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1.
J Anal Toxicol ; 37(4): 233-6, 2013 May.
Article in English | MEDLINE | ID: mdl-23471956

ABSTRACT

Buprenorphine is a potent partial opioid agonist that is analyzed in urine to (i) monitor adherence to maintenance or detoxification therapy and (ii) detect illicit use. Buprenorphine analysis is commonly conducted on urine by immunoassay, but is subject to cross-reactivity from other drugs/drug metabolites, including morphine, codeine and dihydrocodeine. This study reports false-positive buprenorphine analysis [Thermo Fisher Scientific cloned enzyme donor immunoassay (CEDIA)] in patients who denied unauthorized buprenorphine use prior to sampling, but who had been prescribed amisulpride. In two cases, confirmatory analysis by liquid chromatography-tandem mass spectrometry was negative (<0.5 µg/L) for buprenorphine and metabolites and positive for amisulpride. Although the cross-reactivity of amisulpride and sulpiride in the CEDIA buprenorphine assay is low (estimated at 0.003 and 0.002%, respectively), it remains a significant consideration given the likely high concentrations of these compounds in urine relative to the low cutoff of the buprenorphine assay. Neither amisulpride nor sulpiride was listed as potential sources of interference on the CEDIA data sheet when this work was performed. These findings highlight the importance of confirming immunoassay-positive buprenorphine results using a more selective analytical technique.


Subject(s)
Buprenorphine/urine , Immunoenzyme Techniques/methods , Sulpiride/analogs & derivatives , Sulpiride/urine , Adult , Amisulpride , Buprenorphine/administration & dosage , Chromatography, High Pressure Liquid , False Positive Reactions , Humans , Immunoassay/methods , Male , Substance Abuse Detection/methods , Sulpiride/therapeutic use
2.
Biomed Mater ; 7(3): 035016, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22539092

ABSTRACT

Topography and surface chemistry have a profound effect on the way in which cells interact with an implant, which in turn impacts on clinical use and performance. In this paper we examine an electrochemical polishing approach in H2SO4/methanol that can be applied to the widely used orthopaedic/dentistry implant material, Ti6Al4V, to produce structured surfaces. The surface roughness, as characterized by R(a), was found to be dependent on the time of electropolishing but not on the voltage parameters used here. The surface chemistry, however, was dependent on the applied electrochemical potential. It was found that the chemical composition of the surface layer was modified during the electrochemical process, and at high potentials (9.0 V) a pure TiO2 layer of at least 10 nm was created on top of the bulk alloy. Characterization of these surfaces with rat cells from the osteoblast lineage provided further evidence of contact guidance by microscale topography with morphology analysis correlating with surface roughness (R(a) 300­550 nm). Formation of a bone-like matrix after long-term culture on these surfaces was not strongly dependent upon R(a) values but followed the voltage parameter. These findings suggest that the surfaces created by treatment at higher voltages (9.0 V) produced a nanoscale layer of pure TiO2 on the Ti6Al4V surface that influenced the programme of cellular differentiation culminating in osteogenesis.


Subject(s)
Biocompatible Materials/chemistry , Osteoblasts/cytology , Osteoblasts/physiology , Titanium/chemistry , Alloys , Animals , Cell Survival , Cells, Cultured , Electroplating , Materials Testing , Rats , Surface Properties
3.
J Mater Sci Mater Med ; 21(1): 277-82, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19669624

ABSTRACT

Intensive work has been performed on the characterization of the mechanical properties of mineralised tissues formed in vivo. However, the mechanical properties of bone-like tissue formed in vitro have rarely been characterised. Most research has either focused on compact cortical bone or cancellous bone, whilst leaving woven bone unaddressed. In this study, bone-like mineralised matrix was produced by osteoblasts cultured in vitro on the surface of titanium alloys. The volume of this tissue-engineered bone is so small that the conventional tensile tests or bending tests are implausible. Therefore, nanoindentation techniques which allow the characterization of the test material from the nanoscale to the microscale were adopted. These reveal the apparent elastic modulus and hardness of the calcospherulite crystals (a representative element for woven bone) are 2.35 +/- 0.73 and 0.41 +/- 0.15 GPa, respectively. The nanoscale viscoelasticity of such woven bone was further assessed by dynamic indentation analysis.


Subject(s)
Alloys/chemistry , Bone Development/physiology , Tissue Scaffolds/chemistry , Titanium/chemistry , Animals , Animals, Newborn , Biomechanical Phenomena , Bone Substitutes/chemistry , Bone and Bones/cytology , Bone and Bones/physiology , Cells, Cultured , Hardness/physiology , Materials Testing , Nanostructures/chemistry , Nanotechnology/methods , Rats , Rats, Sprague-Dawley , Stress, Mechanical , Tissue Engineering
4.
Biomed Mater ; 1(3): 144-54, 2006 Sep.
Article in English | MEDLINE | ID: mdl-18458396

ABSTRACT

The aims of this study were to fabricate poly(ethylene glycol) (PEG) hydrogel micropatterns on a biomaterial surface to guide osteoblast behaviour and to study how incorporating vascular endothelial growth factor (VEGF) within the adhered hydrogel influenced cell morphology. Standard photolithographic procedures or photopolymerization through a poly(dimethyl siloxane) (PDMS) mould were used to fabricate patterned PEG hydrogels on the surface of silanized silicon wafers. Hydrogel patterns were evaluated by light microscopy and surface profilometry. Rat osteoblasts were cultured on these surfaces and cell morphology investigated by fluorescence microscopy, scanning electron microscopy (SEM) and atomic force microscopy (AFM). Release of protein trapped in the polymerized PEG was evaluated and VEGF-PEG surfaces were characterized for their ability to support cell growth. These studies show that photopolymerized PEG can be used to create anti-adhesive structures on the surface of silicon that completely control where cell interaction with the substrate takes place. Using conventional lithography, structures down to 50 microm were routinely fabricated with the boundaries exhibiting sloping sides. Using the PDMS mould approach, structures were fabricated as small as 10 microm and boundaries were very sharp and vertical. Osteoblasts exhibiting typical morphology only grew on the silicon wafer surface that was not coated with PEG. Adding BSA to the monomer solution showed that protein could be released from the hydrogel for up to 7 days in vitro. Incorporating VEGF in the hydrogel produced micropatterns that dramatically altered osteoblast behaviour. At boundaries with the VEGF-PEG hydrogel, there was striking formation of cellular processes and membrane ruffling indicative of a change in cell morphology. This study has explored the morphogenetic properties of VEGF and the applications of nano/microfabrication techniques for guided tissue (bone) regeneration in dental and orthopaedic applications using osteoinductive PEG hydrogel micropatterns.


Subject(s)
Biocompatible Materials/chemistry , Osteoblasts/physiology , Osteogenesis/physiology , Polyethylene Glycols/chemistry , Tissue Engineering/methods , Vascular Endothelial Growth Factor A/administration & dosage , Animals , Cell Adhesion/drug effects , Cell Adhesion/physiology , Cell Enlargement/drug effects , Cell Size/drug effects , Cells, Cultured , Drug Carriers/chemistry , Hydrogels/chemistry , Materials Testing , Osteoblasts/drug effects , Osteogenesis/drug effects , Rats , Surface Properties , Vascular Endothelial Growth Factor A/chemistry
5.
Biochem Biophys Res Commun ; 335(3): 793-8, 2005 Sep 30.
Article in English | MEDLINE | ID: mdl-16105658

ABSTRACT

Osteoclasts are derived from haematopoietic stem cell precursors of the monocyte/macrophage cell lineage, through interaction with factors that are believed to include M-CSF and RANKL. VEGF is a proangiogenic cytokine that has been shown to promote osteoclast differentiation and survival. In this study, we assessed the role of VEGF and its receptors in osteoclastogenesis, in vitro, by culturing osteoclast precursors in the presence of VEGF, VEGF receptor-specific ligands, and blocking antibodies to VEGF receptors. Activation of VEGFR1 in the presence of RANKL induces osteoclast differentiation. Stimulating the receptors individually induced increased resorption by osteoclasts compared to controls but not to the level observed when stimulating both receptors simultaneously. We have shown that VEGF induces osteoclast differentiation through its action on VEGFR1. The way in which VEGF mediates its effect on mature osteoclast activity, however, may be through its interaction with both receptor subtypes.


Subject(s)
Cell Differentiation/physiology , Osteoclasts/cytology , Receptors, Vascular Endothelial Growth Factor/physiology , Bone Resorption , Cells, Cultured , Humans , Osteoclasts/physiology , Vascular Endothelial Growth Factor A/physiology
6.
Br J Cancer ; 92(8): 1531-7, 2005 Apr 25.
Article in English | MEDLINE | ID: mdl-15812559

ABSTRACT

Vascular endothelial growth factor (VEGF) is a proangiogenic cytokine that is expressed highly in many solid tumours often correlating with a poor prognosis. In this study, we investigated the expression of VEGF and its receptors in bone metastases from primary human breast tumours and further characterised its effects on osteoclasts in vitro. Breast cancer metastases to bone were immunohistochemically stained for VEGF, its receptors VEGFR1 and 2 (vascular endothelial growth factor receptor 1 and 2), demonstrating that breast cancer metastases express VEGF strongly and that surrounding osteoclasts express both VEGFR1 and VEGFR2. RAW 264.7 cells (mouse monocyte cell line) and human peripheral blood mononuclear cells (PBMCs) were cultured with VEGF, RANKL and M-CSF. VEGF and RANKL together induced differentiation of multinucleated, tartrate-resistant acid phophatase (TRAP)-positive cells in similar numbers to M-CSF and RANKL. The PBMCs were also able to significantly stimulate resorption of mineralised matrix after treatment with M-CSF with RANKL and VEGF with RANKL. We have shown that VEGF in the presence of RANKL supports PBMC differentiation into osteoclast-like cells, able to resorb substrate. Vascular endothelial growth factor may therefore play a role in physiological bone resorption and in pathological situations. Consequently, VEGF signalling may be a therapeutic target for osteoclast inhibition in conditions such as tumour osteolysis.


Subject(s)
Bone Neoplasms/metabolism , Bone Neoplasms/secondary , Breast Neoplasms/pathology , Osteolysis/metabolism , Vascular Endothelial Growth Factor A/metabolism , Adult , Aged , Animals , Carrier Proteins/pharmacology , Cell Differentiation/drug effects , Cells, Cultured/drug effects , Cells, Cultured/metabolism , Humans , Immunohistochemistry , Membrane Glycoproteins/pharmacology , Middle Aged , Monocytes/drug effects , Monocytes/metabolism , Osteoclasts/cytology , Osteoclasts/drug effects , Osteoclasts/metabolism , RANK Ligand , Receptor Activator of Nuclear Factor-kappa B , Receptors, Vascular Endothelial Growth Factor , Vascular Endothelial Growth Factor A/pharmacology
7.
Bone ; 36(3): 408-17, 2005 Mar.
Article in English | MEDLINE | ID: mdl-15777654

ABSTRACT

During bone formation, there are numerous pivotal changes in the interrelationships between osteoblasts and molecules of the extracellular matrix (ECM). Consequently, the mechanisms that underlie the temporal and spatial distribution of ECM molecules in bone are of considerable interest in understanding its formation. A subfamily of a disintegrin and metalloproteinase (ADAMs) has been identified, which contain thrombospondin-like motifs (ADAMTS), and can break down several ECM molecules. Using reversed transcribed PCR, we identified ADAMTS-1, -4 and -5 mRNA expression in cultures of rat osteoblasts treated with ascorbic acid, beta-glycerophosphate and dexamethasone, molecules known to drive osteoblast differentiation. Of these, ADAMTS-1 followed most closely the osteogenic marker osteocalcin during in vitro mineralisation. Consequently, we studied, in detail, protein expression of ADAMTS-1 during in vitro osteogenesis together with ADAMTS-1 immunohistochemistry staining of sections from 2- and 10-day-old rat femur. Western analysis of osteoblast proteins showed ADAMTS-1 products that correspond well with both full-length and furin-processed species. In the ECM laid down by osteoblasts, only the mature secreted protein (approximately 90 kDa) and its accumulation during the later stages of osteogenesis in vitro were noticed. Furthermore, immunostaining with an antibody recognising ADAMTS-1 demonstrated strong expression around mineralised nodules and intense focal staining of putative new areas of nodule formation in vitro. Finally, immunohistochemistry of 2- and 10-day-old rat femur localised ADAMTS-1 protein to regions associated with osteogenesis. These data show that ADAMTS-1 protein accumulates in osteoblast ECM during differentiation. Furthermore, the focalised expression of ADAMTS-1 in regions of osteogenesis, both in vitro and in vivo, implicates this multifunctional protein to be involved in mineralised nodule and bone formation.


Subject(s)
Disintegrins/biosynthesis , Gene Expression Regulation, Enzymologic/physiology , Metalloendopeptidases/biosynthesis , Osteoblasts/enzymology , Osteogenesis/physiology , Up-Regulation/physiology , ADAM Proteins , ADAMTS1 Protein , Animals , Cells, Cultured , Disintegrins/genetics , Extracellular Matrix Proteins/biosynthesis , Extracellular Matrix Proteins/genetics , Metalloendopeptidases/genetics , Proteoglycans/metabolism , RNA, Messenger/biosynthesis , Rats , Rats, Sprague-Dawley
8.
J Biomed Mater Res B Appl Biomater ; 71(2): 244-51, 2004 Nov 15.
Article in English | MEDLINE | ID: mdl-15459898

ABSTRACT

The cellular reaction to wear debris may result in the failure of an artificial joint's fixation to the skeleton. The influence of debris opsinization on cell activity has received little attention. This study seeks to establish whether different proteinaceous culture environments may invoke variant cellular responses to debris challenge. Consideration of the zeta potential of a low density polyethylene particle group and an ex vitro ultrahigh molecular weight polyethylene particle group revealed that the nature of the protein adsorbants is related to the concentration of the proteins in solution. Furthermore, the composition of the adsorbed layer was shown to vary with the spectra of proteins in solution. In standard cell culture conditions zeta potential approached zero, indicating the high probability of particle agglomeration. Cell challenge studies with U937 macrophages showed that BSA and FCS protein adsorption mediated increased cell adhesion, while bovine IgG showed little change over control values. No changes in behavior of osteoblastic cells were observed in similar experiments.


Subject(s)
Biocompatible Materials/pharmacology , Macrophages/drug effects , Osteoblasts/drug effects , Polyethylenes/pharmacology , Adsorption , Algorithms , Animals , Cell Line, Tumor , Colloids/pharmacology , Culture Media , Electromagnetic Fields , Humans , Particle Size , Proteins/chemistry , Rats , Serum Albumin, Bovine/chemistry , U937 Cells
9.
Biomaterials ; 25(18): 3991-4000, 2004 Aug.
Article in English | MEDLINE | ID: mdl-15046889

ABSTRACT

A novel micro-cellular polymer with a well-defined and uniform micro-architecture has been developed as a three-dimensional support matrix for in vitro tissue engineering applications. This material is manufactured through a high internal phase emulsion (HIPE) polymerization route and may be modified with hydroxyapatite. The generic form of the support is known as PolyHIPE Polymer (PHP). By changing the chemical composition of the emulsion and the processing conditions, the pore size can be altered from sub-micron range to a few hundred microns and the porosity varied from 70% to 97%. Our work has investigated the use of this micro-porous polymer as a biomaterial to support the growth of osteoblasts, the bone forming cells in vitro. Three groups of polymers were used that had pore sizes of 40, 60 and 100 microm. Results demonstrated in vitro cell-polymer compatibility, with osteoblasts forming multicellular layers on the polymer surface and also migrating to a maximum depth of 1.4mm inside the scaffold after 35 days in culture. PHP was also able to support the differentiation of osteoblasts and the production of a bone-like matrix. The effect of modifying the polymer with hydroxyapatite was also studied and showed that there was a significant increase in osteoblast numbers penetrating into the polymer. There were few differences, between the pore sizes studied, on the overall penetration of osteoblasts into the polymer but the rate of movement into 100 microm PHP was significantly higher compared to the other sizes investigated. This study shows that osteoblasts seeded onto PHP demonstrate cellular attachment, proliferation and ingrowth leading to the support of an osteoblastic phenotype. Therefore this highly porous scaffold has a potential for bone tissue engineering.


Subject(s)
Biocompatible Materials/chemistry , Bone Substitutes/chemistry , Osteoblasts/cytology , Osteoblasts/physiology , Osteogenesis/physiology , Polymers/chemistry , Styrenes/chemistry , Tissue Engineering/methods , Animals , Cell Adhesion/physiology , Cell Culture Techniques/methods , Cell Differentiation/physiology , Cell Division/physiology , Cells, Cultured , Materials Testing , Osseointegration/physiology , Porosity , Rats , Surface Properties
10.
Clin Endocrinol (Oxf) ; 53(5): 621-8, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11106924

ABSTRACT

OBJECTIVE: Fibrous dysplasia, observed in bone lesions in the McCune Albright syndrome (MAS), is thought to result from abnormalities in cells of the osteogenic lineage associated with over-activation of the cAMP signalling pathway in affected cells. The aim of this study was to investigate the role of parathyroid hormone-related protein (PTHrP) in the aetiology of MAS, and to determine a possible therapeutic role for 1,25-dihydroxy vitamin D(3) (1,25(OH)(2)D(3)). DESIGN: The effects of 1,25(OH)(2)D(3) on PTHrP production and mRNA expression were determined in vitro. 1,25(OH)(2)D(3) therapy was administered to three patients with MAS. PATIENTS: Clinical data from four MAS patients (MAS1, 2, 3 and 4), and in vitro studies using bone from three MAS patients (MAS1, 2, and 3), are presented. MEASUREMENTS: Immunoradiometric assay and low-cycle number reverse transcriptase-linked PCR were used to determine PTHrP production and mRNA expression in vitro. Standard clinical biochemistry was recorded pre and post commencement of 1,25(OH)(2)D(3) treatment. RESULTS: We report the elevated secretion of PTHrP, and a concomitant rise in PTHrP mRNA expression, in cultured osteoblasts from three MAS patients. Treatment with 1,25(OH)(2)D(3) produced a dose-dependent decrease in PTHrP protein secretion and mRNA expression. Marked improvement in bone biochemistry in MAS1, 2 and 3 post treatment with 1,25(OH)(2)D(3) is documented. CONCLUSION: This study provides the first evidence suggesting that PTHrP may contribute to the aetiology of fibrous dysplasia in MAS. In addition, the therapeutic administration of 1,25(OH)92)D(3) may provide clinicians with an important new regime for symptomatic relief of bone pain and fracture in some patients with MAS.


Subject(s)
Fibrous Dysplasia, Polyostotic/etiology , Parathyroid Hormone/physiology , Adolescent , Adult , Case-Control Studies , Cells, Cultured , Dose-Response Relationship, Drug , Female , Fibrous Dysplasia, Polyostotic/drug therapy , Fibrous Dysplasia, Polyostotic/metabolism , Gene Expression/physiology , Humans , Immunoradiometric Assay , Male , Osteoblasts/physiology , Proto-Oncogene Mas , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Steroid Hydroxylases/therapeutic use
11.
Int J Exp Pathol ; 81(2): 159-63, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10762443

ABSTRACT

Human osteoblasts were derived in culture from explants of bone from patients who had recently suffered osteoporotic fractures and from patients with no evidence of osteoporosis. The expression of cytokine mRNA in these osteoblasts was subsequently determined by reverse transcriptase-linked polymerase chain reaction (RT-PCR). We have detected mRNA for IL-1beta, IL-3, IL-6, IL-8, TNF-alpha and -beta, and the three TGF-beta isoforms in the cells. The profile of cytokines expressed by osteoblasts derived from patients with osteoporotic fractures was consistent with profiles observed in osteoblasts derived from patients with no evidence of reduced bone mass--the latter included children undergoing corrective surgery and adult subjects ranging from 31 to 80 years undergoing elective surgery for osteoarthritis and other bone pathologies.


Subject(s)
Cytokines/metabolism , Osteoblasts/metabolism , Osteoporosis/metabolism , Spinal Fractures/metabolism , Adolescent , Adult , Aged , Cell Culture Techniques , Cytokines/genetics , Female , Gene Expression , Humans , Male , Middle Aged , Osteoporosis/complications , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Spinal Fractures/etiology
12.
Bone ; 24(6): 571-8, 1999 Jun.
Article in English | MEDLINE | ID: mdl-10375199

ABSTRACT

Bone cells are regulated by interactions with both growth factors and components of the extracellular matrix (ECM). Syndecans are cell-surface heparan sulfate proteoglycans known to play a role in cell adhesion and migration, and binding of growth factors. This study was performed to investigate the expression of syndecans by osteoblasts. Reverse transcription-linked polymerase chain reaction (RT-PCR) and Northern analysis detected syndecan transcripts in the human osteosarcoma cell lines MG-63, TE-85, SaOS-2, and U2OS; human osteoblast-like cells; rat calvarial osteoblasts; and in human bone. Western blot analysis of proteoglycans from MG-63 and TE-85 cells detected multiple heparan sulfate proteoglycan core proteins consistent with syndecan expression. Regulation of syndecan-1, -2, and -4 expression was investigated in TE-85, MG-63, and SaOS-2 cells, in response to interleukin (IL)-1beta, and IL-6, parathyroid hormone [PTH(1-34)], and 1,25(OH)2-vitamin D3. Northern analysis demonstrated that in the osteosarcoma cell lines there was no regulation of syndecan transcript levels in response to PTH(1-34) or 1,25(OH)2-vitamin D3 for 24 or 48 h. In contrast, when MG-63 and SaOS-2 cells were incubated with IL-1beta (0.01-10 ng/mL) and IL-6 (0.1-50 ng/mL) there was a dose-dependent decrease in mRNA levels for syndecan-1 and -2 at 24 and 48 h, but in response to IL-1beta upregulation in the levels of syndecan-4 transcripts. In addition, Northern analysis was performed on RNA isolated from neonatal rat calvarial osteoblasts cultured under conditions that promote osteogenesis for 0, 5, 13, 21, and 35 days. Syndecan-1 expression was observed to decrease during the culture period, syndecan-2 transcript levels increased, and there appeared to be no overall change in syndecan-4 levels. Controlled expression of syndecans by cells of the osteoblast lineage may be important in the regulation of osteoblastic proliferation and differentiation.


Subject(s)
Cytokines/pharmacology , Gene Expression Regulation/drug effects , Hormones/pharmacology , Membrane Glycoproteins/genetics , Osteoblasts/drug effects , Osteoblasts/metabolism , Proteoglycans/genetics , Animals , Base Sequence , Calcitriol/pharmacology , Cell Line , Heparan Sulfate Proteoglycans/biosynthesis , Humans , Interleukin-1/pharmacology , Interleukin-6/pharmacology , Oligonucleotide Probes/genetics , Osteosarcoma/genetics , Osteosarcoma/metabolism , Parathyroid Hormone/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Reverse Transcriptase Polymerase Chain Reaction , Syndecan-1 , Syndecans , Tumor Cells, Cultured
13.
Bone ; 24(4): 297-303, 1999 Apr.
Article in English | MEDLINE | ID: mdl-10221541

ABSTRACT

The adhesive interactions that occur between bone cells and the developing matrix during bone formation help guide coupled remodeling and the maintenance of bone mass. Here, we provide evidence that acetylcholinesterase (AChE) is a novel osteoblast-derived mediator of cell-matrix interactions in bone. These findings complement an increasing body of evidence which suggests that AChE, in addition to its role in terminating cholinergic signaling, may be instrumental in regulating cellular differentiation and adhesion. We have shown, using RT-PCR, that osteosarcoma cell lines and primary cultures of osteoblasts express AChE mRNA. Expression appeared to be differentiation-dependent, and restricted to AChE splice variants containing the T subunit (exon 6). Immunofluorescent localization demonstrated that these osteoblastic cells expressed protein for AChE with an intracellular vesicular distribution. Immunohistochemistry on tissue sections confirmed AChE expression by osteoblasts in vivo, and revealed the presence of AChE along cement lines, also identified by enzyme histochemistry. In vitro functional studies indicated that osteoblast-like cells adhered specifically to and spread on AChE substrates, but did not interact with butyrylcholinesterase, a closely related protein. Our evidence strongly implicates AChE as a novel bone matrix protein, capable of mediating cell-matrix interactions, and as such may be a principal participant in organized bone formation and the regulation of remodeling.


Subject(s)
Acetylcholinesterase/metabolism , Bone Development/physiology , Bone Matrix/physiology , Osteoblasts/enzymology , Animals , Cell Adhesion/physiology , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, Cultured
14.
Bone ; 22(6): 645-9, 1998 Jun.
Article in English | MEDLINE | ID: mdl-9626403

ABSTRACT

Signaling between the various types of cells found in bone is responsible for controlling the activity of osteoblasts and osteoclasts, and therefore the regulation of bone mass. Our identification of a neuronal glutamate transporter in osteoblasts and osteocytes suggests the possibility that bone cells may use the excitatory amino acid glutamate as a signaling molecule. In these studies we report the expression of different subtypes of glutamate receptors in osteoblasts and osteoclasts in vitro and in vivo. We have identified expression in human and rat bone cells of N-methyl-D-aspartate receptor-1 (NMDAR-1) and 2D subunits and PSD-95, the NMDA receptor clustering protein associated with signaling in the central nervous system. In situ hybridization and immunohistochemistry localized NMDAR-1 expression to osteoblasts and osteoclasts in human tissue sections. These findings strengthen the suggestion that glutamate is involved in signaling between bone cells.


Subject(s)
ATP-Binding Cassette Transporters/metabolism , Glutamic Acid/metabolism , Osteoblasts/metabolism , Osteoclasts/metabolism , Receptors, N-Methyl-D-Aspartate/biosynthesis , ATP-Binding Cassette Transporters/genetics , Amino Acid Transport System X-AG , Animals , Base Composition , Biological Transport , Disks Large Homolog 4 Protein , Glutamic Acid/genetics , Guanylate Kinases , Humans , Immunohistochemistry , In Situ Hybridization , Intracellular Signaling Peptides and Proteins , Membrane Proteins , Mice , Nerve Tissue Proteins/biosynthesis , Nerve Tissue Proteins/genetics , Polymerase Chain Reaction , RNA, Messenger/isolation & purification , RNA, Messenger/metabolism , Rats , Receptors, N-Methyl-D-Aspartate/genetics , Signal Transduction/genetics
15.
Carcinogenesis ; 18(1): 23-9, 1997 Jan.
Article in English | MEDLINE | ID: mdl-9054585

ABSTRACT

It has been previously reported that 8701-BC cells, derived from a primary carcinoma of the breast, constitutively express parathyroid hormone (PTH)-related peptide (PTHrP) and PTH/PTHrP receptor (PTH/PTHrP-R) genes, that N-terminal, mid-regional and C-terminal immunoreactive PTHrP can be found in cell conditioned medium and, furthermore, that exogenously added PTHrP (1-34), (67-86) and, to a minor extent, (107-139) are anti-mitogenic but promote Matrigel invasion by this cell line. It has also been reported that PTHrP gene expression is selectively switched on in those 8701-BC clonal lines endowed with a higher proliferation rate and invasive ability in vitro. Here we have first examined the presence of PTH/PTHrP-R transcript in the different 8701-BC clones by PCR and Southern blot analysis. Second, we have studied the growth and invasive response in vitro to PTHrP fragments by some of these clones, i.e. BC-3A, BC-61 and BC-66, selected on the basis of their lower (BC-3A) or higher (BC-1 and BC-66) Matrigel invasion ability and their expression of PTHrP (positive for BC-61 and BC-66) and PTH/PTHrP-R (positive for BC-61). Our data show the existence of clonal heterogeneity for PTH/PTHrP-R mRNA and for the proliferative and invasive responses elicited by treatment with diverse PTHrP fragments. In particular: (i) the sensitivity to PTHrP (1-34) is restricted due to the uneven expression of PTH/PTHrP-R; (ii) BC-3A cells (the less 'aggressive' clone) are resistant to the anti-mitogenic effect of the PTHrP domains and, most noticeably, exhibit a growth-potentiating response to PTHrP (67-86) opposite to that found for both the parental 8701-BC cells and the two other clones; (iii) all PTHrP fragments tested induced the expression of a growth-restraining and invasion-promoting phenotype by BC-61 cells (one of the more 'aggressive' clones). Present data in vitro support the hypothesis that in vivo PTHrP may be a key element in local control of the invasive process during breast carcinoma development and that its role may be, in turn, dependent upon the biological characteristics and the level of malignancy of the target cells within the multiclonal population of a primary tumour.


Subject(s)
Breast Neoplasms/pathology , Neoplasm Invasiveness , Neoplasm Proteins/pharmacology , Parathyroid Hormone/pharmacology , Peptide Fragments/pharmacology , Proteins/pharmacology , Cell Division/drug effects , Female , Humans , Parathyroid Hormone-Related Protein , Tumor Cells, Cultured/drug effects
16.
Br J Cancer ; 72(1): 90-5, 1995 Jul.
Article in English | MEDLINE | ID: mdl-7599071

ABSTRACT

Previous reports have shown the production of parathyroid hormone-related protein (PTHrP) by breast cancer cells in vivo and in vitro. We have investigated the expression of the PTH/PTHrP receptor by the human breast cancer cell lines MCF-7, ZR-75-1, T-47-D, SK-BR-3, Hs578T and MDA-MB231. Using reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot analysis, we detected transcripts for the receptor in MCF-7, SK-BR-3 and MDA-MB231 cells. There was no evidence of receptor mRNA in ZR-75-1 and Hs578T cells. Furthermore, Northern blot analysis of mRNA from MCF-7 cells showed two transcripts of 1.5 and 2.4 kb which coded for the PTH/PTHrP receptor. Expression of PTH/PTHrP receptor mRNA by the breast cancer cell lines was also correlated with the detection of PTHrP transcripts. RT-PCR demonstrated PTHrP mRNA in MCF-7, ZR-75-1, T-47-D and Hs578T cells, but not in SK-BR-3 and MDA-MB231 cells. The detection of receptor transcripts was complemented by [3H]thymidine and bromodeoxyuridine incorporation studies, in which mitogenic responses to PTH and PTHrP were observed in MCF-7 cells but not in Hs578T cells. In response to both PTH(1-34) and PTHrP(1-34), quiescent MCF-7 cells proliferated in a similar dose-dependent manner (1.6-100 ng ml-1). No mitogenic effects of these peptides were observed with Hs578T cells. In addition, levels of intracellular cAMP were measured in MCF-7 and Hs578T cells in response to PTHrP(1-34). In MCF-7 cells there was a significant rise in cAMP with 100 ng ml-1 PTHrP(1-34). The expression of PTH/PTHrP receptor by breast cancer cells suggests that PTHrP may be a paracrine/autocrine regulator of breast carcinoma.


Subject(s)
Breast Neoplasms/chemistry , Receptors, Parathyroid Hormone/analysis , Base Sequence , Cyclic AMP/analysis , Female , Humans , Molecular Sequence Data , RNA, Messenger/analysis , Receptor, Parathyroid Hormone, Type 1 , Receptors, Parathyroid Hormone/genetics , Tumor Cells, Cultured
17.
J Bone Miner Res ; 10(7): 1137-45, 1995 Jul.
Article in English | MEDLINE | ID: mdl-7484291

ABSTRACT

Extracellular ATP acting through purinoceptors may be an important factor in the modulation of bone turnover. In this study we cloned and sequenced the P2U purinoceptor from osteoclastoma, confirming the recently published human sequence. Furthermore, by the reverse transcriptase-linked polymerase chain reaction (RT-PCR) and Southern blotting we demonstrated expression of P2U receptor mRNA in bone, primary cultures of human bone-derived cells, and two osteosarcoma cell lines, Saos2 and Te85. P2U receptor transcripts were identified in alkaline phosphatase-positive human bone-derived cells isolated by flow cytometry providing strong evidence for the expression of the P2U purinoceptor in mature osteoblasts. P2U receptor transcripts were also detected in a purified giant cell population isolated from osteoclastoma, indicating that this receptor is also expressed by osteoclasts. These data suggest that purinergic agonists may play a role in the regulation of bone metabolism.


Subject(s)
Bone Neoplasms/metabolism , Bone and Bones/metabolism , Giant Cell Tumor of Bone/metabolism , Osteoblasts/metabolism , Receptors, Purinergic P2/genetics , Aged , Alkaline Phosphatase/metabolism , Base Sequence , Blotting, Southern , Bone Neoplasms/pathology , Cells, Cultured , Cloning, Molecular , DNA, Complementary/biosynthesis , DNA, Complementary/chemistry , Flow Cytometry , Giant Cell Tumor of Bone/pathology , Giant Cells/metabolism , Giant Cells/pathology , Humans , Male , Molecular Sequence Data , Osteoclasts/metabolism , Osteoclasts/pathology , Osteosarcoma/pathology , Polymerase Chain Reaction , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Purinergic P2/metabolism , Sequence Homology, Nucleic Acid , Tumor Cells, Cultured
18.
Mol Cell Endocrinol ; 111(2): 225-32, 1995 Jun.
Article in English | MEDLINE | ID: mdl-7556886

ABSTRACT

It has been previously reported that 8701-BC cells, derived from a primary carcinoma of the breast, constitutively express parathyroid hormone-related peptide (PTHrP) gene and that N-terminal PTHrP immunoreactivity can be found in cell medium. Here we have firstly measured immunoreactive PTHrP in 8701-BC cell medium using antibodies raised against midregion and C-terminal fragments, and also demonstrated the expression of PTH/PTHrP receptor by 8701-BC cells. Secondly, we have examined the role, if any, elicited by diverse PTHrP domains on 8701-BC cell proliferation, and invasive behaviour in vitro related to production of extracellular proteolytic enzymes. Our data show that PTHrP [1-34], and, to a minor extent, [67-86] and [107-139], are anti-mitogenic but 'invadogenic' for 8701-BC cells, and suggest that diverse enzymatic activities may contribute to cell invasion in response to different PTHrP fragments. In light of the present data on a chemoattractive role for PTHrP in vitro, we hypothesize that this protein might intervene in local control of the invasive process in breast carcinoma.


Subject(s)
Breast Neoplasms/pathology , Proteins/pharmacology , Base Sequence , Cell Division/drug effects , Endopeptidases/metabolism , Humans , Molecular Sequence Data , Neoplasm Invasiveness , Parathyroid Hormone/genetics , Parathyroid Hormone-Related Protein , Polymerase Chain Reaction , Protease Inhibitors/pharmacology , RNA, Messenger/metabolism , RNA-Directed DNA Polymerase , Receptor, Parathyroid Hormone, Type 1 , Receptors, Parathyroid Hormone/genetics , Tumor Cells, Cultured
19.
Int J Cancer ; 61(3): 416-9, 1995 May 04.
Article in English | MEDLINE | ID: mdl-7729956

ABSTRACT

The enzyme 17 beta-hydroxysteroid dehydrogenase (17-HSD) is a key regulator of intracellular 17 beta-estradiol (E2), which is associated with breast cancer and is influenced by paracrine factors released by breast-cancer fibroblasts. Since the incidence of breast cancer is much higher in females than in males, we have used an in vitro cell culture system to investigate whether male fibroblasts may inhibit breast-cancer genesis by restricting the intracellular accumulation of E2. Fibroblasts were obtained from normal males and females undergoing reduction mammoplasty, and from females with benign or malignant breast lesions. Fibroblast-conditioned medium (CM) was incubated with the established breast-cancer cell line, MCF-7, and its effects on 17-HSD activity were assessed. CM (25% v/v) from male breast fibroblasts had a significant inhibitory effect on reductive 17-HSD, decreasing E2 production. This was in direct contrast to the effects of CM from female breast fibroblasts, which had a powerful stimulatory effect on reductive 17-HSD. RT-PCR allowing simultaneous detection of a range of cytokines was performed on each type of fibroblast. IL-3 mRNA was consistently detected in fibroblasts from male but not female breast tissue. Addition of rhIL-3 to cultures of MCF-7 caused a reduction in 17-HSD activity and addition of a polyclonal antibody directed against IL-3 to male CM completely reversed the inhibitory effects of CM. Thus, male breast fibroblasts may be responsible for secreting IL-3-like factors which, given the considerably lower incidence rates of breast cancer in men, may have a protective effect against breast cancer.


Subject(s)
17-Hydroxysteroid Dehydrogenases/antagonists & inhibitors , Breast Neoplasms, Male/prevention & control , Breast Neoplasms/prevention & control , Breast/physiology , Cytokines/biosynthesis , Interleukin-3/metabolism , Sex Characteristics , Breast/immunology , Breast/metabolism , Culture Media, Conditioned , Estradiol/biosynthesis , Female , Fibroblasts/immunology , Fibroblasts/metabolism , Gene Expression , Humans , Male , Polymerase Chain Reaction , Reference Values , Transcription, Genetic
20.
J Bone Miner Res ; 10(1): 17-25, 1995 Jan.
Article in English | MEDLINE | ID: mdl-7747625

ABSTRACT

We investigated the production of parathyroid hormone-related protein (PTHrP) by cells derived from explants of human bone. Using an immunoradiometric assay (IRMA), PTHrP was detected in conditioned medium from cultures of bone-derived cells from 6 of 7 patients investigated in this study. PTHrP mRNA was identified in human bone cells using reverse transcriptase-linked polymerase chain reaction (RT-PCR) and by Northern analysis. Transcripts for PTHrP were detected in a purified population of alkaline phosphatase positive cells isolated from human bone marrow cultures by flow cytometry, confirming the expression of PTHrP mRNA by cells of the osteoblastic lineage. Production of PTHrP was inhibited by 10(-6) M of the glucocorticoids, prednisolone and desacetylated deflazacort, in a dose-dependent manner. In addition, RT-PCR followed by Southern blot analysis detected a decrease in steady-state PTHrP mRNA in cultures of human bone-derived cells treated with 10(-6) M prednisolone.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Osteoblasts/metabolism , Prednisolone/pharmacology , Pregnenediones/pharmacology , Proteins/genetics , Adolescent , Adult , Aged , Alkaline Phosphatase/metabolism , Base Sequence , Blotting, Northern , Blotting, Southern , Bone Marrow Cells , Cells, Cultured , Child , Culture Media, Conditioned , DNA Primers/chemistry , Dose-Response Relationship, Drug , Female , Humans , Male , Middle Aged , Molecular Sequence Data , Osteoblasts/cytology , Parathyroid Hormone-Related Protein , Polymerase Chain Reaction , Protein Biosynthesis , Proteins/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism
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