[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 ¡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 37°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 ¿/Iofrabbit anti- was mixed with 100 //I of rabbit anti-human liver acid phosphatase human liver acid phosphatase or with 3-6 /<!of control serum. Each antiserum and 33 n\ of detergent buffer. All other steps were similar to solution was left at room temperature for 30 min and then for at least those described for precipitation of human prostatic acid phosphatase. 20 h at 4°C.Protein A—bacterial adsorbent (Miles Laboratories) pellet Immunoprecipitations of prostatic and lysosomal acid phosphatases prepared from 75 to 150 p\ of protein A suspension (10%, w/v) was from tissue extracts were performed similarly. Extract volumes were added to the above mixture and the sample was kept on ice for 30 min chosen to contain around 10-20 fig of the enzyme. with intermittent shaking. The mixture was centrifuged for 5 min at Electrophoresis and Fluorography. Electrophoresis in SDS (Sigma 12,000 rpm using a Beckman JA-20 rotor at 4'C. The supernatant was and Bethesda Research Laboratories) with and without /3-mercaptoeth- used for determination of the phosphatase activity in the presence and anol was performed according to Laemmli (27). Fluorography of the the absence of 10 mM L-(+)-tartrate (24). dried gel was carried out as described by Bonner and Laskey (28). Cell Labeling and Preparation of Cell and Medium Extracts. Cells Apparent molecular weights were determined using MC-methylated were grown in 25-cm2 flasks for approximately 9 days to a density of standard 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.