tretinoin and Osteosarcoma

Topics: Anthraquinones; Antibodies, Monoclonal; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Brain Neoplasms; Breast Neoplasms; Cisplatin; Dose-Response Relationship, Drug; Female; Genetic Techniques; Hodgkin Disease; Hormones; Humans; Immunotherapy; Interferons; Male; Mechlorethamine; Methotrexate; Mitoxantrone; Neoplasm Metastasis; Neoplasms; Neoplasms, Germ Cell and Embryonal; Nitrosourea Compounds; Osteosarcoma; Prednisone; Procarbazine; Prognosis; Receptors, Cell Surface; Tretinoin; Vincristine

Osteosarcoma (OS) is the most common primary malignant bone tumor in adolescents. The molecular mechanism behind OS progression and metastasis remains poorly understood, which limits the effectiveness of current therapies. RNA N. Liquid chromatography-tandem mass spectrometry (LC-MS/MS), dot blotting, and colorimetric ELISA were used to detect m. We observed the abundance of m. Our study revealed that METTL14 contributes to OS progression and ATRA resistance as an m. This work was supported by the National Natural Science Foundation of China (Grants 81972510 and 81772864).

Topics: Adolescent; Bone Neoplasms; Cell Line, Tumor; Chromatography, Liquid; Humans; Methyltransferases; Osteosarcoma; RNA; RNA-Binding Proteins; RNA, Messenger; Tandem Mass Spectrometry; Trans-Activators; Tretinoin; Tumor Suppressor Proteins

The prognosis of patients with osteosarcoma in many cases remains poor, and life expectancy with lung metastases is around 12 months. Chemotherapy and radiotherapy can only temporarily control neoplastic progression, followed by developing chemo and radioresistant tumours.. This is a retrospective observational study on 15 patients diagnosed with osteosarcoma and treated by a multitherapy approach. The multitherapy consisted of somatostatin and analogous (octreotide) all-trans-retinoic acid (ATRA), β-Carotene, axerophthol dissolved in vitamin E, vitamin D, vitamin C, melatonin (MLT), proteoglycans, glycosaminoglycans, hydroxyurea, and sodium butyrate.. This multitherapy increased the survival rate and life quality, without overt toxicity, compared to the standard treatment for osteosarcomas. The agents in this approach have several functions. They exert antiproliferative, antiangiogenic, cytostatic, antioxidant, antimetastatic, and immunomodulating features. Moreover, the inclusion of ATRA, MLT, and sodium butyrate has reinforced antitumor properties on cancer stem cells. Furthermore, the non-cytolytic and non-cytotoxic metronomic hydroxyurea dosage increased the biological therapy outcome by strengthening antitumor capability.. This multitherapy approach is effective against osteosarcoma.. The multistrategy of this multitherapy therapy are inhibiting the proliferative-invasiveness and neoplastic angiogenesis, silencing the survival system of cancer stem cells, enhancing the immunomodulatory and antioxidant activities, improving vitality and efficiency of normal cells, and depressing the efficiency and vitality of neoplastic ones.

Topics: Antioxidants; Bone Neoplasms; Butyric Acid; Humans; Hydroxyurea; Melatonin; Osteosarcoma; Tretinoin

The main objective of this study was to analyze changes in the antiproliferative effect of vitamin D3, in the form of calcitriol and calcidiol, via its combined application with all-

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Calcifediol; Calcitriol; Cell Line, Tumor; Drug Screening Assays, Antitumor; Humans; Osteosarcoma; Receptors, Calcitriol; Retinoic Acid Receptor alpha; Tretinoin; Vitamins

Osteosarcoma, a common type of bone cancer in children, and represents an aggressive and fetal cancer worldwide. Osteosarcoma initiating cells are considered to be a subpopulation of cancer cells which contribute to the progression, recurrence, metastasis and multi-drug resistance of osteosarcoma. CD133 is considered to be one marker for osteosarcoma initiating cells. All-trans retinoic acid (ATRA), an active metabolite of vitamin A under the family retinoid, is an up-and-coming drug which was able to effectively treat various cancer initiating cells. Nevertheless, there have been no research that reported the activity of ATRA against osteosarcoma initiating cells. In this research, we hereby examined the potential activity of ATRA in osteosarcoma initiating cells, and developed lipid-polymer nanoparticles with CD133 aptamers for targeted ATRA delivery to osteosarcoma initiating cells. Using the cytotoxicity assay, colony formation assay, tumorsphere formation assay and flow cytometry, the therapeutic effect of ATRA and ATRA-loaded lipid-polymer nanoparticles conjugated with CD133 aptamers (ATRA-PLNP-CD133) against osteosarcoma initiating cells were investigated. The results showed that ATRA exerted potent activity towards osteosarcoma initiating cells. ATRA-PLNP-CD133, which showed a size of 129.9 nm and a sustained release of ATRA during 144 h, was demonstrated to efficiently and specifically promote the ATRA delivery to osteosarcoma initiating cells, and achieve superior therapeutic efficacy in osteosarcoma compared with ATRA and non-targeted nanoparticles. This is the first report of the therapeutic efficacy of ATRA towards osteosarcoma initiating cells, and the increased ATRA delivery by nanoparticles to osteosarcoma initiating cells using CD133 aptamers. ATRA-PLNP-CD133 represent an up-and coming approach for the therapy of osteosarcoma initiating cells.

Topics: AC133 Antigen; Animals; Antineoplastic Agents; Aptamers, Nucleotide; Bone Neoplasms; Dose-Response Relationship, Drug; Drug Delivery Systems; Humans; Lipids; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Nanoparticles; Osteosarcoma; Polymers; Tretinoin; Xenograft Model Antitumor Assays

Emerging evidence indicates that M2-polarized tumor-associated macrophages (TAMs) directly participate in tumor initiation, progression and metastasis. However, to date, few studies have investigated novel strategies for inhibiting TAMs in order to overcome osteosarcoma. In this study, we reported that M2 macrophages were enriched in osteosarcoma tissues from patients, and M2-polarized TAMs enhanced cancer initiation and stemness of osteosarcoma cells, thereby establishing M2-polarized TAMs as a therapeutic target for blocking osteosarcoma formation. We also found that all-trans retinoic acid (ATRA) weakened TAM-induced osteosarcoma tumor formation by inhibiting M2 polarization of TAMs in vivo, and inhibited the colony formation, as well as sphere-formation capacity of osteosarcoma cells promoted by M2-type macrophages in vitro. Furthermore, M2-type macrophages enhanced cancer stem cells (CSCs) properties as assessed by increasing the numbers of CD117

Topics: Animals; Cell Differentiation; Cells, Cultured; Female; Macrophages; Mice; Mice, Inbred BALB C; Neoplastic Stem Cells; Osteosarcoma; RAW 264.7 Cells; Tretinoin

Retinoic acid receptor alpha (RARα) has a critical role in the differentiation process of osteosarcoma cells induced by all-trans retinoic acid (ATRA). However, degradation of RARα through ubiquitin proteasome pathway weakens the differentiation efficiency of osteosarcoma cells. In this study, we discover that murine double minute-2 (MDM2) acts as an E3 ubiquitin ligase to target RARα for degradation. We observe that MDM2 is required for RARα polyubiquitination and proteasomal degradation because downregulation of MDM2 by short hairpin RNA results in the accumulation of RARα, and MDM2 overexpression promotes the degradation of RARα. We also demonstrate that the N-terminal domain of MDM2 (amino acids 1-109) is the major RARα-binding site. Importantly, endogenous MDM2 levels are not only upregulated in human primary osteosarcoma blasts but are also inversely correlated with the level of osteopontin, which is a marker of bone differentiation. Moreover, MDM2 impairs the ATRA-induced osteoblastic differentiation of osteosarcoma cells, whereas an inhibitor of the MDM2 ubiquitin ligase synergizes with ATRA to enhance the differentiation of osteosarcoma cells and primary osteosarcoma blasts. Therefore, our study indicates that MDM2 serves as an E3 ubiquitin ligase to regulate the degradation of RARα and suggests that MDM2 is a novel therapeutic target for ATRA-based differentiation therapeutic approaches in osteosarcoma.

Topics: Antineoplastic Agents; Bone Neoplasms; Cell Differentiation; Cell Line, Tumor; Humans; Osteoblasts; Osteosarcoma; Proto-Oncogene Proteins c-mdm2; Retinoic Acid Receptor alpha; Tretinoin

In this study, gambogic acid (GA) and retinoic acid chlorochalcone (RACC) co-loaded glycol chitosan nanoparticle was successfully developed and studied for its therapeutic efficacy against osteosarcoma cancer cells. The GA/RACC loaded glycol chitosan nanoparticles (RGNP) was nanosized and exhibited a controlled release of drug in either pH 7.4 and pH 5.0. Owing to the strong positive charge on the RGNP surface, efficiency cellular uptake was observed in cancer cells. Moreover, a synergistic combination of GA and RACC were effectively suppressed the tumor growth progression. The half maximal inhibitory concentration (IC50) values in MG63 cells were 0.89μg/ml and 0.35μg/ml for GA and RGNP after 24h. The results clearly suggest the synergist effect of GA and RACC in effectively inhibiting the cancer cell proliferation. The RGNP as expected induced a remarkably higher apoptosis of cancer cells with ∼28%. Overall, combination of GA and RACC encapsulated in a nanocarrier could be an effective strategy to treat osteosarcoma. Future studies will focus on the in vivo evaluation of GA/RACC-loaded polymeric nanoparticles.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cyclohexanones; Drug Liberation; Endocytosis; Humans; Kinetics; Nanomedicine; Nanoparticles; Osteosarcoma; Treatment Outcome; Tretinoin; Xanthones

Osteosarcoma is the most common of all the bone malignancies and accounts for 30-80% of the primary skeletal sarcomas. The overall survival rate of patients with osteosarcoma is < 20% suggesting poor prognosis.. The present study demonstrates the effect of retinoic acid chlorochalcone (RACC) incorporated glycol chitosan (GC) nanoparticle transfection in osteosarcoma cells. MG-63 and Saos-2 osteosarcoma cells were transfected with various concentrations of RACC-incorporated GC nanoparticle for 24 h. The effect on cell proliferation, Ezh2 expression, apoptosis, cell cycle arrest, cell migration and invasiveness, Akt phosphorylation and local tumour growth and metastases were studied.. MG-63 and Saos-2 osteosarcoma cells on RACC-incorporated GC nanoparticle transfection for 24 h showed a concentration-dependent inhibition of cell proliferation. Of the various concentrations of RACC tested, the effective concentration started from 5 μM with an IC50 of 20 μM. Wound healing assay also showed that RACC-incorporated GC nanoparticles inhibited migration of tumor cells more effectively compared to the parent RA. RACC transfection resulted in inhibition of cell proliferation, Ezh2 expression inhibition, apoptosis through mitochondrial pathway by decrease in membrane potential and release of cytochrome c and cell cycle arrest in the G0/G1 phase. The invasiveness of cells treated with 5 and 20 μM RACC was decreased by 49 and 76% respectively, compared to the control. RACC-treated mice showed significantly lower number of metastases compared to that in the control mice.. Thus, RACC-incorporated glycol chitosan nanoparticle strategy can be promising for the treatment of osteosarcoma.

Topics: Animals; Apoptosis; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Cell Proliferation; Chitosan; Cyclohexanones; Enhancer of Zeste Homolog 2 Protein; Humans; Male; Mice; Mitochondria; Nanoparticles; Neoplasm Invasiveness; Neoplasm Metastasis; Osteosarcoma; Phosphorylation; Polycomb Repressive Complex 2; Proto-Oncogene Proteins c-akt; Time Factors; Transfection; Tretinoin

All-trans retinoic acid (ATRA) is a widely used differentiation drug that can effectively induce osteogenic differentiation of osteosarcoma cells, but the underlying mechanism remains elusive, which limits the clinical application for ATRA in osteosarcoma patients. In this study, we identified E2F1 as a novel regulator involved in ATRA-induced osteogenic differentiation of osteosarcoma cells. We observed that osteosarcoma cells are coupled with individual differences in the expression levels of E2F1 in patients, and E2F1 impairs ATRA-induced differentiation of osteosarcoma cells. Moreover, remarkable anti-proliferative and differentiation-inducing effects of ATRA treatment are only observed in E2F1 low to negative expressed primary osteosarcoma cultures. These results strongly suggested that E2F1 may serve as a potent indicator for the effectiveness of ATRA treatment in osteosarcoma. Interestingly, E2F1 is found to downregulate retinoic acid receptor α (RARα), a key factor determines the effectiveness of ATRA. E2F1 specifically binds to RARα and promotes its ubiquitination-mediated degradation; as a consequence, RARα-mediated differentiation is inhibited in osteosarcoma. Therefore, our studies present E2F1 as a potent biomarker, as well as a therapeutic target for ATRA-based differentiation therapeutics, and raise the hope of using differentiation-based approaches for osteosarcoma patients.

Topics: Antineoplastic Agents; Biomarkers, Tumor; Cell Line, Tumor; E2F1 Transcription Factor; Humans; Osteogenesis; Osteosarcoma; Proteolysis; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Tretinoin; Ubiquitination

The induced differentiation of tumor cells into mature phenotypes is a promising strategy in cancer therapy. In this study, the effects of combined treatment with all-trans retinoic acid (ATRA) and lipoxygenase/cyclooxygenase inhibitors were examined in two osteosarcoma cell lines, Saos-2 and OSA-01. Caffeic acid and celecoxib were used as inhibitors of 5-lipoxygenase and of cyclooxygenase-2, respectively. Changes in the cell proliferation, matrix mineralization, and occurrence of differentiation markers were evaluated in treated cell populations at intervals. The results confirmed the capability of caffeic acid to enhance the antiproliferative effect of ATRA in both cell lines. In contrast, celecoxib showed the same effect in Saos-2 cells only. Furthermore, the extension of matrix mineralization was observed after combined treatment with ATRA and celecoxib or caffeic acid. The increased expression of osteogenic differentiation markers was observed in both cell lines after the combined application of ATRA and inhibitors. The obtained results clearly demonstrate the capability of lipoxygenase/cyclooxygenase inhibitors to enhance the antiproliferative and differentiating effect of ATRA in osteosarcoma cells, although some of these effects are specific and depend on the biological features of the respective tumor or cell line.

Topics: Antineoplastic Agents; Bone Neoplasms; Caffeic Acids; Celecoxib; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cyclooxygenase Inhibitors; Humans; Lipoxygenase Inhibitors; Osteosarcoma; Pyrazoles; Sulfonamides; Tretinoin

Natural derivatives of vitamin A, including all-trans-retinoic acid (ATRA), commonly known as retinoids, currently produce favorable results in the treatment of many types of tumors. The rexinoid 6-OH-11-O-hydroxyphenantrene (IIF) is a synthetic derivative of ATRA. Previous in vitro and in vivo studies demonstrated that IIF is able to induce growth inhibition of various cancer cells and is a potent apoptosis-inducing agent with clinical potential. Osteosarcoma (OS) is the most common type of bone cancer, characterized by a rising aggressiveness. Recent evidences suggest that mesenchymal stem cells (MSC) may favour tumor growth and progression. Thus, it is important to investigate whether a compound with potential anti-tumoral properties such as IIF affects not only tumor cells but also MSC. The current study is an attempt to understand the mode of the potential cytotoxicity of IIF on OS cells and MSC. The response to IIF treatment of osteosarcoma SaOS-2, MG63, and U2OS cells and of bone marrow-derived MSC was the subject of investigation. The results showed that IIF significantly inhibited cell growth in OS cell lines and MSC in both a time- and dose-dependent manner, as evaluated by methylene blue assay. This was also associated with altered cell morphology and an increase in cell death with the involvement of apoptosis as demonstrated by NucleoCounter, Hoechst 33342 staining and FACS analysis. No cell death and apoptosis was found in U2OS cells. Analysis of cells treated with 20 and 40μM IIF for 24h by western blot suggests the activation of initiator caspase 9, indicating the involvement of caspases in inducing apoptosis. Furthermore, IIF upregulated the expression of the pro-apoptotic protein Bax and downregulated the anti-apoptotic protein Bcl2. For the first time, our results collectively provide an evidence for cell growth inhibition and activation of apoptosis in human OS cells and MSC by IIF. These results confirm that IIF may be an effective compound for anticancer treatment, including that of OS.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cells, Cultured; Humans; Mesenchymal Stem Cells; Osteosarcoma; Tretinoin

Osteosarcoma (OS) is one of the most common malignant bone tumors. Despite the advancement of diagnosis and treatment for OS, the prognosis remains poor. We investigated the proliferation inhibitory effect of all-trans retinoic acid (ATRA) for human OS and the possible mechanism underlying this effect. We examined the proliferation inhibition and apoptosis-inducing effects of ATRA in 143B OS cells. We validated this effect by exogenously expressing the retinoic acid receptor alpha (RARα) in 143B OS cells and injecting the cells into nude mice. We explored the possible mechanism for the proliferation inhibitory effect of ATRA on OS cells and multipotential progenitor cells by detecting osteogenic markers. We demonstrated that the endogenous retinoic acid receptor and retinoid X receptor are all detectable in the commercially available OS cell lines and in primary osteosarcoma cells. ATRA inhibits the proliferation of OS cells in a concentration-dependent manner, as well as induces apoptosis in 143B OS cells. The exogenous expression of RARα inhibits the tumor growth and cell proliferation in vivo. The alkaline phosphatase activity, protein levels of osteopontin (OPN) and osteocalcin (OCN) are all promoted by ATRA in OS cells and mouse embryonic fibroblasts (MEFs), at least by activating the Smad signaling pathway. Collectively, our results strongly indicate that ATRA can inhibit the tumor growth of OS by promoting osteogenic differentiation in OS cells, which is mediated in part by activating Smad signaling. Therefore, combination of ATRA with other current chemotherapy agents may be a promising therapy strategy for OS treatment.

Topics: Animals; Antigens, Differentiation; Antineoplastic Agents; Apoptosis; Caspase 3; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Female; Fibroblasts; Genes, Reporter; Humans; Luciferases, Firefly; Mice; Mice, Nude; Neoplasm Transplantation; Osteogenesis; Osteosarcoma; Phosphorylation; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoid X Receptor alpha; Signal Transduction; Smad Proteins; Tretinoin

Although retinoic acid (RA) is a potent agent that coordinates inhibition of proliferation with differentiation of many cell types, RA-mediated signaling pathways in osteosarcoma cell differentiation are uncharacterized. In this study, we show that in human U2OS osteosarcoma cells, decreased phosphorylation of RA receptor alpha (RARalpha) by RA treatment or overexpressing a phosphorylation-defective mutant RARalphaS77A results in the inhibition of proliferation and induction of differentiation, and that U2OS cells transduced with RARalphaS77A suppresses tumor formation in nude mice. Moreover, using different human primary osteosarcoma cells and human mesenchymal stem cells for gene expression analysis, we found that either RA or RARalphaS77A induces many of the same differentiation response pathways and signaling molecules involved in U2OS cell differentiation. In addition, overexpression of the fibroblast growth factor 8f (FGF8f), one of the downstream targets induced by both RA and RARalphaS77A in U2OS cells, inhibits proliferation and induces expression of osteoblastic differentiation regulators. Hence, these data strongly suggest that RA-suppressed phosphorylation of RARalpha induces FGF8f expression to mediate differentiation response pathway in U2OS osteosarcoma cells.

Topics: Animals; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase-Activating Kinase; Cyclin-Dependent Kinases; Fibroblast Growth Factor 8; G1 Phase; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Inbred BALB C; Mutation; Osteoblasts; Osteosarcoma; Phosphorylation; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Signal Transduction; Tretinoin

Osteosarcoma is the most common primary malignancy of bone. The long-term survival of osteosarcoma patients hinges on our ability to prevent and/or treat recurrent and metastatic lesions. Here, we investigated the activation of peroxisome proliferator-activated receptor gamma (PPARgamma) and retinoid receptors as a means of differentiation therapy for human osteosarcoma.. We examined the endogenous expression of PPARgamma and retinoid receptors in a panel of osteosarcoma cells. Ligands or adenovirus-mediated overexpression of these receptors were tested to inhibit proliferation and induce apoptosis of osteosarcoma cells. Osteosarcoma cells overexpressing the receptors were introduced into an orthotopic tumor model. The effect of these ligands on osteoblastic differentiation was further investigated.. Endogenous expression of PPARgamma and isotypes of retinoic acid receptor (RAR) and retinoid X receptor (RXR) is detected in most osteosarcoma cells. Troglitazone, 9-cis retinoic acid (RA), and all-trans RA, as well as overexpression of PPARgamma, RARalpha, and RXRalpha, inhibit osteosarcoma cell proliferation and induce apoptosis. A synergistic inhibitory effect on osteosarcoma cell proliferation is observed between troglitazone and retinoids, as well as with the overexpression pairs of PPARgamma/RARalpha, or PPARgamma/RXRalpha. Overexpression of PPARgamma, RARalpha, RXRalpha, or in combinations inhibits osteosarcoma tumor growth and cell proliferation in vivo. Retinoids (and to a lesser extent, troglitazone) are shown to promote osteogenic differentiation of osteosarcoma cells and mesenchymal stem cells.. Activation of PPARgamma, RARalpha, and RXRalpha may act synergistically on inhibiting osteosarcoma cell proliferation and tumor growth, which is at least partially mediated by promoting osteoblastic differentiation of osteosarcoma cells.

Topics: Adenoviridae; Animals; Antineoplastic Agents; Apoptosis; Blotting, Western; Bone Neoplasms; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Drug Synergism; Humans; Luciferases; Mesenchymal Stem Cells; Mice; Osteosarcoma; PPAR gamma; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoid X Receptors; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tretinoin

To identify potential biomarkers of therapy response, we have previously done a large-scale gain-of-function genetic screen to identify genes whose expression confers resistance to histone deacetylase inhibitors (HDACI). This genetic screen identified two genes with a role in retinoic acid signaling, suggesting that HDACIs target retinoic acid signaling as part of their anticancer effect. We study here a third gene identified in this genetic screen, UNC45A, and assess its role in retinoic acid signaling and responses to HDACIs using cell-based proliferation and differentiation assays and transcriptional reporter gene assays. The vertebrate Unc45 genes are known for their roles in muscle development and the assembly and cochaperoning of the muscle motor protein myosin. Here, we report that human UNC45A (GCUNC45) can render transformed cells resistant to treatment with HDACIs. We show that UNC45A also inhibits signaling through the retinoic acid receptor alpha. Expression of UNC45A inhibits retinoic acid-induced proliferation arrest and differentiation of human neuroblastoma cells and inhibits the induction of endogenous retinoic acid receptor target genes. These data establish an unexpected role for UNC45A in causing resistance to both HDACI drugs and retinoic acid. Moreover, our data lend further support to the notion that HDACIs exert their anticancer effect, at least in part, through an effect on retinoic acid signaling.

Topics: Amino Acid Sequence; Animals; Bone Neoplasms; Cell Differentiation; Cell Growth Processes; Cell Line, Tumor; Drug Resistance, Neoplasm; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Intracellular Signaling Peptides and Proteins; Mice; Molecular Sequence Data; Neuroblastoma; Osteosarcoma; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Signal Transduction; Tretinoin

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Survival; Dog Diseases; Dogs; Doxorubicin; Osteosarcoma; Tretinoin

While tissue-nonspecific alkaline phosphatase (TNSALP) is a well-known indicator of bone formation and all-trans-retinoic acid a key regulator of that process, the relationship between TNSALP and retinoic acid has not yet been clearly described. The aim of the present study was therefore to clarify the mechanism by which retinoic acid modulates expression of TNSALP. After culturing SaOS-2 human osteoblastic osteosarcoma cells in the presence or absence of 10(-6) M all-trans-retinoic acid, real-time RT-PCR confirmed that retinoic acid up-regulates expression of TNSALP mRNA. Notably, this time-dependent induction of TNSALP expression was accompanied by a corresponding increase in detected catalytic activity of the enzyme. When we then isolated the 5'-upstream region of the human TNSALP gene and carried out luciferase assays with a set of deletion mutants, we found that the promoter region contains a retinoic acid response element-like motif. Moreover, electrophoretic mobility shift assays showed that the nuclear extract bound to the motif. It thus appears that retinoic acid regulates the expression of human TNSALP via a retinoic acid response element in the genes promoter region.

Topics: Alkaline Phosphatase; Base Sequence; Bone Neoplasms; Cell Line, Tumor; Cloning, Molecular; DNA, Neoplasm; Electrophoretic Mobility Shift Assay; Gene Expression Regulation, Enzymologic; Humans; Molecular Sequence Data; Osteosarcoma; Plasmids; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transcription, Genetic; Transfection; Tretinoin

All-trans-retinoic acid (ATRA) induces bone resorption, but the molecular mechanisms are unknown. We have studied the effect of ATRA on osteoprotegerin (OPG) and receptor activator of NF-kappaB ligand (RANKL) expression in human MG-63 osteosarcoma cells and primary osteoblast-like cultures. ATRA dose-dependently down-regulated protein levels of OPG in MG-63 cells, with a maximum (-56%) observed at a dose of 10(-6)M. This effect was confirmed with quantitative real-time PCR, where OPG mRNA was decreased after 4h (-68%) in primary cultures and after 8h (-87%) in MG-63 cells. The reduction in OPG expression was inhibited by a retinoic acid receptor (RAR)-antagonist and was mimicked by a RARbeta,gamma-agonist, indicating that the ATRA effect is mediated by these receptors. In primary cultures we found a threefold induction of RANKL mRNA expression. Thus, the RANKL/OPG ratio was markedly increased, suggesting a potential mechanism of ATRA-induced bone resorption.

Topics: Carrier Proteins; Cell Line, Tumor; Cells, Cultured; Dose-Response Relationship, Drug; Gene Expression Regulation; Glycoproteins; Humans; Membrane Glycoproteins; NF-kappa B; Osteoblasts; Osteoprotegerin; Osteosarcoma; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Receptors, Tumor Necrosis Factor; Tretinoin

In this report, we studied the relationship between telomerase activity and in vitro differentiation of osteosarcoma cells. Human osteosarcoma cells (HOS-8603) were treated with all-trans-retinoic acid (RA) and dexamethasone (DEX). Cell cycle phase, alkaline phosphatase (AP) activity, telomerase activity, and human telomerase RNA (hTR) in treated cells were detected. The results showed that the treated cells underwent morphologic differentiation. AP activity of the cells increased significantly. The proportion of the cells in S and G2/M phases was increased. A pronounced decline in telomerase activity was observed, but no significant difference in the amount of hTR expressed, when compared with the control. This study demonstrates that: (1) both RA and DEX can inhibit cell growth and induce morphologic and functional differentiation of HOS-8603 cells; (2) telomerase is an enzyme system regulated during induced differentiation of HOS-8603 cells; (3) significantly decreased telomerase activity may be an indicator of differentiation but does not parallel the expression level of hTR; and (4) the regulation of telomerase is directly linked to cell differentiation not cell cycle.

Topics: Alkaline Phosphatase; Blotting, Northern; Bone Neoplasms; Cell Cycle; Cell Differentiation; Cell Division; Dexamethasone; Dose-Response Relationship, Drug; Down-Regulation; Humans; Osteosarcoma; RNA, Messenger; Telomerase; Tretinoin; Tumor Cells, Cultured

This study was designed to investigate whether nuclear receptor agonists can be used as potential differentiation therapy agents for human osteosarcoma.. Four osteosarcoma cell lines (143B, MNNG/HOS, MG-63, and TE-85) were treated with proliferator-activated receptor (PPAR)gamma agonists, troglitazone and ciglitazone, and a retinoid X receptor (RXR) ligand, 9-cis retinoic acid. The proliferation and induction of apoptosis in the treated cells were assessed, as was the induction of alkaline phosphatase, a differentiation marker of osteoblasts.. The expression of PPARgamma was readily detected in all tested osteosarcoma lines. On treatment with the PPARgamma and RXR ligands, all four osteosarcoma lines exhibited a significantly reduced proliferation rate and cell viability. Among the four lines, 143B and MNNG/HOS were shown to be more sensitive to ligand-induced apoptosis, as demonstrated by the Crystal Violet and Hoechst staining assays. Of the three tested ligands, troglitazone was shown to be the most effective in inducing cell death, followed by 9-cis retinoic acid. Moreover, a strong synergistic effect on the induction of cell death was observed when both troglitazone and 9-cis retinoic acid or ciglitazone and 9-cis retinoic acid were administered to osteosarcoma cells. Troglitazone was shown to effectively induce alkaline phosphatase activity, a well-characterized hallmark for osteoblastic differentiation.. Our findings suggest that PPARgamma and/or RXR ligands may be used as efficacious adjuvant therapeutic agents for primary osteosarcoma, as well as potential chemopreventive agents for preventing the recurrence and metastasis of osteosarcoma after the surgical removal of the primary tumors.

Topics: Alitretinoin; Antineoplastic Agents; Apoptosis; Cell Differentiation; Cell Division; Chromans; Dose-Response Relationship, Drug; Drug Synergism; Gene Expression Regulation, Neoplastic; HT29 Cells; Humans; Osteosarcoma; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Retinoid X Receptors; RNA, Neoplasm; Thiazoles; Thiazolidinediones; Time Factors; Transcription Factors; Tretinoin; Troglitazone; Tumor Cells, Cultured

To determine differentiation and growth inhibition effects of retinoids on canine osteosarcoma cells.. 3 osteosarcoma cell lines established from osteosarcomas in dogs.. Osteosarcoma cells were incubated with various concentrations of all-trans-retinoic acid and 9-cis-retinoic acid or control medium, counted daily for 10 days, and evaluated for morphologic changes. Synthesis of DNA was measured by use of a cell proliferation ELISA. To analyze effect of retinoids on colony formation on plastic dishes, cells were cultured for 14 days, fixed, and stained; number of colonies was counted.. In a dose-dependent manner, both retinoids induced morphologic differentiation and growth inhibition in the 3 osteosarcoma cell lines and inhibited each cell's ability to form anchorage-dependent colonies.. Retinoids induced differentiation of osteosarcoma cells of dogs, resulting in altered expression of their malignant phenotype. Induction of differentiation by retinoids may have potential as an adjunctive treatment for osteosarcoma in dogs.

Topics: Alitretinoin; Animals; Antimetabolites; Antineoplastic Agents; Bone Neoplasms; Bromodeoxyuridine; Cell Division; DNA; Dog Diseases; Dogs; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Microscopy, Phase-Contrast; Osteosarcoma; Tretinoin; Tumor Cells, Cultured

The effect of two retinoids, all- trans and 9- cis retinoic acid, on the differentiation of three canine osteosarcoma cells (OOS, HOS, and POS) was examined using markers specifically expressed by phenotypic osteoblasts. Both retinoids induced morphologic differentiation in all the canine osteosarcoma cells. Retinoids enhanced cell flattening and spreading, as well as reduction in cell overlapping. Alkaline phosphatase (ALP) activity and ALP staining was enhanced in OOS, and HOS cells, but decreased in POS cells. These results may suggest that OOS and HOS cells have immature osteoblastic properties and POS cells have mature osteoblastic properties. Retinoids decreased osteocalcin production in all the osteosarcoma cells. They induced an increase in production of type I collagen in HOS and POS cells, but a decrease in OOS cells. These results indicate that retinoids induce differentiation of canine osteosarcoma cells, resulting in an altered expression of their malignant phenotype.

Topics: Alitretinoin; Alkaline Phosphatase; Animals; Antineoplastic Agents; Bone Neoplasms; Cell Differentiation; Collagen; Dog Diseases; Dogs; Osteocalcin; Osteosarcoma; Tretinoin; Tumor Cells, Cultured

Pulmonary metastasis is a major cause of death and a major obstacle to the successful treatment of canine osteosarcoma. However, the residual capacity of the neoplasia for differentiation and its susceptibility to undergo apoptosis may be used to suppress its growth and metastatic properties. The highly metastasizing POS (HMPOS) canine osteosarcoma cell line which preferentially metastasize to the lungs was used to test the possible efficacy of 22-oxa-calcitriol (OCT) and all- trans retinoic acid (ATRA) to inhibit growth and pulmonary metastasis of the subcutaneously grown osteosarcoma in nude mice. Treatments in vitro, morphologically elongated and increased alkaline phosphatase activity and staining of cells. Tumour growth in vivo was inhibited significantly and the combination treatment of OCT and ATRA (OCT + ATRA) exerted a synergistic and stronger suppression at concentration of 1.0 microg kg(-1)body weight when given subcutaneously three times a week for 5 weeks. The subcutaneous tumours of the control mice consisted of osteoblast-like cells and isolated chondroblast-like cells, but formed several areas of osteoid and increased amount of collagen tissue in all treated mice. Pinpoint macrometastatic nodules developed only in all control mice. Micrometastatic nodule developed only in two of six mice treated with ATRA. However, nodule size and number, and lung wet weight were all reduced significantly. Metastasis were not seen in the mice treated with OCT or OCT + ATRA. This study demonstrated that inhibition of growth and pulmonary metastasis was induced by subcutaneous treatment with these drugs and suggest that both its differentiating and apoptotic inducing activities may be responsible for the antitumour effects. These drugs may be useful in the clinic as an adjunct for the treatment of canine osteosarcoma.

Topics: Alkaline Phosphatase; Animals; Antineoplastic Agents; Biomarkers; Bone Neoplasms; Calcitriol; Cell Division; Dog Diseases; Dogs; Female; Lung Neoplasms; Mice; Mice, Nude; Osteosarcoma; Transplantation, Heterologous; Tretinoin; Tumor Cells, Cultured

Retinoids, all-trans-retinoic acid (ATRA) and 9-cis-retinoic acid (9-cis-RA), induced morphological changes and apoptosis-like cell death characterized by cell shrinkage, chromatin condensation and nuclear disintegration in three canine osteosarcoma cells, OOS, HOS and POS, at a concentration of 10(-5) M. Both retinoid receptors, RARs and RXRs, were identified in these cells. 9-cis-RA bound to both the RXRs and the RARs, whereas ATRA bound to only the RARs in these cells. Those results indicate that the induction of apoptosis in canine osteosarcoma cells may be mediated by the specific control of RARs and RXRs.

Topics: Alitretinoin; Animals; Apoptosis; Dog Diseases; Dogs; Osteosarcoma; Receptors, Retinoic Acid; Retinoid X Receptors; Transcription Factors; Tretinoin

To determine effects of all-trans and 9-cis retinoic acid (RA) on tumor growth and metastatic ability of canine osteosarcoma cells transplanted into athymic (nude) mice.. Forty-five 5-week-old female BALB/c nude mice.. 1 X 10(7) POS osteosarcoma cells were transplanted subcutaneously into the intrascapular region of mice. All-trans RA (3 or 30 microg/kg of body weight in 0.1 ml of sesame oil), 9-cis RA (3 or 30 mg/kg in 0.1 ml of sesame oil), or sesame oil (0.1 ml; control treatment) were administered intragastrically 5 d/wk for 4 weeks beginning 3 days after transplantation (n = 4 mice/group) or after formation of a palpable tumor (5 mice/group). Tumor weight was estimated weekly by measuring tumor length and width, and retinoid toxic effects were evaluated daily. Two weeks after the final treatment, mice were euthanatized, and number of mice with pulmonary metastases was determined.. Adverse treatment effects were not detected. Tumor weight was less in mice treated with either dose of 9-cis RA than in control mice, although this difference was not significant. Treatment with 30 mg of 9-cis RA/kg initiated after tumor formation significantly reduced the incidence of pulmonary metastasis, compared with the control group.. 9-cis RA decreased the incidence of pulmonary metastasis in nude mice transplanted with canine osteosarcoma cells and may be a potential adjunct therapy for treatment of osteosarcoma in dogs.

Topics: Alitretinoin; Animals; Antineoplastic Agents; Bone Neoplasms; Dog Diseases; Dogs; Female; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Metastasis; Neoplasm Transplantation; Osteosarcoma; Transplantation, Heterologous; Tretinoin; Tumor Cells, Cultured

A boy age 14 years who was in complete remission from Stage IIB small cell osteosarcoma, which was misdiagnosed as Ewing sarcoma and consequently was treated, developed inoperable lung metastases when he was off therapy. He received second-line treatment for recurrent Ewing sarcoma, including chemotherapy and radiotherapy, and obtained only a temporary response. A compassionate treatment with all-trans retinoic acid (ATRA) and interferon-alpha (IFNalpha) was then undertaken.. The patient initially was treated according to the national SE91 protocol for nonmetastatic Ewing sarcoma. After a bilateral pulmonary recurrence, he received second-line chemotherapy and irradiation of the largest metastasis, with a temporary partial response. The patient was then treated with a combination of oral ATRA (90 mg/m(2) for 3 days per week) and subcutaneous IFNalpha (3 x 10(6) U/m(2) 5 days per week) for 4 months. The same therapy also was administered for the control of residual disease after surgery for a total duration of 1 year of ATRA/IFN treatment. During the first 3 weeks of therapy, ATRA pharmacokinetics were studied.. After progression of the patient's disease, despite the administration of first-line and second-line chemotherapy, combined treatment with ATRA/IFNalpha yielded a partial remission, which allowed surgical resection of the largest metastasis. The same therapy was effective in preventing tumor recurrence after incomplete removal of the remaining metastases. Treatment was well tolerated, and the patient is in stable complete remission 14 months after the end of therapy. The pharmacokinetics results confirmed the indication of an intermittent schedule for oral ATRA therapy.. ATRA/IFNalpha treatment may be considered as an alternative approach in the treatment of patients with metastatic osteosarcoma who have disease that is resistant to conventional chemotherapy and in the treatment of patients with minimal tumor residue.

Topics: Adolescent; Drug Therapy, Combination; Femoral Neoplasms; Humans; Interferon-alpha; Lung; Lung Neoplasms; Male; Osteosarcoma; Treatment Outcome; Tretinoin

The effects of 22-oxacalcitriol (OCT), calcitriol and all-trans retinoic acid (ATRA) on the induction of functional differentiation and growth inhibition of the canine osteosarcoma cell line POS were investigated in vitro via bone differentiation markers and proliferation assays, respectively. The intracellular alkaline phosphatase (ALP) activity and the gamma-carboxyglutamic acid osteocalcin (GLA-OC) and procollagen type I C peptide (PIP) production were used as markers of differentiation. Treatment with 10(-8) M concentrations of all drugs for 72 h significantly inhibited growth (P < 0.0001) and increased ALP activity and GLA-OC and PIP production in POS. OCT, calcitriol and ATRA significantly increased the: ALP activity from 1.58 +/- 0.14 mumol/min/mg protein (mean +/- SD; control) to 2.50 +/- 0.09 (P < 0.0001), 2.30 +/- 0.14 (P < 0.0001) and 2.00 +/- 0.14 (P = 0.0008), respectively; GLA-OC production from 0.71 +/- 0.01 ng/ml (control) to 2.87 +/- 0.01 (P < 0.0001), 2.87 +/- 0.11 (P < 0.0001) and 1.36 +/- 0.06 (P < 0.0001), respectively; and PIP production from 433.91 +/- 23.29 ng/ml (control) to 536.54 +/- 15.46 (P = 0.0002), 497.06 +/- 1.99 (P = 0.0028) and 481.66 +/- 0.01 (P = 0.0104), respectively. This study demonstrated that treatment with these drugs induced a phenotypic maturation of POS cells into cells that exhibit the properties of functionally mature bone cells with parallel growth inhibition. The effects of these drugs on functional differentiation may be useful to complement the progression of a normal osteogenic differentiation process in the sarcoma.

Topics: Alkaline Phosphatase; Animals; Antineoplastic Agents; Bone Neoplasms; Calcitriol; Dog Diseases; Dogs; Osteocalcin; Osteosarcoma; Peptide Fragments; Procollagen; Tretinoin; Tumor Cells, Cultured

Change in the synthesis of type I collagen, the major extracellular matrix component of skin and bone, are associated with normal growth, tissue repair processes, and several pathological conditions. Expression of the COL 1A1 gene is regulated by transcriptional and post-transcriptional mechanisms. However, the hormonal regulation of type I collagen synthesis in human bone has not been well characterized. We have studied the influence of calcitriol, dexamethasone, retinoic acid, and estradiol on the COL 1A1 gene expression by determining the secretion of the C-terminal propeptide (PICP) and the levels of alpha 1(I) procollagen mRNA in cultured human MG-63 and SaOs-2 osteoblast-like osteosarcoma cells. Similar experiments were also performed with respect to expression of the nuclear proto-oncogenes, c-fos and c-jun, in MG-63 cells. In MG-63 cells, calcitriol stimulated the synthesis and secretion of PICP. The alpha 1(I) procollagen mRNA level was elevated with no effect on message stability, indicating a transcriptional mechanism of regulation. In contrast, dexamethasone treatment was accompanied by an accelerated rate of alpha 1(I) procollagen mRNA turnover, observed as decreased amounts of the message and the secreted PICP, implying a posttranscriptional regulation. Retinoic acid, in turn, decreased the levels of alpha 1(I) procollagen mRNA and secreted PICP by slowing down transcription of the COL1A1 gene without any effect on message stability. The ability of these hormones to regulate the alpha 1(I) transcripts was sensitive to puromycin treatment, suggesting an involvement of an induced mediator protein in the action of the hormones on the COL1A1 gene. Both dexamethasone and calcitriol rapidly but transiently increased the expression of the c-fos and c-jun proto-oncogenes. Neither proto-oncogene responded to retinoic acid treatment with significant changes in mRNA levels. Estradiol treatment was found to have no influence on type I procollagen synthesis. In SaOs-2 cells, which are not as well differentiated as the MG-63 cells, calcitriol and dexamethasone did not influence type I procollagen synthesis. Retinoic acid as well as estradiol reduced collagen gene expression in these cells. These findings suggest that hormonal effects on type I procollagen synthesis may depend on the maturational state of the osteoblastic cells that express different regulatory factors and receptors, resulting in, in each case, a finely adjusted rate of gene expression.

Topics: Administration, Topical; Anti-Inflammatory Agents; Calcitriol; Dexamethasone; Dose-Response Relationship, Drug; Estradiol; Gene Expression Regulation; Glucocorticoids; Humans; Osteosarcoma; Procollagen; Proto-Oncogene Mas; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Steroids; Tretinoin; Tumor Cells, Cultured

Bone morphogenetic proteins (BMPs) belong to the transforming growth factor-beta (TGF-beta) superfamily and are crucial factors in the process of bone formation. Despite knowledge on their wide distribution and expression, however, there is very little information on the biological factors that affect gene transcription of these osteoinductive agents. To investigate this aspect of BMP gene regulation we have studied the effect of a number of factors known to affect osteogenic cells. Northern analysis showed modulation of the expression of BMP-2 and BMP-4 mRNAs in two human osteosarcoma cell lines, MG63 and Saos-2, by prostaglandin E2 (PGE2), interleukin-1beta (IL-1beta), interleukin-6 (IL-6), interferon-alpha (IFN-alpha), retinoic acid and 1,25(OH)2 vitamin D3. mRNA expressions of the normally used "housekeeping genes", glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-actin, were found to be susceptible to influence by some of the factors used. Hence, an oligo(dT)15-18 probe was used to reliably estimate the relative quantities of mRNA present for normalization of data. In general, all factors down-regulated mRNA expressions of BMP-2 and BMP-4 in MG63 cells. IL-6 completely abolished detectable expression of BMP-2 mRNA, which was also greatly reduced by IL-1beta, retinoic acid and 1,25(OH)2 vitamin D3. PGE2 had similar influences on BMP-2 and BMP-4 expressions, showing reductions to approximately 60% of normal. In Saos-2 cells only 1,25(OH)2 vitamin D3 had any great effect on BMP-2 expression, which was down-regulated to approximately 60% of control values. BMP-4 was down-regulated by IFN-alpha (approximately 60%) and IL-1beta (approximately 20%). We conclude that BMPs are subject to regulation by a variety of factors and that this is dependent on the stage of the cell in the osteogenic lineage. Furthermore, the use of GAPDH and beta-actin genes as "housekeeping genes" in expression-modulation studies must be treated with care.

Topics: Actins; Blotting, Northern; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 4; Bone Morphogenetic Proteins; Cholecalciferol; Dinoprostone; Gene Expression Regulation; Glyceraldehyde-3-Phosphate Dehydrogenases; Humans; Interferon-alpha; Interleukin-1; Interleukin-6; Oligonucleotide Probes; Osteosarcoma; RNA, Messenger; Transcription, Genetic; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

3,5,3'-Tri-iodothyronine (T3), 1 alpha,25(OH)2-vitamin D3 (D3) and retinoids activate related nuclear receptors which interact by heterodimerisation to regulate gene expression. Actions of each hormone are discrete and may be specified by changes in the relative concentrations of their receptors (T3R, vitamin D receptor (VDR), retinoic acid receptor (RAR), retinoid X receptor (RXR)). T3, D3 and retinoids are essential for skeletal development and maintenance and we have previously shown complex interactions amongst their signalling pathways in osteosarcoma cells. In these studies we demonstrate that similar T3R, VDR, RAR and RXR proteins are co-expressed in both osteoblast lineage cell primary cultures and osteosarcoma cells by Western blotting. We investigated whether hormone interactions in bone result from changes in receptor stoichiometry. Cells were treated with combinations of T3, D3, 9-cis retinoic acid (9-cis RA) and all-trans retinoic acid (RA) that are known from previous studies to produce complex cell specific responses. No alteration in expression of any receptor protein was seen in response to any hormone combination in three phenotypically distinct osteosarcoma cell lines. Thus, in contrast to studies of overexpressed receptors in vitro, changes in the physiological concentrations of endogenous T3R, VDR, RAR and RXR do not specify discrete hormone actions in osteoblastic cells. Other unidentified factors are likely to modulate hormone action in these bone cells.

Topics: Animals; Blotting, Western; Cells, Cultured; Cholecalciferol; Osteoblasts; Osteosarcoma; Rats; Rats, Wistar; Receptors, Calcitriol; Receptors, Retinoic Acid; Receptors, Thyroid Hormone; Signal Transduction; Tretinoin; Triiodothyronine; Tumor Cells, Cultured

The transforming growth factor beta (TGF-beta) family of growth factors control proliferation, extracellular matrix synthesis and/ or differentiation in a wide variety of cells. However, the molecular mechanisms governing ligand binding, receptor oligomerization and signal transduction remain incompletely understood. In this study, we utilized a set of antibodies selective for the extracellular and intracellular domains of the TGF-beta type-II receptor as probes to investigate the intrinsic kinase activity of this receptor and its physical association in multimeric complexes with type-I and type-III receptors. The type-II receptor immuno-precipitated from human osteosarcoma cells exhibited autophosphorylation and casein kinase activity that was markedly stimulated by polylysine yet was insensitive to heparin. Affinity cross-linking of 125I-TGF-beta 1 ligand to cellular receptors followed by specific immunoprecipitation demonstrated that type-II receptors form stable complexes with both type-I and type-III receptors expressed on the surfaces of both human osteosarcoma cells and rabbit chondrocytes. Pretreatment of the cultured cells with an antibody directed against a distinct extracellular segment of the type-II receptor (anti-TGF-beta-IIR-NT) effectively blocked the 125I-TGF-beta labelling of type-I receptors without preventing the affinity labelling of type-II or type-III receptors, indicating a selective disruption of the type-I/type-II hetero-oligomers. The anti-TGF-beta-IIR-NT antibodies also blocked the TGF-beta-dependent induction of the plasminogen activator inhibitor (PAI-1) promoter observed in mink lung epithelial cells. However, the same anti-TGF-beta-IIR-NT antibodies did not prevent the characteristic inhibition of cellular proliferation by TGF-beta 1, as determined by [3H]thymidine incorporation into DNA. The selective perturbation of PAI-1 promoter induction versus cell-cycle-negative regulation suggests that strategic disruption of TGF-beta type-I and -II receptor interactions can effectively alter specific cellular responses to TGF-beta signalling.

Topics: Amino Acid Sequence; Animals; Antibodies; Antibody Specificity; Cartilage, Articular; Casein Kinases; Cell Differentiation; Cell Line; Cells, Cultured; Cross-Linking Reagents; DNA; DNA Replication; Humans; Luciferases; Lung; Mink; Molecular Sequence Data; Osteosarcoma; Peptide Fragments; Phosphorylation; Promoter Regions, Genetic; Protein Kinases; Protein Serine-Threonine Kinases; Rabbits; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Recombinant Proteins; Signal Transduction; Succinimides; Thymidine; Transfection; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

Two human osteosarcoma cell lines, Hu09 and OST, were suspended in Matrigel (Becton Dickinson Labware, Bedford, Massachusetts) and implanted subcutaneously in the backs of nude mice. To study phenotypic changes of tumor cells and host cells, expression of mRNA for osteopontin (OPN), osteocalcin (OC), and osteonectin (ON) was analyzed by in situ hybridization. Bone tissue was formed in the tumors derived from Hu09 cells. OPN mRNA was transcribed predominantly in osteocyte-like cells within the bone, whereas OC mRNA was transcribed in osteoblast-like cells that surrounded the bone. ON mRNA was detected in both types of cells. The similarity of the expression pattern of OPN, OC, and ON during osteogenesis of Hu09 cells to that of normal skeletal development suggests that the bone formed in Hu09-implanted mice is the same as normal bone tissue. By DNA-DNA in situ hybridization using a human-specific Alu probe and a mouse-specific m-L1 probe, osteoblast-like cells in Hu09 tumorous bone were, however, of human origin, whereas osteocyte-like cells were of mouse origin. In the tumors derived from OST cells, no osteogenesis was observed during the experimental period, and the expression of OPN, OC, and ON was not detected in tumor cells. An endochondral bone formation was not evident when these cells were simply implanted into muscle tissue. An endochondral bone was, however, reactively induced in the host mUscle tissue either when 1 alpha-hydroxyvitamin D3 and all-transretinoic acid were administered to OST-implanted mice or when Hu09 cells were pretreated with dexamethasone before implantation. Hu09 implantation seems to be a useful tool not only for the study of the differentiation of osteosarcoma cells but also for the investigation of the mechanism of bone formation. This system, using Hu09 and OST, may provide us with a new tool for the isolation of the unidentified factors that induce or inhibit osteogenesis in vivo.

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 4; Bone Morphogenetic Proteins; Cell Adhesion; Cell Transplantation; Cells, Cultured; Cytokines; Dexamethasone; Humans; Hydroxycholecalciferols; In Situ Hybridization; Mice; Mice, Nude; Neoplasm Transplantation; Osteoblasts; Osteocalcin; Osteocytes; Osteogenesis; Osteonectin; Osteopontin; Osteosarcoma; Phosphoproteins; RNA, Messenger; Sialoglycoproteins; Transforming Growth Factor beta; Transplantation, Heterologous; Tretinoin

Osteocalcin (OC) is a bone matrix protein, synthesized by osteoblasts, which contains three residues of gammacarboxyglutamic acid (GLA). A fraction of circulating OC, which is not fully carboxylated and does not bind to hydroxyapatite, is called undercarboxylated OC (ucOC). In elderly institutionalized women, we have shown an increase of circulating ucOC level which may result not only from vitamin K deficiency but also from vitamin D deficiency (Szulc et al., J Clin Invest 91:1769; 1993). This intriguing finding prompted us to study the effect of vitamin D on the secretion of ucOC by osteoblastic cells in vitro in the presence of warfarin, an inhibitor of gammacarboxylation of GLA-containing proteins. The potential influence of retinoic acid (RA) was also studied, because its mechanism of action involves pathways that are similar to vitamin D. In the presence of warfarin (0.05 microg/mL), 1alpha,25(OH)2D (10(-8)-10(-6) mol/L) decreased dose dependently ucOC secretion by human osteosarcoma MG63 cells (from 3.87 +/- 0.96 to 2.12 +/- 0.13 ng/10(6) cells). When expressed as a fraction of total OC, secretion ucOC decreased from 47.4 +/- 1.4% to 24.8 +/- 3.2% in the MG63 cells. The secretion of total OC was stimulated by RA and by Ro 13-7410, which is a specific ligand of retinoic acid receptor (RAR), but not by 9-cis retinoic acid (9-cisRA), which is a physiologic ligand of retinoid X receptor (RXR). RA and Ro 13-7410 decreased ucOC secretion and ucOC% in warfarin-treated MG63 cells (RA: from 50.4 +/- 13.3% to 13.5 +/- 2.8%; Ro 13-7410: from 28.4 +/- 8.2% to 11.3 +/- 8.4%). 9-cisRA had no effect on OC gammacarboxylation. These results show that vitamin D, RA, and Ro 13-7410, but not 9-cisRA, may modify the gammacarboxylation of OC in human MG63 cells.

Topics: Animals; Benzoates; Carboxylic Acids; Cattle; Humans; Osteocalcin; Osteosarcoma; Retinoids; Tretinoin; Tumor Cells, Cultured; Vitamin D; Warfarin

1 alpha,25-Dihydroxyvitamin D3 (D3), T3, and retinoids are necessary for normal skeletal development, and their actions are interdependent due to the heterodimerization capabilities of their receptors. We investigated the hypothesis that these hormones act on osteoblasts directly to produce complex target gene responses resulting from multiple hormone interactions. Physiological interactions among D3, T3, and retinoid signaling were analyzed in serum-free cultures of the osteosarcoma cell lines ROS 25/1, UMR106, and ROS 17/2.8. These cells express distinct stages of the osteoblast phenotype and coexpress appropriate hormone receptors. Regulation of collagen I alpha 1 and alpha 2, alkaline phosphatase, osteopontin, and osteocalcin messenger RNAs was dependent on the dose and duration of hormone stimulation and modified by cell confluence. Retinoids were required for comprehensive expression of phenotypic responses to D3 and T3 in each cell type and hormone interactions were both cell and target gene specific. Differing responses of target genes in each cell line may provide a molecular basis for discrete hormone actions seen at specific stages of osteoblast differentiation or skeletal development.

Topics: Animals; Calcitriol; Gene Expression Regulation; Osteoblasts; Osteocalcin; Osteopontin; Osteosarcoma; Rats; RNA, Messenger; Sialoglycoproteins; Tretinoin; Triiodothyronine; Tumor Cells, Cultured

The effects of steroid and thyroid hormones are mediated by intracellular hormone receptors. An important mechanism modulating target tissue responsiveness to hormones is homologous and heterologous regulation of the receptors. We have characterized the expression of steroid hormone receptors in human MG-63 osteosarcoma cells. The MG-63 cells express receptor mRNAs for glucocorticoids, estrogen, retinoic acid, and 1,25(OH)2D3. We found that only the vitamin D receptor (VDR) mRNA concentration was influenced by the hormones. The stability of the VDR message was identical in control, dexamethasone- and estradiol-treated cells. On the other hand, both 1,25(OH)2D3 and retinoic acid separately stabilized the VDR mRNA levels increasing the apparent half-life by 11 h and 6 h, respectively. The VDR protein levels, however, as measured by immunoprecipitation, increased only after the 1,25(OH)2D3 treatment.

Topics: Blotting, Northern; Bone Neoplasms; Calcitriol; Cell Line; Dexamethasone; Estradiol; Gene Expression; Humans; Kinetics; Osteosarcoma; Receptors, Calcitriol; Receptors, Estrogen; Receptors, Glucocorticoid; Receptors, Retinoic Acid; Receptors, Steroid; RNA, Messenger; Steroids; Time Factors; Tretinoin; Tumor Cells, Cultured

Treatment of human MG-63 osteosarcoma cells with human recombinant transforming growth factor beta 1 (TGF-beta 1) was found to inhibit cell proliferation. In addition, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-induced osteocalcin synthesis was greatly influenced by TGF-beta 1. Dose- and time-dependent inhibition was seen both in medium osteocalcin and the corresponding mRNA concentrations. Furthermore, TGF-beta 1 decreased osteocalcin synthesis modulated negatively by dexamethasone or positively by retinoic acid. The stability of osteocalcin mRNA was not decreased by the TGF-beta 1 treatment, but in vitro transcription assays demonstrated diminished osteocalcin gene transcription caused by the TGF-beta 1 treatment. Binding of vitamin D receptor (VDR) to an oligonucleotide probe containing the osteocalcin vitamin D response element (VDRE) was not influenced by TGF-beta 1, however. Incubation of the cells with the serine/threonine kinase inhibitor H-7 did not block the ability of TGF-beta 1 to decrease osteocalcin synthesis but caused a further inhibition. Also, the 1,25(OH)2D3-induced osteocalcin synthesis was decreased by H-7 treatment, suggesting that phosphorylation as such is involved in the transcriptional activation mechanism of VDR. These results demonstrate that TGF-beta 1 is a strong inhibitor of the synthesis of osteocalcin, a calcium binding protein participating in bone mineralization, by counteracting the stimulatory effects of other hormones on its synthesis. We further suggest that TGF-beta 1 affects the synthesis of osteocalcin at the level of transcription through mechanism(s) different from the serine/threonine kinase pathway.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Base Sequence; Bone Neoplasms; Calcitriol; Cell Division; Dexamethasone; Dose-Response Relationship, Drug; Drug Interactions; Gene Expression Regulation, Neoplastic; Humans; Isoquinolines; Molecular Sequence Data; Oligonucleotide Probes; Osteocalcin; Osteosarcoma; Phosphorylation; Piperazines; Protein Kinase C; Receptors, Calcitriol; RNA, Messenger; Transcription, Genetic; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured; Vitamin D-Binding Protein

We report a rare case of complete remission for 32 months with continuous treatment with all-trans retinoic acid (ATRA) alone in a patient with acute promyelocytic leukemia which developed as a second malignancy after the treatment of osteosarcoma after failure of conventional chemotherapy. The adverse effects of ATRA were apparently tolerable.

Topics: Adolescent; Antineoplastic Combined Chemotherapy Protocols; Femoral Neoplasms; Humans; Leukemia, Promyelocytic, Acute; Male; Neoplasms, Second Primary; Osteosarcoma; Tretinoin

The rat osteoblastic osteosarcoma cell line UMR 106-01 secretes interstitial collagenase in response to retinoic acid (RA). The present study demonstrates by Northern blot analysis that RA causes an increase in collagenase messenger RNA (mRNA) at 6 h, which is maximal at 24 h (20.5 times basal) and declines toward basal level by 72 h. This stimulation is dose dependent, with a maximal response at 5 x 10(-7) M RA. Nuclear run-on assays show a greater than 20-fold increase in the rate of collagenase mRNA transcription between 12-24 h after RA treatment. Cycloheximide blocks RA stimulation of collagenase mRNA, demonstrating the need for de novo protein synthesis. RA not only causes an increase in collagenase secretion, but is known to decrease collagen synthesis in UMR 106-01 cells. In this study, the increase in collagenase mRNA is accompanied by a concomitant decrease in the level of alpha 1(I) procollagen mRNA, which is maximal at 24 h (70% decrease), with a return to near-control levels by 72 h. Nuclear run-on assays demonstrated that the decrease in alpha 1 (I) procollagen expression does not have a statistically significant transcriptional component. RA did not statistically decrease the stability of alpha 1 (I) procollagen mRNA (calculated t1/2 = 8.06 +/- 0.30 and 9.01 +/- 0.62 h in the presence and absence of RA, respectively). However, transcription and stability together probably contribute to the major decrease in stable alpha 1 (I) procollagen mRNA observed. Cycloheximide treatment inhibits basal level alpha 1 (I) procollagen mRNA accumulation, demonstrating the need for on-going protein synthesis to maintain basal expression of this gene.

Topics: Animals; Blotting, Northern; Collagen; Collagenases; Drug Stability; Enzyme Induction; Osteosarcoma; Rats; RNA, Messenger; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

HOS-8603 is a newly established human osteosarcoma cell line with phenotypic characteristics of osteoblasts. When these cells were grown in monolayer culture in the presence of dexamethasone (Dex) or retinoic acid (RA), there was a significant inhibition of proliferation in a concentration-dependent manner. The combined effects of Dex and RA depended upon the concentrations: at low concentrations (< 10 nM) the effects of Dex and RA were additive, whereas at high concentrations the effects were antagonistic. Anchorage-independent growth studies performed in methylcellulose culture indicated that Dex or RA inhibited colony formation by HOS-8603 cells. Treatment of HOS-8603 cells with 100 nM Dex induced alkaline phosphatase activity in a time-dependent manner, reaching a maximum of about 6.5-fold over basal levels. All these effects of Dex on HOS-8603 cells could be reversed by RU 486, a potent antiglucocorticoid. Based upon saturation of specific binding and Scatchard plot analysis, we demonstrated that a saturable, high-affinity glucocorticoid receptor (GR) existed in HOS-8603 cells, suggesting that the effects of glucocorticoids on HOS-8603 cells are mediated by the specific GR. Finally, we further investigated the homologous and heterologous regulation of GR in HOS-8603 cells. Treatment of these cells with Dex led to a time-dependent decrease in GR concentrations. This homologous GR downregulation occurred not only at the level of hormone binding but also at the level of GR mRNA. In contrast, RA was capable of increasing GR concentrations in a concentration- and time-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Alkaline Phosphatase; Bone Neoplasms; Cell Adhesion; Cell Differentiation; Cell Division; Cell Line; Dexamethasone; Dose-Response Relationship, Drug; Drug Interactions; Humans; Kinetics; Osteosarcoma; Receptors, Glucocorticoid; Tretinoin; Tumor Cells, Cultured

The binding of transcription factor AP-1 and vitamin D receptor (VDR) to the composite AP-1 plus vitamin-D-responsive promoter region (AP-1 + VDRE) of the human osteocalcin gene was characterized in osteocalcin-producing (MG-63) and non-producing (U2-Os, SaOs-2) human osteosarcoma cell lines. In mobility-shift assays with AP-1 + VDRE, AP-1, and VDRE probes and nuclear extracts from these cells, one AP-1-specific and two VDR-specific (fast and slow mobility) interactions were observed. Characterization of the complexes indicated that AP-1 and VDR do not bind simultaneously to the AP-1 + VDRE oligonucleotide. Intensity of the complexes was greatly influenced by cell density: in MG-63 and SaOs-2 cells, AP-1 binding was strong during the proliferative period disappearing at confluency whereas, in U2-Os cells, AP-1 binding was prominent also at the confluent stage. Furthermore, MG-63 cells possessed the faster migrating VDR complex at all stages of confluency whereas, in U2-Os and SaOs-2 cells, it was very weak or absent. There were no detectable differences in the levels of VDR protein between these cell lines. In U2-Os cells, the level of c-jun mRNA was higher than in the other two cell lines, whereas none of these cell lines exhibited detectable levels of c-fos mRNA at the confluent stage. Exogenous c-Jun protein effectively blocked the VDR-DNA interaction. Further, all these cell lines expressed mRNA for retinoid X receptor alpha (RXR alpha), the factor suggested to be required for the VDR-DNA interaction. The presence of an accessory factor in the VDR-DNA complexes was indirectly shown by treatment of the cells with 9-cis retinoic acid and by cycloheximide. Both treatments reduced VDR binding without affecting the VDR protein level. These results suggest that AP-1 interferes with VDR binding to the AP-1 + VDRE element and that the vitamin D responsiveness of the osteocalcin gene correlates with weak AP-1 binding and strong binding of the faster migrating VDR complex.

Topics: Base Sequence; Blotting, Northern; Blotting, Western; Cycloheximide; DNA; Gene Expression; Humans; Molecular Sequence Data; Osteocalcin; Osteosarcoma; Polymerase Chain Reaction; Promoter Regions, Genetic; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Receptors, Calcitriol; RNA, Messenger; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Basic fibroblast growth factor (bFGF) has been detected in bone cells and stimulates osteoblast proliferation; however, its role in the regulation of bone metabolism remains speculative. We demonstrated that the human osteocalcin promoter is activated by bFGF when transfected into rat osteoblastic (ROS 17/2.8) cells. This effect is concentration dependent, with a twofold induction at 10 ng/ml detected after 20 h. The bFGF response is independent of both the 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and retinoic acid activation of the osteocalcin promoter. To identify the promoter sequences through which bFGF exerts its effect, we tested a series of promoter deletion constructs for their response to bFGF. Deletion of the upstream region between -673 and -588 bp results in a significant loss of induction. Gel-shift analysis demonstrates that proteins present in ROS 17/2.8 nuclear extracts bind specifically to these sequences. This region alone was unable to confer the bFGF response on a minimal osteocalcin or an heterologous promoter. However, sequences between -678 and -476 bp, which also includes the vitamin D response element (VDRE), were able to confer bFGF inducibility on both a minimal osteocalcin and a heterologous promoter. These data suggest that induction of the human osteocalcin promoter by bFGF requires the interaction of more than one sequence element.

Topics: Animals; Base Sequence; Calcitriol; Cell Division; Dose-Response Relationship, Drug; Fibroblast Growth Factor 2; Gene Expression Regulation; Humans; Molecular Sequence Data; Mutation; Osteoblasts; Osteocalcin; Osteosarcoma; Promoter Regions, Genetic; Rats; Regulatory Sequences, Nucleic Acid; Sequence Deletion; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

The human osteosarcoma cell culture HOS does not produce matrix metalloproteinases (MPs). However, after transformation with the Ki-ras oncogene, the resulting culture (KHOS) produced readily detectable MPs. The molecular weight of the major MP was 66 kDa, while the molecular weights of two other minor bands were 71 kDa and 60 kDa. The activity of all three enzymes was inactivated by treatment with ethylene diaminetetra acetic acid, indicating that they are probably MPs. The substrate preference of the 66-kDa MP (in decreasing order) was gelatin and collagens V, I, III, and IV. Treatment of the MPs with p-aminophenylmercuric acetate led to the appearance of 62-kDa activated enzyme. The MP produced by KHOS cells did not react with the monoclonal anti-rat stromelysin antibody MC. Treatment of KHOS cells with retinoic acid and dexamethasone, which are known to suppress c-fos/c-jun and AP-1, suppressed the production of the MPs. Therefore, the activation of MPs by Ki-ras in KHOS cells may involve c-fos/c-jun and the AP-1-responsive pathway.

Topics: Genes, ras; Humans; Metalloendopeptidases; Osteosarcoma; Phenylmercuric Acetate; Tretinoin; Tumor Cells, Cultured

Cellular retinoic acid-binding proteins (CRABPs) are thought to play a key role in the regulation of the levels of retinoic acid (RA) available to interact with the nuclear receptors. In this study we have used reverse transcription (RT) and competitive polymerase chain reaction (cPCR) to investigate the effects of RA on CRABP-II expression levels in the human osteosarcoma cell line MG-63. Two different isomers of RA, all-trans (at) and 9-cis RA, were chosen since at-RA but not 9-cis RA binds to CRABP-II. Cells were treated with 10(-6) M at-RA or 9-cis RA for 24 h. Following RNA preparation and RT, cPCR was performed using constructed internal standards for CRABP-II and beta-actin. While no change occurred in the beta-actin control, an approximate two-fold increase of CRABP-II mRNA levels was seen in cells treated with at-RA and at least a four-fold increase with 9-cis RA. Thus, although 9-cis RA does not bind to CRABP-II, it induces CRABP-II expression more efficiently than at-RA in human osteosarcoma cells.

Topics: Base Sequence; DNA Primers; DNA, Complementary; Gene Expression; Humans; Molecular Sequence Data; Osteoblasts; Osteosarcoma; Polymerase Chain Reaction; Receptors, Retinoic Acid; Stereoisomerism; Tretinoin; Tumor Cells, Cultured

Although several studies have been performed on the biological activities of analogs of 1,25-dihydroxyvitamin D3 (1,25-(OH)2 D3) at the whole animal and cellular levels, little work has been done to analyze their transcriptional activation properties. A highly inducible 1,25-(OH)2 D3-responsive promoter composed of three copies of the mouse osteopontin vitamin D3 response element (VDRE3) inserted upstream of a herpes simplex virus thymidine kinase promoter has been constructed, and its transcriptional properties have been analyzed by transient transfection into the monkey kidney cell line COS-7 and the rat osteoblast-like osteosarcoma line ROS 17/2.8. We have studied systematically transcriptional activation by a number of 1,25-(OH)2 D3 analogs, particularly those substituted at positions 16, 23, 26, and 27, sites that are targets for metabolism. Strikingly, except for derivatives that bind the 1,25-(OH)2 D3 receptor (VDR) very weakly, we find no parallel between the potency of action of a derivative as a transcriptional inducer and its affinity for the VDR. Derivatives substituted by multiple bonds at positions 16 and/or 23, although having varying affinities for the VDR, all stimulate transcription more potently than D3, in some cases at 100-fold lower concentrations. The peak transcriptional activity observed varies by only approximately 20% among different active analogs, indicating little difference in the activity of the VDR once bound to ligand. Gel retardation assays with ROS 17/2.8 nuclear extracts suggest that the VDR binds to the mouse osteopontin VDRE predominantly as a heterodimer with retinoid X receptor(s) (RXR(s)). We find that 9-cis-retinoic acid, the cognate ligand for RXRs, does not have a significant effect on the response of the VDRE3 promoter to 1,25-(OH)2 D3 or a number of its derivatives in ROS 17/2.8 or in COS-7 cells, under conditions in which promoters containing retinoid X response elements are activated. This suggests that 9-cis-retinoic acid may not act on the response to 1,25-(OH)2 D3 or its derivatives by directly influencing the transcriptional activity of VDR/RXR heterodimers. This promoter/reporter system should be useful for analyzing the tissue-specific transcriptional activity of 1,25-(OH)2 D3 and its derivatives in any cell type amenable to transient transfection.

Topics: Animals; Base Sequence; Calcitriol; Cell Line; Chloramphenicol O-Acetyltransferase; Chlorocebus aethiops; Gene Expression; Genetic Vectors; Kidney; Kinetics; Mice; Molecular Sequence Data; Oligodeoxyribonucleotides; Osteoblasts; Osteopontin; Osteosarcoma; Phosphoproteins; Promoter Regions, Genetic; Rats; Restriction Mapping; Sialoglycoproteins; Structure-Activity Relationship; Thymidine Kinase; Transcription, Genetic; Transfection; Tretinoin; Tumor Cells, Cultured

We have previously shown that protein S, a vitamin K-dependent protein, is a bone matrix component synthesized and secreted by osteoblasts. Because protein S is a cofactor of protein C in inhibiting factor Va and VIIIa, we have looked for the presence of the proteins related to the anticoagulant protein C system in human MG 63 osteosarcoma cells and in human adult osteoblast-like cells. Using immunoblotting, we have shown that protein C, factor V, and C4b binding protein are not secreted by these cells. We have shown by enzyme-linked immunoassay, immunocytochemistry, and immunoprecipitation of labeled proteins that thrombomodulin, a transmembrane glycoprotein involved with thrombin in the activation of protein C, is present at the cell surface of osteoblasts. Moreover, using a protein C activation system where thrombin and protein C are added to the cells, we have shown that protein C could be activated at the osteoblast cell surface. This activation of exogenous protein C, reflecting the activity of thrombomodulin, as well as the expression of the thrombomodulin antigen, is regulated by some bone resorption-enhancing factors. 1,25-dihydroxyvitamin D3 and retinoic acid increase thrombomodulin expression and activity in a dose-dependent manner whereas tumor necrosis factor alpha and interleukin 1 decrease these parameters. Because thrombomodulin is known to inhibit single-chain urokinase-type plasminogen activator, a molecule present in the osteoblast microenvironment, these findings suggest that thrombomodulin could play a role in the regulation of bone resorption by modulating the plasmin system.

Topics: Calcitriol; Cell Membrane; Cyclic AMP; Enzyme-Linked Immunosorbent Assay; Factor V; Humans; Immunoblotting; Immunosorbent Techniques; Interleukin-1; Osteoblasts; Osteosarcoma; Protein C; Receptors, Cell Surface; Receptors, Thrombin; Tretinoin; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Exposure of ROS 17/2.8 cells to dexamethasone (DEX) or retinoic acid (RA) increases and decreases, respectively, adenylate cyclase activity (ACA) in response to isoproterenol, forskolin, guanylylimidodiphosphate, or NaFl. Despite dramatic changes in ACA, there were no significant changes in levels of cholera toxin- or pertussis toxin (PT)-dependent ADP-ribosylation of membranes prepared from cells after DEX or RA exposure as compared to controls. Similarly, immunochemical detection of alpha S, alpha i1-3, and alpha O, as well as Northern blot analysis of messenger RNA for each of the respective GTP binding proteins, also failed to demonstrate an influence of DEX or RA when contrasted with controls. In a novel use of the cyc- reconstitution assay, wherein the influence of inhibitory guanine nucleotide binding proteins in the extracts of control, DEX-, and RA-treated membranes is removed by a previous 24-h incubation with PT in the intact cell, we demonstrate that this PT treatment markedly enhances ACA in the cyc- reconstitution assay for all three preparations, but that the fold-increase due to PT-treatment is greatest in RA-treated cells. The greater magnitude of the effect of PT on RA-treated ROS 17/2.8 cells, in the absence of any obvious quantitative changes in the levels of the PT substrates, suggests that the effect of RA on ROS 17/2.8 cells appears to be an augmentation of the influence of inhibitory guanine nucleotide binding proteins, ultimately leading to reduced ACA.

Topics: Adenosine Diphosphate Ribose; Adenylyl Cyclases; Amino Acid Sequence; Blotting, Northern; Cell Membrane; Colforsin; Dexamethasone; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanylyl Imidodiphosphate; Isoproterenol; Molecular Sequence Data; Osteosarcoma; RNA, Messenger; Sodium Fluoride; Tretinoin; Tumor Cells, Cultured

In this study we have used a reverse transcription polymerase chain reaction (RT-PCR) to demonstrate that adult primary human osteoblasts and SaOS-2, a human osteosarcoma-derived cell line with osteoblastic properties, express cellular retinol-binding protein I (CRBP I), cellular retinoic acid-binding protein II (CRABP II), and very low levels of CRABP I. We also show that CRABP II is expressed in the adult liver, which does not express CRABP I. The results suggest that CRABP II is the important isoform in the adult bone as well as in the adult liver. Since the 9-cis retinoic acid receptor (RXR) alpha previously has been shown to be expressed predominantly in the liver, CRABP II might be involved in the transport of 9-cis retinoic acid to its nuclear receptor.

Topics: Adult; Base Sequence; Carrier Proteins; Cells, Cultured; Humans; Liver; Molecular Sequence Data; Oligodeoxyribonucleotides; Osteoblasts; Osteosarcoma; Polymerase Chain Reaction; Receptors, Retinoic Acid; RNA; Tretinoin; Tumor Cells, Cultured

Clonal cell lines presumably "arrested" at a particular stage of differentiation are useful models to study the processes of differentiation in osteoblasts. UMR-201 is a presumptive preosteoblastic nontransformed rat clonal cell line with a limited life span in culture. Two immortalized cell lines, UMR-201-10A (10A) and UMR-201-10B (10B), were derived from UMR-201 by stable transfection with simian virus (SV) 40 large T antigen. This study compares the growth and profile of gene expression of the immortalized cell lines with those of UMR-201 and UMR-106-06, a rat clonal cell line with well-defined osteoblast-like phenotypic characteristics. All four cell lines constitutively expressed the mRNA for the gamma, alpha, and beta receptors for retinoic acid (RA), the growth hormone receptor, pro-alpha 1(I) collagen, osteonectin, bone proteoglycan I, and bone morphogenetic proteins (BMP) 1 and 2A. Alkaline phosphatase mRNA was absent in the preosteoblast cell lines but was induced by treatment with 10(-6) M RA, which also increased the steady-state levels of mRNA for osteopontin and BMP1. mRNA for matrix gla protein was constitutively present and further induced by RA in UMR-201 and 10B only. Messenger RNA for bone sialoprotein and bone morphogenetic protein 3 were constitutively expressed in UMR-106-06 and UMR-201 but absent in the immortalized cell lines. None of the cell lines expressed measurable mRNA for bone gla protein or bone proteoglycan II. 10B grew more rapidly than UMR-201, but unlike UMR-201, it was also able to proliferate in serum-free medium and exhibit anchorage-independent growth. In summary, this study identifies novel retinoic acid effects on gene expression in these cells. Differences noted in the expression of mRNAs between UMR-106-06 and the other cell lines may provide some insight into the sequence of expression of these phenotypic characteristics as osteoblasts differentiate.

Topics: Alkaline Phosphatase; Animals; Calcium-Binding Proteins; Carrier Proteins; Cell Differentiation; Cell Line; Cell Line, Transformed; Extracellular Matrix Proteins; Matrix Gla Protein; Osteoblasts; Osteopontin; Osteosarcoma; Receptors, Retinoic Acid; RNA, Messenger; Sialoglycoproteins; Tretinoin; Tumor Cells, Cultured

We have previously shown that osteocalcin synthesis is readily induced by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in MG-63 human osteosarcoma cells (Mahonen et al. (1990) Biochim. Biophys. Acta 1048, 30-37). In the present study, the regulation of osteocalcin synthesis by other hormones of the steroid-thyroid hormone family (retinoic acid, 17 beta-estradiol, triiodothyronine, and dexamethasone) was examined. We found that the other hormones alone had no effects on medium osteocalcin and osteocalcin mRNA concentrations by 96 h of treatment. Compared with 1,25(OH)2D3, however, the combination of 1,25(OH)2D3 with dexamethasone resulted in a greatly reduced medium osteocalcin concentration. Also estradiol and triiodothyronine diminished the stimulatory effect of 1,25(OH)2D3. In contrast, the combination of 1,25(OH)2D3 with retinoic acid resulted in an increased medium osteocalcin concentration. The inhibition of osteocalcin synthesis by dexamethasone and triiodothyronine was accompanied by decreased osteocalcin mRNA levels. Retinoic acid and estradiol, however, did not influence the 1,25(OH)2D3-induced osteocalcin mRNA levels. To examine the specificity of the hormonal effects, the activity of alkaline phosphatase was determined. Both baseline and 1,25(OH)2D3-stimulated alkaline phosphatase activity was found to be inhibited by all other hormones. These results suggest that the steroidal hormones specifically affect osteocalcin synthesis in osteoblastic bone cells, and that complex interactions occur at the level of transcription and/or translation resulting in each case in a finely adjusted rate of osteocalcin synthesis.

Topics: Alkaline Phosphatase; Calcifediol; Cell Division; Dexamethasone; Estradiol; Humans; Osteocalcin; Osteosarcoma; RNA, Messenger; Steroids; Thyroid Hormones; Tretinoin; Triiodothyronine; Tumor Cells, Cultured

Confluent cultures of rat bone cells synthesize several forms of secreted phosphoprotein 1 (SPP-1, osteopontin), the major phosphorylated forms of which migrate at 55 and 44 kDa on 15% cross-linked SDS-PAGE gels and correspond to the transformation-associated proteins pp 69 and pp 62. A clonal rat calvarial cell line (RCA 11), which expressed the highest level of SPP-1, produced only the 55 kDa form of the phosphorylated protein, whereas normal rat calvarial cells enriched in osteoblastic cells (RC IV cells) produced mostly the 55 kDa form, with small amounts of the 44 kDa form. In contrast, a 44 kDa form was the major [32PO4]-labelled SPP-1 synthesized by a rat osteocarcoma cell line (ROS 17/2.8 cells) with lesser amounts of the 55 kDa SPP-1. When [35S]methionine was used to measure protein synthesis, only the 55 kDa SPP-1 could be clearly detected in confluent cultures of each cell population, indicating that the 55 kDa SPP-1 is the prominent form of SPP-1 synthesized by each cell population. Following treatment of the normal rat bone cells for 24 h with osteotropic hormones (vit D3, PTH and RA), growth factors (PDGF, EGF, TGF-beta), a tumor promoter (TPA) and a plant lectin (Con A), the 55 kDa [35S]methionine labelled SPP-1 was increased 1.7-8.3-fold. Similar, but generally lower responses were observed in the clonal RCA 11 cell line, whereas the ROS 17/2.8 cells were more refractory, showing only a strong response to vit D3. In general, vit D3 produced the strongest stimulation in all populations with TGF-beta producing a good response in the non-transformed cells and RA in the RC IV cells. In contrast, PTH was inhibitory in both RCA 11 and ROS 17/2.8 cells. In most, but not all, cases the alteration in SPP-1 synthesis reflected similar changes in SPP-1 mRNA and in the intensity of the [32PO4]-labelled 55 kDa SPP-1. Collectively, these studies demonstrate that bone cells produce several forms of SPP-1 which are differentially regulated in normal and transformed cells through both transcriptional and posttranscriptional mechanisms.

Topics: Animals; Calcitriol; Cell Line; Cell Line, Transformed; Cells, Cultured; Concanavalin A; Epidermal Growth Factor; Growth Substances; Nucleic Acid Hybridization; Osteopontin; Osteosarcoma; Parathyroid Hormone; Platelet-Derived Growth Factor; Precipitin Tests; RNA, Messenger; Sialoglycoproteins; Tetradecanoylphorbol Acetate; Transcription, Genetic; Transforming Growth Factor beta; Tretinoin

We have examined the effects of parathyroid hormone (PTH) and PTH-related peptide (PTH-rP) on intracellular calcium (Ca2+i) in a rat osteogenic sarcoma cell line, UMR106. Synthetic bovine (b)PTH(1-34) caused a small inconsistent rise in Ca2+i in UMR106 cells, whilst cells pretreated with retinoic acid (RA, 1 mumol/l) for 18 h exhibited reproducible, significant and dose-dependent increases in Ca2+i levels in response to bPTH. The effect of RA on PTH-induced changes in Ca2+i were dependent upon both dose and time. Purified human (h)PTH-rP(1-34) increased Ca2+i in the absence of RA in the same cells. However, RA increased the magnitude of PTH-rP-stimulated changes in Ca2+i without affecting the concentration required for a maximal response. RA also prolonged the delay before the Ca2+i response was observed. Maximal responses to PTH-rP were greater in magnitude than those to PTH. These changes appeared not to be due to cyclic AMP (cAMP), since neither dibutyryl cAMP (1 mmol/l) nor forskolin (15 mumol/l) affected Ca2+i. PTH- and PTH-rP-mediated Ca2+i transients were not completely abolished by the absence of extracellular calcium, and both peptides increased basal levels of inositol trisphosphate. PTH and PTH-rP were subject to mutual desensitization, but were not desensitized by prostaglandin E2. PTH(7-34) antagonized PTH- but not PTH-rP-mediated Ca2+i transients. We conclude that there may be some important differences in the mechanism of action of PTH and PTH-rP.

Topics: Animals; Calcium; Dose-Response Relationship, Drug; Osteosarcoma; Parathyroid Hormone; Parathyroid Hormone-Related Protein; Peptide Fragments; Proteins; Rats; Tretinoin; Tumor Cells, Cultured

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3) is a known up-regulator of 1,25(OH)2D3 receptor (VDR) both in vitro and in vivo. However, a 5- to 10-fold increase in plasma 1,25(OH)2D3 induced by dietary calcium deficiency does not result in up-regulation of intestinal VDR, and kidney VDR is down-regulated. Under certain physiological stresses, an increase in plasma PTH precedes increased plasma 1,25(OH)2D3. Therefore, the present study examined the effect of PTH on VDR regulation in vitro in ROS 17/2.8 cells and in vivo in male Holtzman rats. Treatment of ROS cells with PTH (0-5 nM) resulted in a dose and time-dependent decline in VDR from 95 +/- 9 to 35 +/- 5 fmol/mg protein at 18 h of exposure. The ED50 for PTH was 1 nM. This decline in VDR protein was attended by a 50% decline in VDR messenger RNA (mRNA). The PTH-mediated down-regulation of VDR occurred without affecting the affinity of VDR for 1,25(OH)2D3 as determined by Scatchard analysis. Also, the effect of PTH on VDR regulation was specific since cell glucocorticoid receptor concentration was not affected by PTH treatment. In accompanying experiments, 1,25(OH)2[3H]D3 treatment of ROS cells was shown to result in a 3- to 4-fold increased expression of VDR and VDR mRNA. The simultaneous addition of PTH and 1,25(OH)2[3H]D3 resulted in inhibition of the 1,25(OH)2[3H]D3-mediated up-regulation of VDR and VDR mRNA. Similarly, PTH also inhibited heterologous up-regulation of VDR and VDR mRNA induced by retinoic acid. In in vivo experiments, rats infused for 5 days with 1,25(OH)2D3 (1.5 ng/h) increased their expression of intestinal VDR, kidney VDR, and kidney 24-hydroxylase by 31, 336, and 4000%, respectively. Coinfusion of PTH (1.8 IU/h) along with 1,25(OH)2D3 completely inhibited the 1,25(OH)2D3-mediated increases in intestinal VDR and kidney 24-hydroxylase and reduced the 1,25(OH)2D3-mediated up-regulation of kidney VDR by more than half. These data suggest that PTH is a potent down-regulator of VDR and that PTH and 1,25(OH)2D3 have opposing effects on the expression of certain genes.

Topics: Animals; Calcitriol; Cell Line; Cytosol; Down-Regulation; Duodenum; Intestinal Mucosa; Kidney; Kinetics; Male; Osteosarcoma; Parathyroid Hormone; Parathyroid Hormone-Related Protein; Proteins; Rats; Receptors, Calcitriol; Receptors, Glucocorticoid; Receptors, Steroid; RNA, Messenger; Tretinoin; Tumor Cells, Cultured; Up-Regulation

Retinoic acid (RA) inhibits the increases in alkaline phosphatase (AP) and hormone-stimulated adenylate cyclase that accompany the growth of ROS 17/2.8 osteosarcoma cells in culture. The RA effects were first detected 2 days after initiation of treatment and were dose dependent, with an EC50 of 100 nM. The reduction in the hormone-responsive adenylate cyclase activity was associated with lower levels of beta-catecholamine receptors, without a change in apparent receptor affinity and with lower levels of the GTP-binding proteins Gs and Gi, visualized by NAD-dependent [32P]ADP ribosylation. The reduction in AP was correlated with a decrease in the steady state level of AP mRNA. RA had no effect on cell proliferation or saturation density. Retinoids thus inhibit the same features that are promoted by glucocorticoids in ROS 17/2.8 cells. These features seem to be subject to coordinate regulation, probably at the pretranslational level.

Topics: Adenylyl Cyclases; Alkaline Phosphatase; Animals; Cell Division; Cholera Toxin; Cyclic AMP; Diphtheria Toxin; Dose-Response Relationship, Drug; GTP-Binding Proteins; Iodocyanopindolol; Isoproterenol; Microscopy, Phase-Contrast; NAD; Osteosarcoma; Pindolol; Receptors, Adrenergic; Receptors, Catecholamine; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

Retinoic acid has previously been shown to alter 1-25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptors in tumorigenic (ROS 17/2A, UMR 106M) and nontumorigenic (RCJ 1.20) bone-derived cells. The mechanism of this regulation is unclear. In the present series of experiments, we have investigated the mechanism of the retinoic acid-induced increase in 1,25-(OH)2D3 receptors by studying the effects of sodium butyrate on this process. In ROS 17/2A rat osteosarcoma cells, retinoic acid induced a 2-4-fold increase in 1,25-(OH)2D3 receptors in proliferating cells but only a 1.5- to 2-fold increase in nonproliferating cells. The retinoic acid-induced increase in 1,25-(OH)2D3 receptors in proliferating ROS 17/2A cells was inhibited by sodium butyrate, but sodium butyrate had no effect on the retinoic acid-induced increase in 1,25-(OH)2D3 receptors in nonproliferating cells. Pretreatment with hydroxyurea of low density cells decreased the effect of retinoic acid, and abolished the sodium butyrate inhibition, indicating that the differing effects of retinoic acid in high and low density cells are related to cell proliferation and not to cell density or time of exposure to retinoic acid. In low density UMR 106M cells, the effects of retinoic acid and sodium butyrate on the number of 1,25-(OH)2D3 receptors were similar to those in ROS 17/2A cells. However, in RCJ 1.20 cells, a nontumorigenic cell line with some of the characteristics normally attributed to osteoblasts, the effects of retinoic acid and sodium butyrate were opposite: retinoic acid caused a decrease in the number of 1,25-(OH)2D3 receptors, which was inhibited by sodium butyrate. The possibility that the different responses observed between the two osteosarcoma cell lines and the RCJ 1.20 cells constitute differences in response pattern between tumorigenic and nontumorigenic cell lines is of interest, but requires further experimentation to verify.

Topics: Animals; Bone and Bones; Butyrates; Butyric Acid; Calcitriol; Cell Count; Cell Division; Osteosarcoma; Rats; Receptors, Calcitriol; Receptors, Steroid; Tretinoin; Tumor Cells, Cultured

Collagenases that specifically cleave native collagen at neutral pH have been implicated in the maintenance and turnover of connective tissue. In bone, the origin of neutral collagenase has remained equivocal, although recent studies have indicated that it is synthesized by the osteoblast. In the present work, regulation of secretion of neutral collagenase and a collagenase inhibitory activity was investigated using the osteoblastic tumor cell line UMR 106-01 and a variety of bone-resorbing agents. Under basal conditions, UMR 106-01 cells produced very low levels of collagenase but substantial amounts of the inhibitory activity. Exposure to PTH and, to a lesser extent, 1,25-dihydroxyvitamin D3, prostaglandin E2, retinoic acid, and epidermal growth factor stimulated the release of collagenase, an effect not seen with interleukin-1 or heparin. The stimulation of collagenase by PTH was dose dependent, with a half-maximal response occurring at 10(-8) M. Inclusion of isobutylmethylxanthine decreased the concentration of PTH required to produce half-maximal stimulation to 2 X 10(-10) M, indicating action via cAMP. With respect to the inhibitory activity, PTH and epidermal growth factor were the only agents, among those tested, able to enhance its production. Both hormones caused a 50-100% increase over control levels 72 h after hormone administration. There were notable differences in the time courses of production of collagenase and the inhibitor. After treatment with PTH, the enzyme reached maximal concentrations between 12-48 h, but declined to undetectable levels by 96 h. In contrast, the inhibitory activity was secreted in a linear fashion, with the highest concentrations achieved around 72-96 h. These results suggest a complex pattern of regulation of collagenase and inhibitor secretion by the osteoblastic cell, with the steady accumulation of inhibitor perhaps being responsible for the ultimate curtailment of enzyme activity.

Topics: Animals; Bone Resorption; Calcitriol; Cell Line; Cyclic AMP; Dinoprostone; Epidermal Growth Factor; Female; Kinetics; Microbial Collagenase; Osteosarcoma; Parathyroid Hormone; Prostaglandins E; Rats; Tretinoin; Uterus

Two 1,25-dihydroxyvitamin D3-controlled parameters in the osteoblastlike osteosarcoma cell line ROS 17/2, bone gamma-carboxyglutamic acid-containing protein (BGP) and collagen synthesis, were measured after pretreatments with either retinoic acid (RA), or triamcinolone acetate (TRM). RA and TRM both caused double the expected increase in BGP secretion at 16 hr after treatment with 1,25-dihydroxyvitamin D3. Triamcinolone acetate concentrations of 10(-8) and 10(-9) M or 10(-6) M retinoic acid were effective in enhancing the 1,25-dihydroxyvitamin D3 stimulation of BGP secretion. Treatment with RA or TRM alone did not stimulate BGP secretion. RA alone had no effect on BGP secretion, while TRM inhibited BGP secretion. Collagen synthesis is inhibited by 1,25-dihydroxyvitamin D3. Neither retinoic acid nor triamcinolone acetate enhanced the 1,25-dihydroxyvitamin D3-mediated inhibition of collagen synthesis. Retinoic acid by itself inhibited collagen synthesis but did not change the 1,25 dihydroxyvitamin D3-mediated inhibition of collagen synthesis. Triamcinolone acetate by itself or together with 1,25-dihydroxyvitamin D3 increased collagen synthesis. We conclude that, although both triamcinolone acetate and retinoic acid increase the 1,25-dihydroxyvitamin D3 stimulation of BGP secretion by ROS 17/2 cells, they have different effects on the regulation of collagen production. Thus, although both hormones increase the 1,25-dihydroxyvitamin D3 receptor concentration in these cells, their actions are not mediated solely by this mechanism.

Topics: 1-Carboxyglutamic Acid; Animals; Bone and Bones; Calcitriol; Cell Division; Collagen; Glucocorticoids; Osteosarcoma; Rats; Tretinoin; Tumor Cells, Cultured

Based on the finding that retinoic acid (RA) increases 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptor number in ROS 17/2 cells, we investigated the effects of RA on the ability of 1,25-(OH)2D3 to regulate alkaline phosphatase activity and PTH-responsive adenylate cyclase in these cells. A maximally effective dose of 1,25-(OH)2D3 (10(-8) M) caused a 75-80% increase in alkaline phosphatase activity and an approximately 70-75% attenuation of the cAMP response to PTH, while RA (10(-6) M) decreased alkaline phosphatase activity by 30-45% and decreased PTH-stimulated cAMP levels by approximately 20%. Preincubation with RA did not enhance the 1,25-(OH)2D3-induced increases in alkaline phosphatase activity. The ED50 values for control and RA-treated cultures were approximately 8 X 10(-10) M and 6 X 10(-10) M, respectively. With regard to PTH responsiveness, the effects of RA preincubation on the 1,25-(OH)2D3 attenuation of cAMP response varied with the concentration of 1,25-(OH)2D3. At low doses (less than 10(-9) M), the effects of 1,25-(OH)2D3 and RA were additive. At higher doses of 1,25-(OH)2D3, the effects of RA and 1,25-(OH)2D3 were not additive, and there were no differences between control- and RA-treated cultures. The ED50 values for control- and RA-treated cultures were 10(-10) M and 3 X 10(-11) M, respectively. None of the above effects were observed using equimolar doses of the vitamin D3 metabolites 24,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3. The data show that pretreating ROS 17/2A cells with RA to increase 1,25-(OH)2D3 receptors does not correspond with a concomitant increase in the cellular responsiveness to 1,25-(OH)2D3, as measured by increases in alkaline phosphatase activity and decreases in PTH-responsive adenylate cyclase.

Topics: Adenylyl Cyclases; Alkaline Phosphatase; Animals; Calcitriol; Cell Division; Cell Line; Cyclic AMP; Kinetics; Osteoblasts; Osteosarcoma; Parathyroid Hormone; Rats; Tretinoin

Pretreatment with 10(-8) M retinoic acid for 4 days caused changes in three distinct components of the parathyroid hormone (PTH)-stimulated cyclic adenosine 3':5'-monophosphate response in a clonal rat osteogenic sarcoma cell line, UMR 106-06: the amplitude of the cyclic adenosine 3':5'-monophosphate response to PTH was moderately increased after pretreatment with retinoic acid; while the cellular content of the two isoenzymes of the cyclic adenosine 3':5'-monophosphate-dependent protein kinase was approximately equal in control cells, retinoic acid pretreatment was associated with a marked increase in the ratio of type II to type I holoenzyme activity. This change might be due to a decrease in the type I holoenzyme as suggested by immunofluorescence detection of decreased type I regulatory subunit in fixed cells together with the relative decrease in type I holoenzyme determined biochemically; there was a marked alteration of the pattern of PTH-stimulated protein kinase isoenzyme activation from predominantly type I isoenzyme in control cells to almost exclusively type II isoenzyme in retinoic acid-treated cells. Growth inhibition by submaximal amounts of PTH and retinoic acid when added together was greater than that for either agent alone.

Topics: Animals; Cells, Cultured; Cyclic AMP; Enzyme Activation; Histocytochemistry; Humans; Isoenzymes; Osteosarcoma; Parathyroid Hormone; Protein Kinases; Rats; Tretinoin

Topics: Animals; Calcitriol; Cell Line; Osteosarcoma; Rats; Receptors, Calcitriol; Receptors, Steroid; Tretinoin

The distribution of cell surface negatively-charged macromolecules was determined electron microscopically on untreated and on retinoic acid (RA)-treated cultured human osteosarcoma Hs791 and chondrosarcoma Hs705 cells using cationized ferritin (CF), an electron-dense marker of anionic sites. Labeling on the surface of prefixed cells was continuous and uniform whether they were grown in the absence or presence of RA. In contrast, CF distribution on unfixed cells was markedly affected by RA; CF labeling of untreated cells occurred in patches and clusters whereas the label on RA-treated cells was continuous, as on prefixed cells. CF labeling of unfixed cells decreased considerably after incubation of the cells either with hyaluronidase or neuraminidase. There was also a reduction in patching and clustering. Changes induced by RA in the apparent membrane microviscosity, in neuraminidase-releasable sialic acid, or in transglutaminase activity could not be related to the effect of RA on CF-induced anionic site redistribution since these characteristics were modulated differently in the two cell lines. In contrast, RA increased the sialylation of specific cell surface membrane glycoproteins on both cell types. These results suggest that RA prevents redistribution of cell surface sialoglycoconjugates and glycosaminoglycans by CF. This effect may be the result of increased sialylation of specific surface components and may be related causally to the suppression of the transformed phenotype in the sarcoma cells.

Topics: Acyltransferases; Anions; Cell Line; Cell Membrane; Chondrosarcoma; Ferritins; Glycoproteins; Humans; Hyaluronoglucosaminidase; Iron-Binding Proteins; Membrane Proteins; Neuraminidase; Osteosarcoma; Receptors, Cell Surface; Transglutaminases; Tretinoin

Since several aspects of the effects of vitamin A and 1 alpha,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on bone metabolism are quite similar, we examined the possibility that vitamin A effects on bone were mediated through the regulation of cytosolic 1,25-(OH)2D3 receptors. A clonal osteoblast-like cell line derived from rat osteosarcoma (ROS 17/2) was used as a model system. Vitamin A acid (retinoic acid) in concentrations ranging from 10(-8) to 10(-5) M was found to elicit a dose-dependent increase in 1,25-(OH)2D3 binding in these cells. This effect was maximal after 24 h, was independent of cell density, and was inhibited by actinomycin D (0.05-0.5 microgram/ml). The 1,25-(OH)2D3 binding macromolecule in cytosol preparations from both vehicle- and retinoic acid-treated cells had a sedimentation coefficient of 3.2 S and binding specificities for vitamin D3 metabolites in the order: 1,25-(OH)2D3 greater than 25-(OH)-D3 greater than 24,25-(OH)2D3. Sucrose density gradient analysis, vitamin D3 metabolite displacement studies, and saturation and Scatchard analyses all indicated that the specific increase in 1,25-(OH)2[3H]D3 binding in these cells was the result of a selective increase in the number of specific 1,25-(OH)2D3 receptors.

Topics: Animals; Bone and Bones; Calcitriol; Cell Line; Cells, Cultured; Cytosol; Kinetics; Osteosarcoma; Rats; Receptors, Calcitriol; Receptors, Steroid; Tretinoin; Vitamin A

The effect of beta-all-trans-retinoic acid (RA) on the synthesis of cellular, cell surface, and secreted glycoconjugates by human Hs705 chondrosarcoma and Hs791 osteosarcoma cells was investigated in vitro. Untreated and RA-treated cells were labeled either metabolically with radioactive precursors or by oxidation of externally exposed cell membrane glycoprotein(s) (GP) by treatment with NalO4 or neuraminidase and galactose oxidase followed by reduction with NaB[3H]4. The cells were solubilized and analyzed by polyacrylamide gel electrophoresis followed by fluorography. RA enhanced the labeling of sialic acid and galactose residues on the GP of relative molecular weight(s) (Mr) in the range 95,000-300,000 on the surfaces of both cell types. [3H]glycosamine incorporation into GP with Mr of 100,000, 150,000, and 190,000 in both cell lines was also stimulated. In the Hs705 cells there was also an increase in the labeling of a 290,000-Mr GP. In contrast, [3H]glucosamine incorporation into glycoconjugates greater than 400,000 Mr in both the cells and the conditioned medium of Hs705 cells decreased. The latter glycoconjugates were susceptible to hyaluronidase and chondroitinases. [3H]glucosamine incorporation into a secreted 230,000-Mr GP, identified as fibronectin, was also reduced. Analyses of conditioned media of cells labeled with [35S]methionine or [14C]proline demonstrated that RA decreased the secretion of procollagen chains and fibronectin. Immunofluorescence revealed that RA alters the distribution of cell-associated fibronectin. These results demonstrated that RA increases the glycosylation of specific cellular and cell surface GP and decreases the production of secreted GP and glycosaminoglycans by the sarcoma cells.

Topics: Cell Line; Cell Membrane; Chondrosarcoma; Glucosamine; Glycoproteins; Humans; Methionine; Molecular Weight; Osteosarcoma; Proline; Tretinoin

Necessary for growth and differentiation in many normal tissues and capable of inducing differentiation in human promyelocytic cell lines, retinoids were the subject of this study. Specifically, effects of 13-cis-retinoic acid and 13-trans-retinoic acid on the growth of normal human bone marrow cells in soft-agar system were studied. Both short-term incubation and continuous exposure to retinoic acid caused a decreased number of granulocyte colonies and an increased cluster-to-colony ratio. This effect was concentration-dependent. Examination of specimens stained with Wright-Giemsa or nitro blue tetrazolium stains showed a progressive increase in the percentage of immature granulocytic precursors with increasing concentrations of retinoic acid. No effect of retinoic acid was seen on a number of human tumor cell lines. Retinoic acid blocked both differentiation and proliferation and appeared to do so by specific, noncytotoxic mechanisms in normal human bone marrow cells.

Topics: Bone Marrow; Bone Marrow Cells; Burkitt Lymphoma; Cell Division; Cell Line; Clone Cells; Colony-Forming Units Assay; Colony-Stimulating Factors; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Humans; Leukemia, Myeloid; Melanoma; Multiple Myeloma; Osteosarcoma; Tretinoin

Topics: Animals; Cell Adhesion; Cell Division; Cell Line; Clone Cells; Female; Fibrinolysis; Fibrosarcoma; HeLa Cells; Humans; Melanoma; Mice; Neoplasms; Osteosarcoma; Plasminogen Activators; Tretinoin; Vulvar Neoplasms

The ability of retinoic acid (RA) to inhibit the growth of three cell lines (Te85, Hs781, and Hs791) derived from human osteosarcomas and two cell lines (Hs705 and Hs819) derived from human chondrosarcomas was studied in culture. The exposure to 10(-5) M RA resulted, within 4 days, in changes in both cell morphology and cell growth. RA-treated cells appeared flat and spread on the substratum more than untreated cells, their exponential growth rates decreased, and their saturation densities were markedly reduced. All these effects could be reversed by removal of RA from the growth medium. The various cell lines exhibited differential susceptibility to the growth-inhibitory effect of RA. The most sensitive was the Hs705 chondrosarcoma. The proliferation of these cells was inhibited 50% by 10(-9) M RA and was completely blocked by 10(-5) m RA. In contrast, the concentrations of RA required for 50% inhibition of Hs791, Te85, Hs819, and Hs781 were 10(-7), 2 X 10(-7), 2.5 X 10(-7), and 2 X 10(-6) M, respectively. Only the Te85 and the Hs781 osteosarcoma cells and cells derived from a chondrosarcoma biopsy were able to form colonies in a semisolid medium, and this growth was dramatically inhibited by RA. These results demonstrate that RA can suppress in these mesenchymal tumor cells the expression of morphological and growth properties frequently associated with transformed cells.

Topics: Cell Division; Cell Line; Cell Survival; Chondrosarcoma; Drug Evaluation, Preclinical; Humans; Osteosarcoma; Tretinoin