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Biochemistry,GenesandDisease
BiochemistryGenesandDisease
CANCER
MatthewVerheyden
Email:Matthew.Verheyden@uts.edu.au
DrNajahNassif
SchoolLifeSciences,FacultyofScience
L
LectureOverview
O
i
Thecellcycleandapoptosis
Propertiesofcancercells
P
i
f
ll
Theinterplayofgeneticandenvironmentalfactorsin
p y g
thedevelopmentofcancer
Classesofcancergenes(oncogenesandtumour
Cl
f
(
dt
suppressorgenes)
TheroleofPTEN genemutationsincoloncancer
(laboratoryproject)
N.NASSIF
WHATISCANCER?
Cancerisadisorderofuncontrolledcellgrowth
(imbalancebetweencellproliferation/growthandcell
(imbalance
between cell proliferation/growth and cell
death)
Cellsarenormallyprogrammed todevelop,grow,
leadstocancer
N.NASSIF
TheCell
Cycleand
NormalCell
Growth
Occursin4phases:
Occurs in 4 phases:
G1
S
G2
M
TheLifeandDeathofaCell
NormalCellGrowthandDivision
Eukaryoticcellshaveuniversalregulatorymechanisms
for controlling cell division
forcontrollingcelldivision
Proteinkinasesandproteinphosphorylationevents
regulatethetimingmechanismsthatdetermineentry
l
h i i
h i
h d
i
intocelldivisionandorderlypassagethroughthecycle
Innormalcells,cellreplicationisstoppedtoallowthe
repairofanyDNAdamage
Ifthisdoesnotoccur,cellreplicationwillcontinueand
cells will replicate with damaged DNA
cellswillreplicatewithdamagedDNA
N.NASSIF
TheLifeandDeathofaCell
ControlofCellDivision
Checkpoints are
mechanismsto
p g
halttheprogress
ofreplication if
chromosomal
DNAisdamaged
g
orcertain
processesare
aberrant
Checkpoints
ensurethateach
stageof
replicationis
completeand
accurate
N.NASSIF
TheLife
andDeath
ofaCell
Dormant
((G0)
Cell division
Growth
cell
CellDeath
D th
Death
Necrosis
Autophagy
Apoptosis
Extrinsic
receptormediated
N.NASSIF
Intrinsic
Senescence
Granzyme
TheLifeandDeathofaCell
Apoptosis(ProgrammedCellDeath)
Programmedcelldeath ageneticallycontrolled
regulatedintracellularprogram
Playsacriticalroleindevelopmentbyremoving
unwantedcells
Contrasttonecrosis(celldeathduetoinjury)
N.NASSIF
CharacteristicsofApoptosis
Itischaracterisedbydistinctmorphological
h
db d
h l
l
andbiochemicalchanges
Apoptosis
Necrosis
Activeprocess
p
Passiveprocess(?)
Passive process (?)
Cellshrinkage
Progressive
Membraneblebbing
breakdownof
Chromatincondensation cellularstructure
DNAfragmentation
Doesnotinducean
inflammatoryresponse
N.NASSIF
Necrosisvs Apoptosis
N.NASSIF
10
NormalCellularControls
Signals from
environment
GROW
Reductive
capacity
REST
Energy state
Membrane
M
b
integrity
DIE
mitochondria
Level of
denatured
proteins
State of DNA
nucleus
cytoplasm
environment
N.NASSIF
11
CANCERDevelopment
Oncogenesis orTumorigenesis
Cancerincludesaclassofdiseasesinwhichcells
displayuncontrolledgrowth,invasion
display
uncontrolled growth invasion and
and
metastasis (spreadtootherbodylocations)
Oncogenesis ortumorigenesisistheprocess
wherebynormalcellsbecometransformed into
cancercells
Itisusuallyamultistepprocessinvolvinggenetic
It is usually a multi step process involving genetic
mutations/cellularchangesateachstep
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12
CANCERDevelopment
p
Oncogenesis orTumorigenesis
ATUMOUR isapopulationofcellsresultingfrom
localised,unregulatedgrowthanddivisionofasingle
localised
unregulated growth and division of a single
cell
Tumour cellsarecharacterisedbyunregulatedmitotic
activity,underconditionsthatwouldnormallyrestrict
celldivision
Alsoreferredtoasaneoplasmora
Al
f
dt
l
cancer (also
( l
carcinoma,sarcoma,lymphoma,leukaemia)
N.NASSIF
13
CANCERDevelopment
Oncogenesis orTumorigenesis
Factorsnotedtocauseincreasedcancerincidence
include: viruses,chemicalexposure,UVlightexposure,
,
p
,
g
p
,
radiation,somaticmutationsandinheritedgenetic
abnormalities
Howdothesefactorscauseanormalcelltobecome
malignant?
Severaltheoriesincluded:
((a)abnormalcelldifferentiation
) b
l ll d ff
(b)humantumourviruses
(c) mutagens causing damage to cellular DNA
(c)mutagenscausingdamagetocellularDNA
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14
CancerStatistics
CancerRelatedDeathsinUSA(in2008)
F
Females
l
Males
M
l
294,120CancerRelatedDeaths
271,530CancerRelatedDeaths
Prostate
Breast
Colonandrectum
Colonandrectum
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15
HallmarksofCancerCells
1.LossofContactInhibition
Cancercellsandnormalcellscanbecultured inthe
laboratory
Normalcellsgrownontissueculturedishesproliferate
untilthesurfaceofthedishiscoveredbyasinglelayer
il h
f
f h di h i
db
i l l
ofcellstouchingeachother.Thenmitosisceases.This
phenomenon is called contact inhibition
phenomenoniscalledcontactinhibition
CancercellsDONOTshowcontactinhibition.Oncethe
surfaceofthedishiscovered,cellscontinuetodivide
andgrowovereachother
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16
HallmarksofCancerCells
1.LossofContactInhibition
ContactInhibition
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17
HallmarksofCancerCells
2.CellularImmortality
Normal(nontumour)cellshaveonlyalimited
capacityforcelldivision inculture
Tumour cellsplacedincultureconditions,inthe
laboratory,areoftenimmortal
Theywillgrowanddivideindefinitelyaslongas
nutrientconditionsareprovided
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18
HallmarksofCancerCells
3.AbnormalKaryotype
Normal cellsordinarilyhavethenormalsetof
y
chromosomes ofthespecies(i.e.haveanormal
karyotype)
y yp )
Cancer cellsalmostalwayshaveanabnormal
cells almost always have an abnormal
karyotype (e.g.abnormalchromosomenumberor
structure)
structure)
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19
HallmarksofCancerCells
3.AbnormalKaryotype
http://galleryhip.com/aneuploidy.html
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20
The
Hallmarks
ofCancer
1. Failuretostop
growing
2. Evasionof
apoptosis
3. Growth
without
appropriate
signals
4. Cellular
immortality
5. Angiogenesis
6. Tissue
Invasionand
metastasis
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21
InfluenceofGeneticandEnvironmental
FactorsintheDevelopmentofCancer
Cancersresultfromtheinteractionofbothgenetic
andenvironmentalfactors
and
environmental factors leadingtoaccumulation
leading to accumulation
ofmutationsinessentialgenes
EnvironmentalInfluence: Exposuretocertainagents
nowknowntoincreasetherisk ofcancer(e.g.
cigarettesmoke,asbestos,radiation)
k
b
d
)
GeneticInfluence:
Genetic Influence: Levelsofsusceptibilitydiffer
Levels of susceptibility differ
betweenindividuals(determinedbygeneticmakeup)
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22
InfluenceofGeneticandEnvironmental
FactorsintheDevelopmentofCancer
Someindividuals
haveagenetic
makeup
k
th t
that
makesthem
more
susceptible to
certainagents
Otherscan
tolerate moreof
the carcinogen
thecarcinogen
beforetheywill
developcancer
N.NASSIF
23
EnvironmentalFactors:
OccupationalExposureandCancerRisk
Occupation
Agent
Site of Disease
Aromatic amines
Aromaticamines
Bladder
Asbestosminingandhandling
Asbestos
Lung,mesothelium
Cadmiumworkers
Cadmium
Prostate
Uraniumminers
Ionisingg radiation
Lungg
Coalgasmanufacturers,asphalters
andothers
Polycyclichydrocarbons
Skin,lung
Farmers,sailors,sportsmen
UVlight
Skin,lip
Unknown
Nasal sinuses
Nasalsinuses
N.NASSIF
24
EnvironmentalFactors:
CancerRisk
Cigarettesmokingislinkedwithanincreasedriskofthe
following cancer types:
followingcancertypes:
Lungg
Larynx(voicebox)
Oralcavity(mouth,tongue)
Noseandsinuses
Pharynx(throat)
Esophagus
Stomach
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Pancreas
Cervix
Kidney
Bladder
Ovary
Colorectum
l
Acutemyeloidleukemia
25
NonGeneticFactors:
AgeandCancerRisk
N.NASSIF
26
EnvironmentalFactors:
InternationalVariationinCancerIncidence
*
*
* *
*
*
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27
GeneticFactors:
InvolvementofGenesinCancerDevelopment
Subsetsofgenesinthegenomehavebeenfound
tobeimportantintheprevention,development
to
be important in the prevention development
andprogressionofcancer
Thesegeneshavebeenfoundtobeeither
f
g,
f
malfunctioning,ornonfunctionalindifferent
tumour types
Categorised
d into3broadcategoriesbasedontheir
b d
b d
h
cellularfunction(s)
N.NASSIF
28
GeneticFactors:
InvolvementofGenesinCancerDevelopment
1. Geneswhoseproteinproductsstimulateorenhance
cell division, growth and viability (i.e. oncogenes)
celldivision,growthandviability(i.e.oncogenes)
2. Geneswhoseproteinproductsdirectlyorindirectly
p
p
y
y
inhibitorpreventcelldivisionorpromotecelldeath
(i.e.tumour suppressorgenes)
3. Geneswhoseproteinproductsareinvolvedinthe
correctionofacquiredmutationsinDNA(i.e.DNA
i
f
i d
i
i
(i
repairgenesandtheirproteinproducts)
N.NASSIF
29
Cl
ClassesofGenesinCancer
fG
i C
Majorclassesofgenesidentified:
Major classes of genes identified:
1. Oncogenes
2. Tumour suppressorgenes
3. DNArepairgenes
p g
N.NASSIF
30
ClassesofCancerGenes
1.TheOncogenes
Oncogenes =growthpromoting(dominantgainof
function)
Normalversionsaretermedprotooncogenesandtheir
divisionand/orinhibitcelldeath
Protooncogenesareusuallygrowthfactors,growth
Proto oncogenes are usually growth factors growth
factorreceptors,signaltransductionmoleculesor
nucleartranscriptionfactors
p
N.NASSIF
31
ClassesofCancerGenes
1.TheOncogenes (cont.)
Mutatedoncogenes canleadtounregulatedcell
division
Cellsareabletogrowintheabsenceofnormal
growthsignals
Examples:
E
l
ras: asignaltransductionmolecule
i l
d i
l l
myc:
y atranscriptionfactor
p
src:aproteintyrosinekinase
N.NASSIF
32
Oncogenes IdentifiedinCancer
Oncogene
Function
N.NASSIF
CancerType
33
Theras Oncogene
Ras subfamilyisafamilyofsmall
GTPases involvedincellsignal
t
transduction
d ti
Ras communicatesandtranslates
communicates and translates
signalsfromoutsidethecelltothe
nucleus
Activationofras signalling causescellgrowth,andsurvival
Mutationsofras canpermanentlyactivateitandthiscan
leadtocancer
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34
MutatedOncogenes CanLeadto
UnregulatedCellGrowth
Geneexpressionwithout
correctsignals
i
l
NormalGrowth
N.NASSIF
35
ClassesofCancerGenes
2.TheTumour SuppressorGenes
TumourSuppressorgenes=antioncogenes (recessive
lossoffunction)
Tumour suppressorgeneproductsacttoinhibitthe
division ofcellsifconditionsofgrowtharenotmet
of cells if conditions of growth are not met
Conditionstriggeringthebrakesofcelldivision
gg
g
include:DNAdamage,lackofgrowthfactors
Itistheabsence,orlossoffunctionofatumour
It is the absence or loss of function of a tumour
suppressorgeneproductthatleadstotumour
formation
N.NASSIF
36
ClassesofCancerGenes
2.TheTumour SuppressorGenes
Someexamplesinclude:
p53(TP53):atranscriptionfactorregulatingcell
division
Rb: controlscelldivision
APC: controlsavailabilityofatranscriptionfactor
BRCA: InvolvedinrepairofdamagedDNA
N.NASSIF
37
Tumour
Suppressor
Genes
Identifiedin
Cancer
Tumour
Suppressor
Function
N.NASSIF
CancerType
38
GenesinCancer
Thep53Tumour Suppressor
p53(theguardianofthegenome)
functionstopreventcelldivisionofcellswith
damagedDNA
mutationsofp53
mutations of p53 leadtolossoffunction
lead to loss of function
p53mutationsobservedinapproximately50%of
p
f
allcancers(ofvarioustypes)
N.NASSIF
39
GenesinCancer
Thep53Tumour Suppressor
p53isamultifunctionalprotein
which plays a role in:
whichplaysarolein:
modulatinggenetranscription
modulating gene transcription
policingcellcyclecheckpoints
activatingapoptosis
g p p
controllingDNAreplication
andrepair
maintaininggenomicstability
respondingtogeneticinsults
N.NASSIF
40
GenesinCancer
TumourSpecificTumourSuppressorGenes
Breastcancer(inheritedform)
BRCA1andBRCA2genes
Mutatedinahighpercentageofearlyonsetinherited
breast and ovarian cancer cases
breastandovariancancercases
Mutationsincreaseriskofdevelopingbreastand
ovariancancer
N.NASSIF
41
Cancer
Development
isaMultistep
Process
Normal Cell
First Mutation
Second Mutation
Mutations
are
acquired
att every
step
Third Mutation
Fourth or
later
Mutation
Malignant Cell
N.NASSIF
42
Th R l fPTEN Gene
TheRoleofPTEN
G
MutationsinSporadic
ColorectalCancer
ColorectalCancer
l
l
Cancerofthecolon
and/or rectum
and/orrectum
Thirdmostcommonly
diagnosedcancerinmen
(8%),andthesecondin
women(10%)
Thirdmostcommoncause
Third most common cause
ofcancerdeath(9%)
N.NASSIF
44
GeneticChangesIdentifiedin
ColorectalCancer
N.NASSIF
45
GenesInvolvedinColonCancer
Development
N.NASSIF
46
Th PTEN TumourSuppressorGene
ThePTEN
T
S
G
chromosome 10
chromosome10
PTEN geneislocatedatonchromosome10q23.3
gene is located at on chromosome 10q23.3
Thegeneproductisaproteinandlipidphosphatase
g
p
p
p p p
Regulatescellgrowth,survival,celladhesion,cell
g
g
differentiationanddeath(apoptosis)
N.NASSIF
47
PTENGeneandProteinStructure
EncodesNterminalDomain
EncodesCterminalDomain
EncodesPhosphataseDomain
N.NASSIF
48
CellularFunctionsofPTEN
ThemajorsubstrateofPTENisthesecondmessengerlipid
Th
j
b
f PTEN i h
d
li id
PIP3(phosphoinositol3,4,5triphosphate)
PTENdephosphorylatesPIP3toPIP2 (keepsPIP3levelslow)
LossofPTENleadstoincreasedlevelsofPIP3and
consequentactivationofAkt/PKBandthePI3Kcell
survival/anti apoptotic pathway
survival/antiapoptoticpathway
Subsequentprotectionfromapoptoticstimuliandincreased
q
p
p p
cellgrowthandsurvival
PTENisthereforeanegativeregulatorofcellsurvival
PTEN i th f
ti
l t
f ll
i l
N.NASSIF
49
TheCellularRole(s)ofPTEN
MultipleRolesofPTENinTumourSuppression
(PIP 3)
(PIP3)
(
(PIP2)
2)
N.NASSIF
50
ProjectResults
Pairedtumourandcorrespondingnormaltissue
samplesfromsporadicCRCpatientswerescreenedfor
PTEN
PTENgenemutationsanddeletions
t ti
d d l ti
Mutationsweredetectedinapproximately20%of
Mutations were detected in approximately 20% of
primarysporadiccolorectaltumours
Overall,alterations(mutationsanddeletions)ofthe
PTEN genewerepresentin40%ofprimarysporadic
colorectaltumours
N.NASSIF
51
Detection
ofPTEN
Gene
Mutations
inSporadic
Colorectal
Cancer
NormalDNA
TumourDNA
NormalDNA
TumourDNA
N.NASSIF
Patient 1
Exon 5
A>G
Patient 2
Exon 5
G>T
52
PTEN MutationsDetectedinSporadicCRC
E150Q
D153N
K62R
K125X D153Y
K125E
Y65C
E1
E2
E3
E4
E5
E6
319X
N323K
V217A
E7
E8
E9
G129E
C124S
N.NASSIF
53
Project:
Evaluating the Functional
Significance of the Detected
PTEN Mutations
FunctionalAnalysisoftheDetected
PTEN GeneMutations
Wildtype(WT)PTENhasbeencloned,andeachofthe
mutantPTENshavebeenengineeredinmammalian
mutant
PTENs have been engineered in mammalian
expressionvectors
TheWTandmutantPTEN hasbeenexpressedinselected
cellslines (e.g.U87MGglioblastomacells)aftertransfection
DeterminedeffectofWTPTEN andeachPTEN mutanton:
(a)cellproliferation(yourprojectaim)and
(b)PTENsubcellulardistribution
N.NASSIF
55
TheEffectof
PTEN
Mutationson
U87MGCell
Proliferation
*
*
WTPTENisable
tocausea
slowing of cell
slowingofcell
proliferation
AIMofPROJECT:
Todetermine
the effect of
theeffectof
PTEN mutation
oncell
proliferation
N.NASSIF
56
EffectofPTENMutationonPTEN
S b ll l Di t ib ti
SubcellularDistribution
WTPTEN
WT PTEN
expressionis
evenly
di t ib t d
distributed
betweenthe
cytoplasmand
y p
thenucleusin
U87MGcells
SomePTEN
mutantshave
increased
cytoplasmic
localisation
N.NASSIF
57
Conclusions
WTPTENisabletobringaboutaslowingofcell
proliferation ofculturedU87MGcells
of cultured U87MG cells
IsMUTANTPTENabletoslowcellproliferationas
Is MUTANT PTEN able to slow cell proliferation as
effectivelyastheWTPTEN?
(Youwillfindthisoutaspartofyourpractical
project!)
N.NASSIF
58
UsefulWebsitesandContentLinks
Cellcycleanimation
C ll
l
i
i
http://www.cellsalive.com/cell_cycle.htm
Howthecellcycle
workshttp://www.youtube.com/watch?v=diXoAgH3LMk
Cellcyclecheckpointsandtheroleofp53
http://www.youtube.com/watch?v=1EB8q9aR8Hk&feature=related
Howcancerdevelops
http://www.youtube.com/watch?v=j_wRpa2b5XI&feature=related
LiteraturearticledescribingthediscoveryofPTENmutationsin
colorectalcancer(downloadablepdf fileavailableatthissite)
h //
http://www.nature.com/onc/journal/v23/n2/full/1207059a.html
/ /
l/ / /f ll/
h l
N.NASSIF
59
R f
References
NelsonandCox(2008)Lehninger PrinciplesofBiochemistry,6th
Edition
G.Karp(2008)CellandMolecularBiology:Conceptsand
Experiments.5
Experiments
5th Edition
MH.Lodish etal.MolecularCellBiology
ReviewarticlecoveringPTENanditsroleincancer(downloadable
pdf fileavailableatthissite)
file available at this site)
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2710138/?tool=p
ubmed
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60