Biomedical subjects

R S Taichman

Publications and source records attributed to R S Taichman.

17 recordsLinked to original sources

PubMed2000

Secretory products from PC-3 and MCF-7 tumor cell lines upregulate osteopontin in MC3T3-E1 cells.

Tumor cells frequently have pronounced effects on the skeleton including bone destruction, bone pain, hypercalcemia, and depletion of bone marrow cells. Despite the serious sequelae associated with skeletal metastasis, the mechanisms by which tumor cells alter bone homeostasis remain largely unknown. In this study, we tested the hypothesis that the disruption of bone homeostasis by tumor cells is due in part to the ability of tumor cells to upregulate osteopontin (OPN) mRNA in osteoblasts. Conditioned media were collected from tumor cells that elicit either osteolytic (MCF-7, PC-3) or osteoblastic responses (LNCaP) in animal models and their effects on OPN gene expression were compared using an osteoblast precursor cell line, MC3T3-E1 cells. Secretory products from osteolytic but not osteoblastic tumor cell lines were demonstrated to upregulate OPN in osteoblasts while inhibiting osteoblast proliferation and differentiation. Signal transduction studies revealed that regulation of OPN was dependent on both protein kinase C (PKC) and the mitogen-activated protein (MAP) kinase cascade. These results suggest that the upregulation of OPN may play a key role in the development of osteolytic lesions. Furthermore, these results suggest that drugs that prevent activation of the MAP kinase pathway may be efficacious in the treatment of osteolytic metastases.

Animals↗

PubMed2000

Bone marrow accessory cells regulate human bone precursor cell development.

OBJECTIVE: Much remains to be learned about the intimate relationship between bone marrow and its surrounding tissue: the bone. We hypothesized that bone marrow accessory cell populations might regulate the development of human bone precursor cells. MATERIALS AND METHODS: We used immunologic phenotyping, and isolation methods to fractionate subpopulations of nonadherent, low-density (NALD) human bone marrow cells. These cells were examined for their ability to support the serum-free survival, proliferation, and expression of bone proteins by highly purified populations of human bone precursor cells. Quantitative assessment of the accessory cell populations as well as human bone precursor cells phenotype was performed using multiparameter flow cytometry. Bone protein expression was evaluated by immunocytochemistry, Western analysis, and enzymatic analysis (for alkaline phosphatase activity). RESULTS: Human bone marrow contains a cell population that stimulates the development of purified bone precursor cells. Feeder-layer studies demonstrate that these osteopoietic accessory cells (OACs) do not require cell-cell interaction to promote bone precursor cell development but, rather, produce soluble molecules responsible for their effects. Flow cytometric analyses reveal that bone marrow derived B cells, T cells, macrophages, natural killer cells, and endothelial cells do not produce this stimulatory factor. The (growth) factor cannot be replaced by addition of exogenous cytokines. The isolation of human transforming growth factor beta receptor type II (TGF-betaRII)-positive cells increases OAC-specific activity in bone cell ex vivo expansion cultures. Moreover, isolation of OAC bone marrow cells characterized by high TGF-betaRII expression, relatively low cellular complexity, and small size yields a population that is highly enriched for OACs. CONCLUSION: We conclude that human bone marrow contains a population of OACs that are an obligate requirement for the early phases of bone cell development ex vivo.

Antigen-Presenting Cells↗

PubMed2000

Human osteoblast-like cells and osteosarcoma cell lines synthesize macrophage inhibitory protein 1alpha in response to interleukin 1beta and tumour necrosis factor alpha stimulation in vitro.

Recent investigations have demonstrated that macrophage inhibitory protein 1alpha (MIP-1alpha) plays a critical role in haematopoiesis. In part, MIP-1alpha limits the differentiation of early haematopoietic cells, thereby ensuring that sufficient quantities of blood precursors are available to meet haematopoietic demands. MIP-1alpha is produced by cells of the marrow microenvironment (marrow stromal cells) in response to a variety of stimuli, including interleukin 1beta (IL-1beta) and tumour necrosis factor alpha (TNF-alpha). Our recent investigations demonstrated that normal human osteoblast-like cells (HOBs) maintain the early phenotype of haematopoietic precursors, like other members of the bone marrow stroma. Although the precise molecular mechanisms for these observations have not been determined, the production of MIP-1alpha remains one such possibility. In the present study, we investigated whether cells of the osteoblast lineage under basal, IL-1beta and/or TNF-alpha stimulation produce MIP-1alpha. We observed that IL-1beta and TNF-alpha stimulated HOBs and human osteosarcoma cells to rapidly express MIP-1alpha mRNA and to secrete large quantities of the protein. MIP-1alpha mRNA and protein was not, however, detected under basal conditions. Perhaps more importantly, enriched human CD34+ bone marrow cells in co-culture may be capable of stimulating the expression of MIP-1alpha mRNA by HOBs in vitro. These findings suggest that human osteoblast-like cells may produce MIP-1alpha in vivo to support haematopoiesis at sites where osteoblasts and haematopoietic cells are closely associated.

Bone Neoplasms↗

PubMed2000

Induction of the chemokine stromal-derived factor-1 following DNA damage improves human stem cell function.

The chemokine stromal-derived factor-1 (SDF-1) controls many aspects of stem cell function. Details of its regulation and sites of production are currently unknown. We report that in the bone marrow, SDF-1 is produced mainly by immature osteoblasts and endothelial cells. Conditioning with DNA-damaging agents (ionizing irradiation, cyclophosphamide, and 5-fluorouracil) caused an increase in SDF-1 expression and in CXCR4-dependent homing and repopulation by human stem cells transplanted into NOD/SCID mice. Our findings suggest that immature osteoblasts and endothelial cells control stem cell homing, retention, and repopulation by secreting SDF-1, which also participates in host defense responses to DNA damage.

Animals↗

PubMed1998

The role of osteoblasts in the hematopoietic microenvironment.

Bone Marrow Cells↗

PubMed1998

Ultrastructural aspects of cytomegalovirus-infected fibroblastic stromal cells of human bone marrow.

Human cytomegalovirus (HCMV) and bone marrow interactions are important in the pathogenesis of HCMV infections. Human bone marrow fibroblastic stromal cells (HBFM-sc) were infected by Towne strain of cytomegalovirus (CMV) in cell culture. Several cytostructural features of control bone marrow stromal cells are described and compared with those of CMV-infected cells. Under these experimental conditions, HBFM-sc are cell types that can be successfully infected by CMV in vitro. The CMV-infected cells displayed typical features characteristic of DNA virus infections, such as cellular swelling, intranuclear inclusions, nucleolar condensation and disappearance (at the end stage), nuclear envelope proliferation as redundant folds. Other characteristics of CVM-infected cells include mitochondrial enlargement and vacuolization, cytoplasmic dense bodies associated or not with viral particles, accumulation and extrusion of viral particles and dense bodies. These preliminary observations shed some light on human bone-marrow stromal-cell morphology and function, one of the latter being that of a potentially harmful reservoir for CMV virus.

Bone Marrow Cells↗

PubMed1998

Protein extracts of dentin affect proliferation and differentiation of osteoprogenitor cells in vitro.

Proteins associated with the mineral phase of dentin are considered to have the potential to alter cell function within the local environment, during development and regeneration of tooth/periodontal tissues. Cells that may be altered include osteoblasts, ameloblasts, periodontal ligament cells, odontoblasts, and cementoblasts. However, specific factors within dentin controlling cell activity have not been elucidated. To investigate further the role of dentin proteins in regulating cell behavior, MC3T3-E1 cells, a mouse osteoprogenitor cell line, were exposed to guanidine/EDTA extracts of dentin (G/E-D) prepared from bovine teeth. Cells, with or without G/E-D (2 to 50 microg/ml), were evaluated for proliferative activity and for mRNA expression of bone-associated genes. Results indicated that G/E-D suppressed cell proliferation and caused striking morphological changes, including the conversion of cuboidal cells into fibroblastic, spindle-shaped cells. Markers of osteoblast differentiation, osteocalcin and bone sialoprotein mRNA were decreased, while osteopontin mRNA was enhanced in cells exposed to G/E-D. Since transforming growth factor beta (TGFbeta1) has been reported to influence cells in a similar fashion, G/E-D were examined for the presence of and concentration of TGFbeta using slot blot analysis and enzyme immunoassay (ELISA), respectively. These analyses demonstrated that G/E-D contained 6.6 ng/mg of TGFbeta1. Next, cells were exposed to G/E-D in conjunction with anti-TGFbeta1,2,3 antibody. When cells were exposed to antibody, G/E-D-mediated changes in morphology and gene expression were blocked. These results suggest that TGFbeta1 and perhaps other factors in dentin can regulate cell behavior and, therefore, can influence development, remodeling, and regeneration of mineralized tissues.

3T3 Cells↗

PubMed1997

Augmented production of interleukin-6 by normal human osteoblasts in response to CD34+ hematopoietic bone marrow cells in vitro.

Based on anatomic and developmental findings characterizing hematopoietic cells in close approximation with endosteal cells, we have begun an analysis of osteoblast/hematopoietic cell interactions. We explore here the functional interdependence between these two cell types from the standpoint of de novo cytokine secretion. We determined that, over a 96-hour period, CD34+ bone marrow cells had no significant effect on osteoblast secretion of granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, or transforming growth factor-beta1, but in some experiments minor increases in leukemia inhibitory factor levels were observed. However, when CD34+ bone marrow cells were cocultured in direct contact with osteoblasts, a 222% +/- 55% (range, 153% to 288%) augmentation in interleukin-6 (IL-6) synthesis was observed. The accumulation of IL-6 protein was most rapid during the initial 24-hour period, accounting for nearly 55% of the total IL-6 produced by osteoblasts in the absence of blood cells and 77% of the total in the presence of the CD34+ cells. Cell-to-cell contact does not appear to be required for this activity, as determined by coculturing the two cell types separated by porous micromembranes. The identity of the soluble activity produced by the CD34+ cells remains unknown, but is not likely due to IL-1beta or tumor necrosis factor-alpha, as determined with neutralizing antibodies. To our knowledge, these data represent the first demonstration that early hematopoietic cells induce the production of molecules required for the function of normal bone marrow microenvironments, in this case through the induction of hematopoietic cytokine (IL-6) secretion by osteoblasts.

Biological Factors↗

PubMed1997

Human osteosarcomas inhibit hematopoietic colony formation: partial reversal by antibody to transforming growth factor-beta 1.

Recently we found that primary human osteoblast-like cells (HOBs) support hematopoietic progenitor cells (assayed by colony formation in methylcellulose) and long-term culture initiating (LTC-IC) activity in vitro. In the present investigation, we evaluate whether human osteosarcoma cells share in these activities. We observed that relative to controls, significantly fewer hematopoietic colonies were formed in the presence of HOS TE85, MG-63, SaOS-2, or U2-OS human osteosarcomas. In addition, neither MG-63 or SaOS-2 cells supported hematopoietic progenitor cell activity or LTC-IC activity in vitro. We established that the suppressive activity produced by the osteosarcomas is soluble, correlated with osteosarcoma cell number and is partially neutralized with antibody to TGF-beta 1,2,3. While it is clear that the osteosarcomas express several phenotypic characteristics of primary human osteoblasts, these data suggest that they may be functionally disregulated with regard to their ability to support normal hematopoiesis. For these reasons, caution should be exercised when evaluating osteoblastic and hematopoietic cell interactions based purely on the use of osteosarcoma cell lines.

Antibodies↗

PubMed1997

Infection and replication of human cytomegalovirus in bone marrow stromal cells: effects on the production of IL-6, MIP-1alpha, and TGF-beta1.

We have investigated the mechanisms by which hematopoiesis is suppressed in patients suffering from human cytomegalovirus (HCMV) infections. Mixed populations of human bone marrow stromal and hematopoietic progenitor cells were inoculated with the Towne strain of HCMV to determine whether these populations could be infected and support HCMV replication. We found that the Towne strain of HCMV was capable of infecting and replicating in a mixed population of bone marrow stromal cells. We observed no significant alterations in bone marrow stromal cell proliferation or the production of IL-6, GM-CSF, soluble c-kit ligand and TNF-alpha following HCMV replication in either stimulated lipopolysaccharide (LPS) or unstimulated conditions. In samples of culture supernatants from LPS-stimulated HCMV-infected stromal cells, significant elevations in MIP-1alpha were observed. TGF-beta1 levels on the other hand exhibited two patterns following HCMV exposure; either TGF-beta1 levels decreased regardless of LPS stimulation or there was no effect. In addition, we observed that exposure to the Towne strain of HCMV resulted in significant inhibition of both granulocytic and erythrocytic colony formation in methylcellulose progenitor assays. Thus, both the direct effect of HCMV on hematopoietic progenitors as well as altered cytokine production by bone marrow stromal cells (including MIP-1alpha and TGF-beta1, but not IL-6) could contribute to hematopoietic failure during HCMV infection.

Adipose Tissue↗

PubMed1996

Human osteoblasts support human hematopoietic progenitor cells in vitro bone marrow cultures.

Hematopoietic stem cell differentiation occurs in direct proximity to osteoblasts within the bone marrow cavity. Despite this striking affiliation, surprisingly little is known about the precise cellular and molecular impact of osteoblasts on the bone marrow microenvironment. Recently, we showed that human osteoblasts produce a variety of cytokine mRNAs including granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and interleukin-6. We examined here the ability of osteoblasts to support the development of hematopoietic colonies from progenitors as well the ability to maintain long-term culture-initiating cells (LTC-IC) in vitro. Examination of the hematopoietic cells recovered after 2 weeks of culture showed that osteoblasts support the maintenance of immature hematopoietic phenotypes. In methylcellulose assays, osteoblasts stimulate the development of hematopoietic colonies to a level at least 10-fold over controls from progenitor cells. Using limiting dilutional bone marrow cultures, we observed an activity produced by osteoblasts resulting in an threefold to fourfold expansion of human LTC-IC and progenitor cells in vitro. Thus, the presence of hematopoietic stem cells in close proximity to endosteal surfaces in vivo may be due in part to a requirement for osteoblast-derived products.

Adult↗

PubMed1996

Comparison of benzimidazole nucleosides and ganciclovir on the in vitro proliferation and colony formation of human bone marrow progenitor cells.

Recently we have shown that certain benzimidazole ribonucleosides are potent and selective inhibitors of human cytomegalovirus (HCMV) replication. Because antiviral drugs used to treat HCMV and human immunodeficiency virus (HIV) infections can suppress marrow progenitors, we have evaluated the most promising of the new benzimidazoles for their effects on human bone marrow cells in vitro. In an initial study of the bone marrow toxicity of one of the most active compounds, 100 microM 2-bromo-5,6-dichloro-1-(beta-D-ribofuranosyl)-benzimidazole (BDCRB) inhibited cell proliferation by 20% over a 10 d period compared to 52% inhibition by 100 microM ganciclovir, the drug currently most used to treat HCMV infections. The effects of these drugs and selected other benzimidazole nucleosides were evaluated more extensively in haemopoietic progenitor cell colony formation assays. Colony formation was determined at 2 weeks and scored as either burst forming units-erythroid (BFU-E), or colony forming units-granulocyte/macrophage (CFU-GM). At the highest concentration tested, 100 microM BDCRB only moderately affected BFU-E or CFU-GM formation (31% and 47% inhibition, respectively). This concentration is 10-fold higher than that required to produce a 10000-fold reduction in virus titre. Evaluation of the 2-chloro analog of BDCRB (TCRB) which is less potent against HCMV, its 5'-deoxy analog (5'-dTCRB) which is more potent, and the 2-unsubstituted compound (DRB) gave the following order of haemopoietic toxicity: DRB > TCRB > or = 5'-dTCRB > BDCRB. In contrast to the benzimidazoles, ganciclovir decreased colony formation by 84% for BFU-E and 86% for CFU-GM at 100 microM. These results establish that certain benzimidazole nucleosides are less toxic to haemopoietic progenitors than the preferred drug now being used clinically for HCMV infections. The results also establish that different structure-activity relationships exist for antiviral activity and progenitor cell toxicity, thereby suggesting that different mechanisms are involved in the two types of drug action.

Antiviral Agents↗

PubMed1996

Human osteosarcoma cell lines MG-63 and SaOS-2 produce G-CSF and GM-CSF: identification and partial characterization of cell-associated isoforms.

Bone marrow stem cells reside in close proximity to endosteal osteoblasts. To explore the potential role of osteoblasts in hematopoietic differentiation, we measured the mRNA accumulation, protein production, and secretion of hematopoietic growth factors by the nonmineralizing MG-63 and the mineralizing SaOS-2 human osteosarcoma cell lines. mRNA for the osteoblast-specific protein osteocalcin was well as granulocyte colony-stimulating factor (G-CSF), transforming growth factor-beta 1 (TGF-beta 1), and tumor necrosis factor-alpha (TNF-alpha) was produced by the MG-63 and SaOS-2 cells, like primary human cells, in the presence and absence of L-ascorbate and beta-glycerol phosphate. In contrast, both cell lines expressed c-kit ligand mRNA only in the absence of L-ascorbate and beta-glycerol phosphate induction. Granulocyte-macrophage (GM)-CSF and interleukin-6 (IL-6) mRNA appeared to develop with increasing culture age. G-CSF protein was identified in several cell-associated forms including the 28- and 32-kD species, In addition, GM-CSF was found in cell-associated form. These results suggest that osteoblasts might play a central role in the hematopoietic microenvironment as basal producers of G-CSF and GM-CSF and suggest the possibility that osteoblasts may locally present these proteins in an membrane-associated fashion

Base Sequence↗

PubMed1994

Human osteoblasts support hematopoiesis through the production of granulocyte colony-stimulating factor.

Previous attempts at identifying the constitutive source(s) of granulocyte colony-stimulating factor (G-CSF) in human bone marrow have been unsuccessful despite the fact that normal bone marrow supports abundant myelopoiesis in vivo. We hypothesized that the intimate physical association between bone and hematopoietic cells facilitates interactions between osteoblasts and hematopoietic stem cells. Here we provide the first direct evidence that human osteoblasts participate in hematopoiesis by constitutively producing G-CSF and present the protein in a membrane-associated fashion to human hematopoietic progenitors. These results suggest a direct and central role for osteoblasts in normal myelopoiesis.

Base Sequence↗

PubMed1993

A strategy for constructing inducible expression plasmids for T-lymphocytes.

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Animals↗

PubMed1992

Effects of interleukin-1 beta and tumor necrosis factor-alpha on osteoblastic expression of osteocalcin and mineralized extracellular matrix in vitro.

Osteoblasts play a pivotal role during the bioresponse of bone to agents that stimulate bone resorption and/or inhibit bone formation including hormones, polypeptide growth factors, and cytokines. We examined the cytokines interleukin-1-beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) for their effects on osteoblastic proliferation and development and expression of alkaline phosphate and the osteoblast-specific protein osteocalcin in a mineralizing environment. Primary rat osteoblast-like cells (ROB) and osteoblastic cell lines derived from rat (ROS 17/2.8) and human (MG-63) osteosarcomas were studied. IL-1 beta and TNF-alpha were chosen because of their critical importance during the host response to local inflammatory stimuli. Qualitatively similar two- to threefold inhibition of osteocalcin synthesis by IL-1 beta and TNF-alpha were observed in all three postconfluent bone-forming model systems. Because of the readily measurable concentrations of osteocalcin produced in our culture protocol, it was not necessary to enhance osteoblastic synthesis of osteocalcin by supplementation with 1,25(OH)2-vitamin D3, a treatment which exerts pleiotropic effects on osteoblasts. Under the constraints of our protocol, where alkaline phosphatase and mineralization were already elevated at the 14-day onset of treatment, neither of these phenotypic properties was sensitive to a three-day cytokine exposure. Differences were noted in proliferation, where only TNF-alpha stimulated DNA synthesis in ROB cells, while both cytokines stimulated MG-63 cells. IL-1 beta and TNF-alpha failed to alter ROS 17/2.8 DNA synthesis except at the highest doses (25 pM IL-1 beta and 1 nM TNF-alpha) where inhibition was observed. These results further support the view that cytokine-mediated osteoblastic regulation can be relatively selective.

Alkaline Phosphatase↗

PubMed1992

Gene transfer investigations of p56-LCK function in IL-2-dependent T-cell lines: implications for mechanisms of IL-2-signal transduction.

Recently, the SRC-like non-receptor protein tyrosine kinase p56-LCK has been shown to physically associate with the interleukin-2 receptor (IL-2-R) complex and to undergo rapid elevations in its tyrosine kinase activity upon stimulation of T lymphocytes with IL-2. The functional significance of p56-LCK kinase activation for IL-2-mediated lymphocyte responses, however, has never been directly assessed. Using gene transfer approaches, we have achieved markedly elevated levels of p56-LCK kinase activity in the IL-2-dependent cytolytic T-cell line CTLL-2 and the helper line HT-2. CTLL-2 and HT-2 cells that were stably transfected with expression plasmids encoding either the normal human p56-LCK or a constitutively active version of the mouse p56-LCK kinase (LCK[Y505]) contained striking elevations in the levels of tyrosine phosphorylation on several proteins (34-36, 50-60, 62-68, 77-78, 104-110 kDa), as determined by immunoblot analysis using anti-phosphotyrosine antibodies. CTLL-2 and HT-2 LCK- and LCK(Y505F)-transfected cells remained dependent on IL-2 for their growth and survival in culture despite the findings that (i) IL-2 specifically stimulated elevations in the activity of the endogenous p56-LCK in untransfected CTLL-2 cells without affecting the activities of the other SRC-like kinases in these cells (p59-FYN, p62-YES) and that (ii) IL-2-mediated regulation of p56-LCK correlated with IL-2-driven proliferation of these T cells. Specifically, no elevation in the proliferation (DNA synthesis) or growth of these T cells was found at any of the concentrations of IL-2 examined (0.01-25 U/ml), relative to untransfected and control transfected cells. Furthermore, when cultured in the absence of IL-2, transfected T cells whose relative levels of p56-LCK activity were elevated by approximately 20-50-fold died with the same kinetics as control cells and underwent apoptosis, as defined by uptake of trypan blue dye and DNA fragmentation assays, respectively. Taken together, these data indicate that while IL-2 can up-regulate the enzymatic activity of p56-LCK, elevated levels of p56-LCK tyrosine kinase activity are insufficient to stimulate IL-2-mediated pathways required for T-cell growth and survival. These findings thus imply the existence of other signal-transducing molecules, besides p56-LCK, that physically participate in IL-2R complexes and that are necessary for initiation of the biochemical events ultimately responsible for IL-2's pleiotropic actions on lymphocytes.

Animals↗