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