ABSTRACT
BACKGROUND: To identify new epigenetic markers and further characterize Urothelial Cell Carcinoma (UCC), we tested the promoter methylation (PM) status of 19 genes previously identified as cancer specific methylated genes in other solid tumors. METHODS: We used bisulfite sequencing, methylation specific PCR and quantitative methylation specific PCR (QMSP) to test the PM status of 19 genes in urothelial cancer cell lines. RESULTS: Among the 19 genes tested, VGF was found to be completely methylated in several UCC cell lines. VGF QMSP analysis showed that methylation values of almost all the primary 19 UCC tissues were higher than the paired normal tissues (P=0.009). In another cohort, 12/35 (34.3%) of low grade UCC cases displayed VGF methylation. As a biomarker for non-invasive detection of UCC, VGF showed a significantly higher frequency of methylation in urine from UCC cases (8/20) compared to controls (1/20) (P=0.020). After treatment of cell lines with 5-Aza-2'-deoxycytidine, VGF was robustly re-expressed. Forced expression of VGF in bladder cancer cell lines inhibited cell growth. CONCLUSION: Selection of candidates from genome-wide screening approach in other solid tumors successfully identified UCC specific methylated genes.
Subject(s)
Biomarkers, Tumor/genetics , Biomarkers, Tumor/urine , Carcinoma, Transitional Cell/genetics , Epigenesis, Genetic , Nerve Growth Factors/genetics , Urinary Bladder Neoplasms/genetics , Carcinoma, Transitional Cell/urine , Cell Line, Tumor , DNA Methylation , Gene Expression Regulation, Neoplastic , Gene Silencing , Humans , Nerve Growth Factors/urine , Promoter Regions, Genetic/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Urinary Bladder Neoplasms/urineABSTRACT
Cigarette smoking contributes to the development of lung cancer throughout the world, with cases of pulmonary adenocarcinoma (PAC) the most numerous. Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), which is formed from nicotine, has been demonstrated to cause mutations in genes that affect cell regulation and proliferation. Moreover, NNK has been shown to interact directly with and stimulate beta adrenergic receptor (ADRB) signal transduction pathways. Our goal was to determine whether single-nucleotide polymorphisms (SNPs) in the Adrb2 from PAC tumors were induced in golden hamsters by the injection of NNK. Here we report the cloning and sequencing of Adrb2 clones from either dissected lung tumors from NNK-injected animals or whole-lung tissue from water-injected controls. Both sets of animals contained SNPs; however, we found significantly more SNPs in the Adrb2 from NNK-injected animals than in the controls. The majority of these SNPs were novel, nonsynonymous mutations found in regions of the Adrb2 known to be involved in ligand binding, G-protein coupling, and desensitization/down-regulation. Our data verified the mutagenic effects of NNK as well as demonstrated that this animal model provides an outstanding way of identifying mutations not only in the Adrb2, but also in other genes that may play essential roles in the regulation and growth of pulmonary adenocarcinomas.
Subject(s)
Adenocarcinoma/genetics , Carcinogens/toxicity , Lung Neoplasms/genetics , Nitrosamines/toxicity , Polymorphism, Single Nucleotide , Receptors, Adrenergic, beta-2/genetics , Adenocarcinoma/chemically induced , Amino Acid Sequence , Animals , Cricetinae , Lung Neoplasms/chemically induced , Mesocricetus , Molecular Sequence DataABSTRACT
Epidemiologic evidence suggests that pulmonary diseases with a prominent chronic inflammatory component elevate lung cancer risk. Genetic manipulations of mouse models of lung inflammation and tumorigenesis can be used to investigate this association. The genes encoding pro-inflammatory tumor necrosis factor-alpha (TNFalpha) and antiinflammatory IL-10 cytokines map within quantitative trait loci that regulate susceptibility to lung tumor development in mice; sensitive A/J and resistant C57BL/6J (B6) mice have different Tnfa and Il-10 alleles. Genetic ablation studies were performed to examine whether these genes would qualify as candidate tumor modifiers. Tnfa null (-/-) mice on a B6 background and B6.129 Il-10(-/-) mice were intercrossed with A/J mice and subjected to urethane carcinogenesis; lung tumor multiplicity was determined 20 weeks later. In the absence of one copy of Tnfa, tumor number. Male Il-10(+/+) mice developed more tumors than did female mice (P < 0.001), absence of one copy of Il-10 raised tumor number in female mice to that observed in +/+ males, but no change in multiplicity occurred in Il-10 hemizygous males. Thus, a deficit of pro-inflammatory TNFalpha decreased the number of tumors, whereas diminished gene copy number of anti-inflammatory IL-10 increased tumorigenesis; manifestation of an effect of Il-10 haploinsufficiency is gender dependent. These studies support a role for inflammation in lung cancer susceptibility.
