The precursor form of the human kallikrein 2, a kallikrein homologous to prostate-specific antigen, is present in human sera and is increased in prostate cancer and benign prostatic hyperplasia

^a Address correspondence to this author at: Hybritech Incorporated, a subsidiary of Beckman Coulter, Inc., P.O. Box 269006, San Diego, CA 92196. Fax 619-621-4610; e-mail mssaedi{at}beckman.com.

	Abstract

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Prostate-specific antigen (PSA, hK3) is a diagnostic marker for prostatic cancer but lacks the specificity to sufficiently distinguish between prostatic cancer and benign prostatic hyperplasia (BPH). Human glandular kallikrein 2 (hK2) has been proposed as a potential diagnostic marker for prostate cancer that could complement the current PSA test. Recently we demonstrated that proPSA is present in prostate cancer sera. This study examines the expression of prohK2 in prostate cells and its presence in human sera. Western blot analysis was used to assess prohK2 expression in the human carcinoma cell line, LNCaP. A highly specific and sensitive dual monoclonal immunoassay for prohK2 was developed and used to assess the presence of prohK2 in human sera. prohK2 was detected in the spent media of LNCaP cells. Furthermore, prohK2 was present at immunodetectable concentrations in human sera, and its concentration was increased in prostatic cancer and BPH. These results indicate for the first time that prohK2 is secreted by human prostate cells and is a major component of uncomplexed (free) hK2 in human sera. In addition, prohK2 in human sera is associated with prostate disease and thus may be a useful marker for prostatic cancer and BPH.

	Introduction

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Early diagnosis and staging of prostatic cancer is of utmost importance because it is the most frequently diagnosed cancer in American men. The estimate for 1997 indicates that prostatic cancer would account for 42% of all new cancers detected in American men (1). Serum concentrations of prostate-specific antigen (PSA)¹ have been recognized as a reliable marker for prostate disease (2)(3). The current PSA tests, however, do not clearly distinguish between prostatic cancer and benign prostatic hyperplasia (BPH), specifically when the serum PSA concentration is between 4 and 10 µg/L (4).

PSA belongs to the family of human glandular kallikreins, a multigene family of serine proteases (5). Other members of this family include human glandular kallikrein 2 (hK2) and pancreatic/renal glandular kallikrein (hK1). hK2 shares many similarities with PSA and thus has been recognized as a potential diagnostic marker to enhance or complement the sensitivity of the PSA test and to help distinguish between prostatic cancer and BPH [reviewed in Ref. (6)]. Recently, we cloned and expressed hK2 in both bacteria and mammalian cells (7). The recombinant protein was purified to homogeneity and shown to have trypsin-like protease activity (8). The purified protein was used to generate hK2-specific monoclonal antibodies (mAbs), which in turn were used to identify hK2 in LNCaP cells (9) and in serum (10) as well as to show that hK2 expression is prostate-localized (11). Other groups have also identified hK2 in human serum and seminal fluid (12)(13).

PSA exists in serum in different forms (5). Typically ~90% of the immunoreactive PSA in prostatic cancer serum is detected in complex with ₁-antichymotrypsin (ACT), and the remainder is detected as uncomplexed or "free" PSA (14). Recent studies indicate that an increased ratio of free (uncomplexed) to total PSA is more highly correlated with BPH than prostatic cancer (15). The inactive zymogen form of PSA, proPSA, has now been identified as a major fraction of free PSA in prostatic cancer sera (16). Little is known about the molecular forms of hK2 in serum. Western blot analysis indicates that hK2 in serum is also present as the free (uncomplexed) form and in complex with ACT (10). Results from our recombinant mammalian expression system indicate that hK2 is expressed as its zymogen, prohK2, from recombinant mammalian cells (7). This observation suggested that prohK2 could be expressed by prostate cells and, thus, may be present in human sera.

In this study, we report the development of a prohK2-specific immunoassay and show for the first time that prohK2 is expressed by the human prostatic carcinoma cell line, LNCaP. We also demonstrate that analogous to proPSA and free PSA, prohK2 is a major component of free hK2 in human sera and is increased in prostatic cancer and BPH. This observation indicates that prohK2 may be a potentially useful diagnostic marker for prostatic cancer or BPH.

	Materials and Methods

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preparation of prohK2^A217V, hK2, proPSA, and PSA
prohK2^A217V and hK2 were prepared from the spent media of recombinant cell lines using an HK1G586.1 (which detects both prohK2 and mature hK2) immunoaffinity column as described previously (7). The concentration of purified prohK2^A217V and hK2 were determined on the basis of absorbance at 280 nm (17). proPSA was purified from serum-free spent media of recombinant cells, using a PSM773.3 immunoaffinity column (18). proPSA was converted to mature PSA by mild trypsin treatment followed by HPLC purification (18). The concentrations of proPSA and PSA were determined using the Tandem^®-MP PSA immunoassay (Hybritech Incorporated). This immunoassay detects both proPSA and PSA.

propagation of LNCaP cells and western blot analysis
The prostate carcinoma cell line, LNCaP.FGC (ATCC CRL 1740), was cultured in RPMI supplemented with 100 mL/L fetal clone (Hyclone). Cells were grown to ~80–90% confluency, washed with Hanks' basic salt solution, and incubated in serum-free HH4 medium containing 10 nmol/L mibolerone (Amersham). The presence of hK2 in the spent medium was assessed by Western blots using an enhanced chemiluminescence kit (Amersham).

immunoassay
HK1G464.3 (4 mg/L) and HK1G268.1 (2 mg/L) were paired as capture and detect mAbs, respectively. HK1G464.3 detects only prohK2, and HK1G268.1 detects both prohK2 and mature hK2. HK1G464.3 was biotinylated with NHS-LC-Biotin (Pierce), and HK1G268.1 was labeled with europium (Eu³) using a Delfia Eu-labeling kit (Wallac). Streptavidin-coated microplates (Wallac) were incubated with 50 µL of biotinylated HK1G464.3 and 50 µL of calibrator or specimen in duplicate for 3 h at room temperature while rotating at 800 rpm. The wells were washed with phosphate-buffered saline containing 1 mL/L Tween-20 (phosphate-buffered saline-Tween) and incubated with 100 µL of Eu³-labeled HK1G268.1 for 1 h at room temperature. The wells were again washed with phosphate-buffered saline-Tween and then incubated with 120 µL of Delfia enhancement solution. The results were obtained on a time-resolved fluorometer (Wallac) as relative light units. To reduce nonspecific binding by human anti-mouse antibodies, PolyMaK-33 (200 mg/L; Boehringer Mannheim) and mouse immunoglobulin (100 mg/L) were added to the assay diluent. The biological detection limit (BDL) was determined as described by Ellis et al. (19) by measuring the recovery of prohK2 from a pool of patient sera (with known concentration of prohK2) serially diluted in postradical prostatectomy (PRP) sera. PRP serum was used as the diluent because it does not contain hK2 or prohK2. The BDL was defined as the lowest limit of prohK2 detection in patient sera in which 95% confidence remains above the hK2 value in the zero diluent (PRP sera) upon repetitive analysis. Tandem-MP PSA and Tandem-MP free PSA assays (both from Hybritech Incorporated) were used to measure total and free PSA, respectively, in human sera.

	Results

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LNCaP, a metastatic prostate carcinoma cell line that expresses both hK2 and PSA (9), was utilized to assess the presence of prohK2 in a human cell line. The pro region of hK2 is seven amino acids, making it very difficult to distinguish between prohK2 and mature hK2 on the basis of their migration patterns on sodium dodecyl sulfate gels. Thus, we used a mAb specific to prohK2 (HK1G464.3) and a mAb that detects both prohK2 and mature hK2 (HK1G586.1) to distinguish between these two forms of hK2. Spent medium from mibolerone-induced LNCaP cells was collected after 2 days and evaluated for hK2 content by Western blot analysis using HK1G586.1 and HK1G464.3 mAbs (Fig. 1 ). These mAbs do not detect PSA (20), enabling us to specifically monitor hK2 expression. Immunostaining with HK1G464.3 shows that prohK2 (~34 kDa) is present in the spent media of LNCaP cells. Furthermore, the amount of prohK2 is similar to the sum of prohK2 and mature hK2 detected by HK1G586.1, indicating that hK2 is secreted by LNCaP cells as prohK2. Western blot analysis using HK1G586.1 did not show any prohK2 or mature hK2 in the cell lysates of mibolerone-induced LNCaP cells collected from day 1 to day 7, indicating that prohK2 is secreted by the cells and not released as a result of cell lysis (data not shown). An ~22-kDa degradation product resulting from cleavage at Arg 145 was detected in purified hK2.

View larger version (19K): [in this window] [in a new window]   Figure 1. Western blot analysis of LNCaP serum-free spent media. Spent media from LNCaP cells collected on day 2 was concentrated and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 4–20% gel. Proteins were electroblotted and probed with either HK1G586.1 (10 mg/L) or HK1G464.3 (20 mg/L). A goat anti-mouse Fc-horseradish peroxidase conjugate diluted 1:1000 (Jackson ImmunoResearch Laboratories, Inc.) was used as the secondary mAb. Controls included hK2A217V (75 ng) and prohK2A217V (75 ng). kD, kilodalton; phK2, prohK2.

To develop a more sensitive and quantitative assay than Western blot, a monoclonal sandwich assay specific for prohK2 was developed. Purified prohK2^A217V was used to calibrate the assay because it is stable to purification and as a calibrator in solution (17). Moreover, HK1G464.3 immunoreactivity to either prohK2 or prohK2^A217V is equivalent (17). The dose–response range of this assay was established at 0–20 µg/L (Fig. 2 , inset). The average coefficients of variation for between-run imprecision (n = 7) were 8.11% and 4.48% for serum pools with mean prohK2 values of 2.4 and 8.8 µg/L, respectively. The average coefficient of variation for within-run imprecision (n = 8) was 4.0% for a serum pool with a mean prohK2 value of 2.3 µg/L. The specificity of the prohK2 assay was evaluated using purified proteins and displayed <0.1% cross-reactivity to hK2, proPSA, and PSA, respectively (Fig. 2 ). The dose–response curve was linear, with a correlation coefficient of 0.998 (Fig. 2 , inset). The analytical limit of detection of the prohK2 assay was <0.02 µg/L, which corresponds to the concentration of prohK2 that is two standard deviations greater than the mean of 24 replicate determinations of the zero calibrator. The BDL at 95% confidence was established at <0.04 µg/L. This experiment also showed that the assay was linear, with a correlation coefficient of 0.998 (data not shown). Thus, the immunoassay is highly sensitive and specific to prohK2 with negligible cross-reactivity to hK2, proPSA, or PSA.

View larger version (14K): [in this window] [in a new window]   Figure 2. prohK2 assay does not cross-react with proPSA, PSA, or hK2. Cross-reactivity was determined by challenging the prohK2 assay with varying concentrations of purified hK2 (0–1000 µg/L), proPSA (0–7000 µg/L), and PSA (0–7000 µg/L) and comparing the slope for each respective best fit line to that of the prohK2 (0–20 µg/L) calibration curve. (), prohK2; (), proPSA; (), PSA; (), hK2. RLU, relative light units. For clarification, the prohK2 calibration curve is also represented on the 0–20 µg/L prohK2 (phK2) scale (inset).

Because Western blot data indicated that hK2 is secreted as prohK2 from a human prostate cell line, we postulated that prohK2 may be present in biological fluids. To assess the presence of prohK2 in human sera, a panel of healthy male, healthy female, PRP, diagnosed prostatic cancer, and BPH serum samples were tested for prohK2. Fig. 3 shows a scatter plot of the prohK2 values, and Table 1 displays the range and the mean for each of the populations. The mean values for healthy male, healthy female, and PRP samples were less than the BDL (0.04 µg/L). However, the mean values for the prostatic cancer (0.21 µg/L) and BPH (0.09 µg/L) populations were greater than the BDL, with the prostatic cancer population having the highest mean. For reference, the mean PSA and free PSA values are also displayed in Table 1 . These results indicate that prohK2 is present in human sera and is increased in prostate disease. We have also detected prohK2 in human sera by Western blot analysis (21).

View larger version (9K): [in this window] [in a new window]   Figure 3. Detection of prohK2 in human sera. phK2, prohK2; NF, healthy female; NM, healthy male; PCa, prostatic cancer.

	Discussion

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Understanding the biosynthetic processing of the prostate kallikreins PSA and hK2 will enhance our understanding of both their physiological roles and their utility as diagnostic markers. Evidence presented here illustrates for the first time that prohK2 is secreted by the human prostate carcinoma cell line, LNCaP. prohK2 was detected in spent media as assessed by Western blot analysis, whereas neither prohK2 nor mature hK2 was detected inside the cells, indicating that prohK2 is rapidly secreted from the cells and not released as a result of cell lysis. Western blot analysis (Fig. 1 ), confirmed by immunoassay (data not shown), showed that the concentration of mature hK2 prohK2 was the same as the concentration of prohK2 in day 1 spent media, indicating that most of the hK2 was in the prohK2 form. These results are analogous to our previous observations with recombinant cells, which indicated that hK2 and PSA are expressed as their respective zymogen forms and are converted to their mature, enzymatically active forms extracellularly (7)(18).

PSA is currently the most valuable serum marker for prostatic cancer. Increased concentrations of PSA in serum are associated with BPH or prostatic cancer; however, current PSA tests lack the desired specificity to distinguish between BPH and prostatic cancer in the critical PSA range of 4–10 µg/L (4). PSA is present in many forms in human sera [reviewed in Ref. (5)]. The majority of PSA is bound to either ₂-macroglobulin or ACT. Most of the immunodetectable form of PSA is present as a complex with ACT, and the remainder is present as the enzymatically inactive and uncomplexed form, free PSA. Recently we reported that ~25% of the free PSA fraction in serum is composed of proPSA (16). One way to enhance the specificity of the PSA test has been to measure different forms of PSA in serum. Measurement of the free-to-total PSA ratio has enhanced the specificity of the PSA test (15). However, further improvements are still needed to more accurately diagnose prostatic cancer and to evaluate its aggressiveness. We have previously demonstrated that hK2 is present in human serum and that it complexes with ACT and ₂-macroglobulin (8). Here we show that a fraction of the free hK2 in serum is composed of prohK2. Furthermore, we have developed a highly specific and sensitive immunological assay to measure prohK2 in serum and demonstrated that this assay does not cross-react with PSA, proPSA, and mature hK2, providing an accurate measurement of prohK2 concentrations in serum. Evidence presented here demonstrates for the first time that the serum concentration of prohK2 is increased in prostate disease. We have also observed in immunohistochemical studies, using the HK1G464.3 mAb, that the concentration of prohK2 is increased in prostatic cancer tissue as compared with BPH or healthy tissue (20). These results are highly relevant because they demonstrate that prohK2 may be a potentially useful diagnostic marker for prostatic cancer and BPH and that measurement of this protein alone or in combination with various forms of PSA and hK2 may further enhance prostate cancer diagnostics. This clinical evaluation is currently proceeding using the prohK2-specific immunoassay described here.

	Acknowledgments

 
We would like to thank Lisa Millar for purification of hK2 and prohK2.

	Footnotes

 
Hybritech Incorporated, a subsidiary of Beckman Coulter, Inc., San Diego, CA 92126.

¹ Nonstandard abbreviations: PSA, prostate-specific antigen; BPH, benign prostatic hyperplasia; hK2, human glandular kallekrein 2; mAb, monoclonal antibody; ACT, ₁-antichymotrypsin; BDL, biological detection limit; and PRP, postradical prostatectomy.

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