QUANTIFYING C-MYC EXPRESSION IN C-MYC ANTISENSE PHOSPHOROTHIOATE OLIGODEOXYNUCLEOTIDE-TREATED LEUKEMIC AND COLON-CANCER CELL-LINES

Antisense oligodeoxynucleotides (oligo) have been used to inhibit onco gene expression and have potential therapeutic applications. Using a 1 5-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 (Cole 320 DM ) and a promyelocytic leukemic cell line (HL-60). Quantitative analysi s of c-myc mRNA transcript is performed by competitive reverse transcr iption-polymerase chain reaction (RT-PCR). This utilizes an RNA compet itive reference standard (CRS RNA) template that is identical to the n ative c-myc mRNA except for a short segment deletion to allow for diff erentiation of the two by gel electrophoresis. A fixed quantity of tes t mRNA and a series of known concentrations of CRS RNA template placed in the same test tubes under identical conditions are reverse transcr ibed and amplified by PCR. Since the reaction is competitive, the rati o 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 Cole 320 DM and HL-60 cell s 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 d ose dependent and sequence specific (c-myc sense and missense oligo ha ve no effects). The quantitative decrease in c-myc mRNA is associated with corresponding inhibition of c-myc oncoprotein synthesis as demons trated by how cytometry and Western blots. Furthermore, there is inhib ition of cellular proliferation of the respective cell lines. Thus tre ating two cell lines with known c-myc overexpression with c-myc antise nse S-oligo results in: (1) a decrease in c-myc mRNA transcripts, (2) a decrease in c-myc oncoprotein synthesis, and (3) an inhibition of ce llular proliferation. (C) 1995 Academic Press, Inc.