The expression of the six known insulin‐like growth factor binding proteins (IGFBPs) and their corresponding messenger RNAs has been examined in three cell lines established from surgical and biopsy specimens of human prostate carcinoma. All three cell lines produced both IGFBP‐4 and IGFBP‐6 and the respective mRNAs; expression of IGFBP‐6 has not been previously demonstrated in human prostate tumor cells. No other binding proteins were detected. The levels of IGFBP mRNAs were not regulated by androgens or IGF‐1, but the level of IGFBP‐6 mRNA was sharply increased by 1,25‐dihydroxyvitamin D3 (1,25(OH)D3). The stimulation was dose‐dependent with a maximum effect at 10 nM 1,25(OH)D3 and a clearly discernible effect at 0.1 nM. The results support a role for vitamin D in the control of prostate tumor growth, mediated at least in part by interaction with IGFs and specific IGFBPs.

The effect of [D‐Leu6, des‐Gly‐NH210, Proethylamide9]‐GnRH, leuprolide, was determined for the human primary prostate tumor cell line ALVA‐31 by in vitro mitogenic assays. Prostate tumor cell proliferation was inhibited up to 50% by leuprolide. Inhibition was not observed in parallel cultures treated with other low molecular weight bioactive peptides. The incorporation and metabolic reduction of testosterone was not affected by concentrations of leuprolide that were inhibitory in the mitogenic assay. Specific high‐affinity binding of 125I‐labeled leuprolide was also demonstrated on intact tumor cells with an estimated effective median dose (ED50) of <1 × 10–9M. Inhibition of prostate tumor growth was further demonstrated in Balb/c athymic intact and castrate male mice bearing ALVA‐31 tumor xenografts following chronic administration of leuprolide. These data clearly demonstrate that leuprolide can inhibit the growth of a human prostate carcinoma cell line. Studies conducted in castrate animals further suggest an alternative mechanism of growth inhibition that appears to be independent of the suppression of steroid hormone biosynthesis by LHRH analogues.

A new human prostate tumor cell line (ALVA-31) has been established from a biopsy specimen of primary tumor obtained during prostatectomy. The cell line has been maintained for more than 48 months in stable growth. The in vitro doubling time was determined to be approximately 26 hr. The chromosome number ranged from 24-112, with a modal number of 59 tested over several time points throughout continuous culture. Karyotypic analysis of late-passaged cells demonstrated approximately 70 human chromosomes, 8-14 markers, and two X chromosomes without a Y chromosome. Prostatic origin was confirmed by the expression of both prostate specific antigen and prostatic acid phosphatase, using specific antisera and immunoradiolabelling techniques. Prostate tumor xenografts were grown in intact male, castrate male, and female athymic mice; however, the rate of tumor growth was clearly dependent upon serum testosterone levels.

The purpose of this study was to determine what effects sex hormone binding globulin (SHBG) might have on the growth and steroid content of human prostate carcinoma. Two human prostate carcinoma cell lines were used for this study, ALVA-41 and ALVA-101. The first part of the study was to determine the effect of SHBG or albumin on the uptake of [3H]DHT in the cells. In this experiment both SHBG and albumin inhibits the uptake of [3H]DHT into each of the cell lines when studied in vitro. The degree of inhibition was dependent on the binding capacity of the protein. When [3H]thymidine uptake was measured in each of the cell lines following either the addition of SHBG or albumin to the culture media, an increase in uptake and presumably DNA synthesis was noted in the ALVA-41 and ALVA-101 cells for SHBG additions but not for albumin. Further, this stimulation was increased when testosterone was added to the media, however, [3H]thymidine uptake was decreased by high concentrations of dihydrotestosterone (DHT) or if the SHBG was saturated with DHT prior to being added to the media. The cells also demonstrate high affinity cell membrane receptors for SHBG. Finally, using a 3', 550 bp cDNA or SHBG, 1.9 and 2.8 kb mRNAs were detected on Northern analysis of the ALVA-101 and ALVA-41 cells. These data indicate SHBG can inhibit uptake of steroids into the prostate, but also it may act as a stimulus for growth through a SHBG cell surface receptor. In addition, the growth effect may be through an autocrine effect from SHBG or a SHBG-related peptide.

The W3/25+ T cell subset in rats, defined by a xenogeneic monoclonal antibody and separated by using the FACS, was demonstrated to be the proliferating cell type in the in vitro MLC and to provide an amplifier function for the W3/25- T cell subset in the generation of cytotoxic effector cells to alloantigenic targets.

A factor extracted from syngeneic thymic lymphoid cells (thymocytes) is shown to amplify the proliferative (MLC) response of syngeneic lymphoid cells to alloantigen in vitro. The optimal conditions for an effect of the thymus factor are quantitatively defined by kinetic and dose-response studies. Other variables that could potentially influence the activity of the thymus factor, such as the presence of 2-mercaptoethanol and the source of alloantigen, are identified. Factor activity can be recovered from semi-allogeneic thymocytes, as well as syngeneic thymocytes. The factor appears to predominantly effect the proliferative response of T cells localized in peripheral lymphoid organs. As such, this factor appears to be distinct from the variety of previously described factors derived from thymic reticuloepithelial elements that are thought to primarily induce the differentiation of T cell precursors found predominantly in bone marrow. Several possible mechanisms of action of this thymocyte-derived factor are considered.

A synergistic interaction between rat lymphoid cell subsets was found for the in vitro immune response to the syngeneic Gross virus-induced lymphoma (C58NT)D. Mixtures of thymocytes and lymph node cells from inbred WF rats primed in vivo to the lymphoma demonstrated significantly greater proliferative and cytotoxic reactivities in vitro than would be expected from the sum of the reactivities of the two cell types tested separately. A soluble extract of nonimmune syngeneic thymocytes, shown in previous studies to amplify the in vitro responses to alloantigen, was also demonstrated to increase significantly proliferative and cytotoxic responses in vitro to syngeneic lymphoma cells. Preliminary evidence indicated that this soluble thymus amplifier factor differed from previously described thymus factors.

A synergistic interaction in the proliferative response to alloantigen is described for mixtures of rat thymus and lymph node cells. The optimal conditions for synergy are quantitatively defined. Regression analysis of the slope of the dose-response curve has been utilized to estimate the degree of interaction in thymus-lymph node cell mixtures. The slope of the response of cell mixtures was noted to be significantly greater than the slope for the response of lymph node cells alone. Irradiation was shown to have a differential effect on the response of thymus and lymph node cells in mixtures. Irradiated thymus cells retained the capacity for synergy in mixtures, whereas irradiated lymph node cells did not. Additional studies have demonstrated that both de novo protein synthesis and specific antigen recognition by both responding cell populations in mixtures was required for maximal synergy. These studies demonstrate that synergy cannot be explained as an artifact of altered cell density in vitro. They establish that thymus cells and lymph node cells represent distinct subsets which manifest qualitatively different functions in the proliferative response to alloantigen. Thymus cells can respond directly to alloantigen by proliferation but also have the capacity to amplify the proliferative response of lymph node cells—a capacity which is resistant to X irradiation but requires recognition of alloantigen and de novo protein synthesis. Lymph node cells may similarly respond by proliferation to alloantigen but lack the amplifier activity of thymus cells. Synergy for rat lymphoid cells, like mouse lymphoid cells, has been shown to involve an interaction of thymus-derived lymphocytes.

A synergistic interaction in the proliferative response to alloantigen has been previously noted when intact thymus cells are cultured with post-thymic (peripheral) lymphoid cells. In the present study, a factor extracted from the thymus has been shown to similarly enhance the reactivity of syngeneic lymph node cells and thus to retain the amplifier activity of intact thymus cells. The factor has no effect on lymphoid cell proliferation in the absence of alloantigen. Cells with amplifier activity are found in highest concentration in the thymus but also may be detected in spleen cells that are nonadherent to nylon wool. The factor is shown in these experiments to be derived from thymic lymphoid cells and to act primarily upon post-thymic (peripheral) lymphoid cells. As such, this factor appears to be distinct from various other thymus factors that have been localized to thymic reticuloepithelial elements and that are thought to effect predominantly the differentiation of T-cell precursors.

Mixed leukocyte culture reactivity was studied in adult W/Fu rats judged to be highly tolerant after the inoculation of (W/Fu X BN)F1 hybrid spleen and bone marrow cells at birth. Reactivity was observed in a majority of tolerant donors tested and documented by quantitative dose-response and kinetic studies. Allograft tolerance cannot be explained by a complete lack of specific immune reactivity to tolerated alloantigens.