Transforming growth factor beta-1 and beta-2 and type II receptor functional regulation of ALVA-101 human prostate cancer cells.

Transforming growth factor beta-1 (TGFbeta-1) causes apoptosis of many epithelial cells, including the prostate, but other secondary effects of TGFbeta-1 may be important in carcinogenesis. In a human prostate cancer cell line (ALVA-101), we determined the effects of TGFbeta-1 and TGFbeta type I and II receptor antibody on cell proliferation and TGFbeta-1 receptor binding. TGFbeta-1 and -2 and TGFbeta type II receptor mRNA expression levels were determined by polymerase chain reaction (PCR) and Northern blot analysis. A dose-responsive suppression (0.03 to 10 ng/mL) was observed for cells treated with TGFbeta-1 from 3 to 7 days (P < .01). Untreated cells had 1.1 x 10(3) (n = 3) TGFbeta receptors per cell, with a Kd of 0.20 nmol/L (n = 3) as determined by Scatchard analysis; treatment for 3 days with TGFbeta-1 (1 ng/mL) reduced the receptor number (0.9 x 10(3)) and the Kd (0.12 nmol/L). Antibodies to TGFbeta type I and II receptor stimulated proliferation with or without added TGFbeta-1 (50% +/- 5% above control, P < .01, n = 6). TGFbeta-1 and -2 and TGFbeta type II receptor mRNA expression was observed in untreated cells. In cells treated with TGFbeta-1, TGFbeta-1 mRNA was not affected by treatment, but expression levels of the TGFbeta type II receptor and TGFbeta-2 mRNA were moderately suppressed after 72 hours of treatment. Control cells actively produced TGFbeta-1 as measured by radioimmunoassay. The active and inactive forms of TGFbeta-1 were approximately equal, but TGFbeta-2 was secreted in smaller quantities than TGFbeta-1 and the inactive form of TGFbeta-2 predominated, with very small amounts of the active form. Our results suggest that the human prostate cancer cell line ALVA-101 retains negative control of proliferation in response to TGFbeta-1. Inhibition of endogenous TGFbeta action by antibodies to its receptor enhances the growth of ALVA-101 human prostate cancer cells, suggesting that endogenous TGFbeta exerts an inhibitory control on their growth and cellular function.

Growth inhibition of human prostate tumor cells by an agonist of gonadotrophin-releasing hormone.

The effect of [D-Leu6,des-Gly-NH2(10),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 x 10(-9)M. 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.

IGF-binding proteins in human prostate tumor cells: expression and regulation by 1,25-dihydroxyvitamin D3.

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.

Carboxy-truncated insulin-like growth factor binding protein-5 stimulates mitogenesis in osteoblast-like cells.

Recently we demonstrated that a 23 kDa form of IGFBP-5, derived from osteoblast-like cells, stimulates osteoblast mitogenesis and enhances IGF-I action. Because osteoblast-derived IGFBP-5 is smaller than recombinant intact IGFBP-5 (23 vs 30 kDa) and has decreased binding affinity for IGF-I, we proposed that the native 23 kDa form of IGFBP-5 was truncated at a carboxy-terminal position. We now show that a recombinant form of carboxy-truncated IGFBP-5 binds IGF-I with reduced affinity and stimulates mitogenesis in mouse osteoblasts. We also show that 125I-truncated IGFBP-5 specifically binds to osteoblast monolayers with low binding affinity, similar to that seen with native 23 kDa IGFBP-5. These data indicate that carboxy-truncated IGFBP-5 stimulates osteoblast mitogenesis and suggest that reduced IGF-binding and cell-surface attachment are local mediators of this response.

Human primary prostate tumor cell line, ALVA-31: a new model for studying the hormonal regulation of prostate tumor cell growth.

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.

Effects of sex hormone binding globulin (SHBG) on human prostatic carcinoma.

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.

Two human tumor-associated antigens, p155 and p210, detected by monoclonal antibodies.

BALB/c mice were immunized with human melanoma cells and their spleen cells hybridized with NS-1 myeloma cells. The hybrids were screened for the production of antibodies that bound to melanoma cells. Two hybridomas of interesting specificity were identified and cloned. Hybridoma 5.1 produce an IgG1 antibody that binds to about half of the melanomas and carcinomas tested. The target is a polypeptide with an apparent molecular weight of 210 kilodaltons on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The antigen, denoted p210, is also expressed in normal adult brain and in certain fetal tissues. Hybridoma 6.1 produces an IgM antibody that binds to about 50% of the melanomas, and 80% of the kidney carcinomas tested. The antigen defined by this antibody in melanomas has an apparent molecular weight of 155 kilodaltons and is denoted p155. It has not been observed on any normal adult or fetal tissues. The antigen present in the kidney carcinomas was not p155, but rather consisted of two proteins of approximately 60,000 and 250,000-300,000 daltons. This observation suggests the possibility that the antigenic determinant recognized by antibody 6.1 may be present on several distinct protein molecules.

Analysis of normal neoplastic human tissues for the tumor-associated protein p97.

We have used immunoprecipitation to test tumor biopsies and normal adult and fetal human tissues for p97, a tumor-associated protein. Five of nine melanoma biopsies contained p97 in low to very high levels. Three of seven non-melanoma tumors contained p97, but in smaller amounts. No p97 was detected in any of the normal adult tissues examined. The protein was, however, observed in samples of fetal colon and umbilical cord, and in one sample of fetal lung. One of two benign nevi contained high levels of p97, whole on benign angiofibroma was negative. We conclude that the presence of p97, in levels detectable by our method, appears to be characteristic of certain neoplastic and fetal tissues.

T cell synergy in the rat: serologic characterization of T cell subsets.

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.

Synergy in proliferative and cytotoxic responses of WF rats to syngeneic Gross virus-induced lymphoma cells in vitro.

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.

Amplification of the proliferative response to alloantigen by a factor present in an extract of syngeneic thymic lymphoid cells: demonstration of optimal conditions and target cell for factor activity.

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.

Amplification of the proliferative response to alloantigen by a factor present in an extract of syngeneic thymic lymphoid cells.

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.

In vitro reactivity in allograft tolerance: persistence of mixed leukocyte culture reactivity in highly tolerant rats.

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.