| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Tumor Biology |
Hybritech Incorporated, a subsidiary of Beckman Coulter, Inc., San Diego, CA 92121 [S. D. M., L. S. Mi., T. J. W., H. G. R.]; University of California, Los Angeles, CA, 90095 [L. S. Ma.]; and Scott Department of Urology, Baylor College of Medicine and the Methodist Hospital, Houston, TX 77030 [W. S., T. M. W., K. M. S.]
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
1-antichymotrypsin (ACT; "complexed
PSA") (5
, 6)
. From 70 to 95% of the measurable total
serum PSA is complexed with ACT (4)
. In patients with a
mildly elevated PSA, the ratio of free to total PSA in serum has been
demonstrated to significantly improve the discrimination of prostate
cancer (PCa) from benign prostatic hyperplasia (BPH), with higher
ratios correlating with a lower risk of prostate cancer (7
, 8)
. The biological mechanism resulting in the increased ratio of
serum free PSA in patients with BPH is unknown. It is generally assumed that the free PSA in serum is enzymatically inactive because it would otherwise complex with endogenous serum protease inhibitors. Free forms of PSA may include mature, inactive PSA (9) and various forms of clipped, inactivated PSA (10 , 11) as well as precursor or zymogen forms of PSA (12) . The molecular forms comprising "free" PSA in the serum of prostate cancer patients may differ from those in BPH patients. Previously, we reported that pro PSA (pPSA) was a component of the free PSA in PCa serum (12) . Identification of free PSA forms in the serum of BPH patients has not been feasible because the lower levels of PSA are insufficient for current analytical techniques. To determine whether specific molecular forms of pPSA or clipped PSA were preferentially associated with prostate cancer or BPH tissues, we examined matched sets of tissues harvested from patients undergoing radical prostatectomy. From each patient samples of prostate cancer and adjacent noncancerous tissues from the peripheral zone (PZ-C and PZ-N, respectively) as well as a sample of benign transition zone tissue (TZ) were selected for analysis. Most cancers are localized to the peripheral zone (13) , whereas pathological BPH occurs almost exclusively in the transition zone of the prostate (14) . We have previously reported that TZ tissues exhibiting nodular hyperplasia (pathological BPH) contain elevated levels of a specific, clipped form of PSA, called BPSA (BPH-associated form of PSA), when compared with levels in both cancerous and noncancerous PZ tissues (15) . We now report that pPSA is associated primarily with PZ prostate cancer tissues but not with benign TZ tissues, indicating that an assay specific for pPSA in serum may help further distinguish PCa from BPH.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Isolation of PSA from Prostate Tissue.
Prostate tissues were frozen in liquid nitrogen, pulverized to a fine
powder, and homogenized in PBS containing a protease inhibitor cocktail
(Complete, Boehringer Mannheim, Indianapolis, IN). Samples were then
centrifuged to remove cell debris, and the supernatant solution was
filtered through a 0.2 µm membrane. The supernatant solution was
passed over two immunoaffinity columns in series, a column containing
the hK2-specific mAb HK1G586.1, and the PSA-specific mAb, PSM773.
HK1G586.1 has been shown to have no cross-reactivity with PSA
(17
, 18)
. PSM773 has been shown previously to have no
cross-reactivity with hK2 and to have specificity for mature, clipped,
and precursor forms of PSA (19, 20, 21)
. The columns were
washed with 40 volumes of PBS containing 0.1% reduced Triton X-100 and
bound protein eluted with 100 mM glycine (pH 2.5) containing 200 mM
sodium chloride. The eluant was immediately neutralized with 10%
vol/vol 1 M Tris (pH 8.0).
Immunoassay of PSA.
The concentration of PSA in the extracts was determined by Tandem-MP
PSA and Tandem-MP free PSA assays (Hybritech, San Diego,
CA). The percentage of inactive PSA was determined by measuring the
level of noncomplexed PSA in the original extract and comparing it to
the level of noncomplexed PSA remaining after incubation with female
serum for 3 h at 37°C. Incubation with female serum allowed
active PSA to complex with serum proteins. The total protein in the
extracts was determined using the Bradford protein assay (Pierce,
Rockford, IL)
High Performance Hydrophobic Interaction Chromatography
(HIC-HPLC) of PSA.
HIC-HPLC was performed using a polypropylaspartamide column (PolyLC,
Western Analytical Products, Temecula, CA). The column was
4.6 x 250 mm in length with a 1000 Å pore size.
Samples were applied in 1.5 M ammonium sulfate and eluted with a
gradient. Buffer A contained 1.2 M sodium sulfate and 25 mM
sodium phosphate (pH 6.3). Buffer B contained 50 mM sodium
phosphate and 5% v/v 2-propanol. The gradient was 0–35% B for 1 min,
30–80% B for 12 min, and then isocratic at 80% B for 2 min before
equilibration in Buffer A. High-sensitivity peak detection was obtained
with a Varian model 9070 scanning fluorescence detector using an
excitation of 232 nm and emission of 334 nm to detect the tryptophan
residues in protein.
Amino Acid Sequencing of PSA.
N-terminal sequence of the samples was performed through 9 cycles on a
PE-Applied Biosystems model 492 amino acid sequencer (Perkin-Elmer,
Applied Biosystems Division, Foster City, CA). Purified PSA and peaks
collected by HIC-HPLC were applied directly to polyvinylidene
difluoride membranes using the Prosorb cartridges (PE-Applied
Biosystems), washed 3 times with 0.1 ml 0.1% trifluoroacetic acid, and
applied to the model 492 sequencer.
Statistical Analysis.
All statistical analyses were performed using SAS software. The median
percentages of pPSA in TZ, PZ-N, and PZ-C tissues were evaluated for
statistical significance using the nonparametric Kruskal-Wallis test.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
shows the HIC-HPLC profile of the affinity-purified PSA from the PZ-C
of patient #1 (Table 1)
. PSA typically elutes at 10 min under the conditions used. The
identities of inactive forms of PSA in this peak were determined by
performing HIC-HPLC after incubation of the purified PSA with ACT (Fig. 2)
and after sequencing individual fractions to confirm their identities.
Peak 1 in Fig. 2A
is the inactive, clipped ACT that is
formed during complex formation with PSA (22)
. Peak 2
represents the residual excess active ACT remaining after incubation.
Peak 3 is the PSA-ACT complex, and peak 4 comprises a mixture of
inactive forms of PSA that failed to complex with ACT. Under these
conditions, the PSA-ACT complex eluted at 8 min and was clearly
resolved from the residual, unreactive forms of PSA, which remained at
10 min.
|
|
|
shows the relative elution
positions of these different forms of inactive PSA. The front shoulder
of the inactive PSA peak contained primarily mature PSA, PSA clipped at
Lys145 and Arg85, and PSA clipped after Gly4. The second half of
the inactive PSA peak contained the majority of the [-2]pPSA. The
small peak eluting at 12 min contained the [-4]pPSA. The retention
time of [-4]pPSA has been reported previously (12)
. The
[-2] and [-4] forms of pPSA are truncated forms of pPSA. proPSA
is normally expressed with a heptapeptide pro leader sequence
consisting of APLILSR. The [-2]pPSA contains the SR pro dipeptide on
the N-terminal isoleucine of mature PSA, and [-4]pPSA contains an
ILSR leader peptide. As indicated by their inability to form a complex
with ACT, each of these clipped or truncated forms of pPSA was
enzymatically inactive. The [-2]pPSA constituted 65% of the
inactive PSA in this sample, and [-4]pPSA was 6%. Other samples of
purified PSA were incubated with ACT as described above and showed
varying percentages of the different inactive forms of PSA (data not
shown).
The Distribution of pPSA in Prostate Tissues.
The distribution of the different percentages of clipped PSA and pPSA
in the 18 sets of matched tissues was determined by N-terminal
sequencing of whole immuno-affinity purified PSA. The clips at
[+5]PSA, Lys145, and Arg85 showed no significant trends in the PZ-N,
PZ-C, and TZ tissues (data not shown). However, [-2]pPSA was
elevated in almost all of the PZ-C and in many of the PZ-N tissues but
was largely undetectable in the TZ (<0.2% pPSA). The [-2] form of
pPSA was the predominant form of pPSA observed in the purified PSA,
although very minor levels of other proforms could be detected in some
samples.
Table 1
shows the percentage of [-2]pPSA in the 18 sets of matched
tissue specimens. When grouped according to tissue type, levels of
[-2]pPSA were significantly higher in PZ tissues than in TZ tissues
(Fig. 3)
. Only 5 TZ samples showed measurable pPSA. Ten of eighteen TZ were
shown to have pathological nodular BPH (15)
, but they were
not statistically different in their levels of pPSA from the eight TZ
tissues without nodules. The median value of pPSA in TZ was 0, compared
with 1.5% for PZ-N and 3.0% of the total PSA for PZ-C
(p < 0.0026). The mean values for TZ, PZ-N,
and PZ-C were 1.1, 3.9, and 5.6% of the total PSA, respectively. The
mean values of total PSA in the three tissues, as determined by
immunoassay, were 15.6, 12.4 and 9.0 µg PSA per mg of total protein
in the TZ, PZ-C, and PZ-N, respectively. Eight samples of transition
zone tissue, obtained during TURP for BPH, also revealed no detectable
pPSA (data not shown).
|
also shows the percentage of total inactive PSA in each sample
as determined by immunoassay after incubation with female serum (see
"Materials and Methods"). The pPSA in PZ-N and TZ showed no
statistical correlation with the percentage of inactive PSA, whereas
the pPSA in PZ-C showed a mild correlation (p < 0.012). The median percentages of inactive PSA for PZ-N, PZ-C,
and TZ were 38, 35, and 37%, respectively. |
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
These tissue studies add important new insight into our previous work,
in which we reported that pPSA is a component of the PSA in PCa serum
(12)
. In that work we identified [-4]pPSA by HIC-HPLC
as a component of the free PSA in PCa serum. The serum may also have
contained [-2]pPSA, the form we have found most abundantly in
tissues, but [-2]pPSA cannot be readily distinguished from mature
forms of PSA by HIC-HPLC (see Fig. 2
) and requires sufficient
quantities of protein for sequence analysis to confirm the identity of
these species. Therefore, the analysis of serum PSA forms is limited by
the necessity of using serum with highly elevated levels of PSA,
typically from 100 to 1000 ng/ml or higher.
Because high PSA levels are found primarily in patients with prostate
cancer but not BPH, it remains unknown whether pPSA can be detected in
the serum of patients with BPH only. However, high levels of PSA are
released into the blood during TURP, and a recent study analyzed the
PSA in TURP serum and found no pPSA (23)
. This is
consistent with our finding that pPSA is largely absent in the
transition zone (Fig. 3)
. In the same study, attempts were made to
detect pPSA in the PSA purified from PCa serum by probing Western blots
with an antibody raised against the N-terminal propeptide moiety of
pPSA. However, this aspect of those studies remains inconclusive in
light of our findings because the antibodies were not specific for the
[-2] and [-4] pPSA forms we have found in the tissues and serum
(12)
. Comprehensive analysis of serum samples awaits the
availability of immunoassays that can specifically detect the [-2]
and [-4] pPSA forms.
The current study of prostate tissues provides some evidence for the origin of pPSA in serum. Because transition zone tissues contain little or no pPSA, it is more likely that any pPSA found in the serum is derived from cancerous prostate PZ tissue. This distinction between PZ and TZ becomes critical in the early stages of prostate cancer in which comparable levels of total serum PSA may be derived from both cancer and BPH. In the diagnostic gray zone of 4–10 ng/ml, the measurement of pPSA in serum may help distinguish PCa from BPH.
The reasons for the higher levels of these truncated pPSA forms in the
PZ are not known. PSA, like most tissue kallikreins, is secreted from
cells as the precursor form (21
, 24
, 25)
, but it has not
been established how or where it is converted to the enzymatically
active form in vivo. The truncated forms of pPSA are likely
to result from post-translational proteolytic modification and may
reflect an altered biochemical pathway. An analysis of seminal plasma
PSA, as in Fig. 2
, found no detectable pPSA forms (data not shown).
Because the homologous prostate kallikrein, hK2, has been shown to
activate pPSA in vitro, it has been speculated that hK2 may
be involved in the in vivo regulation of PSA activity
(21
, 26
, 27)
. hK2 is currently the subject of studies to
determine its relationship to prostate cancer (17)
.
In these studies, we simultaneously purified the hK2 from the tissue extracts by immunoaffinity chromatography using an hK2-specific mAb. hK2 was present at 2–4% of the PSA (data not shown) and is largely inactive in tissue extracts due to internal peptide bond cleavage, as shown previously (28) . The [-2] form of pPSA contains the same N-terminal amino acids as [-2]phK2, SRIVGGWEC, whereas the [-4] pro forms are different from each other. Any significant contamination by [-2]phK2 was ruled out by the use of both hK2-specific and PSA-specific mAbs, together with the observation that hK2 represents only a minor percentage of the PSA in these extracts.
Monoclonal antibodies specific for different pPSA forms are currently under development. If the levels of pPSA in serum reflect the presence of pPSA in prostate cancer PZ tissues but not in the transition zone, then an immunoassay that measures pPSA may improve the discrimination of prostate cancer from BPH. The combination of an assay for cancer-associated pPSA, together with an assay for the nodular BPH-associated form of PSA that we recently reported (15) , may enhance our ability to distinguish cancer from BPH.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 To whom requests for reprints should be
addressed, at Hybritech Incorporated, a subsidiary of Beckman Coulter,
Inc., P.O. Box 269006, San Diego, CA 92196-9006. Phone:
(858) 621-4601; Fax: (858) 621-4610; E-mail: sdmikolajczyk{at}beckman.com 
2 The abbreviations used are: PSA, prostate
specific antigen; ACT, 1-antichymotrypsin; PCa, prostate
cancer; BPH, benign prostatic hyperplasia; pPSA, pro PSA; TZ, benign
transition zone tissue; PZ-C, peripheral zone cancer; PZ-N, peripheral
zone noncancer; TURP, transurethral resection of the prostate;
HIC-HPLC, high performance hydrophobic interaction chromatography.
Received 8/ 5/99. Accepted 12/ 2/99.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
1-antichymotrypsin. Clin. Chem., 37: 1618-1625, 1991.1-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.This article has been cited by other articles:
|
|
 |
|
  G. Tabares, C. M. Radcliffe, S. Barrabes, M. Ramirez, R. N. Aleixandre, W. Hoesel, R. A. Dwek, P. M. Rudd, R. Peracaula, and R. de Llorens Different glycan structures in prostate-specific antigen from prostate cancer sera in relation to seminal plasma PSA Glycobiology, February 1, 2006; 16(2): 132 - 145. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Jung, J. Reiche, A. Boehme, C. Stephan, S. A. Loening, D. Schnorr, W. Hoesel, and P. Sinha Analysis of Subforms of Free Prostate-Specific Antigen in Serum by Two-Dimensional Gel Electrophoresis: Potential to Improve Diagnosis of Prostate Cancer Clin. Chem., December 1, 2004; 50(12): 2292 - 2301. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. D. Mikolajczyk, W. J. Catalona, C. L. Evans, H. J. Linton, L. S. Millar, K. M. Marker, D. Katir, A. Amirkhan, and H. G. Rittenhouse Proenzyme Forms of Prostate-Specific Antigen in Serum Improve the Detection of Prostate Cancer Clin. Chem., June 1, 2004; 50(6): 1017 - 1025. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Peracaula, G. Tabares, L. Royle, D. J. Harvey, R. A. Dwek, P. M. Rudd, and R. de Llorens Altered glycosylation pattern allows the distinction between prostate-specific antigen (PSA) from normal and tumor origins Glycobiology, June 1, 2003; 13(6): 457 - 470. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. J. Linton, L. S. Marks, L. S. Millar, C. L. Knott, H. G. Rittenhouse, and S. D. Mikolajczyk Benign Prostate-specific Antigen (BPSA) in Serum Is Increased in Benign Prostate Disease Clin. Chem., February 1, 2003; 49(2): 253 - 259. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. P. Balk, Y.-J. Ko, and G. J. Bubley Biology of Prostate-Specific Antigen J. Clin. Oncol., January 15, 2003; 21(2): 383 - 391. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. D. Mikolajczyk, K. M. Marker, L. S. Millar, A. Kumar, M. S. Saedi, J. K. Payne, C. L. Evans, C. L. Gasior, H. J. Linton, P. Carpenter, et al. A Truncated Precursor Form of Prostate-specific Antigen Is a More Specific Serum Marker of Prostate Cancer Cancer Res., September 1, 2001; 61(18): 6958 - 6963. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. M. Yousef and E. P. Diamandis The New Human Tissue Kallikrein Gene Family: Structure, Function, and Association to Disease Endocr. Rev., April 1, 2001; 22(2): 184 - 204. [Abstract] [Full Text]
|
|
|
|
|
|
J. Peter, C. Unverzagt, T. N. Krogh, O. Vorm, and W. Hoesel Identification of Precursor Forms of Free Prostate-specific Antigen in Serum of Prostate Cancer Patients by Immunosorption and Mass Spectrometry Cancer Res., February 1, 2001; 61(3): 957 - 962. [Abstract] [Full Text]
|
|
|
|
 |
|
 C. Stephan, K. Jung, M. Lein, P. Sinha, D. Schnorr, and S. A. Loening Molecular Forms of Prostate-specific Antigen and Human Kallikrein 2 as Promising Tools for Early Diagnosis of Prostate Cancer Cancer Epidemiol. Biomarkers Prev., November 1, 2000; 9(11): 1133 - 1147. [Abstract] [Full Text]
|
|
|
|
|
|
P. Nurmikko, V. Vaisanen, T. Piironen, S. Lindgren, H. Lilja, and K. Pettersson Production and Characterization of Novel Anti-Prostate-specific Antigen (PSA) Monoclonal Antibodies That Do Not Detect Internally Cleaved Lys145-Lys146 Inactive PSA Clin. Chem., October 1, 2000; 46(10): 1610 - 1618. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
