[Effect of adenomatous polyposis coli(APC) promoter methylation on gene transcription in lung cancer cell lines].

Center of Clinical Laboratory, The First Affiliated Hospital, Nanjing Medical University, Nanjing, Jiangsu 210029, P. R. China.
Ai zheng = Aizheng = Chinese journal of cancer (Impact Factor: 2.16). 06/2007; 26(6):576-80.
Source: PubMed


Hypermethylation of CpG islands in adenomatous polyposis coli (APC) gene has been detected in a variety of human tumors, which is involved in the pathogenesis of these tumors. In previous research, we detected APC promoter methylation in 47% lung tumor tissues. This study was to analyze the effect of APC promoter methylation on the gene transcription in 3 lung cancer cell lines.
The methylation status of APC promoter 1A in lung adenocarcinoma cell line SPCA1, small cell lung cancer cell line NCI-H446, and big cell lung cancer cell line NCI-H460 was detected by methylation-specific polymerase chain reaction (MSP) and microarray methylated cord blood DNA served as positive control, and unmethylated cord blood DNA served as negative control. The expression of APC was examined by real-time quantitative polymerase chain reaction (PCR) with Sybr-Green I staining. After treatment of 1, 5, 10, 15 micromol/L DNA methyltransferase inhibitor 5-aza-2-deoxycytidine (5-aza-dC), the expression of APC in NCI-H460 cells was detected by real-time PCR.
APC promoter 1A was methylated in NCI-H460 cells, and unmethylated in NCI-H446 and SPC-A1 cells. Hypermethylation was detected in all 5 CpG islands (687, 707, 714, 719, 726) of APC promoter 1A in NCI-H460 cells. The expression of APC in NCI-H460 cells was decreased by 26.04% of that in NCI-H446 cells and by 32.36% of that in SPCA1 cells. After treatment of 1, 5, 10, 15 micromol/L 5-aza-dC, the expression of APC promoter 1A in NCI-H460 cells was enhanced by 4.59, 5.78, 9.58, 5.98 folds, respectively.
APC gene is hypermethylated in HCI-H460 cells, and its transcription coud be activated by 5-aza-dC.

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    • "SNPs could influence miRNA as seen with rs334348 associated with germline allele specific expression of TGFBR1 correlated to colorectal cancer (Nicoloso et al., 2010). Methylated cytosines have been experimentally proven in CpG islands of APC gene promoter region (Genbank accession: U02509, CpG Position 687) (Zhang et al., 2007) and rs35417795, which is another SNP, occurs at close proximity in this island. Further, the tumor suppressor gene, Tet1, is known for its conversion of methylated cytosine to hydroxymethylcytosine. "
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    ABSTRACT: DNA methylation maintains allele specific gene expression (Chan et al., 2003) in which miRNA influences allele-specific protein expression and SNPs, found inside miRNA, in turn influences tumor susceptibility (Nicoloso et al., 2010). Further, methylated CpGs have been correlated to APC gene in colorectal cancer (Zhang et al., 2007) and retrotranspositions have also been correlated to APC gene (Miki et al., 1992). Another aspect unrelated to it is Tet1 gene, which has been associated with the conversion of methylcytosine to hydroxymethyl cytosine (Tahiliani et al., 2009). DNA methylation might also have a role in the prevention of normal differentiation in pediatric cancers (Diede et al., 2009). As the difference between a nucleobase and its methylated form is its structure and its molecular weight, this research article is focussed on using the molar mass of nucleobases to find out if there is any uniqueness as for the position of occurrence of a nucleotide in a given model. SNPs occurrence position was used as a model in this research for addressing the cytosine trios (cytosine, 5methyl cytosine, hydroxy methyl cytosine) based on molar mass of the nucleobase. As the conditions for occurrence of SNP, at a given position in a sequence, were found to uniformly conform with all 140,000 SNPs analysed, including all clinically associated SNPs from NCBI SNP database, it intrigued conformity to be cross checked with SNPs near experimentally proven methylation sites.
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    ABSTRACT: Previous findings have suggested that methylation of the APC gene may be associated with some tumors including lung cancer. To explore the pattern of APC methylation and the effect of APC gene methylation on its protein expression in lung cancer cell lines, we investigated APC promotor methylation by methylation specific PCR (MSP) and bisulfite sequencing and analyzed the APC protein levels by western blot in three lung cancer cell lines. Monoallelic methylation and 20 methylated CpGs in CpG island near the open reading frame (ORF) of the APC gene were found in the NCI-H460 cell line, and were stablely inherited within 10 generations of the cell line in culture. Our results showed that two special CpG sites (794, 797) might be binding sites for proteins that regulate APC expression. Protein expression of the APC gene in the NCI-H460 cell line declined, but was enhanced after the treatment with 5-aza-2-deoxycytidine (5-aza-dC). Inherited monoallelic methylation of the APC gene may play an important role in lung cancer. Demethylation of the APC gene by 5-aza-dC may be useful for the treatment of lung cancer.
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