Expression and purification of recombinant active prostate-specific antigen from Escherichia coli

Human prostate-specific antigen (PSA), a 33 kDa serine protease with comprehensive homology to glandular kallikrein, is secreted from prostatic tissue into the seminal fluid and enters into the circulation. The level of PSA increases in the serum of patients with prostatic cancer and hence is widely employed as a marker of the disease status. In particular, an enzymatically active PSA that is a form cleaved at the N-terminal seven-amino-acids prosequence, APLILSR, of proPSA may play an important roll in the progression of prostate cancer. Thus, the presence of the active form would selectively discriminate the cancer from benign prostatic hyperplasia. In this study, we developed a convenient purification method for the acquisition of active PSA and proPSA. Recombinant proPSA and active PSA were expressed directly in Escherichia coli, easily and efficiently isolated from inclusion bodies, refolded, and purified. Moreover, the enzymatic activity of the recombinant active PSA was confirmed as serine protease using chromogenic chymotrypsin substrate. This purified active PSA could be further applied to scrutinize the biological or conformational characteristics of the protein and to develop specific diagnostic and/or therapeutic agents against prostate cancer.     

Combination of Autoantibody Signature with PSA Level Enables a Highly Accurate Blood-Based Differentiation of Prostate Cancer Patients from Patients with Benign Prostatic Hyperplasia

Although an increased level of the prostate-specific antigen can be an indication for prostate cancer, other reasons often lead to a high rate of false positive results. Therefore, an additional serological screening of autoantibodies in patients’ sera could improve the detection of prostate cancer. We performed protein macroarray screening with sera from 49 prostate cancer patients, 70 patients with benign prostatic hyperplasia and 28 healthy controls and compared the autoimmune response in those groups. We were able to distinguish prostate cancer patients from normal controls with an accuracy of 83.2%, patients with benign prostatic hyperplasia from normal controls with an accuracy of 86.0% and prostate cancer patients from patients with benign prostatic hyperplasia with an accuracy of 70.3%. Combining seroreactivity pattern with a PSA level of higher than 4.0 ng/ml this classification could be improved to an accuracy of 84.1%. For selected proteins we were able to confirm the differential expression by using luminex on 84 samples. We provide a minimally invasive serological method to reduce false positive results in detection of prostate cancer and according to PSA screening to distinguish men with prostate cancer from men with benign prostatic hyperplasia.     

Selective recognition of enzymatically active prostate-specific antigen (PSA) by anti-PSA monoclonal antibodies

Prostate-specific antigen (PSA) is widely used as a serum marker for the diagnosis of prostate cancer. To evaluate two anti-free PSA monoclonal antibodies (mAbs) as potential tools in new generations of more relevant PSA assays, we report here their properties towards the recognition of specific forms of free PSA in seminal fluids, LNCaP supernatants, 'non-binding' PSA and sera from cancer patients. PSA from these different origins was immunopurified by the two anti-free PSA mAbs (5D3D11 and 6C8D8) as well as by an anti-total PSA mAb. The composition of the different immunopurified PSA fractions was analysed and their respective enzymatic activities were determined. In seminal fluid, enzymatically active PSA was equally purified with the three mAbs. In LNCaP supernatants and human sera, 5D3D11 immunopurified active PSA mainly, whereas 6C8D8 immunopurified PSA with residual activity. In sera of prostate cancer patients, we identified the presence of a mature inactive PSA form which can be activated into active PSA by use of high saline concentration or capture by an anti-total PSA mAb capable of enhancing PSA activity. According to PSA models built by comparative modelling with the crystal structure of horse prostate kallikrein described previously, we assume that active and activable PSA could correspond to mature intact PSA with open and closed conformations of the kallikrein loop. The specificity of 5D3D11 was restricted to both active and activable PSA, whereas 6C8D8 recognized all free PSA including intact PSA, proforms and internally cleaved PSA.     

Cancer de la prostate : les marqueurs biologiques

Normal prostate cells and prostate cancer cells produce prostate-specific antigen (PSA): thus, it is frequently increased in non-malignant conditions such as prostatitis and benign prostatic hyperplasia. Indeed, PSA is an excellent biomarker to monitor disease progression. The low diagnostic specificity of PSA leads to many false-positive and a large number of biopsies. These well-recognized limitations of PSA suggest that new prostate cancer biomarkers could play a useful role in reducing the number of unnecessary biopsies.     

Prostate cancer vs hyperplasia: relationships with prostatic and adipose tissue fatty acid composition

The objective of the present study was to study whether adipose tissue and prostatic tissue fatty acid composition differentiates between prostate cancer and benign hyperplasia patients. In addition, the present investigation aimed at exploring the extent to which prostatic tissue fatty acid composition differentiates between prostate-confined cancer and extraprostatic disease including possible metastasis. The subjects were 71 male patients from the island of Crete. Half the patients (n=35) had been diagnosed with benign hyperplasia of the prostate, half with prostatic malignancy (n=36). Patients were examined at the outpatient clinic of the urology unit, University Hospital, Medical School, University of Crete. Relative to benign hyperplasia patients, cancer patients had elevated adipose tissue saturated and reduced monounsaturated fatty acid levels. Cancer patients had reduced prostate tissue stearic to oleic acid ratios and stearic acid levels as opposed to hyperplasia patients. The most pronounced difference between cancer patients and hyperplasia patients was a 3-fold elevated prostatic palmitoleic acid in the former group. Relative to benign hyperplasia patients, cancer patients had reduced prostate tissue arachidonic and docosahexaenoic acid levels. Finally, there was a significantly reduced omega-3/omega-6 polyunsaturated fatty acid ratio in the prostate cancer patient as opposed to the benign hyperplasia group. The pronounced elevations in prostatic tissue palmitoleic acid in cancer patients highlight a possible role of this fatty acid in neoplastic processes. The decreased arachidonic acid levels in cancer patients possibly stem from enhanced metabolism of arachidonic acid via lipoxygenase and cyclooxygenase pathways, and the formation of derivatives such as 5-HETE, 15-HETE, 12(S)-HETE and PGE(2).     

Proteomic analysis of formalin-fixed prostate cancer tissue

Proteomic analysis of formalin-fixed paraffin-embedded (FFPE) tissue would enable retrospective biomarker investigations of this vast archive of pathologically characterized clinical samples that exist worldwide. These FFPE tissues are, however, refractory to proteomic investigations utilizing many state of the art methodologies largely due to the high level of covalently cross-linked proteins arising from formalin fixation. A novel tissue microdissection technique has been developed and combined with a method to extract soluble peptides directly from FFPE tissue for mass spectral analysis of prostate cancer (PCa) and benign prostate hyperplasia (BPH). Hundreds of proteins from PCa and BPH tissue were identified, including several known PCa markers such as prostate-specific antigen, prostatic acid phosphatase, and macrophage inhibitory cytokine-1. Quantitative proteomic profiling utilizing stable isotope labeling confirmed similar expression levels of prostate-specific antigen and prostatic acid phosphatase in BPH and PCa cells, whereas the expression of macrophage inhibitory cytokine-1 was found to be greater in PCa as compared with BPH cells.     

Characterization of prostate-specific antigen binding peptides selected by phage display technology

Prostate-specific antigen (PSA) is an important marker for the diagnosis and management of prostate cancer. Free PSA has been shown to be more extensively cleaved in sera from benign prostatic hyperplasia patients than in sera from prostate cancer patients. Moreover, the presence of enzymatically activatable PSA was characterized previously in sera from patients with prostate cancer by the use of the specific anti-free PSA monoclonal antibody (mAb) 5D3D11. As an attempt to obtain ligands for the specific recognition of different PSA forms including active PSA, phage-displayed linear and cyclic peptide libraries were screened with PSA coated directly into microplate wells or presented by two different anti-total PSA mAbs. Four different phage clones were selected for their ability to recognize PSA and the inserted peptides were produced as synthetic peptides. These peptides were found to capture and to detect specifically free PSA, even in complex biological media such as sera or tumour cell culture supernatants. Alanine scanning of peptide sequences showed the involvement of aromatic and hydrophobic residues in the interaction of the peptides with PSA whereas Spotscan analysis of overlapping peptides covering the PSA sequence identified a peptide binding to the kallikrein loop at residues 82-87, suggesting that the peptides could recognize a non-clipped form of PSA. Moreover, the PSA-specific peptides enhance the enzymatic activity of PSA immobilized into microplate wells whereas the capture of PSA by the peptides inhibited totally its enzymatic activity while the peptide binding to PSA had no effect in solution. These PSA-specific peptides could be potential tools for the recognition of PSA forms more specifically associated to prostate cancer.     

The androgen receptor CAG repeat length polymorphism associates with prostate volume in Finnish men with benign prostatic hyperplasia

The development of benign prostatic hyperplasia requires the presence of testicular androgens during prostate development, puberty, and ageing. We thus examined the association of three polymorphisms, namely, CYP3A5 6986A>G, CYP19A1 1531C>T, and androgen receptor (AR) gene CAG repeat length, which have previously been linked to the androgen pathway and with clinical characteristics of benign prostatic hyperplasia. Tissue samples from 262 consecutive prostate operations were used for genotyping. Prostate volumes and prostate-specific antigen values were collected from patient records. Linear regression analysis was performed to study the polymorphisms in an age-adjusted model. We did not find any association between the CYP3A5 6986A>G polymorphism and clinical characteristics of benign prostatic hyperplasia. Further, the previously published CYP19A1 1531C>T polymorphism association with an enlarged prostate could not be confirmed with this material. However, we detected an association between short AR gene CAG repeat length and a small prostate volume, which confirms a previous finding in the Finnish population. The data presented suggest a negligible role for the CYP3A5 6986A>G polymorphism in benign prostate enlargement in the Finnish population. However, the results presented do provide further evidence for potentially different genetic mechanisms behind benign prostatic hyperplasia in Finnish and other Caucasian populations. This is based on the conflicting results for AR gene CAG repeat length associations with benign prostatic hyperplasia found in published works.     

The contribution of prostatic acid phosphatase and prostatic specific antigen in the diagnosis of prostatic cancer

Prostatic acid phosphatase (PAP) and prostatic specific antigen (PSA) were measured by immunochemical methods using test preparations from two different companies. In 66 patients with benign hyperplasia of the prostate a good correlation was found only between PSA levels (orthogonal regression analysis: y = 1.77 x -0.68; r = 0.995). Discrimination analysis between benign hyperplasia and new prostatic cancer (28 patients), using ROC curves, revealed a sensitivity for prostatic cancer of about 30 percent using both PAP methods and of about 58 percent using both PSA methods at the 95-percentile of benign hyperplasia. The PSA methods were both more sensitive in detecting prostatic cancer than the PAP methods.     

Overexpression of JKTBP1 induces androgen-independent LNCaP cell proliferation through activation of epidermal growth factor-receptor (EGF-R)

Heterogenous nuclear ribonucleoprotein D-like protein (JKTBP) belongs to a new member of hnRNPs. Previous studies implied that JKTBP1 may be associated with the progression of androgen-independent (AI) prostate cancer. In this study, we generated three stable LNCaP cell lines which expressed exogenous JKTBP1. Furthermore, the effect of ectopic JKTBP1 on the proliferation of LNCaP cells and its mechanism was investigated. We originally found that the ectopic JKTBP1 expression resulted in the proliferation of LNCaP cells in an AI way, as well as inducing the upregulated expression of EGF-R and prostate-specific antigen (PSA), but did not influence the expression level of AR. Moreover, AG1478 suppressed the effect of proliferation induced by JKTBP1. In addition, immunohistochemistry showed that JKTBP1 expression was significantly elevated in AI prostate cancer tissues when compared with the androgen-dependent (AD) prostate cancer and benign prostatic hyperplasia. Our data indicated that overexpression of JKTBP1 in LNCaP cells leads to abnormal cell proliferation and may be involved in the process of AD to AI through induction of EGF-R expression.     

The application of the RT-PCR method for the staging of the prostate cancer progression

Molecular biology seems to bring more convincing markers for the detection of prostate cancer as well as the development of metastases than immunohistochemistry. The main goal of present work was to detect the expression of prostate specific antigen (PSA) and prostate-specific membrane antigen (PSM) genes in the micrometastases by the RT-PCR to assess the progression of prostate cancer. We analyzed 50 patients: 28 patients with clinically localized or locally advanced prostate cancer who underwent radical prostatectomy, 7 patients with clinically proven metastases, 8 patients with benign prostatic hyperplasia, and 7 healthy young men. The results of RT-PCR in the first group of 28 patients varied, however, they were in good correlation with the health status of the patients. Positive results of PSA and notably for PSM were good predictors of beginning metastasing process. Seven patients with metastatic disease had positive RT-PCR results both for PSA and PSM. All of the patients with benign prostatic hyperplasia and healthy young men had negative RT-PCR results for PSA and PSM. The study showed that positive RT-PCR results for PSA and especially for PSM correlated well with the progression of the disease and negative results reflected good health status of the patients.     

Beyond prostate-specific antigen: new serologic biomarkers for improved diagnosis and management of prostate cancer

The use of total prostate-specific antigen (tPSA) measurement has dramatically improved the ability to detect prostate cancer at earlier stages. However, as the number of men presenting with advanced disease (and high tPSA levels) has decreased, and given the fact that tPSA is highly reflective of benign prostatic hyperplasia, the need has emerged for novel biomarkers specifically associated with prostate cancer in order to improve predictive models. Several new biomarkers have shown promise, and studies continue to investigate the role of these markers in the detection, staging, and prognosis of prostate cancer. As new useful biomarkers continue to emerge, guidelines for their employment, as well as coordination of further research studies, are needed; a systematic, phased, nomogram-based model is a rational way to manage these efforts.     

Analysis of relations between serum levels of epitestosterone, estradiol, testosterone, IGF-1 and prostatic specific antigen in men with benign prostatic hyperplasia and carcinoma of the prostate

Epitestosterone competes with testosterone for androgen receptors and inhibits several enzymes of steroidogenesis. Insulin-like growth factors (IGFs) stimulate the growth of prostate cells and directly activate androgen receptors in prostatic tumor cell lines. The prostate-specific antigen (PSA) decreases the affinity of IGF-binding protein-3. The samples were collected from 71 patients suffering from various diseases of the prostate (56 patients without prostate cancer but with benign prostatic hyperplasia and 15 patients with prostate cancer). Correlations between age and IGF-1 (r = -0.281, p<0.05), age and serum epitestosterone (r = -0.261, p<0.05), estradiol and testosterone (r = 0.367, p<0.01), and between estradiol and epitestosterone (r = -0.414, p<0.001) were found. After age adjustment, IGF-I correlated with epitestosterone (r = -0.277, p<0.05). The age correlated positively with PSA (r = 0.286, p<0.05) and negatively with IGF-1 (r = -0.377, p<0.01) in partial correlations. PSA levels were higher in patients with prostate cancer (p<0.00001). Epitestosterone, which is negatively correlating with estradiol and IGF-1, may modulate the development of prostate diseases.     

Nuclear volume estimates in prostatic atypical adenomatous hyperplasia

OBJECTIVE: To investigate nuclear volume estimates by the point-sampled intercepts method in atypical adenomatous hyperplasia (AAH) as compared with nodular hyperplasia and well-differentiated prostatic adenocarcinoma. STUDY DESIGN: The study group consisted of 27 formalin-fixed, paraffin-embedded, whole-mounted radical prostatectomy specimens that contained foci of nodular hyperplasia, atypical adenomatous hyperplasia and well-differentiated adenocarcinoma (Gleason pattern 1 and 2). Representative sections were selected for stereologic estimation of volume-weighted mean nuclear volume by the point-sampled intercepts method. On each focus, an average of five fields of vision were systematically chosen. RESULTS: The quantitative results indicate an increase in nuclear volume from nodular hyperplasia (209 +/- 65 micron 3) to AAH (237 +/- 85 micron 3) and prostate adenocarcinoma (436 +/- 106 micron 3). Significant differences were found (F = 39.0, P < .001) with two group comparisons (Scheffe's procedure) between prostate cancer and AAH (P < .001) or nodular hyperplasia (P < .001). The difference between AAH and benign hyperplasia was not signifcant. CONCLUSION: The results indicate that three-dimensional estimates of the nuclear size discriminate AAH and nodular hyperplasia from well-differentiated prostate adenocarcinoma. These findings suggest that AAH is probably a histologic variant of benign prostatic hyperplasia the exact relationship of which to prostatic adenocarcinoma remains to be determined.     

Adiponectin as a potential marker of prostate cancer progression: studies in organ-confined and locally advanced prostate cancer

Serum levels of adiponectin were measured in patients with benign prostatic hyperplasia and prostate cancer of pT2 and pT3 stage. Adiponectin ELISA assay, immunohistochemistry, and selected metabolic and biochemical parameters measurement was performed in 25 patients with benign prostatic hyperplasia and 43 with prostate cancer (17 patients with organ-confined and 26 patients with locally advanced disease). Serum adiponectin levels did not differ between prostate benign hyperplasia and cancer clinical stage T2, but was significantly higher in pT3 relative to pT2 group (14.51+/-4.92 vs. 21.41+/-8.12, P = 0.003). Tissue immunohistochemistry showed enhanced staining in neoplastic prostate glands and intraepithelial neoplasia relative to benign prostatic hyperplasia without distinction between disease grade and stage. Serum adiponectin levels are higher in locally advanced relative to organ-confined prostate cancer and may thus serve as an auxiliary marker providing further improvement for discrimination between pT2 and pT3 stages.     

Carbohydrate structure and differential binding of prostate specific antigen to Maackia amurensis lectin between prostate cancer and benign prostate hypertrophy

Serum prostate-specific antigen (PSA) assay is widely used for detection of prostate cancer. Because PSA is also synthesized from normal prostate, false positive diagnosis cannot be avoided by the conventional serum PSA test. To apply the cancer-associated carbohydrate alteration to the improvement of PSA assay, we first elucidated the structures of PSA purified from human seminal fluid. The predominant core structure of N-glycans of seminal fluid PSA was a complex type biantennary oligosaccharide and was consistent with the structure reported previously. However, we found the sialic acid alpha2-3 galactose linkage as an additional terminal carbohydrate structure on seminal fluid PSA. We then analyzed the carbohydrate moiety of serum PSA from the patients with prostate cancer and benign prostate hypertrophy using lectin affinity chromatography. Lectin binding was assessed by lectin affinity column chromatography followed by determining the amount of total and free PSA. Concanavalin A, Lens culinaris, Aleuria aurantia, Sambucus nigra, and Maackia amurensis lectins were tested for their binding to the carbohydrates on PSA. Among the lectins examined, the M. amurensis agglutinin-bound fraction of free serum PSA is increased in prostate cancer patients compared to benign prostate hypertrophy patients. The binding of PSA to M. amurensis agglutinin, which recognizes alpha2,3-linked sialic acid, was also confirmed by surface plasmon resonance analysis. These results suggest that the differential binding of free serum PSA to M. amurensis agglutinin lectin between prostate cancer and benign prostate hypertrophy could be a potential measure for diagnosis of prostate cancer.     

fucosyltransferase1 and H-type complex carbohydrates modulate epithelial cell proliferation during prostatic branching morphogenesis

The prostate undergoes branching morphogenesis dependent on paracrine interactions between the prostatic epithelium and the urogenital mesenchyme. To identify cell-surface molecules that function in this process, monoclonal antibodies raised against epithelial cell-surface antigens were screened for antigen expression in the developing prostate and for their ability to alter development of prostates grown in serum-free organ culture. One antibody defined a unique expression pattern in the developing prostate and inhibited growth and ductal branching of cultured prostates by inhibiting epithelial cell proliferation. Expression cloning showed that this antibody binds fucosyltransferase1, an alpha-(1,2)-fucosyltransferase that synthesizes H-type structures on the complex carbohydrate modifications of some proteins and lipids. The lectin UEA I that binds H-type 2 carbohydrates also inhibited development of cultured prostates. These data demonstrate a previously unrecognized role for fucosyltransferase1 and H-type carbohydrates in controlling the spatial distribution of epithelial cell proliferation during prostatic branching morphogenesis. We also show that fucosyltransferase1 is expressed by epithelial cells derived from benign prostatic hyperplasia or prostate cancer; thus, fucosyltransferase1 may also contribute to pathological prostatic growth. These data further suggest that rare individuals who lack fucosyltransferase1 (Bombay phenotype) should be investigated for altered reproductive function and/or altered susceptibility to benign prostatic hyperplasia and prostate cancer.     

Gonadotropin-releasing hormone receptors in prostate tissue

OBJECTIVE: To perform an immunohistochemical analysis of gonadotropin-releasing hormone receptors (GnRH-Rs) in archival prostate tissue. STUDY DESIGN: Thirteen benign prostatic hyperplasia (BPH) specimens from open surgery, 48 radical prostatectomy specimens (30 surgery only and 18 neoadjuvant hormone treatment and surgery) and 14 prostate needle biopsies were examined. The avidin-biotin-peroxidase technique and monoclonal antibody A9E4 against the extracellular domain of GnRH-Rs were employed. Cases with > 5% immunoreactive cells (IR) were considered positive. RESULTS: The epitheliumfrom all 13 cases of BPH was immunoreactive. Most tumor cellsfrom biopsies were IR positive. Twenty-seven of 30 surgery-only specimens were IR positive vs. 8/18 in the surgery and neoadjuvant hormone treatment group. CONCLUSION: GnRH-Rs have been histochemically demonstrated in normal lutenizing hormone/follicle-stimulating hormone pituitary cells. In cell lines LN-CaP and DU-145, Gn-RH-R was identical to that of the pituitary. GnRH-Rs in the prostate can be quite easily assessed immunohistochemically in archival tissue samples, and hormone treatment significantly decreases the immunoreactivity of GnRH-Rs in prostate cancer tissue. This strongly suggests that GnRH agonists bind to BPH and prostate cancer cells.