The Effects of Deleting Cytosolic Thioredoxin Reductase on p53 Target Gene Expression - PowerPoint PPT Presentation

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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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The effects of deleting cytosolic thioredoxin reductase on p53 target gene expression l.jpg

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

Sydney Radding

Dr. Gary MerrillDept. Of Biochemistry/Biophysics


Cancer l.jpg

Cancer

  • Responsible for 25% of all deaths

  • Causes:

    • Carcinogens

    • Random errors in DNA replication

    • Inherited abnomalities


P53 in preventing cancer l.jpg

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 l.jpg

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 l.jpg

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.


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NADPH

NADP+

Thioredoxin reductase

Thioredoxin

Transcriptionfactors

Thioredoxin

peroxidase

Ribonucleotide

reductase

p53

DNA synthesis

Antioxidant

Gene transcription

P21, mdm2, Gadd45, Bax, PUMA


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Hypothesis

Cytosolic thioredoxin reductase is needed for efficient target gene activation by p53


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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


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Experiment

Mutant

Wild-Type

Liver mRNA

Reverse Transcriptase

cDNA

qRT-PCR

mRNA levels

p21

Gadd45

mdm2

Bax

PUMA


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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


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    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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    *

    *


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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


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    Acknowledgements

    • Howard Hughes Medical Institute

    • Dr. Gary Merrill

    • Dr. Kevin Ahern

    • Cameron Long

    • CGRB Core Lab


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