Naturally occurring germline and tumor-associated mutations within the ATP-binding motifs of PTEN lead to oxidative damage of DNA associated with decreased nuclear p53 by He, X., Ni, Y., Wang, Y., Romigh, T. and Eng, C.

1
Naturally occurring germline and
tumor-associated mutations within the ATP-binding
motifs of PTEN lead to oxidative damage of DNA
associated with decreased nuclear p53 by He, X.,
Ni, Y., Wang, Y., Romigh, T. and Eng, C.

• Citation
• He, X., Ni, Y., Wang, Y., Romigh, T. and Eng, C.
  (2011) Naturally occurring germline and
  tumor-associated mutations within the ATP-binding
  motifs of PTEN lead to oxidative damage of DNA
  associated with decreased nuclear p53. Human
  Molecular Genetics, 20, 8089.
• Brittany Byerley
• Biol 506
• December 5, 2011

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Outline

• Background information
• PTEN (function, connection with breast cancer)
• Objective
• Experimental Approach and Results
• Conclusion
• Future research

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Background Information

• PTEN (phosphatase and tensin homolog)
• Function tumor supression (regulate cell cycle
  and apoptosis)
• Mutant forms shown to be involved in breast cancer

Courtesy of http//ghr.nlm.nih.gov
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Background Information, Cont.

• Cowden Syndrome (CS)
• Greater risk of breast cancer (25-50 13 for
  general population)
• Earlier age of onset
• Caused by mutations in PTEN
• PTEN with mutation in ATP-binding motif
• PTENK62R, PTENY65C, and PTENK125E
• Cause mislocalization in the nucleus

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Objective

• Researchers sought to analyze the functional
  consequences of nuclear-cytoplasmic
  mislocalization of these PTEN ATP-binding
  mutants, and whether these consequences may
  induce breast carcinogenesis.

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Experimental Approach and Methods

• Effects on cell cycle regulation pathways
• Effects on p53 levels
• Amount of double stranded breaks (DSBs)
• Levels of reactive oxygen species (ROS)
• Effects on antioxidant activity

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ATP-binding motif mutants abrogate PTENs
tumor-suppressive capabilities on cell signaling
pathways

• Purpose Determine the effects of increased
  levels of PTEN on cell cycle regulation pathways
• Method immunoblotting
• P-AKT cell cycle arrest, lipid phosphatase
• Cyclin D1 G1/S checkpoint

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Levels of P-AKT and Cyclin D1

• Only wild type is able to decrease levels of
  P-AKT and cyclin D1

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Mislocalized PTEN ATP-binding mutants reduce p53
levels in the nucleus

• Test 1
• Purpose Determine effect of overexpression of
  ATP-binding mutants on apoptosis and G1/S cell
  ratio
• Method Subcellular fractionation of MCF-7 cells
  (human breast carcinoma line)
• p53 induces apoptosis

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Levels and location of p53

• Nuclear p53 level decreases in mutant

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Mislocalized PTEN ATP-binding mutants reduce p53
levels in the nucleus

• Test 2
• Purpose Reconfirm results of test 1
• Method same cells were examined using
  immunofluorescence confocal analyses to determine
  p53 levels

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Intensity and location of p53

• DAPI used to stain nucleus
• 85 increase in staining intensity in wild type
• Mutants have decreased intensity

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Mislocalized PTEN ATP-binding mutants reduce p53
levels in the nucleus

• Test 2, continued
• Purpose Determine whether p53 is translated in
  ATP-binding mutants
• Method Add proteasome inhibitor and use
  immunofluorescence confocal analyses

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Without proteasome inhibitor
With proteasome inhibitor
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Mislocalized PTEN ATP-binding mutants reduce p53
levels in the nucleus

• Test 3
• Purpose Investigate mechanism of p53 degradation
• Method observed level of MDM2 and phospho-MDM2
  in MCF-7 cells using immunoblotting

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Levels of p53, MDM2, and P-MDM2

• High levels of MDM2 and P-MDM2 in wild type PTEN
• Low levels in ATP-binding mutants

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PTEN ATP-binding mutations cause accumulated DNA
double-stranded breaks

• Purpose Test DNA for double-stranded breaks
  (DSBs)
• Method senile cells were treated with histone
  H2AX, which binds to broken DNA. DSBs were then
  detected using immunofluorescence microscopy

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Show amount of Double-Stranded Breaks (DSBs)

• DAPI stains nucleus
• ?-H2AX is phosphoylated form of histone H2AX
  which signals DSBs

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Breast caner cells overexpressing PTEN
ATP-binding mutations are under oxidative stress

• Test 1
• Purpose Examine basal levels of reactive oxygen
  species (ROS) production in cancer cells
• Method immunofluorescence

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Reactive Oxygen Species (ROS) levels

• Hoechst used to stain DNA
• DCF represents ROS
• Reduced ROS level in wild type
• Increased ROS level in mutant

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Breast caner cells overexpressing PTEN
ATP-binding mutations are under oxidative stress

• Test 2
• Purpose Compare mRNA levels of tumor protein
  53-induced nuclear protein (TP53INP), which
  mediates p53 antioxidant function
• Method qRT-PCR

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mRNA level of TP53INP

• mRNA level reduced in PTEN mutants
• Suggests reduced p53 antioxidant activity

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Breast caner cells overexpressing PTEN
ATP-binding mutations are under oxidative stress

• Test 3
• Purpose Compare quantities of SOD1, a
  superoxide dismutase (an antioxidative enzyme)
• Method Western Blot

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Levels of SOD1 enzyme

• Increased levels in mutant cells
• Researchers conclude that mutation leads to
  increased levels, but aberrant expression

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Conclusions

• PTEN ATP-binding mutants have a reduced ability
  to regulate cell cycle, as evidenced by P-AKT and
  cyclin D levels
• Mutation reduces nuclear p53 levels through a
  MDM2 independent mechanism
• Researchers speculate that this is due to an
  inability to bind to p300 and stabilize p53
  through acetylation
• Mutant cells show increased levels of ROS,
  leading to oxidative stress.
• May lead to DSBs
• Important role in breast tumorigenesis

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Further Research

• Determine mechanism by which mutation increases
  frequency of DSBs
• Determine mechanism by which p53 is degraded in
  mutant cells
• Research mechanism by which oxidative stress is
  increased in cells

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Questions?