Background information › PTEN (function, connection with breast cancer) Objective Experimental...
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Transcript of Background information › PTEN (function, connection with breast cancer) Objective Experimental...
![Page 1: Background information › PTEN (function, connection with breast cancer) Objective Experimental Approach and Results Conclusion Future research.](https://reader035.fdocuments.in/reader035/viewer/2022062801/56649e4c5503460f94b418bd/html5/thumbnails/1.jpg)
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Background information› PTEN (function, connection with breast
cancer) Objective Experimental Approach and Results Conclusion Future research
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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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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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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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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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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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Only wild type is able to decrease levels of P-AKT and cyclin D1
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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
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Nuclear p53 level decreases in mutant
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Test 2Purpose: Reconfirm results of test 1Method: same cells were examined using immunofluorescence confocal analyses to determine p53 levels
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DAPI used to stain nucleus
85% increase in staining intensity in wild type
Mutants have decreased intensity
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Test 2, continuedPurpose: Determine whether p53 is translated in ATP-binding mutantsMethod: Add proteasome inhibitor and use immunofluorescence confocal analyses
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Test 3Purpose: Investigate mechanism of p53 degradationMethod: observed level of MDM2 and phospho-MDM2 in MCF-7 cells using immunoblotting
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High levels of MDM2 and P-MDM2 in wild type PTEN
Low levels in ATP-binding mutants
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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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DAPI stains nucleus γ-H2AX is
phosphoylated form of histone H2AX which signals DSBs
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Test 1Purpose: Examine basal levels of reactive oxygen species (ROS) production in cancer cellsMethod: immunofluorescence
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Hoechst used to stain DNA
DCF represents ROS Reduced ROS level
in wild type Increased ROS level
in mutant
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Test 2Purpose: Compare mRNA levels of tumor protein 53-induced nuclear protein (TP53INP), which mediates p53 antioxidant functionMethod: qRT-PCR
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mRNA level reduced in PTEN mutants
Suggests reduced p53 antioxidant activity
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Test 3Purpose: Compare quantities of SOD1, a superoxide dismutase (an antioxidative enzyme)Method: Western Blot
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Increased levels in mutant cells
Researchers conclude that mutation leads to increased levels, but aberrant expression
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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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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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