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
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.
NADPH
NADP+
Thioredoxin reductase
Thioredoxin
Thioredoxin
peroxidase
Transcription factors
Ribonucleotide
reductase
p53
DNA synthesis
Antioxidant
Gene transcription
P21, mdm2, Gadd45, Bax, PUMA
Hypothesis
Cytosolic 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
Experiment
Mutant
Wild-Type
Liver mRNA
Reverse Transcriptase
cDNA
qRT-PCR
mRNA levels
p21
Gadd45
mdm2
Bax
PUMA
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
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Howard Hughes Medical Institute
Dr. Gary Merrill
Dr. Kevin Ahern
Cameron Long
CGRB Core Lab