To evaluate the significance of alterations in cell adhesion-related genes methylation during lung multistep
carcinogenesis induced by the genotoxic
carcinogens 3-methylcholanthrene (MCA) and
diethylnitrosamine (DEN), tissue samples microdissected from MCA/DEN-induced rat lung
carcinogenesis model were subjected to methylation-specific PCR to evaluate the DNA methylation status of CADM1, TIMP3,
E-cadherin and
N-cadherin. Immunohistochemistry was used to determine
protein expression of CADM1, TIMP3,
N-cadherin and the
DNA methyltransferases (DNMTs) 1, 3a and 3b.
E-cadherin hypermethylation was not detected in any tissue. CADM1, TIMP3 and
N-cadherin hypermethylation was correlated with the loss of their
protein expression during the progression of pathologic lesions. The prevalence of DNA methylation of at least one gene and the average number of methylated genes increased with the histological progression. DNMT1 and
DNMT3a protein expression increased progressively during the stages of lung
carcinogenesis, whereas DNMT3b overexpression was only found in several samples. Furthermore, DNMT1
protein expression levels were correlated with CADM1 methylation, and
DNMT3a protein expression levels were correlated with CADM1, TIMP3 and
N-cadherin methylation. The average number of methylated genes during
carcinogenesis was significantly correlated with DNMT1 and
DNMT3a protein expression levels. Moreover,
mRNA expression of CADM1 significantly increased
after treatment with DNMT inhibitor
5-aza-2'-deoxycytidine in CADM1-methylated primary tumor cell lines. Our findings suggest that an accumulation of hypermethylation accounts for cell adhesion-related gene silencing is associated with dynamic changes in the progression of MCA/DEN-induced rat lung
carcinogenesis. We suggest that DNMT1 and
DNMT3a protein overexpression may be responsible for this aberrant DNA methylation.