Abstract
BackgroundThe poly Q polymorphism in AIB1 (amplified in breast cancer) gene is usually assessed by fragment length analysis which does not reveal the actual sequence variation. The purpose of this study is to investigate the sequence variation of poly Q encoding region in breast cancer cell lines at single molecule level, and to determine if the sequence variation is related to AIB1 gene amplification.MethodsThe polymorphic poly Q encoding region of AIB1 gene was investigated at the single molecule level by PCR cloning/sequencing. The amplification of AIB1 gene in various breast cancer cell lines were studied by real-time quantitative PCR.ResultsSignificant amplifications (5–23 folds) of AIB1 gene were found in 2 out of 9 (22%) ER positive cell lines (in BT-474 and MCF-7 but not in BT-20, ZR-75-1, T47D, BT483, MDA-MB-361, MDA-MB-468 and MDA-MB-330). The AIB1 gene was not amplified in any of the ER negative cell lines. Different passages of MCF-7 cell lines and their derivatives maintained the feature of AIB1 amplification. When the cells were selected for hormone independence (LCC1) and resistance to 4-hydroxy tamoxifen (4-OH TAM) (LCC2 and R27), ICI 182,780 (LCC9) or 4-OH TAM, KEO and LY 117018 (LY-2), AIB1 copy number decreased but still remained highly amplified. Sequencing analysis of poly Q encoding region of AIB1 gene did not reveal specific patterns that could be correlated with AIB1 gene amplification. However, about 72% of the breast cancer cell lines had at least one under represented (<20%) extra poly Q encoding sequence patterns that were derived from the original allele, presumably due to somatic instability. Although all MCF-7 cells and their variants had the same predominant poly Q encoding sequence pattern of (CAG)3CAA(CAG)9(CAACAG)3(CAACAGCAG)2CAA of the original cell line, a number of altered poly Q encoding sequences were found in the derivatives of MCF-7 cell lines.ConclusionThese data suggest that poly Q encoding region of AIB1 gene is somatic unstable in breast cancer cell lines. The instability and the sequence characteristics, however, do not appear to be associated with the level of the gene amplification.
Highlights
The poly Q polymorphism in AIB1 gene is usually assessed by fragment length analysis which does not reveal the actual sequence variation
Amplification of AIB1 gene Real-time quantitative PCR (Q-PCR) analysis allows the measurement of actual copy number of AIB1 gene using a single copy β2microglobulin gene as a reference
We developed real time quantitative PCR method to more accurately assess the amplification of AIB1 gene in breast cancer cell lines
Summary
The poly Q polymorphism in AIB1 (amplified in breast cancer) gene is usually assessed by fragment length analysis which does not reveal the actual sequence variation. While predisposition to breast cancer is largely due to mutations in high penetrance tumor suppressor genes such as BRCA1 and BRCA2, progression of cancer is the result of accumulation of genetic alterations. The AIB1 gene is a member of the SRC-1 (steroid receptor coactivator) family and is known as RAC3, TRAM-1 or ACTR [7,9,10]. It is located at chromosome 20q12 region and encodes a protein of 1420 amino acids containing bHLHPAS dimerization domain, a hormone receptor interaction domain, a CBP interaction domain, and histone acetyltransferase domain [11]. Since AIB1 bridges between nuclear receptors and other coactivators or the transcriptional machinery, its amplification and overexpression may play crucial roles in the development of breast cancer and may potentially have influence on the hormonal prevention and treatment for breast cancer
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