Dynamic changes in DNA methylation during multistep rat lung carcinogenesis induced by 3-methylcholanthrene and diethylnitrosamine.
3-methylcholanthrene (MCA) and diethylnitrosamine (DEN) are typical genotoxic carcinogens that can induce tumors in a variety of human and rodent tissues. However, the epigenetic mechanisms underlying their tumorigenesis are unclear. In this study we used a MCA/DEN-induced multistep lung carcinogenesis rat model to study the evolution of alterations in DNA methylation. Rats were treated with a single dose of MCA and DEN in iodized oil by left intra-bronchial instillation. The animals were killed on days 15, 35, 55, 65 and 75 and samples of various pathological phases during carcinogenesis were obtained on these days. The status of global methylation was analyzed for each sample using a monoclonal antibody specific for 5-methycytosine (5-mC) and quantified by image analysis software. We found that the degree of global methylation was, in general, higher in basal cells compared to luminal cells of normal, precancerous and tumor tissues. The combined 5-mC scores of different types of tissues decreased gradually during the progression of carcinogenesis. We also used methylation-sensitive arbitrarily primed PCR (MS-AP-PCR) to screen a total of eight differentially methylated DNA fragments in both precancerous and tumor tissues isolated using laser capture microdissection (LCM), and observed that both unique hypomethylation and hypermethylation fragments coexist after exposure to genotoxic carcinogens. Remarkably, epigenetic alterations in p16 (CDKN2A), but not in p15 (CDKN2B), were observed, and these correlated with the presence of pathologic lung lesions and loss of p16 protein expression. Moreover, defective expression of p16 in methylated primary tumor cell lines recovered markedly after treated with 5-aza-2'-deoxycytidine (5-aza-dC). These results suggest that DNA methylation alterations are an early event in tumorigenesis and play an important role during MCA/DEN-induced multistep rat lung carcinogenesis.
Liu
Wen-bin
WB
Department of Hygienic Toxicology, Preventive Medical College, Third Military Medical University, Key Laboratory of Medical Protection for Electromagnetic Radiation, Ministry of Education of China, Chongqing 400038, PR China. jinyiliutmmu@163.com
Liu
Jin-yi
JY
Ao
Lin
L
Zhou
Zi-yuan
ZY
Zhou
Yan-hong
YH
Cui
Zhi-hong
ZH
Yang
Huan
H
Cao
Jia
J
D000818:Animals; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D002460:Cell Line; D003043:Cocarcinogenesis; D050762:Cyclin-Dependent Kinase Inhibitor p15 / Q000235:genetics; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics; D019175:DNA Methylation / Q000187:drug effects*; D000077209:Decitabine; D004052:Diethylnitrosamine / Q000633:toxicity*; D005260:Female; D007150:Immunohistochemistry; D008168:Lung / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D008175:Lung Neoplasms / Q000139:chemically induced* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D008297:Male; D008748:Methylcholanthrene / Q000633:toxicity*; D042282:Microdissection; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction
C493969:Cdkn2b protein, rat; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16; D004052:Diethylnitrosamine; D008748:Methylcholanthrene; D000077209:Decitabine; D001374:Azacitidine