The use of
surrogate end-point biomarkers could help in the development of chemopreventive agents. To define potential
surrogate end-point biomarkers, the ability of
budesonide to decrease
mRNA expression of the
insulin-like growth factor-2 (
Igf-II) and c-myc genes and to cause the remethylation of the genes was investigated in lung
tumors. Lung
tumors were induced in female strain A mice by administering i.p. 16 mg/kg
vinyl carbamate for 2 consecutive wk or by a single dose of 100 mg/kg
benzo[a]pyrene (B[a]P). Thirty-four weeks later, the mice given
vinyl carbamate received
budesonide (0.6 or 2.4 mg/kg diet) for 7 d and then were killed. Mice were killed 24 wk after administration of B[a]P. The
mRNA expression of the
Igf-II and c-myc genes was increased in lung
tumors relative to normal lung tissue.
Budesonide decreased
mRNA expression of both genes in
tumors. The methylation status of 27 CpG sites in the differentially methylated region 2 in the
Igf-II gene was determined with the
bisulfite-treated
DNA-sequencing procedure. The numbers of methylated CpG sites were 17-21 in normal lung (70.4 +/- 2.6%); 0-2, and 1-2 in lung
tumors induced by
vinyl carbamate and B[a]P (4.9 +/- 1.2% and 4.6 +/- 1.2%, respectively); and 4-5 or 7-16 in
tumors after treatment with 0.6 or 2.4 mg/kg
budesonide (16.0 +/- 1.2% and 46.2 +/- 5.1%, respectively). Thus, lung
tumors had strikingly less methylated CpG sites than normal lung tissue, while even limited treatment with
budesonide resulted in remethylation of the CpG sites in
tumors. With HpaII digestion followed by Southern blot analysis, the internal
cytosine of CCGG sites in the c-myc gene was found to be methylated in normal lung tissue, whereas some of the sites were unmethylated in lung
tumors. Treatment for 7 d with
budesonide resulted in the remethylation of these sites. In conclusion, mouse lung
tumors showed decreased methylation of the
Igf-II and c-myc genes that was associated with increased expression of these genes.
Budesonide treatment caused remethylation and decreased expression of both genes. The results support the possibility of using decreased
mRNA expression and remethylation of the
Igf-II and c-myc genes as
biomarkers for the efficacy of
budesonide.