2001 CTOS Annual Meeting Posters— Medical Oncology

NON-VIRAL GENE TRANSFER INTO HUMAN OSTEOSARCOMA CELLS USING THE NON-LIPOSOMAL LIPID EFFECTENE - A PRIMER FOR MALIGNANT BONE TUMOR GENE THERAPY STRATEGIES.
Detlev Gottschalk¹,  B. Schmitz^2
1Department of Orthopedics - Connective Tissue Research, Universityclinic and Medical School, RWTH Aachen, and Interdisciplinary Centre for Clinical Research on Biomaterials (IZKF "Biomat.), RWTH Aachen,  ²Department of Clinical Chemistry and Pathobiochemistry, RWTH Aachen

METHODS: For transfection, a 4.7 kB plasmid (pEGFP-C1, Clontech) was applied containing a CMV promotor and the enhanced green fluorescent protein (EGFP) gene sequence, a red shifted variant of the wild type GFP. For transfection of human primary osteoblast like cells (hOB) and a osteogenic, osteosarcoma cell line (HOS58), using a modified standard protocol by quiagen, two different plasmid concentrations (0.4 ug, 1.0 ug) were used. Controlls were treated with Effectene reagent without prior DNA application. For microscopic analysis, cells were washed in HBSS and fixed in 4% paraformaldehyde for 20 min at RT, washed again in HBSS and analyzed by standard microscopy and confocal laser scan microscopy. For measuring transfection efficacy by FACScan analysis cells were washed in HBSS, trypsinized and subsequently analyzed on FACScalibur. PI (0.1 mg/ml) was added to each tube to exclude dead cells. The amount of EGFP-expression was determined in the life gate.

RESULTS: A linearity of transfection efficacy could be observed with increasing amounts of the used plasmid concentrations in hOB. Transfection with 0.4 ug plasmid DNA results in mean of about 6.5% of EGFP expressing cells. The EGFP expression varied from weak to very strong fluorescence intensities. The amount of PI-positive cells was determined as about 20%. A higher plasmid DNA concentration (1.0 ug) resulted in increased amount of EGFP expressing viable cells (transfection rate about 13%), but effected also in a negative way the total amount of EGFP expressing living cells (about 18%). The transfection rate of HOS58 was about 11% and 13% respectively but higher plasmid concentration had no cell-toxic effect as in the non-malignant transformed hOB system (living cells 79% and 82% respectively). An increase of plasmid concentration had no significant effect on the transfection rate in HOS58 but significantly decreased the amount of viable hOBs. Therefore, future gene therapy strategies for malignant bone tumors should be performed with caution, even using non-viral vectors.

CONCLUSION: Effectene used as new non-viral transfection agent , here proved to be succesful on human primary osteoblasts (hOB)and a human osteosarcoma cell line (HOS58)has a high reproducibility and a low cytotoxicity compared to other systems. Moreover, Effectene works in the presence of serum without restriction allowing longer incubation time. In addition, lower concentrations of DNA are needed. These items are of advantage in cancer treatment protocolls. Gene therapy for malignant bone disease is a challenge for the new decade of cancer treatment and could support neoadjuvant chemotherapy and / or could help to decrease intrinsic tumor cell resistance to chemotherapeutic agents.