Joy L. Ware

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.

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.

cDNA microarray analysis identifies genes induced in common by peptide growth factors and androgen in human prostate epithelial cells

Prostate cancer cells initially require androgen for continued proliferation, but invariably become androgen independent or unresponsive and recur after treatment by androgen ablation. Exploitation of common signaling components downstream of their specific receptors (i.e., androgen receptor (AR), insulin‐like growth factor 1 (IGF‐1) receptor, and epidermal growth factor (EGF) receptor) could provide a mechanism by which androgen independent cells survive and proliferate. Our objective was to design and implement prostate enriched cDNA microarrays to identify genes induced in prostate epithelial cells in a similar temporal pattern by both androgen and IGF or EGF. AR positive and AR negative human prostate epithelial cells of the M12 line were exposed in parallel to DHT, EGF, or IGF for 0, 6, or 24 h. RNA extracted from each of these groups was analyzed by cDNA microarrays composed of a unique set of 6373 prostate‐derived cDNA clones from the Prostate Expression Database (PEDB). We observed statistically significant changes in 20 genes induced in common after 6 and 24 h exposure to androgen or these growth factors, and validated the microarray results by RT‐PCR for three or four of these genes: v‐myc, isocitrate dehydrogenase, and calnexin. Androgen response element binding motifs were identified in the upstream sequence in 16 of these 20 genes. These results provide comprehensive and unique insights into potential mechanisms by which peptide growth factors provide alternate pathways to control prostate epithelial cell proliferation in malignant states.

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.

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.

A review: prostate monoclonal antibodies ― the magic bullet

SummaryTarget-oriented monoclonal antibodies have been promoted as cell-specific anti-cancer agents. However, target specificity and cell-specific toxicity are two problems which have provided difficulty. The production of a hybrid monoclonal antibody, bimolecularly specific for a tumor cell surface antigen and the cell toxin ricin A-chain, has provided a mechanism by which the theoretic cell-kill specific potential of monoclonal antibodies may be exploited.