2.  Background information › PTEN (function, connection with breast cancer)  Objective  Experimental Approach and Results  Conclusion  Future research

3.  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

4.  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 › PTEN K62R, PTEN Y65C, and PTEN K125E › Cause mislocalization in the nucleus

5.  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.

6.  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

7.  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

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

9. 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

10.  Nuclear p53 level decreases in mutant

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

12.  DAPI used to stain nucleus  85% increase in staining intensity in wild type  Mutants have decreased intensity

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

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

16.  High levels of MDM2 and P- MDM2 in wild type PTEN  Low levels in ATP-binding mutants

17.  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

18.  DAPI stains nucleus  γ -H2AX is phosphoylated form of histone H2AX which signals DSBs

19. Test 1  Purpose: Examine basal levels of reactive oxygen species (ROS) production in cancer cells  Method: immunofluorescence

20.  Hoechst used to stain DNA  DCF represents ROS  Reduced ROS level in wild type  Increased ROS level in mutant

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

22.  mRNA level reduced in PTEN mutants  Suggests reduced p53 antioxidant activity

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

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

25.  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

26.  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