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Prostate-specific antigen expression in neoplastic human myeloid cell lines.

Prostate-specific antigen (PSA) is a 33-kDa serine protease produced by prostatic epithelial cells under the influence of the androgen receptor (1). Although PSA previously was thought to be a prostatic tissue-specific protein not expressed in any other tissue in men or women, at present it is considered to be present in many nonprostatic tissues and fluids (2, 3). PSA expression in both physiological and pathological conditions is not organ- or sex-specific; however, it is steroid hormone-mediated (4). In fact, several cell lines (from breast, ovary, prostate, and lung cancer) showed specific PSA production modulated by steroid hormones (3, 5, 6). Recently, nested PCR has shown PSA messenger RNA in a variety of non-prostate cells, including hematological cell lines (7, 8), nondiseased peripheral blood (9,10), and bone marrow specimens (11,12). The finding of PSA expression within cells that are outside of the prostate is of great clinical utility in establishing the nonspecificity of PSA as an indicator of micrometastatic malignant disease (13-15).

To establish the presence of PSA immunoreactivity in nondiseased human peripheral blood unfractionated leukocytes (PBLs) and in leukemic cell lines, we undertook the present study on PSA content and immunoreactivity in blood samples from 10 healthy female and male volunteers, ages 25-42 years (mean, 33 [+ or -] 4 years). After collection, the blood samples were enriched in unfractionated mononuclear cells by centrifugation at 5008 for 20 min at 4 [degrees]C on Ficoll-Hypaque (Pharmacia). Erythrocyte-depleted blood samples were prepared and counted after treatment with a lysing solution (Coulter Electronics). Sera from PSA-negative healthy control females were used as the negative control.

The leukemic cell lines HL-60 (promyelocytic), K562 (erythromyeloid), and U937 (premonocytic), all mycoplasma free, were maintained in RPMI supplemented with 100 mL/L heat-inactivated fetal calf serum (Irvine Scientific), 2 mmol/L glutamine, 100 000 units/L penicillin, and 100 mg/L streptomycin (Seromed, Biochrom KG) in a 5% C[O.sub.2] humidified incubator at 37 [degrees]C. The prostate carcinoma cell line LNCaP was grown in RPMI medium supplemented with 50 g/L fetal calf serum in a separate C[O.sub.2] humidified incubator at 37 [degrees]C to avoid the possibility of cross-contamination. This cell line is known to produce PSA and was used as the positive control. The cell pellets were lysed for 30 min on ice with 1 mL of lysis buffer containing 50 mmol/L Tris, pH 7.5, 150 mmol/L sodium chloride, 5 mmol/L EDTA, 10 g/L nonidet NP-40 surfactant, 1 mmol/L phenylmethylsulfonyl fluoride, and 1 mg/L each of aprotinin and leupeptin as proteinase inhibitors (16). The suspensions were frozen at -80 [degrees]C and thawed at 37 [degrees]C three times and sonicated for 5 cycles at 40 W of output for 30 s on ice (Heat-System Ultrasonic Inc.). Cell debris was pelleted at 90008 at 4 [degrees]C for 30 min, and the supernatants were collected and immediately assayed for PSA and total protein concentration.

The total protein content was determined with either the Coomassie G-250 or the bicinchoninic methods (with commercially available kits from Bio-Rad Laboratories and Pierce Chemical Co., respectively). Total and free PSA concentrations were measured by microparticle enzyme immunoassay (16,17), using a monoclonal mouse antihuman PSA antibody (AxSYM[R] from Abbott Laboratories). The detection limits of the assay were 0.02 [micro]g/L for total PSA and 0.01 [micro]g/L for free PSA. To exclude the possibility of matrix artifacts, PBLs and leukemic cell extracts were serially diluted in PSA-negative female serum and reanalyzed for the response linearity. The analytical recovery of at least two concentrations of purified PSA added to the leukemic cellular extracts (3.5 and 7.0 [micro]g/L) was tested (18).

All the assays performed on the cellular extracts were carried out in replicates of three in at least six independent experiments.

Reagents and equipment for Western blotting were purchased from Bio-Rad Laboratories. Our protocols were followed throughout, using the primary anti-human PSA monoclonal mouse antibody from Dako (16). Results, expressed as mean [+ or -] SD, were considered to be statistically significant when P <0.01. All statistical analyses were performed with the StatView, Ver. 4.1, package (Abacus Concepts Inc.) on a Macintosh Power PC (Apple Computer).

The present work was carried out in accordance with the ethical standards of the Helsinki Declaration of 1975, as revised in 1983.

The prostate cancer cell line LNCaP produces and secretes PSA in the culture medium; in our conditions, the extract from [10.sup.7] LNCaP cells showed a high total PSA content, of which ~95% was free (Table 1). The mean PSA concentration in extracts of PBLs from healthy subjects (n = 8) was 0.04 [+ or -] 0.02 [micro]g/L, close to the limit of detection of the assay and below the biological limit of detection (0.06 [micro]g/L) previously reported for this assay (19). However, total PSA content in unfractionated PBLs showed no significant difference between men and women, and it was not statistically different from the zero calibrator. On the other hand, the total PSA content in extracts from neoplastic myeloid cell lines was significantly greater than in nondiseased PBLs (P = 0.0058, P = 0.0008, and P = 0.0004 for the HL-60, K562, and U937 cell lines, respectively). The percentage of noncomplexed PSA detectable in leukemic cell extracts was 97%, 95%, and 93% for the HL-60, K562, and U937 cell lines, respectively.

The linearity studies revealed a good linear correlation between PSA concentration and dilution ([r.sup.2] = 0.97, [r.sup.2] = 0.99, and [r.sup.2] = 0.99 for the HL-60, K562, and U937 cell lines, respectively). The average analytical recovery of purified PSA added to cytosolic extracts from mononuclear leukemic cell lines was 97% [+ or -] 2%. The reproducibility of the assay (CV), determined by assaying leukocyte extracts in replicates of two in at least three independent analytical runs, was 2.8% and 4.8% for within-run and between-run, respectively.

The different patterns of PSA expression in leukemic cells reflect the peculiar characteristics of these cell lines; in fact, these cell lines show a considerable variation in both cell size (ranging from 9 to 55 [micro]m in diameter) and expression of specific proteins (20-22).

In all leukemic cell lines examined, Western blotting detected a major characteristic immunoreactive band of ~33 kDa of molecular mass, reflecting the specific presence of free PSA (Fig. 1).

A previous paper convincingly demonstrated that PSA messenger RNA is expressed in the HL-60 promyelocytic leukemic cell line, whereas the related serine protease is below the detection limit in cell lysates under the extraction conditions used (7). On the other hand, a recent paper showed that hematological cell lines (including HL-60) were negative for PSA mRNA, even if the frequency of positivity varied widely from line to line (8). Immununoreactivity for PSA in bone marrow extracts was recently reported (23); these data confirmed previous evidence (utilizing mRNA PCR method, immunohistochemistry, and antibody detection on blots) that PSA-positive cells may be present in the blood mononuclear cell fraction, in lymph nodes, and in bone marrow mononuclear cells from a variety of patient populations, including females (9,11-14). However, this is a matter of a recent debate; in fact, other studies failed to detect PSA mRNA in the peripheral blood of benign prostatic patients and healthy subjects (10,15, 24). To our knowledge, this is the first reported expression of total and free PSA in neoplastic myeloid cells obtained from different and well-established leukemic cell lines and detected by a commercial method. The increased PSA immunoreactivity in human neoplastic leukemic cell lines, compared with the nearly undetectable values from nondiseased unfractionated peripheral leukocytes, gives further evidence of the presence of this biochemical marker in non-prostate sources as a widely distributed serine protease. Although the biological effects and the physiologic role of PSA in leukemic cells are currently unknown, the presence of PSA messenger RNA and the active PSA protease in some cells of non-prostate origin might interfere with methods to detect micrometastases (7, 8, 11, 25, 26).



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Ferdinando Mannello, [1] * Francesca Luchetti, [2] Domenico Lancioli [3] Serafina Battistelli, [1] Stefano Papa, [2] and Giancarlo Gazzanelli [1]

([1] Instituto di Istologia ed Analisi di Laboratorio, Facolta di Scienze, MFN Universita, Via E. Zeppi, 61029 Urbino, Italy; [2] Instituto di Scienze Morfologiche, Universita Studi, 61029 Urbino, Italy; [3] Laboratorio Analisi, Ospedale Civile, 61029 Urbino, Italy; * author for correspondence: fax 39-722-322370, e-mail
Table 1. PSA and protein content in various cell lines.

 Total protein, mg/[10.sup.7] PSA, ng/[10.sup.7]
 cells cells

LNCaP (n = 6) 1.18 [+ or -] 0.19 26.20 [+ or -] 1.89
HL-60 (n = 6) 0.83 [+ or -] 0.24 0.43 [+ or -] 0.11
K562 (n = 6) 2.34 [+ or -] 0.42 0.77 [+ or -] 0.15
U937 (n = 6) 1.45 [+ or -] 0.33 0.37 [+ or -] 0.08
PBLs (n = 8) 0.75 [+ or -] 0.46 0.12 [+ or -] 0.09
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Title Annotation:Technical Briefs
Author:Mannello, Ferdinando; Luchetti, Francesca; Lancioli, Domenico; Battistelli, Serafina; Papa, Stefano;
Publication:Clinical Chemistry
Date:Sep 1, 1998
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