Background information› PTEN (function, connection with breast

cancer) Objective Experimental Approach and Results Conclusion Future research

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

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

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.

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

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

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

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

› p53: induces apoptosis

Nuclear p53 level decreases in mutant

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

DAPI used to stain nucleus

85% increase in staining intensity in wild type

Mutants have decreased intensity

Test 2, continuedPurpose: Determine whether p53 is translated in ATP-binding mutantsMethod: Add proteasome inhibitor and use immunofluorescence confocal analyses

Test 3Purpose: Investigate mechanism of p53 degradationMethod: observed level of MDM2 and phospho-MDM2 in MCF-7 cells using immunoblotting

High levels of MDM2 and P-MDM2 in wild type PTEN

Low levels in ATP-binding mutants

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

DAPI stains nucleus γ-H2AX is

phosphoylated form of histone H2AX which signals DSBs

Test 1Purpose: Examine basal levels of reactive oxygen species (ROS) production in cancer cellsMethod: immunofluorescence

Hoechst used to stain DNA

DCF represents ROS Reduced ROS level

in wild type Increased ROS level

in mutant

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

mRNA level reduced in PTEN mutants

Suggests reduced p53 antioxidant activity

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

Increased levels in mutant cells

Researchers conclude that mutation leads to increased levels, but aberrant expression

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

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