The effects of a cigarette carcinogen on glucocorticoid receptor methylation.
Small cell lung cancer (SCLC) is an aggressive disease with an extremely poor prognosis. Lung cancer has been linked to cigarette smoking. SCLCs are glucocorticoid insensitive due to reduced levels of glucocorticoid receptor (GR) expression. Re-expression of the GR, both in vitro and in vivo, induces apoptosis. The loss of GR expression therefore provides a survival advantage in SCLC cells. The GR gene in SCLC cells may be epigenetically silenced by DNA hypermethylation of its promoter region. DNA methylation is carried out by DNA methyltransferase 1 (DNMT1), shown to be over-expressed in lung cancer patients who smoke. The first aim of this study was to determine which GRα isoforms are re-expressed in SCLC cells after exposure to a demethylation agent. The SCLC cell line, DMS 79; the glucocorticoid-sensitive non-SCLC cell line, A549; and the glucocorticoid-resistant HEK 293 cell lines were treated with the DNMT1inhibitor, 5-Aza-2’-deoxycytidine (5-Aza), to determine which GRα isoforms are re-expressed in SCLC cells, compared to the control cell lines. Other studies have found certain GRα isoforms to be more effective at inducing apoptosis, such as GRα-C. DMS 79 cells and HEK cells showed GRα protein expression of isoform -A, -B and GRα-C, whilst only isoforms -A and -B were seen in the A549 cells. A key carcinogen in cigarette smoke, nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), has been linked to lung carcinoma development. NNK has been linked in the nuclear accumulation of DNMT1 and is thus responsible for the hypermethylation of multiple TSGs in lung cancers. The second aim was to determine whether the NNK tobacco carcinogen induces DNMT1 accumulation and TSG hypermethylation at the GR gene. The MRC-5 normal lung fibroblast cell line was treated with NNK. There was a significant down-regulation of GRα mRNA expression after 2 hours of NNK treatment (p < 0.05). Long term treatment with NNK showed a further significant decrease in GRα mRNA expression in the MRC-5 cells (p < 0.001). GRα protein expression differed in the MRC-5 cell line between the vehicle (control) and the cells treated with NNK for 24, 48 and 72 hours (p < 0.001). These data indicated that long term treatment with NNK appeared to further decrease GRα protein expression. This is consistent with other studies which showed prolonged NNK or cigarette exposure inactivated tumour suppressor genes (TSGs). To determine whether down-regulation is due to NNK-induced recruitment of DNMT1 to the GR promoter, Chromatin Immunoprecipitation (ChIP) analysis was performed. Preliminary ChIP analysis data showed possible recruitment of DNMT1 to promoter 1F and 1J of the GR, suggesting that NNK treatment resulted in the silencing of GR expression via promoter 1F and 1J. Recent data in our laboratory has indicated that when the GR is re-expressed the use of promoters 1F and 1J may be responsible for inducing apoptosis in SCLC cells. These preliminary data may provide insight into the mechanism of cigarette carcinogen induced SCLC carcinogenesis. GRα appears to be an important TSG in SCLC and may be fundamental in SCLC development. Elucidating the pathways involved in SCLC development and GRα expression could lead to a breakthrough in oncology and novel ways in which therapies can target this deadly disease.