Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0029463 (osteosarcoma)
 16,637 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Skeletal alkaline phosphatase (ALP) is anchored to membrane inositol-phosphate on the outer surface of osteoblasts. Although skeletal ALP activity in serum is, essentially, all in an anchorless (soluble) form, in vitro studies indicate that ALP can be released in either an anchorless, soluble form (e.g., by a phospholipase) or an anchor-intact, insoluble form (e.g., by vesicle exocytosis). The current studies were intended to define the contributions of each of these putative processes of ALP release and to assess the significance of regulation by calcium (Ca) and skeletal effectors. ALP activity was measured in serum-free medium from replicate cultures of human osteosarcoma (SaOS-2) cells and normal human bone cells. Temperature-sensitive phase distribution (in Triton X-114) allowed separation of soluble from insoluble ALP activity. Our studies revealed that most of the ALP activity released from SaOS-2 cells was in an insoluble form (78% +/- 8%), a percentage that was constant between 2 and 96 hours. A similar result was seen for normal human bone cells. Calcium had a negative, biphasic dose-dependent effect on net release of ALP activity: r = -0.85, P < 0.001 at 24 hours, with KIapparent values for biphasic inhibition of 20 and 300 mumol/l Ca. Of the skeletal effectors tested, insulin-like growth factor-II (IGF-II) had the greatest effect, decreasing the net release of ALP activity in a dose-dependent manner (r = -0.82, P < 0.005). Neither Ca nor IGF-II affected the distribution of soluble/insoluble ALP activity by more than 9%. IGF-II had no effect on extracellular ALP stability, but the addition of Ca to Ca-free cultures resulted in parallel losses of extracellular ALP activity and ALP immunoreactive protein (P < 0.001 for each). A similar effect was seen when Ca was added to Ca-free, cell-free, conditioned medium, but not when Ca was added to purified ALP, which is consistent with the general hypothesis that a Ca-dependent protease might be present in the cell-conditioned medium. Together, these data suggest that most of the ALP activity released from osteoblasts is insoluble (and, presumably, anchorless), net release of ALP activity is negatively regulated by Ca and skeletal growth factors, the effect of Ca may reflect Ca-dependent protease activity, and an exogenous (e.g., serum) phospholipase may be responsible for releasing ALP from its insoluble anchor.
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PMID:Skeletal alkaline phosphatase activity is primarily released from human osteoblasts in an insoluble form, and the net release is inhibited by calcium and skeletal growth factors. 950 59

Cationic lipid-mediated gene transfer has been shown to be a competent albeit inefficient mechanism of promoting cellular gene transfer. One way to improve the efficacy of cationic lipid-mediated transgene expression is to optimize conditions for complex formation between the lipids and nucleic acids. In this report we describe the beneficial effects of using phosphate buffer to precondition lipofectin (a 1:1 (w/w) mixture of N-[1-(2,3-dioleyloxy)propyl]-n,n, n-trimethylammonium chloride (DOTMA), and dioleoyl phosphatidylethanolamine (DOPE)) prior to complexing with plasmid DNA or mRNA. Under such optimized conditions we studied the kinetics of DNA- and RNA-mediated transgene expression in a human osteosarcoma cell line (HOS). Preincubation of lipofectin in phosphate buffer resulted in up to 26- and 56-fold increases in luciferase expression from plasmid DNA and mRNA, respectively. Addition of chloroquine (50 microM), which enhanced plasmid-mediated gene delivery 3-fold, was synergistic with phosphate resulting in an additional 46-fold increase in luciferase expression. The preincubation with phosphate shortened both the time required for cellular uptake and the time to achieve maximal transgene expression. Optimal transfection was achieved in the presence of 30-80 mM phosphate, at pH 5.6-6.8 under which the phosphate anion is divalent. The effect of phosphate anion was specific in that monovalent Cl- and acetate anions were not stimulatory. These results demonstrate that divalent phosphate anion plays a stimulatory role during complex formation and transfection when cationic lipids come in contact with negatively charged nucleic acids and cell membranes. These findings delineate specific conditions which dramatically enhance transfection efficiency for both DNA and mRNA, and provide an effective procedure for gene transfection studies.
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PMID:Phosphate-enhanced transfection of cationic lipid-complexed mRNA and plasmid DNA. 951 70

Stanniocalcin (STC) is a Ca- and phosphate-regulating hormone produced by the corpuscles of Stannius in bony fishes. The mammalian homologue of STC has recently been reported (STC1), which stimulates the phosphate uptake of kidney. Here we report the cloning of a second mammalian stanniocalcin (STC2) from the human osteosarcoma cDNA library. STC2 has 302 amino acid residues with 34% identity with STC1 and eel STC. STC2 has a conserved N-glycosylation site and is rich in cysteines as is the case with other stanniocalcins. STC2 has the same exon-intron boundaries as STC1. The culture medium of STC2-transfected CHO cells inhibited the promoter activity of Na-phosphate cotransporter (NaPi-3) and also inhibited the phosphate uptake of a kidney cell line (OK cells). Therefore, the function of STC2 seems to be opposite to that of STC1 on Na-phosphate cotransporter. Northern blot analysis revealed multiple transcripts in number of human tissues with high levels being present in skeletal muscle and heart. STC2 was also expressed in mice widely and its expression was lower in hypophosphatemic mice (Hyp mice) in many organs. We have cloned a widely expressed new human stanniocalcin homologue which suppressed the expression of renal Na-phosphate cotransporter.
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PMID:Molecular cloning of a second human stanniocalcin homologue (STC2). 975 16

Hyaluronidase has gained increasing interest as an adjuvant in local and systemic cancer therapy, despite the incomplete knowledge of its physiological function. To this end, direct intratumoral injection of bovine testicular hyaluronidase (500, 1600 or 7500 U in 50 microl phosphate-buffered saline (PBS)) was performed in orthotopic (o.t.) osteosarcoma xenografts grown in the hind leg of nude mice. Control tumours received 50 microl PBS alone or supplemented with 10% bovine serum albumin (BSA). Central tumour interstitial fluid pressure (IFP) and mean arterial blood pressure (MABP) were measured using the wick-in-needle technique and after cannulation of the carotid artery, respectively. IFP was 32 +/- 8 mmHg (n = 44, mean +/- SD) in untreated tumours and there was a significant correlation between tumour IFP and volume (P < 0.01). The hyaluronidase injection reduced IFP to 63-84% after 1 h compared with controls (P < 0.05) and in a non-linear concentration-dependent manner. MABP was not affected significantly. In conclusion, an intratumoral hyaluronidase injection might reduce IFP temporally in solid osteosarcoma xenografts.
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PMID:Hyaluronidase reduces the interstitial fluid pressure in solid tumours in a non-linear concentration-dependent manner. 983 21

Bone tissue has been shown to contain numerous cell-to-cell signalling peptides called growth factors. These growth factors are thought to have important regulating effects for bone remodeling and bone healing, due to their potent effects on bone cell metabolism. In vivo studies over the last half decade have demonstrated that growth factors candidates for future clinical use in orthopedic surgery. In numerous clinical situations enhanced bone formation and bone healing could lead to improved results of surgical procedures. This thesis describes the most important bone growth factors and their actions in vitro and in vivo. In vitro investigations of growth factor effects on osteoblast chemotaxis and metabolism are described as well as in vivo studies with growth factor stimulation of fracture healing and bone healing to prosthetic-like implants. In vitro results: Several growth factors exhibited chemotactic effects towards human osteoblasts. TGF-beta 1 and PDGF-BB had the strongest chemotactic effects, whereas PDGF-AA, IGF-1, and IGF-2 had less but significant chemotactic effects towards human osteoblasts. TGF-beta 1 exhibited the highest chemotactic potency with maximal activity at 100 pg/mL, whereas the other growth factors had maximal effects at 10-100 ng/mL. BMP-2 was found to have chemotactic effects toward human osteoblasts, human bone marrow osteoprogenitor cells, and U2-OS osteosarcoma cells. BMP-4 and BMP-6 were without any chemotactic effects towards these celltypes. Human bone marrow osteoprogenitor cells were the most responsive celltype to BMP-2 stimulation. Growth factor combinations resulted in synergic stimulative effects on different metabolic functions on human osteoblasts. Combinations with TGF-beta 1 and PDGF-BB strongly stimulated proliferation and chemotaxis. Combinations with TGF-beta 1, PDGF-BB and BMP-2 strongly stimulated an osteoblast differentiation parameter (alkaline phosphatase activity). The different growth factor combinations had no effect on collagen synthesis in human osteoblasts. In vivo results: Continuous application of 1 and 10 micrograms natural TGF-beta to a plated tibial osteotomy in rabbits increased mechanical bending strength and callus formation at 6 weeks observation. Diaphyseal cortical bone remodeling was not affected by the local growth factor application. In a dog model with unloaded implants surrounded by a gap, 0.3 microgram rhTGF-beta 1 adsorbed to gritblasted tricalcium phosphate coated implants, was able to enhance mechanical fixation, bone ingrowth and gap bone formation. 3.0 micrograms rhTGF-beta 1 had less but significant stimulative effect. In a weight-loaded model, 0.3 microgram rhTGF-beta 1, adsorbed to gritblasted tricalcium phosphate coated implants, was able to enhance bone ingrowth, without enhancement of mechanical fixation. In the unloaded model, 0.3 microgram rhTGF-beta 1, adsorbed to gritblasted hydroxyapatite coated implants, was able to enhance bone ingrowth, without enhancement of mechanical fixation. 3.0 micrograms rhTGF-beta 1 had no stimulative effects. The establishment of a biological implant fixation concept with growth factor absorbed to ceramic coatings of implants was successful. These data are promising for a possible future clinical usage of growth factors, especially for enhancement of bone healing to cementless prosthetic components.
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PMID:Growth factor stimulation of bone healing. Effects on osteoblasts, osteomies, and implants fixation. 985 74

Inorganic phosphate (Pi) can regulate the level of skeletal alkaline phosphatase (ALP) activity in human osteoblast-like cells by stabilizing the enzyme (without affecting transcription, ALP release from the cell surface, or the amount of ALP protein). These observations suggest that Pi determines the level of ALP activity by modulating a process of irreversible inactivation. The current studies were intended to examine the hypothesis that this inactivation of ALP activity is caused by the dissociation of an active center Zn and that Pi inhibits that dissociation. Initial studies showed that Zn, like Pi, could increase ALP specific activity in human osteosarcoma SaOS-2 cells in a time- and dose-dependent manner (e.g., a 50% increase at 0.2 micromol/liter Zn, P < 0.005). This effect was specific for Zn (i.e., no similar effect was seen with Ca, Fe, Co, Mg, Mn, or Cu), but not for SaOS-2 cells. Zn also increased ALP specific activity in (human osteosarcoma) MG-63 cells and in cells derived from normal human vertebrae (P < 0.001 for each). The effect of Zn to increase ALP activity was not associated with parallel increases in total protein synthesis, collagen production, or tartrate-resistant acid phosphatase activity (no change in any of these indices), net IGF-2 synthesis (a Zn-dependent decrease, P < 0.005), or PTH-dependent synthesis of cAMP (a biphasic increase, P < 0.02). Kinetic studies of Pi and Zn as co-effectors of ALP activity showed that Zn was a mixed-type effector with respect to Pi, whereas Pi was competitive with respect to Zn. Mechanistic studies showed that (1) Zn reversed the effect of Pi withdrawal to decrease ALP activity, but not by reactivating inactive ALP protein (the process required protein synthesis, without increases in ALP mRNA or the level of ALP immunoreactive protein); (2) Zn increased the half-life of ALP activity in intact cells and after a partial purification; and (3) Pi inhibited the process of ALP inactivation by EDTA (which chelates active center Zn). All these findings are consistent with the general hypothesis that Pi increases the half-life of skeletal ALP by preventing the dissociation of active center Zn and with a mechanistic model of skeletal ALP activity in which active center Zn participates in Pi-ester binding and/or hydrolysis.
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PMID:Effects of zinc on human skeletal alkaline phosphatase activity in vitro. 991 26

Novel bioactive ceramic hollow microspheres with an apparent density in the range 0.8-1.0 g cm(-3) have been developed as microcarriers for 3-D bone tissue formation in rotating-wall vessels (RWV). Hollow ceramic microspheres with a composition of 58-72% SiO2, 28-42% Al2O3 (wt%) and an apparent density 0.8-1.0 g cm(-3) were pretreated in 1.0 N NaOH for 2 h before being coated with synthesized calcium hydroxyapatite (HA) particulate sol. The HA-coated hollow microspheres were sintered for 1 h at 600, 800 and 1000 degrees C. SEM analysis revealed that the grain size and pore size of the calcium phosphate coating increased with the sintering temperature. FTIR analysis showed that crystalline calcium hydroxyapatite was present in the coatings sintered at 600 and 800 degrees C. When sintered at 1000 degrees C, the coating consisted of alpha-tricalcium phosphate. All the coatings adhered well, independent of sintering temperature. The trajectory analysis revealed that the hollow microsphere remained suspended in a rotating-wall vessel (RWV), and experienced a low shear stress (approximately 0.6 dyn cm(-2)). Cell culture studies using rat bone marrow stromal cells and osteosarcoma cells (ROS 17/2.8) showed that the cells attached to and formed 3-D aggregates with the hollow microspheres in a RWV. Extracellular matrix was observed in the aggregates. These data suggest that these hollow bioactive ceramic microspheres can be used as microcarriers for 3-D bone tissue formation in vitro, as well as for the study of the effects of microgravity on bone cell functions.
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PMID:Fabrication, characterization and evaluation of bioceramic hollow microspheres used as microcarriers for 3-D bone tissue formation in rotating bioreactors. 1037 99

Insulin-like growth factor (IGF) I is a potent mitogen for human osteosarcoma cells such as the Saos-2/B-10 cell line. IGF binding proteins (IGFBPs) prevent stimulation of DNA synthesis by IGFs. In contrast to recombinant human (rh) IGFBP-2, -3, -4, and -5, 10-100 nM rhIGFBP-6 stimulated [(3)H]thymidine incorporation into DNA and multiplication of Saos-2/B-10 cells. Upon withdrawal of serum, 30 nM IGFBP-6 also decreased apoptosis (within 4 h) and increased protein content and sodium-dependent phosphate uptake (within 24 h), but less potently than IGF I. (125)I-labeled rhIGFBP-6 did not bind to the cells, and cold IGFBP-6 did not affect (125)I-labeled IGF I binding. Production of IGF I, IGF II, and IGFBP-6 by the cells or significant degradation of rhIGFBP-6 could not be detected within 24 h of incubation. Thus, among the rhIGFBPs tested, rhIGFBP-6 is unique in stimulating osteosarcoma cell growth. Furthermore, it has an antiapoptotic effect.
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PMID:Mitogenic and antiapoptotic effects of insulin-like growth factor binding protein-6 in the human osteoblastic osteosarcoma cell line Saos-2/B-10. 1051 58

Oncogenic osteomalacia is a rare paraneoplastic syndrome. It is characterized by bone pain, muscle weakness, gait disturbance, fractures and skeletal deformities. Hypophosphatemia, diminished renal phosphate reabsorption, decreased 1,25 dihydroxy Vitamin D and elevated alkaline phosphatase are the biochemical hallmarks of this disorder. Most tumors are of mesenchymal origin. We report the case of a 39-year-old woman with oncogenic osteomalacia caused by osteosarcoma of the right scapula which was unrecognized for several years. She subsequently developed tertiary hyperparathyroidism after treatment with oral phosphate and Vitamin D. This case illustrates that oncogenic osteomalacia may persist for many years before the tumor is discovered. This is because the tumors are frequently very small and are in obscure locations. The uniqueness of this case is the coexistence of hyperparathyroidism and oncogenic osteomalacia. Five other cases have been reported up to date. All patients had received phosphate supplement, ranging from 10 to 14 years prior to their diagnosis. Interestingly, our patient was on the treatment for only 2 years. The proposed mechanism is that exogenous phosphate stimulates parathyroid activity through sequestration of calcium.
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PMID:Development of tertiary hyperparathyroidism after phosphate supplementation in oncogenic osteomalacia. 1085 15

We describe a new, highly sensitive semiquantitative method for rapid measurement of in vitro mineralization using calcein. Fluorescence analysis of the calcein bound to the calcium phosphate (hydroxyapatite) allows direct quantitation of extracellular matrix mineral content in monolayer cultures of bone-forming cells such as primary osteoblasts or osteosarcoma cells. Osteosarcoma cell lines UMR 106 and SaOS-2 were used to demonstrate that qualitatively, calcein was bound to the same regions of the mineralized cell monolayer as seen by conventional histological staining with von Kossa or Alizarin Red S. Moreover, total bound calcein could be quantitated by direct fluorescence analysis using a Cytofluor II plate reader. Changes in cell monolayer calcein fluorescence were shown to correlate well with direct colorimetric measurement of acid-solubilized Ca+2 from parallel cultures. Relative mineral quantitation by calcein fluorescence is rapid and more sensitive than colorimetric Ca+2 assays, can be performed directly on unfixed or fixed cell monolayers, and does not require the use of radioisotopes. The cell monolayer remains intact and potentially available for further analysis.
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PMID:Semi-quantitative fluorescence analysis of calcein binding as a measurement of in vitro mineralization. 1090 18


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