Shelley Hay

Molecular and cellular characterisation of highly purified stromal stem cells derived from human bone marrow

Previous studies have provided evidence for the existence of adult human bone marrow stromal stem cells (BMSSCs) or mesenchymal stem cells. Using a combination of cell separation techniques, we have isolated an almost homogeneous population of BMSSCs from adult human bone marrow. Lacking phenotypic characteristics of leukocytes and mature stromal elements, BMSSCs are non-cycling and constitutively express telomerase activity in vivo. This mesenchymal stem cell population demonstrates extensive proliferation and retains the capacity for differentiation into bone, cartilage and adipose tissue in vitro. In addition, clonal analysis demonstrated that individual BMSSC colonies exhibit a differential capacity to form new bone in vivo. These data are consistent with the existence of a second population of bone marrow stem cells in addition to those for the hematopoietic system. Our novel selection protocol provides a means to generate purified populations of BMSSCs for use in a range of different tissue engineering and gene therapy strategies.

The differences in toxicity and release of bone-resorbing mediators induced by titanium and cobalt-chromium-alloy wear particles.

We investigated the relationship between the toxic effects of metal wear particles and their ability to stimulate the release of inflammatory mediators implicated in bone resorption. In vitro studies were carried out with use of rat peritoneal macrophages, which were exposed to either cobalt-chromium-alloy or titanium-aluminum-vanadium particles, milled from the metal components of hip prostheses. The particles were in the size-range of, and at concentrations similar to, those found in the tissues surrounding failed prostheses in humans. The titanium-aluminum-vanadium particles showed little toxicity even at high concentrations, while the cobalt-chromium particles were very toxic. The titanium-aluminum-vanadium particles induced significantly more release of prostaglandin E2 than did the cobalt-chromium particles, and this was true for a wide range of concentrations. Exposure to titanium-aluminum-vanadium increased the release of prostaglandin E2, interleukin-1, tumor necrosis factor, and interleukin-6. In contrast, exposure to cobalt-chromium particles was associated with a decreased release of prostaglandin E2 and interleukin-6, and it had little effect on the release of interleukin-1 and tumor necrosis factor.

Differential Cell Surface Expression of the STRO-1 and Alkaline Phosphatase Antigens on Discrete Developmental Stages in Primary Cultures of Human Bone Cells

Human osteoblast‐like cells can be readily cultured from explants of trabecular bone, reproducibly expressing the characteristics of cells belonging to the osteoblastic lineage. Dual‐color fluorescence‐activated cell sorting was employed to develop a model of bone cell development in primary cultures of normal human bone cells (NHBCs) based on the cell surface expression of the stromal precursor cell marker STRO‐1 and the osteoblastic marker alkaline phosphatase (ALP). Cells expressing the STRO‐1 antigen exclusively (STRO‐1+/ALP−), were found to exhibit qualities preosteoblastic in nature both functionally by their reduced ability to form a mineralized bone matrix over time, as measured by calcium release assay, and in the lack of their expression of various bone‐related markers including bone sialoprotein, osteopontin, and parathyroid hormone receptor based on reverse trancriptase polymerase chain reaction (PCR) analysis. The majority of the NHBCs which expressed the STRO‐1−/ALP+ and STRO‐1−/ALP− phenotypes appeared to represent fully differentiated osteoblasts, while the STRO‐1+/ALP+ subset represented an intermediate preosteoblastic stage of development. All STRO‐1/ALP NHBC subsets were also found to express the DNA‐binding transcription factor CBFA‐1, confirming that these cultures represent committed osteogenic cells. In addition, our primer sets yielded four distinct alternative splice variants of the expected PCR product for CBFA‐1 in each of the STRO‐1/ALP subsets, with the exception of the proposed preosteoblastic STRO‐1+/ALP− subpopulation. Furthermore, upon re‐culture of the four different STRO‐1/ALP subsets only the STRO‐1+/ALP− subpopulation was able to give rise to all of the four subsets yielding the same proportions of STRO‐1/ALP expression as in the original primary cultures. The data presented in this study demonstrate a hierarchy of bone cell development in vitro and facilitate the study of bone cell differentiation and function.

Expression of Osteoclast Differentiation Signals by Stromal Elements of Giant Cell Tumors

The mechanisms by which primary tumors of the bone cause bone destruction have not been elucidated. Unlike most other lytic bone tumors, osteoclastomas, otherwise known as giant cell tumors (GCT), contain osteoclast‐like cells within the tumor stroma. A new member of the TNF‐ligand superfamily member, osteoclast differentiation factor (ODF/OPGL/RANKL/TRANCE), was recently identified. ODF was shown to directly stimulate osteoclastogenesis, in the presence of M‐CSF. In this study, the expression of ODF was examined in a number of tumor samples associated with bone lysis in vivo. In addition, we investigated expression of the ODF receptor on osteoclast precursors, RANK, as well as the ODF inhibitor osteoprotegerin (OPG), and another TNF‐ligand superfamily member, TRAIL, previously shown to abrogate the inhibitory effects of OPG. We report here the novel finding that GCT stromal cells contain abundant ODF mRNA, whereas the giant cell population exclusively expresses RANK mRNA. These results are consistent with the osteoclast‐mediated bone destruction by these tumors. We also report the expression of OPG and TRAIL mRNA in GCT samples. A comparison with other lytic and nonlytic tumors of bone showed that GCT express more ODF and TRAIL mRNA relative to OPG mRNA. In addition, GCT were found to express a number of cytokines previously reported to play central roles in osteoclastogenesis, namely, IL‐1, −6, −11, −17, as well as TNF‐α. Importantly, GCT were also found to express high levels of M‐CSF mRNA, a cytokine shown to be an essential cofactor of ODF, and a survival factor for mature and developing osteoclasts. Furthermore, expression of these molecules by stromal cells isolated from GCT continued in vitro. Thus GCT constitutively express all of the signals that are currently understood to be necessary for the differentiation of osteoclasts from precursor cells.

Chemotherapeutic agents sensitize osteogenic sarcoma cells, but not normal human bone cells, to apo2l/trail-induced apoptosis

Apo2L/TRAIL is a member of the tumor necrosis factor (TNF) family of cytokines that induces death of cancer cells but not normal cells. Its potent apoptotic activity is mediated through its cell surface death domain‐containing receptors, DR4 and DR5. Apo2L/TRAIL interacts also with 3 “decoy” receptors that do not induce apoptosis, DcR1, DcR2, which lack functional death domains, and osteoprotegerin (OPG). The aim of our study was to investigate the cytotoxic activity of Apo2L/TRAIL on established osteogenic sarcoma cell lines (BTK‐143, HOS, MG‐63, SJSA‐1, G‐292 and SAOS2) and in primary cultures of normal human bone (NHB) cells. When used alone, Apo2L/TRAIL at 100 ng/ml for 24 hr induced greater than 80% cell death in only 1 (BTK‐143) of the 6 osteogenic sarcoma cell lines. In contrast, Apo2L/TRAIL‐resistant cells were susceptible to Apo2L/TRAIL‐mediated apoptosis in the presence of the anticancer drugs, Doxorubicin (DOX), Cisplatin (CDDP) and Etoposide (ETP) but not Methotrexate (MTX) or Cyclophosphamide (CPM). Importantly, neither Apo2L/TRAIL alone nor in combination with any of these drugs affected primary normal human bone cells under equivalent conditions. Apo2L/TRAIL‐induced apoptosis, and its augmentation by chemotherapy in the resistant cell lines was mediated through caspase‐8 and caspase‐3 activation. Furthermore, Apo2L/TRAIL‐induced apoptosis and its augmentation by chemotherapy was effectively inhibited by caspase‐8 zIETD‐fmk and caspase‐3 zDEVD‐fmk protease inhibitors and by the pan‐caspase inhibitor zVAD‐fmk. The pattern of basal Apo2L/TRAIL receptor mRNA expression, or expression of the intracellular caspase inhibitor FLICE‐inhibitory protein, FLIP, could not be readily correlated with resistance or sensitivity to Apo2L/TRAIL‐induced apoptosis. However, the augmentation of Apo2L/TRAIL effects by chemotherapy was associated with drug‐induced up‐regulation of death receptors DR4 and DR5 mRNA and protein. No obvious correlation was seen between the expression of OPG mRNA or protein and susceptibility of cells to Apo2L/TRAIL‐induced apoptosis. Stable over‐expression of a dominant negative form of the Fas‐associated death domain protein (FADD) in the Apo2L/TRAIL‐sensitive BTK‐143 cells completely inhibited Apo2L/TRAIL‐induced cell death. Our results indicate that chemotherapy and Apo2L/TRAIL act synergistically to kill cancer cells but not normal bone‐derived osteoblast‐like cells, which has implications for future therapy of osteosarcoma.

Osteoprotegerin inhibits osteoclast formation and bone resorbing activity in giant cell tumors of bone

Osteolysis is a common complication of tumors that arise in, or metastasize to, bone. The recent discovery of key regulators of osteoclast formation and activity, including receptor activator of nuclear factor of kappaB ligand (RANKL), RANK, and osteoprotegerin (OPG), may facilitate new treatment regimes for certain tumors associated with excessive bone loss. We recently showed that the stromal cells of osteolytic giant cell tumors (GCT) of bone express high levels of mRNA encoding RANKL, relative to mRNA for the RANKL antagonist, OPG, compared with the expression patterns of other lytic and nonlytic bone tumors. In this study, we found that expression of RANKL and OPG mRNA continued by the stromal element of these tumors in a constitutive manner for at least 9 days in the absence of giant cells. Immunostaining of unfractionated GCT cultured in vitro revealed punctate cytoplasmic/membranous staining for RANKL and both cytoplasmic and extracellular matrix staining for OPG in stromal cells. Giant cells (osteoclasts) were negative for RANKL staining, but stained brightly for cytoplasmic OPG protein. We also investigated the functional relevance of these molecules for GCT osteolysis by adding recombinant OPG and RANKL to cultured GCT cells. We found that OPG treatment potently and dose-dependently inhibited resorption of bone slices by GCT, and could also inhibit the formation of multinucleated osteoclasts from precursors within the GCT. These effects of OPG were reversed by stoichiometric concentrations of exogenous RANKL. These data indicate that both the processes of osteoclast formation and activation in GCT are promoted by RANKL. Therefore, GCT represent a paradigm for the direct stimulation of osteoclast formation and activity by tumor stromal cells, in contrast to the mechanisms described for osteolytic breast tumors and multiple myeloma. The demonstration of these relationships is important in developing approaches to limit tumor-induced osteolysis.

REGULATION OF BONE CELLS BY PARTICLE-ACTIVATED MONONUCLEAR PHAGOCYTES

Bone loss around replacement prostheses may be related to the activation of mononuclear phagocytes (MNP) by prosthetic wear particles. We investigated how osteoblast-like cells were regulated by human MNP stimulated by particles of prosthetic material. Particles of titanium-6-aluminium-4-vanadium (TiAIV) stimulated MNP to release interleukin (IL)-1beta, tumour necrosis factor (TNF)alpha, IL-6 and prostaglandin E2 (PGE2). All these mediators are implicated in regulating bone metabolism. Particle-activated MNP inhibited bone cell proliferation and stimulated release of IL-6 and PGE2. The number of cells expressing alkaline phosphatase, a marker associated with mature osteoblastic cells, was reduced. Experiments with blocking antibodies showed that TNFalpha was responsible for the reduction in proliferation and the numbers of cells expressing alkaline phosphatase. By contrast, IL-1beta stimulated cell proliferation and differentiation. Both IL-1beta and TNFalpha stimulated IL-6 and PGE2 release from the osteoblast-like cells. Our results suggest that, particle-activated mononuclear phagocytes can induce a change in the balance between bone formation and resorption by a number of mechanisms.

Induction of cell death of human osteogenic sarcoma cells by zoledronic acid resembles anoikis

The aim of this study was to investigate the cytotoxic activity of the third-generation nitrogen-containing bisphosphonate zoledronic acid (ZOL) as a single agent, and in combination with clinically relevant anticancer drugs, in a panel of human osteogenic sarcoma cell lines (HOS, BTK-143, MG-63, SJSA-1, G-292, and SAOS2). We found that ZOL, when used alone, reduced cell number in a dose- and time-dependent manner, due either to cell cycle arrest in S-phase or to the induction of apoptosis. In the sensitive HOS, BTK-143, and G-292 cell lines, genomic DNA fragmentation and morphological changes characteristic of apoptosis were evident, and cells became nonadherent. Induction of apoptosis in osteosarcoma cells by ZOL was associated with caspase activation. However, coaddition of the broad-spectrum caspase inhibitors, z-VAD-fmk, Boc-D-fmk, or the caspase-3-specific inhibitor z-DEVD fmk, failed to protect these cells from ZOL-induced apoptosis. Our data support a ZOL-specific induction of cell apoptosis that involves cell detachment (anoikis), and in which caspase activation occurs secondarily to, and is redundant as a mediator of cell death. The addition of geranylgeraniol, an intermediate of the mevalonate pathway, suppressed the ZOL-induced apoptosis, suggesting that the cytotoxic effects of ZOL in osteosarcoma cells were mediated by the mevalonate pathway. While treatment of osteosarcoma cells with the chemotherapeutic agents doxorubicin or etoposide decreased cell viability, combination of these agents with ZOL did not significantly augment apoptosis in any of the cell lines tested. These observations suggest that ZOL has direct effects on the proliferation and survival of osteosarcoma cells in vitro, which has implications for future therapy of osteosarcoma.

Progressive resistance of BTK-143 osteosarcoma cells to Apo2L/TRAIL-induced apoptosis is mediated by acquisition of DcR2/TRAIL-R4 expression: resensitisation with chemotherapy

Apo2 ligand (Apo2L, also known as TRAIL) is a member of the tumour necrosis factor (TNF) family of cytokines that selectively induces the death of cancer cells, but not of normal cells. We observed that recombinant Apo2L/TRAIL was proapoptotic in early-passage BTK-143 osteogenic sarcoma cells, inducing 80% cell death during a 24 h treatment period. Apo2L/TRAIL-induced apoptosis was blocked by caspase inhibition. With increasing passage in culture, BTK-143 cells became progressively resistant to the apoptotic effects of Apo2L/TRAIL. RNA and flow cytometric analysis demonstrated that resistance to Apo2L/TRAIL was paralleled by progressive acquisition of the decoy receptor, DcR2. Blocking of DcR2 function with a specific anti-DcR2 antibody restored sensitivity to Apo2L/TRAIL in a dose-dependent manner. Importantly, treatment of resistant cells with the chemotherapeutic agents doxorubicin, cisplatin and etoposide reversed the resistance to Apo2L/TRAIL, which was associated with drug-induced upregulation of mRNA encoding the death receptors DR4 and DR5. BTK-143 cells thus represent a useful model system to investigate both the mechanisms of acquisition of resistance of tumour cells to Apo2L/TRAIL and the use of conventional drugs and novel agents to overcome resistance to Apo2L/TRAIL.

Human osteoblasts are resistant to Apo2L/TRAIL-mediated apoptosis

Apo2 ligand (Apo2L/TRAIL) is a member of the tumor necrosis factor (TNF) cytokine family. Apo2L/TRAIL can selectively induce programmed cell death in transformed cells, although its wide tissue distribution suggests potential physiological roles. We have investigated the expression, in human osteoblast-like cells (NHBC), of Apo2L/TRAIL and the known Apo2L/TRAIL death receptors, DR4 and DR5, and the Apo2L/TRAIL decoy receptors, DcR-1, DcR-2, and osteoprotegerin (OPG). NHBC expressed abundant mRNA corresponding to each of these molecular species. Immunofluorescence staining demonstrated that Apo2L/TRAIL protein was abundant within the cytoplasm of NHBC and OPG was strongly expressed at the cell surface. DR5 and DcR-2 were present in the cell membrane and cytoplasm and DcR-1 was confined to the nucleus. DR4 staining was weak. Neither Apo2L/TRAIL alone, nor in combination with chemotherapeutic agents of clinical relevance to treatment of osteogenic sarcoma, induced cell death in NHBC, as assessed morphologically and by activation of caspase-3. In contrast, the human osteogenic sarcoma cell lines, BTK-143 and G-292, were sensitive to exogenous Apo2L/TRAIL alone, and to the combined effect of Apo2L/TRAIL/cisplatin and Apo2L/TRAIL/doxorubicin treatments, respectively. In NHBC, we observed strong associations between the levels of mRNA corresponding to the pro-apoptotic molecules, Apo2L/TRAIL, DR4, and DR5, and those corresponding to pro-survival molecules, DcR-1, DcR-2, OPG, and FLIP, suggesting that the balance between pro-survival and pro-apoptotic molecules is a mechanism by which NHBC can resist Apo2L/TRAIL-mediated apoptosis. In contrast, osteogenic sarcoma cells had low or absent levels of DcR-1 and DcR-2. These results provide a foundation to explore the role of Apo2L/TRAIL in osteoblast physiology. In addition, they predict that therapeutic use of recombinant Apo2L/TRAIL, in combination with chemotherapeutic agents to treat skeletal malignancies, would have limited toxic effects on normal osteoblastic cells.