Related Experiment Videos
Quantifying c-myc expression in c-myc antisense phosphorothioate oligodeoxynucleotide-treated leukemic and colon
B D Li1, R M Budnick, C A Russo
1Department of Surgical Oncology, Roswell Park Cancer Institute, Buffalo, New York 14263, USA.
Abstract:
Antisense oligodeoxynucleotides (oligo) have been used to inhibit oncogene expression and have potential therapeutic applications. Using a 15-mer antisense phosphorothioate oligo (S-oligo), inhibition of c-myc oncogene expression and cellular proliferation is studied in two cell lines with c-myc overexpression: a colon cancer cell line (Colo 320 DM) and a promyelocytic leukemic cell line(HL-60). Quantitative analysis of c-myc mRNA transcript is performed by competitive reverse transcription-polymerase chain reaction (RT-PCR). This utilizes an RNA competitive reference standard (CRS RNA) template that is identical to the native c-myc mRNA except for a short segment deletion to allow for differentiation of the two by gel electrophoresis. A fixed quantity of test mRNA and a series of known concentrations of CRS RNA template placed in the same test tubes under identical conditions are reverse transcribed and amplified by PCR. Since the reaction is competitive, the ratio of the PCR products reflects the ratio of the initial concentrations of the two templates. After gel electrophoresis, the two PCR products are quantified densitometrically. Treating Colo 320 DM and HL-60 cells with c-myc antisense oligo and S-oligo results in a 20- to 100-fold decrease in c-myc mRNA transcripts, respectively. This inhibition is dose dependent and sequence specific (c-myc sense and missense oligo have no effects). The quantitative decrease in c-myc mRNA is associated with corresponding inhibition of c-myc oncoprotein synthesis as demonstrated by flow cytometry and Western blots. Furthermore, there is inhibition of cellular proliferation of the respective cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Antisense oligodeoxynucleotides (oligo) effectively inhibit c-myc oncogene expression and cellular proliferation in cancer cell lines. This research demonstrates significant therapeutic potential for these targeted genetic therapies.
Area of Science:
- Molecular Biology
- Cancer Research
- Oligonucleotide Therapeutics
Background:
- Antisense oligodeoxynucleotides (oligos) show promise for inhibiting oncogene expression.
- Targeting c-myc oncogene is a strategy for cancer therapy.
- Phosphorothioate oligos (S-oligos) are utilized for enhanced stability and efficacy.
Purpose of the Study:
- To investigate the inhibition of c-myc oncogene expression and cellular proliferation using antisense oligos.
- To evaluate the efficacy of a 15-mer antisense phosphorothioate oligo (S-oligo) in colon cancer (Colo 320 DM) and leukemia (HL-60) cell lines.
- To establish a quantitative method for assessing c-myc mRNA transcript levels.
Main Methods:
- Utilized a 15-mer antisense phosphorothioate oligo (S-oligo) targeting c-myc.
- Employed competitive reverse transcription-polymerase chain reaction (RT-PCR) with a c-myc RNA competitive reference standard (CRS RNA) for quantitative analysis.
- Assessed c-myc oncoprotein synthesis via flow cytometry and Western blots.
- Monitored cellular proliferation rates.
Main Results:
- A 20- to 100-fold decrease in c-myc mRNA transcripts was observed in Colo 320 DM and HL-60 cells, respectively, following treatment with c-myc antisense oligo and S-oligo.
- Inhibition was dose-dependent and sequence-specific, with sense and missense oligos showing no effect.
- Reduced c-myc mRNA levels correlated with decreased oncoprotein synthesis and inhibited cellular proliferation.
Conclusions:
- Antisense phosphorothioate oligos are effective in inhibiting c-myc oncogene expression and cellular proliferation in relevant cancer cell lines.
- The findings support the therapeutic potential of antisense oligo technology for targeting c-myc-driven cancers.
- Quantitative RT-PCR provides a reliable method for measuring the efficacy of antisense oligonucleotide-based therapies.