Breast Neoplasms ; genetics ; metabolism ; pathology ; Carcinoma, Ductal, Breast ; genetics ; metabolism ; pathology ; Carcinoma, Intraductal, Noninfiltrating ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; DNA Methylation ; Female ; Gene Expression Regulation, Neoplastic ; Genes, Tumor Suppressor ; Humans ; Precancerous Conditions ; genetics ; metabolism ; pathology ; Promoter Regions, Genetic ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; rho GTP-Binding Proteins ; biosynthesis ; genetics

OBJECTIVETo investigate the promoter methylation status and mRNA expression of APC gene in MCF10 model of breast cancer progression.

METHODSMethylation specific PCR and sodium bisufite genomic sequencing were employed to detect the methylation status of APC promoter 1A in normal breast tissues, conventional breast cancer cell line MCF-7 and MCF10 model cell lines including MCF10A (breast hyperplastic cell line, non-tumorigenic), MCF10AT (pre-malignant cell lines, producing slowly progressing hyperplastic and dysplastic lesions), MCF10DCIS.com (breast ductal carcinoma in-situ cell line, producing ductal carcinoma in-situ), MCF10CA1a, MCF10CA1d, MCF10CA1h cell lines (invasive breast carcinoma cell line, forming aggressive tumors of different morphology and metastatic potential). In addition, mRNA expression of APC was determined by reverse transcriptase PCR and real-time PCR assays.

RESULTSHypomethylation of APC promoter 1A was identified in hyperplastic cell line MCF10A, pre-malignant cell line MCF10AT, ductal carcinoma in-situ cell line MCF10DCIS.com, invasive carcinoma cell lines MCF10CA1a, MCF10CA1d, MCF10CA1h and normal breast tissue. MCF-7 showed partial methylation at the promoter. Statistically significant reduction of APC mRNA expression was not found in all MCF10 cell lines and MCF-7, compared with that of normal breast tissue (MCF10AT, MCF10CA1a, MCF10CA1d, MCF10CA1h and MCF10DCIS.com showed reduced mRNA expressions of APC at 0.27, 0.96, 1.78, 2.70, and 2.03 times respectively. MCF10A and MCF-7 even showed an increase of APC mRNA expression at 0.02 and 0.33 times, respectively).

CONCLUSIONThe aberrant promoter methylation of APC is not related to the breast cancer progression, at least in the MCF10 model system.

Adenomatous Polyposis Coli Protein ; biosynthesis ; genetics ; Breast ; pathology ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; DNA Methylation ; Female ; Gene Expression Regulation, Neoplastic ; Genes, APC ; Humans ; Hyperplasia ; genetics ; metabolism ; pathology ; Precancerous Conditions ; genetics ; metabolism ; pathology ; Promoter Regions, Genetic ; genetics ; RNA, Messenger ; biosynthesis ; genetics

OBJECTIVETo investigate the role of WT1 gene in breast carcinogenesis by analyses of the promoter methylation status and mRNA expression of WT1 gene in MCF10 model system of breast cancer progression.

METHODSMethylation specific PCR and sodium bisufite genomic sequencing were employed to detect methylation status of WT1 promoter in normal breast tissue, traditional breast cancer cell line MCF7 and MCF10 model series, including MCF10A (breast hyperplastic cell line, non-tumorigenic), MCF10AT (pre-malignant cell line, forming slowly progressing hyper and dysplastic lesions), MCF10DCIS.com (breast ductal carcinoma in situ cell line, forming ductal carcinoma in situ), and three invasive cell lines with metastatic potential (MCF10CA1a, MCF10CA1d, and MCF10CA1h). Real time reverse transcription PCR assay was used to determine the mRNA expression levels of WT1 in various cell lines.

RESULTSHypermethylation of WT1 promoter was identified in MCF7 and all MCF10 model cell lines (MCF10A, MCF10AT, MCF10DCIS.com, MCF10CA1a, MCF10CA1d, and MCF10CA1h). Unexpectedly, an increased expression of WT1 mRNA was found in all MCF10 cell lines and MCF7 comparing with normal breast tissue [folds of overexpression: 3.23 (MCF10A), 1.94 (MCF10AT), 4.20 (MCF10CA1a), 1.53 (MCF10CA1d), 4.20 (MCF10CA1h), 4.35 (MCF10DCIS) and 28.69 (MCF7)].

CONCLUSIONSPromoter methylation does not silence the mRNA expression of WT1 during the development of breast cancer. Overexpression of WT1 occurs in the early stages of breast cancer development, suggesting its role as an oncogene rather than a tumor suppressor gene.

Base Sequence ; Breast ; pathology ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Carcinoma, Ductal, Breast ; genetics ; metabolism ; pathology ; Carcinoma, Intraductal, Noninfiltrating ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; DNA Methylation ; DNA, Neoplasm ; genetics ; Disease Progression ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Hyperplasia ; genetics ; metabolism ; pathology ; Molecular Sequence Data ; Precancerous Conditions ; genetics ; metabolism ; pathology ; Promoter Regions, Genetic ; RNA, Messenger ; metabolism ; WT1 Proteins ; genetics ; metabolism