BIOMOL 2013 Cell Cycle Apoptosis 2 (1)

8/11/2019 BIOMOL 2013 Cell Cycle Apoptosis 2 (1)

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CELL CYCLE DAN

APOPTOSISEDY MEIYANTO

CCRC FARMASI UGM


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CHAPTER 17:

CELL CYCLE


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Hanahan & Weinberg 2011

Summary of 40 years of research (1971-2011)


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review

Fase-fase cell cycle? Apa yang disebut sel quiscence dan senescence?

Regulator cell cycle: Positif vs negatif

Bagaimana Go-> G1? Apa peran pRb?

Bagaimana transisi G1/S ?

Bagaimana mengamati profil cell cycle?

Apa peran p53?

Apa peran CDK-1/cyc B?

Apa peran APC?


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Continuity of life

Only from existing cells come new cells.

We are all decedents of the first cells on the

planet.

A cell reproduces by duplicating its contentsand then dividing into two

This cycle of events is known as the cell cycle


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Consideration

Bagaimana sel menduplikasi

kandungannya?

Bagaimana sel membagi kandungannyamenjadi 2 dan membelahnya?

Bagaimana hal itu diatur?


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Stein, et al., Ed. The molecular basis of cell cycle

and growth control, Wiley-Liss, Inc. 1999.

Cooper, GM, The Cell, a Molecular Approach,

ASM Press, 2001.

Three types of proliferative cells:

1. Always dividing

2. Quiescent (Go)

3. Terminally differentiated

Proliferation, establishment, differentiation

and reversal of terminal differentiation are

dependent on serum factors and

oncoproteins

CELL CYCLE REGULATION


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Cell division timing

Different cells have cell cycles of

different lengths;

Nerve Cells = never

Human Liver Cells = 1 year

Intestinal epithelial cells = 12 hours

Yeast cells = 1.5 to 3 hours Bacteria = 90 minutes


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Eukaryotic ChromosomesAll eukaryoticcells store genetic

information in chromosomesMost eukaryotes have between 10 and50 chromosomesin their body cells

Human body cellshave 46chromosomesor 23 identical pairs


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Eukaryotic ChromosomesEach chromosome is composed of a

single,tightly coiled DNAmoleculeChromosomes cant be seenwhencells aren

t dividingand are calledchromatin


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Compacting DNA intoChromosomes

DNA istightly

coiledaroundproteins

calledhistones


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Chromosomes in Dividing Cells

Duplicatedchromosomes arecalled

chromatids&are heldtogether by thecentromere

Called Sister Chromatids


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KaryotypeA pictureof thechromosomes froma human cellarranged in pairs by

sizeFirst 22 pairs arecalled autosomes

Last pair are thesex chromosomes

XXfemale or XYmale

B G l


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Boy or Girl?

Y - Chromosome

X - Chromosome

The Y Chromosome Decides


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Cell Reproduction


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Types of Cell ReproductionAsexual reproduction involves a

single cell dividing to make 2 new,identical daughter cellsMitosis & binary fission are

examples of asexual reproductionSexual reproduction involves twocells (egg & sperm) joining to make a

new cell (zygote) that is NOTidentical to the original cellsMeiosis is an example


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Cell Division inProkaryotes


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Cell Division in Prokaryotes Prokaryotes such as

bacteria divide into 2identical cells by theprocess of binaryfission

Single chromosomemakes a copy ofitself

Cell wall formsbetween thechromosomes dividingthe cell

Parentcell

2 identical daughter cells

Chromosomedoubles

Cell splits


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Prokaryotic CellUndergoing Binary Fission


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Animation of Binary Fission


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The CellCycle

F Ph f h ll l


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Five Phases of the Cell Cycle

G1- primary growth phaseS synthesis; DNA replicatedG2- secondary growth phase

collectively these 3 stages arecalled interphase

M - mitosisC - cytokinesis

ll l


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Cell Cycle

I h G S


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Interphase - G1Stage

1stgrowth stageafter celldivision

Cells matureby making morecytoplasm & organelles

Cell carries on its normal

metabolic activities

I t h S St


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Interphase S StageSynthesisstage

DNAis copied or replicated

Twoidenticalcopiesof DNA

Original

DNA

I t h G St


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Interphase G2Stage

2

nd

GrowthStageOccurs after DNA has been copied

All cell structures needed fordivisionare made (e.g. centrioles)Both organelles & proteinsare

synthesized

Wh H h


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Whats Happening in Interphase

What the cell looks like

Animal Cell

What

s occurr ing

Sk t h th C ll C l


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Sketch the Cell Cycle

DaughterCells

DNA Copied

CellsMature

Cells prepare forDivision

Cell Divides intoIdentical cells


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http://www.biologymad.com/CellDivision/CellDi1.gif


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Mitosis

Mit i


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MitosisDivision of the

nucleusAlso calledkaryokinesis

Only occurs ineukaryotesHas fourstages

Doesn

t occur insome cells suchas brain cells

F Mit ti St


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Four Mitotic Stages

Prophase

Metaphase

Anaphase

Telophase

E l P h


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Early ProphaseChromatinin nucleus condenses to

form visible chromosomesMitotic spindle forms from fibers incytoskeleton or centrioles (animal)

Chromosomes

Nucleolus Cytoplasm

Nuclear Membrane

L t P h


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Late ProphaseNuclear membrane & nucleolusare

broken downChromosomescontinue condensing &are clearly visible

Spindle fibers called kinetochoresattach to the centromereof each

chromosomeSpindlefinishes forming between thepoles of the cell

L t P h


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Late Prophase

Nucleus & Nucleolus have disintegrated

Chromosomes

Spindle Fiber attached to


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Spindle Fiber attached toChromosome

Kinetochore Fiber

Chromosome

R i f P h


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Review of Prophase

What the celllooks like

What

s happening

S i dl Fib


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Spindle FibersThe mitotic spindleform from the

microtubulesin plantsand centriolesin animal cellsPolar fibers extend from one pole of

the cell to the opposite poleKinetochore fibersextend from thepole to the centromere of the

chromosome to which they attachAstersare short fibers radiatingfrom centrioles

Sk t h Th Spindl


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Sketch The Spindle

M t ph s


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MetaphaseChromosomes, attached to the

kinetochore fibers, move to the centerof the cell

Chromosomes are now lined up at the

equator

Pole of

the Cell

Equator of Cell

Metaphase


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Metaphase

Chromosomeslined at theEquator

Asters atthe poles

SpindleFibers

Metaphase


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Metaphase

Aster

Chromosomes at Equator

Review of Metaphase


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Review of Metaphase

What the cell lookslike

What

soccurring

Anaphase


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Anaphase

Occurs rapidly

Sisterchromatidsare

pulled apart toopposite polesof the cell by

kinetochorefibers

Anaphase


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Anaphase

Sister

Chromatidsbeingseparated

Anaphase Review


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Anaphase Review

What thecell looks

like

Whatsoccurring

Telophase


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Telophase

Sister chromatids at opposite

polesSpindle disassemblesNuclear envelopeforms around

each set of sister chromatidsNucleolusreappearsCYTOKINESISoccursChromosomes reappear aschromatin

Comparison of Anaphase & Telophase


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Comparison of Anaphase & Telophase

Cytokinesis


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CytokinesisMeans division of the cytoplasm

Divisionof cell into two,identical halves called daughtercellsIn plant cells, cell plate formsat the equator to divide cellIn animal cells, cleavage furrowforms to split cell

Cytokinesis


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Cytokinesis

Cleavage furrowin animal cell Cell plate inanimal cell

Mitotic Stages


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Mitotic Stages

Daughter Cells of Mitosis


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Daughter Cells of MitosisHave the same number of

chromosomes as each otherand asthe parent cellfrom which theywere formed

Identicalto each other, but smallerthan parent cell

Must grow in sizeto become maturecells (G1of Interphase)

Identical Daughter Cells


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Identical Daughter Cells

Chromosome number the same, but cellssmallerthan parent cell

What isthe 2n

ordiploid

number?

2


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Review

ofMitosis


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Draw & Learn these Stages


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Draw & Learn these Stages

Name the Mitotic Stages:


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Interphase

Prophase

Metaphase

Anaphase

Telophase

Name the Mitotic Stages:

Name this?

Name this?

Eukaryotic Cell Division


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Eukaryotic Cell Division

Used for growth andrepair Produce two new cells

identical to the original

cell Cells are diploid (2n)

Chromosomes duringMetaphase of mitosis

Prophase Metaphase Anaphase TelophaseCytokinesis

Mitosis Animation


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Mitosis AnimationName each stage as you see it occur?

Mitosis in Onion Root Tips


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Mitosis in Onion Root Tips

Do you see any stages of mitosis?


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Test Yourself

over Mitosis

Mitosis Quiz


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Mitosis Quiz

Mitosis Quiz


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Mitosis Quiz

Name the Stages of Mitosis:


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Name the Stages of Mitosis:

Interphase

Early prophase

Mid-ProphaseLateProphase

Metaphase

LateAnaphase

Early Anaphase

EarlyTelophase,

Begincytokinesis

Late telophase,Advancedcytokinesis

Identify the Stages


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Identify the Stages

Early, Middle, & Late Prophase

Late Prophase Metaphase Anaphase

Late Anaphase Telophase

Telophase &

Cytokinesis

?

? ? ?

? ? ?

Locate the Four Mitotic


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Locate the Four MitoticStages in Plants

Metaphase

Prophase

Anaphase

Telophase


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Eukaryotic Cell Cycle


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18_02_four_phases.jpgEukaryotic Cell Cycle



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Four phases

M phase (mitotic phase)

G1 phase (Gap 1) S phase (synthesis phase)

G2 phase (Gap 2)

l


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Flowcytometer

Instrumen Flowcytometer


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Sel

Antibodi

marker

Sel


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tiap individu sel dilewatkan

melalui celah sempit


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Penembakan sel oleh sinar laser

Menghasilkan hamburan cahaya

Detektor


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Central Control
http://www.esb.utexas.edu/DOCUME~1/surangi/LOCALS~1/Temp/FrontPageTempDir/textart.ppts/1440.ppt


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Central Control

The cell-cycle control system regulates

the entire process

A great analogy is to think of it as a

washing machine control knob

Cannot implement the next program

until the first one in completed.

Feedback systems operate to provide

information on how processes areprogressing.


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FAKTOR UTAMA


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The master controllers of events in cellcycle are a small number ofheterodimeric protein kinases that

contains: regulatory subunit (cyclin) and

catalytic subunit (cyclin-dependentkinase; CDK)

http://medweb.bham.ac.uk/bmedsci/bms2/chime/structure/2A.gif


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Lodish et al., 2004


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Lodish et al., 2004


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Level beberapa jenis cyclin selama 1 putaran cell cycle


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Figure 17-16 Molecular Biology of the Cell( Garland Science 2008)

Fig 17-16 Cyclin-Cdk complex of the cell-cycle control system.


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Cycle regulation


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Cycle regulation

The key players are the kinases and

phoshatases


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IMPORTANT PRINCIPLE


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IMPORTANT PRINCIPLE Proteins may have phosphate groups added to

certain amino acids These phosphates are added by enzymes calledKINASES

Proteins may have phosphate groups removed fromcertain amino acids

These phosphates are removed by enzymes calledPHOSPHATASES

The same proteins may be repeatedly activated anddeactivated by simply adding or removing phosphategroups


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The cell-cycle is regulated by the phosphorylation of special

t i ll d Cdk ( h i d) H th t i


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18_05_ Cdks.jpgproteins called Cdks(shown in red). However, these proteins

must associate with other peptides before they become

functional. These other peptides are known as cyclins(shown

in green).

The cell just regulates the concentration of different cyclins

(hence their name - as they vary in concentration during the

cell cycle)


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18_25_growth_factors.jpg


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Checkpoints


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Checkpoints

The cell cycle halts at various points

until signals are sent to proceed


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18_03_control_system.jpg


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A. CDK inhibitorsactivated by:

P53 (p21)

VitaminD (p21)

Adhesion (p27)

TGFb (p15, p27)

B. Rb protein family:

pRb, p107, p130


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The cyclin which drives cells into M phase is called M-cyclin. Itinteracts with a kinase called M-Cdk.


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18_06_M_Cdk.jpgLevels of M-cyclin build steadily.

Then it is rapidly removed from the cell by rapid degradation by

the proteasome

Removal of M-cyclin results in the inactivation of M-Cdk andthe cell divides.

APC(anaphase promoting complex) decides when to remove

M-cyclin from the cell.

The Go (G zero) stateresults in the dismantling of most of thereplication machine. Nerve and muscle cells are in this state.


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18_16_G1_checkpoint.jpgMany other cells come to this important checkpoint each time

after they have divided.

In conclusion, the cell is faced with a number of points in the cellcycle where it has to satisfy certain molecular requirements before


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18_17_arrest_checkpt.jpg

it is permitted to continue along the cell cycle.

Control of cell numbers and


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Control of cell numbers and

cell size Three processes operate to control theeventual form a body part takes Cell growth

Cell division Cell death

Single celled organisms grow as fast as theyare able to limited by factors such a food

availability Multicellular organisms receive signals from

other cells in the body


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18_04_Feedback.jpg


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Fig 17-62 How DNA damage arrests the cell cycle in G1


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Fig 17-64 Cell-cycle arrest or apoptosis induced by

excessive stimulation of mitogenic pathways.


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Fig 17-65 Stimulation of cell growth by extracellular factors and nutrients.


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MPF:

Maturation PromotingFactor
http://www.esb.utexas.edu/DOCUME~1/surangi/LOCALS~1/Temp/FrontPageTempDir/textart.ppts/14250.ppthttp://www.esb.utexas.edu/DOCUME~1/surangi/LOCALS~1/Temp/FrontPageTempDir/textart.ppts/14250.ppthttp://www.esb.utexas.edu/DOCUME~1/surangi/LOCALS~1/Temp/FrontPageTempDir/textart.ppts/14250.ppthttp://www.esb.utexas.edu/DOCUME~1/surangi/LOCALS~1/Temp/FrontPageTempDir/textart.ppts/14250.ppt


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The key molecular event that marks many proteins fordestruction by the proteasome is ubiquitation - a type of

modification


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18_07_cyclin_degradat.jpg

modification.

Here the M-cyclin is ubiquitinated and is thus quickly destroyed.


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Fig 17-21 An overview of the cell-cycle control system.


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APOPTOSIS

Cell Death


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The body is very good at maintaining a

constant number of cells. So there has toexist mechanisms for ensuring other cells in

the body are removed, when appropriate.

Two formsApoptosis- suicide - programmed cell death

Necrosis- killing - decay and destruction

Necrosis Apoptosis

Programmed death


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Accidental death

Severe & sudden

injuryischaemia, physical

or

chemical trauma

Cellular and

organelleswelling

Random spillage of

cellular content

Inflammatory

responseMajor site of damage

plasma membrane

Programmed death

Process is more subtle,

and

more physiologically

determined.

Cell shrinkage

Plasma & nuclearmembrane

blebbing

Production of membrane

enclosed apoptotic

Clear by macrophages

No inflammatory response


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Cells are born, live for a given

Cells are born, live for


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Cells are born, live for a given

period of time and then dieBowen,

1998

--- Physiological celldeathdeath

--- Cell suicide

--- Cell deletion

--- Programmed cell

APOPTOSIS

,

a given period

of time and then dieBowen, 1998

Apoptosis in Development


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Digit development

Elongation of long

bone

Amphibian

metamorphosis


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18_27_nerve_target.jpg


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KELUARGA PROTEIN BCl 2


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KELUARGA PROTEIN BCl-2

Caspases


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Proteins which degrade other proteins

are employed by apoptosis - caspases Made as inactive precursors -

procaspases

These are activated by other proteinswhen the right signal is received

One caspase cleaves the lamin proteins

resulting in the irreversible breakdownof the nuclear membrane.


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18_28_regulating_Bcl2.jpg


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DNA Fragmentation


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DNA Fragmentation

In normal cells, DNA

are wired around

protein spindle called

histones DNA and histones form

units called

nucleosomes

In apoptotic cells,cleaved by DNase,

nucleosomes are cut

loose, like beads come

off a string

DNA Fragmentation


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DNA Fragmentation

DNA of apoptotic cells

subject to

electrophoresis

The DNA innucleosomes cut loose

have lower MW than

intact DNA, thus move

faster in electrophoresis

Result in a ladder in

the gel

Comet assay


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DNA fragments are released from nuclei usingelectrophoresis

Isolated nuclei are mounted into electrophoretic gelafter electrophoresis are stained with fluorescent dye.

If DNA fragments are present a comet tailis presentobserved in the vicinity of the nuclei.

+

Comet assay


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Comet assay

Possible results of a comet assay


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Possible results of a comet assay

Normal nucleus without

fragments

(DNA is not damaged

mutagenicity excluded)

Two nuclei with DNA

damage

Detection of Apoptosis by Flow


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y

Cytometry

Early stage Annexin V/7-AAD(PI)

Mid stage TUNEL assay

Late stage < Go/G1 DNA content

PENGAMATAN APOPTOSIS DENGAN ANNEXIN V

DAN PROPIDIUM IODIDE (PI)


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Phosphatidylserine Translocation(Detected by Flow Cytometry)


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(Detected by Flow Cytometry)


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Sel

Antibodi

marker

Sel


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tiap individu sel dilewatkan

melalui celah sempit

Penembakan sel oleh sinar laser


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Menghasilkan hamburan cahaya

Detektor

PENGAMATAN APOPTOSIS DENGAN ANNEXIN V


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DAN PROPIDIUM IODIDE (PI)

TUNEL ASSAY

Apoptosis


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TdT-mediated dUTP Nick-End Labeling

Incorporation of fluorescein-12-dUTP to

3-OH DNA ends using

enzyme Terminal deoxynucleotidyl Transferase (TdT)

5

3

OH

dUTP****

DNA degradation


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TUNEL Assay

with BrdUrd labeling

Apoptosis


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with BrdUrd labeling

anti-BrdUrd antibody

Cell number (2 x 106)

Chromatin denaturation

Acid Pepsin

Separation between neg. and pos. signals

Data analysis TUNEL assay

Apoptosis (TUNEL) from Rat Lavage Fluid

Control, 11 months Sterling V, 11 months


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DNA Content DNA Content

0.97 %

20.82%

G1

SG2M

G1

SG2M

Sub-G1


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SEMA3B induces caspase-3-dependent apoptosis and VEGF165 antagonizes this effect.


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Documents

BIOMOL 2013 Cell Cycle Apoptosis 2 (1)