Figure 2. Conformational change of S100P structure caused by two Ca 2+ ions binding to EF- hand calcium binding motifs (Madej et al. 2012). Create a novel S100P-mCherry fusion protein using the pMcherry-N1 mammalian expression vector. Transfect HT29 colon carcinoma cells with S100P- pmCherry-N1 fusion plasmid. Compare chemotherapy drug effectiveness - treat wild type, S100P overexpressed, and S100P silenced (via siRNA) HT29 cells with cisplatin, doxorubicin, and etoposide. Objectives S100P is a 95-amino acid protein that belongs to the 21 protein S100 family. The nomenclature S100P refers to the protein’s ability to solubilize in 100% saturated ammonium sulfate solution. The specific designation “P” is used because it was first purified from placenta (Becker et al. 1995). The gene coding for S100P is located on chromosome 4p16 where some fatal disease-related genes are also mapped including: Huntington disease, Crohn’s disease and cervical cancer (Arumugam and Logsdon 2011). S100P interacts with its cellular targets via a Ca 2+ conformational change (Becker et al. 1995; Figure 2). S100P regulates many intracellular and extracellular activities including: protein phosphorylation, enzyme activity, gene transcription, and cell proliferation and differentiation (Donato 2001; Figure 3). Several studies revealed that S100P was overexpressed in human cancer cells including: colon (Bertram et al. 1998), breast, and prostate (Basu et al. 2008). Due to its overexpression and role in carcinogensis and cell proliferation in cancer cells, S100P serves as a potential molecular target for cancer treatment. Targeted therapies interfere with cell proliferation by focusing on proteins involved in signal transduction. Doxorubicin (topoisomerase II inhibitor/free radical generator), cisplatin (DNA crosslinker), and etoposide (topoisomerase II inhibtor) act via different cellular mechanisms. Introduction HT29 cell treatment was unsuccessful with all three chemotherapy drugs. Was not able to treat overexpressed and siRNA knockout cells. S100P gene was isolated from cDNA, ligated into pmCherry-N1 vector, and verified by sequencing. HT29 cells were successfully transfected with the constructed mCherry-S100P fusion plasmid. The nuclear translocation of S100P-mCherry may have been induced by UV light and/or heat during fluorescence microscopy. Previous research has reported Ca 2+ and binding interaction induced translocation of S100P but there has yet to be research reporting a UV or heat induced translocation. Jiang et al. (2005) stated that S100P is associated with enhanced survival in cells under stress, while Al-Baker et al. (2004) reported that UV-light induced DNA damage can cause a nuclear translocation of proteins. In this case, S100P may be translocating to the nucleus to serve as a protective/repairing DNA agent. Conclusions Cloning and expression of a novel S100P-mCherry fusion protein in HT29 colon carcinoma cells Joseph T. Orlando Department of Biological Sciences, York College of Pennsylvania Acknowledgments I would like to thank Dr. Ronald Kaltreider and Dr. Jeffrey Thompson for their constant support and advice throughout the duration of this project. References Arumugam, T., and Logsdon, C. D S100P: a novel therapeutic target for cancer. Amino Acids. 41: Becker, T., Gerke, V., Kube, E., and Weber, K S100P a novel Ca 2+ - binding protein from human placenta cDNA cloning, recombinant protein expression and Ca 2+ binding properties. European Journal of Biochemistry 207: Donato, R S100: a multigenic family of calcium-modulated proteins of the EF-hand type with intracellular and extracellular functional role. International Journal of Biochemistry and Cell Biology. 33: Jiang, H., Hu, H., Tong, X., Jiang, Q., Zhu, H., and Zhang, S Calcium- binding protein S100P and cancer: mechanisms and clinical relevance. Journal of Cancer Research and Clinical Oncology. 138:1-9. Madej, T., Addess, K. J., Fong, J. H., Geer, L. Y., Geer, R. C., Lanczycki, C. J., Liu, C., Lu, S., Marchler-Bauer, A., Panchenko, A. R., Chen, J., Thiessen, P. A., Wang, Y., Zhang, D., and Bryant, S. H MMDB: 3D structures and macromolecular interactions. Nucleic Acids Research. 40: Methods Chemotherapy Drug Treatment 5x10 4 HT29 cells were plated in 200µL medium (DMEM, 10% FBS, and 1% pen/strep) in a 96-well plate and incubated overnight. Medium was replaced with varying doses ( µM) of either cisplatin, doxorubicin, or etoposide, and incubated for 24 hours. Medium was removed and cells were washed 3x with PBS. 50µL MTT (5mg/ml) added to each well and incubated for 4 hours. 50µL DMSO added to dissolve formed formazan crystals. Cell viability was measured by using an MTT assay (absorbance at 590nm measured). Plasmid Construction RT-PCR of human mammary epithelial cell RNA to generate cDNA. Developed original primers to isolate S100P from cDNA (BglII and EcoRI restriction sites). 5’-ggactcagatctgccgccaccatgacggaactagagacagccatgggc-3’ 5’-catgcagaattcctttgagtcctgccttctcaaagtact-3’ Digested PCR fragment with BglII and EcoRI to generate sticky ends (Figure 5A). Ligated the PCR product into a linearized pmCherry-N1 mammalian expression vector (Clontech: Figure 7) with Instant Sticky-end Ligase Master Mix® (New England Biolabs). HT29 cell transfection Transformed pMcherry-N1 vector (Clontech) containing S100P-mCherry fusion into E. coli. Isolated and purified plasmid from four transformed E. coli colonies (selection with kanamycin). S100P gene presence was verified by a restriction digest (BglII and EcoRI) of the isolated plasmids (Figure 5B). Sequenced isolated clone plasmids to verify correct orientation of gene (P CMV IE primer). Transfected HT29 colon carcinoma cells using FuGENE 6 (Promega) and analyzed cells via fluorescent microscopy (Figure 6). b. Results a. c.d. b. Figure 6. Transfected HT29 cells viewed under fluorescent microscope A) HT29 cells transfected with pmCherry-N1 control vector photographed immediately after being brought into field of view B) Same group of cells as photo A photographed 40 seconds later. C) HT29 cells transfected with S100P-pmCherry-N1 vector photographed immediately after being brought into field of view D) Same group of cells as photo C photographed 40 seconds later. a. b. Figure 5. Gel images A. Isolation of S100P gene from cDNA. Digested (BglII and EcoRI) S100P fragment was run on 2% agarose gel with 100 bp ladder. B. Transformed E. coli colonies digested with BglII and EcoRI and electrophoresed on a 2% agarose gel. Digestion was performed to verify that isolated colonies contained the 319bp S100P gene. Figure 4. Mean (and SEM: n=3) percentage of cell viability of cisplatin, doxorubicin, and etoposide treated HT29 colon cancer cells determined by MTT assay. Cells were treated with chemotherapy drugs (1, 10, 50, 100 and 1000 µM) for 24 hours at 37°C followed by three PBS washes prior to addition of MTT. Cells were incubated for 4 hours then absorbance at 590nm was measured. Figure 3. The complexity of S100P signal transduction pathways and regulatory molecules (Jiang et al. 2012). Ca 2+ Future Studies Knockout S100P expression (via siRNA), overexpress (via transfection) and treat cells – compare cell viability. Treat transfected cells with CaCl 2 to study Ca 2+ interactions. Co-transfection of HT29 cells with H2B-GFP fusion protein to confirm nuclear translocation. Observation of translocation with confocal microscopy. Figure 7. Plasmid map of pmCherry- N1(right). S100P (stop codon excluded) inserted 50bp upstream of mCherry gene. 15 AA linker sequence inserted between S100P and mCherry gene. MCS (below). b.b. Clone 100bp ladder 319bp 100bp PCR Ladder product 319bp Figure 1. Ribbon structure of mCherry Hypothesis A decreased expression level of S100P in HT29 cells will significantly decrease the LD 50 whereas an overexpression of S100P will significantly increase the LD bp