Cationic lipids reduce time and dose of c-myc antisense oligodeoxynucleotides required to specifically inhibit Burkitt's lymphoma cell growth.
Burkitt's lymphoma is characterized by a translocation of the c-myc gene 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 study, a polycationic lipid reagent, Lipofectamine (LFM), was utilized as a vehicle to increase efficiency of delivery, decrease the time needed 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 strategy to inhibit proliferation of c-myc-dependent lymphoma cells and to test the specificity of Burkitt's lymphoma cell line-directed AS-ODNs for potential use as molecular purging agents in bone marrow transplantation. Proliferation assays were performed to determine the inhibitory effect of the AS-ODNs on two Burkitt's lymphoma cell lines with different chromosomal translocations, Daudi and ST486, in medium containing 8.5 microM LFM. AS-ODNs at a concentration of 0.36 microM induced a significant decrease in proliferation for both cell lines using the specific AS-ODN for each respective translocation. Within 5 h, Daudi responded to its specific AS-ODN/lipid complexes with a 35% decrease in proliferation, compared to cells which received no treatment or Daudi-specific AS-ODN without LFM (P = 0.0001). Daudi showed an insignificant decrease in proliferation when treated with an AS-ODN specific for the ST486 translocation (4%, P = 0.26). ST486 proliferation was decreased by 52% when treated with the specific antisense for ST486 compared to no treatment or ST486-specific AS-ODN without LFM (P < 0.003). Treatment with the AS-ODN specific for Daudi showed an insignificant 4% decrease (P = 0.42). Controls, including sense ODN for structure, reverse AS-ODN for structure and base composition, and AS-ODN without LFM, did not produce a significant change in cells treated with LFM alone or cells receiving no treatment. Clonogenic assays of both Daudi and ST486 treated with their specific AS-ODNs revealed a 50% inhibition of colony formation after the 5 h incubation as compared to no treatment. Confocal laser scanning microscopy verified that cellular uptake of AS-ODN 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 administered. Normal bone marrow progenitors were unaffected by the ODN/LFM complexes. These results suggest that the specific c-myc AS-ODN/LFM complexes inhibit c-myc-dependent tumor proliferation at an earlier time and 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 approach to autologous stem cell transplantation in Burkitt's lymphoma.
Williams
S A
SA
Division of Hematology/Oncology and Blood and Bone Marrow Transplantation, Children's Hospital of Orange County, CA 92668, USA.
Chang
L
L
Buzby
J S
JS
Suen
Y
Y
Cairo
M S
MS
D000328:Adult; D000818:Animals; D002051:Burkitt Lymphoma / Q000188:drug therapy* / Q000235:genetics* / Q000473:pathology; D002411:Cation Exchange Resins / Q000008:administration & dosage / Q000494:pharmacology*; D002412:Cations; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D004334:Drug Administration Schedule; D004357:Drug Synergism; D016259:Genes, myc; D006801:Humans; D007700:Kinetics; D008055:Lipids / Q000008:administration & dosage / Q000494:pharmacology*; D016376:Oligonucleotides, Antisense / Q000008:administration & dosage / Q000493:pharmacokinetics / Q000494:pharmacology*; D012680:Sensitivity and Specificity; D014178:Translocation, Genetic
D002411:Cation Exchange Resins; D002412:Cations; D008055:Lipids; C086724:Lipofectamine; D016376:Oligonucleotides, Antisense