The Cellular Basis of Disease

Cell Injury 3

Apoptosis and Necrosis

Cellular Aging

Christine Hulette MD
 This lecture is pretty

Objectives straightforward. You

should be able to
address these
objectives by the
end.
• Discriminate cell adaptation, reversible cell
injury and irreversible cell injury (cell death)
based on etiology, pathogenesis and
histological and ultrastructural appearance.
• Compare and contrast pathologic features and
the clinical settings in which necrotic and
apoptotic cell death occurs.
• List in temporal order the genetic and
biochemical steps in apoptosis.
• Contrast and compare physiologic and
pathologic apoptosis.
• Describe the mechanisms and implications of
cellular aging.
 Necrosis
• Morphologic expression of cell death
• Progressive disintegration of cell structure
• Initiated by overwhelming stress
• Usually elicits an acute inflammatory cell
response (neutrophils may be present).
Just because Dr. H
mentioned it, inflammatory
response doesn't occur in
immunocompromised
patients.
 Apoptosis
• Pathway of cell death induced by a tightly
regulated suicide program.
• Controlled by specific genes.
• Fragmentation of DNA. In a regular pattern.
• Fragmentation of nucleus.
• Blebs form and apoptotic bodies are released.
• Apoptotic bodies are phagocytized.
• No neutrophils. apoptosis.
No inflammation in
• Necrosis or
Apoptosis?
Necrosis. Why? Lots of Leukocytes.
Does Apoptosis involve an
inflammatory response? No!
 Consequences of Cell Death

Necrosis
Loss of functional tissue
Impaired organ function, transient or permanent

Apoptosis
Removal of damaged or unnecessary cells
 Necrosis Apoptosis

NORMAL
CELL
I NORMAL
CELL
J

Re vers ible
Recovery
injury

~~3__ Condeosation
01 chroma tin

:--"" Membrane blobs

Cllliula r
fragmentation

Bf9akdown 01
plasma membr"""
I A POPTOSIS 1
organolills and .

_ ...
....:IIIUS ; hla kage
of contents

N ECROSIS 1
of apoptotic calls
Amofpnous densities and fragments
., m~ochoodria

Capyrighl c>"",o ~ s - . .
""-"--
 Causes of Apoptosis This slide is
incredibly hi-yield.

• Physiologic
• Pathologic
 Physiologic Apoptosis
Think
• Embryogenesis and fetal development. development of
hands and feet.

• Hormone dependent involution.

Prostate glandular epithelium after castration
Regression of lactating breast after weaning

• Cell loss in proliferating cell populations.

• Immature lymphocytes
• Epithelial cells in the GI tract Lymphocytes
that act against
• Elimination of self-reactive lymphocytes. host. If not
eliminated, you
get autoimmune
• Death of cells that have served their diseases.

function.
• Neutrophils, Lymphocytes
 Apoptosis in Pathologic
Conditions
• DNA damage due to radiation, chemotherapy.

• Accumulation of misfolded proteins leads to

ER stress which ends with apoptosis.

• Cell death in viral infections that induce

apoptosis such as HIV and Adenovirus or by
the host immune response such as hepatitis.

• Organ atrophy after duct obstruction.

 General Characteristics
Compare and contrast Necrosis and
Apoptosis.. Look at the chart.

NECROSIS APOPTOSIS

Usually affects large areas Usually affects scattered

of contiguous cells individual cells

Control of intracellular Control of intracellular

environment is lost environment maintained
early in early stages

Cells swell and Cells contract

organelles swell
 General Characteristics

NECROSIS APOPTOSIS

Nuclear chromatin Nuclear chromatin

marginates early, marginates and
while injury is still chromatin condenses,
reversible becoming very compact

When DNA is cleaved, Chromatin condensation

which is usually a late and DNA fragmentation
event, fragments are occur together; DNA
random in size cleaved into multiples of
(smear pattern in gels) 200 base pair units We will see
this soon.
(ladder pattern in gels)
 General Characteristics

NECROSIS APOPTOSIS

Cell membrane ruptures as Blebs form and apoptotic

terminal event and cell bodies containing
contents are released, nuclear fragments
which are chemotactic are shed

Chemotactic factors lead to Phagocytosis of intact

neutrophil infiltration to apoptotic bodies, no
degrade dead cells chemotactic factors
are generated
 Apoptosis
Section of skin with Regular margination
immunologic mediated and break up of DNA
disease
 Apoptotic cell with
ladder pattern,
reflecting
cleavage at 200bp
intervals.

Normal cell

Necrosis. DNA in
all different
lengths, smear
pattern.

e ElioM'r 2005
A - early apoptosis; chromatin
margination & condensation

B - later in apoptosis; nucleus

is fragmented

C - phagocytosis of apoptotic
cellular remnants by
adjacent cell

D - swollen, necrotic cell for

comparison disruption and
dissolution of
cytoplasmic
components
 Apoptosis What do you think?

A. Refers to the process of cellular disintegration

A. Caused by ischemia and inflammation.
B. Responsible for changing cell type in response to
stress
C. Beneficial process to eliminate damaged cells
D. Reduces the size of an organ
E. Induced by retinoic acid
 Apoptosis
A. Refers to the process of cellular disintegration
A. Caused by ischemia and inflammation. Necrosis
B. Responsible for changing cell type in response to
stress Metaplasia

C. Beneficial process to eliminate damaged cells

D. Reduces the size of an organ Atrophy

E. Induced by retinoic acid Patterning during

embryogenesis
 Mechanisms of Apoptosis
• Death receptor (Extrinsic) pathway
• Mitochrondrial (Intrinsic) pathway
• Execution Phase
• Removal of dead cells
This slide indicates that
there are 2 pathways
(Intrinsic/Extrinsic) that lead
to the execution phase and
removal of cells.
 I think this slide is pretty
MITOCHONDRIAL (INTRINSIC) self explanatory. Any DEATH RECEPTO R (EXTRINSIC)
PATHWAY important points are PATHWAY
brought up again.
Receptor-li ga nd interactions
o Fas
o TNF '908ptOf
Cell injury Mitocr.on<!na Cytoc hrome c
oG rowth lacto, and olh&r
-Mthdrawal JIIO-apoptoltc
oONA damage proIains

",_......J
(by radiation.
toUls, froo Inilialo< caspaS<lS
radtcals) \ caspas...
o P'oI~~~::~~ , caspases
_ _ _ A,--~'.

~ndoo\ldOOS<l
activation
Br8akdown 01
cytosk8letoo

lig ands 10,

phagocytic
c81 ' 9C8p1orS
 Mechanisms of Apoptosis
• Cells contain intrinsic death and survival
signals that are genetically regulated.
• Genes are highly conserved across
species and are homologous to ced (cell
death abnormal) genes in nematodes that
initiate or inhibit apoptosis.
 For more detailed
information, check out the
Molecule and Cells Apoptosis
lecture.

Intrinsic -Mitochrondrial pathway

• Increased mitochondrial permeability with release of pro-apoptotic

molecules into the cytoplasm (cytochrome c).

• Synthesis of anti-apoptotic molecules (Bcl-2) promoted by Growth factors.

•
• When cells are deprived of growth factors or subjected to stress anti-
apoptotic molecules (Bcl-2) are lost.

• Bcl-2 is over expressed in most follicular B-cell lymphomas – allowing

abnormal cells to proliferate. Clinical significance.

• Mitochondrial membrane becomes permeable and proteins that activate

caspase leak out.
 • Intrinsic
(Mitochondrial)
Pathway of
Apoptosis
Don't memorize
details unless you
plan on getting a
PhD.

Hi MSTPs!!

Normal Cell
 Essence of intrinsic
(mitochondrial) pathway

• Pro-apoptotic and protective molecules that

regulate mitochondrial permeability and the
release of death molecules sequestered in
the mitochrondria are maintained in balance
normally.

• Imbalance initiates the death pathway.

 Extrinsic (Death receptor
initiated) pathway

• Death receptors are members of the tumor

necrosis factor receptor family and a
related protein called Fas (CD95).

• These molecules contain a death domain.

 • Extrinsic (Death
Receptor-initiated)
Fas Expressed on cell
Pathway of
surface linked to Death
domain. If activated,
apoptosis is initiated.
Apoptosis
 Execution Phase

• The intrinsic and extrinsic pathways converge to a

caspase activation cascade.

• Caspases (cysteine-aspartic-acid-proteases) are

conserved across species.

• Synthesized as inactive precursors; activated by

proteolytic cleavage.

• Family of at least 12 proteases, a few of which

are involved in inflammation, and many of which
are involved in apoptosis
 How Caspases Disassemble a Cell

• Cleave structural proteins leading

to nuclear breakdown.

• Converts cytoplasmic DNase to

active form.

• DNase causes characteristic

internucleosomal cleavage of DNA.
 Removal of Dead Cells

• Dying cells secrete factors the recruit

phagocytes.
• This facilitates prompt clearance before
they undergo secondary necrosis.
• Dead cells disappear without a trace and
do not produce inflammation.
 Which of the following features is
seen in apoptosis but not in
necrosis?
Thoughts?

A. Internucleosome cleavage of DNA

B. Inflammation
C. Pyknosis
D. Cytoplasmic hypereosinoplilia
E. Karyolysis
 Which of the following features is
seen in apoptosis but not in
necrosis?
A. Internucleosome cleavage of DNA
B. Inflammation
C. Pyknosis Features of
Necrosis
Reflection of cell
injury and not D. Cytoplasmic hypereosinoplilia
death.
E. Karyolysis
 Examples of Apoptosis
Lack of growth factor or hormone

• Hormone sensitive cells deprived of hormone.

• Lymphocytes that are not stimulated.

• Neurons deprived of growth factor.

Remember Brain and Behavior?
 Examples of Apoptosis
Specific activation of death receptors
• DNA damage - Tumor suppressor gene p53 accumulates in
damaged cells and arrests the cell cycle. p53 is mutated or
absent in some cancers and can not initiate apoptosis in
malignant cells.

• Protein misfolding – unfolded protein response and ER

stress – Alzheimer, Parkinson and Huntington diseases.

• TNF Receptor family.

• Cytotoxic T lymphocyte
 Apoptosis in Pathologic
Conditions
• Ionizing radiation
• Cytotoxic chemotherapeutic drugs
• Mild thermal injury
• Cell injury in some viral diseases
• Pathologic atrophy after duct obstruction
• Cell death in tumors
• Glucocorticoids induce apoptosis in
lymphocytes
 You've got this..

Apotosis Summary
“Programmed cell death” can be activated by
moderate stress which has damaged the cell
beyond its ability to recover fully or by viral
infection.
This has the desirable effect of removing
damaged or infected cells.
Selective manipulation of apoptotic pathways
may be an important approach for treating
cancer in the future.
We recognize that cell death has occurred
by morphologic manifestations which are
often influenced by the environment.

Is the distinction between necrosis and

apoptosis absolute? NO!!!
 Cellular Aging
We're done with Apoptosis now. Here are the
main points to focus on for cellular aging:

• Structural and Biochemical Changes with Aging

• Decreased cellular replication
• Telomere shortening causes cell cycle arrest
• Accumulation of Metabolic and Genetic Damage
• Calorie restriction delays aging
 Well understood

Not well
understood

Telomere Environmental DNA repair Abnormal growth

shortening insults defeels flllctor si;:!nllliing
t-......LJ t....,,;,;, (e.g .. insulinl1GF)

Free ____• DNA Aelivation of I

radialis • damage Sirtuins
Mechanism?

Roplicativo Damago to protoins

l
Accumulation
I
Caloric rcctrielion
?

senescence and organelles of mutat:ons

Unfortunately "?"
is not a sufficient
explanation in
CELLULAR AGING science.
 Structural and Biochemical Changes
with Aging

• Oxidative phosphorylation is reduced

• Synthesis of nucleic acids, structural proteins,
enzymes, cell receptors and transcription factors
are reduced
• Decreased capacity for nutrient uptake and repair
of DNA damage
• Cytologic changes
• Accumulation of abnormally folded proteins
 Replicative Senescence

• Cells have a limited capacity for replication.

• Cultured human fibroblasts have limited division

potential.

• Werner’s syndrome is a rare disease characterized

by premature senescence. patients
Werner's syndome
often die as
early as age 20, but
may look 100.
 Oh hey, Robbins.
20 ,---------------------------------,

.n 15 Newborn
o

-
x
IT
W Werner
m 10
:;; syndrome Fibroblasts from
=>
z individuals, # of times
they can reproduce.
---'
---'
w
U 5

o-t--,------,r-----r- :?---r-----' ;r-----r~ '>-,------1

o 10 20 30 40 50 60 70 80
POPULATION DOUBLING LE VEL
 Replicative Senescence
• With each cell division there is incomplete
replication of telomeres.
• Broken telomeres signal cell cycle arrest.
• As cells age, the telomere becomes shorter.

• Telomerase normally adds nucleotides.

• Telomerase is active in germ cells and stem cells but
absent in somatic tissue.
• Telomerase may be reactivated in cancers
 Telomerase

Telomerase
activated
Parental strand 5' i i i i i i i I I i I I i I I i II' 3'
TT· AGGG TT AGGG TT AG
. . .. .
. . ....
AAtccc
!
Newly synthesized 3' .. I ! I I I I 5'
(lagging) strand
Binding of telomerase

5'-r,T"'-'-
' T"'""1'TTrT'T"'-'-'T"'-"rT
' T'...... 3'
TT
· AGGG
...... AGGG TT AG
. .
AATCCC AU CCC AAU C Telomerase
enzyme

Telomerase
Extension of 3' end
by telomerase

5' ~!"''I' ,'"'T'!' "'!':-:'rT'" "'!':T'!', ':-:', ,''I'"'!':T''I',':-T'!' ......3~'~

T TAG~G TT AGGG T T~~~YTT~Y /
3' ..
AAtccc
I I I I I I 5'

Telomerase
A
(D&o_.-... .... .... _ .......... c.o. _ _ _ _ _ . lfIItt.l
 Genes that influence Aging
• Insulin growth factor receptor. Active area of
research.

• Decreased signaling of IGF -1 receptor is the result of

decreased caloric intake or mutations and results in
longer lifespan in C. elegans.

• Evidence suggests that aging is genetically determined.

Some families have more
individuals that seem to live
longer.
 Accumulation of Metabolic and
Genetic Damage Occurs with aging.

• Reactive oxygen species (lipofuscin). likely

In older person, more
to have lipofuscin
in cytoplasm

• Over expression of SOD extends life span in

Drosophila.
• Werner’s syndrome – defective helicase.
• Ataxia telangiectasia – ineffective repair of dsDNA
breaks.
• Damaged organelles accumulate.
Which of the following cells has the highest
telomerase activity?

A. Endothelial cells
B. Germ cells
C. Neurons
D. Neutrophils
E. Erythrocytes
Which of the following cells has the highest
telomerase activity?

A. Endothelial cells
B. Germ cells
C. Neurons Don't replicate,
remember?

D. Neutrophils
E. Erythrocytes Don't even have
chromatin!
 Summary Don't memorize
molecular
mechanisms.

• Necrosis and Apoptosis

• Molecular mechanisms
• Role in health and disease

• Cellular aging