cell biol 2008s - QUT · Tonicity • the effect that a solution will have on a cell • hypertonic...
Transcript of cell biol 2008s - QUT · Tonicity • the effect that a solution will have on a cell • hypertonic...
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Cells and cancerDr Lisa Chopin
School of Life Sciences, QUT
Leader, Ghrelin Research Group, IHBI,
QUT
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Cells and cancer
• The building blocks of cells
• The basic cell• Cancer• Cancer processes• Hormone dependent
cancer
http://www.newscentre.bham.ac.uk/press/2007/10/Cancer_Cold_Virus_03_10_07.shtml4
Cells• The building blocks of cells
– Proteins, carbohydrates and fats (lipids)
• The basic cell– The plasma membrane– Organelles– The cytoskeleton
• Cells form tissues– The nucleus
• DNA and RNA
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Building blocks of life
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Levels of structural organisation• many levels of organisation
• smallest = chemical level
• atoms
• molecules
•Cells
•Tissues
•organs
• largest = organism
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Building blocks – we are what we eat
• The body– Carbohydrates– Proteins– Lipids (fat)– Nucleic acids– Water – Vitamins and
minerals
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• sugar and starches
• Large part of our diet
• make up 1-2% of a cell•C = carbon
•H = Hydrogen
•O = oxygen
• form a ring structure
•Monosaccharide•Simple sugar
•Building blocks of sugars
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•Disaccharides •= 2 sugars joined•eg lactose (milk)
•Polysaccharides•Poly = many•Long chains of simple sugars
•Starch = plant polymer of glucose
•potatoes 10
Protein
• We eat 125g/day• adults need 40-50g/day• replace proteins• 20-60g/day lost
– secretions– shedding of gut cells into
lumen
• In diet– Fish, meat, eggs, nuts
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•Building blocks = amino acids
•20 amino acids in animals
•Joined to make proteins
•Can form very large polymers = proteins•Can form complex structures •or function as amino acids•Short = peptides
http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2005/Heiner/hemoglobin.html 12
•Basic material making up the body structure
•Hair •Fingernails•Skin•Muscle
•Also important for function•Hormones•Enzymes•Neurotransmitters•Oxygen transport•antibodies
http://srs.dl.ac.uk/Annual_Reports/AnRep95_96/Protein%20crystallography.html
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Lipids (fats)•25-160g/day
•Plant and animal fat
•Not soluble in water
•C, H, O (P)
•Lipids
•Triglycerides
•Phospholipids
•steroids14
Lipids (fats)
•Triglycerides•Most common fats•Building blocks
•Fatty acids•3 Long chains
•+ 1 Glycerol
http://www.chemistryland.com/ElementarySchool/BuildingBlocks/BuildingOrganic.htm
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Phospholipids
• Modified triglycerides• 2 fatty acid chains• Phosphate group (P)• Special properties• Very important in biology
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Cells• all living things made of cells
– (if they can replicate)
•Trillions in the body•Hundreds of different types
http://www.icr.ac.uk/ieu/projects/CancerScienceWebsite/AnimGallery/AnimGallery.htm
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The basic cellPlasma membrane
(cell membrane)
Cytoplasm
Semi-fluid
Nucleus
Control centre18
The plasma membrane• made up of phospholipids
•Head = likes water
•Tail = hates water
• In water (in the body)
•tails repel water
•Clump together
http://www.aber.ac.uk/gwydd-cym/cellbiol/cellmembrane/index.htm
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Plasma membrane
• contains other lipids
• cholesterol
•Proteins
•Sugars
Cytoplasm = watery
Extracellular fluid = watery environment
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• Cytoplasm– Intracellular fluid
• Outside the cell– Extracellular fluid– Different to
intracellular fluid– Ions– Nutrients– Waste
• Plasma membrane– Barrier between
intracellular fluid + extracellular fluid
Extracellular fluid
Intracellular fluid
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• Some substance must pass between 2– Nutrients into cell– Wastes out of cell
• Not freely as they would become equal
• Plasma membrane = selective barrier
• = selectively permeable
Extracellular fluid
Intracellular fluid
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Passive transport –simple diffusion
• Important in all cells
• Molecules always moving– Thermal motion
• Collide with each other
• Scatter evenly throughout
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Brownian motion
http://www.microscopy-uk.org.uk/dww/home/hombrown.htm24
Passive transport - Diffusion• Molecules move from higher
concentration lower concentration– collisions
• Until equally distributed– Reach equilibrium
• concentration inside and outside cell and inside cell are equal
• Molecules diffuse along their concentration gradient– Chemical driving force
http://bio.winona.edu/berg/ANIMTNS/Directry.htm
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Osmosis
• http://www.biologycorner.com/resources/osmosis.gif
• Water diffuses from high water concentration to low water concentration– Water diffuses by
osmosis towards higher solute concentration
• Membrane is semi-permeable– Not freely
permeable to solutes
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Tonicity• the effect that a solution will have on a cell
• hypertonic (Hyper = above)•Higher solute concentration than in the cell
•Hyperosmotic
•Water moves out by osmosis
•Iso-tonic (iso = the same)•Iso-osmotic
•Same solute concentration
•Hypotonic (hypo = below)•Less solute in the solution than in the cell
•Hypo-osmotic
•Water moves in by osmosis
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The cytoplasm • Cytoplasm
– Cellular material – Between nucleus and
plasma membrane• cytosol
– Fluid• Cytoplasmic organelles• cytoskeleton
http://www.aber.ac.uk/gwydd-cym/cellbiol/transport/index.htm
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Cytoplasmic organelles• little organs• Cell machinery• Include
– Mitochondria– ribosomes– Lysosomes– Endoplasmic
reticulum– Golgi apparatus
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Cytoplasmic organelles -Mitochondria
• power plant of cell•energy source for cell
•Makes ATP
•Ancient Bacterial origin•Has its own DNA
•Inherit from mother
http://www.mitochondrial.net/ 30
Energy
• ATP is made in mitochondria – adenosine triphosphate– Energy stored in the cell as ATP
• Made from energy from food– Carbohydrates– Fats products• Amino acids
• Products – energy (ATP) + O2+ water
adenosine P P P
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Energy
adenosine P P P
adenosine P P P+ + EnergyADP
•Break High energy bonds
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Cytoplasmic organelles •Organelles work
together • Produce• Store• export substances• Degrade substances
http://cmweb.pvschools.net/~bbecke/newell/Cells.html
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Endoplasmic reticulum (ER)• “within cell
network”• Network of tubes
and membranes• Fluid filled sacs • Continuous with
nuclear membrane
• 2 types– Smooth – Rough
http://www.unipv.it/webbio/anatcomp/freitas/2006-2007/smooth%20&%20rough.jpg
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• studded on outside with ribosomes
•Proteins synthesised
•On ribosomes
•Outside of RER
•Enter RER
•Processed and modified
Rough Endoplasmic reticulum (RER)
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Cytoplasmic organelles -ribosomes
• small
•Dark staining granules
•Contain RNA
•Ribosomal RNA
•Site of protein synthesis
• bound or free
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• extension of RER
• No ribosomes
• No protein synthesis
•Many roles •Lipid metabolism and synthesis
•Steroid synthesis
•Drug detoxification
Smooth endoplasmic reticulum (SER)
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Golgi apparatus• membranous sacs
• Stacks of dinner plates
•Tiny vesicles•Fluid filled spheres
•Function:•Modify
•Concentrate
•Package
ER products
• Proteins
•lipids
http://www.nih.gov/sigs/cellbio/38
Golgi• protein/lipid vesicles reach Golgi from RER
•Fuse with receiving side of Golgi
•Contents emptied into Golgi
•Move through Golgi
•Enzymes modify proteins
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3 Golgi pathways
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The cytoskeleton• Cell skeleton• Support
structures of the cell
• Attach cells to each other
• Allows cells to move
• To move things in the cell
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The cytoskeleton
• 3 types of cytoskeletal elements– 1. microtubules– 2. microfilaments– 3. intermediate
filaments• Support rods• Network in the
cell42
The cytoskeleton-microfilaments•actin
•Functions:
•Mechanical strength for cell
•Attach cytoplasm to the cytoplasmic membrane
•Needed for cell movement (muscle)
http://www.cerebrospinalfluidresearch.com/content/3/1/13/figure/F1
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The cytoskeleton-microtubules
• Function– Shape of cell– Strength– Motor- move organelles
along microtubules– Form structures
• Cilia• flagella
– Cell divisionMicrotubules in green, actin in red
http://migration.wordpress.com/2007/07/11/basics-the-cytoskeleton/
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The cytoskeleton-intermediate filaments
• intermediate between the 2
• rope-like
•Fibrous
•Very strong
•Resist forces applied to cell
•Cell shapehttp://www.olympusfluoview.com/applications/protocols/cellsintermediatefilaments.html
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Cytoskeleton – joining cells together
• Epithelium = a tissue– 90% of human
cancers are epithelial• Sheets of cells
– Skin– Mucous membranes
• linings• Nests of cells
– glands
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Epithelium• Made from epithelial cells• Forms glands• Covers all surfaces of the
body• Boundary between
environments– Skin and outside body– Bladder wall and urine in
the bladder
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Epithelium - characteristics
• Many cells, tightly packed
• Make special contacts with each other– Tight junctions– Desmosomes
• Connect with cytoskeleton
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Tight junctions and
desmosomes–attach epithelial
cells together
• Tight junctions - like a zipper, Proteins in the plasma membrane, Joins plasma membranes• Desmosomes – Anchors, Like buttons and thread,
Attach to cytoskeleton
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Extracellular matrix (ECM)• Epithelium sits
on ECM• Lots of space • Extracellular
matrix secreted by cells– Water– Proteins– Carbohydrates
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Basement membrane
• Specialised extracellular matrix
• Connects cells to connective tissue underneath– Glycoprotein
• Contains no cells• Protects epithelial
layer• Helps resist forces• Anchors cells• Affects cell
behaviour
Basement membrane
Epithelial cells
http://fig.cox.miami.edu/~cmallery/150/memb/epithelial_cell.jpg
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Collagen
• Major component– Basement
membrane– ECM
• Protein– Long polymer
http://www.accessexcellence.org/RC/VL/GG/ecb/collagen_elastin.html
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Glands• Cell or group of
epithelial cells that make a product
• Product is exported – Secreted
• Product = a secretion• 2 types of glands
– Endocrine• Produce hormones
– Exocrine• Many types• Sweat, digestive enzymes,
saliva, mucus
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Mucus• Slimy coating protects
epithelial surfaces• Protective
– Eg stomach protected from acid
• Lubricating– Saliva– Swallow food
• Mucins– Protein with many sugar
groups coating it
http://712designs.com/animations.htm 54
The nucleusPlasma membrane
(cell membrane)
Cytoplasm
Semi-fluid
Nucleus
Control centre
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The nucleus
• Cell’s computer• Stores genetic
information• Instructions for
making proteins• Usually round or
oval• Centre of the cell
http://www.york.ac.uk/res/sots/activities/diydna.htm56
Nucleic acids
• DNA• RNA• Largest
molecules in the body
• building blocks = nucleotides
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DNA and RNA• nucleotides
•phosphate
•Sugar•Deoxyribose
•ribose
•Base = code•AGCT – DNA
•AGCU - RNA 58
• Double stranded• Double helix• carries genetic
material• Genes
http://www.scar.utoronto.ca/~olaveson/A01F-Fall2007.html
http://www.biologycorner.com/bio1/DNA.html
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RNA• No double helix• Single stranded
– Can base-pair with itself
• Different roles– Protein synthesis– Gene regulation
• May be very important– Differences between
species
http://www.biologycorner.com/bio1/DNA.html60
Nucleus -chromatin
•Chromatin•DNA stored in the nucleus as chromatin•Packaged DNA•Very long molecules
http://openlearn.open.ac.uk/file.php/2645/S377_1_039i.jpg
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Chromosomes
• DNA stored as chromatin• Even more packaged into chromosomes
for cell division• Chromosomes 50 000 times shorter than
DNA unpackaged
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Cell division = mitosis• Cell proliferation
– Happens about 25 million times per second
• Depends on cell type– Embryo
• Very rapid– Gut cells and skin cells
• Rapidly turnover• Prone to damage
– Brain cells• Up to 100 years old
– Liver cells• In response to signal
http://www.cancerhelp.org.uk/help/default.asp?page=95http://www.biophysik.uni-bremen.de/radmacher/animations.html 64
Cell division
• blue = DNA
• green = Microtubules
• red = intermediate filaments http://www.biophysik.uni-bremen.de/radmacher/animations.html
• cells divide into 2 daughter cells
•Double DNA content
• DNA condenses into chromosomes
•Change in cytoskeleton
•Forms mitotic spindle
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Cell division
• chromosomes moved to opposite poles
• nucleus reforms
• cells form 2 daughter cells
•Equal number of chromosomes
•Cells growhttp://www.biologycorner.com/bio1/cellcycle.html http://www.learner.org/channel/courses/essential/life/session3/closer1.html#animation
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The cell cycle – control of cell division
• Very tight control
• Growth factors• If DNA
damaged cycle is halted
http://www.emc.maricopa.edu/faculty/farabee/BIOBK/BioBookmito.html
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Protein synthesis
• DNA (genes) code for all proteins
• Must be decoded• DNA RNA protein
• Controlled by nucleus
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DNA RNA• Transcription• DNA copied into
RNA• mRNA
– Messenger RNA• RNA is further
processed• Exported from the
nucleus
http://www.biologycorner.com/bio1/DNA.html
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RNA protein• Translation• RNA (nucleic acids)
translated proteins (amino acids)
• Made by ribosomes• Into rough
endoplasmic reticulum
• To Golgi for processing
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DNA damage causes cancer• DNA damage
– Sunlight– radiation– Chemicals– Smoke– Car exhaust– Normal life processes
• Using oxygen!
• A few cancers inherited– Mutation in gene– Further damage required
• Often no clear causehttp://www.dmacdigest.com/cancer.html
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DNA damage• Repeated damage to DNA
– Constantly occurring• DNA in one cell damaged 10 000
times a day– More damage with age
• Protection against cancer– DNA repair mechanisms– Cells commit suicide
• apoptosis– Immune system destroys cells
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Apoptosis• Active process• RNA and proteins
made• Break down of
cytokeleton• Breakdown of nucleus
and DNA laddering• Cytoplasm thrown off• Mitochondria involved• Engulfed by white
blood cellshttp://www.dadamo.com/wiki/wiki.pl/Apoptosis http://student.ccbcmd.edu/courses/bio141/lecguide/unit5/cellular/cmidefense/ctls/ctlapop.html
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• Proliferation
• Invasion
• Migration
• Anti-apoptosis
• Angiogenesis
• Metastasis
Changes in cancer cell behaviour – the hallmarks of cancer
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Proliferation• Cell division usually
tightly controlled• Cancer = uncontrolled
cell division– Divide rapidly– Don’t mature properly
• Tumour = mass of cells– Benign– malignant
http://www.bme.gatech.edu/vcl/Tissue_Engineering/Background/5_cell_seeding.htm
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Cell death
• Cancer cells become immortal
• Evade apoptosis– Don’t undergo cell
death when DNA is damaged
• = survival
http://www.cat.cc.md.us/courses/bio141/lecguide/unit3/intro/nk/nkapop.html 78
Angiogenesis
• Synthesis of new blood vessels– Angio– genesis
• Normal – Growth as children
• Abnormal– Cancer
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Angiogenesis • Cells need a blood supply for oxygen to generate energy
• Cancer cells outgrow their blood supply– Start to starve– Make energy without
oxygen– Need a lot of energy
due to proliferation• Cancer cells send
signals• Encourage more
blood vessels to form and grow = angiogenesis
http://www.gene.com/gene/research/focusareas/oncology/angiogenesis.html
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Cell Migration• Some cells can move• Cytoskeleton
changes• Forms new anchors
– adhesions• to crawl along
extracellular matrix– Action of actin– Part of cytoskeleton
http://www.biolsci.org/v03p0303.htm
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Some normal human cells can crawl
• Cells in the embryo• Brain development• Cells in wound healing
and blood clotting– Platelets– Skin cells
• White blood cells
http://www.rockefeller.edu/labheads/hatten/mechanism.htmlhttp://www.youtube.com/watch?v=eTWc4PFSpzQ
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Cancer cell migration• Cancer cells migrate
– Most normal cells dont• Must break anchors
– Neighbouring cells– Extracellular matrix
• Invasion– through
• the basement membrane• Extracellular matrix
– Enzymes break down structures
• Pathway for cell migration
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Metastasis• Migrating cells
reach blood vessel or lymphatic vessel– Travel to distant
sites– = metastasis
• Few survive• Exit vessel• Colonise distant
sites– Form metastases
http://www.nature.com/embor/journal/v7/n11/full/7400839.html
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Metastasis
• Major cause of cancer death
• Spread of cancer cells to distant sites
• No cure
http://www.nature.com/nrc/journal/v3/n1/fig_tab/nrc967_F1.html
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Hormone dependent cancers
• Prostate and breast cancer
• Prostate cancer – most commonly diagnosed
cancer in men– Affects 1 in 11 Australian
men by the age of 80• Androgens stimulate
growth– testosterone
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Hormone dependent cancers
• Breast cancer – most commonly
diagnosed cancer in women
– Affects about 1 in 11 Australian women
• Oestrogens stimulate growth
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ANDROGENS in ANDROGENS in males males (testosterone)(testosterone)
EPITHELIUM
Extracellular matrix
GROWTH FACTORSGROWTH FACTORS
Basement membrane OESTROGEN OESTROGEN
in femalesin females
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Ghrelin
• Peptide hormone • Discovered in 1999• Causes growth hormone release• Role in increasing appetite
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• http://www.gghjournal.com/volume20/1/featureArticle.cfm
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Ghrelin action• Acts by binding
a receptor on the cell surface– Protein
embedded in the plasma membrane
– Cannot pass through the lipid bilayer
• Signal passed on through the receptor– Signaling
molecules in the cell
• Study role of receptor in cancer
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Ghrelin is made in prostate and breast cancer cells
• Stain cells in cancer specimens – ghrelin peptide present in tissues– Epithelial cells
negative
positive92
ANDROGENS in ANDROGENS in males males (testosterone)(testosterone)
EPITHELIUM
Extracellular matrix
GhrelinGhrelin
Basement membrane OESTROGEN OESTROGEN
in femalesin females
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Proliferation assays
• Breast and prostate cancer cells produce ghrelin– Cancer growth factor?
• Grow breast and prostate cancer cells – 37C– Media with nutrients– Immortal– Sterile conditions
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Grow in normal culture conditions for 4, 24, 48, 72 and 96 hrs
2000cells
Media blank
Cells
Add WST-1
2 hrs
Read plate at 650-405 nm
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Proliferation assays
• Breast and prostate cancer cells grow faster in the test tube when treated with ghrelin
05
101520253035404550
0 0.1 1 10 100 1000
Ghrelin concentration (nM)
% A
bove
con
trol
*
**
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Migration - ghrelin
• Migration assays• Double well
– Cells migrate through a membrane
– 8μm pores – Cells rearrange
cytoskeleton to pass through
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Tissue cultureinsert
Well
Cells in medium
20% FCS
Remove unmigrated cellsFix and stain migrated cells
Readplate at595 nm
24hr Incubation
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Migration assays
• Cancer cells migrate faster in response to ghrelin treatment– Breast cancer– Ovarian cancer
Mean SKOV3 Migration Over 24hr
00.10.20.30.40.50.60.70.80.9
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media 10nMGhrelin
100nMGhrelin
10nM Ghr10nM Obe
10nM Ghr100nM Obe
10nMObestatin
100nMObestatin
Treatment
Abs
orba
nce * * * * *
B. Mean SKOV3 Migration Over 24hr
00.10.20.30.40.50.60.70.80.9
1
media 10nMGhrelin
100nMGhrelin
10nM Ghr10nM Obe
10nM Ghr100nM Obe
10nMObestatin
100nMObestatin
Treatment
Abs
orba
nce * * * * *
B.
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Invasion assays
Matrigel
PBSTissue cultureinsert
Well
Cells in medium
20% FCS
Remove uninvaded cells and MatrigelFix and stain invaded cells
Readplate at595 nm
24hr Incubation
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Ghrelin
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Cancer biomarkers• Compare proteins
produced by normal breast and breast cancer
• Breast cancer very varied disease
• At least 5 different diseases– Need to be able to diagnose
better– Find new tests– New treatments
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References and source of figures
• Marieb (2004) Human anatomy and physiology 6th edition
• Martini (2004) Fundamentals of human anatomy and physiology
• http://www.cancerquest.org/index.cfm?page=3102#
• http://cellbase.ascb.org/education.html• Thanks to: Carina Walpole, Rachael Murray,
Penny Jeffery,