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MDM2 antagonist Nutlin-3 suppresses the proliferation and differentiation of human pre-osteoclasts through a p53-dependent pathway
- Publication Year :
- 2007
- Publisher :
- Blackwell Science Incorporated:350 Main Street, Sixth Floor:Malden, MA 02148:(888)661-5800, (781)388-8250, EMAIL: csjournals@blacksci.com, INTERNET: http://www.blackwell-science.com, Fax: (781)388-8232, 2007.
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Abstract
- Exposure of human pre-osteoclasts to the MDM2 antagonist Nutlin-3 activated the p53 pathway and significantly decreased the entry of pre-osteoclasts in the S phase in response to RANKL. Moreover, repeated exposure to Nutlin-3 suppressed osteoclastic differentiation, without affecting cell survival at any culture time. Introduction: The p53 oncosuppressor coordinates an intracellular network involved in protection from malignant transformation and cell cycle control; its activation is tightly regulated by the murine double minute 2 (MDM2) gene and p53-MDM 2 interaction can be disrupted by selective small molecule inhibitors, the Nutlins. Although the ability of Nutlins to suppress the growth of wildtype p53 tumors has been clearly established, their biological activity in normal cells and tissues has not been extensively studied. Materials and Methods: Peripheral blood mononuclear cell pre-osteoclasts were cultured with macrophage-colony stimulating factor (M-CSF ) + RANKL or co-cultured with SaOS-2 osteosarcoma cells in the presence of IL-1β to induce osteoclastic differentiation. Cell cycle was analyzed by BrdU incorporation. The degree of osteoclastic differentiation was monitored at different culture times by TRACP and DAPI staining, as well as by TRACP-5b ELISA. Finally, the role of p53 in mediating the biological activity of Nutlin-3 was studied using specific siRNA. Results: Exposure of human pre-osteoclasts to RANKL induced an early (24 h) increase in the percentage of cells in the S phase, followed by the exit from the cell cycle at later time-points. The simultaneous addition of Nutlin-3 and RANKL dose-dependently decreased the percentage of pre-osteoclasts in the S phase and induced a rapid accumulation of p53 protein coupled with the induction of p53 target genes. Unexpectedly, the administration of Nutlin-3 to pre-osteoclasts at early culture times significantly suppressed the final output of osteoclasts at day 14 of culture. The role of p53 in mediating this biological activity of Nutlin-3 was underscored by gene knockdown experiments, in which the anti-osteoclastic activity of Nutlin-3 was significantly counteracted by siRNA specific for p53. Nutlin-3 also significantly decreased the formation of osteoclasts in a co-culture system of SaOS-2 osteosarcoma and pre-osteoclastic cells. Conclusions: These findings indicate that Nutlin-3 abrogates both pre-osteoclastic proliferation and differentiation through a p53-dependent pathway and may have therapeutic implications for those neoplastic diseases characterized by an abnormal osteoclastic activity.
- Subjects :
- musculoskeletal diseases
medicine.medical_specialty
p53-pathway
Time Factors
Transcription, Genetic
Endocrinology, Diabetes and Metabolism
Piperazines
chemistry.chemical_compound
Osteoclast
Internal medicine
Cell Adhesion
medicine
Humans
Cell Lineage
Orthopedics and Sports Medicine
DAPI
Cells, Cultured
Cell Proliferation
Osteosarcoma
biology
Cell growth
Macrophage Colony-Stimulating Factor
RANK Ligand
Imidazoles
RANKL
Nutlin-3
Cell Differentiation
Proto-Oncogene Proteins c-mdm2
Biological activity
Nutlin
Cell cycle
Coculture Techniques
osteoclasts
cell cycle
Up-Regulation
Cell biology
medicine.anatomical_structure
Endocrinology
chemistry
biology.protein
Tumor Suppressor Protein p53
Intracellular
Subjects
Details
- Language :
- English
- Database :
- OpenAIRE
- Accession number :
- edsair.doi.dedup.....22046372d470ae536778f4913ae6c2ad