Gene silencing in cancer cells using siRNA : genetic and functional studies

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Title: Gene silencing in cancer cells using siRNA : genetic and functional studies
Author: Abdel Rahim, Ma'en Ahmad
Abstract: Sequence -specific small interfering RNA (siRNA ) duplexes can be used for gene silencing in mammalian cells and as mechanistic probes for determining gene function . Transfection of siRNA for specificity protein 1 (Sp1 ) in MCF -7 or ZR -75 cells decreased Sp1 protein in nuclear extracts , and immunohistochemical analysis showed that Sp1 protein in transfected MCF -7 cells was barely detectable . Decreased Sp1 protein in MCF -7 was accompanied by a decrease in basal and estrogen -induced transactivation and cell cycle progression . These results clearly demonstrate the key role of Sp1 protein in regulating growth and gene expression of breast cancer cells . The aryl hydrocarbon (AhR ) is a ligand -activated nuclear transcription factor . siRNA for the AhR decreased TCDD -induced CYP1A1 protein , CYP1A1dependent activity , and luciferase activity in cells transfected with an Ah -responsive construct . 17 ? -Estradiol (E2 ) induces proliferation of MCF -7 cells , and this response is inhibited in cells cotreated with E2 plus TCDD . The effects of TCDD on E2 -induced cell cycle progression were partially blocked in MCF -7 cells transfected with siRNA for AhR . The decrease in AhR protein in MCF -7 cells was also accompanied by increased G0 /G1 ? S phase progression . Surprisingly , TCDD alone induced G0 /G1 ? S phase progression and exhibited estrogenic activity in MCF -7 cells transfected with siRNA for the AhR . In contrast , degradation of the AhR in HepG2 liver cancer cells resulted in decreased G0 /G1 ? S phase progression , and this was accompanied by decreased expression of cyclin D1 , cyclin E , cdk2 and cdk4 . In the absence of ligand , the AhR exhibits growth inhibitory (MCF -7 ) and growth promoting (HepG2 ) activity that is cell context -dependent . Sp family proteins play a complex role in regulation of pancreatic cancer cells growth and expression of genes required for growth , angiogenesis and apoptosis . Sp1 , Sp3 and Sp4 cooperatively activate VEGF promoter constructs in these cells ; however , only Sp3 regulates cell proliferation . siRNA for Sp3 inhibits phosphorylation of retinoblastoma protein , blocks G0 /G1 ? S phase progression of Panc -1 cells , and upregulates p27 protein /promoter activity . Thus , Sp3 plays a critical role in angiogenesis (VEGF upregulation ) and the proliferation of Panc -1 cells by a novel mechanism of Sp3 -dependent suppression of the cyclin -dependent kinase inhibitor p27 .
URI: http : / /hdl .handle .net /1969 .1 /218
Date: 2004-09-30

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Gene silencing in cancer cells using siRNA : genetic and functional studies. Available electronically from http : / /hdl .handle .net /1969 .1 /218 .

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