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Letters |
1
Istituto di Istologia ed Analisi Laboratorio, Facoltà di Scienze Matematiche e Naturali, Università degli Studi, Via Zeppi sn, 61029 Urbino (PS), Italy,
2
Istituto di Scienze Morfologiche,
3
Istituto di Anatomia e Fisiologia, Università degli Studi, Polo Scientifico c/o Ex-Sogesta, 61029 Urbino (PS), Italy
To the Editor:
We read with particular interest the article of Melegos et al. (1) on the prostate-specific antigen (PSA) immunoreactivity of cerebrospinal fluids, in which the authors report positive results for this kallikrein-like serine protease in ~7% of patients affected by various neurological disorders, suggesting that PSA could originate from the brain tissue. In light of this hypothesis, we undertook to study the expression, subfraction distribution, and secretion of PSA from two neuroblastoma cell lines: the SK-N-BE cell line, established in culture from a bone marrow biopsy specimen (2); and the SK-N-MC cell line, derived from a metastatic tumor mass (3). Both cell lines have tumor-producing capacity as well as the morphological patterns of malignant neuronal cells (4). These cell lines, all Mycoplasma free, were cultured in Eagle's minimum essential medium supplemented with nonessential amino acids, 15% fetal bovine serum, and antibiotics (10 IU/L penicillin and 100 mg/L streptomycin) and maintained according to conditions reported previously (2)(3). After growing, the cells were scraped and resuspended in the culture media. After a centrifugation at 5000g for 15 min at 4 °C, the supernatants were stored at -80 °C until assayed, while the cell pellets were lysed according to a described previously (5).
Total and free PSA determinations on culture media and supernatants of cell extracts, expressed in µg/L, were carried out in triplicate by using an automated microparticle enzyme immunoassay with a mouse anti-human monoclonal antibody (AxSYM®, Abbott Labs). The PSA determination procedure, performed according to manufacturer's recommendations, was described in detail elsewhere (5). The detection limit of the AxSYM PSA assay, defined as the concentration at two SDs above the zero calibrator, was reported to be 0.02 and 0.01 µg/L for total and free PSA, respectively. To exclude the possibility of "matrix" artifacts due to interfering substances present in cell lysate, neuroblastoma cell extracts were serially diluted in PSA-negative healthy female serum and reanalyzed for the response linearity. The analytical recovery of at least two concentrations of purified human seminal fluid PSA (Sigma Chemical Co.) added to the neuroblastoma cellular extracts was tested, then the recovered amount of PSA was calculated by subtracting the concentration initially present from the measured concentration after the addition.
The linearity and interference studies revealed good linear correlation between PSA concentration and dilution performed (Y = -0.183 68.9x, r = 0.987), thus demonstrating that neuroblastoma cell matrix extract did not affect the PSA analysis with the widely used immunoassay for serum samples. The mean analytical recovery of purified seminal fluid PSA added to cytosolic extracts from neuroblastoma cell lines was 95 ± 3%. Imprecision (CV) was 3.9% within-run and 5.4% between-run.
The cellular extract of the SK-N-BE cell line showed a markedly lower
total PSA content than that found in the SK-N-MC cell line extract,
with a different proportion of the PSA subfractions as well. In fact,
the cellular extract of the SK-N-MC neuroblastoma cell line showed a
predominant complexed form of PSA with a minor fraction (~30% of the
total PSA) of free uncomplexed form (Table 1
). Moreover, the higher amount of both the PSA fractions in
culture media of the SK-N-MC cells is consistent with the capability of
this cell line to more actively secrete this kallikrein-like serine
protease in respect to the other neuroblastoma cell line, after a
period 3–5 days of in vitro culture.
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In addition to neuroectodermal tumor-derived cell lines, SK-N-MC and SK-N-BE are typically composed of heterogeneous cellular subpopulations, including neuroblastic and nonneuronal cells with different neuronal enzyme and protein expression (4). This preliminary report adds further experimental data to the notion that PSA is an ubiquitous kallikrein-like serine protease with biological functions much more complex than recently thought (6)(7). Although the role of PSA in neuroblastoma cells is still unknown, our data are consistent with the proposal that the brain tumor tissue can actively produce and secrete this kallikrein-like serine protease.
A more detailed study, performed through additional methodological approaches (biochemical characterization and ultrastructural immunocytochemical localization of immunoreactive PSA protein) is currently in progress.
Footnotes
1 Author for correspondence. Fax 39-722-322370; e-mail mannello{at}bio.uniurb. it. 
References
The following articles in journals at HighWire Press have cited this article:
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  F. Mannello, M. Malatesta, F. Luchetti, S. Papa, S. Battistelli, and G. Gazzanelli Immunoreactivity, Ultrastructural Localization, and Transcript Expression of Prostate-specific Antigen in Human Neuroblastoma Cell Lines Clin. Chem., January 1, 1999; 45(1): 78 - 84. [Abstract] [Full Text] [PDF]
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