1. The Effects of Deleting Cytosolic Thioredoxin Reductase on p53 Target Gene Expression Sydney Radding Dr. Gary Merrill Dept. Of Biochemistry/Biophysics

2. Cancer Responsible for 25% of all deaths Causes: – Carcinogens – Random errors in DNA replication – Inherited abnomalities

3. p53 in preventing cancer Once p53 is activated it can – Hold the cell in one phase of the cell cycle – Activate DNA repair if damage is minor and restart cell cycle – Or if DNA damage is irreparable, it will initiate programmed cell death (apoptosis)

4. Thioredoxin The p53 protein may be controlled by another type of protein known as thioredoxin Thioredoxins reduce other proteins by electron donation – Thioredoxin becomes inactive due to loss of electrons

5. Thioredoxin reductase Only known enzymes that reduce thioredoxin to its active state Mammals contain three types of thioredoxin reductase – Txnrd1: cytosolic and is in all tissues – Txnrd3: cytosolic but is only in testes – Txnrd2: mitochondrial and is in all tissues.

6. Thioredoxin reductase Thioredoxin NADPHNADP+ Transcription factors p53 Ribonucleotide reductase DNA synthesis Thioredoxin peroxidase Antioxidant P21, mdm2, Gadd45, Bax, PUMA Gene transcription

7. Hypothesis Cytosolic thioredoxin reductase is needed for efficient target gene activation by p53

8. Mutant mice Mice which do not produce any cytosolic thioredoxin reductase die in the womb after 7.5 days Dr. Merrill has designed a mouse that expresses Txnrd1 in all cells but liver cells Allowed for isolation of txnrd1 null liver cells for experimental use

9. Liver mRNA MutantWild-Type cDNA Reverse Transcriptase qRT-PCR mRNA levels p21Gadd45mdm2BaxPUMA Experiment

10. Data analysis Actin and GAPDH were used as controls to normalize for variation in mRNA recovery from each mouse Compared ratio of mRNA levels of each mouse by dividing gene mRNA levels by control mRNA levels Compared values of mutants to wildtypes to determine if there was a significant difference p <.05 by Student’s t-Test was judged to be significant

11. Predicted Results Higher mRNA levels of Gadd45, Bax-a, PUMA, p21, and mdm2 were predicted in wild-type mice when compared to the mutant mice

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14. Next step Induce the p53 pathway by giving the mice a dose of ionizing radiation known to activate the p53 response Repeat the same procedure as before to determine mRNA levels of the same proteins

15. Acknowledgements Howard Hughes Medical Institute Dr. Gary Merrill Dr. Kevin Ahern Cameron Long CGRB Core Lab