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 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