PAMIDRONATE INDUCES CELL DEATH OF STROMAL CELLS OF GIANT CELL TUMORS
J. D. Doppelt1, S. S. Chang1, F. Y. Lee1*, H. Z. Zhang2, R. J. Winchester2
1Department of Orthopaedic Surgery, Columbia University, USA
2Center for Autoimmune and Molecular Diseases, Department of Pediatrics, Columbia Univeristy, USA
INTRODUCTION: Giant cell tumor of bone (GCT) is a primary neoplasm consisting of stromal cells and large, multinucleated giant cells that are phenotypically similar to osteoclasts. In this study we show that Pamidronate, a nitrogen-containing BP, induces apoptosis in cultured GCT stromal cells as indicated by morphological characteristics and flow cytometry measurements of annexin V binding.
METHODS: Specimens were freshly minced with scissors in Dulbecco's minimum essential medium (DMEM) producing a cell suspension with small fragments of tissue. After 3-4 passages, primary GCT cell cultures mainly consisting of proliferating stromal cells were briefly trypsinized and transferred to petri dishes. Zero, 50, 100, or 200 mM Pamidronate disodium was added to the culture medium when the cells reached 70-80% confluency. Five ml of annexin V-FITC (A.G. Scientific) was added to 195 ml of cell suspension and incubated in the dark for 10 minutes. The cells were pelleted and washed with PBS and resuspended again in 190 ml of binding buffer. Ten ml of 20 mg/ml propidium iodide (A.G. Scientific) was added to the cell suspension and flow cytometry was performed within 30 minutes using a FACS Calibur (Becton Dickson). Monoparametric cytograms of annexin V- FITC fluorescence (FL1) versus number of events were created using the CellQuest program gating for living cells and excluding dead cells on the basis of their propidium iodide uptake.
RESULTS: Pamidronate decreases cell proliferation and induces cell death in cultured GCT stromal cells. Exposure to all 3 concentrations of bisphosphonate produces morphological changes and a decrease in the number of adherent cells after only 24 hours of treatment compared to controls. Similarly cultured and treated normal fibroblasts are not affected by Pamidronate, providing evidence of specificity in the drug's action. Analysis of annexin V binding by flow cytometry shows that Pamidronate treatment increases the occurrence of apoptosis.
DISCUSSION: The tumor-induced osteolysis seen with GCT might be treated with Pamidronate based on its ability to both inhibit giant cell mediated bone resorption as well as induce apoptosis in the neoplastic stromal cells.
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