Hepatocyte growth factor and vitamin D cooperatively inhibit androgen-unresponsive prostate cancer cell lines.

Expression of MET, the receptor for hepatocyte growth factor (HGF), has been associated with androgen-insensitive prostate cancer. In this study we evaluated MET activation by HGF and HGF action in prostate cancer cell lines. HGF causes phosphorylation (activation) of the MET receptor in three androgen-unresponsive cell lines (DU 145, PC-3, and ALVA-31) together with morphological change. Although HGF is known to stimulate the growth of normal epithelial cells, including those from prostate, we found that HGF inhibited ALVA-31 and DU 145 (hormone-refractory) cell lines. Moreover, HGF and vitamin D additively inhibited growth in each androgen-unresponsive cell line, with the greatest growth inhibition in ALVA-31 cells. Further studies in ALVA-31 cells revealed distinct cooperative actions of HGF and vitamin D. In contrast to the accumulation of cells in G1 seen during vitamin D inhibition of androgen-responsive cells (LNCaP), growth inhibition of the androgen-unresponsive ALVA-31 cell line with the HGF and vitamin D combination decreased, rather than increased, the fraction of cells in G1, with a corresponding increase in the later cell cycle phases. This cell cycle redistribution suggests that in androgen-unresponsive prostate cancer cells, HGF and vitamin D act together to slow cell cycle progression via control at sites beyond the G1/S checkpoint, the major regulatory locus of growth control in androgen-sensitive prostate cells.

Gap-junctional communication in normal and neoplastic prostate epithelial cells and its regulation by cAMP.

Gap-junctional communication and expression of gap junction-forming proteins were investigated in normal human prostate epithelial cells and in several malignant prostate cell lines. In comparison with normal cells, gap-junctional communication in malignant cells, as assayed by the transfer of 443-Da fluorescent tracer Lucifer yellow, was either reduced or not detected. Malignant cells expressed mRNA transcripts for connexin (Cx) 43, whereas normal cells expressed mRNA transcripts for Cx32 and Cx40. In both normal and malignant cells, gap-junctional communication was enhanced twofold to fivefold by treatment with forskolin, an agent known to increase intracellular levels of cAMP. Immunocytochemical staining with a Cx43-specific antibody revealed that in malignant cells this enhancement correlated with the number of gap junctions and occurred without any qualitative or quantitative alteration in Cx43 mRNA or protein. Moreover, western blot analyses showed that both control and forskolin-treated malignant cells expressed only one form of Cx43. Our data suggest that gap-junctional communication in both normal and malignant prostate cells may be regulated by hormones that work via a cAMP-dependent signal transduction pathway. Thus, both normal and malignant cells offer a new experimental model system in which interactions between a hormonal form of cellular communication and intercellular communication mediated via gap junctions can be studied.

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.

Comparison of prostate specific antigen, prostate specific membrane antigen, and LNCaP-based enzyme-linked immunosorbent assays in prostatic cancer patients and patients with benign prostatic enlargement.

Serum assays for prostate specific antigen (PSA; monoclonal), for prostate specific membrane antigen (PSM; Western blot), and a LNCaP/7E11.C5-based competitive enzyme-linked immunosorbent assay (ELISA) were evaluated in a small number of prostate cancer patients with localized or disseminated disease, and judged to be in clinical progression or remission based on National Prostate Cancer Project (NPCP) criteria. PSA values recognized the presence of clinical progression in localized disease (B1-C) and to a lesser degree disseminated disease (D1-D2). In contrast, to a limited degree the ELISA test recognized clinical progression mainly in disseminated disease and chiefly in stage D2. PSM values were elevated in both D1 and D2 but not in a linear fashion as observed with PSA. The ELISA and PSM results may be assessing a different clinical response to prostatic cancer than that recognized by PSA. This could reflect a developing clone of resistant prostatic cells as previously postulated. To further pursue this possibility a secondary generation of monoclonal antibodies to PSM is being developed. The ELISA levels for benign prostatic enlargement were not elevated above normal. In contrast both with PSA and PSM the assays reflected levels significantly above the normal range in benign prostatic hypertrophy (BPH).

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.

Effect of 5-alpha-reductase inhibition and dexamethasone administration on the growth characteristics and intratumor androgen levels of the human prostate cancer cell line PC-3.

The endocrine treatment of metastatic prostate cancer includes castration which reliably lowers the serum testosterone (T); however, the effect on intratumor levels of T and dihydrotestosterone (DHT) is less predictable. In vitro work demonstrated that the human prostate cancer cell line PC-3 had significant 5-a-reductase activity that could be inhibited with 17b-N,N-diethylcarbamoyl-4-aza-5a-androstan-3-one (4MA). In this study, we examined the effect of 5-a-reductase inhibition with 4MA and androgen suppression with dexamethasone on the growth characteristics and intratumor androgen levels in the PC-3 cell line in male athymic nude mice (Balb/c). The mice were randomized into six treatment groups: 1) noncastrate vehicle control, 2) 4MA, 0.25 mg/day, 3) 4MA, 1 mg/day, 4) dexamethasone, 25 micrograms/day, 5) 4MA, 1 mg/day, and dexamethasone, 25 micrograms/day, and 6) castrate control group. After 21 days of treatment the animals were sacrificed, serum collected, and tumors harvested. Each treatment produced intratumor DHT levels equivalent to the castrate group. Only the low dose 4MA caused a reduction in intratumor DHT without producing castrate levels of circulating T. The combination of dexamethasone and 4MA was less effective in lowering the intratumor DHT/T ratio than 4MA alone. No significant differences in tumor growth parameters were noted between intact control animals and any of the treatment arms. Serum T levels correlated poorly with intratumor androgen levels. Five-a-reductase inhibition produced castrate levels of intratumor DHT in the nonandrogen-dependent prostate cancer cell line PC-3. The combination of dexamethasone and 5-a-reductase inhibition with 4MA appears to be less effective in lowering intratumor androgen levels than either therapy alone.

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.

In vitro differences in lymphocyte subpopulation reactivity in lung cancer patients: purified protein derivative-specific suppressor T lymphocytes in patients who have received Bacillus Calmette-Guerin.

Antigen-specific responses of lymphocyte to purified protein derivative (PPD) were studied in seven patients with non-small cell lung cancer who had undergone complete resection and received postoperative intrapleural Bacillus Calmette-Guerin. In addition, one patient with unresected lung cancer and reactive tuberculin skin test (PPD +) and five normal PPD- individuals were also studied. Lung cancer patients had significantly fewer responsive peripheral blood lymphocytes, unfractionated T cells, and T cells bearing Fc-IgG receptors (TG + populations) than normal controls. These deficits were most pronounced in the group who had received intrapleural Bacillus Calmette-Guerin but who had failed to develop reactive tuberculin skin tests. In contrast, the TG-populations (FC-IgG receptor-negative T cells) from all patients responded to PPD. TG + cells specifically inhibited TG- -cell responses to PPD in both proliferation and immunoglobulin secretion. Radiosensitive suppressor monocytes were found in other patients. This study shows interesting immune deficits in early lung cancer patients. These patients appear to have PPD-specific suppressor TG + cells which may contribute to the immune deficits in these patients.

Immunoglobulin production after marrow transplantation. III. The functional heterogeneity of FC-IgG receptor positive and negative T cell subpopulations.

Ten patients with chronic graft-versus-host disease (GVHD) after HLA-identical marrow transplantation were studied between 372 and 1649 days post-transplant for their T cell subset functions in pokeweed mitogen-stimulated immunoglobulin (Ig) synthesis. In vitro Ig synthesis was assessed using an indirect haemolytic plaque assay after 6 days of culture. T cells, TG+ cells (Fc-IgG receptor positive), TG- cells (Fc-IgG receptor negative), and B cell-enriched populations from the patients were co-cultured with normal T and/or B cells. Such cultures in patients with chronic GVHD showed deficient B cell activity (eight of 10); and deficient helper activity in T cells (six of 10), TG+ cells (five of nine), and TG- cells (three of nine). Greater than 50% suppression of Ig synthesis was detected with T cells (four of 10), TG+ cells (three of 10), and TG- cells (three of 10). This study provides evidence for variable regulatory function of Fc receptor T cell subsets in patients with chronic GVHD. The unexpected finding was that TG+ and TG- subpopulations can lack helper activity or actively suppress Ig synthesis.

Invasive aspergillosis presenting as pericarditis and cardiac tamponade.

A 38-year-old leukemic patient developed pericarditis and cardiac tamponade due to Aspergillus niger one month after undergoing bone marrow transplantation. She failed to improve even though amphotericin B and rifampin therapy had been initiated before infection was evident. Her unique case illustrates both the unusual presentations of invasive aspergillosis and the difficulty of diagnosing and treating this increasingly common disease.

Polymyxin B and rifampin: new regimen for multiresistant Serratia marcescens infections.

Polymyxin B and rifampin were given to 12 patients with multi-drug-resistant nosocomial Serratia marcescens infections. Eight cures were achieved; drug hepatotoxicity occurred once; one fatal suprainfection was encountered; and two patients died during therapy of causes related to severe underlying illnesses. Polymyxin B and rifampin were uniformly synergistic in vitro against the infecting strains and against 40 additional clinical isolates of S. marcescens.