CATIONIC LIPIDS REDUCE TIME AND DOSE OF C-MYC ANTISENSE OLIGODEOXYNUCLEOTIDES REQUIRED TO SPECIFICALLY INHIBIT BURKITTS-LYMPHOMA CELL-GROWTH

Burkitt's lymphoma is characterized by a translocation of the c-myc ge ne with one of the immunoglobulin loci which activates overexpression of the c-myc oncogene. Antisense-oligodeoxynucleotides (AS-ODNs) offer the potential to block specific c-myc gene expression within lymphoma cells, but often exhibit a low efficiency of AS-ODN uptake. In this s tudy, a polycationic lipid reagent, Lipofectamine (LFM), was utilized as a vehicle to increase efficiency of delivery, decrease the time nee ded to observe an inhibitory effect, and decrease the AS-ODN dose. The objective was to develop a more efficient and rapid in vitro AS-ODN s trategy to inhibit proliferation of c-myc-dependent lymphoma cells and to test the specificity of Burkitt's lymphoma cell line-directed AS-O DNs for potential use as molecular purging agents in bone marrow trans plantation. Proliferation assays were performed to determine the inhib itory effect of the AS-ODNs on two Burkitt's lymphoma cell lines with different chromosomal translocations, Daudi and ST486, in medium conta ining 8.5 mu M LFM. AS-ODNs at a concentration of 0.36 mu M induced a significant decrease in proliferation for both cell lines using the sp ecific AS-ODN for each respective translocation. Within 5 h, Daudi res ponded to its specific AS-ODN/lipid complexes with a 35% decrease in p roliferation, compared to cells which received no treatment or Daudi-s pecific AS-ODN without LFM (P = 0.0001). Daudi showed an insignificant decrease in proliferation when treated with an AS-ODN specific for th e ST486 translocation (4%, P = 0.26). ST486 proliferation was decrease d by 52% when treated with the specific antisense for ST486 compared t o no treatment or ST486-specific AS-ODN without LFM (P < 0.003). Treat ment with the AS-ODN specific for Daudi showed an insignificant 4% dec rease (P = 0.42). Controls, including sense ODN for structure, reverse AS-ODN for structure and base composition, and AS-ODN without LFM, di d not produce a significant change in cells treated with LFM alone or cells receiving no treatment. Clonogenic assays of both Daudi and ST48 6 treated with their specific AS-ODNs revealed a 50% inhibition of col ony formation after the 5 h incubation as compared to no treatment. Co nfocal laser scanning microscopy verified that cellular uptake of AS-O DN was enhanced by cationic lipids. Immunoblot analysis showed a 63 +/ - 5% and a 50 +/- 3% reduction in intracellular c-myc levels for Daudi and ST486, respectively, when their respective AS-ODNs were administe red. Normal bone marrow progenitors were unaffected by the ODN/LFM com plexes. These results suggest that the specific c-myc AS-ODN/LFM compl exes inhibit c-myc-dependent tumor proliferation at an earlier time an d at a lower dose compared to no lipid facilitation. This approach may form the basis for utilizing specific AS-ODN/LFM therapy either alone or in a cocktail of other agents as an ex vivo molecular purging appr oach to autologous stem cell transplantation in Burkitt's lymphoma.