The Establishment Of Arsenite-transformed Cells(TLECs)and The Study On Ethylation Of Non-CpG Island Promoter Repressing NQO1 Expression In TLECs

Arsenic and its derivatives are widely distributed in the environment and are seriously harmful to human health.Although many epidemiological studies have shown that inorganic arsenicals are human lung carcinogens,there is currently no accepted mechanism for arsenic-induced carcinogenesis,which requires further investigations in this area.Since arsenic-induced carcinogenesis is chronic and very complicated,it is most important to establish a cell model to emulate this process.So,we executed the transformation of normal rat lung epithelial cells(LECs)with chronic arsenite treatment and gained the arsenite-transformed LECs(TLECs).Then,a series of relevant experiments were carried out to verify the successful establishment of TLECs for the following studies.Reactive oxygen species(ROS)pathway was supposed to play an important role in arsenite-induced cell transformation with the intracellular redox system being disrupted.NAD(P)H:quinone oxidoreductase 1(NQ01),one significant member of the system,is a phase II flavoenzyme catalyzing reduction reactions that protect cells against electrophiles and oxidants.For its detoxification of quinones and the effect on increasing the stability of p53 in cells,NQ01 was also confirmed to be involved with the cell tumorigenesis.Here we first report the NQ01 exhibited expression differentiation in arsenite-transformed LEC(TLEC)whose ROS level was elevated.That is,the gene became either inactive or hyperactive in the subclones.It indicated that the change of NQ01 expression was an important event in arsenite-induced cell transformation and carcinogenesis.So,the study on the mechanism of its aberrant expression is necessary and meaningful.Our laboratory have found that the NQ01 gene of rat displays tissue-specific methylation in the non-CpG island promoter and it seemed to be inversely correlated with the transcript level in corresponding tissue.In addition,more and more studies have shown that the epigenetic signatures of such promoter are almost identical to CpG island promoter.We speculated that the changes of TLEC NQ01 could result from the aberrant methylation of non-CpG island promoter.Next,Bisulfite-sequencing was carried out to revealed that non-CpG island promoter is hyper-or hypo-methylation in subclones with inactive or hyperactive transcription of NQ01 respectively.Treated with demethylating drug 5-aza-2'-deoxycytidine(5-Aza-CdR),NQ01 in the hypermethylated subclones was restored drastically in transcript level.Further,the NQ01 promoter(-306 to +229)containing the ARE(Antioxidant response element)enhancer in 5'end was fused into the CpG-less vector pCpGL-Basic with luciferase gene to generated the reporter as pCpGL-NQ01.Luciferase assay showed that the pCpGL-NQ01 construct in vitro methylation was completely silenced in contrast to its mock methylaton control.Together,our results demonstrate for the first time that hypermethylation of the non-CpG island promoter could repress NQ01 expression in rat arsenite-transformed lung epithelial cells.