Abstract 1954: In vitro effects on gene expression of Casp9, GRB10, and CDKN1B following rG3 treatment of human breast cancer cells

Abstract

Abstract Introduction: Integrins can stimulate cellular growth and proliferation, as well as apoptosis, and are important factors in AKT cell signaling. To assess their binding, a recombinant protein of the G3 domain of laminin-5 (rG3) that specifically binds the alpha subunit expressed on metastatic MDA-MB-231 cells in vitro was produced. Studying changes in expression of genes involved in cell signaling and the apoptosis cascade, specifically CDKN1B, GRB10, and Caspase 9, can help improve understanding of signaling pathways of metastatic cells and the AKT pathway. Methods: rG3 was produced using ArcticExpress Competent Cells (Stratagene). Plasmid DNA was transformed into competent cells and transferred to ampicillin plates for colony production of successful transformation. A single colony was used to inoculate LB broth containing gentamycin and ampicillin for an overnight incubation at 37°C. LB without antibiotics was inoculated with the overnight culture and incubated at 30°C for 3 hours. The temperature was lowered to 11.5°C before the expression was induced using IPTG at a final concentration of 1mM. SDS-PAGE/immunoblotting confirmed successful soluble rG3 protein expression. MDA-MB-231 human breast cancer cells from ATCC were grown in tissue culture flasks and maintained until confluent. Cells were plated at 3 × 106 cells/well in a 6-well tissue culture plate in a 2 ml volume of DMEM without pen/strep in duplicate. rG3 was added to wells at a final concentration of 175 µg/ml for treated cells and corresponding control buffer for untreated cells. After incubation overnight, cells were collected at time points within 0-60 hours and RNA was isolated using Qiagen RNeasy. An eppendorf Realplex 96-well plate reader was used for PCR analysis using a β-actin standard curve. Differences in gene expression were shown in fold change using Pfaffl's Method. Results: Our preliminary data show there are changes in gene expression after treatment with rG3.We are further investigating these changes in expression. Preliminary results suggest that changes in gene expression after treatment may be informative in understanding the AKT signaling pathway. Conclusion: The study of these changes in gene expression after altering the AKT pathway from treatment with rG3 could prove to be a helpful tool in understanding the signaling mechanisms of metastatic cells and the details of the signaling pathways involved in apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1954. doi:10.1158/1538-7445.AM2011-1954