Quantifying c-myc expression in c-myc antisense phosphorothioate oligodeoxynucleotide-treated leukemic and colon cancer cell lines.

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)