BES2005 Poster Presentations Endocrine tumours and neoplasia (46 abstracts)
Long term culture of MCF-7 breast cancer cells with siRNA to the IGF-I receptor causes continued transcriptional silencing of the IGF-I receptor and decreased cell proliferation
CJ McVittie 1 , W Ogunkolade 2 , S Khalaf 2 , V Macaulay 3 , SA Bustin 4 , R Carpenter 1 & PJ Jenkins 2
1Department of Breast & Endocrine Surgery, St Bartholomew's Hospital, London, UK; 2Department of Endocrinology, St Bartholomew's Hospital, London, UK; 3Weatherall Institute of Molecular Medicine, Oxford, UK; 4Department of Academic Surgery, Barts and the London, London, UK.
Introduction: The GH/IGF-I axis has been implicated in the pathogenesis of breast cancer and development of tamoxifen resistance. We have previously used siRNA to successfully silence the IGF-I receptor (IGF-IR) in MCF-7 breast cancer cells. To further characterize the growth changes that occur when elements of the GH/IGF-I axis are disrupted we investigated the use of siRNA to cause long term silencing of IGF-IR.
Methods: MCF-7 cells were cultured in phenol red-free medium (RPMI 1640) with 10% fetal calf serum (FCS). We used a specific siRNA duplex designed to target IGF-IR mRNA. MCF-7 cells (104 cells/well) were washed and transfected in serum-free medium with Oligofectamine alone (mock-transfection), or with Oligofectamine plus 200nM siRNA. Every six days, cells were re-transfected with siRNA in the same manner. Every three days, cell proliferation was assessed using the colorimetric MTS assay, cells were lysed and RNA extracted for quantitative RT-PCR for IGF-IR mRNA.
Results: Compared with untransfected cells, proliferation was decreased in IGF-IR siRNA-transfected cells to 45.5% at day 3, 42.2% at day 6, 33.6% at day 9, 10.5% at day 15 and 4.1% at day 18. Mock-transfected cells also showed decreased cell proliferation to 73.8% at day 3, 87.3% at day 6, 62.6% at day 9, 54.1% at day 15 and 44.8% at day 18. This was probably due to an element of non-specific toxicity of Oligofectamine. PCR confirmed the siRNA decreased IGF-IR mRNA: IGF-IR mRNA copy number (% of media only) is 5.9% at day 3, 3.7% at day 6 and 0.08% at day 15.
Conclusions: Repeated transfection of MCF-7 cells with IGF-IR siRNA every six days, enables us to induce prolonged IGF-IR knock down and cause a sustained decrease in cell proliferation. This anti-proliferative effect of IGF-IR siRNA is confirmed by the decrease in IGF-IR mRNA expression in siRNA-transfected cells.
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Endocrine Abstracts
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