FIBRONECTIN FRAGMENT-FACILITATED RETROVIRAL TRANSFER OF THE GLUTATHIONE-S-TRANSFERASE-PI GENE INTO CD34(-AGENTS() CELLS TO PROTECT THEM AGAINST ALKYLATING)

To protect bone marrow cells from the toxicity of chemotherapy, a mult idrug resistant gene or a dihydrofolate reductase gene has been introd uced into stem cells. These genes, however, are not capable of conferr ing refractoriness to alkylating agents (AA), which are some of the mo st commonly used agents in chemotherapy regimens. In the present study , an attempt was made to endow human stem cell (CD34(+) cells) with re sistance to cyclophosphamide, a well-known AA, and adriamycin (ADM) by transducing the glutathione-S-transferase pi (GST-pi) gene whose prod uct is thought to detoxify AA by conjugating them with glutathione and to remove a toxic peroxide formed by ADM. The gene transduction was c arried out retrovirally with a virus titer of 1 x 10(5) FFU/ml, employ ing a recombinant fibronectin fragment; transduction efficiency was ex tremely low without the fragment. Incubation with interleukin-6 and st em cell factor enhanced the expression of fibronectin ligands VLA4 and VLA5 on CD34(+) cells. This enhanced expression of VLA4 and VLA5 was considered to facilitate a close contact of the CD34(+) cell to the re troviral vector via fibronectin fragments and the subsequent transduct ion process. The GST-pi gene-transduced CD34(+) cells formed almost 3- and 2.5-fold more CFU-GM than neo gene-transduced CD34(+) cells in th e presence of 2.5 mu g/ml of 4-hydroperoxycyclophosphamide (4-HC), an active form of cyclophosphamide, and 30 ng/ml ADM, respectively. The t ransfectants formed an appreciable number of colonies, even at higher concentrations of these drugs (5.0 mu g/ml of 4-HC, 50 ng/ml of ADM) w hereas neo gene-transduced or nontransduced CD34(+) cells formed no co lonies at all, indicating the possibility of selecting out the transfe ctants by exposing them to these anticancer drugs. Thus, me were able to demonstrate that transduction of the GST-pi gene confers resistance to cyclophosphamide as well as to ADM, and therefore this approach ca n be applied clinically for high-dose chemotherapy.