1. 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 )
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