Lentivirus-mediated osteosarcoma-selective chemogene therapy employing the human folylpolyglutamate synthetase/methotrexate system and the human osteocalcin promoter

Abstract

The antifolate methotrexate (MTX) is an important chemotherapeutic agent for treatment of osteosarcomas. This drug usually converts intracellulary into polyglutamate derivates by the enzyme folylpolyglutamate synthetase (FPGS). MTX polyglutamates show an enhanced and prolonged cytotoxicity in comparison to the monoglutamate. We, therefore, investigated in the present study if transfer of the human fpgs gene to osteosarcoma cells may augment their MTX sensitivity. For this purpose, we employed the human osteocalcin (OC) promoter which had shown marked osteosarcoma specificity in promoter studies using different luciferase assays in osteosarcoma and non-osteosarcoma cell lines. A recombinant lentiviral vector was generated with the OC promoter driving the expression of fpgs and the gene for enhanced green fluorescent protein (egfp) which was linked to fpgs by an internal ribosomal entry site (IRES). Since the vector backbone contained only a self-inactivating viral LTR promoter, any interference of the OC promoter by unspecific promoter elements was excluded. We tested the expression of fpgs and egfp after lentiviral transduction in various osteosarcoma (murine ROS, human MG-63, human TM791) and non osteoblastic cell lines (293T embryonic human kidney cells, HeLa human cervix carcinoma cells) by assessing egfp expression and MTX sensitivity in comparison to non-transduced controls. Whereas the OC promoter failed to enhance MTX sensitivity via fpgs expression in non osteoblastic cell lines, the OC promoter mediated a markedly increased MTX cytotoxicity in all osteosarcoma cell lines after lentiviral transduction. In future animal studies, we will investigate if our lentiviral-mediated chemogene therapy approach also confers osteosarcoma-selective cytotoxicity in vivo.