Human Prostatic Acid Phosphatase Research Articles

Tyrosine Phosphorylation of a 185 kDa Phosphoprotein (pp185) Inversely Correlates with the Cellular Activity of Human Prostatic Acid Phosphatase

Human prostatic acid phosphatase (PAcP), a prostate epithelium-specific differentiation antigen, was determined to exhibit the endogenous protein tyrosine phosphatase activity. We investigated the phosphoprotein(s) that might be dephosphorylated by PAcP in human prostate carcinoma cells. Several lines of evidence were presented to show that the tyrosine phosphorylation level of a 185 kDa phosphoprotein (pp185) is negatively correlated with the cellular activity of PAcP. (i) In DU145, PC-3 and high passaged LNCaP prostate carcinoma cells that have no or low PAcP expression, the phosphotyrosine (p-tyr) level of pp185 was higher than that in low passaged LNCaP cells that express an endogenous PAcP. (ii) In LNCaP cells grown in the presence of L(+)-tartrate, an inhibitor of PAcP, the tyrosine phosphorylation of pp185 was increased. (iii) Mediated by Lipofectin, a cationic liposome, the incorporation of purified PAcP protein into DU145 cells resulted in the decreased phosphorylation of pp185. Thus, the results taken collectively demonstrated that the p-tyr level of pp185 is inversely correlated with the cellular activity of PAcP and indicated that the pp185 may be a putative substrate of PAcP in prostate carcinoma cells.

Human prostatic acid phosphatase and prostate specific antigen: protein structure, gene organization, and expression in neoplastic and benign tissues.

Human prostatic acid phosphatase (ACPP) and prostate specific antigen (PSA) have been used as diagnostic and prognostic markers of prostate cancer. The structure of ACPP and PSA genes and their encoded proteins are described. The expression of both genes was shown to be elevated significantly in neoplastic tissue when compared to benign prostatic hyperplasia. The prospect of developing new molecular DNA/RNA markers to diagnose prostate cancer is discussed.

The folding intermediate of reversibly denatured human prostatic acid phosphatase

Human prostatic acid phosphatase (hPAP) [EC 3.1.3.2], a homodimer of ca. 50 kDa subunit molecular weight, shows reversible denaturation in 6 M urea at pH 2.5. Rapid dilution of the denatured enzyme allowed partial renaturation of hPAP, as measured by enzyme activity, to a level which depended on the composition of the dilution solution employed and time of the reaction. The renaturation reaction of hPAP was examined using spectral analysis (circular dichroism and fluorometry), fast size-exclusion chromatography and proteolysis by trypsin. The observed results are in agreement with the concentration-dependent kinetics of hPAP reactivation, assuming that the reconstitution of the active enzyme requires the association of subunits in dimeric form. Moreover, it suggests formation of an inactive intermediate during refolding of the denatured PAP. A mechanism of renaturation of the active enzyme from denatured PAP is proposed.

Benzylphosphonic acid inhibitors of human prostatic acid phosphatase

A series of α-substituted benzylphosphonic acids is described as inhibitors of human prostatic acid phosphatase, an enzyme which has been used as a model to study aryl phosphatases. The most potent inhibitors in this series are 2-trifluoromethylbenzhydrylphosphonic acid (9 μM), and α-(2-phenylethyl)benzylphosphonic acid (14 μM). The structure-activity studies suggest that bulk tolerance beyond the phosphate binding area limits the steric or hydrophobic contribution to inhibitor potency achieved through α-carbon substitution.

Phosphotyrosine Antibodies Preferentially React with Basal Epithelial Cells in the Dog Prostate

Purpose Considering the hypothesis that androgen-independent but growth factor dependent epithelial cell division may be important in the development and progression of prostate cancer and that protein tyrosine kinases and phosphotyrosine protein phosphatases are key enzymes modulating the levels of specific phosphotyrosylated proteins implicated in several growth factor regulated signal transduction pathways, our aim was to study the cellular distribution of phosphotyrosine proteins in normal and hyperplastic dog prostates as well as in those of castrated dogs supplemented with either androgens or estrogens in order to modify the relative proportion of basal versus secretory epithelial cells. Materials and Methods Following the determination of optimal conditions to specifically detect phosphotyrosine proteins by a rabbit polyclonal antibody directed against phosphotyrosine, immunohistochemistry was performed on prostate tissue sections from these experimental animals. In addition to morphological criteria, an antibody to high molecular weight cytokeratins and antisera against arginine esterase were used to selectively identify basal and secretory cells. Since prostatic acid phosphatase may be involved in the local regulation of phosphotyrosine proteins, its distribution was also evaluated with a human prostatic acid phosphatase antiserum. Results In all prostatic tissues examined, basal epithelial cells were preferentially and specifically stained with antiphosphotyrosine. The staining intensity per basal cell was highest in the estrogen-supplemented dogs. In addition, basal cells were numerically increased and all were highly immunoreactive for high molecular weight cytokeratins. In prostates displaying a well-differentiated glandular epithelium, the number of positive basal cells and their staining intensity varied in the following order: normal less than hyperplastic less than androgen-supplemented dogs. At all times, the levels of phosphotyrosine proteins in prostatic acid phosphatase and arginine esterase positive cells (secretory) remained low. Fibroblasts and smooth muscle cells were unreactive to antiphosphotyrosine, even though estrogen supplementation increased the prostatic stromal volume. Conclusions The preferential localization of phosphotyrosine proteins in basal cells, their increased level per cell and the number of positive cells in the different experimental animals support the concept that basal cells represent the stem cells of the prostate. The sex steroid-mediated up-regulation of protein phosphorylation on tyrosine residues in these cells suggests that their proliferation is likely to involve growth factor regulated signal transduction pathways. In this respect, the lack of maturation of basal cells and the differentiation of secretory cells induced by androgen deprivation, combined with estrogen stimulation, favors the activation of these pathways and cell growth. On the other hand, the activation of glandular cell differentiation and the increase of stromal volume do not alter the threshold level of protein tyrosine kinase and phosphatase activities in secretory cells, fibroblasts and smooth muscle cells.

Single-step purification of prostatic acid phosphatase: immunoaffinity chromatography with a monoclonal antibody.

Prostatic acid phosphatase (PAP) is an important protein which should be studied further as a tumor marker or as a biologically functional molecule. The purpose of the study was to establish a simple and reliable method to obtain highly pure PAP. Spleen cells from mice immunized with prostatic epithelial cells prepared from benign prostatic hyperplasia tissue were fused with myeloma cells X63Ag8-653. Hybrid cells of interest were selected using the indirect immunofluorescence method with unfixed frozen tissue sections. One clone of the hybrid cell lines was established which secreted the monoclonal antibody specifically reactive to prostatic acid phosphatase. Using this monoclonal antibody, we purified the antigen from human prostatic tissue by means of single-step immunoaffinity chromatography. SDS-PAGE profiling under reducing conditions indicated that the protein recognized by this antibody consisted of several components of molecular weight 41,000-45,000. Partial amino acid sequence analysis of this protein indicated that these components involved a heterogeneously modified single polypeptide, and that this antigen is identical to human prostatic acid phosphatase. This single-step method saves the time needed to purify prostatic acid phosphatase and requires only half a day for the whole procedure. Moreover, the purity of the isolated protein was extremely high. This method seems to be useful not only for purifying prostatic acid phosphatase but also for purifying other proteins from the prostate gland and for analysis of antigenic macromolecules.

Highly sensitive immunological assays for human chorionic gonadotrophin and prostatic acid phosphatase using phenacyl phosphate as a chemiluminescent label

A highly sensitive assay for alkaline phosphatase (ALP) was developed using phenacyl phosphate as a substrate for chemiluminescent assays. This substrate is applied to two types of immunological assays based on ALP labelling, an assay for human chorionic gonadotrophin (hCG) in urine by a two-site immunometric assay and an assay for human prostatic acid phosphatase (PAP) adsorbed on a solid phase by an antibody. The results of the validation on these assays were good and detection limits were 15–40 times better than for conventional assays, viz. 6.25 × 10 −6 IU for hCG and 1.25 × 10 −18 mol PAP.

Generation and characterization of putative monoclonal and polyclonal antibodies against tartrate-resistant acid phosphatase.

The generation of monoclonal antibodies (MAbs) specific for tartrate-resistant acid phosphatase (TRAP) may aid development of a serological immunoassay for this marker of bone resorption. The lack of MAbs to TRAP largely reflects the difficulty in obtaining sufficient antigen for in vivo immunization strategies. We have circumvented this problem by using in vitro immunization, requiring a small amount of TRAP isolated from osteoclastoma tumor. Two MAbs designated 312D and 310A were generated that exhibited weak anti-TRAP activity in enzyme-linked immunosorbent assay and immunocytochemistry. A polyclonal antibody to TRAP (Ab8023) was also raised in rabbit, using synthetic peptide. Ab8023 and MAbs 310A and 312D exhibited no activity against TRAP in dot-blotting experiments. Further characterization in enzyme-liked immunosorbent assay showed that Ab8023 was remarkably specific for TRAP whereas MAb 312D cross-reacted with another metalloenzyme, human prostatic acid phosphatase.

Human prostatic acid phosphatase: Selected properties and practical applications

Human prostatic acid phosphatase (EC 3.1.3.2) is a non-specific phosphomonoesterase, synthetized and secreted into seminal plasma under androgenic control. The enzyme is a dimer of molecular weight around 100 kDa. Gene coding this protein is localized on chromosome 3. Since many years prostatic phosphatase has been used as a marker for diagnosis and therapy control of cancer of the prostate gland. The biological role of this enzyme, however, remains unknown and needs further exploration.

Suicide Inactivation of Human Prostatic Acid Phosphatase and a Phosphotyrosine Phosphatase

4-Difluoromethylphenyl bis(cyclohexylammonium) phosphate was synthesized in 4 steps starting from dibenzyl phosphite and shown to be a time-dependent suicide inactivator of human prostatic acid phosphatase and the SHP protein tyrosine phosphatase. The inactivation of human prostatic acid phosphatase followed pseudo-first-order kinetics with inactivation constants of Ki = 1.0 mM; ki = 0.15 min−1 (t12 = 4.6 min at saturation). Phenyl phosphate protected the enzyme against inactivation, indicating that inactivation occurs in the active site. The inactivation of SHP also followed pseudo-first-order kinetics, with a t12 = ∼15 min in the presence of 8.2 mM inhibitor. The mechanism of inactivation likely involves the enzymatic release of difluoromethyl phenol which rapidly eliminates fluoride,generating a quinone methide. This potent electrophile then reacts with residues at the active site of the enzyme. This inhibitor and peptidic derivatives thereof have excellent potential for selective inactivation and labeling of protein tyrosine phosphatases.

Effect of cell density on androgen regulation of the mRNA level of human prostatic acid phosphatase

The expression of human prostatic acid phosphatase (PAcP), a differentiation antigen of prostate epithelial cells, has been proposed to be regulated by androgen. We investigated this regulatory mechanism at the post-transcriptional level in LNCaP human prostate carcinoma cells, the only cultured cells that express PAcP. Our results demonstrated that the level of PAcP mRNA decreased when the cell density increased. Further, 5α-dihydrotestosterone, an active form of the endogenous androgen, stimulated the PAcP mRNA level in low-density cells; while, caused a decrease in high-density cells. Thus, in LNCaP human prostate carcinoma cells, cell density could modulate PAcP expression at the mRNA level including androgen regulation.

Analysis of the promoter of the human prostatic acid phosphatase gene

From the analysis of two overlapping cosmid clones prepared from human genomic DNA libraries, a contig of 44 kb containing a 5′ portion of the PAP gene and 17 kb of the upstream region was established. It was characterized by restriction mapping and sequence analysis of 2.5 kb upstream of the initiation codon. Two major transcription initiation sites were found to be located around 56 and 91 bp upstream of the initiation codon, as determined by nuclease S1 and primer extension mapping. Expression of the PAP gene was measured by Northern blots in the androgen responsive LNCaP cell line. It was found to be induced 2–3-fold by the addition of the synthetic androgen mibolerone to the cells. The induced mRNA levels were approx. 10-times lower than those for the prostate-specific antigen (PSA) in LNCaP cells.

Heterologous expression of human prostatic acid phosphatase and site-directed mutagenesis of the enzyme active site.

Earlier covalent modification experiments indicated that histidine, arginine, and carboxylic acid residues were involved in the catalytic mechanism of the acid phosphatase enzyme from human prostate. The present study utilizes site-directed mutagenesis to evaluate the catalytic importance of 7 residues of human prostatic phosphatase, namely His12, His257, Asp258, Arg11, Arg15, Arg54, and Arg79, which are highly conserved in the sequences of high molecular weight acid phosphatases. Both wild type and mutant enzymes were expressed in the yeast Saccharomyces cerevisiae as fusions to a leader peptide of prepro-alpha-factor, which resulted in the secretion of processed proteins into the culture media. The kinetic parameters of the recombinant wild type enzyme were shown to be effectively identical with those of the native protein. The circular dichroism spectra of wild type and mutant proteins were effectively superimposable. Replacement of His12, which was previously shown to be transiently phosphorylated during catalysis, as well as the adjacent Arg11, both resulted in > 10(4) reductions of enzyme activity. Consistent with a postulated involvement of arginine residues in substrate binding, large reductions in Vmax caused by the R15A and R79A mutations were accompanied by marked increases in Km. However, the mutation R54A had only small effects on kinetic parameters. Essential roles for both His257 and Asp258 were also established. The D258A mutation not only caused larger decreases of Vmax toward p-nitrophenyl phosphate compared to the effect of the H257A mutation, but it also caused the reaction to become more sensitive to the nature of the leaving group. It is concluded that His12 and Asp258 are involved in the formation of the phosphoenzyme intermediate as an acceptor of the phospho group and a proton donor for the substrate leaving group, respectively, while His257 may participate in substrate binding or may facilitate the breakdown of the phosphoenzyme by maintaining the protonated state of the phosphorylated His12 residue.

Human prostate acid phosphatase is carrier protein for ABH blood group antigens of semen.

Prostate acid phosphatase (PAcP) was characterized as one of carrier proteins for ABH blood group antigens in semen. The diluted semen was sensitized with anti-PAcP. The resulting immunocomplex was subjected to SDS-PAGE after being washed with saline. SDS-PAGE patterns of the immunocomplex consisted of four bands. After electroblotting from gel to nitrocellulose filter, immunostaining using anti-PAcP or monoclonal anti-blood group antibodies was performed. The result showed that the protein of 50 kD was the subunit of PAcP and the band of 94 kD was identified with blood group antigens respectively. Other two bands were rabbit IgG against PAcP and its fragment. When semen was treated with the chemicals such as papain, SDS, 2-mercaptoethanol and dithiothreitol, the ABH of antigen which was captured by anti-PAcP were considerably reduced. The reduction of ABH activity by these treatments indicated that ABH antigens were located on PAcP in semen which was inactivated by the treatment. Therefore, PAcP was one of proteins carrying ABH epitopes in semen in addition to alpha 2-seminoglycoprotein.

The Prostatic Acid Phosphatase (ACPP) Gene Is Localized to Human Chromosome 3q21-q23

Human prostatic acid phosphatase (ACPP) has been used as a diagnostic marker for prostate cancer. It is synthesized under androgen regulation and secreted by the epithelial cells of the prostate gland. We have confirmed the previous assignment of the ACPP gene to chromosome 3 by probing a panel of 25 human-Chinese hamster somatic cell hybrids, and we have further localized the ACPP gene to chromosome 3q21-q23 by fluorescence in situ hybridization.

Fluorometric analysis of native, urea-denatured and refolded human prostatic acid phosphatase

Human prostatic acid phosphatase (EC 3.1.3.2) was denatured in 6 M urea at pH 2.5, but was refolded by dilution at pH 7.0, as demonstrated by the recovery of nearly complete enzyme activity and dimeric structure. The conformational changes among the native, denatured and refolded states were monitored by means of steady-state and nanosecond pulse fluorometry of tryptophan residues of the enzyme. The relative quantum yield of the fluorescence was highest in the native enzyme and lowest in the denatured one, and was intermediate in the refolded enzyme, although the emission peak was reproducible after refolding. The observed decay curves of tryptophan fluorescence of the native, denatured and refolded states were analyzed by decay functions of three lifetimes. The fluorescence lifetimes of the refolded enzyme were shorter than those of the native one. The fluorescence of the denatured enzyme decayed much faster than that of the other forms. The fluorescence excitation spectra revealed that the excitation energy of phenylalanine was transferred to tryptophan(s) in the native and refolded forms, but not in the denatured form. The efficiency of the energy transfer was higher in the native enzyme than in the refolded one. It was found by excitation polarization spectra that the freedom of internal motion of tryptophans was greater in the refolded enzyme than in the native enzyme. In the denatured enzyme the polarization anisotropies were very low. These results indicate that the higher structure with respect to tryptophans of the refolded enzyme is delicately but definitely different from that of the native enzyme and that local conformation of the active center is recovered upon refolding.

5162504 Monoclonal antibodies to a new antigenic marker in epithelial prostatic cells and serum of prostatic cancer patients: Julius Horoszewicz assigned to Cytogen Corporation

Stable clones of murine hybridomas 7E11-C5 and 9H10-A4 were obtained following immunization with LNCaP cells. The LNCaP cells were isolated from a human prostatic cancer (Ca). Both hybridomas secreted monoclonal antibodies (MoAb) of the IgG1 subclass which were reactive with the insoluble, cytoplasmic, membrane rich fractions of the immunogen. Neither MoAb reacted with the soluble cytosol of LNCaP cells nor with purified human prostatic acid phosphatase (PAP) nor prostate specific antigen (PSA). MoAb 9H10-A4 reactivity was very narrow and limited to the surfaces of LNCaP cells only. MoAb 7E11-C5 specificity was restricted to human prostatic epithelium, both normal and malignant. Except LNCaP, none of the 32 lines of human normal or neoplastic cells reacted with MoAb 7E11-C5. In a survey of frozen sections from 175 human specimens, positive indirect immunoperoxidase staining was limited to epithelium in all 11 specimens of localized and metastatic CaP, 7 benign prostatic hypertrophy (BPH) cases and 7 normal prostates. None of the 26 various nonprostatic tumors nor 120 out of 122 specimens from 28 different normal organs were reactive. Positive staining occurred in 2 out of 14 normal kidneys. Competitive binding with MoAb 7E11-C5 or its F(ab')2 fragments demonstrated the presence of circulating epitope 7E11-C5 in 20 out of 43 sera from CaP patients. Only 3 out of 66 sera from nonprostatic malignancies reacted. None of 30 normal blood donors sera nor 7 BPH sera were positive. Thus, highly significant (p less than 0.0001) association between diagnosed prostatic cancer and circulating molecules expressing the epitope reactive with MoAb 7E11-C5 was established. Significant probability (p less than 0.05) also suggested that patients with positive ELISA test are more likely to be in progression, than those who are negative. These results suggest that this apparently new antigenic marker may be of clinical potential in CaP.

Regulation of Prostatic Acid Phosphatase Expression and Secretion by Androgen in LNCaP Human Prostate Carcinoma Cells

The expression and the secretion of human prostatic acid phosphatase (PAcP), a differentiation antigen which is the major acid phosphatase in prostate epithelial cells, are thought to be regulated by an androgen. We investigated this regulatory mechanism at the post-transcriptional level in LNCaP human prostate carcinoma cells using a cDNA clone for the secretory form of PAcP. 5α-Dihydrotestosterone (DHT, an active form of endogenous androgen) stimulated the secretion of PAcP from cells grown in a steroid-reduced medium and in a defined serum-free medium, respectively. The secreted PAcP activity was increased following a DHT dose in a dose-dependent fashion at concentrations of up to 1 μM. Further, the stimulation of PAcP secretion occurred following the period of exposure to DHT. During a 5-day treatment period, with 10 nM of DHT in the steroid-reduced medium, the secretion of PAcP was stimulated approximately 150% over that from control cells. Nevertheless, PAcP was secreted from cells grown in the absence of added DHT. First, the androgen dependency of PAcP expression was examined. The expression and the secretion of PAcP were observed in cells that were grown in a defined serum-free medium and grown in a steroid-reduced medium, in the absence of DHT. The increased secretion by DHT was further demonstrated to be in part due to an increase in PAcP mRNA level, as evidenced by Northern blot analysis. PAcP mRNA levels were elevated approximately 2-fold and corresponded to an increase of approximately 2.5-fold in the secreted level of newly synthesized 35S-PAcP. Then, the effect of DHT on the secretory process was investigated. Results of pulse-chase labeling experiments indicated that the secretory rate of PAcP was stimulated by about 50% on average by DHT. In conclusion, our data demonstrated that, in LNCaP cells, the expression and the secretion of PAcP regulated by androgen are apparently hormone-responsive processes. Further, DHT stimulation of PAcP secretion operates within at least two levels: increased PAcP mRNA and stimulation of the secretory pathway.

Radioimmunodetection of prostate cancer by 111In-labeled monoclonal antibody against prostatic acid phosphatase.

Purified human prostate acid phosphatase (PAP) was used to generate a specific monoclonal antibody (FC 3001) for detection of PAP expressed by some prostatic carcinomas. DTPA derivatives of MoAb-F(ab')2-fragments were labeled with indium-111 chloride. This labeled antibody was tested in 15 prostate cancer patients who underwent staging pelvic lymphadenectomy; 9 of them received labeled antibody alone whereas 6 received simultaneous injections of labeled and unlabeled antibody with two dose levels (40 or 80 mg). Biodistribution data obtained by direct blood measurements and imaging procedures indicated that simultaneous injection of unlabeled antibody reduced both the blood elimination rate and the accumulation in the liver. Accumulation of the radionuclide in pelvic lymph node metastases was observed in some patients but in a couple of patients accumulation was noted also in normal lymph nodes. The method cannot in its present design replace staging pelvic lymphadenectomy and further studies are needed for elaboration of clinically useful radioimmunodetection methods.

Overexpression, site-directed mutagenesis, and mechanism of Escherichia coli acid phosphatase

Site-directed mutagenesis was used to examine the catalytic importance of 2 histidine and 4 arginine residues in Escherichia coli periplasmic acid phosphatase (EcAP). The residues that were selected as targets for mutagenesis were those that were also conserved in a number of high molecular weight acid phosphatases from eukaryotic organisms, including human prostatic and lysosomal acid phosphatases. Both wild type EcAP and mutant proteins were overproduced in E. coli using an expression system based on the T7 RNA polymerase promoter, and the proteins were purified to homogeneity. Examination of the purified mutant proteins by circular dichroism and proton NMR spectroscopy revealed no significant conformational changes. The replacement of Arg16 and His17 residues that were localized in a conserved N-terminal RHGXRXP motif resulted in the complete elimination of EcAP enzymatic activity. Critical roles for Arg20, Arg92, and His303 were also established because the corresponding mutant proteins exhibited residual activities that were not higher than 0.4% of that of wild type enzyme. In contrast, the replacement of Arg63 did not cause a significant alteration of the kinetic parameters. The results are in agreement with a previously postulated distant relationship between acid phosphatases, phosphoglycerate mutases, and fructose-2,6-bisphosphatase. These and earlier results are also consistent with the conclusion that 2 histidine residues participate in the catalytic mechanism of acid phosphatases, with His17 playing the role of a nucleophilic acceptor of the phospho group, whereas His303 may act as a proton donor to the alcohol or phenol.