Biochemical nature of the prostate-associated antigen identified by the monoclonal antibody, KR-P8.

The KR-P8 monoclonal antibody identifies an organ-specific antigen that is associated with normal as well as malignant specimens of human prostate tissue. The antigen is secreted by cells of the prostate and is present in samples of seminal plasma. Data presented here describe the biochemical nature of the antigen that is recognized by KR-P8 as it occurs in seminal plasma and in extracts prepared from cells of the prostate tumor line, PC3. Antigen contained in seminal plasma migrated as a broad band on SDS-polyacrylamide gels in the molecular weight range of 48,000-75,000 d. A similar pattern was observed for antigen prepared from detergent extracts of PC3 cells. The antigen was found to be sensitive to treatment with trypsin and chymotrypsin and the contribution of carbohydrate residues to the structure of the molecule was shown by studies that demonstrated binding of the antigen to Concanavalin A and Soybean Agglutinin lectins. Loss of antigenicity subsequent to periodate oxidation suggested that carbohydrate units are involved in the recognition site for KR-P8 on the antigen.

Localization of a normal prostatic secretory product using the monoclonal antibody KR-P8.

We previously reported that the monoclonal antibody KR-P8 detects a prostate organ-specific antigen that is distinct from other markers such as prostatic acid phosphatase and prostate specific antigen. In this report we demonstrate that the antigen recognized by KR-P8 is among the secretory products of both normal and malignant prostatic epithelium. Immunoperoxidase staining patterns showed that the antigen was concentrated on the luminal surfaces of the glandular epithelial cells of prostate, and also that the antigen was localized within secretory vacuoles of cells of the prostate tumor line PC3. In addition, using an immunoblotting assay the KR-P8 binding antigen was detected in the growth media of PC3 cells and also found to be present in human urine and seminal plasma. The data suggest that the antigen recognized by KR-P8 may be a useful marker for studying the secretory processes of the prostate gland.

Ultrastructural analysis of acute lymphoblastic cells: peanut lectin binding correlates with degree of differentiation of the leukemic cells.

Peanut agglutinin (PNA) has been shown to bind selectively to immature cells. Bone marrow cells from some children having acute lymphocytic leukemia (ALL) bind PNA while cells from other ALL patients do not bind, the significance being that the patients whose cells bind PNA have a poorer prognosis than those not binding PNA. In the present study, PNA was conjugated to horseradish peroxidase and the two cell types were compared. Cells binding PNA are immature compared with the non-PNA-binding cells.

Characterization of a monoclonal antibody, KR-P8, that detects a new prostate-specific marker.

This report characterizes a prostate-specific monoclonal antibody, KP-P8, which was prepared against the human prostate cell line PC3. The antigen detected by KR-P8 was identified on the surfaces of 90% of cells of the PC3 line, as well as on 67% of cells of the Du-145 prostate line, but it was absent from the surfaces of normal peripheral blood leukocytes and cells of a number of lymphoblastoid lines. As judged by immunoperoxidase staining techniques, KR-P8 reacted with the glandular epithelium of all specimens of normal, benign hypertrophic, and malignant prostate glands tested. However, no reactivity was noted with numerous other human tissues including normal bladder, lung, liver, kidney, testis, colon, parotid gland, thyroid gland, and spleen. These results indicate that the antigen detected by KR-P8 is prostate organ-specific. Competitive blocking studies showed that the antibody did not recognize the previously described prostate-specific antigen or the alpha-Pro-3 antigen described by other investigators. The KR-P8 antibody also did not bind to purified prostatic acid phosphatase. The presence of the KR-P8 antigen was demonstrated in cell-free preparations of dilute seminal plasma by radioimmunoassay, indicating that this antigen is secreted by the glandular cells of the prostate gland. The clinical significance of this marker was demonstrated by its ability to identify prostate metastases of the lymph node.

Effect of fibronectin on macrophage-induced tumor cell cytostasis.

Purified fibronectin (Fn) mediated attachment of MCG-T14 cells, a mouse adenocarcinoma, to culture vessel surfaces. Concentrations of Fn less than 30 micrograms/ml enhanced the growth rate of these cells as judged by 3H-thymidine incorporation, whereas higher levels of Fn were inhibitory. Concentrations of Fn and macrophages, which had little or no effect on the growth rate of the T14 cells when added individually, mediated a 64% decrease in the rate of growth of these cells when cultured together. Free Fn was not required for this effect since target cells pretreated with Fn and then washed also were susceptible to growth inhibition by macrophages. These results indicate that Fn is able to mediate an enhancement of macrophage antitumor activity, probably by supporting the binding of target cells to the macrophage effector cells.

Fibronectin binding to protein A-containing staphylococci.

Fibronectin (Fn) was found to bind to protein A-containing isolates of Staphylococcus aureus, but not to mutant strains devoid of this protein nor to clinical isolates of S. epidermidis. Fn was purified from human plasma by affinity chromatography on gelatin-Sepharose. After elution with 4 M urea, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified material detected no immunoglobulin contamination. This purified Fn was radiolabeled with 125I and used in binding assays. Quantitatively, Fn binding was directly correlated with the cellular protein A content of the various strains tested. Mannitol salt broth preculture or organisms resulted in a reduction of their cellular protein A and a decrease in Fn binding by these cells. However, soluble protein A maximally inhibited the binding of radiolabeled Fn to protein A-positive strains of staphylococci by only 50%, indicating the possibility of multiple Fn binding sites. Fn's binding to protein A-containing S. aureus strains may play a role in the pathogenicity of these organisms by promoting their attachment to and subsequent invasion of host tissues.

Oxacillin-induced lysis of Staphylococcus aureus.

Six clinical isolates of Staphylococcus aureus were compared for their relative susceptibilities to the killing effects of oxacillin. Three of the strains had minimum bactericidal concentrations which were >10 times the minimum bacteriostatic concentration for this antibiotic and were designated tolerant (Tol(+)). The other strains had minimum bactericidal concentrations which were comparable to the minimum bacteriostatic concentration (Tol(-)). Lysis curves of these strains revealed that the Tol(+) strains exhibited a diminished rate of lysis when inhibited by oxacillin. This reduced rate of lysis was reflected also in a reduced rate of viability loss when the cells were exposed to oxacillin. During log growth the uptake of [(14)C]glycerol by Tol(+) cells was 1.5-fold greater than that by Tol(-) cells. Glycerol-labeled cells of each phenotype secreted radioactivity when inhibited by oxacillin. However, the Tol(+) strains released over twice as much label as the Tol(-) strains. No difference in the proportion of lipid secreted by the two phenotypes was found. The behavior of 60 to 65% of the labeled material released by inhibited cells during both sodium dodecyl sulfate gel electrophoresis and Sepharose 6B chromatography corresponded to that of lipoteichoic acid. When the major component of secreted material was added to oxacillin-inhibited Tol(-) strains, an inhibition of the lytic response was observed. These results suggest that oxacillin tolerance in S. aureus could be related to the enhanced secretion of an autolysin inhibitor, such as lipoteichoic acid.