MOLECULAR BASİS
OF CELL AGİNG
Prof. Dr. Turgut ULUTİN
The main factors acting in aging process and the functional relationship between them

The aging of higher organisms is multifactorial process. It is influenced and
modified by various genetic, biochemical,
regulation and other systems working at
once in close contact. Each system can
make direct impact on aging process or
act indirectly (e.g. through other
pathway). That is why interaction between
mentioned systems is important too. Here
we have tried to overview the influence of
each factor to the aging process and to
discuss how all mentioned aging-factors
could act as the whole complex.

Mitochondriae are the main unit of chemical power supply
in the cell. During the synthesis of macroergical biomolecules free radicals are being produced as the byproduct. Free radicals when released in large quantities
cause intercellular oxidative stress (e.g. oxidative damage
of DNA, proteins and other bio-molecules). Oxidative stress
is the main reason of accelerated senescence. Free radicals
can result tissue degeneration by damaging mitochondria
genome and cause early apoptosis (programmed cell
death) through the damage of nuclear genome.
Endogenous oxidative damage and repair systems play
a big role in spontaneous mutagenesis. Mutated genes
usually encode nonfunctional products, which disturb
biochemical or/and signaling pathways leading to more or
less expressed pathological state. Free radicals attack
proteins and modify them. It usually disturbs protein
function and can accelerate the aging process.

Cell cycle is regulated by different
specific proteins. At this moment we know
lots of different proteins which regulate
cell cycle, phase change (cancer
supressors, cyclins, and MAP kinases).
When these proteins are damaged by
mutations cell cycle regulation can be
disturbed. Cells could die or become not
controlled depending on the nature of
mutation- this could lead to cancer. Cell
cycle regulation disorders leads to
accelerated aging and/or cell malignancy.

We know genes concerned with pathological
aging. When they are damaged organism ages
much faster. These genes are named
gerontogenes - aging genes. Genetic
polymorphisms (determining individual's
longevity) are found. The existence of longevity
gene is still very real. Some age linked diseases
are known in medical practice (Werner's,
Bloom's, Cocaine's syndromes, progery and
other). Patents had damaged various
gerontogenes. It was observed that these genes
encoded replication, transcription and repair
machinery components of the cell.

Telomeres are the terminal parts of
eukaryotic chromosomes. The influence to
aging of telomeres is highly discussed.
They are called "molecular clock" of the
cell. Cell division times are correlated with
telomere length. After each cell division
telomeres get shorter. When telomere
shortens to the critical stage, the intensity
of cell division significantly decreases, and
then cell differentiates and ages.
Telomeres are persistent in the not aging
cells: cancer and germ line.

The influence of transcription,
translation and posttranslational
modification systems to the cell is not
static but highly regulated. For example,
when synthesized protein is modified
incorrectly (wrong phosphorylation) its
function alters. If protein function is
important, appropriate intracellular
processes or regulation could be
disturbed. Such errors lower vitality of
organism and accelerate aging.

Intracellular processes are accordant and
rigorous; it means cell is highly organized and
integrated system. Information (signal
transduction) and the regulation of bioprocess
are the main players in the development and the
maintenance of this system and aging. When
mutations or modification disturb proteins/genes
of signal systems, signal transduction and other
bioprocesses proceed abnormally. We should not
forget that organism is integrated system and all
factors mentioned above act in-between with
others. Mitochondrial metabolism process
stimulates oxidative damage, but each cell has
repair systems defeating it (reparative systems,
apoptosis, etc.).


Aging is a natural process, living organisms are
highly adapted to the laws of nature, and
senescent cells are being changed with juvenile.
The existence of not differentiated stem cells in
every living organism has a deep meaning; they
act as a depot in the regeneration of damaged
cells.
In the higher organisms, aging and renovation
process is strictly regulated, anyway the source
of aging and renovation signal are of material
nature (biomolecules), which, changes during the
life cycle. Because of these changes
(modifications and mutations) organism
necessarily lose its battle with aging.
CHROMOSOME
TELOMERE
TTAGGGTTAGGGTTAGGGTTAGGGTTAGGG
AATCCCAATCCC
5’
3’
Telomere




senescent cells have shorter telomeres
length differs between species
in humans 8-14kb long
telomere replication occurs late in the cell cycle
Functions



Provide protection from enzymatic degradation
and maintain chromosome stability
Organisation of the cellular nucleus by serving
as attaching points to the nuclear matrix
Allows end of linear DNA to be replicated
completely
Replicative senescence
• Telomeres shortens progressively with each
cell division
• 100 base pair lost with each cell division
• Growth arrest
S
C-FOS, ID-1,ID-2,E2F1
G1
E2F5
G2
P21,p16
Go
M
Aging
* extremely complex
process
*senescence associated gene expression
*oxidative damage, replicative senescence
*cell senescence can be reversed
Cancer



incidence increases with age
replicative senescence tumor suppressive
mechanism
marker of malignancy
What are telomeres?

Telomeres are…
• Repetitive DNA sequences at the ends of all
human chromosomes
• They contain thousands of repeats of the sixnucleotide sequence, TTAGGG
• In humans there are 46 chromosomes and thus 92
telomeres (one at each end)
• senescent cells have shorter telomeres
• length differs between species
• in humans 8-14kb long
• telomere replication occurs late in the cell cycle
Telomere function...

Telomeres are also thought to be the
"clock" that regulates how many times an
individual cell can divide. Telomeric
sequences shorten each time the DNA
replicates.
How are telomeres linked to aging?

Once the telomere shrinks to a certain
level, the cell can no longer divide. Its
metabolism slows down, it ages, and dies.
How Does Telomerase Work?


Telomerase works by adding back
telomeric DNA to the ends of
chromosomes, thus compensating
for the loss of telomeres that
normally occurs as cells divide.
Most normal cells do not have this
enzyme and thus they lose
telomeres with each division.
Telomeres & Aging

Healthy human cells are mortal
because they can divide only a finite
number of times, growing older each
time they divide. Thus cells in an
elderly person are much older than
cells in an infant.
Think of it like this…
• For the cell, having a long telomere can
be compared to having a full tank of gas
in your automobile; having a short
telomere is like running on empty. Each
time a cell divides, its telomeres
become a little shorter until the cells
simply can no longer divide (e.g., it runs
out of fuel).
CELL DEATH
(APOPTOSİS)
Prof. Dr. Turgut ULUTİN
More than one way to die:
Necrosis and Apoptosis
cytoplasmic, nuclear
condensation
chromatin margination
rapid membrane
membrane blebbing
permeablization
swelling of the
cytoplasm
Necrosis
Swelling of the
nucleus
osmotic shock
release of intracellular
content
Apoptosis
cell implosion and
formation of
apoptotic bodies
recognition and engulfment of apoptotic
bodies by phagocytic cells
More than one way to die:
Necrosis and Apoptosis
cytoplasmic, nuclear
condensation
chromatin margination
swelling of the
cytoplasm
rapid membrane
permeablization
membrane blebbing
Swelling of the
nucleus
Necrosis
osmotic shock
release of intracellular
content
cell implosion and
formation of
apoptotic bodies
recognition and engulfment by
phagocytic cells
Apoptosis
Apoptosis and Phagocytosis
• Phagocytes recognize “eat-me”
or cell corpse signals on the
apoptotic cell surface. These
signal the phagocyte to activate
cellular engulfment machinery.
Scavenger
Receptors
Phagocyte
?
Oxidized LDL-like Site
PS
Phosphatidylserine
Receptors
C1q
Binding
Site
C1q
Bridge
Apoptotic
Cell
RAC-1
DOCK 180
C1q Receptor
ELMO
Cytoskeletal
Reorganization for
Engulfment
CRKII
• Phosphatidylserine exposure on
the target cell surface and the
phosphatidylserine receptor on
the phagocyte are essential for
phagocytosis.
• Defining other receptors, bridge
molecules, “eat-me” signals and
signaling molecules involved in
initiating the cytosolic changes
needed for engulfment are very
active areas of research. The
articles listed below review
current knowledge and are the
sources for this diagram.
Savill, J. and Fadok, V. 2000. Nature. 407:784.
Canradt, B. 2002. Nature Cell Biol. 4:E139.
Apoptosis
Oxygen Society Education Program
Tome & Briehl 5
Apoptosis and Phagocytosis
• The first pathway shows
the engulfment of an
apoptotic cell exposing
“eat-me” signals.
1.
Phagocyte
Apoptotic
Cell
With "eat me"
signals
• Data from mammalian
systems and genetic
studies from
Caenorhabditis elegans
have shown that
phagocytes and target
cells have several types
of interactions.
2.
Phagocyte
Healthy
Cell
Phagocyte induces
apoptotic machinery
in healthy cell
3.
Engulfment
Phagocyte
Apoptotic
Cell
With "eat me"
signals
Apoptotic cell induces
phagocytic machinery
in phagocyte
Phagocyte
Precursor
Apoptotic
Cell
With "eat me"
signals
Apoptotic cell induces
maturation of precursor
into phagocyte
• Conradt has proposed
several models (2-4) to
indicate the more
complex phagocytetarget interactions.
4.
Apoptosis
Conradt, B. 2002. Nature Cell Biol. 4:E139.
Greene, D.R. and Beere, H.M. 2001. Nature. 412:133.
Oxygen Society Education Program
Tome & Briehl 6
THE APOPTOTIC PATHWAY
Triggers
. Growth factor
Deprivation
. Hypoxia
. Loss of adhesion
. Death receptors
. Radiation
. Chemotherapy
Modulators Effectors
. FADD
. TRADD
. FLIP
. Bcl-2 family
. Cytochrome c
. p53
. Mdm2
. Caspases
Substrates DEATH
. Many cellular
proteins
. DNA
CASPASES
Caspase-1 (ICE)
Caspase-2 (ICH-1, Nedd-2)
Caspase-3 (CPP32, Apopain, Yama)
Caspase-4 (ICH-2, TX, ICEreıı)
Caspase-5 (ICErelııı, TY)
Caspase-6 (Mch2)
Caspase-7 (ICE-LAP3, Mch3, CMH1)
Caspase-8 (FLICE, Mch5, MACH)
Caspace-9 (Mch6, ICE-LAP6)
Caspase-10 (Mch4)
SUBSTRATES for CASPASES
... PARP
... DNA-PK
... pRb
... Lamins
... NuMA
... Fodrin
... -Aktin
... Mdm2
... Cyclin A2
... Presenilin
... Others
Why should a cell commit suicide?
There are two different reasons.
1. Programmed cell death is as needed for proper
development as mitosis is.
Examples:
- The resorption of the tadpole tail at the time of its metamorphosis
into a frog occurs by apoptosis.
- The sloughing off of the inner lining of the uterus
(the endometrium) at the start of menstruation occurs by apoptosis.
- The formation of the proper connections (synapses) between
neurons in the brain requires that surplus cells be eliminated by
apoptosis
2. Programmed cell death is needed to destroy cells that represent
a threat to the integrity of the organism.
Examples:
-
Cells infected with viruses
One of the methods by which cytotoxic T lymphocytes (CTLs)
kill virus-infected cells is by inducing apoptosis. (And some
viruses mount countermeasures to thwart it.)
-
Cells with DNA damage
Damage to its genome can cause a cell
* to disrupt proper embryonic development leading to birth defects
* to become cancerous.
Cells respond to DNA damage by increasing their production of p53.
p53 is a potent inducer of apoptosis. Is it any wonder that mutations in
the p53 gene, producing a defective protein, are so often found in
cancer cells (that represent a lethal threat to the organism if permitted
to live)?
What makes a cell decide to commit suicide?
The balance between:
- the withdrawal of positive signals; that is,
signals needed for continued survival
- the receipt of negative signals
Withdrawal of positive signals
The continued survival of most cells requires that they
receive continuous stimulation from other cells and, for many,
continued adhesion to the surface on which they
are growing. Some examples of positive signals:
- growth factors for neurons
- Interleukin-2 (IL-2), an essential factor for the mitosis
of lymphocytes
Receipt of negative signals
- increased levels of oxidants within the cell
- damage to DNA by these oxidants or other agents like
* ultraviolet light
* x-rays
* chemotherapeutic drugs
- molecules that bind to specific receptors on the cell surface and
signal the cell to begin the apoptosis program.
These death activators include:
* Tumor necrosis factor - alpha (TNF-a ) that binds to the
TNF receptor;
* Lymphotoxin (also known as TNF-b) that also binds to the
TNF receptor;
* Fas ligand (FasL), a molecule that binds to a cell-surface
receptor named Fas (also called CD95)
Apoptosis
For every cell, there is a time to live and a time to die.
There are two ways in which cells die:
• they are killed by injurious agents
• they are induced to commit suicide
Death by injury
Cells that are damaged by injury, such as by
•
•
mechanical damage
exposure to toxic chemicals
undergo a characteristic series of changes:
•
•
•
they (and their organelles like mitochondria) swell
the cell contents leak out, leading to
inflammation of surrounding tissues
Death by suicide
Cells that are induced to commit suicide:
- shrink
- have their mitochondria break down with the release of cytochrome c
- develop bubble-like blebs on their surface
- have the chromatin (DNA and protein) in their nucleus degraded
- break into small, membrane-wrapped, fragments
- The phospholipid phosphatidylserine, which is normally hidden within
the plasma membrane is exposed on the surface.
- This is bound by receptors on phagocytic cells like macrophages and
dendritic cells which then engulf the cell fragments.
- The phagocytic cells secrete cytokines that inhibit inflammation.
The pattern of events in death by suicide is so orderly that the process is often
called programmed cell death or PCD. The cellular machinery of programmed cell
death turns out to be as intrinsic to the cell as, say, mitosis. Programmed cell death
is also called apoptosis
The Mechanisms of Apoptosis
There are 2 different mechanisms by which a cell
commits suicide by apoptosis.
- one generated by signals arising within the cell
- the other triggered by death activators binding to
receptors at the cell surface.
* TNF-a
* Lymphotoxin
* Fas ligand (FasL)
Apoptosis triggered by internal signals
- In a healthy cell, the outer membranes of its mitochondria
express the protein Bcl-2 on their surface.
- Bcl-2 is bound to a molecule of the protein Apaf-1.
- Internal damage in the cell causes Bcl-2
* to release Apaf-1
* to no longer keep cytochrome c from leaking
out of the mitochondria
- The released cytochrome c and Apaf-1 bind to
molecules of caspase 9.
Major Apoptotic Pathways in Mammalian Cells
Mitochondrial Pathway
Death Receptor Pathway
FasL
oxidants ceramide others
Fas/Apo1
/CD95
D
DNA
damage
D
D
D
D
Bcl-2
FADD
DISC
Procaspase 8
dATP
Apaf -1
BID
Caspase 8
Procaspase 9
Procaspase 3
Cytochrome
c
dATP
Apaf -1
Caspase 9
Cellular targets
Caspase 3
apoptosome
Hengartner, M.O. 2000. Nature. 407:770.
Green, D. and Kroemer, G. 1998. Trends Cell Biol. 8:267.
Apoptosis
Oxygen Society Education Program
Tome & Briehl 3
- The resulting complex of
* cytochrome c
* Apaf-1
* caspase 9
* (and ATP)
is called the apoptosome.
- These aggregate in the cytosol.
- Caspase 9 is one of a family of over a dozen caspases.
They are all proteases. They get their name because they
cleave proteins - mostly each other - at
aspartic acid (Asp) residues).
- Caspase 9 cleaves and, in so doing, activates other caspases.
- The sequential activation of one caspase by another creates
an expanding cascade of proteolytic activity
(rather like that in blood clotting and complement
activation) which leads to
* digestion of structural proteins in the cytoplasm
* degradation of chromosomal DNA and
- phagocytosis of the cell
Apoptosis triggered by external signals
- Fas and the TNF receptor are integral membrane proteins with
their receptor domains exposed at the surface of the cell
- binding of the complementary death activator (FasL and
TNF respectively) transmits a signal to the cytoplasm that leads to
activation of caspase 8
- caspase 8 (like caspase 9) initiates a cascade of caspase
activation leading to
- phagocytosis of the cell.
The early steps in apoptosis are reversible - at least in C. elegans.
In some cases, final destruction of the cell is guaranteed only with
its engulfment by a phagocyte.