Isolation and immunochemical characterization of rabbit 7S anti-IgG with restricted heterogeneity.

7S anti-IgGs were isolated from four rabbit antistreptococcal antisera by the use of immunoabsorbent columns. The isolated proteins were of restricted molecular heterogeneity; they formed a monodisperse band on microzone electrophoresis and had a limited number of light chain bands when analyzed on urea polyacrylamide gel. The binding site of the 7S anti-IgGs was detected in the F(ab')(2) portion of the molecule. The binding site has antibody specificity for the Fc portion of IgG. For one 7S anti-IgG the combining site on the Fc portion could further be defined. A pepsin fragment of Fc, described as Pep-III', was a potent inhibitor of the coprecipitation of 7S anti-IgG with antigen-antibody complexes. An idiotypic cross-reaction was detected between the 7S and 19S anti-IgGs isolated from the same rabbit with anti-idiotype sera prepared in guinea pigs. This idiotypic specificity was not detected in the 7S anti-IgGs of 20 other rabbits.

Induction of in vitro proliferation and maturation of human aneuploid myelogenous leukemic cells.

Human leukemic cells were induced to proliferate and mature to macrophage-like cells in primary cultures supplemented with conditioned medium (CM) from phytohemagglutinin and alloantigen-stimulated normal T lymphocytes. Blast and promyelocyte-enriched preparations, isolated after depletion of adherent phagocytic cells and lymphoid cells from samples of myelogenous leukemia patients, were suspended in liquid cultures with 30% CM. Cell cycle analysis was performed throughout the course of induced cellular maturation. Within 24 h of exposure to CM, cells with macrophage-like morphology were identified among the developing adherent cells. Approximately 15-30% of the cells in culture suspensions also developed macrophage-like morphology and esterase reactivity with alpha-napthyl acetate after incubation for 2 d. The number of these nonproliferating cells increased and became predominant in the later culture period. Flow cytometric measurement of DNA content showed that these mature cells had the same aneuploid stemline as the undifferentiated leukemic cells, indicating that genetically abnormal leukemic cells can be induced to differentiate. Reduction in the total RNA content of the macrophage-like cells was also determined by flow cytometry. Reduction in RNA and development of adherent cells served as early markers of maturation, in addition to the later acquisition of complement receptors and phagocytic capacity. Cell cycle analysis showed that CM stimulated the proliferation of immature cells. This initial proliferation may precede intertwined events of proliferation and concurrent maturation of immature cells. Later in the culture period, cellular proliferation decreased, leading to termination of the cultures.

Homogenous rabbit 7S anti-IgG with antibody specificity for peptidoglycan.

The relationship between 7S anti-IgG and antibodies to streptococcal cell wall peptidoglycan was examined for four streptococcal Group C antisera. Homogeneous 7S anti-IgG components in these sera were isolated by means of an IgG immunoadsorbent column. For two of the four antisera, the anti-peptidoglycan activity of the 7S anti-IgG had specificity for the pentapeptide, L-Ala-D-Glu-gamma-L-Lys-D-Ala-D-Ala, the antigenic determinant of peptidoglycan, as well as for the Fc of IgG. Detailed studies on the 7S anti-IgG from one of the antisera revealed that the pentapeptide inhibited the coprecipitation reaction of 7S anti-IgG R3387 with antigen-antibody complexes and the precipitin reaction between 7S anti-IgG R3387 and its anti-idiotype serum.

Deficient histone acetylation in acute leukemia and the correction by an isothiocyanate.

OBJECTIVE: Since histone hypoacetylation due to excess histone deacetylases (HDACs) has been associated with transcriptional repression in leukemia, we aimed to determine deficient histone acetylation in patients with acute leukemia and the effect of its correction by an isothiocyanate. METHODS: The acetylation status of histones H3 and H4 in cells from patients with untreated acute leukemia was determined by Western blot. Deficient histone acetylation was analyzed in relation to the disease state. Bone marrow cells from 10 patients with acute myeloid leukemia (AML) were cultured in phenylhexyl isothiocyanate (PHI) to evaluate correction of the deficiency. RESULTS: Acetylation of histones H3 and H4 was virtually undetectable or significantly lower in acute leukemia. This deficiency was consistent among all the patients examined. Histone acetylation was up-regulated in the presence of PHI, revealing an excess of deacetylation activity in AML. PHI treatment induced apoptosis, indicating HDAC inhibition was able to correct the deficiency. CONCLUSIONS: Deficient histone acetylation may represent an aberration at the epigenetic level in acute leukemia. PHI might represent a target for correcting deficient acetylation, and potential epigenetic regulators for preventing the progression of leukemia.

De-repression of the p21 promoter in prostate cancer cells by an isothiocyanate via inhibition of HDACs and c-Myc.

Natural isothiocyanates from cruciferous vegetables have been described as important dietary factors for prostate cancer prevention. Phenethyl isothiocyanate (PEITC), found rich in watercress, induces growth arrest and apoptosis in prostate cancer cells, and also inhibits the testosterone-mediated growth of prostates by regulating the androgen receptor and cell cycle progression in rats. PEITC has been recently identified as an inhibitor of histone deacetylases (HDACs). Herein we describe the mechanism of PEITC-mediated growth attenuation in relation to HDAC inhibition in human prostate cancer cells. Exposure of androgen-dependent prostate cancer cells LNCaP to PEITC resulted in cell cycle arrest and a p53-independent up-regulation of the inhibitors of cyclin-dependent kinases, including p21WAF1 and p27. The mechanism of p21 activation was investigated. PEITC significantly enhanced histone acetylation and induced selective modification of histone methylation for chromatin remodeling. Chromatin immunoprecipitation revealed that the p21 gene was associated with the PEITC-induced hyperacetylated histones. As a result, the chromatin unfolding permitted the transcription activation of the p21 gene. PEITC also significantly reduced the expression of c-Myc which represses p21. Pull-down assays using Sp1 affinity oligo beads of the p21 promoter, showed decreased c-Myc binding to the Sp1 transcriptional complexes in the p21 promoter, resulting in reduced p21 repression. The quantity of PEITC (0.5-1 micro M) effective to mediate cell cycle arrest was less than that for inhibiting c-Myc (2-5 micro M), suggesting that the inhibition of HDACs may be the primary mechanism for p21 activation. The PEITC-mediated growth attenuation of prostate cancer cells includes an interactive mechanism involving HDAC and c-Myc inhibition.

Phenylhexyl isothiocyanate inhibits histone deacetylases and remodels chromatins to induce growth arrest in human leukemia cells.

Natural isothiocyanates, present in cruciferous vegetables and synthetic phenylhexyl isothiocyanate (PHI) are chemopreventive agents which act by blocking the initiation of carcinogen-induced tumors in rodents. We have demonstrated that isothiocyanates are also growth regulators, inhibiting cell cycle cdk activity and up-regulating inhibitor p21WAF1 (p21) in cancer cells. The up-stream mechanism to modulate cell cycle progression remained to be elucidated. Here, we have demonstrated that exposure of HL-60 leukemia cells to PHI induced G1 arrest and apoptosis. The hypothesis that PHI inhibits cell growth via chromatin remodeling was investigated. PHI mediates the complex cross talk between chromatin and DNA, and it was demonstrated for the first time as an inhibitor of histone deacetylases (HDAC). Thus, the HDAC activity in PHI-exposed HL-60 cells was reduced. Additionally, PHI reduced the expression of HDAC and increased the level of acetyl transferase p300, in favor of accumulation of acetylated histones. Within hours, global acetylation of histones was enhanced. PHI further mediated selective alterations of histone methylation, with a pattern consistent to the marks of transcription competent chromatins. The relationship between acetylated histones and p21 was examined by chromatin immunoprecipitation (ChIP) assay. Chromatins from cells exposed to PHI contained more p21 DNA in the precipitates of hyperacetylated histones, indicating more accessibility of transcription machinery to the p21 promoter after chromatin unfolding. The cell cycle inhibitors were activated as a result. In contrast to the PHI-induced apoptosis in HL-60 cells, which was mediated by caspase-9 up-regulation and bcl-2 reduction, PHI did not induce significant apoptosis in the mononuclear cells from normal peripheral blood and bone marrow. The results revealed a potential selective effect of isothiocyanates to inhibit the growth of malignant cells.

The phenylhexyl isothiocyanate induces apoptosis and inhibits leukemia cell growth in vivo.

Isothiocyanates are potent chemopreventive agents for carcinogen-induced cancers in rodents. The major mode of action for chemoprevention is cytoprotection i.e. inducing detoxifying enzymes to remove the carcinogens, thus blocking the initiation of carcinogenesis. Analysis has indicated that isothiocyanates also act at the post-initiation levels of carcinogenesis. We have also reported that the phenylhexyl isothiocyanate (PHI) induced growth arrest and apoptosis in human leukemia HL-60 cells in culture. Since then we have investigated the in vivo efficacy of PHI. The effects of PHI were evaluated in immunodeficient mice, with xenografts of human leukemia HL-60 cells. The maximum tolerated dose (MTD) was determined. The experimental mice received 80% of the MTD. Oral feedings of PHI significantly reduced tumor incidence (p<0.05) without overt toxicity. PHI inhibited cell cycle progression through the down-regulation of cyclin expression, Rb phosphorylation and the up-regulation of the cdk-inhibitors. Apoptosis was significantly increased in the treated tumors but not in the normal mouse tissues. In conclusion, PHI induced apoptosis and inhibited the growth of xenografts by targeting the cell cycle regulators. PHI induced selective apoptosis effects in the rapidly growing tumor cells but not in the normal tissues.

Differentiation antigens of HL-60 promyelocytes during induced maturation.

The human promyelocytic leukemic cell line HL-60 can be induced to mature monocytes and macrophages in vitro by lymphocyte-conditioned medium. We are reporting sequential changes in surface antigenic expressions, which are sensitive markers of the characteristic events in the process of cell differentiation. The promyelocyte membrane antigen, detected by a monoclonal antibody produced using HL-60 cells as an immunogen, was shown to be associated with immature myeloid cells and was used to determine HL-60 cell development. The expression of this membrane antigen, determined to have a molecular weight of 85,000 was lost early in the differentiation period. In the following stage, in which the promyelocytes developed into monocytic cells, a steady increase of cells bearing the OKM1 normal monocyte antigen was observed. When macrophages became predominant in the final culture period, the expression of the OKM1 antigen decreased. The usefulness of these differentiation antigens in studying cellular development is discussed.

Inhibition of benzo(a)pyrene-induced lung tumorigenesis in A/J mice by dietary N-acetylcysteine conjugates of benzyl and phenethyl isothiocyanates during the postinitiation phase is associated with activation of mitogen-activated protein kinases and p53 activity and induction of apoptosis.

Recent studies in cell culture have shown that isothiocyanates (ITCs) induce apoptosis via activation of mitogen-activated protein (MAP) kinases and p53 pathways, suggesting a potential for ITCs or their conjugates to inhibit tumorigenesis during the postinitiation phase. To evaluate whether ITC compounds administered after carcinogen treatment inhibit lung tumorigenesis, we investigated in A/J mice the effects of the N-acetylcysteine (NAC) conjugates of benzyl (BITC-NAC) and phenethyl ITC (PEITC-NAC) in the diet (15 micromol/g) administered after a single dose of 20 micromol benzo(a)pyrene [B(a)P]. The formation of lung adenomas was examined 140 days after B(a)P dosing. Both the BITC-NAC and PEITC-NAC-treated groups showed a significant reduction in lung tumor multiplicity from 6.1 +/- 3.1 tumors/mouse in the B(a)P group fed the control diet to 3.7 +/- 2.9 and 3.4 +/- 2.7 tumors/mouse (P = 0.018 and 0.006, respectively). To investigate the mechanisms of tumor inhibition, lung tissues were obtained at 21, 84, and 140 days at interim sacrifices during the bioassay. These tissues showed a significant increase in apoptosis as determined by in situ end-labeling for both ITC-NAC-treated groups. The MAP kinase pathway was activated in the ITC-NAC-treated groups. The activation of c-Jun NH(2)-terminal kinase was higher in the BITC-NAC and PEITC-NAC groups when compared with B(a)P-treated control. The phosphorylation of p38 and extracellular signal-regulated kinases (ErKs) 1 and 2 was also induced by these treatments. To determine the downstream target of MAP kinases, activator protein-1 (AP-1) and nuclear factor-kappaB activities were evaluated by gel shift assay. The AP-1 binding activity was remarkably increased in lung tissue from both the BITC-NAC and PEITC-NAC groups. No change in nuclear factor-kappaB binding activity was found, however. Phosphorylation of p53 was also higher than the constitutive levels in both ITC-NAC-treated groups, but no induction of p53 expression was detected. This study demonstrates the chemopreventive efficacy of the NAC conjugates of PEITC and BITC administered in the diet after a single dose of B(a)P for lung tumorigenesis and provides the first in vivo evidence that activation of MAP kinases, AP-1 transcription factors, p53 phosphorylation, and the induction of apoptosis may be involved in the chemopreventive activity of these compounds.

Hypoacetylation, hypomethylation, and dephosphorylation of H2B histones and excessive histone deacetylase activity in DU-145 prostate cancer cells.

BACKGROUND: Hypoacetylation on histone H3 of human prostate cancer cells has been described. Little is known about the modifications of other histones from prostate cancer cells. METHODS: Histones were isolated from the prostate cancer cell line DU-145 and the non-malignant prostatic cell line RC170N/h. Post-translational modifications of histone H2B were determined by liquid chromatography-mass spectrometry (LC-MS)/MS. RESULTS: The histone H2B of the prostate cancer cell line DU-145 was found to have hypoacetylation, hypomethylation, and dephosphorylation as compared to the non-malignant prostatic cell line RC170N/h. H2B regained acetylation on multiple lysine residues, phosphorylation on Thr19, and methylation on Lys23 and Lys43 in the DU-145 cells after sodium butyrate treatment. CONCLUSIONS: The histone H2B of DU-145 prostate cancer cells are hypoacetylated, hypomethylated, and dephosphorylated. Histone deacetylase inhibitor reversed this phenotype. Epigenetic agent may therefore be useful for prostate cancer therapy and worth further investigation.

Interaction of a small molecule Natura-α and STAT3-SH2 domain to block Y705 phosphorylation and inhibit lupus nephritis.

A small molecule, Natura-α, a clinical stage investigational new drug for certain inflammatory diseases, has been evaluated for drug interaction with STAT3 and inhibiting systemic lupus erythematosus (SLE). Studies have revealed that it selectively inhibits STAT3-Y705 phosphorylation and, suppresses pro-inflammatory cytokines, stimulates anti-inflammatory cytokine IL-10, thereby skewing T cell differentiation from the Th1/Th17 lineages toward the Treg lineage. The potential binding of the drug to STAT3 protein has been investigated with a computational modeling and docking simulation using X-ray crystal structure of the STAT3ß homodimer. Natura-α was shown to directly bind to SH2 domain of STAT3 and forms H-bonds with amino acids Glu594 and Arg609. The phosphorylation of Y705 was prevented and making the formation of STAT3 homodimer impossible, thereby blocking STAT3 activation. The in vivo efficacy of Natura-α in SLE was evaluated in a bioassay with NZB/W female mice. Mice at week 19 were given orally Natura-α at 25 or 75 mg/kg, once a day, 5 days per week for 29 weeks. Mice were monitored weekly until 52 weeks of age. Both dosages were effective to reduce proteinuria and significantly improved animal survival rate. The renal functions were preserved with glomerular lesions reversed, which paralleled with decreased C3 deposit. The numbers of kidney cells stained with phosphorylated STAT3-Y705 remarkably decreased, demonstrating blocking of Y-705 phosphorylation by the treatment. Since NZB/W mice develop nephritis which resembles SLE in men, the data strongly suggests that Natura-α may be a potential effective therapeutic agent for lupus.

Establishment of prostate cancer spheres from a prostate cancer cell line after phenethyl isothiocyanate treatment and discovery of androgen-dependent reversible differentiation between sphere and neuroendocrine cells.

Prostate cancer can transform from androgen-responsive to an androgen-independent phenotype. The mechanism responsible for the transformation remains unclear. We studied the effects of an epigenetic modulator, phenethyl isothiocyanate (PEITC), on the androgen-responsive LNCaP cells. After treatment with PEITC, floating spheres were formed with characteristics of prostate cancer stem cells (PCSC). These spheres were capable of self-renewal in media with and without androgen. They have been maintained in both types of media as long term cultures. Upon androgen deprivation, the adherent spheres differentiated to neuroendocrine cells (NEC) with decreased proliferation, expression of androgen receptor, and PSA. NEC reverse differentiated to spheres when androgen was replenished. The sphere cells expressed surface marker CD44 and had enhanced histone H3K4 acetylation, DNMT1 down-regulation and GSTP1 activation. We hypothesize that PEITC-mediated alteration in epigenomics of LNCaP cells may give rise to sphere cells, whereas reversible androgenomic alterations govern the shuttling between sphere PCSC and progeny NEC. Our findings identify unrecognized properties of prostate cancer sphere cells with multi-potential plasticity. This system will facilitate development of novel therapeutic agents and allow further exploration into epigenomics and androgenomics governing the transformation to hormone refractory prostate cancer.

Cell-cycle differences of HL-60 leukemia cells fractionated by centrifugal elutriation.

HL-60 leukemia cells were fractionated into G1 and S/G2 populations using a rapid centrifugal elutriation technique, and studied for differences between the cell-cycle phases. The G1 fraction was found to contain smaller cells with a sedimentation velocity of 7 mm/h. The S/G2 fraction consisted of larger cells with a sedimentation velocity of 125 mm/h. The latter fraction was found to have a peak level of the enzyme (2'-5')An-binding protein, as compared to the G1 fraction, indicating a possible role for (2'-5')An-binding protein and its products in the growth regulation of these leukemic cells. In addition, cytofluorometric analysis of fractionated HL-60 cells indicates that elutriation is an effective fractionation method, rapidly yielding large numbers of cells for study, without the use of chemical treatments.

In vitro effects and clinical evaluation of a human chorionic gonadotrophin preparation in acute leukemia.

Commercial human chorionic gonadotrophin (HCG) preparations decrease the tumorigenicity of human tumors in immunodeficient mice and induce apoptotic cell death in animal tumor models. Preliminary studies in humans have demonstrated tumor regression in patients with Kaposi's sarcoma given intralesional injections of HCG. To further evaluate HCG's antitumor activity we conducted in vitro and clinical evaluations of HCG in acute myeloid leukemia (AML). In HL-60 leukemic cell lines, a 20-40% inhibition of cell density was demonstrated by trypan blue exclusion method at low concentrations of an HCG preparation (2 x 10(-3)-2 x 10(-2)). Similar concentrations also resulted in a reduction in the proportion of cells in G2M phase of the cell cycle, as well as enhanced differentiation compared to control cells. Fifteen patients with advanced AML with marrow blast counts >30%, and five with marrow blast counts between 10 and 26% were given daily subcutaneous injections of HCG 2-4 IU and oral levamisole 50 mg weekly. Five patients with absolute blast counts in the blood ranging from 0 to 3500/microl and percent blasts in the marrow ranging from 16 to 81% were observed to have no progressive increase in either marrow or peripheral blast counts for 70-121 days. One patient with a pretreatment blast count of 10% in the marrow, no circulating blasts and minor cytopenias had a decrease in marrow blasts to less than 5% which has persisted at 550 days. No significant improvement from baseline levels of neutrophils, hemoglobin or platelets were observed in any nl the patients treated. Increases in apoptotic cell death were observed in over 50% of patients' cells with some demonstrating peak levels similar to experiences in patients treated with DNA-damaging chemotherapy. A decreased expression of bcl-2 was seen in the majority of patients ranging from 6 to 62%. These new observations suggest that HCG preparations may inhibit leukemic cell growth through enhancement of cell death mechanisms and could be used in judicious combinations with other approaches. The results confirm the pro-apoptotic effects of HCG preparations reported in patients with Kaposi's sarcoma. Identification of the active component of HCG preparations and further understanding of its growth modulatory action will be important in its development as a clinically useful agent.

Human T lymphocytes and myeloid colony forming cells share common antigen.

Human peripheral blood T lymphocyte antigen has been detected by goat antisera raised to thymocytes of Rhesus monkey. An absorbed antiserum reacted in complement-mediated cytotoxicity reactions and absorption experiments with isolated human peripheral blood T lymphocytes, thymocytes, cells of T lymphocyte line, but not with B lymphocytes or cells of B lymphocyte line. Anti-T lymphocyte antibodies blocked the T lymphocyte rosette formation with sheep erythrocytes. By cytotoxicity test an average of 85% cells of unfractionated lymphocyte preparations and 98% cells of isolated T lymphocyte preparations were positive for the antigen. Peripheral blood granulocytes and monocytes were shown to lack the antigen by cytotoxicity and absorption tests. Lysis of bone marrow cells by the antiserum abolished granulocyte-macrophage colony formation in vitro. Absorption studies showed that myeloid colony forming cells express antigen common to T lymphocytes. The common antigen may be the membrane structure located close to the lymphocyte receptors for spontaneous binding of sheep erythrocytes. This common antigen probably represents an antigen characteristic of the T lymphocyte line which is shared with and thus may be inherited from the pluripotent stem cells.

Antiproliferative effect of a prostatic cell-derived activity on the human androgen-dependent prostatic carcinoma cell line LNCaP.

We have identified a new antiproliferative activity from the conditioned medium of two androgen-independent prostatic cancer cell lines, PC3 and DU-145. This antiproliferative activity selectively inhibited cell proliferation of an androgen-dependent prostate cancer cell line LNCaP in a dose-dependent manner. No antiproliferative activity was observed against mouse fibroblast 3T3, normal human lymphocytes, human leukemic cells, including promyelocyte HL-60 or T cell HUT-78, or human adenocarcinoma cell lines, including prostatic cells JCA-1, ovary NIH:OVCAR-3, cervix C-33A, or breast MDA-MB-231. Cell cycle analysis revealed that the antiproliferative activity did not induce apoptosis in LNCaP cells, but it prevented some G1 LNCaP cells from entering into the S phase of the cell cycle. The antiproliferative activity was sensitive to high temperature (100 degrees C) and to proteinase digestion; however, it was resistant to 56 degrees C, pH 2.0, and reducing agent treatment, as well as to DNase and RNase digestion. The antiproliferative activity was partially purified by gel filtration, ion-exchange chromatography, and SDS-PAGE, with an apparent molecular weight of 50 kD. The antiproliferative activity was not affected by neutralizing antibody against TGF-beta 1,2,3, TNF-alpha, PDGF, EGF, IL-1, IL-2, IL-3, IL-4, or IL-6.

Separate T cell subclasses inducing or attenuating graft-versus-host reaction.

The subclasses of chicken T cells capable of initiating or regulating graft-versus-host reaction (GVHR) were studied. T cells isolated from spleens of bursectomized agammaglobulinemic chickens were rosetted to separate the TG cells bearing the chicken IgG-Fc membrane receptor from those TG- cells lacking the receptor. The GVH reactivity of these subclasses was quantified by their capacity to induce pock lesions on chorioallantoic membranes of allogeneic chick embryos after their implantation on the membranes. Cells of TG- and unseparated T cell preparations but not TG cells were shown to be capable of inducing GVHR. TG- cells, obtained after the depletion of TG from unseparated T cell preparations exhibited increased GVH reactivity over that of unseparated T cells. Addition of TG cells to TG- cell preparations attenuated the TG- cell-induced GVHR. The degree of suppression was in proportion to the number of TG added to the inoculum. The GVH inducer cells were shown to be radiosensitive, being incapable of initiating the reaction after exposure to 300 rad of gamma-radiation. The suppressive activity of TG cells was unchanged after high dosage radiation. The possibility that TG cells regulate GVHR via a suppression of cellular proliferation is discussed.

Identification of a co-stimulatory factor for human T cell proliferation.

A factor enhancing proliferation of human peripheral blood lymphocytes (PBL) was identified in the culture supernatant of a human myeloid cell line HL-60 after ion exchange separation. Partially purified lymphocyte growth enhancing factor (LGEF) was found to enhance mitogen-stimulated PBL or purified T cell proliferation in a dose dependent fashion. LGEF alone did not stimulate PBL or T cell proliferation and its activity was dependent on the presence of mitogen. LGEF mediated T cell proliferation was neither accompanied by an increase in the level of IL-2 receptor expression, nor was dependent on the presence of monocytes. In ELISA assay antibodies against IL-1, IL-2, IL-3 and IL-6 did not recognize the LGEF preparation. The comparison of LGEF with other lymphokines is discussed.

Growth modulation of human prostatic cancer cells by interleukin-1 and interleukin-1 receptor antagonist.

The effects of interleukin-1 (IL-1 alpha or IL 1 beta) on androgen-dependent LNCaP and androgen-independent JCA-1 human prostatic cancer cell proliferation were investigated. IL-1 alpha or IL-1 beta induced a dose dependent growth reduction by 50-80% as determined by cell cycle phase distribution, cell number and clonal growth in short and long term cultures. IL-1 negated the androgen growth effect on equal molar basis but the androgen receptors were not blocked by IL-1. The presence of intracellular IL-1 receptors was detected by flow cytometry and the IL-1 mediated growth reduction could be blocked with a 500 fold excess of the IL-1 receptor antagonist (hIL-1ra), revealing a growth control involving IL-1, HIL-1ra, androgen and their receptors.

Detection of an inhibiting activity for osteoclast bone resorption from human prostatic cancer cells.

An inhibiting activity for isolated osteoclasts and bone resorption was demonstrated in culture supernatants from androgen-independent, but not androgen-dependent, human prostatic cancer cell lines. It causes a dose-dependent osteoclast inhibition as quantified with a bone resorbing pit formation assay. The constitutively released activity was determined to be protein (>50 kDa) distinct from some of the known cytokines in bone resorption. The activity does not affect osteoclast morphology and viability. Time-lapse video microscopy revealed an osteoclast motility increase, disrupting their anchorage to the bone and resorbing processes. The association of the activity with androgen-independent cancer cells that disrupt bone remodeling is discussed.