosterone and tunica- 110 K- -â€¢Â« mycin on the synthesis of prostatic and lysosomal acid phosphatases in PC-3SF12 cells. 93 K- The cells were labeled with ['Hjleucine for 6 h -77 K in the presence of 20 iiM testosterone or tuni- T7K â€”â€” 71 K â€¢62K68 K camycin. 10 >ig/ml. The cell and medium ex â€¢I â€” 6l K tracts were used to immunoprecipitate the prostatic and lysosoma! enzyme, respectively. 55K â€¢Â» A. molecular weight in thousands. 49 K * M

20 K-

DISCUSSION I 234567

â€” 62 K These results demonstrate that the prostatic epithelial cell â€” 55 K line PC-3SF12 synthesizes both types of acid phosphatases, PROSTATIC â€” 49 K consistent with earlier observations that both acid phosphatases are present in other prostatic epithelial cells (22). However, the l prostatic acid phosphatase is synthesized in approximately 6- -68 K fold smaller amounts than is the lysosomal type. 62 K LYSOSOMAL 49 K Prostatic Acid Phosphatase Characterization of the higher-molecular-weight polypeptides of prostatic-type acid phosphatase (A/r 110,000, 91,000, and 77,000) as precursors for the mature forms of the polypep- ~ tides with molecular weights of 55,000 and 49,000 and degraded polypeptides of low molecular weight (M, 20,000) is based on the following evidence: (a) immunoprecipitation of large, ma IHM ture, and small polypeptides occurs with a monospecific antiserum raised against a homogeneous prostatic acid phospha 80 1.16 o tase; (b) immunoprecipitation of these polypeptides is competed against by homogeneous enzyme from prostatic tissue or sem IÂ£S 60 U2Â¡ inal fluid; (c) no precipitation is observed when preimmune g serum is used; (d) similar high-molecular-weight polypeptides g 40 1-08 <] are observed in human prostatic tissue extract and in seminal fluid (17); and (e) in longer term (6-48 h) pulse-chase experi OD ments, the mature and finally a unique peptide with a molecular LO4 weight of 20,000 predominates in PC-3SF12. Therefore, our results suggest that the prostatic acid phosphatase in PC-3SF12 O l 2 3 4 5 6 7 is synthesized as high-molecular-weight precursors that are processed to mature polypeptides with molecular weights of Fig. 6. Subcellular localization of prostatic and lysosomal acid phosphatasc in PC-3SF12 cells. The postnuclear supernatant of the cell homogenate was 55,000 and 49,000 via several intermediates. Both within the subjected to centrifugation in a Percoli density gradient. Fractions of 5 ml were cells and during homogenization, the mature polypeptide is collected and monitored for density (A), acid phosphatase activity (D). and degraded in PC-3SF12 cells to fragments, including an immu- radioactivity (O). The membranes were recovered from each fraction by centrif ugation, solubilized, and used for immunoprecipitation of both acid phosphatases. noreactive polypeptide with a molecular weight of 20,000. The immunoprecipitates were subjected to electrophoresis and fluorography. The proteolytic degradation of mature prostatic phosphatase Electrophoresis lanes 1-7 correspond to gradient fractions 1-7. K, molecular weight in thousands. polypeptides is much more extensive in the prostatic carcinoma cell line PC-3SF12 than in normal lung cells (17). This appears 4801

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1986 American Association for Cancer Research. ACID PHOSPHATASES IN PROSTATE CARCINOMA CELLS to result from higher amounts of proteolytic enzymes in PC- polypeptides is accompanied by the appearance of new, low- 3SF12 cells, since the present results show that PC-3SF12 cells molecular-weight polypeptides; (d) secretion of related high- have around 5 times more proteolytic activity than do normal molecular-weight polypeptides into the medium is observed. cells such as WI-38. Elevated protease activities have been Thus, in the human prostatic carcinoma cell line PC-3SF12, an observed in other cancer tissues and cells (35). The plasminogen initial precursor polypeptide with a molecular weight of 68,000 activator content is known to be higher in many cancer tissues is synthesized and then subjected to two different cellular proc and cells (34). In turn, it activates higher amounts of plasmin essing events. One involves the processing of the polypeptide, ogen to form plasmin. Therefore, we used purified human and the other one involves the processing of the oligosaccharide plasmin in order to test a possible mechanism of degradation side chains. The proteolytic processing of the precursor poly of homogeneous prostatic acid phosphatase isolated from hu peptide results in a mature enzyme polypeptide with a molecular man seminal fluid. The results show that the acid phosphatase weight of 49,000 via several precursor intermediates. Finally, from seminal fluid is quickly degraded to lower-molecular- smaller immunologically active polypeptides are seen (Fig. 4). weight polypeptides by human plasmin (see Fig. 2), suggesting Characterization of the mature polypeptide was confirmed by that a plasmin like enzyme may contribute to the degradation immunoblotting experiments using prostatic tissue extract from of the enzyme in PC-3SF12 cells and in other carcinoma tissues which a major polypeptide with a molecular weight of 48,000 and cells. This is consistent with the protection of the acid was seen (18). The human liver acid phosphatase used to raise phosphatase in PC-3SF12 cell extracts that is afforded by the antisera has been shown to contain identical M, 50,000 subunits addition of BPTI, which is known to inhibit plasmin (Fig. 3). (14). Several lines of evidence support the significance of these The other cellular event worth emphasizing is the conversion studies with respect to normal human tissues. Precursor (.\t, of part of the M, 68,000 precursor polypeptide pool to higher- 120,000-77,000), mature (M, 55,000-43,000), and low-molec molecular-weight polypeptides with molecular weights of ular-weight (M, 26,000) polypeptides are detectable in human 71,000 and 77,000; these are stored in the cell. Some of the M, prostatic tissue extracts and in seminal fluid using an immu- 71,000 precursor, together with M, 68,000 and 62,000 polypep noblotting technique. These observations also support the con tides, is secreted into the medium. Tunicamycin, an inhibitor clusion that the polypeptides with molecular weights of 55,000 of protein glycosylation (40), apparently prevents the transfer and 49,000 in PC-3SF12 represent the mature form of the of oligosaccharide chains to the polypeptide of lysosomal acid enzyme molecule and a similar biosynthetic route is followed phosphatase. The sizes and the total number of precursor in normal tissues. Further purification and characterization of polypeptides in both the cell and medium extracts are reduced immunologically active high-molecular-weight polypeptides when tunicamycin is present (Fig. 5). Thus, it can be concluded from human seminal fluid is in progress. that the precursor polypeptides are quickly glycosylated, that Prostatic acid phosphatase is normally released from the the multiple forms of the precursor are due at least in part to prostate gland into the seminal fluid, but the present studies differences in the extent of glycosylation, and that the oligosac- indicate that prostatic acid phosphatase is not secreted from charides are linked to asparagine residues in the polypeptide. PC-3SF12 cells. In fact, our results suggest that PC-3SF12, Similarly, placenta! alkaline phosphatase in choriocarcinoma like PC-3 cells (38), may be deficient in androgen receptor. cells is synthesized as a M, 61,500 polypeptide and after l h of Conflicting results on the effect of testosterone on the biosyn chase the initial polypeptide (M, 61,500) is converted to a thesis and secretion of the acid phosphatase in cultured human polypeptide with a molecular weight of 64,500 (41). That cell lines and tissues (10, 39) prompted us to examine the effect observation was further supported by the demonstration that of testosterone on the biosynthesis and secretion of the enzyme the increase in the apparent molecular weight of the polypeptide in PC-3SF12 cells. The presence of 20 ^M testosterone does was due to the addition of glucosamine residues on the oligo not affect the biosynthesis or secretion of prostatic acid phos saccharide side chains. phatase in this cell line. Although androgen has been found to influence the isoelectric In the PC-3SF12 cell, a majority of the prostatic acid phos pattern of the lysosomal acid phosphatase in rat prostate gland phatase is localized in low-density membrane fractions which (11), we observed no effect of 20 Â¿Â¿Mtestosterone on the generally constitute the Golgi apparatus, endoplasmic reticu- biosynthesis, glycosylation, and secretion of lysosomal acid lum, primary lysosomes, and secretory granules (37). The high- phosphatase in PC-3SF12 cells (Fig. 5). At least to the extent density membrane fraction, composed mainly of secondary that the charge heterogeneity of such an enzyme arises from lysosomes (1.112-1.114 g/ml), does not contain prostatic acid posttranslational modifications that are unaffected by hor phosphatase. Localization of prostatic acid phosphatase in hu mones, no change would be expected. man tissues by immunofluorescent spectroscopy also indicate In PC-3SF12 cells, lysosomal acid phosphatase is located in that prostatic acid phosphatase is not present in the lysosomes, the low-density membrane fraction (1.033-1.066 g/ml), which but is instead present in a different organelle.4 generally constitutes Golgi endoplasmic reticulum lysosome (GERL) membranes, as well as in the high-density membrane Lysosomal-type Acid Phosphatase fraction (1.114 g/ml), which is rich in secondary lysosomes (37). These results are consistent with the conclusion that the Lysosomal acid phosphatase in PC-3SF12 cells is also syn human liver acid phosphatase (14), used to raise the antiserum, thesized as a high-molecular-weight precursor that is processed to the mature tissue enzyme and finally to smaller-molecular- is a lysosomal acid phosphatase. Thus, the present studies demonstrate that: (a) prostatic and weight polypeptides. This conclusion is based on the following lysosomal acid phosphatases are synthesized as high-molecular- experimental evidence: (a) immunoprecipitation of larger and weight precursor molecules following a pattern similar to that smaller polypeptides occurs with an antiserum raised against observed for other lysosomal enzymes and proteins (42); (/>) homogeneous tissue enzyme; (/>)preimmune serum precipitates precursor or mature forms of the prostatic enzyme are not no polypeptide; (c) the decrease in the intensity of the larger secreted into the medium; (c) in contrast, precursor or mature 4 D. Drenckhahn, A. Waheed, and R. L. Van Etten, unpublished results. forms of the lysosomal enzyme are secreted into the medium; 4802

(Prostate Cancer, pp. 671-692. New York: Marcel Dekker, their amino acid compositions (13,14) and their immunological 1979. 22. Choe, B. K., Pontes, J. E., Lillehoj, H. S., and Rose, M. R. Immunohistolog- cross-reactivities (4, 15, 16), one must conclude that prostatic ical approaches to human prostatic epithelial cells. Prostate, /: 383-398, acid and lysosomal acid phosphatases are products of two 1980. different genes in PC-3SF12 cells; and (g) although the biosyn 23. Van Etten, R. L., and Saini, M. S. Selective purification of tartrate-inhibitable acid phosphatases: rapid and efficient purification (to homogeneity) of human thesis and transport of both acid phosphatases share a common and canine prostatic acid phosphatases. Clin. Chem., 24:1525-1530, 1978. route through the GERL (37), they are stored (at least in part) 24. Kumarasamy, R., and Symons, R. H. 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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1986 American Association for Cancer Research. Biosynthesis and Processing of Prostatic and Lysosomal Acid Phosphatases in a Prostate Carcinoma Cell Line PC-3SF12

Abdul Waheed and Robert L. Van Etten

Cancer Res 1986;46:4796-4803.

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