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The Effects of Deleting Cytosolic Thioredoxin Reductase on p53
Target Gene Expression
Sydney RaddingDr. Gary Merrill
Dept. Of Biochemistry/Biophysics
Cancer
• Responsible for 25% of all deaths
• Causes:– Carcinogens– Random errors in DNA
replication– Inherited abnomalities
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)
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
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.
Thioredoxin reductase
Thioredoxin
NADPH NADP+
Transcription factors
p53
Ribonucleotidereductase
DNA synthesis
Thioredoxinperoxidase
Antioxidant
P21, mdm2, Gadd45, Bax, PUMA
Gene transcription
HypothesisCytosolic thioredoxin reductase is needed for efficient target gene activation by p53
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
Liver mRNA
Mutant Wild-Type
cDNA
Reverse Transcriptase
qRT-PCR
mRNA levels
p21 Gadd45 mdm2 Bax PUMA
Experiment
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
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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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
Acknowledgements
• Howard Hughes Medical Institute• Dr. Gary Merrill• Dr. Kevin Ahern• Cameron Long• CGRB Core Lab