The effect of cisplatin on Ribosome Biogenesis Olive D. Anagu, Emily Sutton, Victoria J. DeRose Department of Chemistry and Biochemistry, University of Oregon, Eugene, OR 3. Question 1. Cisplatin Function 5. Discussion Cisplatin (cis-diammine-dichloro-platinum), is a neutral inorganic, square planar complex that reacts with the purine bases of DNA to form DNA adducts, primarily intra strand crosslink adducts Question: How does cisplatin effect the transcription of rDNA in the nucleolus of the cell? How does cisplatin affect early processing of rRNA? Hypothesis: If cisplatin disrupts the transcription of rDNA to rRNA in ribosome biogenesis, then this disruption may cause a cytotoxic effect in the cell After multiple trials and error, was finally able to optimize primers that will be indicative of the amount of primary transcript present. Cancer is an abnormal growth of cells which tend to proliferate (grow) in an uncontrolled way 1 Cisplatin enters cell Ready to begin treatments: Cisplatin Oxaliplatin No treatment Control Actinomycin D Transcription 3 Apoptosis 2 Cisplatin-DNA Adducts rDNA with cisplatin-DNA adducts rRNA (Ribosomes) These intrastrand DNA adducts are form lesions that are partially responsible for the cytotoxic activity of cisplatin by interfering with processes such as replication and transcription 6. Future Directions 4. Methods Will look for the further processed forms of transcript and see if these stages amounts are affected. This will tell if ribosome biogenesis stress is in part inflicted during the processing steps rather than the transcription steps. Examine downstream effects on p53 activation and other indicators of ribosome biogenesis stress. Question: Is there a reduced level of rRNA primary transcript upon cisplatin treatment? Evidence indicates that there are many other ways that cisplatin may exert its cytotoxic effects. Elucidating these can help us better understand cisplatin’s mechanism of action, resistance to cisplatin, and its off-target effects. I. Cell Culture and Treatment Grew and treated Hela cells with differing concentration of cisplatin for different increments of time. 2. Ribosome Biogenesis II. Quantitative Reverse Transcipt-PCR Hela cells Reverse Transcriptase 7. Acknowledgements Ia. RNA Extraction The DeRose lab has generated “clickable” platinum reagents that allow for post-treatment fluorescent labeling of platinum in cells. These platinum compounds have been observed to localize to the nucleolus, the site of ribosome biogenesis in the cell. Thanks To: Emily Sutton DeRose Lab Dr. DeRose Summer Program for Undergraduate Research (SPUR) Chemistry Department at Whittier College NSF REU Site Program in Molecular Biosciences at the University of Oregon: NSF DBI/BIO 1460735) Reverse Transcribed extracted rRNA into cDNA, then used this to quantify relative amount of rRNA that has been transcribed DAPI Platinum Wirth, White, Moghaddam, et al (2015). Journal of the American Chemical Society rRNA cDNA 5 uM azidoplatin, 3h 1kb ladder 1kb ladder Primary Transcript TX Water Ctrl ETS1 ETS2 Water Ctrl ribosomalDNA 5’ ETS gone The main events of the nucleolus are pre-rRNA transcription, processing, and partial ribosomal assembly Processing Bands represent the presence and successful probing of ETS2 and ETS1 Assembled ribosome