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A precursor form of PSA (pPSA) is a component of the free PSA in prostate cancer serum
ELSEVIER
A PRECURSOR FORM OF PSA (pPSA) IS A COMPONENT OF THE FREE PSA IN PROSTATE CANCER SERUM STEPHEN D. MIKOLAJCZYK, LANA S. GRAUER, LISA S. MILLAR, TIMOTHY M. HILL, ABHAY KUMAR, HARRY G. RITTENHOUSE, ROBERT L. WOLFERT, AND MOHAMMAD S. SAEDI
ABSTRACT Objectives. Prostate-specific antigen (PSA) is a widely used serum marker for human prostate cancer (PCs). The majority of PSA in serum is present as a complex with alpha-l -antichymotrypsin (ACT). In recent years, the ratio of free (uncomplexed) to total PSA has shown improved discrimination of PCs from benign prostatic hyperplasia. This study examines the nature of the free PSA form detected in PCs serum and shows that some of the uncomplexed PSA is an inactive precursor of PSA (pPSA). Methods. Western blot analysis was used to detect clipped, fragment forms of PSA in sera and seminal fluid. Hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC) was used to identify forms of PSA present in the free PSA population. Pooled sera was passed over a PSA immunoaffinity column, and the eluted PSA components were further resolved by HIC-HPLC. Results. Western blot analysis of whole sera showed complexed PSA and the intact, approximately 34 kilodalton free PSA. Only negligible levels of clipped or degraded forms of PSA, as found in seminal fluid, were detected. Column fractions measured for uncomplexed PSA using the Tandem-MP free PSA assay showed that about 25% of the free PSA eluted as pPSA beginning at the [-4]amino acid. Studies with purified recombinant [-4]pPSA showed that this proenzyme form is inactive and does not complex with ACT. Conclusions. These results suggest that the uncomplexed PSA in PCs serum is primarily unclipped PSA that contains a significant fraction of pPSA. UKOLOGY 50: 710-714, 1997. 0 1997, Elsevier Science Inc. All rights reserved.
M
easurement of prostate-specific antigen (PSA) in serum has been widely used as a diagnostic marker for human prostate cancer (PCa).lp3 PSA is a serine protease,4-6 and in serum it forms complexes with endogenous protease inhibitors, primarily alpha-2-macroglobulin and alpha-I-antichymotrypsin (ACT).7X8 Typically, 80% to 95% of the immunoreactive PSA in PCs sera is detected in complex with ACT, the remainder as uncomplexed, or free, PSA. In recent years the measurement of both free and complexed PSA in serum has become important in better distinguishing PCs from benign prostatic hyperplasia (BPH).9-‘2 An increased ratio of free to total (free plus complexed) PSA is more highly correlated with BPH than total PSA alone. Reasons for the persistence of uncomplexed PSA in the presence From Hybritech Incorporuted, San Diego, California Reprint requests: Stephen D. Mikolajczyk, P.O. Box 269006, San Diego, CA 92 196-9006 Submitted (Rapid Communication): June 24, 1997, accepted (with revisions): July 1.5, 1997 0
7 10
1997,
ALL RIGHTS
ELSEVIER SCIENCE RESERVED
INC.
of a large excess of serum protease inhibitors is the subject of speculation. Because inhibitor complex formation requires proteolytic activity, it is likely that free PSA is enzymatically inactive. It is known that approximately 30% of the PSA in seminal fluid is inactive because of internal peptide bond cleavages and that this clipped form does not complex with ACT.7 Recent studies have shown that BPH nodules in the prostate contain elevated levels of clipped PSA compared to seminal fluid PSA, which could account for the elevated ratio of free PSA in the serum of patients with BPH.13 It has been assumed that such clipped forms would also predominate in PCs serum. Another possibility for uncomplexed, inactive PSA is the presence of zymogen, or precursor PSA (pPSA). In the current study we examined pooled PCs serum for precursor forms of PSA using hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC) to resolve the free PSA fraction into its component parts. We found that pPSA is a significant subpopulation of the free PSA in serum. This finding has 0090-4295/97/$17.00 PII soogo-4295(97)00449-4
potentially important implications for the detection of PCs because it identifies a novel form of PSA that may enhance the discrimination of PCs from BPH. MATERIAL
AND
METHODS
RESOLUTION OF PSA FORMS BY HIC-HPLC The PSA gene was expressed in a hamster cell line, as described for human kallikrein 2.t4 PSA is secreted as its pro form from this cell line (AVlZ-PSA).r5 The pPSA expressed by the AVlZ-PSA cells was purified from the spent media by immunoaffinity chromatography. The column consisted of the PSA-specific monoclonal antibody (mAb) PSM773 covalently bound to Aminolink resin (Pierce) at a concentration of 5 mg mAb/mL resin. PSA was eluted with 100 mM glycine, 0.5 M sodium chloride, pH 2.6. The different forms of immunoreactive PSA (PSA-ACT, PSA, proPSA forms) were resolved from each other by HPLC using HIC (PolyLC, polypropyl aspartamide column, 100 nm pore size, 4.6 mm X 200 mm, distributed by Western Analytical, Temecula, Calif). Buffer A was 20 mM sodium phosphate, 1.2 M sodium sulfate pH 6.3, and buffer B was 50 mM sodium phosphate, 5% 2propanol, pH 7.4. The elution gradient was 0% to 35% buffer B from 0 to 1 minute and 35% to 80% Buffer B from 1 to 14 minutes before re-equilibration in Buffer A. The flow rate was 1 mumin. Purified forms of recombinant pPSA were confirmed by amino acid sequencing using an ABI Precise Model 492 sequencer.
PSA FORMS IN PCs SERAAND SEMINAL FLUID Human PCs sera (75 mL) was pooled from patient samples containing 50 to 100 ng PSA/mL, and contained approximately 63 ng/mL total PSA and 3 ng/mL free PSA. Solid ammonium sulfate was added to a final concentration of 2 M; then, the sample was dialyzed versus 2 M ammonium sulfate for 16 hours at 4°C. The serum was clarified by centrifugation, and the supernatant solution was dialyzed 3 X 1 hour against 2 L of 20 mM sodium phosphate, pH 7. The sample was then filtered through a 0.2~pm membrane filter and passed over a 0.5-mL PSM773 affinity column at 1 ml/min. The affinity column was washed with 50 mL phosphate-buffered saline (PBS), and the PSA was eluted with 100 mM glycine, 0.5 M sodium chloride, pH 2.5, containing 1 mg/mL bovine serum albumin. The eluant (3 mL) was neutralized with 300 PL of 1 M Tris, pH 8. Ammonium sulfate was added to the eluant to a final concentration of 2 M, and this sample was analyzed by HIC-HPLC, as described above. Fractions from HIC-HPLC were collected at 0.5-mL intervals and measured for free PSA using the Tandem-MP free PSA assay. Immunoaffinity-purified PSA from seminal fluid was obtained by passage of seminal fluid diluted 1:lO in PBS over a 0.5-mL PSM773 immunoaffinity column, as described above. Western blot analysis was performed after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 4% to 20% gradient gels under reducing conditions. The immunoreactive bands were detected by the ECL system (Amersham, Buckinghamshire, UK) according to the manufacturer’s instructions. Where designated, SuperSignal Ultra (Pierce Chem. Co., Rockford, Ill) was used for highly enhanced signal detection.
RESULTS WESTERN BLOT ANALYsrs OF PCs SERUM Whole serum from patients with PCs was subjected to SDS-PAGE and Western blot analysis to UROLOGY
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Clipped PSA
FIGURE 1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis Western blot analysis of PSA forms in prostate cancer (PCs) serum. Blots were probed with PF 102 15.1, which recognizes both intact and fragment forms of PSA. Lane 1, 1 -pL PCs serum, which contained 28 ng of total PSA. Lane 2, 1 PL (28 ng) of purified seminal fluid PSA in buffer. Lane 3, 1 )JL female serum spiked with 28 nglpL purified seminal fluid PSA. (r2MC = alpha2-macroglobulin; ACT = alpha- 1-antichymotrypsin. kDa = kilodaltons.
test for the presence of internally clipped forms of PSA. Figure 1 shows a Western blot probed with mAb PF lD2 15.1, which has been previously demonstrated to detect degradation products as well as intact PSA.16 Lane 2 shows the clipped forms of PSA present in seminal fluid. Aside from the primary band of intact PSA at approximately 34 kilodaltons (kDa), a series of lower PSA fragment bands are detected. By contrast, PCs sera (lane 1) contains no bands lower than the approximately 34-kDa band of intact, free PSA. The higher molecular mass bands in lane 1 correspond to PSA complexed to alpha-2-macroglobulin (approximately 200 and 115 kDa) and ACT (approximately 90 kDa). The positions of the complexed forms of PSA in sera are identical to purified PSA incubated with purified alpha-2-macroglobulin and ACT, respectively (data not shown). As a control, seminal fluid PSA was spiked into female sera and examined for the presence of clipped forms of PSA. The amount of PSA spiked into the female sera was 28 ng/pL, equivalent to the endogenous level of PSA present in the PCs sera. Lane 3 shows that fragment forms seen in 711
lane 2 were conserved after being spiked into serum. Four additional samples of PCs sera ranging from 2 to 8 ng PSA/pL were also analyzed by Western blot. Strong bands of complexed PSA and intact 34-kDa PSA were detected by PFlD215.1 in these samples using ECL detection. Faint bands consistent with chpped PSA became visible only after prolonged exposures while using the more sensitive Pierce Ultra detection (data not shown). These results indicate that clipped forms of PSA are minimal compared with intact free PSA in PCs serum with highly elevated total PSA. RESOLUTION OF PSA FORMS BY HIC-HPLC HIC-HPLC was used to resolve and identify different forms of PSA. Studies with pPSA expressed in AVlZ-PSA cells showed that the majority of the PSA was secreted into the spent media as the pro form. HIC-HPLC was able to resolve the pPSA into two different peaks that contain [ - 5, - 71 pPSA and [ -4]pPSA, as confirmed by amino acid sequencing. Recombinant, active PSA was prepared from pPSA by treatment with 1% trypsin for 15 min at 37°C as described for pro human kallikrein 2 (phK2).17 The purified pPSA forms were mixed with the mature PSA to establish the relative retention times of these different forms of PSA. Figure 2 (top) shows that PSA, [-4]pPSA, and [ -5,-7lpPSA are clearly resolved from each other. Clipped and unclipped mature seminal fluid PSA are eluted as a single peak identically with recombinant mature PSA (Fig. 2, bottom). PSAACT elutes earlier at 7 minutes (data not shown). DETECTIONOF pPSA INPC~ SERUM PCs serum was passed over a PSM773 immunoaffinity column to enrich the levels of PSA forms for further analysis by HIC-HPLC. Figure 2 (middle) shows column fractions eluted from HICHPLC as measured with the Tandem-MPfree PSA assay. The slight peak of activity at 7 minutes corresponds to the retention time of PSA-ACT (data not shown). This reflects a minimal crossreactivity of PSA-ACT in the free PSA assay. PSA-ACT was present at levels approximately 20 times higher than free PSA. The peak at 10 minutes corresponds to mature, uncomplexed PSA. The peak at 12 minutes corresponds to the retention time of the [ -41 form of pPSA. The [-41 form of the recombinant pPSA was tested further to verify that it is inactive. ACT was incubated with PSA, [--4]pPSA, and [-5,-7lpPSA. The mature PSA formed a PSAACT complex as determined by HIC-HPLC, whereas none of the precursor forms complexed with ACT (data not shown). 712
PCs Serum
Seminal
Fluid II
h--/,&-y-J 0
10
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FIGURE 2. Hydrophobic
interaction chromatographichigh performance liquid chromatographic (H/C-HPLC) resolution of PSA forms in prostate cancer (PCs) serum and seminal fluid. (Top panel) Profile of a mixture of PSA forms prepared with individually purified recombinant PSA, [-4lprecursor PSA, (pPSA), and (-5, - 7]pPSA, as detected by A280. Recombinant, active PSA was prepared from pPSA by treatment with 1% trypsin. (Middle panel) PSA forms in pooled PCs serum (63 nglmL total PSA) eluted from PSM773 immunoaffinity column. HICHPLC column fractions were analyzed by Tandem-MP free PSA assay. The maximum peak height is 12 nglmL. PSA activity at 12 minutes is consistent with [-4IpPSA. (Bottom panel) Seminal fluid PSA forms eluted from PSM773 immunoaffinity column as detected by A280. No precursor forms of PSA were detected.
The immunoaffinity-purified PSA forms in seminal fluid were compared with the free PSA profile found in PCs serum. The high levels of PSA in seminal fluid (greater than 1 mg/mL) were monitored by A280 (Fig. 2, bottom). No precursor forms were detected. The peak at 10 minutes, corresponding to mature PSA, was confirmed by amino acid sequencing. In addition, no precursor forms of PSA were detected when 1 /JL of whole seminal fluid was applied directly to HIC-HPLC and the fractions monitored by Tandem-MP free PSA (data not shown). These results show that precursor forms of PSA are not a measurable fraction of the free PSA pool in seminal fluid. UROLOGY 50 61, 1997
COMMENT This study shows that pPSA is a significant fraction of the free PSA in PCs sera, as determined from a pool containing 63 ng/mL total PSA. Approximately 25% was resolved by HIC-HPLC as pPSA, whereas the remainder was eluted as mature PSA. The presence of pPSA as a component of free PSA is a novel finding and potentially important as a marker for the enhancement of the free PSA assay. Additional studies are necessary to determine whether pPSA in individual patient samples correlates more closely with BPH or PCs than the entire pool of free PSA. The reasons for the presence of free PSA in serum that appears to elute as mature, inactive PSA are not clear. Western blot analysis showed that clipped PSA, as found in seminal fluid, was negligible in PCs serum. Other studies have failed to detect clipped PSA in PCs serum.l* It is possible that this inactive form is similar to a component of mature PSA found in seminal fluid that is not clipped but does not complex with ACT.19 The present study cannot rule out the possibility that clipped forms of PSA may be significant in BPH samples or in PCs serum when the PSA level is much lower than was tested in our study. The finding of pPSA is consistent with studies in which we showed that human kallikrein 2 (hK2), a homologous kallikrein in the prostate, is found as the pro form in LNCaP cells and in PCs serum.2o hK2 has been shown to activate both phK2l’ and pPSAi5 in vitro, although a physiologic relation has yet to be established. Immunohistochemical studies of hK2 in prostate tissues have shown that hK2 may add important new information to prostate cancer diagnosis.21 hK2 is highly expressed in cancer epithelium compared with BPH or normal prostatic tissue.22 The measurement of the precursor and mature forms of hK2 and PSA together may also lead to improved cancer diagnosis. Although HIC-HPLC allowed resolution of pPSA from PSA in pooled serum of patients with advanced disease, detection at levels commensurate with more limited disease will be possible only through the use of immunoassays for pPSA. The measurement of pPSA levels in individual patient samples must necessarily wait for sensitive, pPSAspecific mAbs that are currently under development. CONCLUSIONS pPSA and unclipped PSA constitute the predominant forms of free PSA in PCs serum. REFERENCES 1. Brawer MK, Chetner MP, Beatie J, Buchner DM, Vessella RL, and Lange PH: Screening for prostate carcinoma with prostate specific antigen. J Urol 147(3 Pt 2): 841-845, 1992. UROLOGY
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2. Catalona WJ. Smith DS, Ratliff TL, Dodds KM, Coplen DE, Yuan JJ, Tetros JA, and Andriole GL: Measurement of prostate-specific antigen in serum as a screening test for prostate cancer. N Engl J Med 324: 1156-1161, 1991. 3. Labrie F, DuPont A, Suburu R, Cusan L, Tremblay M, Gomez J-L, and Emond J: Serum prostate specific antigen as pre-screening test for prostate cancer. J Urol 147 (3 Pt 2): 846-852, 1992. 4. Watt KWK, Lee P-J, M’Timkulu T, Chan W-P, and Loor R: Human prostate-specific antigen: structural and functional similarity with serine proteases. Proc Nat1 Acad Sci USA 83: 3166-3170, 1986. 5. Bridon DP, and Dowel1 BL: Structural comparison of prostate-specific antigen and human glandular kallikrein using molecular modeling. Urology 45: 801-806, 1995. 6. Villoutreix BO, Getzoff ED, and Griffin JH: A structural model for the prostate disease marker, human prostate-specific antigen. Protein Sci 3: 2033-2044, 1994. 7. Christensson A, Laurel1 C-B, and Lilja H: Enzymatic activity of prostate-specific antigen and its reactions with extracellular serine proteinase inhibitors. Eur J Biochem 194: 755-763, 1990. 8. Lilja H, Christensson A, Dahlen U, Matikainen M-T, Nilsson 0, Pettersson K, and Lovgren T: Prostate-specific antigen in serum occurs predominantly in complex with alphal-antichymotrypsin. Clin Chem 37: 1618-1625, 1991. 9. Stenman UH, Leinonen J, Alfthan H, Rannikko S, Tuhkanen K, and Alfthan 0: A complex between prostate specific antigen and alpha-l-antichymotrypsin is the major form of prostate-specific antigen in serum of patients with prostatic cancer: assay of the complex improves clinical sensitivity for cancer. Cancer Res 51: 222-226, 1991. 10. Christensson A, Bjork T, Nilsson 0, Dahlen U, Matikainen T, Cockett A, Abrahamsson P-A, and Lilja H: Serum prostate specific antigen complexed to alpha-l-antichymotrypsin as an indicator of prostate cancer. J Urol 150: lOO105,1993. 11. Catalona WJ: Clinical utility of measurements of free and total prostate-specific antigen (PSA): a review. Prostate Suppl 7: 64-69, 1996. 12. van Iersel MP, Witjes WPJ, Thomas CMG, Segers MFG, Oosterhoff GON, and Debruyne FMJ: Review on the simultaneous determination of total prostate-specific antigen and free prostate-specific antigen. Prostate 7(suppl): 48-57, 1996. 13. Chen Z, Chen H, and Stamey TA: Prostate specific antigen in benign prostatic hyperplasia: purification and characterization. J Urol 157: 2166-2170, 1997. 14. Kumar A, Goel A, Hill T, Mikolajczyk S, Millar L, Kuus-Reichel K, and Saedi M: Expression of human glandular kallikrein, hK2, in mammalian cells. Cancer Res 56: 53975402, 1996. 15. Kumar A, Mikolajczyk SD, Goel AS, Millar LS, and Saedi MS: Expression of pro form of prostate-specific antigen by mammalian cells and its conversion to mature, active form by human kallikrein 2. Cancer Res 57: 3111-3114, 1997. 16. Wang TJ, Linton HJ, Sokoloff RL, Grauer LS, Rittenhouse HG, and Wolfert RL: Antibody specificities for PSA and PSA fragments by SDS-PAGE Western blot analysis. Tumor Biol (in press). 17. Mikolajczyk SD, Millar LS, Marker KM, Grauer LS, Goel AS, Cass MMJ, Kumar A, and Saedi MS: Ala217 is important for the catalytic function and autoactivation of prostate-specific human kallikrein 2. Eur J Biochem 246: 440446, 1997. 18. Qian Y, Sensibar JA, Zelner DJ, Schaeffer AJ, Finlay JA, Rittenhouse HG, and Lee C: Two-dimensional gel electrophoresis detects prostate-specific antigen-alpha-l-antichymotrypsin complex in serum but not in prostatic fluid. Clin Chem 43: 352-359, 1997.
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19. Zhang W-M, Leinonen J, Kalkkinen N, Dowel1 B, and Stenman U-H: Purification and characterization of different molecular forms of prostate-specific antigen in human seminal fluid. Clin Chem 41: 1567-1573, 1995. 20. Saedi MS, Hill TM, Goel AS, and Kumar A: Assessment of the precursor form of human glandular kallikrein (pro hK2) as marker for prostate cancer (abstract). J Urol 157(suppl): 442, 1997. 21. Tremblay RR, Deperthes D, Tetu B, and Dube JY: Im-
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munohistochemical study suggesting a complementary role of kallikreins hK2 and hK3 (PSA) in the functional analysis of human prostate tumors. Am J Path01 15: 455-459, 1997. 22. Darson MF, Parcelli A, Roche P, Rittenhouse HG, Wolfert RL, Young CYF, Klee GG, Tindall DJ, and Bostwick DG: Human glandular kallikrein 2 (hK2) expression in prostatic intraepithelial neoplasia and adenocarcinoma: a novel prostate cancer marker. Urology 49: 857862, 1997.
UROLOGY
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1997
A PRECURSOR FORM OF PSA (pPSA) IS A COMPONENT OF THE FREE PSA IN PROSTATE CANCER SERUM STEPHEN D. MIKOLAJCZYK, LANA S. GRAUER, LISA S. MILLAR, TIMOTHY M. HILL, ABHAY KUMAR, HARRY G. RITTENHOUSE, ROBERT L. WOLFERT, AND MOHAMMAD S. SAEDI
ABSTRACT Objectives. Prostate-specific antigen (PSA) is a widely used serum marker for human prostate cancer (PCs). The majority of PSA in serum is present as a complex with alpha-l -antichymotrypsin (ACT). In recent years, the ratio of free (uncomplexed) to total PSA has shown improved discrimination of PCs from benign prostatic hyperplasia. This study examines the nature of the free PSA form detected in PCs serum and shows that some of the uncomplexed PSA is an inactive precursor of PSA (pPSA). Methods. Western blot analysis was used to detect clipped, fragment forms of PSA in sera and seminal fluid. Hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC) was used to identify forms of PSA present in the free PSA population. Pooled sera was passed over a PSA immunoaffinity column, and the eluted PSA components were further resolved by HIC-HPLC. Results. Western blot analysis of whole sera showed complexed PSA and the intact, approximately 34 kilodalton free PSA. Only negligible levels of clipped or degraded forms of PSA, as found in seminal fluid, were detected. Column fractions measured for uncomplexed PSA using the Tandem-MP free PSA assay showed that about 25% of the free PSA eluted as pPSA beginning at the [-4]amino acid. Studies with purified recombinant [-4]pPSA showed that this proenzyme form is inactive and does not complex with ACT. Conclusions. These results suggest that the uncomplexed PSA in PCs serum is primarily unclipped PSA that contains a significant fraction of pPSA. UKOLOGY 50: 710-714, 1997. 0 1997, Elsevier Science Inc. All rights reserved.
M
easurement of prostate-specific antigen (PSA) in serum has been widely used as a diagnostic marker for human prostate cancer (PCa).lp3 PSA is a serine protease,4-6 and in serum it forms complexes with endogenous protease inhibitors, primarily alpha-2-macroglobulin and alpha-I-antichymotrypsin (ACT).7X8 Typically, 80% to 95% of the immunoreactive PSA in PCs sera is detected in complex with ACT, the remainder as uncomplexed, or free, PSA. In recent years the measurement of both free and complexed PSA in serum has become important in better distinguishing PCs from benign prostatic hyperplasia (BPH).9-‘2 An increased ratio of free to total (free plus complexed) PSA is more highly correlated with BPH than total PSA alone. Reasons for the persistence of uncomplexed PSA in the presence From Hybritech Incorporuted, San Diego, California Reprint requests: Stephen D. Mikolajczyk, P.O. Box 269006, San Diego, CA 92 196-9006 Submitted (Rapid Communication): June 24, 1997, accepted (with revisions): July 1.5, 1997 0
7 10
1997,
ALL RIGHTS
ELSEVIER SCIENCE RESERVED
INC.
of a large excess of serum protease inhibitors is the subject of speculation. Because inhibitor complex formation requires proteolytic activity, it is likely that free PSA is enzymatically inactive. It is known that approximately 30% of the PSA in seminal fluid is inactive because of internal peptide bond cleavages and that this clipped form does not complex with ACT.7 Recent studies have shown that BPH nodules in the prostate contain elevated levels of clipped PSA compared to seminal fluid PSA, which could account for the elevated ratio of free PSA in the serum of patients with BPH.13 It has been assumed that such clipped forms would also predominate in PCs serum. Another possibility for uncomplexed, inactive PSA is the presence of zymogen, or precursor PSA (pPSA). In the current study we examined pooled PCs serum for precursor forms of PSA using hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC) to resolve the free PSA fraction into its component parts. We found that pPSA is a significant subpopulation of the free PSA in serum. This finding has 0090-4295/97/$17.00 PII soogo-4295(97)00449-4
potentially important implications for the detection of PCs because it identifies a novel form of PSA that may enhance the discrimination of PCs from BPH. MATERIAL
AND
METHODS
RESOLUTION OF PSA FORMS BY HIC-HPLC The PSA gene was expressed in a hamster cell line, as described for human kallikrein 2.t4 PSA is secreted as its pro form from this cell line (AVlZ-PSA).r5 The pPSA expressed by the AVlZ-PSA cells was purified from the spent media by immunoaffinity chromatography. The column consisted of the PSA-specific monoclonal antibody (mAb) PSM773 covalently bound to Aminolink resin (Pierce) at a concentration of 5 mg mAb/mL resin. PSA was eluted with 100 mM glycine, 0.5 M sodium chloride, pH 2.6. The different forms of immunoreactive PSA (PSA-ACT, PSA, proPSA forms) were resolved from each other by HPLC using HIC (PolyLC, polypropyl aspartamide column, 100 nm pore size, 4.6 mm X 200 mm, distributed by Western Analytical, Temecula, Calif). Buffer A was 20 mM sodium phosphate, 1.2 M sodium sulfate pH 6.3, and buffer B was 50 mM sodium phosphate, 5% 2propanol, pH 7.4. The elution gradient was 0% to 35% buffer B from 0 to 1 minute and 35% to 80% Buffer B from 1 to 14 minutes before re-equilibration in Buffer A. The flow rate was 1 mumin. Purified forms of recombinant pPSA were confirmed by amino acid sequencing using an ABI Precise Model 492 sequencer.
PSA FORMS IN PCs SERAAND SEMINAL FLUID Human PCs sera (75 mL) was pooled from patient samples containing 50 to 100 ng PSA/mL, and contained approximately 63 ng/mL total PSA and 3 ng/mL free PSA. Solid ammonium sulfate was added to a final concentration of 2 M; then, the sample was dialyzed versus 2 M ammonium sulfate for 16 hours at 4°C. The serum was clarified by centrifugation, and the supernatant solution was dialyzed 3 X 1 hour against 2 L of 20 mM sodium phosphate, pH 7. The sample was then filtered through a 0.2~pm membrane filter and passed over a 0.5-mL PSM773 affinity column at 1 ml/min. The affinity column was washed with 50 mL phosphate-buffered saline (PBS), and the PSA was eluted with 100 mM glycine, 0.5 M sodium chloride, pH 2.5, containing 1 mg/mL bovine serum albumin. The eluant (3 mL) was neutralized with 300 PL of 1 M Tris, pH 8. Ammonium sulfate was added to the eluant to a final concentration of 2 M, and this sample was analyzed by HIC-HPLC, as described above. Fractions from HIC-HPLC were collected at 0.5-mL intervals and measured for free PSA using the Tandem-MP free PSA assay. Immunoaffinity-purified PSA from seminal fluid was obtained by passage of seminal fluid diluted 1:lO in PBS over a 0.5-mL PSM773 immunoaffinity column, as described above. Western blot analysis was performed after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 4% to 20% gradient gels under reducing conditions. The immunoreactive bands were detected by the ECL system (Amersham, Buckinghamshire, UK) according to the manufacturer’s instructions. Where designated, SuperSignal Ultra (Pierce Chem. Co., Rockford, Ill) was used for highly enhanced signal detection.
RESULTS WESTERN BLOT ANALYsrs OF PCs SERUM Whole serum from patients with PCs was subjected to SDS-PAGE and Western blot analysis to UROLOGY
50
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30-
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20+
Clipped PSA
FIGURE 1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis Western blot analysis of PSA forms in prostate cancer (PCs) serum. Blots were probed with PF 102 15.1, which recognizes both intact and fragment forms of PSA. Lane 1, 1 -pL PCs serum, which contained 28 ng of total PSA. Lane 2, 1 PL (28 ng) of purified seminal fluid PSA in buffer. Lane 3, 1 )JL female serum spiked with 28 nglpL purified seminal fluid PSA. (r2MC = alpha2-macroglobulin; ACT = alpha- 1-antichymotrypsin. kDa = kilodaltons.
test for the presence of internally clipped forms of PSA. Figure 1 shows a Western blot probed with mAb PF lD2 15.1, which has been previously demonstrated to detect degradation products as well as intact PSA.16 Lane 2 shows the clipped forms of PSA present in seminal fluid. Aside from the primary band of intact PSA at approximately 34 kilodaltons (kDa), a series of lower PSA fragment bands are detected. By contrast, PCs sera (lane 1) contains no bands lower than the approximately 34-kDa band of intact, free PSA. The higher molecular mass bands in lane 1 correspond to PSA complexed to alpha-2-macroglobulin (approximately 200 and 115 kDa) and ACT (approximately 90 kDa). The positions of the complexed forms of PSA in sera are identical to purified PSA incubated with purified alpha-2-macroglobulin and ACT, respectively (data not shown). As a control, seminal fluid PSA was spiked into female sera and examined for the presence of clipped forms of PSA. The amount of PSA spiked into the female sera was 28 ng/pL, equivalent to the endogenous level of PSA present in the PCs sera. Lane 3 shows that fragment forms seen in 711
lane 2 were conserved after being spiked into serum. Four additional samples of PCs sera ranging from 2 to 8 ng PSA/pL were also analyzed by Western blot. Strong bands of complexed PSA and intact 34-kDa PSA were detected by PFlD215.1 in these samples using ECL detection. Faint bands consistent with chpped PSA became visible only after prolonged exposures while using the more sensitive Pierce Ultra detection (data not shown). These results indicate that clipped forms of PSA are minimal compared with intact free PSA in PCs serum with highly elevated total PSA. RESOLUTION OF PSA FORMS BY HIC-HPLC HIC-HPLC was used to resolve and identify different forms of PSA. Studies with pPSA expressed in AVlZ-PSA cells showed that the majority of the PSA was secreted into the spent media as the pro form. HIC-HPLC was able to resolve the pPSA into two different peaks that contain [ - 5, - 71 pPSA and [ -4]pPSA, as confirmed by amino acid sequencing. Recombinant, active PSA was prepared from pPSA by treatment with 1% trypsin for 15 min at 37°C as described for pro human kallikrein 2 (phK2).17 The purified pPSA forms were mixed with the mature PSA to establish the relative retention times of these different forms of PSA. Figure 2 (top) shows that PSA, [-4]pPSA, and [ -5,-7lpPSA are clearly resolved from each other. Clipped and unclipped mature seminal fluid PSA are eluted as a single peak identically with recombinant mature PSA (Fig. 2, bottom). PSAACT elutes earlier at 7 minutes (data not shown). DETECTIONOF pPSA INPC~ SERUM PCs serum was passed over a PSM773 immunoaffinity column to enrich the levels of PSA forms for further analysis by HIC-HPLC. Figure 2 (middle) shows column fractions eluted from HICHPLC as measured with the Tandem-MPfree PSA assay. The slight peak of activity at 7 minutes corresponds to the retention time of PSA-ACT (data not shown). This reflects a minimal crossreactivity of PSA-ACT in the free PSA assay. PSA-ACT was present at levels approximately 20 times higher than free PSA. The peak at 10 minutes corresponds to mature, uncomplexed PSA. The peak at 12 minutes corresponds to the retention time of the [ -41 form of pPSA. The [-41 form of the recombinant pPSA was tested further to verify that it is inactive. ACT was incubated with PSA, [--4]pPSA, and [-5,-7lpPSA. The mature PSA formed a PSAACT complex as determined by HIC-HPLC, whereas none of the precursor forms complexed with ACT (data not shown). 712
PCs Serum
Seminal
Fluid II
h--/,&-y-J 0
10
5
Retention
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20
Time (min)
FIGURE 2. Hydrophobic
interaction chromatographichigh performance liquid chromatographic (H/C-HPLC) resolution of PSA forms in prostate cancer (PCs) serum and seminal fluid. (Top panel) Profile of a mixture of PSA forms prepared with individually purified recombinant PSA, [-4lprecursor PSA, (pPSA), and (-5, - 7]pPSA, as detected by A280. Recombinant, active PSA was prepared from pPSA by treatment with 1% trypsin. (Middle panel) PSA forms in pooled PCs serum (63 nglmL total PSA) eluted from PSM773 immunoaffinity column. HICHPLC column fractions were analyzed by Tandem-MP free PSA assay. The maximum peak height is 12 nglmL. PSA activity at 12 minutes is consistent with [-4IpPSA. (Bottom panel) Seminal fluid PSA forms eluted from PSM773 immunoaffinity column as detected by A280. No precursor forms of PSA were detected.
The immunoaffinity-purified PSA forms in seminal fluid were compared with the free PSA profile found in PCs serum. The high levels of PSA in seminal fluid (greater than 1 mg/mL) were monitored by A280 (Fig. 2, bottom). No precursor forms were detected. The peak at 10 minutes, corresponding to mature PSA, was confirmed by amino acid sequencing. In addition, no precursor forms of PSA were detected when 1 /JL of whole seminal fluid was applied directly to HIC-HPLC and the fractions monitored by Tandem-MP free PSA (data not shown). These results show that precursor forms of PSA are not a measurable fraction of the free PSA pool in seminal fluid. UROLOGY 50 61, 1997
COMMENT This study shows that pPSA is a significant fraction of the free PSA in PCs sera, as determined from a pool containing 63 ng/mL total PSA. Approximately 25% was resolved by HIC-HPLC as pPSA, whereas the remainder was eluted as mature PSA. The presence of pPSA as a component of free PSA is a novel finding and potentially important as a marker for the enhancement of the free PSA assay. Additional studies are necessary to determine whether pPSA in individual patient samples correlates more closely with BPH or PCs than the entire pool of free PSA. The reasons for the presence of free PSA in serum that appears to elute as mature, inactive PSA are not clear. Western blot analysis showed that clipped PSA, as found in seminal fluid, was negligible in PCs serum. Other studies have failed to detect clipped PSA in PCs serum.l* It is possible that this inactive form is similar to a component of mature PSA found in seminal fluid that is not clipped but does not complex with ACT.19 The present study cannot rule out the possibility that clipped forms of PSA may be significant in BPH samples or in PCs serum when the PSA level is much lower than was tested in our study. The finding of pPSA is consistent with studies in which we showed that human kallikrein 2 (hK2), a homologous kallikrein in the prostate, is found as the pro form in LNCaP cells and in PCs serum.2o hK2 has been shown to activate both phK2l’ and pPSAi5 in vitro, although a physiologic relation has yet to be established. Immunohistochemical studies of hK2 in prostate tissues have shown that hK2 may add important new information to prostate cancer diagnosis.21 hK2 is highly expressed in cancer epithelium compared with BPH or normal prostatic tissue.22 The measurement of the precursor and mature forms of hK2 and PSA together may also lead to improved cancer diagnosis. Although HIC-HPLC allowed resolution of pPSA from PSA in pooled serum of patients with advanced disease, detection at levels commensurate with more limited disease will be possible only through the use of immunoassays for pPSA. The measurement of pPSA levels in individual patient samples must necessarily wait for sensitive, pPSAspecific mAbs that are currently under development. CONCLUSIONS pPSA and unclipped PSA constitute the predominant forms of free PSA in PCs serum. REFERENCES 1. Brawer MK, Chetner MP, Beatie J, Buchner DM, Vessella RL, and Lange PH: Screening for prostate carcinoma with prostate specific antigen. J Urol 147(3 Pt 2): 841-845, 1992. UROLOGY
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19. Zhang W-M, Leinonen J, Kalkkinen N, Dowel1 B, and Stenman U-H: Purification and characterization of different molecular forms of prostate-specific antigen in human seminal fluid. Clin Chem 41: 1567-1573, 1995. 20. Saedi MS, Hill TM, Goel AS, and Kumar A: Assessment of the precursor form of human glandular kallikrein (pro hK2) as marker for prostate cancer (abstract). J Urol 157(suppl): 442, 1997. 21. Tremblay RR, Deperthes D, Tetu B, and Dube JY: Im-
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munohistochemical study suggesting a complementary role of kallikreins hK2 and hK3 (PSA) in the functional analysis of human prostate tumors. Am J Path01 15: 455-459, 1997. 22. Darson MF, Parcelli A, Roche P, Rittenhouse HG, Wolfert RL, Young CYF, Klee GG, Tindall DJ, and Bostwick DG: Human glandular kallikrein 2 (hK2) expression in prostatic intraepithelial neoplasia and adenocarcinoma: a novel prostate cancer marker. Urology 49: 857862, 1997.
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