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370 HUMAN OSTEOSARCOMA CELLS AS A CONTROL FOR GENE AND PROTEIN EXPRESSION
  1. P. Dube
  1. Miami, FL; S. Coleman UCLA Los Angeles

Abstract

Purpose Bone disease is a universal complication in Chronic Renal Failure patients and includes disorders ranging from high to low turnover bone lesions. Normal bone remodeling requires appropriate bone formation (osteoblastic cells) and bone resorption (osteoclastic cells). Receptor Activator of Nuclear Factor Kappa B Ligand (RANKL) and Osteoprotegerin (OPG) regulate osteoclastic and osteoblastic activity, respectively. This experiment evaluates HOS cells as an immortalized bone cell line to serve as a control for various experiments. HOS cells are grown in +/- Growth Hormone (GH) and/ or Bone Morphogenic Protein - 7 (BMP7) and tested for changes in expression of RANKL and OPG.

Methods The Human Osteosarcoma (HOS) cells (13 y.o. Caucasian female) are from American Tissue Type Culture. HOS cells are cultured using a modified DMEM media (10% Fetal Calf Serum, Pen/Strep, L-glutamine, 50 mg/ L ascorbic acid, pH 7.45). HOS cells were plated to 8- 6 well plates. Cells were fed using modified DMEM +/- growth factors which included BMP7 (100ng/ ml) and GH (200ng/ml). Cells are harvested using Trizol Method for RNA and protein extract. RNA was quantified by OD 260/280 and 5μg total RNA was converted to cDNA by reverse transcriptase (Invitrogen SuperScript II). Gene expression is evaluated by PCR. Gene copy numbers were obtained for housekeeper gene Glyceraldehyde 3-phosphate dehydrogenase (GAPD), Osteoprotegerin (OPG), and RANKL using Light Cycler Real Time PCR. PCR products are verified by a single correct melting point (Light Cycler Program) and size (#base pair) by Polyacrylamide Gel Electrophoresis.

Results Statistical analysis was by ANOVA using a Tukey Post- Hoc Test. P values ≤ 0.05 are reported as significant. For all treatments, there were no differences for OPG expression. This matches previous data for normal bone cells. RANKL expression increased for cells treated with BMP7/GH compared to normal media (p≤ 0.001) and increased for cells treated with BMP7 compared to BMP7/GH (p≤ 0.01). The ratio of RANKL/OPG increased for BMP7/GH compared to normal media (p≤ 0.001) and BMP7/GH compared to GH (p≤ 0.05).

Conclusion Prior experiments using 16 y.o. normal bone cells display a decrease in OPG, no change in RANKL, resulting in an increase in RANKL/ OPG when treated with BMP7 (not published). This clearly shows that HOS cells do not react in the same manner as normal bone cells, but for purposes of having an internal experiment to experiment control may still be of significant value.

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