- Email: [email protected]
Non-viral osteoprotegerin gene transfer inhibits the tumor progression and prolongs survival in a mouse experimental model of osteosarcoma
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ABSTRACTS / Bone 38 (2006) S23 – S32
osteoclastogenesis via its receptor RANK. OPG is able to form a tertiary complex and that OPG must be also considered as a direct effector of osteoclast functions. As OPG contains a heparin binding domain, the present study investigated the interactions between OPG and glycosaminoglycans (GAGs) by surface plasmon resonance and their involvement in the OPG functions. Kinetic data confirmed that OPG binds to heparin with a high affinity and that the pre-incubation of OPG with heparin inhibits in a dose-dependent manner the OPG binding to the complex RANK-RANKL. GAGs (heparan sulfate, dermatan sulfate, chondroitin sulfate) exert similar activity on OPG binding. The results demonstrated that sulfation is essential in the OPG-blocking function of GAGs since a totally desulfated heparin loose its capacity to bind to block OPG-binding to RANKL. Moreover a decasaccharide is the minimal structure that totally inhibits the OPG binding to the complex RANKRANKL. Western blot analysis performed in 293 cells surexpressing RANKL, revealed that the pre-incubation of OPG with these GAGs inhibits the OPG-induced decrease of membrane RANKL half-life. These data support an essential function of the related glycosaminoglycans heparin and heparan sulfate in the activity of the triad RANK-RANKL-OPG. doi:10.1016/j.bone.2006.01.111
52 Non-viral osteoprotegerin gene transfer inhibits the tumor progression and prolongs survival in a mouse experimental model of osteosarcoma F. Lamoureux a, P. Richard b, Y. Wittrant a, V. Trichet a, B. Pitard b, D. Heymann a, F. Re´dini a a EA 3822-INSERM ERI 7 Physiopathologie de la Re´sorption Osseuse b INSERM U533, Faculte´ de Me´decine, 44035 Nantes cedex 1, France As Osteoprotegerin (OPG) is a potent inhibitor of osteoclast differentiation and activation, the therapeutic effects of OPG gene transfer are examined in a murine osteolytic model of osteosarcoma (POS-1). In vitro, we can efficiently transfer and express either the GFP marker or the murine OPG 1-194 genes into murine musculoskeletal C2C12 cells but not osteosarcoma POS-1 cells, by using the cationic non-viral vector polyethylenimine (PEI). OPG administered as exogenous molecule or by transgene expression does not modify the cell proliferation nor the gene expression in both cell types. In vivo, a transplantable model of osteosarcoma in C3H/He mice was used, leading to the development of a primary osteolytic tumor and pulmonary metastases dissemination within three weeks. An alternative molecular structure was used named lutrol, as a carrier for DNA delivery. Three groups of mice were assigned respectively as controls (no injection), control vectors (lutrol/pcDNA3 alone) and OPG (lutrol/pcDNA3-OPG 1–194). The lutrol/DNA formulations were injected into the skeletal muscle once a week. The osteosarcoma incidence was significantly diminished in the OPG
group as compared to control groups (respectively 33 and 83%) and the mean tumor volume in this group was also decreased as compared to controls (respectively 616 and 3060 mm3 at day 23, P < 0.01). This was accompanied by a significant increase of animal survival in the OPG group (67% versus 16% at day 28, P < 0.005). The efficacy of OPG as anti-resorptive agent was confirmed by evaluating the prevention of tumor-associated bone degradation, as revealed by radiograph and micro-scanner analyses. doi:10.1016/j.bone.2006.01.112
53 The effects of tumor necrosis factor-A on osteoclast formation depend on the haematopoietic precursors Jeannette Portenier 1, Silvia Dolder 1, Antoinette Wetterwald, Willy Hofstetter Group for Bone Biology, Department Clinical Research, University of Berne, Murtenstrasse 35, CH-3010 Berne, Switzerland E-mail address: [email protected]. Previously we have demonstrated that tumor necrosis factora (TNFa) not only is a stimulator of osteoclast formation, but may also function as a potent inhibitor of osteoclastogenesis. Within the present study we investigated the effects of the cytokine on the differentiation of various populations of haematopoietic precursors to osteoclasts. The formation of osteoclasts was followed in vitro in cocultures of primary murine osteoblasts and (I) total bone marrow cells (BMC); (II) spleen cells; (III) CSF-1 dependent, non-adherent osteoclast precursors (OCP); and (IV) CSF-1 dependent bone marrow macrophages (BMMA). Furthermore, the four populations of haematopoietic precursors were grown alone in media supplemented with CSF-1 (3 and 30 ng/ml) and RANKL (0, 0 – 5, 5, and 50 ng/ml). To assess the effects of TNFa on the development of osteoclasts, the cultures were grown with various doses of the cytokine, ranging from 0.1 to 50 ng/ml. In co-cultures of osteoblasts and BMC or spleen cells, TNFa inhibited the development of osteoclasts dose-dependently, significant inhibition being detected at TNFa concentrations as low as 0.1 ng/ml. In the absence of 1,25(OH)2D3, no osteoclasts developed in these cultures. If TNFa was added to co-cultures of osteoblasts and OCP or BMMA, addition of TNFa in the presence of 1,25(OH)2D3 induced an increase in the number of osteoclasts at low and an decrease at high concentrations. In the absence of 1,25(OH)2D3 high doses of TNFa (5 ng/ ml and higher) caused a stimulation of osteoclastogenesis. In cultures of haematopoietic precursors supplemented with CSF-1 and RANKL, TNFa supported osteoclastogenesis at low concentrations of RANKL (0, 0.5 ng/ml). At RANKL concentrations of 5 and 50 ng/ml, TNFa exerted no significant effect on the development of osteoclasts. The data demonstrates that TNFa, in vitro, exerts complex effects on the development of osteoclasts. Depending on the nature of the haematopoietic precursors and on the composition of the haematopoietic microenvironment, TNFa stimulates or
ABSTRACTS / Bone 38 (2006) S23 – S32
osteoclastogenesis via its receptor RANK. OPG is able to form a tertiary complex and that OPG must be also considered as a direct effector of osteoclast functions. As OPG contains a heparin binding domain, the present study investigated the interactions between OPG and glycosaminoglycans (GAGs) by surface plasmon resonance and their involvement in the OPG functions. Kinetic data confirmed that OPG binds to heparin with a high affinity and that the pre-incubation of OPG with heparin inhibits in a dose-dependent manner the OPG binding to the complex RANK-RANKL. GAGs (heparan sulfate, dermatan sulfate, chondroitin sulfate) exert similar activity on OPG binding. The results demonstrated that sulfation is essential in the OPG-blocking function of GAGs since a totally desulfated heparin loose its capacity to bind to block OPG-binding to RANKL. Moreover a decasaccharide is the minimal structure that totally inhibits the OPG binding to the complex RANKRANKL. Western blot analysis performed in 293 cells surexpressing RANKL, revealed that the pre-incubation of OPG with these GAGs inhibits the OPG-induced decrease of membrane RANKL half-life. These data support an essential function of the related glycosaminoglycans heparin and heparan sulfate in the activity of the triad RANK-RANKL-OPG. doi:10.1016/j.bone.2006.01.111
52 Non-viral osteoprotegerin gene transfer inhibits the tumor progression and prolongs survival in a mouse experimental model of osteosarcoma F. Lamoureux a, P. Richard b, Y. Wittrant a, V. Trichet a, B. Pitard b, D. Heymann a, F. Re´dini a a EA 3822-INSERM ERI 7 Physiopathologie de la Re´sorption Osseuse b INSERM U533, Faculte´ de Me´decine, 44035 Nantes cedex 1, France As Osteoprotegerin (OPG) is a potent inhibitor of osteoclast differentiation and activation, the therapeutic effects of OPG gene transfer are examined in a murine osteolytic model of osteosarcoma (POS-1). In vitro, we can efficiently transfer and express either the GFP marker or the murine OPG 1-194 genes into murine musculoskeletal C2C12 cells but not osteosarcoma POS-1 cells, by using the cationic non-viral vector polyethylenimine (PEI). OPG administered as exogenous molecule or by transgene expression does not modify the cell proliferation nor the gene expression in both cell types. In vivo, a transplantable model of osteosarcoma in C3H/He mice was used, leading to the development of a primary osteolytic tumor and pulmonary metastases dissemination within three weeks. An alternative molecular structure was used named lutrol, as a carrier for DNA delivery. Three groups of mice were assigned respectively as controls (no injection), control vectors (lutrol/pcDNA3 alone) and OPG (lutrol/pcDNA3-OPG 1–194). The lutrol/DNA formulations were injected into the skeletal muscle once a week. The osteosarcoma incidence was significantly diminished in the OPG
group as compared to control groups (respectively 33 and 83%) and the mean tumor volume in this group was also decreased as compared to controls (respectively 616 and 3060 mm3 at day 23, P < 0.01). This was accompanied by a significant increase of animal survival in the OPG group (67% versus 16% at day 28, P < 0.005). The efficacy of OPG as anti-resorptive agent was confirmed by evaluating the prevention of tumor-associated bone degradation, as revealed by radiograph and micro-scanner analyses. doi:10.1016/j.bone.2006.01.112
53 The effects of tumor necrosis factor-A on osteoclast formation depend on the haematopoietic precursors Jeannette Portenier 1, Silvia Dolder 1, Antoinette Wetterwald, Willy Hofstetter Group for Bone Biology, Department Clinical Research, University of Berne, Murtenstrasse 35, CH-3010 Berne, Switzerland E-mail address: [email protected]. Previously we have demonstrated that tumor necrosis factora (TNFa) not only is a stimulator of osteoclast formation, but may also function as a potent inhibitor of osteoclastogenesis. Within the present study we investigated the effects of the cytokine on the differentiation of various populations of haematopoietic precursors to osteoclasts. The formation of osteoclasts was followed in vitro in cocultures of primary murine osteoblasts and (I) total bone marrow cells (BMC); (II) spleen cells; (III) CSF-1 dependent, non-adherent osteoclast precursors (OCP); and (IV) CSF-1 dependent bone marrow macrophages (BMMA). Furthermore, the four populations of haematopoietic precursors were grown alone in media supplemented with CSF-1 (3 and 30 ng/ml) and RANKL (0, 0 – 5, 5, and 50 ng/ml). To assess the effects of TNFa on the development of osteoclasts, the cultures were grown with various doses of the cytokine, ranging from 0.1 to 50 ng/ml. In co-cultures of osteoblasts and BMC or spleen cells, TNFa inhibited the development of osteoclasts dose-dependently, significant inhibition being detected at TNFa concentrations as low as 0.1 ng/ml. In the absence of 1,25(OH)2D3, no osteoclasts developed in these cultures. If TNFa was added to co-cultures of osteoblasts and OCP or BMMA, addition of TNFa in the presence of 1,25(OH)2D3 induced an increase in the number of osteoclasts at low and an decrease at high concentrations. In the absence of 1,25(OH)2D3 high doses of TNFa (5 ng/ ml and higher) caused a stimulation of osteoclastogenesis. In cultures of haematopoietic precursors supplemented with CSF-1 and RANKL, TNFa supported osteoclastogenesis at low concentrations of RANKL (0, 0.5 ng/ml). At RANKL concentrations of 5 and 50 ng/ml, TNFa exerted no significant effect on the development of osteoclasts. The data demonstrates that TNFa, in vitro, exerts complex effects on the development of osteoclasts. Depending on the nature of the haematopoietic precursors and on the composition of the haematopoietic microenvironment, TNFa stimulates or