Effects of phosphodiester and phosphorothioate antisense oligodeoxynucleotides on cell lines which overexpress c-myc: Implications for the treatment of Burkitt's lymphoma

Abstract

The product of the c-myc proto-oncogene is a highly conserved nuclear phosphoprotein whose expression is closely linked to cellular proliferation and differentiation. We have been interested in developing an antisense oligodeoxynucleotide (ODN) strategy to inhibit the proliferation of c-myc-dependent malignancies for use in future specific therapies and bone marrow purging regimens. Our experimental approach was to incubate either antisense or sense ODNs, spanning the 5' cap region of the c-myc gene, with c-myc overexpressing cell lines (HL-60) Raji, MJBL, CA-46) for up to seven days. Proliferation assay to test the inhibitory effect of an unmodified antisense ODN 15-mer (GCACAGCTCGGGGGT) showed that concentrations as low as 50 micrograms/ml significantly decreased proliferation of HL-60 cells by approximately 40% (P < 0.0001; n = 6) compared to controls. Clonogenic assays showed that the same antisense ODN inhibited colony formation by MJBL (40%0 and Raji (52%) cells. Subsequent experiments to study the effect of a more nuclease-stable, phosphorothioate-modified antisense ODN 18-mer (GCAGCACAGCTCGGGGGT) revealed 66% inhibition of HL-60 cell proliferation at 96 and 120 hours at 50 micrograms/ml, whereas sense ODN control had no effect. However, tenfold less of the modified antisense ODN (1 micrograms/ml) was required to inhibit proliferation of HL-60 cells by 50% compared to the unmodified antisense ODN. A decrease in the HL-60 native c-myc protein level was also observed with 100 micrograms/ml of modified antisense ODN, but not with the sense ODN control, by immunoblot analysis. Additionally, concentrations up to 10 micrograms/ml of either modified antisense or sense ODN did not decrease CFU-GM formation (145 +/- 35%, P = 0.27) in human bone marrow, suggesting that these levels of ODN would have a negligible effect on normal hematopoietic cells. These pilot data suggest that modified antisense ODN directed at the cap region of the c-myc gene could specifically inhibit c-myc expression at a single, lower dose than unmodified ODN and may play a future role in inhibiting the growth of c-myc-dependent malignant cells.