Psoralen-modified clamp-forming antisense oligonucleotides reduce cellularc-Myc protein expression and B16-F0 proliferation

The c-myc protooncogene plays an important role in the abnormal growth patt ern of melanoma cells. In an attempt to inhibit c-Myc expression and the gr owth of an established murine melanoma cell line, we targeted homopurine se quences within the mouse myc mRNA with modified antisense oligonucleotides (AS ODNs). Psoralen was conjugated to the 5 ' -end of these clamp-forming o ligonucleotides (clamp ODNs). Gel mobility shift analysis demonstrated a se quence-specific interaction between the active clamp ODNs (Myc-E2C and Myc- E3C) and the 1.4 kb c-myc mRNA, but no interaction with the control clamp O DN (SCR**). This association was further confirmed by thermal denaturation studies. In vitro translation assays demonstrated that both Myc-E2C and Myc -E3C at 5 muM inhibited c-Myc expression > 99% after UV activation at 366 n m. Immunostaining of B16-F0 cells with a c-Myc monoclonal antibody revealed a significant reduction in c-Myc after clamp ODN treatment compared with t he untreated or SCR** control-treated cells. This result was corroborated b y western blot analysis. Utilizing the MTT assay to determine the effects o f ODN-mediated c-Myc reduction on B16-F0 growth, we observed 60 and 64% red uctions in growth after treatment with 5 muM Myc-E3C and Myc-E2C, respectiv ely. We attribute the enhanced effectiveness of the clamp ODNs to psoralen activation. Our preliminary data suggest that inhibiting c-Myc overexpressi on results in a significant reduction in abnormal proliferation of B16-F0 m elanoma cells and that the increased efficiency of clamp ODNs may provide a n important advantage for their use in antisense therapies.