Zuxiong Chen

Prostate specific antigen cleaves parathyroid hormone-related protein in the PTH-like domain: inactivation of PTHrP-stimulated cAMP accumulation in mouse osteoblasts.

PURPOSE To determine whether parathyroid hormone-related protein (PTHrP) is a substrate of prostate-specific antigen (PSA) and how the biological activity of PTHrP may be altered by cleavage with PSA. MATERIALS AND METHODS Prostate-specific antigen cleavage of recombinant human PTHrP 1-141 was conducted in vitro at 37C and analyzed by SDS-PAGE. Five rounds of automated amino-terminal amino acid sequence analysis were performed on blotted PSA-cleaved PTHrP peptide fragments to determine the PSA cleavage sites. The mouse osteoblast cell line MC3T3-E1 was used to test whether PSA cleavage of PTHrP 1-141 altered its ability to stimulate cAMP production. RESULTS Prostate-specific antigen was found to specifically cleave PTHrP 1-141 in a time- and dose-dependent manner. Cleavage of PTHrP 1-141 by PSA generated fragments on Coomassie-stained acrylamide gels that migrated with mobilities that corresponded to 19.5, 17, 15 and < 7 kd. The preferred PSA cleavage site of PTHrP 1-141 was determined to be at the carboxyl-terminus of phenylalanine 23, consistent with chymotryptic-like enzymatic activity of PSA. Cleavage of PTHrP by PSA completely abolished the ability of PTHrP to stimulate cAMP production. CONCLUSIONS Cleavage of PTHrP 1-141 by PSA carboxyl-terminal to phenylalanine 23 represents a unique pattern of PTHrP processing that may be specific to the prostate. Prostate-specific antigen inactivation of the cAMP-inducing activity of PTHrP 1-141 demonstrates that PSA cleavage regulates the biological activity of PTHrP. These results have implications for the role of PTHrP in prostate cancer metastasis to bone and its subsequent regulation of bone remodeling. Study of the biological activities of the PSA-generated PTHrP peptides identified in this study merits further investigation.

Prostate Specific Antigen in Benign Prostatic Hyperplasia: Purification and Characterization

PURPOSE The ratio of free-to-total prostate specific antigen (PSA) in serum is greater in patients with benign prostatic hyperplasia (BPH) than in those with prostate cancer, and it provides a means of partially discriminating these 2 diseases. To understand the molecular mechanism of why the free-to-total PSA ratio is greater in BPH than in prostate cancer we analyzed PSA obtained directly from nodules of BPH tissue. MATERIALS AND METHODS PSA from BPH nodule fluids was first purified by gel filtration and ion exchange chromatography. Purified BPH PSA was characterized by gel electrophoresis, enzyme assay and N-terminal sequence analysis of the amino acids. RESULTS A single band at 30 kDa. on sodium dodecyl sulfate polyacrylamide gel electrophoresis under nonreducing conditions was identical to that of seminal fluid PSA. Under reducing conditions most BPH PSA was degraded, whereas most seminal fluid PSA existed as an intact molecule. BPH PSA had multiple internal cleavages in addition to the common cleavage site between lysines 145 and 146 of seminal fluid PSA. Cleavage sites at C-terminals of histidine 54 and phenylalanine 57 were also detected. Enzymatic activity studies with different substrates showed that PSA from seminal fluid and BPH nodules had similar specific trypsin-like activity. However, BPH PSA had much lower specific chymotrypsin-like activity than seminal fluid PSA. N-terminal sequence analysis showed that BPH PSA was neither in the pre-proenzyme form (261 amino acids) nor the zymogen proenzyme form (244 amino acids) of PSA, both of which are known precursors of mature PSA (237 amino acids). CONCLUSIONS Most PSA in BPH nodules is in the nicked form with low chymotrypsin-like activity. When it leaks into the circulation it will form fewer PSA-antichymotrypsin complexes and more will remain in the free form. We predict that a protease with trypsin-like activity in BPH nodule fluid is probably responsible for the nicked form of BPH PSA. These findings suggest that antibodies produced against PSA in BPH nodules may be useful in discriminating prostate cancer from BPH.

ISOLATION AND CHARACTERIZATION OF FREE FORM PROSTATE SPECIFIC ANTIGEN (f-PSA) IN SERA OF MEN WITH PROSTATE CANCER

PURPOSE The free uncomplexed form of prostate specific antigen (f-PSA) from prostate cancer sera was partially isolated and characterized because the molecular form of f-PSA in the serum is unknown. MATERIALS AND METHODS 230 ml. of sera from 59 men with bone metastasis and individual PSA values of >2000 ng./mL were combined and centrifuged for 60 minutes at 30,000 RPM (4C). The sera were fractionated by gel filtration column chromatography (Sephacryl S-200, 2.5 cm. x 92 cm.). Free and complexed PSA in the eluted fractions were isolated by measuring immunoreactivity of PSA (Tosoh AIA-600 assay); f-PSA from 23 separate runs were combined, concentrated and re-chromatographed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to immobilize the isolated proteins onto a nitrocellulose membrane and a polyvinylidene difluoride (PVDF) membrane. Monoclonal antibody (F5) was used to probe PSA on nitrocellulose membrane and the PSA band was detected by Emission Chemoluminescence (ECL) kit. Amino terminal sequence analysis of the isolated f-PSA was performed with a gas-phase sequentor (Applied Biosyntens 4760 A) using the program designed by the manufacturer. RESULTS 0.5 cc of f-PSA (27,000 ng./mL) was obtained from serums after rechromatography. SDS-PAGE showed one double band around 30 kDa; with ECL technique, one major band at 30-kDa was identified as PSA. The amino terminal sequence analysis of this band showed residue 1 through 9 and 146 through 152. CONCLUSIONS In our preliminary experiment, the free form of serum PSA is partially isolated directly from human sera. Amino terminal sequence analysis has shown that serum f-PSA is not a pre-mature or zymogen form of PSA because serum f-PSA has a N-terminus identical to that of seminal fluid PSA. A nicked form of f-PSA is also found in these patient sera.

Serum Prostate Specific Antigen Binding α1-Antichymotrypsin: Influence of Cancer Volume, Location and Therapeutic Selection of Resistant Clones

We examined by gel filtration chromatography (Sephacryl 200) sera from 73 untreated patients with peripheral zone prostatic cancer volumes of 1 to 17 cc as well as patients with clinical stages C and D2 cancer. We also examined the sera from 40 patients who had failed radiation or hormonal therapy to determine if clonal cell selection by these 2 therapies altered the binding of prostate specific antigen (PSA) to alpha 1-antichymotrypsin. Finally, we compared sera from 10 patients with benign prostatic hyperplasia (BPH) and 14 with large transition zone-BPH cancer. Without exception, of the total serum PSA recognized by the Hybritech Tandem-R, Yang Pros-Check, Abbott IMx and Ciba Corning ACS assays, 88 to 98% were complexed with alpha 1-antichymotrypsin in all cancer patients. The 10 patients with BPH showed less complexation (73 to 84%). These studies suggest that much of the quantitative differences among assays is determined more by relative differences in recognition of the free and complex forms of PSA than by calibration differences between assays.

Physiologic (intraindividual) variation of serum prostate-specific antigen in 814 men from a screening population*

OBJECTIVES We measured the intraindividual variation of prostate-specific antigen (PSA) in the serum of healthy men screened for prostate cancer. METHODS We used a fully automated PSA assay system (ACS: 180 assay) to evaluate a screening population of 814 men (mean age, 63.3 years; range, 50 to 79 years) without documented prostate cancer or prostate surgery. A second blood sample was drawn 15 to 183 days after the first specimen (mean, 80 days). RESULTS In the ACS PSA ranges of 0 to 7.2 ng/mL, 7.3 to 17.9 ng/mL, and 18.0 ng/mL or greater (O to 4 ng/mL, 4 to 10 ng/mL, and 10.0 ng/mL or greater by the Tandem-R assay), the mean coefficient of variation of the first and second blood drawn was 20%, 12%, and 10%, respectively. In 435 men whose first blood samples were measured twice for PSA difference (interassay or run-to-run variation), the intraindividual variation in the range of 0 to 7.2 ng/mL was significantly larger than the interassay variation, which was also true the 7.3 to 17.9 ng/mL range. In the range of 0 to 7.2 ng/mL, 251 of 695 (36%) showed a 20% or greater relative increase and 69 of 695 (10%) showed a 1.3 ng/mL (0.75 ng/mL by the Tandem-R assay) or greater absolute increase of PSA at the second blood sample. CONCLUSIONS We conclude that in the low ranges of PSA concentrations, one should consider the possibility of substantial intraindividual variation when interpreting serial PSA measurements.

Tissue Slice Grafts : An in Vivo Model of Human Prostate Androgen Signaling

We developed a tissue slice graft (TSG) model by implanting thin, precision-cut tissue slices derived from fresh primary prostatic adenocarcinomas under the renal capsule of immunodeficient mice. This new in vivo model not only allows analysis of approximately all of the cell types present in prostate cancer within an intact tissue microenvironment, but also provides a more accurate assessment of the effects of interventions when tissues from the same specimen with similar cell composition and histology are used as control and experimental samples. The thinness of the slices ensures that sufficient samples can be obtained for large experiments as well as permits optimal exchange of nutrients, oxygen, and drugs between the grafted tissue and the host. Both benign and cancer tissues displayed characteristic histology and expression of cell-type specific markers for up to 3 months. Moreover, androgen-regulated protein expression diminished in TSGs after androgen ablation of the host and was restored after androgen repletion. Finally, many normal secretory epithelial cells and cancer cells in TSGs remained viable 2 months after androgen ablation, consistent with similar observations in postprostatectomy specimens following neoadjuvant androgen ablation. Among these were putative Nkx3.1(+) stem cells. Our novel TSG model has the appropriate characteristics to serve as a useful tool to model all stages of disease, including normal tissue, premalignant lesions, well-differentiated cancer, and poorly differentiated cancer.

Addition of Purified Prostate Specific Antigen to Serum from Female Subjects: Studies on the Relative Inhibition by alpha 2-Macroglobulin and alpha 1-Antichymotrypsin

PURPOSE Two forms of prostate specific antigen (PSA), 1 complexed to alpha 1-antichymotrypsin and the other a free PSA, are recognized by current commercially available immunoassays. A third form of PSA complexed to alpha 2-macroglobulin also is present in the serum. To study these 3 different molecular forms of PSA in vivo, we simulated leakage of PSA from the prostate into the serum in vitro. MATERIALS AND METHODS Purified seminal fluid PSA was incubated with fresh sera from female subjects at different concentrations. Following gel filtration chromatography, the 3 forms of PSA were studied by immunoassays and Western blot analysis. RESULTS Using a commercial immunoassay, 60% of immunoreactivity of seminal fluid PSA was lost after incubation with sera from female subjects. Western blot analysis showed that most of this loss in PSA signal was caused by complexation to alpha 2-macroglobulin. Minimal, if any, complexation to alpha 1-antichymotrypsin occurred even when excess alpha 1-antichymotrypsin was added to the serum. CONCLUSIONS Our studies demonstrated that alpha 2-macroglobulin is a much stronger inhibitor to PSA than alpha 1-antichymotrypsin. Further studies of these complexes may be important. They clearly explain why spiking PSA into sera from female subjects to be used as quality controls for PSA assays leads only to the free form of enzymatically inactive PSA in the serum, and not to the dominant form of complexed PSA and alpha 1-antichymotrypsin present in human serum.

Inhibition of monoamine oxidase A promotes secretory differentiation in basal prostatic epithelial cells

Monoamine oxidase A (MAO-A) expression is associated with high-grade prostate cancer. Immunohistochemistry showed that MAO-A is also expressed in the basal epithelial cells of normal prostate glands. Using cultured primary prostatic epithelial cells as a model, we showed that MAO-A prevents basal epithelial cells from differentiating into secretory cells. Under differentiation-promoting conditions, clorgyline, an irreversible MAO-A inhibitor, induced secretory cell-like morphology and repressed expression of cytokeratin 14, a basal cell marker. More importantly, clorgyline induced mRNA and protein expression of androgen receptor (AR), a hallmark of secretory epithelial cells. In clorgyline-treated cells, androgen induced luciferase activity controlled by the promoter of prostate-specific antigen, an AR target gene, in a dose-dependent manner. This activity was blocked by the AR antagonist Casodex, showing that AR is functional. In turn, androgen decreased MAO-A expression in clorgyline-treated, secretory-like cells. Our results demonstrated that cultured basal epithelial cells have the potential to differentiate into secretory cells, and that inhibition of MAO-A is a key factor in promoting this process. Increased expression of MAO-A in high-grade prostate cancer may be an important contributor to its de-differentiated phenotype, raising the possibility that MAO-A inhibition may restore differentiation and reverse the aggressive behavior of high-grade cancer.

Increased expression of GCNT1 is associated with altered O‐glycosylation of PSA, PAP, and MUC1 in human prostate cancers

Protein glycosylation is a common posttranslational modification and glycan structural changes have been observed in several malignancies including prostate cancer. We hypothesized that altered glycosylation could be related to differences in gene expression levels of glycoprotein synthetic enzymes between normal and malignant prostate tissues.

MONOCLONAL ANTIBODIES 2F5 AND 4G10 AGAINST PROSTATE SPECIFIC ANTIGEN (PSA) COMPLEXED TO alpha 1-ANTICHYMOTRYPSIN

OBJECTIVES We have shown that an immunoassay (Chugai) for the PSA-ACT complex in serum has 2 to 3 times better specificity than total PSA at sensitivities of 85 to 97% in distinguishing biopsy positive men from biopsy negative men undergoing transrectal ultrasound (TRUS) examination. To increase the specificity of PSA immunoassay for prostate cancer, we produced specific antibodies exclusively against our purified PSA-ACT complex for development of assay kits for PSA-ACT complex. METHODS PSA-ACT complex was used as antigen to immunize BALB/c mice. PSA-ACT complex, free PSA, and ACT were used for hybridoma screening. To characterize the new monoclonal antibodies, we used Western blot, immunohistochemistry, and an in house immunoassay. RESULTS Two monoclonal antibodies, 2F5 and 4G10 were produced exclusively against PSA-ACT complex without any immunoreactivity to ACT or PSA alone. Western blot analysis indicated that 2F5 and 4G10 recognize conformation-dependent epitopes on PSA-ACT complex. Immunohistochemistry studies showed that 2F5 reacted with prostate cancer in about 30% of the cancer cells (sensitivity), but almost never stained normal prostate glands in the peripheral or transition zone tissue (about 100% specificity). Our in-house assay showed that 2F5 can be used as a tracer antibody specifically to detect PSA-ACT complex. CONCLUSIONS Using monoclonal antibody 2F5 as tracer antibody, we have developed a PSA immunoassay exclusively against PSA-ACT complex. This assay should maximize specificity in distinguishing BPH from prostate cancer.