Karen S. Webb

Spontaneous metastasis of cells of the human prostate carcinoma cell line PC-3 in athymic nude mice.

Spontaneous metastasis and extensive invasiveness were observed in athymic nude mice injected with human prostatic carcinoma cells of the PC-3 line or heterotransplants of nude mouse supported PC-3 tumor. In 3 experimental series, 60, 63 and 50 per cent of the nude mice receiving subcutaneous inoculations of PC-3 cells or tumor heterotransplants developed 1 or more lymphatic tumor metastases. Examination of metaphase-arrested cells recovered from the metastatic sites confirmed the tumor origin as human in each case. Cells recovered from 1 nude mouse supported subline, MPC-3-10, frequently exhibited double minute chromosomes in addition to the typical PC-3 chromosomal profile. These observations provide the foundation for a study of the relationship between prostate carcinoma cell characteristics and lymphatic metastasis in the nude mouse.

Establishment and Characterization of a New Human Prostatic Carcinoma Cell Line (DuPro-1)

A new human prostate adenocarcinoma cell line (DuPro-1) has been established from the athymic nude mouse supported xenograft DU5683. This was accomplished by embedding dispersed xenograft cells in 0.1 by 5.0 cm. spaghetti-like strands of Basement Membrane MATRIGEL [BMM (Collaborative Research, Inc.)], a unique technique facilitating the transition to tissue culture. Now passed over 30 times, the cells display anchorage and serum concentration independent growth with a doubling time of 22 to 24 hours. Cells exhibit pronounced morphological differences when grown on BMM coated culture dishes, assuming a pseudoglandular configuration, in contrast to typical homogeneous monolayer growth on plastic culture dishes. Light and electron microscopy show cohesive sheets of anaplastic epithelial cells, consistent with prostate carcinoma. Karyotypic analysis revealed all human chromosomes, near tetraploidy, 10 to 12 markers, and 3 to 4 X chromosomes, without a Y chromosome. Cells injected s.c. or embedded in BMM and implanted in the subrenal capsule space are equally tumorigenic in male and female athymic mice, suggesting that DuPro-1 cells are hormonally insensitive. Embedding cells in BMM may be useful in developing other tissue culture cell lines from neoplasms difficult to initiate in vitro. DuPro-1 should provide a valuable means to study the biology, immunology, and chemosensitivity of human prostate cancer.

Monoclonal antibodies to different epitopes on a prostate tumor-associated antigen

SummaryMouse monoclonal antibodies αPro3 and αPro5 bind to different epitopes on an antigen (p54) of 54 kD reduced and 175 kD nonreduced MW. p54 antigen has been characterized previously with regard to tissue distribution using αPro3 monoclonal antibody; the p54 antigen is present in substantially greater quantities in malignant prostatic tissue extracts than in benign prostatic and nonmalignant nonurogenital tissue extracts. In this report, we have established that αPro5 and αPro3 monoclonal antibodies exhibit same molecule-different epitope recognition. That both antibodies recognize the same molecular entity has been established by partial physicochemical characterization of the antigen recognized by the two antibodies and by sequential immunoprecipitation experiments. Different determinant recognition was established by lack of competitive surface binding between αPro3 and αPro5 to a prostatic carcinoma cell line. The p54 antigen can be labeled with glucosamine and immunoprecipitated from urea-solubilized membrane proteins; however, p54 cannot be detected by immunoprecipitation in a glycosylated form in spent culture medium removed from glucosamine-labeled cells. Experiments using indirect cellular immunoassays and directly radioiodinated monoclonal antibody have shown that both αPro3 and αPro5 form stable complexes with p54 antigen on the prostatic carcinoma cell surface. To the extent that modulation occurs upon interaction of p54 with αPro3 and αPro5, endocytosis of the immune complex appears to be the primary route of modulation. Furthermore, modulation by endocytosis is more intense when αPro3 and αPro5 are used in combination than when either monoclonal antibody is used alone. Although in vivo biologic behavior does not invariably correlate with in vitro behavior, careful in vitro analysis of monoclonal antibodies with respect to cell surface behavior, nevertheless, should precede in vivo evaluation. The data presented in this report indicate that preliminary in vitro analyses will expedite the effectiveness of in vivo immunotherapeutic trials; preliminary in vitro evaluations are absolutely essential if monoclonal-toxic agent (e.g., ricin A) conjugates, which must be internalized by the tumor cell to achieve cytotoxicity, are employed as immunotherapeutic agents.

Characterization of prostate-tissue-directed monoclonal antibody, α-Pro 13

SummaryThe α-Pro 13-secreting hybridoma was produced by immunizing mice with an equal mixture of PC-3, DU145, and LNCaP established prostatic carcinoma cell lines. The specificity of α-Pro 13 monoclonal antibody was evaluated by the criteria of differential binding to cultured cells; differential binding to extracts of malignant prostate, nonmalignant prostate, and malignant and nonmalignant tissues of various histiotypes in solid phase radioimmunoassay; and by immunoperoxidase staining of primary surgical tissues of varied histiotypes. The data generated by multiple assay investigation indicate that α-Pro 13 exhibits preferential binding to the ductal epithelium of prostate tissue; immunoperoxidase evaluation indicates a considerable heterogeneity of staining of ductal epithelial cells. The most prevalent cross-reactivity of α-Pro 13 monoclonal antibody with non-prostate tissue occurs with blood vessel endothelium of restricted tissues. Electrophoretic analysis of immunoprecipitates from radioiodinated prostatic tumor extracts indicates that the molecule recognized by α-Pro 13 is of 120,000 dalton apparent nonreduced molecular weight. Under reducing conditions, the antigen (p40) consists of a major component of 40,000 dalton apparent MW and a minor component of 17,000 dalton MW. p40 has an isoelectric point of 3.5–4.5. The antigen is intrinsically stable on the PC-3 cell surface; its release into spent culture medium is negligible. p40 is also stable upon complexation with α-Pro 13 antibody in that it is not shed from the cell surface as an immune complex nor is it endocytosed to any extent as an immune complex.

In vitro synergism between hybrid immunotoxins and chemotherapeutic drugs: relevance to immunotherapy of prostate carcinoma

SummaryCultured prostate carcinoma cells incubated in the presence of a novel hybrid immunotoxin and ricin A chain exhibited synergy with the chemotherapeutic drugs vinblastine, methotrexate, and bleomycin. No cooperative effect was noted with adriamycin. Under conditions where individual components of immunotoxin or chemotherapeutic drug mixtures were nontoxic or minimally toxic the immunotoxin-drug mixture exhibited marked impact on 14C amino acid incorporation into prostate carcinoma cells. Analysis of drug-treated cells by flow cytometry indicated that cells exposed to vinblastine and bleomycin bound hybrid immunotoxin antibody to a greater extent than cells not exposed to these drugs. Adriamycin did not exhibit synergistic cytotoxicity with hybrid immunotoxin. Also, adriamycin did not enhance antibody binding as evaluated by flow cytometry. The fact that hybrid monoclonal antibody-ricin A chain (HIT-RAC) conjugates inhibited uptake of 14C amino acids 3 to 10-fold within 48 h of incubation with target cells and that this inhibition was further increased 2 to 3-fold in conjunction with three out of four chemotherapeutic drugs tested may be attributed to the unique cytotoxicity imposed by the hybrid immunotoxins. The RAC moiety is not chemically coupled to antibody but instead occupies one of the antigen-combining sites of the molecule. In this manner, RAC is closely juxtaposed to the cell membrane of the target cell and is anchored in this position via binding of the remaining antigen-combining site to p40 prostate restricted antigen.

Metastatic phenotype of human prostate tumor cells in athymic nude mice: Alteration by exposure to ethyl methanesulfonate and “reversion” by 5-azacytidine

SummaryThe human prostate tumor subline 1-LN-PC-3-1A (1-LN) is reproducibly metastatic in adult athymic nude mice. Cells surviving a brief in vitro exposure to ethyl methanesulfonate (EMS) exhibited a profound decrease in capacity for experimental lung metastasis in nude mice. Thirty days after EMS treatment, 1×106 uncloned EMS-treated 1-LN cells (1-LN-EMS-10) were injected IV into groups of 6 to 8-week-old male athymic nude mice (BALB/cAnBOM). A median of 8.5 colonies/lung was observed among 20 1-LN-EMS-10-injected mice, which was significantly different from the median of 51 colonies/lung produced among 14 1-LN-injected mice (P=0.0002). This altered phenotype remained stable during 150 days of continuous culture. However, the 1-LN-EMS-10 cells were tumorigenic in 10/10 nude mice injected SC. Single lung tumor colonies recovered from 1-LN-EMS-10-injected mice and reinjected IV into nude mice produced medians of 32–63 colonies/lung. The altered metastatic phenotype resulting from treatment of 1-LN with EMS was reversed by exposure to a noncytotoxic dose of 5-azacytidine, but unaffected by a second exposure to EMS. Collectively these data demonstrate that the metastatic phenotype of these human tumor cells in athymic nude mice can be heritably altered by in vitro exposure to EMS and 5-azacytidine. Analysis of the mechanisms underlying these phenotypic changes may provide insight into parts of the complex process of tumor cell evolution.

Rationale for immunotoxin therapy of metastatic prostate carcinoma formatted as a multi-stage delivery system.

A family of triple hybridomas secreting hybrid monoclonal antibodies has been developed in our laboratory. The hybrid monoclonal antibodies exhibit bimolecular specificity towards both antigenic determinants on the prostate carcinoma cell surface as well as toxin or toxic moieties (ricin A chain and pokeweed antiviral protein). These hybrid antibodies, when bound univalently to their cognate toxin, constitute the primary reagents responsible for selective in vitro prostate carcinoma cell kill; oncolytic impact is achieved by binding of the hybrid antibody-toxin complex (primary hybrid immunotoxin) to a prostate carcinoma cell surface-expressed antigen by the remaining univalent binding site of the hybrid antibody, allowing access of the toxin to the cytosol by internalization of the hybrid antibody-toxin complex. Internalization by endocytosis of a hybrid antibody-toxic subunit has been strikingly enhanced by the use of secondary monoclonal antibody reagents alone or in conjunction with other biomodifier reagents. For example, use of a second monoclonal antibody specific for ricin A chain to which ricin B chain (binding subunit) is chemically coupled results in selective and synergistic cell kill of targeted cancer cells. In vitro studies involving temporally staggered exposure of the cells to the individual components (primary hybrid antibody, toxin, and secondary antitoxin monoclonal antibody biomodifier) have been performed in a manner allowing maintenance of cytotoxic efficacy. It is concluded that sequential administration of these immunotherapeutic components, individually nontoxic, is a feasible strategy to develop an effective immunotherapeutic treatment of human prostate carcinoma.

Correlation of apparent molecular weight and antigenicity of viral proteins: An SDS-PAGE separation followed by acrylamide-agarose electro-phoresis and immunoprecipitation☆

A simple method is described which combines a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SOS-PAGE) in the first demension with a second electrophoresis, at right angles to the first, into an agarose matrix. The proteins, separated by SDS-PAGE, are exposed to appropriate antisera after the second stage electrophoresis and immunoprecipitates form in the agarose corresponding to the relative electrophoretic mobilities of proteins in the first stage SDS-PAGE separation. The method thus provides a simple, reproducible means for correlating antigenicity with apparent molecular weight of proteins. The technique is qualtitative, but requires smaller quantities of antisera than more conventional immunoelectrophoretic methods such as rocket electrophoresis.

1,10-Phenanthroline reversibility inhibits proliferation of two human prostate carcinoma cell lines (PC-3 and DU145)

The metal chelator 1,10-phenanthroline reversibly inhibits proliferation of two human prostate carcinoma cell lines, PC-3 and DU145. Inhibition is dose-dependent, and persists for several days after removal of the chelator. The ability to induce reversible quiescence in human prostate carcinoma cells in vitro may facilitate the study of their growth cycle biochemistry.

Surface proteins of a transitional carcinoma cell line (KS-31E).

SummaryBiosynthetic and post-synthetic labeling procedures have been used to examine the cell surface proteins of a cell line (KS-31) originating from a human transitional cell carcinoma. One isolate of this cell line (KS-31E) is epithelial in appearance and has retained the characteristic of the original tumour with regard to synthesis of a viscous mucin-like material. A 220–250, 000 (250K) dalton large external transformationsensitive (LETS) glycoprotein, and often a 95, 000 (95K) dalton glycoprotein, is released into the medium of KS-31E in association with the mucinlike material produced by the cell line. The finding of LETS and a 95K glycoprotein expressed on the cell surface and extruded into the growth medium of epithelial cells originating from a spontaneous human carcinoma is of interest in relation to the role of cell surface glycoproteins in normal cellular association and the antisocial behaviour exhibited by cancer cells.