cell biology module 3

7/24/2019 Cell Biology Module 3

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The

cell

cycle,

is

the

series

of

events

that

takes

place

in

a

cell

leading

to

its

divisionandtheproductionoftwodaughtercellseachcontainingchromosomes

identicaltothoseoftheparentalcell.

Cellcycle

Incellswithoutanucleus(prokaryotic), thecellcycleoccursviaaprocess

termedbinaryfission.

Incellswithanucleus(eukaryotes),thecellcyclecanbedividedintwobrief

periods:interphase andthemitosis(M)phase

EUKARYOTICCELLCYCLE

Theeukaryoticcellcyclecommonlyisrepresentedas

fourstages

Insomaticcells,cellssynthesizeRNAsandproteins

duringtheG1phase,preparingforDNAsynthesisand

Inthemitotic(M)phase,thechromosomesareevenlypartitionedtotwodaughtercells,

andthecytoplasmdividesroughlyinhalfinmostcases.

Overallduringinterphase,whichconsistsoftheG1,S,andG2phases,thecellroughly

doublesitsmassaccumulatingnutrientsneededformitosisandduplicatingitsDNA

c romosome

rep ca on

ur ng

e

syn es s

p ase.ReplicationofDNAduringSleavesthecellwithfour

copiesofeachtypeofchromosome.


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Various Phases of Cell CycleOverview of Cell Cycle in Eukaryotic cells

DuringcelldivisioneachchromosomeiscomposedofthetwodaughterDNA

moleculesresultingfromDNAreplicationplusthehistones andother

chromosomalproteinsassociatedwiththem.

Theidenticaldau hterDNAmoleculesandassociatedchromosomal roteins

thatformonechromosomearereferredtoassisterchromatids.

Sisterchromatids areattachedtoeachotherbyproteincrosslinksalongtheir

lengths.

Invertebrates,thesebecomeconfinedtoasingleregionofassociationcalled

thecentromere aschromosomecondensationprogresses.

Duringinterphase,theportionofthecellcyclebetweentheendofoneM

phaseandthebeginningofthenext,theouternuclearmembraneiscontinuous

w

e

en op asm c

re cu um.


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Withtheonsetofmitosisinprophase,thenuclearenveloperetractsintothe

endoplasmicreticulum

in

most

cells

from

higher

eukaryotes,

and

Golgi

membranesbreakdownintovesicles.

Cellular microtubulesdisassemble andreassemble intothemitoticapparatus

cons s ngo a oo a s ape un eo m cro u u es esp n e w as ar

shapedclusterofmicrotubulesradiatingfromeachend,orspindlepole.

Duringthemetaphaseperiodofmitosis,amultiprotein complex,the

kinetochore,assemblesateachcentromere.

Thekinetochores ofsisterchromatids thenassociatewithmicrotubulescoming

fromoppositespindlepoles.

Duringtheanaphaseperiodofmitosis,sisterchromatidsseparate.

The initiall are ulledb motor roteinsalon thes indlemicrotubules

towardtheoppositepolesandthenarefurtherseparatedasthemitoticspindle

elongates

MITOSIS

Beforeaeucaryotic cell

divides,

it

must

duplicate

its

centrosome to

provide

one

for

each

ofitstwodaughtercells.

InearlyMphasethecentrosomal complexseparates

intotwoandeachcentrosome nucleatesaradialarrayof

microtubules,calledanaster.

Thetwoasters,whichinitiallyliesidebysideandclose

tothenuclearenvelope,moveapartbyafew

micrometers.

thebaseofeacholdcentriole andatarightangletoit.Bylateprophasethebundlesofpolarmicrotubulesthat

interactbetweenthetwoasterspreferentiallyelongateas

thetwocentersmoveapartalongtheoutsideofthe

nucleus.

Theelongationofthedaughtercentriole isusually

completedbyG2phase.


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Different Stages of MitosisThe first

five stages of the M phase constitute mitosis (originally defined as the

period in which the

PROMETAPHASE

PROPHASE

MITOSIS

ANAPHASE

TELOPHASE

Prometaphase

NUCLEUS

Interphase

Chromosomal Alignment and spindle organization during mitosis

MT

MTOC

Centrosome

Metaphase

Prophase

KT Fibre

KT MT

Polar MT

Ast ral

MT

Prometaphase

Metaphase


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MTDynamicsPlayCriticalRoleInTheFormationOfThe

SpindleApparatus

Alberts et al; MBC


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The two processes that separate sister chromatids at anaphase

InanaphaseAthechromatids arepulledtowardoppositepolesbyforcesassociatedwith

shorteningoftheirkinetochore microtubules.

Theforcedrivingthismovementisthoughttobegeneratedmainlyatthekinetochore.

Inana haseBthetwos indle olesmovea art.

ItislikelythattheforcesdrivinganaphaseBaresimilartothosethatcausethecentrosome

tosplitandseparateintotwospindlepolesatprophase.

Thereis

evidence

that

two

separate

forces

are

responsible

for

anaphase.

Theplusenddirectedmotorproteinsofthekinesin familycrosslinkadjacent,overlapping,

antiparallel polarmicrotubulesandslidethemicrotubulespasteachother,therebypushing

thespindlepolesapart.

Theminusenddirectedmotorproteinsbindtothecellcortexandtothoseastral

microtubulesthatpointawayfromthespindleandpullthespindlepolesapart.

Functionsofkinetochore microtubulesatmetaphaseandanaphase

Duringmetaphase,subunitsareaddedtotheplusendofamicrotubuleatthe

kinetochore andareremovedfromtheminusendatthespindlepole.

,

maintainaconstant

length

and

remain

under

tension.

Atanaphasethechromatid isreleasedfromattachmenttoitssisteratthemetaphase

plateandthekinetochore movesrapidlyupthemicrotubule,removingsubunitsfromits

plusendasitgoes.

Itsattachedchromatid istherebycarriedtothespindlepole.

Partofthechromatid movementisduetothesimultaneouslossoftubulin subunits

fromtheminusendofthemicrotubulesatthepole.


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Duringtelophase,theeffectsofprophaseand

prometaphase eventsarereversed.

Twodaughternucleiforminthecell.

Thenuclearenvelopesofthedaughtercellsare

formedfromthefragmentsofthenuclearenvelope

oftheparentcell.

Asthenuclearenvelopeformsaroundeachpairof

chromatids,thenucleolireappear.

TheKTmicrotubulesdisappearandthePolarMTs

elongate

Telophase accountsforapproximately2%ofthecell

cycle'sduration.

Cytokinesis

Initiationofcytokinesis takesplacebeforetheendofAnaphase


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Cytokinesis

Duringcytokinesis thecytoplasmdividesbyaprocesscalledcleavage.

Normally,themitoticspindledetermineswherecleavageoccursaswellaswhen.

Thefirstvisiblesignofcleavageinanimalcellsisapuckeringandfurrowingofthe

plasmamembraneduringanaphase.

Thefurrowinginvariablyoccursintheplaneofthemetaphaseplate,atrightangles

tothelongaxisofthemitoticspindle.

Thecontractileringassembleslateranddividesthecellintwo.

Cleavageisaccomplishedbythecontractionofathinringcomposedmainlyofan

overlappingarray

of

actin filaments

and

bipolar

myosin

IIfilaments.

Itconsistsofcircumferentiallyorientedfilamentsboundtothecytoplasmic faceof

theplasmamembranebyuncharacterizedattachmentproteins

Meiosisinvolves

two

nuclear

divisionsratherthanone.

Itproduceshaploidgametesfrom

diploidparentalcells

Manythingshappenduringthefirst

Meiosis

p aseo me os s ca e prop ase.

lastsforover90%oftimerequiredfor

meiosis.

Duringthistimehomologous

chromosomescometogetheraspairsin

aprocesscalledsynapsis toformfour

chromatid structurecalledastetrad.

Atthispointgeneticinformationis

exchan ed between nonsister

chromatids byaprocess

called

crossing

over.

TheXshapedsitesofsuchcross

overs arecalledchiasmata


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InthenextphaseofmeiosisI,i.e.,metaphaseI,thetetradsmovetothe

equatorofthecell(randomlypositioningofthehomologues).

Thekinetochore microtubulesexpandfromeachchromosomeinopposite

directionstowards

the

nearest

pole

NextinanaphaseI,thespindleandthekinetochore microtubulesinteractto

separatethehomologouschromosomes.

Thecentromeres joiningsister chromatids donotsplit,butinsteadentire

doublechromatid chromosomesaremovedtowardthetwopoles

Atthispointcytokinesis beginswhichresultsin twocellseachwitheither

thematernalorpaternalhomologueofeachtypeofchromosome.

Thefinalstage,i.e.,telophase differsamongspecies.

AftertheendofmeioticdivisionI,nuclearmembranesreformaroundthe

twodaughternucleiandabriefinterphase begins.

,

theysoonrecondense andprophaseIIbegins.

AsthereisnoDNAsynthesisduringthisinterval,insomeorganismsthe

chromosomesseemtopassalmostdirectlyfromonedivisionphaseintothenext.

MeioticDivisionIIResemblesaNormalMitosis

InallorganismsprophaseIIisbrief:thenuclearenvelopebreaksdownasthe

newspindleforms,afterwhichmetaphaseII,anaphaseII,andtelophase IIusually

MeiosisII

o ow nqu c success on.

Asinanordinarymitosis,aseparatesetofkinetochore fibersformsoneach

sisterchromatid,andthesetwosetsoffibersextendinoppositedirections.

Moreover,thetwosisterchromatids arekepttogetheronthemetaphaseplate

untiltheyarereleasedbythesuddenseparationoftheirkinetochores at

anaphase

Cytokinesis iscompletedbytheendoftelophase IIresultingin4haploid

dau htercells

Eachdaughter

cell

has

complete

set

of

chromosomes

and

each

set

is

unique

becauseofindependentassortmentandcrossingoverduringprophaseof

meiosisI


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Twomajorcontributionstothereassortment ofgeneticmaterialthatoccursduringmeiosis.

1) Theindependentassortmentofthematernalandpaternal

GeneticReassortment IsEnhancedbyCrossingover

BetweenHomologousNonsister Chromatids

homologuesduringthefirstmeioticdivisionproduces2n

differenthaploidgametesforanorganismwithn

chromosomes.

Heren=3,andthereare8differentpossiblegametes.

2) CrossingoverduringmeioticprophaseIexchangessegments

ofhomologouschromosomesandtherebyreassorts genesin

individualchromosomes.

BecauseofthemanysmalldifferencesinDNAsequencethat

,

increasethegeneticvariabilityoforganismsthatreproduce

sexually.

During

prophase

I,

DNA

is

exchanged

between

homologous

chromosomes

in

a

processcalledhomologousrecombination.

Thisoftenresultsinchromosomalcrossover.

ThenewcombinationsofDNAcreatedduringcrossoverareasignificantsource

ofgeneticvariation,andmayresultinbeneficialnewcombinationsofalleles.

Prophase I

,

havetwochromosomesandfourchromatids,withonechromosomecomingfrom

eachparent.

Thetwoduplicatedhomologues(maternalandpaternal)areseentobe

physicallyconnectedatspecificpoints.

Eachconnection,calledachiasma (pluralchiasmata),correspondstoa

crossoverbetweentwononsister chromatids

Manybivalentscontainmorethanonechiasma,indicatingthatmultiple

crossoverscan

occur

between

homologues.

Thisprophaseistraditionallydividedintofivesequentialstagesleptotene,

zygotene,pachytene,diplotene,anddiakinesis definedbythesemorphological

changes.


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Different stages of meiosis

ThefirststageofprophaseIistheleptotenestage,alsoknownasleptonema

Duringthisstage,individualchromosomesbegintocondenseintolongstrandswithinthe

nucleus.

Themoststrikingeventistheinitiationofintimatechromosomesynapsisatzygotene,

whenacomplexstructurecalledthesynaptonemalcomplexbeginstodevelopbetweenthe

two sets of sister chromatids in each bivalent. .

Pachyteneissaidtobeginassoonassynapsisiscomplete,anditgenerallypersistsfor

days,untildesynapsisbeginsthediplotenestage,inwhichthechiasmataarefirstseen

Geneticrecombination

requires

aclose

apposition

between

the

recombining

chromosomes.

Thesynaptonemalcomplex,whichformsjustbeforepachyteneanddissolvesjust

afterward,keepsthehomologouschromosomesinabivalenttogetherandcloselyaligned,

andithasbeensuggestedthatitmayplayapartintherecombinationprocess.

Diakinesisisthestageoftransitiontometaphase inwhichthechromosomes

recondense andtranscriptionhalts.

esynap onema cons s so a ong a er epro e ncore,onoppos es eso w c

thetwohomologuesarealignedtoformalonglinearchromosomepair.


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Althoughthesynaptonemal

complexmayprovidethestructural

framework

for

recombination

events,

itprobablyisnottheenginethat

bringsthemabout.

Recombination Nodules

Recombinationnodulessitatintervalsonthesynaptonemalcomplex,placed

likebasketballsonaladderbetweenthetwohomologouschromosomes.

eac verecom na onprocess s

thoughttobemediatedinsteadby

recombinationnodules,whichare

verylargeproteincontaining

assemblieswithadiameterofabout

90nm.

Theyarethoughttomarkthesiteofalargemultienzyme"recombination

machine,"whichbringslocalregionsofDNAonthematernalandpaternal

chromatidstogether

across

the

100

nm

wide

synaptonemal

complex.

Thetotalnumberofnodulesisaboutequaltothetotalnumberofchiasmata

seenlaterinprophase.

Thenodulesaredistributedalongthesynaptonemalcomplexinthesame

waythatcrossovereventsaredistributed

Thetwoduplicatedhomologues(maternalandpaternal)arephysically

connectedatspecificpointscalledaschiasma (plural:chiasmata)

Thechiasma createdbyeachcrossovereventplaysaroleanalogoustothat

Chiasmata

,

paternalhomologuestogetheronthespindleuntilanaphaseI

Inmutantorganismsthathaveareducedfrequencyofmeioticchromosome

crossingover,someofthechromosomepairslackchiasmata.


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Thepairingofhomologous

chromosomes(homologues)isunique

tomeiosis.

Eachchromosome

has

been

duplicatedandexistsasattachedsister

chromatids beforethepairingoccurs.

Asshownbytheformationofchromosomesthatare

partredandpartblack,thechromosomepairingin

meiosisinvolvescrossingover(geneticrecombination)

betweenhomologouschromosomes.

Pairedhomologouschromosomesduringthe

transitiontometaphaseofmeioticdivisionI.

Asinglecrossovereventhasoccurredearlierin

prophasetocreateonechiasma.

Notethat

the

four

chromatids

are

arranged

as

two

distinctpairsofsisterchromatidsandthatthetwo

chromatidsineachpairaretightlyalignedalongtheir

entirelengthsaswellasjoinedattheircentromeres.

Theentireunitoffourchromatidsisreferredtoasa

bivalent.

Chromosomescondensefurtherduringthediakinesis stage,fromGreekwordsmeaning

"movingthrough".Thisisthefirstpointinmeiosiswherethefourpartsofthetetradsare

actuallyvisible.Sitesofcrossingoverentangletogether,effectivelyoverlapping,making

chiasmata clearlyvisible.Otherthanthisobservation,therestofthestageclosely

resemblesprometaphase ofmitosis;thenucleolidisappear,thenuclearmembrane

Diakinesis

disintegratesintovesicles,andthemeioticspindlebeginstoform.


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Formationoftheactualgametenucleiproceedssimplythroughasecondcelldivision,

divisionIIof

meiosis,

without

further

DNA

replication.

Thechromosomesnowalignonasecondspindleandthesisterchromatids separate,

MeiosisII

, .

InprophaseIIweseethedisappearanceofthenucleoliandthenuclearenvelope

againaswellastheshorteningandthickeningofthechromatids.

Centrioles movetothepolarregionsandarrangespindlefibersforthesecondmeiotic

division.

InmetaphaseII,thecentromeres containtwokinetochores thatattachtospindle

fibersfromthecentrosomes ateachpole.

ThisisfollowedbyanaphaseII,wherethecentromeres arecleaved,allowing

microtubulesattachedtothekinetochores topullthesisterchromatids apart.

Thesisterchromatids byconventionarenowcalledsisterchromosomesastheymove

towardopposingpoles.

Theprocess

ends

with

telophase II,

which

is

similar

to

telophase I,

and

is

marked

by

uncoilingandlengtheningofthechromosomesandthedisappearanceofthespindle.

Nuclearenvelopesreformandcleavageorcellwallformationeventuallyproducesa

totaloffourdaughtercells,eachwithahaploidsetofchromosomes.Meiosisisnow

completeandendsupwithfournewdaughtercells.

Meiosis thus consists of two cell divisions following a single phase of DNA

replication, so that four haploid cells are produced from each cell that enters

meiosis.

longer than a normal S phase, and cells can then spend days, months, or even

years in the first meiotic prophase, depending on the species and on the

gamete being formed.


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The two progeny of this division (division I of meiosis) therefore contain a diploid

amount of DNA. But, they differ from normal diploid cells in two ways:

(1) both of the two DNA copies of each chromosome derive from only one of the

two homologous chromosomes in the original cell (except where there has beengenetic recombination), and

(2) these two copies are inherited as closely associated sister chromatids, as if

23pairs 2N

23

2N

23

N

23

N

23

chromosomes

induplicate

First meiotic

division23pairsin

duplicate

c romosomes

induplicate

4N

2N N

23 23

N

2nd

meioticdivision

Regulationofcellcycle

Cellsreproduce

by

duplicating

their

contents

and

then

dividing

intotwo

TheDNAmustbefaithfullyreplicated,andthereplicatedchromosomesmustbesegregatedintotwoseparatecells

Re ulationofthecellc cleiscriticalforthenormaldevelopmentofmulticellularorganisms,andlossofcontrolultimatelyleadstocancer

Regulationofcellcycleisachievedbytheactionofcyclinsandcyclindependentkinases

Thecyclinsaretheregulatorysubunits,whilethekinasesarethecatalyticsubunits

EachCDKcanassociatewithdifferentcyclins,andtheassociated

cyclindetermines

which

proteins

are

phosphorylated

by

aparticularcyclinCDKcomplex.


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CyclinsandCycleDependentKinases(CDKs)

TherearethreemajorclassesofcyclinCDKcomplexesthat

controlpassage

through

the

cell

cycle:

the

G1

phase,

Sphase,

andmitoticcyclinCDKcomplexes.

G1cyclinCDKcomplexesareexpressedfirst.

ThesepreparethecellfortheSphasebyactivatingtranscriptionfactorsthatpromotetranscriptionofgenesencodingenzymesrequiredforDNAsynthesisandthegenesencodingSphasecyclinsandCDKs.

TheactivityofSphasecyclinCDKcomplexesisinitiallyheldincheckbyinhibitors.

LateinG1,theG1cyclinCDKcomplexesinducedegradationof

consequentlystimulatingtheirpolyubiquitinationbythe

multiprotein. SubsequentdegradationofthepolyubiquitinatedSphaseinhibitorbyproteasomesreleasesactiveSphasecyclinCDKcomplexes.

Differentcyclins bindspecificallytodifferentCdks toformdistinctcomplexesat

specificphasesofthecellcycleandtherebydrivethecellfromonephaseto

another.

Thecyclins areafamilyofproteins,which,astheirnamesuggests,are

Cyclins in cell cycle regulation

synthesizedanddestroyedduringeachcellcycle.

Todate,eightcyclins havebeendescribedthatdirectlyaffectcellcycle

progression:cyclins A1andA2,B1,B2,andB3,C,D1,D2,andD3,E1andE2,F,G1

andG2,andH,whichallshareanapprox150aminoacidregionofhomology

calledthecyclin boxthatbindstotheNterminalendofspecificCdks.

Littleinformationiscurrentlyavailableregardingtherecentlydescribedcyclins F

andG,whereascyclin HhasbeenshowntoformcomplexesspecificallywithCdk7

activationofCDC2andCdk2kinases byphosphorylating Thr160andThr161,respectively


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.

Upon stimulation of quiescent cells (G0), different cyclins are expressed in

an orderly way.

Cyclin D is absent in quiescent cells but rapidly accumulates after growthfactor stimulation.Cyclin D is subsequently expressed constitutively throughout subsequent

cell cycles, whereas expression of cyclins E, A and B is related to a specific

cell cycle phase.

cyclin EisrequiredtoovercometheG1/ScheckpointandcommenceDNAreplication,

The otherG1cyclins areidentifiedtobecyclin D,andcyclin C.


Mem erso t ecyc in D ami y unctiontoregu atep osp ory ation o t e

retinoblastomageneproduct,therebyactivatingE2Ftranscriptionfactors.

cyclin BisrequiredfortraversingtheG2/Mcheckpointtoinitiatemitosis

Cyclin AseemstoberequiredforbothSphaseandMphase.Itaccumulatespriorto

cyclin Binthecellcycle,appearstobeinvolvedincontrolofSphaseandhasbeen

showntoassociatewithcyclindependentkinase2(Cdk2).

Cyclin Aavailability

is

apparently

the

rate

limiting

step

for

entry

into

mitosis,

and

cyclin

Aisrequiredforcompletionofprophase.

Atlateprophase,cyclin Amaynolongerbenecessaryascdc2/cyclinB1becomesactive.

Anumberofstudieshavedescribedtheabilityofoverexpressedcyclin Atoaccelerate

theG1toStransitioncausingDNAreplication,andcyclin AantisenseDNAcanprevent

DNAreplication.


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TheCdks areafamilyofserine/threonine proteinkinases thatbindto,andareactivated

by,specificcyclins.

Todate,

at

least

nine

Cdks have

been

described:

CDC2

(Cdk1),

Cdk2,

Cdk3,

Cdk4,

Cdk5,

Cdk6,Cdk7,Cdk8,andCDK9.

i. Cdks 4,5,and6complexmainlywiththecyclin Dfamilyandfunctionduringthe

G0/G1phasesofthecycle;

CycleDependentKinases(CDKs)

ii. Cdk2canalsobindwithmembersofthecyclin Dfamilybutmorecommonly

associateswithcyclins AandEandfunctionsduringtheG1phaseandduringtheG1/

Stransition.

iii. Cdk7isfoundinassociationwithcyclin Handisabletophosphorylate eitherCDC2,

Cdk2,ortheCterminaldomainofthelargestsubunitofRNApolymeraseII,in

additiontotheTATAboxbindingproteinorTFIIE

iv. CDC2isthemitoticCdk andformscomplexeswithcyclins AandBthatfunctioninthe

G2andMphasesofthecellcycle.

v. Cdk8pairswithcyclin Candisfoundinalargemultiprotein complexwithRNA

polymeraseII,whereitisthoughttocontrolRNApolymeraseIIfunction

vi. Finally,

Cdk9

is

a

serine/threonine kinase related

to

CDC2

that

pairs

with

T

type

cyclins.Theactivityofthecyclin T/Cdk9complexisnotcellcycleregulatedbutis

involvedinmanyprocessessuchasdifferentiationandbasaltranscription

SpecificCdks bindtospecificcyclins toformanactivecomplexthatintegratessignals

fromextracellular

molecules

and

controls

progression

through

the

cell

cycle.

TheCdk subunitonitsownhasnodetectablekinase activityandrequiressequential

activationbycyclin bindingandsubsequentphosphorylation byCAKand

dephosphorylation byCDC25proteinphosphatase

Thisactivationprocessoccursinatwostepmanner,asfollows:

1. Bindingofthecyclin totheCdk conferspartialactivitytothekinase.Cyclin binding

causesaconformationalchangeintheCdk molecule,therebybringingtogether

specificresiduesinvolvedinorientingATPphosphateatomsreadyforcatalysiswithin

thecatalyticcleft.Theseconformationalchangesalsosetthestageforsubsequent

phosphorylation andfullactivation.

2. Phosphorylation ofthecyclin/Cdk complexisperformedbyCAKwhichincreasesCdk

activityapproximately100fold.Phosphorylation occursonaconservedthreonine

residuewithin

the

Tloop

region

of

the

Cdk (Thr160

in

CDC2

and

Thr161

in

other

Cdks).Cyclin bindingmovestheTlooptoexposethephosphorylation site,allowing

fullactivationoftheCdk.

Onceactivated,thevariouscyclin/Cdk complexesphosphorylate anumberofspecific

substratesinvolvedincellcycleprogression.SuchsubstratesincludetheRb pocket

proteins,lamins,andhistones.Evidenceexiststosuggestthatcyclins maybeinvolved

indeterminingthesubstratespecificityofCdks


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Thecyclins and

Cdks often

are

referred

to

as

positive

regulators

of

the

eukaryoticcellcycle.

Afamilyofnegativeregulatorsalsoexists,theCDKIs

Cyclin-Dependent Kinase Inhibitors

,

Cdk4)andCIP(Cdkinteractingprotein)/KIP(kinase inhibitorprotein)

TheINK4familyincludesp14,p15(INK4B),p16(INK4A),p18(INK4C),andp19

(INK4D),whichspecificallyinhibitG1cyclin/Cdk complexes(cyclin D/CDK4and

cyclin D/CDK6)andareinvolvedinG1phasecontrol.

TheCIP/KIPfamilyincludesp21(CIP1/WAF1/SDI1), p27(KIP1),andp57(KIP2),

whichare3844%identicalinthefirst70aminoacidregionoftheiramino

terminiaregionthatisinvolvedincyclinbindingandkinaseinhibitoryfunction.

TheCIP/KIPfamil dis la sabroaders ecificit thantheINK4famil ,since

membersinteractwith,andinhibitthekinaseactivitiesof,cyclinE/Cdk2,cyclin

D/Cdk4,cyclinD/Cdk6,cyclinA/Cdk2,andcyclinB/CDC2complexesandalso

functionthroughoutthecellcycle

Thus,CIP/KIPfamilymembersbindto,andinhibittheactivityof,theentire

cyclin/Cdkcomplex

Thetumoursuppressorproteinp53alsoplaysanimportantroleincellcycle

arrestattheG1andG2checkpointssubsequenttoinducingapoptosis.

e

p

pro e n

as

a

cen ra

sequence

spec c

n ng

oma n

an

a

transcriptionalactivationdomainatitsaminoterminus;inresponsetoDNA

damage,itcaninducethetranscriptionoftheCDKIp21,whichinhibitsthe

activationofvariousG1cyclin/Cdkcomplexes


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2

Bindingof

agrowth

factor

molecule

to

its

cell

surface

receptor

stimulate

the

Rasdependentmitogenactivatedproteinkinase (MAPK)pathway

OncecellsenterG1,synthesisofthemRNAsandproteinsnecessaryforDNA

G1Regulation

synt es soccurs,a ow ngce stoenterSp ase.

Theactivationofcyclin D/Cdk4andcyclin D/Cdk6complexesisessentialfor

passagethroughtheG1phase,andtheyexerttheirregulationoncellcycle

progressionbyphosphorylating Rb pocketproteins.

TheRb pocketproteinfamilyservestorepresstheactivityoftheE2F

transcriptionfactorsthatthemselvesareessentialfortranscriptionofgenes

necessaryforentryintoSphase.

The Ras MAPK athwa has been shown to control c clin D ene ex ression

directly.

Oneof

the

most

extensively

studied

substrates

of

the

cyclin/Cdks is

the

retinoblastoma (Rb)familyofpocketproteins.

TheRb familyofpocketproteinscomprisesagroupoftumorsuppressor

proteinsconsistingofthreemembers;pRb,p107,andp130

TheRb roteinisabundantinthenucleusofmammaliancells.

G1/STransition

Itbindstomanyotherproteins,includingseveralimportantgeneregulatory

proteins,butitsbindingcapacitydependsonitsstateofphosphorylation.

WhenRb isdephosphorylated,itbindsasetofregulatoryproteins

thatfavorcellproliferation,holdingthemsequesteredandoutofaction;the

phosphorylation ofRb makesitreleasetheseproteins,allowingthemtoact

IntheG0cellitcontainslittlephosphateandappearstohinderthe

transcriptionofgenes,suchasfos andmyc,thatarerequiredforproliferation.

ese

genes

are

ranscr e

a

a

g

eve

n

mu an

ce s

a

ac

a

functionalcopyoftheRb geneandatamuchlowerlevelinthesecellswhena

functionalcopyoftheRb geneisputbackintothembytransfection.

GrowthfactorsrelievetheinhibitionexertedbyRb bycausingtheproteinto

becomephosphorylated onmultipleserines andthreonines.Thecellsnow

begintoexpressCdk protein,passtheG1checkpoint,andembarkonDNA

synthesis.


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Pre-replication complex (pre-RC) assembly, also called DNA replication

licensing, is strictly regulated to ensure that DNA replication occurs only once per

cell cycle to maintain genomic stability.

Cyclin E has been shown to have a role in pre-replication complex (Pre-RC)

assembly in cells re-entering the cell cycle from quiescence.

InitiationofTranscription

The initiation phase of DNA replication is divided into two distinct steps that

occur at different times in the cell cycle.

The first step occurs in late mitosis and early G1, when a large complex of

initiator proteins, called the prereplicative complex, or pre-RC, assembles at

origins of replication (licensing).

This step is sometimes called licensing of replication origins because initiation

of DNA synthesis is permitted only at origins containing a pre-RC.

The second step occurs at the onset of S phase, when components of the pre-

nucea e e orma on o a arger pro e n comp ex ca e e pre- n a oncomplex.

This complex then unwinds the DNA helix and loads DNA polymerases and

other replication enzymes onto the DNA strands, thereby initiating DNA synthesis.


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Onceactivated,theSphasecyclinCDKcomplexesphosphorylateregulatorysitesin

theproteinsthatformDNAprereplicationcomplexes,whichareassembledon

replicationoriginsduringG1

PhosphorylationoftheseproteinsnotonlyactivatesinitiationofDNAreplicationbut

alsopreventsreassemblyofnewprereplicationcomplexes.

S-Phase

Becauseofthisinhibition,eachchromosomeisreplicatedjustonceduringpassage

throughthecellcycle,ensuringthattheproperchromosomenumberismaintainedin

thedaughtercells.

AnumberofcheckpointsexisttoensurethatDNAisreplicatedonlyoncepercycle,

thatitisfullyandcorrectlyreplicated,andthatreplicationoccursbeforecelldivision.

AnotherimportanteventduringSphase,otherthanDNAreplication,iscentrosome

duplication.

CyclinE/Cdk2playsanimportantroleincentrosomeduplication

duplicationwasdependentonthepresenceofcyclinE/Cdk2complexes.

Inaddition,cyclinE/Cdk2wasshowntophosphorylatenucleophosmininthismodel,

causingdissociation

from

centrosomes

and

subsequent

initiation

of

centrosome

duplication

TheG2/MTransition

MitoticcyclinCDKcomplexesaresynthesizedduringtheSphaseandG2,buttheiractivitiesareheldincheckbyphosphorylationatinhibitorysitesuntilDNAsynthesisiscompleted.

Onceactivatedbydephosphorylationoftheinhibitorysites,mitoticcyclin

condensation,retractionofthenuclearenvelope,assemblyofthemitoticspindleapparatus,andalignmentofcondensedchromosomesatthemetaphaseplate.

TheG2phaseisanothergapphaseinthecellcycleinwhichthecellassesses

thestateofchromosomereplicationandpreparestoundergomitosisand

cytokinesis.

CyclinB/CDC2isthekeymitoticregulatoroftheG2/Mtransitionandwas

or g na y

en e

as

e

ma ura on

promo ng

ac or,

a

ac or

capa e

o

inducingMphaseinimmatureXenopusoocytes.

ThemoleculesthatregulatecyclinB/CDC2activityreceivesignalsfromthe

checkpointmachinery


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2

RoleoftheCyclin B/CDC2ComplexintheG2/MTransition

Cyclin BsynthesisbeginsattheendofSphase

Twocyclin Bisoforms existinmammaliancells,cyclin B1andB2.

Studiesin

cyclin B1

and

cyclin B2

null

mice

have

confirmed

that

cyclin B2

is

nonessentialfornormalgrowthanddevelopment.

Thisparticularisoform associateswiththeGolgiandmayplayaroleinGolgi

remo e ng ur ngm os s

Incontrasttocyclin B2,cyclin B1isthoughttoberesponsibleformostofthe

actionsofCDC2inthecytoplasmandnucleusanditappearstocompensatefor

thelossofcyclin B2inB2nullmiceimplyingthatcyclin B1iscapableoftargeting

CDC2kinase totheessentialsubstratesofcyclin B2

Cyclin B/CDC2complexesareregulatedbothpositivelyandnegativelyby

phosphorylation

Phos hor lation ofCDC2ontheconservedTloo re ion Thr160 isre uired

foractivation,asisthecasewithallCdks,andthisphosphorylation eventis

mediatedbyCAK.

DuringG2,

cyclin B/CDC2

complexes

are

held

in

an

inactive

state

by

phosphorylation ofCDC2atThr14andTyr15.

Phosphorylation onThr14preventsATPbinding,whereasthatonTyr15

interfereswithphosphatetransfertothesubstrateowingtoitspositioninginthe

ATPbindingsiteonCDC2

Theseinhibitoryphosphorylation eventsarecarriedoutbythekinases Wee1andMyt1;

Wee1specifically

phosphorylates Tyr15,

and

Myt1

phosphorylates both

Tyr15

and

Thr14,

withastrongeraffinityforThr14.

Cyclin B/CDC2becomesfullyactivatedfollowingdephosphorylation ofthesesitesbythe

proteinphosphatase CDC25C

RegulationoftheCDC2/cyclin Bcomplex

Theserine/tyrosinekinase,Wee1

onCDC2.Wee1itselfis

phosphorylatedandinactivatedby

Nim1andotherunidentified

kinasestoinducemitosis.

Thr14phosphorylationcanbe

mediatedbyWee1butonlyonce

Tyr15hasbeenphosphorylated.

Itappears

that

the

Thr/Tyr

kinase Myt1

is

the

critical

kinase involved

here.

InhibitionofCDC2bywee1iscounteractedbytheCDC25dualspecificityphosphatases.

CDC25isphosphorylated andactivatedbyCDC2/cyclin B(amplificationpathway).

Proteinphosphatase 1(PP1)inactivatesCDC25bydephosphorylation ofthesameresidue

thatisphosphorylated byCDC2/cyclin B.

FullactivationofCDC2requiresThr161phosphorylation byCdkactivatingkinase (CAK),

whichthenstabilizesCDC2associationwithcyclin B.


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Activatedcylcin B/cdc2complexphosphorylates asetof

proteinsleading

to

the

events

like

nuclear

envelope

breakdown,

rearrangementoftheactin andtubulin cytoskeleton,

condensationofthechromosomes,rearrangementofGolgi

apparatus.

EachoftheseprocessesisthoughttobetriggeredwhenMcdk

phosphorylates specificproteinsinvolvedintheprocess,

althoughmostoftheseproteinshavenotyetbeenidentified.

MCdk doesnotactalonetophosphorylate keyproteins

involvedinearlymitosis.

woa t ona am eso prote n nases,t epo o e nases

andtheAurorakinases,alsomakeimportantcontributionstothe

controlof

early

mitotic

events

Metaphase/Anaphasetransitioncheckpoint

Duringmitosis,

cells

have

evolved

asurveillance

mechanism

called

the

spindleassemblyalsoknownasthemitoticcheckpointwhichiscrucialfor

ensuringfidelityinchromosomesegregation.

Thespindleassemblycheckpointexamineswhetherprerequisitesfor

chromosomesegregationhavebeenmetandtherebydetermineswhetherto

executeortodelaychromosomesegregation.

Ittriggersanaphaseonsetonlywhenallthechromosomesareproperly

attachedandalignedattheequatorialplane,allowingthesplittingofsister

chromatidsandtheirdeliverytoeachspindlepole

ActivationofspecificcyclinCdkcomplexesdrivesprogressionthroughthe

StartandG2/Mcheckpoints,progressionthroughthemetaphaseto

anaphasetransitionistriggerednotbyproteinphosphorylationbutby

proteindestruction,

leading

to

the

final

stages

of

cell

division.


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2

Themajorcomponentsinvolvedinthespindleassemblycheckpointwere

identifiedintwosimilargeneticscreensinbuddingyeastformutantsthatfail

toarrestinmitosisinthepresenceofspindledamagingagents.

ThesecheckpointcomponentsincludeMad1,Mad2,Mad3(mitoticarrest

deficient)Bub1andBub3(buddinguninhibitedbybenzimidazole) Mps1

(monopolar spindle1)initiallyidentifiedasakinase functioningin

duplicationofthespindlepolebody

ThiscomplexisestablishedduringDNAreplicationandmaintainsthe

linkagebetweensisterchromatids.

TheAPC/Ccatalyzestheubiquitylation anddestructionoftwomajor

proteins.

,

sisterchromatid pairstogetherinearlymitosis.

Destructionofsecurin atthemetaphasetoanaphasetransitionactivatesa

proteasethatseparatesthesistersandunleashesanaphase.TheSandMcyclins arethesecondmajortargetsoftheAPC/C.

Unattachedkinenetochores act

as

catalytic

sites

for

the

activation

of

Mad2.

ActivatedMad2thendiffusesandpreventsanaphaseonsetbyinhibitingthe

activityofCdc20APC.

Inaddition,BubR1functionssynergistically withMad2ininhibitingCdc20

APCactivity.

MPhaseRegulation

Afterallthechromosomesareproperlyattachedbykinetochore

microtubulesandalignedatthemetaphaseplate,thespindleassembly

checkpointisturnedoff.

OncethechromosomesarealignedatthemetaphaseplateMad2*isno

longergenerated,andBubR1doesnotinteractwithCdc20APC,resultinginthe

activationofCdc20APC.

ActivatedCdc20APCcatalyzestheubiquitination ofsecurin,leadingtoits

.

Degradationof

securin in

turn

causes

the

release

of

separin.

Thefreeseparin isthenabletocleavetheSCC1subunitofthesister

chromatid cohesioncomplex,triggeringtheseparationofsisterchromatids and

theonsetofanaphase.


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2

Spindle assembly checkpoint signaling

JournalofCellScience115,35473555

At the metaphaseanaphase transition the APC ubiquitinates proteins

whose subsequent degradation by the 26S proteasome is essential for the

initiation of sister chromatid separation.

Later in anaphase and telophase the APC promotes the inactivation of the

mitotic cyclin-dependent protein kinase 1 by ubiquitinating its activating

subunit cyclin B.

The APC also mediates the ubiquitin-dependent proteolysis of several

other mitotic regulators, including other protein kinases, APC activators,

spindle-associated proteins, and inhibitors of DNA replication.

EndofCellcycleRegulation


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2

Control of cell numbers in multi- cellular organisms

During normal development and throughout adult life, intricate genetic

control systems regulate the balance between cell birth and death inresponse to growth signals, growth-inhibiting signals, and death signals.

In tissues such as skin, intestine and the hematopoietic system, there is

continuous loss of cell either by surface abrasion, by damage of due to

senescence

The cell loss has to be compensated for by cell production.

The number of cells produced by cell division precisely balances cell

loss in order for the tissue to maintain its size and mass

In tissues such as the liver, breast, prostate and connective tissues,

there is little or no re lacement under normal conditions takes lace onl

when the integrity is compromised).

In some tissues, e.g., the female germ line and the central nervous

system there is little or no cell replacement or capacity for regeneration

Control of cell division is important in rapidly growing tissues.

The mechanism used by the tissues is unclear

It is obvious it must be regulated by a complex network of signals and

messages including growth factors, cytokines and hormones.

,may be produced by neighboring cell os either similar or unrelated cell types

(paracrine, eg., epithelial-mesenchymal interactions) and circulating

hormones.


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The losses of cellular regulation that give rise to most or all cases of cancer are

due to genetic damage

Mutations in two broad classes of genes have been implicated in the onset of

cancer: proto-oncogenes and tumorsuppressor genes. Proto-oncogenes are

activated to become oncogenes by mutations that cause the gene to be

excessivel active in rowth romotion. .

Tumor suppressor genes normally control growth, so damage to them allows

inappropriate growth.

Many of the genes in both classes encode proteins that help regulate cell birth

or cell death by apoptosis; others encode proteins that participate in

repairing damaged DNA.

Cancer commonly results from mutations that arise during a lifetimes exposure

to carcinogens, which include certain chemicals and ultraviolet radiation.

Cancer-causing mutations occur mostly in somatic cells, not in the germ-line

cells, and somatic cell mutations are not passed on to the next generation.

In contrast, certain inherited mutations, which are carried in the germ line,increase the probability that cancer will occur at some time.

In a destructive partnership, somatic mutations can combine with inherited

mutations to cause cancer.

Most cancers arise after genes are altered by carcinogens or by errors in the

copying and repair of genes.

Even if the genetic damage occurs only in one somatic cell, division of this cell

Rarely, however, does mutation in a single gene lead to the onset of cancer.

More typically, a series of mutations in multiple genes creates a progressively

more rapidly proliferating cell type that escapes normal growth restraints,

creating an opportunity for additional mutations.

will transmit the damage to the daughter cells, giving rise to a clone of altered

cells.

.

primary tumor migrate to new sites (metastasis), forming secondary tumors

that often have the greatest health impact.


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Properties of Tumor Cells

Cancer cells proliferate without anexternal inducing signal.

They fail to sense signals that

restrict cell division and continue to

live when they should die.

They often change their attachment

to surrounding cells or the

extracellular matrix, breaking loose to

divide more rapidly.

A cancer cell may, up to a point, resemble a particular type of normal, rapidly

dividing cell, but the cancer cell and its progeny will exhibit inappropriate immortality.

To grow to more than a small size, tumors must obtain a blood supply, and they

often do so by signaling to induce the growth of blood vessels into the tumor.As cancer progresses, tumors become an abnormal organ, increasingly well

adapted to growth and invasion of surrounding tissue

Tumors that are localized and are of small size are called benign tumors

In contrast, cells composing a malignant tumor, or cancer, usually grow

and divide more rapidly than normal, fail to die at the normal rate (e.g.,

chronic lymphocytic leukemia, a tumor of white blood cells), or invade nearby

tissue without a si nificant chan e in their roliferation rate e. . lessharmful

tumors of glial cells).

Some malignant tumors, such as those in the ovary or breast, remain

localized and encapsulated, at least for a time.

When these tumors progress, the cells invade surrounding tissues, get into

the bodys circulatory system, and establish secondary areas of proliferation,

a process called metastasis.


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3

Subsequent studies showed that the cloned segment included a mutantversion of the cellular ras gene.

Ras protein in its GTP-bound form is active and stimulates a cascade of

protein phosphorylations in the cell. Most of the time, however, the protein is

in its inactive, GDP-bound form.

Transgenicmice

carrying

both

the

mutant

rasD oncogene and

the

cmyc

protooncogene controlledbyamammarycellspecificpromoter/enhancer from

aretroviruswasgenerated.

Whenlinkedtothispromoter,thenormalcmyc geneisoverexpressed in

breasttissuebecausethepromoterisinducedbyendogenoushormonelevels

Multiplemutationsarerequiredfortumorinduction

andtissuespecificregulators.

Thisheightenedtranscriptionofcmyc mimicsoncogenic mutationsthatturn

upcmyctranscription,convertingtheprotooncogeneintoanoncogene.

Byitself,thecmyc transgene causestumorsonlyafter100days,andthenin

onlyafewmice;clearlyonlyaminutefractionofthemammarycellsthat

overproducetheMycproteinbecomemalignant.

Similarly,productionofthemutantRasD proteinalonecausestumorsearlier

.

Whenthe

cmyc and

rasD transgenics are

crossed,

however,

such

that

all

mammarycellsproducebothMycandRasD,tumorsarisemuchmorerapidly

andallanimalssuccumbtocancer


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Steps in the process of metastasis

Metastasisistheescapeofcancercellsfromaprimarysiteandtheirreestablishmentat

distantsecondarylocations.

Metastasisrequiresthedisruptionoflocalcellcellinteractions,invasion,penetrationof

bloodor

lymphatic

vessels

(intravasation),

escape

from

those

vessels

(extravasation),

migrationandgrowth.

1. Invasion 2.Intravasation 3.Extravasation 4.Colonizenewsite

Tumorgrowthisangiogenesisdependent


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TheconversionofaNormalcelltoaTumorcell

Acell

becomes

converted

from

anormal

to

aneoplastic cancer

cell

when

the

regulationofoneormoreprocessesislost.

Lossofregulationoccurswhenmutationsariseintwobroadfamiliesofgenes

a regu a egrow : ncogenes,w c ac aspos ves gna s orgrow an

Tumorsuppressorgenes,whichactasbrakesorcheckpointsonacells

progressionthroughthecellcycle.

Thesemutationsmaybecausedbyenvironmental,chemicalorbiological

agentsoreventsthatresultinirreversiblealterationsinthegenomeofacell,

sothatprogenycellsalsocarrythesamemutationsthatallowforuncontrolled

growth.

Thisisthefirstste ona athwa thatcaneventuall leadtoana ressive,

metastatictumor.

Fortunately,organismspossessseveralmeansofdealingwithenvironmental

insultsand

genetic

alterations.

Morethanonemutationsisrequiredforthedevelopmentofcancer

In

1911,

Peyton

Rous

laid

the

groundwork

for

the

oncogene theory

of

cancer,

a

theorythatbecamethebasisforallmoderncellularsignalingandgenetic

research.

Heidentifiedaspindlecelledsarcomainchickensthatwastransplantable from

onebirdtoanother,usingafiltrateofthetumor.

Oncogenes and the Oncogene Hypothesis

Sarcomavirus.

BuildingontheearlyworkofRous,Shope providedfurtherevidencethatfor

theviralbasisofoncogenesis byhisdemonstrationthatapapillomalikegrowth

wastransmissiblefromanimaltoanimal.

Grossshowedthatmiceinoculatedwithleukaemic extractsdeveloped

neoplams.

Fromthese

extracts,

the

Gross

murine leukaemiavirus

was

isolated.

Sevenyearslater,TeminandRubinshowedthatculturedchickenfibroblasts

withtheRoussarcomaviruscausesneoplastic transformationofthecells.

Martinandotherslateridentifiedtheoncogenic portionoftheRSVgenomeas

vsrc,theviralsrconcogene.

Theseearlyresultssuggestedatransmissiblemechanismfortumorinitiation


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Oncogene Hypothesis

Thebestknowntheoryofoncogenesis wasputforwardbyBishopandVarmus

whohavehypothesizedthatcancercausinggenes,oroncogenes,thatare

carriedby

tumor

inducing

viruses

have

normal

counterparts

that

are

present

in

thegenomesofallvertebratecells.

Thesenormalgenesaretermedprotooncogenes.

Evidenceforthishypothesiscamefromhybridizationstudies,where

radiolabelledvsrcDNAwasfoundtobind,orhybridize,toitscomplementary

counterpart(csrc)innormalaviancellularDNA.

Thevsrcandcsrcgenesencodeatyrosinekinase,anenzymethattransfers

phosphatefromATPtotheaminoacidtyrosinefoundincellularproteins.

Thesephosphorylations haveprofoundeffectsoncellgrowth.

Similarstudieseventuallyledtotheidentificationofafamilyofviral

,

Several

lines

of

evidence

indicate

that

viral

oncogenes arise

when

an

RNA

virus

integratesitsgenomenearthecodingsequenceforaprotooncogeneandincorporates

theprotooncogenesDNAintoitsowngeneticmaterialduringthevirusreplicationcycle.

Throughmultipleroundsofinfectionsandgenomereplication,deletionsandother

mutationsoccurintheprotooncogene,conferringonthegenetumorigenic properties.

EnsuinginfectionofanormalcellbyanRNAtumorviruscarryingsuchanoncogene

Oncogenes

causesmalignanttransformationofthatcell.

Example of human oncogenes


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Proto-Oncogenes

Protooncogenes canbeclassifiedaseithercytoplasmic ornuclear,depending

onwhereinthecelltheyarelocalized.

Manyof

the

cytoplasmic proto

oncogenes code

for

tyrosine

kinase molecules,

enzymesthatareabletophosphorylate substrateproteinsontyrosineresidues

andthatareknowntobeessentialforcontrollingthesiganaling cascadesthat

regulatemitosis.

OtherssuchasRas,transmitcellulargrowthsignalsbybindingguanine

nucleotidesintheformofGTPorGDP.

Ras isoftenfoundmutatedatsinglesitessuchthatitisconstantlyboundto

GTP,whichcausesthemoleculetobeconstitutivelyactive.

MutationsinRas arefoundinapproximately30%ofhumancancers.

,

protooncogenes thatregulateproliferation.Nuclearoncogenes suchasmyc

regulategenetranscription

Tumour suppressor genes

Tumour suppressor genes (TSGs)encode

proteins whose

absence,

repression,

expressioninactivation,ormutationpromotesoncogenesis.

Retinoblastoma (Rb)genewasthefirsttumorsuppressorgenetobeidentified.

Othertumorsuppressorgenesinclude,adenomatous polyposis coli(APC)gene,

thedeletedincoloncancer(DCC)geneandthemutatedincoloncancer(MCC)

gene.

APCmapstochromosome5q21andismutatedin70%ofpatientswitha

hereditaryformofcoloncancer.

TheMCCwhichisalsolocatedinthesamechromosomeismutatedin55%ofall

coloncancers.

DCCwasmappedtochromosome18andisdeletedin73%ofcoloncancers.

APCandDCCcodeforproteinsthatplayrolesinregulatingcelladhesionin

.

Itis

speculated

that

loss

of

these

genes

can

lead

to

increases

in

cell

motility,

a

keycharacteristicofmetastasis

p53 isinvolvedinsensingDNAdamageandregulatingcelldeath.

Itismutatedordeletedinover70%ofhumancancersincludingosteosarcomas,

carcinomasofthebreast,colon,lungandprostate


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BRCA1andBRCA2

BRCA1,wasfoundtobelinkedtoanincreasedriskofhereditarybreastcancer.

Lossofchromosome17qhadlongbeenknowntooccurinfamilialbreastcancer.

ManyheritablemutationswereidentifiedinBRCA1frombreastcancerpatientsand

includean

11

bp

deletion,

a1bp

insertion,

astop

codon and

amissense substitution.

BRCA1genecodesforaproteincalledbreastcancertype1susceptibilityprotein,

responsibleforrepairingDNA.

BRCA1isexpressedinthecellsofbreastandothertissue,whereithelpsrepair

damagedDNA,ordestroycellsifDNAcannotberepaired.

IfBRCA1itselfisdamaged,damagedDNAisnotrepairedproperlyandthisincreases

risksforcancers.

PTENgene

PTEN(phosphatase andtensin homologuedeletedonchromosometen),alsoknownas

MMAC1(mutatedinmultipleadvancedcancers)andTEP1(TGFregulatedandepithelial

cellenrichedphosphatase)wasfirstidentifiedasatumours uppressor genelocalizedon

chromosome10q23.

PTENmutationsoccurinmanytumour typesandareassociatedwithdifferentstagesof

tumorigenesis.

In

response

to

mitogen or

cytokine

stimulation,

phosphoinositide 3

kinase (PI3K)

activationleadstotheproductionofPtdIns(3,4,5)P3andsubsequentrecruitmentofboth

PKB(Akt/RAC)andPDKstotheplasmamembrane.

Theresultingcolocalization ofkinases facilitatesPDK(PHdomaincontainingkinase)

phosphorylation (activation)ofPKB,whichinturnphosphorylates substratesinvolvedin

cellgrowthandsurvival.

Tumor suppressor gene and their functions and associated cancers


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3

The Genetic Basis of Cancer

Conversion,or

activation,

of

aproto

oncogene

into

an

oncogene generally

involves

a

gainoffunctionmutation.

Atleastfourmechanismscanproduceoncogenes fromthecorrespondingproto

Pointmutation(i.e.,changeinasinglebasepair)inaprotooncogenethatresultsin

aconstitutivelyactiveproteinproduct

Chromosomaltranslocationthatfusestwogenestogethertoproduceahybridgene

encodingachimeric proteinwhoseactivity,unlikethatoftheparentproteins,oftenis

constitutive

Chromosomaltranslocationthatbringsagrowthregulatorygeneunderthecontrol

ofadifferentpromoterthatcausesinappropriateexpressionofthegene

Amplification(i.e.,abnormalDNAreplication)ofaDNAsegmentincludingaproto

oncogene,sothatnumerouscopiesexist,leadingtooverproductionoftheencoded

protein

Cancer-Causing Viruses Contain Oncogenes or Activate Cellular Proto-

oncogenes


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3

Thecausesofcancercouldbeanythinglikethefollowing

Tobacco,Infections,

Dietary

related

factors,

Reproductive

and

hormonal

factors,Radiation,Occupationalcarcinogens,Medicalcarcinogens(non

radiation),Environmentalpollution,Geneticpredisposition,Mutagens

Apoptosis

Celldeath:

Cellscan

die

by

either

of

two

major

mechanisms:

necrosisor

apoptosis.

Apoptosisisaprocesswherebycellsdieinacontrolled,programmedmannerinresponse

tospecificstimuli.

Itischaracterizedbydistinctchangesincellvolume,nuclearorganizationandthe

.

Therearethreestagesofapoptosisthatarebestdescribedbymorphologicalcriteria

First,nuclearchromatin,whichisacomplexofnucleicacidsandproteinsinthecell

nucleus,condenses.Thenucleusdecreasesinsize,andcellvolumedecreasesbyabout

onethird.

Thesecondstageofapoptosisinvolvescharacteristicmembraneblebbing,which

meansthecellmembraneformsfoldsandbecomesinvaginated.Inthisphase,boththe

nucleusandthecytoplasmbegintobreakapartintosmall,membraneboundapoptotic

bodies.

Inthe

third

stage,

the

apoptotic

bodies

bud

off

of

the

cell

and

are

degraded

as

they

are

ingestedbyphagocytesandneighboringepithelialcells.Ataboutthistime,an

endonuclease (orendonucleases)beginstocleaveDNA.ThecleavedDNAformsa

characteristicladderpatternoffragmentsof100+n(100)basepairsinsize(1


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Incontrast,theothercommonpathwayforcelldeath,necrosis,ischaracterized

byagradualdissolutionofcellstructureandeventualruptureofthecell

membrane.

Necrosisgenerallyoccursaftermajortoxicinsult,whencellsaretoodamagedtosurvive.

Necrosisbe inswithaninitial haseof eneralizedcellswellin .

Necrosis

Overaperiodofhourstodays,thereisgradualdissolutionoforganellesand

ruptureoftheplasmamembrane.

DNAisnotfragmentedinapatternedway,butsomeDNAdegradationmay

occur.

Necrosistypicallyoccursingroupsofcells,andtakesmanyhoursordaysto

complete.

IfdegradationofDNAoccurs,occasionalsmallfragmentsofDNAare

pro uce ,resu ng na smeary a erpa ernw en e s

electrophoresed onagarose gels.Necrosisinorganismsistypicallyaccompanied

by

acute

inflammation

and

secondary

scarring.

Necrosis

is

never

physiological,

it

isalwayspathological,meaningitiscausedbyanexternalinsult.Itcanbe

complementmediated,

causedbyseverehypoxia,orcausedbyhighlevelsofvarioustoxiccompounds

Hallmarksoftheapoptoticandnecroticcelldeathprocess.


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Apoptosisisatightlyregulatedprocess

Itrequireshighlyefficientcelldeathprogramwhichrequirestheinterplayofa

multitudeof

factors

Apoptosisisanactive,energydependentprocess

IthasbeenproposedthattheATPintracellularlevelisthecrucialconditionto

Molecular Mechanisms of Apoptosis

makedecisionofactivatingtheenergydependentapoptoticpathwayorthe

passivenecrosis.

Poly(ADPribosylation)isaposttranslationalmodificationofproteinsthat,in

eukaryoticcells,playsacrucialroleinDNArepairandreplication,transcription

andcelldeath,andrepresentsacellularemergencyreaction

ThecleavageofPARP1duringapoptosis,oneoftheearlierstepsinapoptosis

wasattributedtoaproteaseresemblingICE

PARP1cleavageproducesan89kDa Cterminalfragment(containingthe

catalyticdomain),andthe24kDa NterminalfragmentwiththeDBD.The89

kDa fragmentretains

the

basal

enzymatic

activity

due

to

the

presence

of

the

catalyticdomain,althoughitcannotbestimulatedbyDNAstrandbreaks.After

cleavage,

PARP1losesthenicksensorfunctionandisinactivetowardsDNAdamage.

Therefore,NAD+ consumptionisprevented.

LaterseveralproteasesoftheICEfamilysuchasCPP32/YAMA/Apopain were

foundtocleavePARP1duringapoptosis.

Suchenzymeswithcysteine proteaseactivityPolymerase1cleavageduring

apoptosishavebeendesignedascaspases,thisnamebeingcomposedbyc

Caspases

(cysteine)andaspase(abilitytocleaveafteranasparticacid).

Caspase activationoccursinacascadelikeway.Theproteolytic cleavageofa

regulatorcaspase activatesdownstreameffector caspases topromotethe

cleavageofanumberofsubstrates.

Todate,afamilyofatleast10caspases havebeenidentified,butitisnot

knownpreciselywhichofthesecaspase(s)areactivatedinvivoandwhichare

responsibleforthecleavageofparticularsubstrates.

Caspases maybe

divided

into

initiator

caspases with

long

prodomains

(caspases8,9,and10),whichactivateeffectorcaspases withshort

prodomains (caspases3,6,and7),whichinturncleaveintracellular

substrates,resultinginthedramaticmorphologicalandbiochemicalchangesof

apoptosis.


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There are two major pathways of apoptosis

The p53 dependent pathway and a p53 independent pathway involving the

apoptotic regulators like the Bcl-2 family of proteins.

Pathways of Apoptosis

p53 was widely recognized as a tumor suppressor gene, mutated or lost in

,50% of all human cancer cases worldwide

The Bcl-2 family includes the anti-apoptotic members such as Bcl-2, Bcl-XL,

Bcl-W, Bfl-1, Mcl-1, etc., which act as pro-survival proteins and

The pro-apoptotic members such as Bax, Bad, Bak, Bik etc., which induce

apoptosis when over expressed

The family members form both homodimers and heterodimers

In both the pathways activation of caspases play an important role in the

execution of apoptosis

A model for p53-mediated apoptosis

p53,inresponsetostressorDNAdamagetranscriptionally activatesthe

checkpointproteinslikep21andarreststhecellcycletofacilitateDNArepair

involvingthenucleotideexcisionrepairandbaseexcisionrepair

Ifthedamageisbeyondrepairitactivatesapoptosisbythetranscriptional

ac va ono se ec or genes e , , ax an .

p53inducesapoptosisbothbyextrinsicpathwayandintrinsicpathway

involvingthemitochondria

Intheextrinsicpathway,p53activatestheexpressionandtraffickingofthe

membranereceptors,FASandDR(deathreceptor).

FASandDRgetactivatedbybindingoftheircorrespondingligands.Activation

ofFASandDRactivatestheprocaspase 8whichinturnactivatestheeffector

cas ases likecas ase 3,cas ase 6andcas ase 7


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Model for p53 Induced Pathway of Apoptosis

YFAS

FAS-LTRAIL

DR5

p53

Extrinsic

Pathwayp53

Mitochondria

p53

aspase-

Caspase-9

Apoptosome

Noxa

Puma

Bax-Bax

Intrinsic

Cyt c

Apaf 1

Caspase-6

Caspase-3

Caspase-7

Apop tos is

Pathway

Haupt et al 2003, Hofseth et al 2004

The intrinsic apoptotic pathway is dominated by the Bcl-2 family of proteins,which governs the release of cytochrome c from the mitochondria .

The Bcl-2 family comprises anti-apoptotic (pro-survival) and pro-apoptotic

members.

Family members are classified on the basis of structural similarity to the Bcl-2

Intrinsic Pathway

, , ,

domain.

The BH3 domain, which is present in all members and is essential for

heterodimerization among members, is the minimum domain required for the

proapoptotic function

The Bcl-2 family is divisible into three classes: prosurvival proteins, whose

members are most structurally similar to Bcl-2, such as Bcl-XL;

Pro-apoptotic proteins, Bax and Bak, which are structurally similar to Bcl-2

and Bcl-XL and antagonize their pro-survival functions; and the pro-apoptotic

BH3-only proteins.A key subset of the Bcl-2 family genes are p53 targets, including Bax, Noxa,

PUMA and the most recently identified, Bid.


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In the Intrinsic pathway, p53 activates the expression of the effector

proteins like bax which upon binding to mitochondria cause release ofcytochrome c (cyt c) from the mitochondria

y oc orme c n s o e apop os s ac va ng ac or pa - an

caspase 9 and this complex activates the downstream effector caspases

leading the execution of apoptosis

The bcl2 pathway involves the bcl2 family of proteins consisting of the

proapototic proteins like bax, bad, bid, bak and the antiapoptotic proteins like

bcl2, bcl-XL, bcl-W, and Bfl-1.

The bcl2 family of proteins has the tendency to form homodimers or

heterodimers. In the unphosphorylated form, bcl-2 forms a complex with bax and

The Bcl2-Bax pathway of apoptosis

phosphorylation of bcl-2 inhbits the formation of bcl2-bax complex.

Microtubule targeted agents suppress the microtubule dynamics and block the

cell cycle progression at mitosis.

This leads to the activation of bcl2 specific kinase which causes

phosphorylation of bcl2.

Upon phosphorylation of bcl-2, bax is released from the bcl-2-bax complex and

undergoes bax-bax homodimerization.

This Bax-Bax homodimer integrates into the mitochondrial membrane and alters

apoptosis as mentioned above.

Hence, a balance between bcl2-bax heterodimer and bax-bax homodimer is

required to maintain the cell survival


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Mitotic

block

Microtubuletargeted drugs

Bcl2

P

Bcl2

P

Bax Bcl2BaxBax

Bax Bcl2

Bcl2

P

Bcl2

P

Bcl2

P

BaxBcl2

Act ivation of Bcl2-

specific Kinase

Model for Bcl2-Bax Pathway of Apoptosis

Bcl2 hyper-phosphorylation

Bcl2

P

ax ax

BaxBax

ax c

Act ivation of

Dissociation of

bcl2-bax

Apoptosi s

Increased ratio of bax-bax

homodimer

Bcl2

Bax

Bax-Bax

Release of Cyt c

Mitochondria

Caspase cascade

Basu and Haldar 1998, Oltvai et al 1993

cell biology module 3

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