Related Experiment Videos
Psoralen-modified clamp-forming antisense oligonucleotides reduce cellular c-Myc protein expression and B16-F0
D A Stewart1, S D Thomas, C A Mayfield
1Departments of Internal Medicine and Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
Abstract:
The c-myc protooncogene plays an important role in the abnormal growth pattern of melanoma cells. In an attempt to inhibit c-Myc expression and the growth of an established murine melanoma cell line, we targeted homopurine sequences within the mouse myc mRNA with modified antisense oligonucleotides (AS ODNs). Psoralen was conjugated to the 5'-end of these clamp-forming oligonucleotides (clamp ODNs). Gel mobility shift analysis demonstrated a sequence-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 ODN (SCR**). This association was further confirmed by thermal denaturation studies. In vitro translation assays demonstrated that both Myc-E2C and Myc-E3C at 5 microM inhibited c-Myc expression >99% after UV activation at 366 nm. Immunostaining of B16-F0 cells with a c-Myc monoclonal antibody revealed a significant reduction in c-Myc after clamp ODN treatment compared with the untreated or SCR** control-treated cells. This result was corroborated by western blot analysis. Utilizing the MTT assay to determine the effects of ODN-mediated c-Myc reduction on B16-F0 growth, we observed 60 and 64% reductions in growth after treatment with 5 microM Myc-E3C and Myc-E2C, respectively. We attribute the enhanced effectiveness of the clamp ODNs to psoralen activation. Our preliminary data suggest that inhibiting c-Myc overexpression results in a significant reduction in abnormal proliferation of B16-F0 melanoma cells and that the increased efficiency of clamp ODNs may provide an important advantage for their use in antisense therapies.
Insights
Modified antisense oligonucleotides (AS ODNs) conjugated with psoralen effectively inhibit c-Myc expression in melanoma cells. This targeted approach significantly reduces melanoma cell proliferation, offering a promising strategy for antisense therapies.
Area of Science:
- Oncology
- Molecular Biology
- Biotechnology
Background:
- The c-myc protooncogene is crucial for the abnormal proliferation of melanoma cells.
- Targeting c-Myc expression is a potential therapeutic strategy for melanoma treatment.
Purpose of the Study:
- To inhibit c-Myc expression and reduce the growth of murine melanoma cells.
- To evaluate the efficacy of psoralen-conjugated antisense oligonucleotides (clamp ODNs) in targeting c-myc mRNA.
Main Methods:
- Development and application of modified antisense oligonucleotides (AS ODNs) conjugated with psoralen.
- Utilizing gel mobility shift analysis, thermal denaturation, in vitro translation assays, immunostaining, western blot, and MTT assay to assess efficacy.
- Testing specific clamp ODNs (Myc-E2C, Myc-E3C) against B16-F0 murine melanoma cells.
Main Results:
- Sequence-specific binding of active clamp ODNs (Myc-E2C, Myc-E3C) to c-myc mRNA was confirmed.
- UV activation of clamp ODNs resulted in >99% inhibition of c-Myc expression in vitro.
- Treatment with clamp ODNs led to significant reductions in c-Myc levels and melanoma cell growth (60-64% reduction).
Conclusions:
- Psoralen-activated clamp ODNs effectively inhibit c-Myc overexpression in melanoma cells.
- Targeting c-Myc with these modified ODNs significantly reduces melanoma cell proliferation.
- The enhanced efficiency of psoralen-conjugated clamp ODNs presents a promising advantage for antisense therapy development.