Thiophilic interaction chromatography of prostate-specific antigen.

Prostate-specific antigen (PSA) protein and complexes of PSA with alpha1-antichymotrypsin (PSA-ACT) or alpha2-macroglobulin (PSA-A2M) prepared in vitro, have strong affinity for different thiophilic gels (T-gel). Free PSA, and these PSA complexes can be isolated due to their affinity for T-gels. The average recovery of PSA from several of the T-gels, based upon ELISA measurements, was 84 to 94%. The data suggest that T-gel affinity can be explored for the purification of free and complexed PSA from various biologic fluids.

Plasmin, substilisin-like endoproteases, tissue plasminogen activator, and urokinase plasminogen activator are involved in activation of latent TGF-beta 1 in human seminal plasma.

We reported previously that TGF-beta 1 is a major immunosuppressive agent in human seminal plasma. TGF-beta 1 in seminal plasma is so abundant that it may represent the highest physiologic concentration of TGF-beta 1 reported for a biological fluid. The in vitro activation of TGF-beta 1 is detected at acidic pH. The acidic environment of the vagina is suggested as an in vivo physiological condition for the activation of seminal plasma latent TGF-beta 1. The present study demonstrates that Pefabloc [4-(2-aminoethyl)-benzenesulfonyl fluoride AEBSF]-inhibitable serine proteases are involved in the activation of latent TGF-beta 1. Pefabloc inhibits latent TGF-beta 1 activation in a dose- and time-dependent manner. The use of other protease inhibitors and specific antibodies reveals that, in addition to plasmin, substilisin-like endoproteases and tissue- and urokinase-type plasminogen activators participate in the activation of latent TGF-beta 1 in human seminal plasma.

Localization of seminal plasma transforming growth factor-beta1 on human spermatozoa: an immunocytochemical study.

OBJECTIVE: To localize immunocytochemically transforming growth factor beta1 (TGF-beta1) in human spermatozoa. DESIGN: Incubation of spermatozoa with anti-TGF-beta1 antiserum at various pH. SETTING: Human volunteers in an academic research institute. PATIENTS: Young healthy fertile men. INTERVENTIONS: Semen specimens were collected. RESULTS: At neutral pH and at physiological pH of seminal plasma, TGF-beta1 immunostaining was detected predominantly at the postacrosomal region of the head, at the neck, and at the middle piece of the tail. Transforming growth factor-beta1 also was found occasionally at the axial filament complex of the connecting piece and the ring. The acrosomal cap section of the head, the principal piece, or the end piece of the tail were immunocytochemically negative for TGF-beta1. The TGF-beta1 immunostaining pattern at acidic pH was similar to that at neutral pH and at physiological pH of seminal plasma, but a greater intensity of immunostaining was found at acidic pH than that at neutral pH. CONCLUSION: These results suggest that an in vivo activation of seminal plasma latent TGF-beta1 may take place in the acidic environment of vagina, which results in a greater amount of activated TGF-beta1 and, in turn, with an enhanced "coating" of TGF-beta1 to spermatozoa.

Detection of soluble P-glycoprotein in culture media and extracellular fluids.

Multidrug resistance (MDR) is a unique phenomenon in cancer patients and is commonly associated with an overexpression of the human MDR gene mdr1, which encodes an energy-dependent Mr 180 kDa membrane bound protein, known as P-glycoprotein. P-glycoprotein serves as a membrane efflux to pump the drugs out of the cancer cells. Western blot analysis, using a newly generated monoclonal antibody F4 which recognizes specifically an extracellular epitope of human MDR1 P-glycoprotein, reveals that soluble P-glycoprotein is detected in the cultured media of viable adriamycin-resistant human ovarian carcinoma 2780AD cells, whereas those of the drug-sensitive parent A2780 cells contain no detectable level of soluble P-glycoprotein. Soluble P-glycoprotein also is detected in extracellular fluids of cancer patients, such as malignant ascites and serum, and is not detectable in serum samples of normal healthy individuals. The Mr of soluble P-glycoprotein is the same as that of membrane bound P-glycoprotein. The presence of soluble P-glycoprotein in extracellular fluids may provide the basis for its use as a quantitative parameter of MDR and as a means to lessen or reverse MDR.

Characterization of a new monoclonal antibody F4 detecting cell surface epitope and P-glycoprotein in drug-resistant human tumor cell lines.

Using viable adriamycin resistant human ovarian carcinoma cells 2780AD and colchicine resistant human oral epidermoid carcinoma cells KB-24 as the immunogen in primary and subsequent i.p. immunizations, followed by i.v. boostings with crude plasma membranes of 2780AD, KB-24, Chinese hamster lung cells resistant to vincristine DC-3F/VCRd-5L, and resistant to daunorubicin DC-3F/DMXX, we have generated a new murine monoclonal antibody (McAb), designated F4, of IgG1 isotype. McAb F4 reacted strongly with a cell surface epitope of drug resistant cells and insignificantly with their drug sensitive counterparts. Cell surface localization of F4 epitope was determined by immunofluorescence and laser scanning confocal imaging system. Results obtained from immunoprecipitation and immunoblot analyses using F4 and mdr1 P-glycoprotein specific McAb JSB-1 demonstrated the reactivity of P-glycoprotein with F4. These results along with those obtained from competitive binding-inhibition, chemical modification, and enzyme hydrolysis, revealed that McAb F4 detects an extracellular epitope of P-glycoprotein, and is different from other major McAbs directed against P-glycoprotein, e.g. C219, MRK16, JSB-1, HYB-241 and C494. Deduced from the putative structure of mdr1 protein and its orientation in cell membrane, it is proposed that F4 epitope is localized in or near the 3rd, and/or 6th extracellular transmembrane loops of P-glycoprotein.

Mitogenic response of osteoblast cells to prostate-specific antigen suggests an activation of latent TGF-beta and a proteolytic modulation of cell adhesion receptors.

During studies of mitogens in prostate, PSA quantities as low as 2.5 ng/mL caused cultured osteoblast cells to proliferate beyond controls (p = 0.05). Investigation of this novel mitogenicity suggested the use of several mechanisms by PSA, namely: 1) the activation of latent hTGF-beta in PC-3 conditioned medium, PSA treated conditioned medium stimulated DNA uptake in UMR-106 cells to 78% of acid treated conditioned medium, while DNA incorporation was less than controls with anti-hTGF-beta neutralizing IgG; and 2) the proteolytic modulation of cell surface receptors with temporary contact inhibition, PSA significantly stimulated cell detachment while hTGF-beta enhanced cell attachment of confluent Saos-2 cells above controls. Clinically, these results suggest that PSA may provide a mechanism for both tumor spread and the osteoblastic metastasis so common to prostate cancer.

Prostate-specific antigenic domain of human prostate specific antigen identified with monoclonal antibodies.

With the use of five murine monoclonal antibodies (1A5, 2A4, 3F1, F5 and 3A12) and an antigen-affinity purified goat polyclonal IgG antibody, the presence of a prostate-specific antigenic domain in human prostate-specific antigen molecule was identified. The results were based upon a series of quantitative competitive inhibition assays of each 125I-labeled monoclonal antibody and polyclonal antibody binding to prostate-specific antigen by unlabeled monoclonal antibodies as inhibitors, and immunohistochemical examination of an extensive panel of human tissue specimens. A cluster of two epitopes that are spatially related or in close topographical proximity and represent a prostate-specific antigenic domain are defined by the monoclonal antibodies 1A5, 2A4, 3F1, and F5, 3A12, respectively.

Monoclonal antibodies to a new antigenic marker in epithelial prostatic cells and serum of prostatic cancer patients.

Stable clones of murine hybridomas 7E11-C5 and 9H10-A4 were obtained following immunization with LNCaP cells. The LNCaP cells were isolated from a human prostatic cancer (Ca). Both hybridomas secreted monoclonal antibodies (MoAb) of the IgG1 subclass which were reactive with the insoluble, cytoplasmic, membrane rich fractions of the immunogen. Neither MoAb reacted with the soluble cytosol of LNCaP cells nor with purified human prostatic acid phosphatase (PAP) nor prostate specific antigen (PSA). MoAb 9H10-A4 reactivity was very narrow and limited to the surfaces of LNCaP cells only. MoAb 7E11-C5 specificity was restricted to human prostatic epithelium, both normal and malignant. Except LNCaP, none of the 32 lines of human normal or neoplastic cells reacted with MoAb 7E11-C5. In a survey of frozen sections from 175 human specimens, positive indirect immunoperoxidase staining was limited to epithelium in all 11 specimens of localized and metastatic CaP, 7 benign prostatic hypertrophy (BPH) cases and 7 normal prostates. None of the 26 various nonprostatic tumors nor 120 out of 122 specimens from 28 different normal organs were reactive. Positive staining occurred in 2 out of 14 normal kidneys. Competitive binding with MoAb 7E11-C5 or its F(ab')2 fragments demonstrated the presence of circulating epitope 7E11-C5 in 20 out of 43 sera from CaP patients. Only 3 out of 66 sera from nonprostatic malignancies reacted. None of 30 normal blood donors sera nor 7 BPH sera were positive. Thus, highly significant (p less than 0.0001) association between diagnosed prostatic cancer and circulating molecules expressing the epitope reactive with MoAb 7E11-C5 was established. Significant probability (p less than 0.05) also suggested that patients with positive ELISA test are more likely to be in progression, than those who are negative. These results suggest that this apparently new antigenic marker may be of clinical potential in CaP.

Cell cultures derived from Wilms' tumour animal model.

Several cell cultures were established from a transplantable Wistar/Furth rat Wilms' tumour. Following cloning by the limiting dilutions method, three morphologically distinct types of cells were identified and preserved for further study of growth regulation in the nephroblastoma model.

A high-resolution study of chromosome changes in a human prostatic carcinoma cell line (LNCaP).

A high-resolution study of chromosomal rearrangements in a human prostatic cancer cell line, LNCaP, has been performed. The cytogenetic analysis revealed a pseudodiploid karyotype and the presence of seven marker chromosomes resulting from five aberrational events. The analysis of four clones derived from the original line showed a near-tetraploid chromosome number and the presence of the same seven markers observed in the original line. This is the first complete description of karyotypic rearrangements in a prostatic cancer cell line.

LNCaP model of human prostatic carcinoma.

The LNCaP cell line was established from a metastatic lesion of human prostatic adenocarcinoma. The LNCaP cells grow readily in vitro (up to 8 x 10(5) cells/sq cm; doubling time, 60 hr), form clones in semisolid media, are highly resistant to human fibroblast interferon, and show an aneuploid (modal number, 76 to 91) human male karyotype with several marker chromosomes. The malignant properties of LNCaP cells are maintained. Athymic nude mice develop tumors at the injection site (volume-doubling time, 86 hr). Functional differentiation is preserved; both cultures and tumor produce acid phosphatase. High-affinity specific androgen receptors are present in the cytosol and nuclear fractions of cells in culture and in tumors. Estrogen receptors are demonstrable in the cytosol. The model is hormonally responsive. In vitro, 5 alpha-dihydrotestosterone modulates cell growth and stimulates acid phosphatase production. In vivo, the frequency of tumor development and the mean time of tumor appearance are significantly different for either sex. Male mice develop tumors earlier and at a greater frequency than do females. Hormonal manipulations show that, regardless of sex, the frequency of tumor development correlates with serum androgen levels. The rate of the tumor growth, however, is independent of the gender of hormonal status of the host.