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The Effects of Deleting Cytosolic Thioredoxin Reductase on p53 Target Gene Expression

The Effects of Deleting Cytosolic Thioredoxin Reductase on p53 Target Gene Expression. Sydney Radding Dr. Gary Merrill Dept. Of Biochemistry/Biophysics. Cancer. Responsible for 25% of all deaths Causes: Carcinogens Random errors in DNA replication Inherited abnomalities.

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The Effects of Deleting Cytosolic Thioredoxin Reductase on p53 Target Gene Expression

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  1. The Effects of Deleting Cytosolic Thioredoxin Reductase on p53 Target Gene Expression Sydney Radding Dr. Gary MerrillDept. 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. NADPH NADP+ Thioredoxin reductase Thioredoxin Transcriptionfactors Thioredoxin peroxidase Ribonucleotide reductase p53 DNA synthesis Antioxidant Gene transcription P21, mdm2, Gadd45, Bax, PUMA

  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. Experiment Mutant Wild-Type Liver mRNA Reverse Transcriptase cDNA qRT-PCR mRNA levels p21 Gadd45 mdm2 Bax PUMA

  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

  12. * *

  13. 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

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

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