cell biology module 3
TRANSCRIPT
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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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Cyclins in cell cycle regulation
.
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.
Cyclins in cell cycle regulation
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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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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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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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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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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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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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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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