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Clinical Cancer Research Vol. 6, 353-356, February 2000
© 2000 American Association for Cancer Research

Advances in Brief

Characterization of Intracellular Prostate-specific Antigen from Laser Capture Microdissected Benign and Malignant Prostatic Epithelium

David K. Ornstein1, Chad Englert, John W. Gillespie, Cloud P. Paweletz, W. Marston Linehan, Michael R. Emmert-Buck and Emanuel F. Petricoin, III

Urologic Oncology Branch [D. K. O., W. M. L.], Pathogenetics Unit, Laboratory of Pathology [D. K. O., C. E.], and Cancer Genome Anatomy Project, Office of the Director [J. W. G., M. R. E-B.], National Cancer Institute, NIH, Bethesda, Maryland 20892; Division of Cytokine Biology, Center for Biologics Evaluation Research, Food and Drug Administration, Bethesda, Maryland 20892 [C. P. P., E. F. P.]; and Department of Chemistry, Georgetown University, Washington, D.C. 20057 [C. P. P.]


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
The proportion of unbound serum prostate-specific antigen (PSA; percent-free PSA) is reported to be lower in men with prostate cancer compared to men with benign prostates (U. H. Stenman et al., Cancer Res., 51: 222–226, 1991; H. Lilja et al., Clin. Chem., 37: 1618–1625, 1991; D. L. Woodrum et al., J. Urol., 159: 5–12, 1998; W. J. Catalona et al., J. Am. Med. Assoc., 279: 1542–1547, 1998). The majority of immunoreactive PSA in serum is complexed to {alpha}-1-antichymotrypsin (ACT). Two major mechanistic questions have previously been unknown: (a) Does PSA in human prostate cancer cells in tissue exist in a free or bound form? and (b) Is PSA produced by malignant cells in the free form because it has lost the ability to form a complex with ACT? Laser capture microdissection (LCM) enables the acquisition of pure populations of defined cell types from tissue (M. R. Emmert-Buck et al., Science, 274: 998-1001, 1996; R. F. Bonner et al., Science, 278: 1481–1483, 1997). This technology provides a unique opportunity to study intracellular protein composition and structure from human cells. In this study, we used LCM to assess the bound versus free form of intracellular PSA in both benign and malignant epithelium procured from prostate tissue.

One-dimensional and two-dimensional PAGE were performed on cellular lysates from LCM-procured benign and malignant prostate epithelium from frozen tissue specimens. Western blotting analysis of one-dimensional PAGE gels revealed a strong band at Mr 30,000 (expected molecular weight of unbound PSA) in all cases demonstrating that the vast majority of intracellular tumor and normal PSA exists within cells in the "free" form. Binding studies showed that PSA recovered from LCM-procured cells retained the full ability to bind ACT, and two-dimensional PAGE Western analysis demonstrated that the PSA/ACT complex was stable under strong reducing conditions. We conclude that intracellular PSA exists in the "free" form and that binding to ACT occurs exclusively outside of the cell.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
Serum total PSA2 measurement is the most useful and widely used test for the early diagnosis of prostate cancer (1) . Despite this, limitations exist, and there is a critical need to improve both sensitivity and specificity of PSA testing. In serum, immunoreactive PSA consists of PSA bound to ACT and unbound ("free") PSA (2, 3, 4) . Recent studies have reported that serum percent-free PSA is lower in men with cancer than in the men with benign prostates and have proposed that its measurement can be used to save 20% of biopsies in men with total serum PSA levels of 4.0–10.0 ng/ml while missing <5% of the cancers (5) . Although the biological reason for the differences in percent-free PSA levels among men with and without prostate cancer is not known, it has been suggested that malignant epithelial cells produce more ACT than benign epithelial cells, leading to an increased proportion of cellular PSA that is bound to ACT in cells (6, 7, 8) . Although it has been shown that prostate cells produce ACT (9) , the site at which the PSA/ACT complex forms has remained unknown.

The infiltrative nature of prostate cancer and the difficulty in generating a biologically representative cell line have previously made it difficult to study intracellular PSA in human tissue. LCM is a recently developed tool that allows isolation and molecular analysis of a defined population of cells from a stained tissue section (10 , 11) . In this study, we used LCM to isolate both normal and malignant epithelial cells from the same prostate cancer patient and then determined whether intracellular PSA is in the "free" form or exists as a complex with ACT.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
LCM of Benign and Malignant Epithelium.
Frozen tissue was obtained from radical prostatectomy specimens and imbedded in OCT. Eight-µm sections were made with a standard cryostat and stained with H&E using standard protocols. Benign and malignant histology was identified by a pathologist, and LCM was performed as previously described except 4-(2-aminoethyl)-benzensulfonylfluoride (Boehringer Mannheim) was added to the staining baths at a final concentration of 2 mM to inhibit proteases (10 , 11) . For one-dimensional and two-dimensional electrophoresis analysis, 2000 (~8000–10,000 cells) and 5000 (~20,000–25,000 cells) 30-µm laser shots of each cell population were used, respectively. Based on careful review of histological sections, each dissection is estimated to contain >95% of desired cells.

Western Blot Analysis of Intracellular PSA.
For one-dimensional PAGE analysis, 2x SDS buffer was used to lyse the procured cells, the lysates were boiled, and the proteins were separated on 4–20% tris-glycine nondenaturing gels and then transferred to a nylon membrane (Novex). A murine monoclonal antibody (Scripps Laboratories) was used as the primary antibody at a concentration of 1:1000, and a murine horseradish peroxidase-tagged anti-IgG antibody (Sigma) was used as the secondary antibody. The colorimetric reaction was generated by the ultra-ECL (Pierce). Purified free PSA and PSA bound to ACT (Scripps) were used as controls. For two-dimensional electrophoresis (two-dimensional PAGE), IEF buffer [7 M urea, 2 M thiurea, 4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, 1% decanoly-N-methylylucamide-10, 1% octyl-b-glucopyranoside, 40 mM Tris, 50 mM DTT, and 2 mM tri-butyl phosphine and 0.5% (v/v) pharmalytes] was used to extract the proteins from the LCM, and the lysates were used to swell precast immobilized pH gradient strips (18 cm, 3–10 nonlinear from Pharmacia) overnight. The purified free and bound PSAs were similarly diluted in IEF buffer. First-dimensional separation was performed for a total focusing time of 120 kV/h. The strips were re-equilibrated with a solution containing SDS and Tris (pH 6.9), reduced with tri-n-butylphosphine (2 mM), alkylated with iodoactemide (2.5% w/v), and directly applied to a 9% isocratic SDS-PAGE gel for electrophoresis overnight at 40 V. The protein was transferred to a nylon membrane, and Western blot was performed using the previously described reagents. Twenty 8-µm cryostat sections containing both malignant and benign epithelium were suspended in IEF buffer and analyzed by two-dimensional PAGE and Western blotting as just described.

PSA/ACT Binding Studies.
Malignant and normal prostatic epithelium were dissected using LCM and lysis buffer (1% NP-40 containing protease inhibitors and 100 mM NaCl) was used to extract and solubilize the proteins from the procured cells. One µg of purified ACT (Scripps) was added to 10 µl of lysis buffer containing ~10,000 cells of normal or malignant epithelium and incubated for 2 h at 37°C.


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
One-dimensional PAGE anti-PSA Western analysis of LCM-derived cellular lysates revealed a single band at Mr 30,000 (the expected molecular weight of unbound PSA) from both normal and malignant epithelium (Fig. 1)Citation . The total abundance of cellular PSA varied such that in some cases, the malignant cells contained more PSA, whereas in other cases, the benign cells contained more PSA.



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  		Fig. 1. One-dimensional PAGE anti-PSA Western of cell lysates from LCM-derived tissue from radical prostatectomy specimens. Lanes 1 and 3, benign epithelium; Lanes 2 and 4, malignant epithelium.

 
One-dimensional PAGE anti-PSA Western analysis of cellular lysates incubated for 120 min revealed a single band at Mr 30,000, whereas the same analysis of cellular lysates incubated with ACT for 60 and 120 min revealed a strong band at Mr 87,000 (the expected weight of the PSA/ACT complex) and a much weaker band at Mr 30,000 (Fig. 2)Citation . The band pattern was similar for cellular lysates derived from both benign and malignant epithelium. The molecular weight of the PSA/ACT complex is ~Mr 87,000, and therefore, results shown in Fig. 2Citation demonstrate that the majority of cellular PSA complexes with ACT after 2 h of incubation.



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  		Fig. 2. A, One-dimensional PAGE anti-PSA Western of cell lysates from LCM-derived benign (Lanes 1–4) and malignant (Lanes 5–8) prostatic epithelium. Lanes 1 and 5, cells were lysed and added directly to SDS buffer. Lanes 2 and 6, ACT was added to cell lysates and added directly to SDS buffer. Lanes 3 and 7, cell lysates were incubated for 120 min at 37°C and then added to SDS buffer. Lanes 4 and 8, ACT was added to cell lysates and incubated for 120 min at 37°C and then added to SDS buffer. B, One-dimensional PAGE anti-PSA Western of cell lysates from LCM-derived benign (Lanes 1–3) and malignant (Lanes 4–6) prostatic epithelium. Lanes 1 and 4, ACT was added to cell lysates and added directly to SDS buffer. Lanes 2 and 5, ACT was added to cell lysates and incubated for 60 min at 37°C and then added to SDS buffer. Lanes 3 and 6, ACT was added to cell lysates and incubated for 120 min at 37°C and then added to SDS buffer.

 
Two-dimensional PAGE anti-PSA Western analysis of cellular lysates from LCM-derived normal and malignant epithelial and from whole tissue sections similarly revealed three distinct Mr 30,000 isoforms (Fig. 3A)Citation . Two-dimensional PAGE anti-PSA Western analysis of purified free PSA and PSA/ACT complexes revealed several Mr 30,000 and Mr 87,000 isoforms, respectively (Fig. 3B)Citation . These results demonstrate that there are no apparent differences in the isoforms of PSA in normal and malignant prostate epithelium.



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  		Fig. 3. A, Two-dimensional PAGE anti-PSA Western of cellular lysates from LCM-derived normal and malignant prostatic epithelium and whole tissue sections. B, Two-dimensional PAGE anti-PSA Western of purified unbound (PSA) and complexed PSA (PSA/ACT).

 

   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
PSA is a serine protease that is produced almost exclusively by prostatic epithelial cells. In serum, the majority of the PSA exists bound to protease inhibitors such as ACT. It has been suggested that the proportion of serum PSA bound to ACT is in part determined by the amount of prostate-derived ACT. To address this question, we sought to determine the form in which intracellular PSA exists. By using LCM technology, we were able to procure prostate epithelial cells from human tissue and to determine by one-dimensional PAGE Western blot analysis that the vast majority of intracellular PSA was in the "free" form. Because LCM captures proteins from the intracellular and pericellular space, this finding suggests that PSA binds to ACT either within the extracellular space or in serum.

A possible limitation of our study could have occurred if tissue procurement and LCM procedures altered the PSA structure and its ability to bind ACT. We therefore diluted purified "free" and complexed PSA in IEF buffer and analyzed these molecules by two-dimensional PAGE. We found that the PSA/ACT complex was stable under strong reducing conditions, thereby supporting our assumption that our methodology would have detected complexed PSA if it indeed existed within prostatic epithelium. We then performed binding experiments that determined that PSA recovered by LCM was still able to fully bind to ACT.

In comparing PSA derived from normal and malignant epithelial, we did not find any differences in molecular weight or ability to bind ACT. In addition, we did not find any differences in isoforms of benign- and malignant-derived PSA as determined by two-dimensional PAGE analysis. These findings suggest that PSA produced by malignant prostate epithelium is not mutated and not differentially processed, at least in the present study set. This statement, however, needs to be confirmed by two-dimensional PAGE analysis of LCM-derived normal and malignant prostate epithelium from several more patients.

Because we did not detect complexed PSA in the cellular lysates from LCM-derived tissue, it is unlikely that the prostate production of ACT is a major determinant of serum percent-free PSA. One explanation for this observation is that the two proteins are isolated in different cellular compartments and that binding does not occur until both are released into the extracellular space. However, one would expect that once the cells were lysed, any compartmentalization would be disrupted, and in vitro binding would occur. When we performed anti-ACT Western blot analysis on cellular lysates obtained from LCM-derived normal and malignant prostate epithelium, we were unable to detect ACT (data not shown). Therefore, a more likely explanation is that even cancerous prostate cells produce a relatively small amount of ACT and that the vast majority of ACT bound to PSA in serum is derived from other noncancerous host sources.

Conclusion.
As demonstrated, LCM is a valuable tool that can by used to compare protein expression and processing in normal and malignant epithelium. By using this new technology, we demonstrated that the majority of immunoreactive cellular PSA is not bound and that binding to ACT takes place after PSA leaves the cell. We have shown that the PSA produced by normal and malignant prostatic epithelium does not differ in regards to molecular weight, isoform, charge, or ability to bind ACT. Our findings also suggest that the difference in serum percent-free PSA levels are not the result of altered ACT production by malignant prostatic epithelium. More comprehensive studies are being conducted to elucidate why prostate cancer is associated with low serum percent-free PSA.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at Urologic Oncology Branch, National Cancer Institute, NIH, Building 10, Room 2B47 Bethesda, MD 20892. Back

2 The abbreviations used are: PSA, prostate-specific antigen; ACT, {alpha}-1-antichymotrypsin; LCM, laser capture microdissection. Back

Received 8/ 6/99; revised 11/ 1/99; accepted 11/15/99.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

  1. Catalona W. J., Richie J. P., Ahmann F. R., Hudson M. A., Scardino P. T., Flanigan R. C., DeKernion J. B., Ratliff T. L., Kavoussi L. R., Dalkin B. L., Waters W. B., MacFarlane M. T., Southwick P. C. Comparison of digital rectal examination and serum prostate specific antigen in the early detection of prostate cancer: results of a multicenter clinical trial of 6,630 men. J. Urol., 151: 1283-1290, 1994.[Medline]
  2. Stenman U. H., Leinonen J., Alfthan H., Rannikko S., Tuhkanen K., Alfthan O. A complex between 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.[Abstract/Free Full Text]
  3. Lilja H., Christensson A., Dahlen U., Matikainen M. T., Nilsson O., Pettersson K., Lovgren T. Prostate-specific antigen in serum occurs predominately in complex with {alpha}1-anticymptrypsin. Clin. Chem., 37: 1618-1625, 1991.[Abstract/Free Full Text]
  4. Woodrum D. L., Brawer M. K., Partin A. W., Catalona W. J., Southwick P. C. Interpretation of free prostate specific antigen clinical research studies for the detection of prostate cancer. J. Urol., 159: 5-12, 1998.[CrossRef][Medline]
  5. Catalona W. J., Partin A. W., Slawin K. M., Brawer M. K., Flanigan R. C., Patel A., Richie J. P., deKernion J. B., Walsh P. C., Scardino P. T., Lange P. H., Subong E. N., Parson R. E., Gasior G. H., Loveland K. G., Southwick P. C. Use of percentage of free prostate-specific antigen to enhance differentiation of prostate cancer from benign prostatic disease. J. Am. Med. Assoc., 279: 1542-1547, 1998.[Abstract/Free Full Text]
  6. Chen Z., Chen H., Stamey T. A. Monoclonal antibodies 2F5 and 4G10 against prostate specific antigen (PSA) complexed to {alpha}1-antichymotrypsin. J. Urol., 160: 870-875, 1998.[CrossRef][Medline]
  7. Bjork T., Bjartell A., Abrahamsson P. A., Hulkko S., di Sant’Agnes A., Lilja H. {alpha}1-antichymotrypsin production in PSA-producing cells is common in prostate cancer but rare in benign prostatic hyperplasia. Urology, 43: 427-434, 1994.[CrossRef][Medline]
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