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题目:: Transcription profiling of human HT29 cells sensitive and resistant to 10e-5M methotrexate
ID:
状态:: 发布时间June 16, 2008 , 更新时间 March 27, 2012 , 提交时间 Oct. 23, 2007,
物种:: Homo sapiens
摘要:: The abstract of the associated publication (Selga E, Noé V, Ciudad CJ. Biochemical Pharmacology 2007 Sep 8; Article in press) is the following:; While studying differentially expressed genes between sensitive and 10-5 M Methotrexate (MTX) resistant HT29 human colon cancer cells, we identified some members of the aldo-keto reductase (AKR) superfamily. The study was followed with the member AKR1C1 (EC 1.1.1.213), validating its increase in mRNA and protein levels in MTX resistant cells. The genomic content for AKR1C1 remained unchanged between sensitive and resistant cells, thereby excluding a mechanism of AKR1C1 gene amplification. Thus, we cloned the AKR1C1 human promoter and performed luciferase experiments that revealed a transcriptional regulation of the gene in the resistant cells. Computational studies showed a putative binding site for the transcription factor Sp1. The co-transfection of Sp1 or Sp3 with different constructs of AKR1C1 promoter deletions, including and excluding the proximal GC-box, demonstrated a key role for these factors in regulating AKR1C1 transcriptional activity. Gel-shift assays revealed an increase in Sp1 and Sp3 binding in resistant compared to sensitive cells, without differences in Sp1 protein levels. Dephosphorylation of the extracts coincided with a decrease in Sp1 binding, which is consistent with a process of regulation of Sp1 by phosphorylation. We also investigated the possible relationship between AKR1C1 expression and MTX action. Overexpression of AKR1C1 counteracted the S-phase accumulation of cells and apoptosis caused by MTX treatment. This suggests a role of AKR1C1 in cell proliferation. Finally, overexpression of AKR1C1 in MTX sensitive HT29 cells conferred resistance to the chemotherapeutic agent and silencing of AKR1C1 by means of iRNA technology sensitized the cells to MTX. Experiment Overall Design: Two cell lines are compared in the study, which are HT29 colon cancer cells sensitive to methotrexate and HT29 cells resistant to 10e-5M methotrexate. Six samples are provided, which correspond to triplicates of each cell line. The samples provided were subsequently normalyzed and analyzed using the specific software GeneSpring GX 7.3.1
实验种类:: transcription profiling by array
样本量:: 6
实验设计:
: 无设计数据
数据号:: E-GEOD-9412, GSE9412
数据状态:: 无法自动分析，您可以尝试手动分析数据。

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