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1.
Eur J Cancer ; 177: 33-44, 2022 12.
Article in English | MEDLINE | ID: mdl-36323051

ABSTRACT

BACKGROUND: Metastatic castration-resistant prostate cancer (mCRPC) patients with positive AR-V7 expression in their circulating tumour cells (CTCs) rarely derive benefit from abiraterone and enzalutamide. DESIGN: We performed a prospective, multicenter, single arm phase II clinical trial (CABA-V7) in mCRPC patients previously treated with docetaxel and androgen deprivation therapy. OBJECTIVE: In this trial, we investigated whether cabazitaxel treatment resulted in clinically meaningful PSA response rates in patients with positive CTC-based AR-V7 expression and collected liquid biopsies for genomic profiling. RESULTS: Cabazitaxel was found to be modestly effective, with only 12% of these patients obtaining a PSA response. Genomic profiling revealed that CTC-based AR-V7 expression was not associated with other known mCRPC-associated alterations. CTC-based AR-V7 status and dichotomised CTC counts were observed as independent prognostic markers at baseline. CONCLUSIONS: AR-V7 positivity predicted poor overall survival (OS). However, cabazitaxel-treated AR-V7 positive patients and those lacking AR-V7 positivity, who received cabazitaxel as standard of care, appeared to have similar OS. Therefore, despite the low response rate, cabazitaxel may still be an effective treatment in this poor prognosis, AR-V7 positive patient population.


Subject(s)
Neoplastic Cells, Circulating , Prostatic Neoplasms, Castration-Resistant , Male , Humans , Prostatic Neoplasms, Castration-Resistant/pathology , Prostate-Specific Antigen , Receptors, Androgen/metabolism , Androgen Antagonists/therapeutic use , Protein Isoforms/genetics , Neoplastic Cells, Circulating/pathology , Nitriles/therapeutic use
2.
Angew Chem Int Ed Engl ; 60(19): 10935-10941, 2021 May 03.
Article in English | MEDLINE | ID: mdl-33620140

ABSTRACT

We report on the wavelength-selective photopolymerization of a hybrid acrylate-oxetane cholesteric liquid crystal monomer mixture. By controlling the sequence and rate of the orthogonal free-radical and cationic photopolymerization reactions, it is possible to control the degree of phase separation in the resulting liquid crystal interpenetrating networks. We show that this can be used to tune the reflective color of the structurally colored coatings produced. Conversely, the structural color can be used to monitor the degree of phase separation. Our new photopolymerization procedure allows for structuring liquid crystal networks in three dimensions, which has great potential for fabricating liquid crystal polymer materials with programmable functional properties.

3.
Cancer Chemother Pharmacol ; 85(3): 547-553, 2020 03.
Article in English | MEDLINE | ID: mdl-31893292

ABSTRACT

PURPOSE: Cabazitaxel, used in patients with metastatic castration-resistant prostate cancer (mCRPC), is associated with adverse events which may require dose reductions or discontinuation of treatment. We investigated the potential association of single-nucleotide polymorphisms (SNPs) in genes encoding drug transporters and drug-metabolizing enzymes with cabazitaxel toxicity, overall survival (OS) and pharmacokinetics (PK). METHODS: A total of 128 cabazitaxel-treated mCRPC patients, of whom prospectively collected data on toxicity and OS were available and 24 mCRPC patients with available cabazitaxel PK measurements, were genotyped using genomic DNA obtained from EDTA blood. The SLCO1B1 (388A > G; *1B; rs2306283 and 521 T > C; *5; rs4149056 and haplotype SLCO1B1*15), SLCO1B3 (334 T > G; rs4149117), CYP3A4 (*22; rs35599367), CYP3A5 (*3; rs776746), ABCB1 (3435C > T; rs1045642), and TUBB1 (57 + 87A > C; rs463312) SNPs were tested for their association with clinical and PK parameters by univariate/multivariate logistic regression, log-rank test, or Kruskal-Wallis test. RESULTS: The SLCO1B1*15 haplotype was significantly associated with a lower incidence of leukopenia and neutropenia (p = 0.020 and p = 0.028, respectively). Patients harboring a homozygous variant for SLCO1B1*1B experienced higher rate ≥ grade 3 (p = 0.042). None of the SNPs were associated with pharmacokinetics or OS. CONCLUSIONS: In this study, SLCO1B1 (SLCO1B1*15 and SLCO1B1*1B) was associated with cabazitaxel-induced adverse events in mCRPC patients. As the associations were opposite to previous studies in other drugs and contradicted an underlying pharmacokinetic rationale, these findings are likely to be false-positive and would ideally be validated with even larger (pharmacokinetic) cohorts.


Subject(s)
Antineoplastic Agents/adverse effects , Antineoplastic Agents/therapeutic use , Polymorphism, Single Nucleotide/genetics , Prostatic Neoplasms, Castration-Resistant/drug therapy , Prostatic Neoplasms, Castration-Resistant/genetics , Taxoids/adverse effects , Taxoids/therapeutic use , Aged , Disease-Free Survival , Haplotypes/genetics , Humans , Leukopenia/chemically induced , Leukopenia/genetics , Male , Middle Aged , Neutropenia/chemically induced , Neutropenia/genetics , Prospective Studies , Prostatic Neoplasms, Castration-Resistant/mortality , Treatment Outcome
4.
Eur J Cancer ; 121: 48-54, 2019 11.
Article in English | MEDLINE | ID: mdl-31542641

ABSTRACT

BACKGROUND: The interpretation of the presence of AR-V7 in circulating tumour cells (CTCs) in men with metastatic castration-resistant prostate cancer (mCRPC) remains to be elucidated. AR-V7 may hold promise as a predictive biomarker, but there may be prognostic impact of AR-V7 positivity as well. To investigate the clinical value of AR-V7, we determined whether AR-V7 detection in CTCs in patients with mCRPC is associated with CTC counts and survival. METHODS: Between December 2011 and January 2019, three prospective clinical trials collected clinical data of patients with mCRPC, who progressed after docetaxel and/or enzalutamide or abiraterone. Baseline (and follow-up) blood samples were withdrawn determining CTC count and AR-V7 expression. The majority of patients started cabazitaxel as the next line of treatment after AR-V7 characterisation. RESULTS: A total of 127 samples were evaluable for the analysis of CTC count versus AR-V7 status. Although an association was observed between AR-V7 and CTC count in all patients with mCRPC (p = 0.017), no such association was found in the prognostic unfavourable subgroup of patients with ≥5 CTCs. After adjusting for clinical prognostic factors, AR-V7 expression in CTCs was not associated with overall survival (hazard ratio = 1.33, 95% confidence interval = 0.81-2.15, p = 0.25). CONCLUSION: We found that AR-V7 expression in CTCs had no additional prognostic value in patients with mCRPC, mostly treated with cabazitaxel. In patients with mCRPC with a predefined worse prognosis of a higher CTC count (≥5), a predictive biomarker is an important unmet medical need. Prospective trials should investigate whether AR-V7 detection in CTCs may guide treatment selection for these adverse prognosis patients.


Subject(s)
Biomarkers, Tumor/blood , Neoplastic Cells, Circulating/metabolism , Neoplastic Cells, Circulating/pathology , Prostatic Neoplasms, Castration-Resistant/blood , Prostatic Neoplasms, Castration-Resistant/diagnosis , Receptors, Androgen/blood , Aged , Biomarkers, Tumor/metabolism , Blood Cell Count , Clinical Trials as Topic/statistics & numerical data , Follow-Up Studies , Humans , Male , Middle Aged , Neoplasm Metastasis , Prognosis , Prostatic Neoplasms, Castration-Resistant/mortality , Prostatic Neoplasms, Castration-Resistant/pathology , Protein Isoforms/blood , Protein Isoforms/genetics , Protein Isoforms/metabolism , Receptors, Androgen/genetics , Receptors, Androgen/metabolism , Retrospective Studies , Survival Analysis , Taxoids/therapeutic use
5.
Br J Clin Pharmacol ; 85(5): 986-992, 2019 05.
Article in English | MEDLINE | ID: mdl-30737835

ABSTRACT

AIMS: Docetaxel has been approved for the treatment of metastatic prostate cancer in combination with prednisone. Since prednisone is known to induce the cytochrome P450 iso-enzyme CYP3A4, which is the main metabolizing enzyme of docetaxel in the liver, a potential drug-drug interaction may occur. In this prospective randomized pharmacokinetic cross-over study we investigated docetaxel exposure with concomitant prednisone, compared to docetaxel monotherapy in men with metastatic prostate cancer. METHODS: Patients scheduled to receive at least 6 cycles of docetaxel (75 mg/m2 ) and who gave written informed consent were randomized to receive either the 1st 3 cycles, or the last 3 consecutive cycles with prednisone (twice daily 5 mg). Pharmacokinetic blood sampling was performed during cycle 3 and cycle 6. Primary endpoint was difference in docetaxel exposure, calculated as area under the curve (AUC0-inf ) and analysed by means of a linear mixed model. Given the cross-over design the study was powered on 18 patients to answer the primary, pharmacokinetic, endpoint. RESULTS: Eighteen evaluable patients were included in the trial. Docetaxel concentration with concomitant prednisone (AUC0-inf 2784 ng*h/mL, 95% confidence interval 2436-3183 ng*h/mL) was similar to the concentration of docetaxel monotherapy (AUC0-inf 2647 ng*h/mL, 95% confidence interval 2377-2949 ng*h/mL). Exploratory analysis showed no toxicity differences between docetaxel monotherapy and docetaxel cycles with prednisone. CONCLUSION: No significant difference in docetaxel concentrations was observed. In addition, we found similar toxicity profiles in absence and presence of prednisone. Therefore, from a pharmacokinetic point of view, docetaxel may be administrated with or without prednisone.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Cytochrome P-450 CYP3A Inducers/pharmacology , Docetaxel/pharmacology , Prednisone/pharmacology , Prostatic Neoplasms, Castration-Resistant/drug therapy , Aged , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Area Under Curve , Cross-Over Studies , Cytochrome P-450 CYP3A/metabolism , Cytochrome P-450 CYP3A Inducers/therapeutic use , Docetaxel/therapeutic use , Drug Interactions , Humans , Male , Middle Aged , Prednisone/therapeutic use , Prospective Studies , Treatment Outcome
6.
Cancer Chemother Pharmacol ; 82(3): 457-468, 2018 09.
Article in English | MEDLINE | ID: mdl-29974203

ABSTRACT

PURPOSE: Phase Ib study evaluating the effect of apalutamide, at therapeutic exposure, on ventricular repolarization by applying time-matched pharmacokinetics and electrocardiography (ECG) in patients with castration-resistant prostate cancer. Safety of daily apalutamide was also assessed. METHODS: Patients received 240 mg oral apalutamide daily. Time-matched ECGs were collected via continuous 12-lead Holter recording before apalutamide (Day - 1) and on Days 1 and 57 (Cycle 3 Day 1). Pharmacokinetics of apalutamide were assessed on Days 1 and 57 at matched time points of ECG collection. QT interval was corrected for heart rate using Fridericia correction (QTcF). The primary endpoint was the maximum mean change in QTcF (ΔQTcF) from baseline to Cycle 3 Day 1 (steady state). Secondary endpoints were the effect of apalutamide on other ECG parameters, pharmacokinetics of apalutamide and its active metabolite, relationship between plasma concentrations of apalutamide and QTcF, and safety. RESULTS: Forty-five men were enrolled; 82% received treatment for ≥ 3 months. At steady state, the maximum ΔQTcF was 12.4 ms and the upper bound of its associated 90% CI was 16.0 ms. No clinically meaningful effects of apalutamide were reported for heart rate or other ECG parameters. A concentration-dependent increase in QTcF was observed for apalutamide. Most adverse events (AEs) (73%) were grade 1-2 in severity. No patients discontinued due to QTc prolongation or AEs. CONCLUSION: The effect of apalutamide on QTc prolongation was modest and does not produce a clinically meaningful effect on ventricular repolarization. The AE profile was consistent with other studies of apalutamide.


Subject(s)
Electrocardiography, Ambulatory/drug effects , Prostatic Neoplasms, Castration-Resistant/drug therapy , Prostatic Neoplasms, Castration-Resistant/physiopathology , Thiohydantoins/administration & dosage , Aged , Aged, 80 and over , Androgen Receptor Antagonists/administration & dosage , Androgen Receptor Antagonists/adverse effects , Androgen Receptor Antagonists/pharmacokinetics , Heart Rate/drug effects , Heart Ventricles/drug effects , Heart Ventricles/physiopathology , Humans , Male , Middle Aged , Prostatic Neoplasms, Castration-Resistant/metabolism , Thiohydantoins/adverse effects , Thiohydantoins/pharmacokinetics
7.
Clin Cancer Res ; 24(3): 541-546, 2018 02 01.
Article in English | MEDLINE | ID: mdl-29150561

ABSTRACT

Purpose: In ongoing clinical research on metastatic castration-resistant prostate cancer (mCRPC) treatment, the potential enhanced efficacy of the combination of taxanes with AR-targeted agents, that is, enzalutamide and abiraterone, is currently being explored. Because enzalutamide induces the CYP3A4 enzyme and taxanes are metabolized by this enzyme, a potential drug-drug interaction needs to be investigated.Experimental Design: Therefore, we performed a pharmacokinetic cross-over study in mCRPC patients who were scheduled for treatment with cabazitaxel Q3W (25 mg/m2). Patients were studied for three consecutive cabazitaxel cycles. Enzalutamide (160 mg once daily) was administered concomitantly after the first cabazitaxel cycle, during 6 weeks. Primary endpoint was the difference in mean area under the curve (AUC) between the first (cabazitaxel monotherapy) and third cabazitaxel cycle, when enzalutamide was added.Results: A potential clinically relevant 22% (95% CI, 9%-34%; P = 0.005) reduction in cabazitaxel exposure was found with concomitant enzalutamide use. The geometric mean AUC0-24h of cabazitaxel was 181 ng*h/mL (95% CI, 150-219 ng*h/mL) in cycle 3 and 234 ng*h/mL (95% CI, 209-261 ng*h/mL) in cycle 1. This combination did not result in excessive toxicity, whereas PSA response was promising.Conclusions: We found a significant decrease in cabazitaxel exposure when combined with enzalutamide. In an era of clinical trials on combination strategies for mCRPC, it is important to be aware of clinically relevant drug-drug interactions. Because recent study results support the use of a lower standard cabazitaxel dose of 20 mg/m2, the clinical relevance of this interaction may be substantial, because the addition of enzalutamide may result in subtherapeutic cabazitaxel exposure. Clin Cancer Res; 24(3); 541-6. ©2017 AACR.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Prostatic Neoplasms, Castration-Resistant/drug therapy , Prostatic Neoplasms, Castration-Resistant/pathology , Taxoids/pharmacokinetics , Aged , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Benzamides , Combined Modality Therapy , Drug Interactions , Humans , Male , Middle Aged , Neoplasm Metastasis , Neoplasm Staging , Nitriles , Phenylthiohydantoin/administration & dosage , Phenylthiohydantoin/analogs & derivatives , Randomized Controlled Trials as Topic , Taxoids/administration & dosage , Treatment Outcome
8.
Oncotarget ; 8(63): 106468-106474, 2017 Dec 05.
Article in English | MEDLINE | ID: mdl-29290963

ABSTRACT

BACKGROUND: Treatment selection for men with metastatic castration-resistant prostate cancer (mCRPC) has become increasingly challenging with the introduction of novel therapies at earlier disease stages. The purpose of this study was to identify prognostic factors for overall survival (OS) and PSA response in patients with mCRPC treated with cabazitaxel. RESULTS: 224 mCRPC patients were included in the current analysis. In multivariable analysis, WHO performance status, baseline hemoglobin, alkaline phosphatase and albumin were all significantly associated with OS. Hemoglobin and alkaline phosphatase were significantly associated with PSA response. CONCLUSIONS: This study identified prognostic factors for OS and PSA response of men with mCRPC treated with cabazitaxel. In an increasingly complicated treatment landscape with several treatment options available our findings might serve to estimate the chance of survival of men qualifying for treatment with second-line chemotherapy in daily practice. Furthermore, these data can be used to risk-stratify patients in clinical trials. METHODS: We performed a post-hoc analysis of a randomized phase II trial of mCRPC patients treated with cabazitaxel. Cox and logistic regression models were used to investigate the influence of clinical and biochemical variables on OS and PSA response. Nomograms were developed to estimate the chance of PSA response and OS.

11.
Bone ; 34(2): 320-9, 2004 Feb.
Article in English | MEDLINE | ID: mdl-14962810

ABSTRACT

Glucocorticoids have marked effects on bone metabolism, and continued exposure of skeletal tissue to excessive amounts of these steroids results in osteoporosis. Therefore, in the present proteomic study, we characterized the potential effects of glucocorticoids on protein expression in human osteoblastic cells. Using two-dimensional gel electrophoresis and mass spectrometry, we identified an increased expression of glutamine synthetase (GS) in dexamethasone (Dex)-treated human MG-63 osteosarcoma cells. GS is an enzyme catalyzing the conversion of glutamate and ammonia to glutamine. Intracellular and extracellular glutamate levels may be important in cell signalling mediated by glutamate transporters and receptors which have recently been found in bone cells. The induction of GS protein by Dex was accompanied by an increase in mRNA level and enzyme activity. Dex induction of GS was also mediated by glucocorticoid receptors (GRs) because it was blocked by the GR antagonist RU-38486. In addition, Dex induction of GS expression was partially blocked by cyclohexamide indicating that it at least partly required new protein synthesis. GS induction by Dex was not associated with apoptosis as determined by Bax/Bcl-2 ratio and DNA staining. In addition to MG-63 cells, Dex induction of GS was also observed in human G-292 osteosarcoma cells as well as conditionally immortalized human preosteoblastic (HOB-03-C5) and mature osteoblastic (HOB-03-CE6) cells. However, in two other human osteosarcoma cell lines, SaOS-2 and U2-OS, GS expression was not affected by Dex. This observation may be explained by the lower levels of GR protein in these cells. In summary, this is the first report of the regulation of GS expression by glucocorticoids in bone cells. The role of GS in bone cell metabolism and glucocorticoid action on the skeleton is not yet known, but as a modulator of intracellular glutamate and glutamine levels, it may have an important role in these processes.


Subject(s)
Dexamethasone/pharmacology , Glucocorticoids/pharmacology , Glutamate-Ammonia Ligase/biosynthesis , Osteoblasts/drug effects , Apoptosis/physiology , Cells, Cultured , Electrophoresis, Gel, Two-Dimensional , Glutamate-Ammonia Ligase/drug effects , Hormone Antagonists/pharmacology , Humans , Immunoblotting , Mass Spectrometry , Osteoblasts/metabolism , Protein Synthesis Inhibitors/pharmacology , Reverse Transcriptase Polymerase Chain Reaction
12.
J Cell Biochem ; 89(2): 389-400, 2003 May 15.
Article in English | MEDLINE | ID: mdl-12704802

ABSTRACT

Osteoblast differentiation is a key aspect of bone formation and remodeling. To further our understanding of the differentiation process, we have developed a collection of conditionally immortalized adult human osteoblast cell lines representing discrete stages of differentiation. To evaluate changes in gene expression associated with differentiation, polyA((+)) RNA from pre-osteoblasts, early and late osteoblasts, and pre-osteocytes was subjected to gene chip analysis using the Affymetrix Hu6800 chip in conjunction with an Affymetrix custom chip enriched in bone and cartilage cDNAs. Overall, the expression of 47 genes was found to change threefold or more on both chips between the pre-osteoblastic and pre-osteocytic stages of differentiation. Many of the observed differences, including down-regulation of collagen type I and collagen-processing enzymes, reflect expected patterns and support the relevance of our results. Other changes have not been reported and offer new insight into the osteoblast differentiation process. Thus, we observed regulation of factors controlling cell cycle and proliferation, reflecting decreased proliferation, and increased apoptosis in pre-osteocytic cells. Elements maintaining the cytoskeleton, extracellular matrix, and cell-cell adhesion also changed with differentiation reflecting profound alterations in cell architecture associated with the differentiation process. We also saw dramatic down-regulation of several components of complement and other immune response factors that may be involved in recruitment and differentiation of osteoclasts. The decrease in this group of genes may provide a mechanism for controlling bone remodeling of newly formed bone. Our screen also identified several signaling proteins that may control osteoblast differentiation. These include an orphan nuclear receptor DAX1 and a small ras-related GTPase associated with diabetes, both of which increased with increasing differentiation, as well as a high mobility group-box transcription factor, SOX4, that was down-regulated during differentiation. In summary, our study provides a comprehensive transcriptional profile of human osteoblast differentiation and identifies several genes of potential importance in controlling differentiation of osteoblasts.


Subject(s)
Cell Differentiation , Gene Expression Profiling , Osteoblasts/metabolism , Transcription, Genetic , Base Sequence , Cell Line , DNA Primers , Humans , Osteoblasts/cytology , Reverse Transcriptase Polymerase Chain Reaction
13.
J Cell Biochem ; 80(3): 424-40, 2001.
Article in English | MEDLINE | ID: mdl-11135373

ABSTRACT

The runt family transcription factor (AML-3/PEBP2alphaA1/Cbfa1/RUNX2) plays a crucial role in formation of the mineralized skeleton during embryogenesis and regulates maturation of the osteoblast phenotype. Because steroid hormones and growth factors significantly influence growth and differentiation properties of osteoblasts, we addressed Cbfa1 as a target gene for regulation by dexamethasone (Dex), 1,25(OH)D(3) (vitamin D(3)), 17beta-estradiol, and transforming growth factor-beta1 (TGF-beta1). The representation of functional protein levels by Western blot analyses and gel mobility shift assays was examined during the growth and mineralization of several conditionally immortalized human osteoblast cell lines HOB 04-T8, 03-CE6, and 03-CE10, each representing different stages of maturation. In situ immunofluorescence demonstrates Cbfa1 is associated with nuclear matrix in punctate domains, some of which are transcriptionally active, colocalizing with phosphorylated RNA polymerase II. Although each of the cell lines exhibited different responses to the steroid hormones and to TGF-beta1, all cell lines showed a similar increase in Cbfa1 protein and DNA binding activity induced only by Dex. On the other hand, Cbfa1 mRNA levels were not altered by Dex treatment. This regulation of Cbfa1 by steroid hormones in human osteoblasts contrasts to modifications in Cbfa1 expression in primary rat calvarial osteoblasts and the mouse MC3T3-E1 osteoblast cell line. Thus, these results reveal multiple levels of regulation of Cbfa1 expression and activity in osteoblasts. Moreover, the data suggest that in committed human osteoblasts, constitutive expression of Cbfa1 may be required to sustain the osteoblast phenotype.


Subject(s)
Cell Differentiation , Cell Division , Neoplasm Proteins , Osteoblasts/metabolism , Transcription Factors/metabolism , Animals , Cell Line , Core Binding Factor Alpha 1 Subunit , DNA/metabolism , Dexamethasone/pharmacology , Humans , Mice , Nuclear Matrix/metabolism , Osteoblasts/cytology , Osteoblasts/drug effects , Phenotype , Protein Binding , Rats , Up-Regulation/drug effects
14.
Bone ; 25(5): 535-43, 1999 Nov.
Article in English | MEDLINE | ID: mdl-10574573

ABSTRACT

Osteocalcin (OC) is an abundant noncollagenous bone matrix protein, yet its function is largely unknown. However, targeted ablation of two OC genes in mice lead to increased bone formation (Ducy et al. Nature 382:448-452; 1996). This implied that OC inhibits osteoblast activity, and that these cells express an OC receptor. In order to characterize the putative OC receptor, we used the Cytosensor microphysiometer to measure responses of a proliferative-stage, conditionally immortalized human osteoblast cell line (HOB-03-C5) to purified bovine OC (bOC). The Cytosensor measures a change in the extracellular acidification rate, which is primarily a measurement of metabolic activity. Treatment of the HOB cells for 5-60 sec with 0.17 micromol/L bOC generated a time-dependent, transient increase in the acidification rate that became optimal after 25 sec. Likewise, treatment of the cells for 25 sec with 0.021 to 1.9 micromol/L bOC caused a dose-dependent 70% increase in the acidification rate. Pre-treatment of the cells for 2 h with inhibitors of adenylyl cyclase, phospholipase C, and intracellular calcium release inhibited the response of the cells to bOC by 50%-100%, which suggested that the putative OC receptor was coupled to a G-protein. These observations from the Cytosensor were confirmed by measuring intracellular cyclic-adenosine monophosphate (cAMP) concentrations in response to bOC. Treatment of the cells for 10 min with bOC decreased basal cAMP levels by 65% in a dose-dependent manner with an IC50 of 0.22 microM. However, cotreatment of the cells with forskolin, which activates adenylyl cyclase, blunted this suppression. Moreover, pretreatment of the cells with pertussis toxin for 48 h, which inhibits G(alpha)i proteins, reversed the suppressive effects of bOC on cAMP production. Treatment of the HOB cells for 48 h with 0.19 to 1.5 micromol/L bOC caused a dose-dependent 40% decrease in alkaline phosphatase activity with an IC50 of 0.21 micromol/L, which suggested that OC may inhibit HOB activity. Finally, although the maturation stage, conditionally immortalized HOB-02-C1 cells also responded to bOC as measured by the Cytosensor, two osteosarcoma cell lines, SaOS-2 and ROS 17/2.8, exhibited a 5- to 10-fold lower response to the bone matrix protein, suggesting that the putative OC receptor was downregulated in these cells. However, all of these bone cell lines responded to parathyroid hormone treatment. In conclusion, these results provide evidence that the HOB cells express an OC receptor, and that this receptor appears to be coupled to a G(alpha)-protein.


Subject(s)
Osteoblasts/metabolism , Osteocalcin/metabolism , Receptors, Peptide/biosynthesis , Adenylate Cyclase Toxin , Adult , Alkaline Phosphatase/antagonists & inhibitors , Alkaline Phosphatase/metabolism , Animals , Biosensing Techniques , Cattle , Cell Line, Transformed , Colforsin/pharmacology , Cyclic AMP/biosynthesis , Cyclic AMP/metabolism , Dose-Response Relationship, Drug , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Humans , Mice , Osteoblasts/cytology , Osteoblasts/drug effects , Osteoblasts/enzymology , Osteocalcin/pharmacology , Osteocalcin/physiology , Pertussis Toxin , Rats , Receptors, Peptide/physiology , Signal Transduction/physiology , Tumor Cells, Cultured , Virulence Factors, Bordetella/pharmacology
15.
Endocrinology ; 140(6): 2439-51, 1999 Jun.
Article in English | MEDLINE | ID: mdl-10342828

ABSTRACT

Estrogens are important for bone homeostasis and are classified as antiresorptive agents. One of the mechanisms for this effect is the inhibition of cytokine-induced bone resorption, which is mediated in part through an interaction between the estrogen receptor (ER) and nuclear factor (NF)-kappaB in osteoblasts. We present evidence that bone-resorbing cytokines that activate NF-kappaB conversely inhibit ligand-dependent ER activity in the conditionally immortalized human osteoblast cell line, HOB-03-CE6. Treatment of HOB-03-CE6 cells with 17beta-estradiol (17beta-E2) up-regulated reporter gene activity [ERE-thymidine kinase (tk)-luciferase] 3- to 5-fold in a dose-dependent manner (EC50 = 1.0 pM). However, cotreatment of the cells with 17beta-E2 and increasing concentrations of either tumor necrosis factor-alpha (TNF alpha), interleukin-1alpha (IL-1alpha), or IL-1beta completely suppressed ERE-tk-luciferase activity in a dose-dependent manner (IC50 = 0.05-5.0 pM). On the other hand, treatment of the cells with growth factors either up-regulated or had no effect on ERE-tk-luciferase expression. Neither TNF alpha, IL-1alpha, nor IL-1beta treatment affected basal reporter gene activity in the cells, and the TNF alpha effect was reversed by a neutralizing antibody to the cytokine. TNF alpha treatment also suppressed ligand-dependent ER activity in MCF-7 human breast cancer cells, but not in Chinese hamster ovary cells that overexpressed human ER alpha, even though both cell lines responded to the cytokine as measured by the up-regulation of NFkappaB-tk-luciferase activity. TNF alpha treatment did not affect the steady state levels of either ER alpha or ER beta messenger RNA expression by the HOB-03-CE6 cells, nor did it reduce [125I]17beta-E2 binding. Moreover, TNF alpha treatment only weakly inhibited ligand-dependent glucocorticoid receptor activity in the HOB-03-CE6 cells. Bone-resorbing cytokines, which do not signal through the NF-kappaB pathway, did not suppress ERE-tk-luciferase activity in HOB-03-CE6 cells. Treatment of the cells with 17beta-E2 partially suppressed the activation of NF-kappaB by TNF alpha, but did not block cytokine-induced IL-6 secretion. Finally, cotreatment of HOB-03-CE6 cells with an antisense oligonucleotide to NF-kappaB p50 partially reversed the suppression of ERE-tk-luciferase activity by TNF alpha. In summary, these data provide evidence for a potent feedback inhibition of estrogen action in human osteoblasts that is at least partly mediated by the activation of NF-kappaB.


Subject(s)
Bone Resorption/etiology , Cytokines/pharmacology , Estradiol/pharmacology , Osteoblasts/drug effects , Receptors, Estrogen/drug effects , Animals , CHO Cells , Cells, Cultured , Cricetinae , Humans , NF-kappa B/physiology , NF-kappa B p50 Subunit , RNA, Messenger/analysis , Receptors, Estrogen/analysis , Receptors, Estrogen/genetics , Receptors, Glucocorticoid/analysis , Tumor Necrosis Factor-alpha/pharmacology
16.
Endocrinology ; 139(4): 2048-57, 1998 Apr.
Article in English | MEDLINE | ID: mdl-9528993

ABSTRACT

Estrogen responsiveness of bone is a fundamental regulatory mechanism operative in skeletal homeostasis. We examined the expression of estrogen receptor-alpha (ER) messenger RNA (mRNA) in cultured rat calvarial-derived osteoblasts during progressive development of the osteoblast phenotype. Levels of ER message were compared with the expression of traditional osteoblastic markers that have been mapped throughout the differentiation process of these cells. ER transcripts, measured using semiquantitative RT-PCR analysis, were expressed at low levels in early stage proliferating osteoblasts and increased at confluence upon initial expression of bone cell phenotypic genes. A 23-fold up-regulation of ER mRNA expression coincided with the initiation of alkaline phosphatase activity (day 8). ER mRNA levels progressively increased 70-fold, reaching a maximum level on days 22-25 in fully differentiated osteoblasts when osteocalcin expression peaked, but declined precipitously by day 32 in osteocytic cells. Analysis of RNA isolated directly from rat calvaria confirmed these in vitro results and demonstrated that ER message levels become more abundant postnatally as bone becomes more mineralized. We also examined the responsiveness of osteoblasts to 17beta-estradiol (17beta-E2) at two periods of maturation: the nodule-forming stage (day 14) and the late mineralization stage (day 30). Estradiol suppressed the levels of alkaline phosphatase, osteocalcin, osteonectin, and ER mRNAs on day 14, but up-regulated these messages on day 30. In contrast, 17beta-E2 treatment regulated the steady state levels of transforming growth factor-beta1 and type I procollagen mRNAs only in the late mineralization stage, whereas histone H4 message was unaffected by the steroid at either stage of differentiation. Thus, the observed developmental expression of ER mRNA correlates with progressive osteoblast differentiation and may be a contributing factor to differential regulation of bone cell gene expression by 17beta-E2.


Subject(s)
Cell Differentiation , Gene Expression Regulation, Developmental , Osteoblasts/cytology , Receptors, Estrogen/genetics , Animals , Bone and Bones/embryology , Calcification, Physiologic , Cells, Cultured , Collagen/genetics , Estradiol/pharmacology , Female , Gene Expression Regulation, Developmental/drug effects , Male , Polymerase Chain Reaction , RNA, Messenger/metabolism , Rats , Time Factors , Transforming Growth Factor beta/genetics , Up-Regulation
18.
J Cell Biochem ; 65(3): 368-87, 1997 Jun 01.
Article in English | MEDLINE | ID: mdl-9138093

ABSTRACT

Osteoblasts are established targets of estrogen action in bone. We screened 66 conditionally immortalized clonal human osteoblast cell lines for estrogen receptors (ERs) using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis for ER alpha mRNA and transactivation of adenovirus-estrogen response element (ERE)-tk-luciferase by 17 beta-estradiol (17 beta-E2) for functional ER protein. One of these cell lines, termed HOB-03-CE6, was chosen for further characterization. The cells, which were conditionally immortalized with a temperature-sensitive SV40 large T antigen, proliferated at the permissive temperature (34 degrees C) but stopped dividing at the nonpermissive temperature (> or = 39 degrees C). Alkaline phosphatase activity and osteocalcin secretion were upregulated by 1 alpha, 25-dihydroxyvitamin D3 in a dose-dependent manner. The cells also expressed type I collagen and other bone matrix proteins, secreted a variety of growth factors and cytokines, formed mineralized nodules based on alizarin red-S and von Kossa histochemical staining, and responded to dexamethasone, all-trans retinoic acid, and transforming growth factor-beta 1. This cell line expressed 42-fold less ER message than MCF-7 human breast cancer cells, as determined by quantitative RT-PCR. However, adenovirus-ERE-tk-luciferase activity was upregulated three- to fivefold in these cells by 17 beta-E2 with an EC50 of 64 pM. Furthermore, this upregulation was suppressed by co-treatment with the anti-estrogen ICI-182, 780. Cytosolic extracts of these cells specifically bound [125I]-17 beta-E2 in a concentration-dependent manner with a Bmax of 2.7 fmoles/mg protein (approximately 1,200 ERs/cell) and a Kd of 0.2 nM. DNA gel-shift analysis using a [32P]-ERE demonstrated the presence of ERs in nuclear extracts of these cells. Moreover, binding of the extracts to this ERE was blocked by a monoclonal antibody to the human ER DNA-binding domain. We evaluated these cells for 14 of 20 reported endogenous responses to 17 beta-E2 in osteoblasts. Although most of these responses appeared to be unaffected by the steroid, 17 beta-E2 suppressed parathyroid hormone-induced cAMP production, as well as basal interleukin-6 mRNA expression; conversely, the steroid upregulated the steady-state expression of alkaline phosphatase message in these cells. In summary, we have identified a clonal, conditionally phenotypic, human osteoblast cell line that expresses functional ERs and exhibits endogenous responses to 17 beta-E2. This cell line will be a valuable in vitro model for exploring some of the molecular mechanisms of estrogen action in bone.


Subject(s)
Estradiol/pharmacology , Osteoblasts/drug effects , Phenotype , Aged , Alkaline Phosphatase/metabolism , Antigens, Polyomavirus Transforming , Calcitriol/pharmacology , Cell Division , Cell Line , Cell Line, Transformed , Female , Gene Expression/drug effects , Humans , Osteoblasts/cytology , Osteoblasts/metabolism , Osteocalcin/metabolism , Polymerase Chain Reaction , Receptors, Estradiol/genetics , Temperature
19.
Endocrinology ; 137(11): 4592-604, 1996 Nov.
Article in English | MEDLINE | ID: mdl-8895322

ABSTRACT

Osteocytes are differentiated forms of osteoblasts that arise upon entrapment within the bone matrix. In this report, we describe the establishment and hormonal regulation of the first conditionally transformed human preosteocytic cell line. Primary adult bone cells were obtained from protease cell line. Primary adult bone cells were obtained from protease digestion of cancellous chips. The cells were infected with adenovirus-ori- SV40 tsA 209, which encodes for a temperature-sensitive large T-antigen. After immortalization, we isolated a clone designated HOB-01-C1. This cell line expressed the mutant T-antigen and proliferated at the permissive temperature (34 C) but stopped dividing at the nonpermissive temperature (39-40 C). Electron microscopy of cells incubated at 39 C demonstrated the presence of preosteocytic cellular processes, some of which appeared to form gap junctions or were rich in microfilaments. The clone expressed alpha 1 type (I) procollagen messenger RNA (mRNA) and secreted type I procollagen C peptide at both temperatures, and this expression was elevated 1.6-fold to 1.8-fold at 40 degrees C. The cells expressed very low basal levels of alkaline phosphatase activity (approximately 0.02 nmol/min.mg), which was increased 2- to 5-fold in a dose-dependent manner by 0.1-100 nM 1 alpha,25-dihydroxyvitamin D3 (vitamin D3) at both temperatures. Vitamin D3 also increased osteocalcin secretion in a dose-dependent manner when the clone was maintained at 34 C (approximately 6-fold), and this stimulation was enhanced > 5 fold at 40 C. In contrast to the low expression of alkaline phosphatase, the cells secreted high amounts of osteocalcin in response to vitamin D3 (approximately 15 ng/mg cell protein); this biochemical profile also resembled that of preosteocytes. Alizarin red-S histochemical staining demonstrated that these cells rapidly produced mineralized nodules at both temperatures. PTH (10 and 100 nM) had no effect on the intracellular accumulation of cAMP at 34 C but stimulated a 14- to 18-fold increase in the production of this second messenger at 40 C. In contrast, 100 nM prostaglandin E2 and 1 microM forskolin stimulated cAMP synthesis better at 34 C. Western blot analysis indicated that the cells expressed CD44, a putative osteocytic marker, at both temperatures. Finally, interleukin-1 beta and tumor necrosis factor-alpha (1-1000 pM) stimulated dose-dependent increases in the secretion of interleukin-6 and monocyte chemoattractant protein-1 at 34 C and 40C. We conclude that the HOB-01-C1 cell line has a preosteocytic phenotype. Moreover, these cells respond to calcitropic hormones and bone resorbing cytokines.


Subject(s)
Alkaline Phosphatase/metabolism , Calcitriol/pharmacology , Osteocytes/cytology , 1-Methyl-3-isobutylxanthine/pharmacology , Adult , Antigens, Viral, Tumor/biosynthesis , Biomarkers , Bone Matrix , Bone and Bones , Calcification, Physiologic , Cell Division/drug effects , Cell Line, Transformed , Clone Cells , Cyclic AMP/metabolism , Cytokines/biosynthesis , Humans , Hyaluronan Receptors/analysis , Hyaluronan Receptors/biosynthesis , Interleukin-1/pharmacology , Kinetics , Osteocalcin/biosynthesis , Osteocytes/drug effects , Osteocytes/metabolism , Parathyroid Hormone/pharmacology , Procollagen/metabolism , Simian virus 40 , Temperature , Tumor Necrosis Factor-alpha/pharmacology
20.
J Bone Miner Res ; 11(6): 806-19, 1996 Jun.
Article in English | MEDLINE | ID: mdl-8725178

ABSTRACT

Many osteoblastic cell lines are currently in use, but these have limitations either in terms of their relevance to adult human biology and disease or in terms of their suitability for biochemical and molecular analyses. Consequently, we undertook the development of conditionally transformed adult human osteoblastic cell lines. Osteoblasts were obtained from a normal explant cancellous bone chip culture. These cells were infected with adenovirus-ori-SV40 tsA 209, which encodes a temperature-sensitive large T-antigen mutant. Cells immortalized with this virus express a transformed phenotype at the permissive temperature of 34 degrees C but revert to a normal phenotype at the nonpermissive temperature of 40 degrees C. Using this approach, we have isolated several cell clones and describe the characterization of one that was designated HOB-02-C1. Immunocytochemistry revealed that > 95% of the cells express the large T-antigen at both temperatures. These cells exponentially proliferate at 34 degrees C with a doubling time of approximately 2 days but irreversibly stop dividing at 40 degrees C. However, cell volume increases > 2-fold when the cells are maintained for 6 days at the higher temperature. This clone expresses alpha 1 type (I) procollagen mRNA and secretes type I procollagen C-peptide at both temperatures, although the levels were slightly elevated at 40 degrees C. The cell line expresses alkaline phosphatase activity at 34 degrees C, and the basal level of this enzyme increases 2- to 6-fold at 40 degrees C. Alkaline phosphatase activity is induced 4- to 8-fold by 1 alpha,25-dihydroxyvitamin D3 (vitamin D3) at both temperatures, but transforming growth factor-beta 1 (TGF-beta 1) suppresses enzyme expression > 90% at 40 degrees C. Vitamin D3 also induces a 10-fold increase in osteocalcin secretion when the clone is maintained at 34 degrees C, and this induction is enhanced > 8-fold at 40 degrees C. Parathyroid hormone and forskolin stimulate a 4- to 6-fold increase in the production of intracellular cyclic AMP (cAMP) by the cells at 34 degrees C, and this stimulation is enhanced 2- to 4-fold at 40 degrees C. In contrast, prostaglandin E2 stimulates a 7- to 8-fold increase in cAMP only when the cells are maintained at 34 degrees C. This cell line secretes TGF-beta 1 and interleukin-6 (IL-6) at 34 degrees C, but only the basal secretion of IL-6 increases 70% at 40 degrees C. Finally, alizarin red-S histochemical staining demonstrates that these cells produce mineralized nodules at both temperatures. In summary, the results of this study indicate that the HOB-02-C1 cells have a mature osteoblastic phenotype. Consequently, this new cell line and others obtained in a similar fashion should be valuable in vitro tools for cellular, biochemical, and molecular studies of adult human osteoblast biology.


Subject(s)
Cell Line, Transformed , Osteoblasts/cytology , Adenoviridae/genetics , Aged , Alkaline Phosphatase/analysis , Antigens, Viral, Tumor/biosynthesis , Antigens, Viral, Tumor/genetics , Blotting, Western , Calcification, Physiologic/drug effects , Calcification, Physiologic/physiology , Cell Division , Cell Size , Cholecalciferol/pharmacology , Collagen/genetics , Cyclic AMP/biosynthesis , Dinoprostone/pharmacology , Humans , Immunohistochemistry , Interleukin-6/metabolism , Male , Mutation/genetics , Osteocalcin/drug effects , Osteocalcin/metabolism , Parathyroid Hormone/pharmacology , Simian virus 40/genetics , Temperature , Transforming Growth Factor beta/metabolism , Transforming Growth Factor beta/pharmacology
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