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 andbone 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 μg/ml significantly decreased proliferation of HL-60cells by approximately 40% ( P < 0.0001; n = 6) compared to controls. Clonogenic assays showed that the same antisense ODN inhibited colony formationby MJBL (40%) and Raji (52%) cells. Subsequent experiments to study the effect of a more nuclease-stable, phosphorothioate-modified antisense ODN 18-mer (GCAGCACAGCTCGGGGGT) revealed66% inhibition of HL-60 cell proliferation at 96 and 120 hours at 50 μg/ml, whereas sense ODN control had no effect. However, tenfold less of the modified antisense ODN (1 μg/mi) 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 μg/ml of modified antisense ODN, but not with the sense ODN control, by immunoblot analysis. Additionally, concentrations up to 10 μg/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 effecton 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.