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

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.

Figure 1

Endogenous c-myc mRNA and c-Myc protein expression in the B16-F0 cell line. (A) Detection of the 2.4 kb c-myc mRNA by northern blot analysis with a 1.8 kb cDNA probe. The lower autoradiogram is the same blot reprobed with a 2.1 kb β-actin probe to compare relative levels of expression. (B) Western blot detection of the c-Myc 67 kDa primary protein product with the c8 c-Myc monoclonal antibody.

Figure 2

Inhibition of in vitro translation of c-myc mRNA following treatment with AS and clamp ODNs in a cell-free rabbit reticulocyte lysate system. Lane 1, lysate only control, containing neither mRNA nor ODN; lane 2, c-myc mRNA alone translation control; lanes 3–6, treated with ODN Myc-E2 at 1, 2.5, 5 and 7.5 µM concentrations, respectively; lanes 7–10, treated with ODN Myc-E3 at 1, 2.5, 5 and 7.5 µM concentrations, respectively; lanes 11–14, treated with ODN Myc-E2C at 1, 2.5, 5 and 7.5 µM concentrations, respectively; lanes 15–18, treated with ODN Myc-E3C at 1, 2.5, 5 and 7.5 µM concentrations, respectively; lanes 19–22, treated with SCR**, the control clamp ODN, at 1, 2.5, 5 and 7.5 µM concentrations, respectively. c-Myc translation after Myc-E2 and Myc-E3 treatment (non-clamp AS ODNs) was not affected at any concentration (lanes 3–10). Compared to control translation of the 1.4 kb transcript, both Myc-E2C and Myc-E3C exerted concentration-dependent translation inhibition on c-Myc expression (lanes 11–18). After treatment with 5 µM of either clamp ODN (lanes 13 and 17) Myc translation was no longer detected, suggesting 100% inhibition. SCR** treatment produced no effect on the translation of Myc protein in this system (lanes 19–22).

Figure 3

Gel mobility shift analysis of clamp ODNs with c-myc mRNA. Gel mobility shift analysis demonstrates sequence-specific binding of Myc-E2C and Myc-E3C to the 1.4 kb c-myc mRNA. The first and third lanes are the labeled active clamp ODNs alone. No shift occurred between the control clamp ODN, SCR**, which migrates at the same mobility in the presence of the mRNA as it does alone. The last lane is HaeII-cleaved pBR322 marker DNA.

Figure 4

Thermal denaturation curves of clamp ODNs and c-myc RNA oligomers. Melting curve of Myc-E2C alone (A), 35 bp RNA oligomer E2R alone (B), Myc-E2C with E2R (C) and SCR** with E2R (D). Melting studies were done in 50 mM Tris–acetate buffer (10 mM MgCl₂ and 50 mM NaCl), pH 5.5, with an equimolar amount of each ODN (3 µM). The absorbance (OD) was measured at 260 nm.

Figure 5

Representative photographs of B16-F0 cells after ODN treatment and immunohistochemical analysis with the c8 c-Myc monoclonal antibody. Cells were treated in slide plates for 4 days, 6 h each day at 37°C, with Opti-MEM medium (a and b) or with 5 µM Myc-E2 (c), Myc-E3 (d), Myc-E2C (e), Myc-E3C (f) or SCR** (g). Endogenous c-Myc expression is detected as a brown stain against a blue hematoxylin counterstain. The cells in (a) were treated with medium alone and were not stained with primary Myc antibody, thus serving as a background control for antibody reactivity. The cells in (b) were also treated with medium only, but were stained with primary antibody to serve as the non-ODN-treated control. The cells in (c) and (d) were treated with Myc-E2 and Myc-E3, respectively, and both show immunoreactivity consistent with the untreated control cells (b). In contrast, the cells treated with Myc-E2C (e) and Myc-E3C (f) exhibit a drastic reduction in cellular Myc detection, as well as marked changes in cell morphology and cell death. The cells treated with the control clamp ODN, SCR** (g), also underwent some cell death, but maintained immunoreactivity for the Myc antibody comparable to the untreated control (b), which implies non-specific effects. The results of the immunohistochemical analysis further substantiate that Myc-E2C and Myc-E3C can specifically target the c-myc mRNA to reduce cellular c-Myc expression, an effect also confirmed in the in vitro translation assay.

Figure 6

ECL detection of western blot analyzed B16-F0 cellular lysates treated with clamp ODNs and UV irradiation (366 nm). After four 5 µM ODN treatments total cell lysates were harvested, resolved by 10% SDS–PAGE and underwent western analysis using the c8 c-Myc and β-actin monoclonal antibodies (Santa Cruz Biotechnology Inc.). The blot was exposed to ECL reagents (Amersham) for c-Myc and actin protein detection. The lanes are labeled with the treatment (i.e. lane 1, CTL, is the UV alone control for B16-F0 cellular c-Myc expression). Myc-E2C treatment resulted in >78% reduction in c-Myc protein expression, while Myc-E3C resulted in >65% reduction. The lanes treated with Myc-E2 and Myc-E3 resulted in 35 and 7% reductions in c-Myc expression, respectively. There was no change in Myc expression after treatment with SCR**.

Figure 7

B16-F0 murine melanoma growth inhibition by c-myc non-clamp and clamp AS ODNs, as measured by the MTT assay. Cells were treated with ODN (filled square) or a combination of ODN and UV irradiation (open square). Myc-E2C and Myc-E3C resulted in 64 and 60% reductions in cellular proliferation, respectively. All other ODNs resulted in no significant effects, as compared to control growth. Means ± SE, defined by bars, of triplicate wells for all data points, representative of four independent experiments, are given.