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CHAPTER 11
APOPTOSIS AND AGING
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I. Aging of cell
The sorting of human cells by their recruitment:
Human natural life time is about 120 years. But, the cells of that human
body is composed are very different in their life time. We can sort them as 4
types as the follows:
Recruiting tissue: The recruiting tissue needs to be replaced always,
for examples, intestine endothelial cells (IEC).
Stable tissue cells: The cells were highly differentiated with special
function. Usually, no obvious aging can be found in the stable tissue cells.
For examples, liver cells, kidney cells.
Consistent cells: There is no cell replacement in the consistent
tissue. For examples, neurons, skeleton muscle cells, heart cell, and others.
Exhausting cells: ovary cells can be exhausted out without any
supplement.
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The signs of cell aging
The changes of forms:
Aged cell appears with increased permeability and fragility, cell atrophy,
decreased organelle quantity (especially for mitochondria), and accumulation of
lipofuscin in cell.
Form changes of an aged cell
Nucleus
Chromatin
Plasma
membrane
Plasma
Enlarged, Stained darkly, Inclusion contained in
nucleus
Condensed, Agglutinated, Broken, Dissolved
Increased viscosity (mucosity), Decreased mobility
Mitochondrion
Accumulation of pigments, Formation of vesicles
Decreased quantity, Enlarged size, Mutated and lost
mtDNA
Golgi Body
Broken
Nissl Body
Disappeared
Inclusion
Nuclear
Membrane
Decreased glycogen, Accumulated fat
Invaginated
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The changes of molecules:
DNA: The DNA replication and transcription will be inhibited, but some genes
will be abnormally activated. Telomere DNA lost. The mtDNA is specifically
absented, and the cell DNA is oxygenated, broken, absented, and
desmethylated.
RNA: The quantity of mRNA and tRNA is decreased.
Protein: Synthesis is inhibited. The proteins are modified with glycosylation and
others resulting in decreased stability, antigenicity, and digestability.
Accumulated free radicals break the peptide chains, and peptide chains are
conjugated together resulting in denatured proteins.
Enzyme: The activity core is oxygenated. Ca2+, Zn2+, Mg2+, and Fe2+ lost.
The secondary structure, solubility, and isoelectric point are changed. So,
enzyme molecules are inactivated.
Lipid: Unsaturated fatty acids are oxygenated resulting in membrane mobility
decreased.
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Mechanisms of cell aging
There are many theories about the mechanism of aging. Briefly, error
theories and genetic/programmed theories are important.
Error theories:
After the cell suffered from damages, repairing system is working not
efficiently resulting in the accumulation of errors that cause cell aging.
1. Waste product accumulation:
Lipofuscin is a macromolecule conjugated by proteins, DNA, and lipids in
lysosomes. Accumulated lipofuscins can inhibit signal transduction and
molecules exchanging. For example, Alzheimer’s disease (AD) is caused by
the accumulation of β- amyloid protein (β-AP). So, β-AP detection can be used
to diagnose AD.
2. Cross linking of large molecules:
Excessive crossly linked macromolecules is an important pathogen to
aging. For examples, The linkage of DNA and collagen will damage their function.
Linkage of collagens is associated with arteriosclerosis and vascular diseases.
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3．Free radical theories:
The free radicals in human body is formed by two ways: Some harmful extraenvironments cause the level of free radicals raised; Some metabolism reaction in
vivo form free radicals. The latter is the major way to raise the free radical level in
vivo.
Usually, the free radicals with the normal level are beneficial to body to clean
the pathogen microorganisms in vivo. But the excessive free radical accumulation
is very harmful to cells. Free radical can cause excessive linkage reaction
between DNA and proteins, fatty lipid, especially polyunsaturated fatty acids
(PUFA), and damage DNA, bio-membrane, structural and functional proteins.
Excessive free radicals can inhibit some important bio-reaction in vivo, and cause
hard erythrocytes formed that is associated with some important and severe
diseases, such as cardiovascular diseases and severe cerebral malaria.
The level of free radicals is raised significantly with becoming. The older, the
higher level of free radicals, that is why the elders ‘ skins and skin color present
special features.
There are free radical cleaning systems in body including superoxide
dismutase (SOD) and others. But in old people, the expression of the proteins for
these system are not enough or deficient, or the receptors for these systems are
inhibited or expressed lowly, so, the level of free radicals in old people is higher
than young people.
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Melatonin is the commercial bio-products in America that can clean
the excessive free radical accumulation. But, for the elders, melatonin
receptor expression is not enough, that is core problem for them.
4. Mitochondrial DNA mutation:
Reactive oxygen species (ROS) can be accumulated in mitochondrion
because of its features. mtDNA is naked DNA that is easy to be damaged by
ROS, and the DNA polymerase for mtDNA replication lacks repairing function, so,
mtDNA is very easy to be mutated by excessive ROS. The mutation of mtDNA
can block the respiratory chain of cell that can cause further accumulation of free
radical. mtDNA mutation and absence is much more obvious in elder than in
young. The changes of mtDNA is closely associated with many diseases in elder,
for example, AD and other degeneration diseases.
Brain, heart is the organs where the oxidative stress is the most high. So,
these organs are easy to become old, that is why the cerebral and
cardiovascular diseases are major killers for old people.
Caloric restriction is beneficial to a long life probably!
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5．Somatic mutation and DNA repair:
Some extra-environmental effects of physics or chemistry can damage DNA,
and all of intra-environmental free radicals can damage DNA. Usually, damaged
DNA can be repaired by the DNA repairing system immediately. But, in old
people, the repairing mechanism is obviously degenerated. So, the mutation
and replication mistake will be accumulated to a high level that causes cell old
and death. The damage DNA of the cells with active function has priority to be
repaired firstly, and the complete repairing happens in DNA replication phase of
cell cycle. That is why the stem cells can keep young and powerfully potential
proliferation and differentiation ability.
6．The inactivation of repeated genes:
The repeated genes in eukaryotic genome can both increase the gene
information and protect the gene damage from the gene disappeared. In
another hand, the repeated genes can delay the early death of the important cell.
If one copy of repeated gene is damaged and blocked, other copies can be
activated immediately till to last copy is used. Experimental data shows that the
repeated gene copy number for rRNA in liver can be decreased with aging.
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Genetic/programmed theories:
1．Programmed senescence:
This theory believes that all of growth, development, senescence, and death
are regulated by some existed genetic programs. The senescence can presents
some senescence markers, such as the senescence marker protein-2 in liver. In
addition, senescence is associated with the programmed degeneration of nerveendocrine system and immune system.
2．Replicative senescence:
The proliferation of normal cells in vivo or vitro is not unlimited. The number
of proliferation is a limit that is called as Hayflick limit, highest division
frequency, or passage number. For example, human embryonic fibroblast can
be cultured in vitro with a passage number of 60-70.
Hayflick limit is associated with the length of telomere DNA. The telomere
can be cut off a part at each time of DNA replication. When it become short to
the Hayflick limit, the DNA damage checkpoint will be started, p53 will be
activated, p21 will be expressed, and the cell will stop the cell cycle to turn to
apoptosis. The telomere of human fibroblast becomes short by 14-18bp/year.
The length of telomere is closely associated with telomerase activity.
Telomerase can synthesize telomere DNA with its RNA as template. In old body,
the telomere activity is not so active.
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3．Longevity genes:
The statistics data shows that the life time of son/daughter is associated with
his/her parents. Each species of animal is of consistent average of life time and
longest life time. The patients of Werner's syndrome present obvious
senescence syndromes at thirty nine years old, and die from this disease at forty
seven years old. The kids of Hutchinson-Gilford syndrome present obvious
senescence syndromes at one year old, and die from this disease at twelve to
eighteen years old.
The life time of species is depended on some genes in species’ genome. We
call these genes as longevity genes or senescence genes.
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A patient of Werner's syndrome at 37 years old
(From http://www.nejm.org on)
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Normal kid
(Left)
Patient of
HutchinsonGilford
syndrome
(Right)
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When a cell turns to aging way, some senescence associated genes (SAG)
are frequently expressed in it, and the level of these expressions are much higher
than normal. Investigators have found some SAGs on 1st, 4th chromosomes and
X chromosome.
At least, there are four mutated genes are associated with AD. The mutation
of the gene of amyloid protein precursor (APP) causes its product, β- amyloid
protein (β-AP) accumulated in brain tissue and AD developed.
II. Necrosis and apoptosis
The cell death happens frequently in normal tissue.
The cell death ways include ① necrosis; ② apoptosis and programmed cell
death (PCD).
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Necrosis:
Necrosis means the cell death caused by chemical, physical, and biological
effects. The pathological changes for necrosis are enzyme digestion and protein
denaturation. If the enzymes are from the same cell, we call this digestion as
autolysis, otherwise, called as heterolysis.
Primary stage of necrosis
Later stage of necrosis
Latest stage of necrosis
Mitochondria
ER
Other organelles
Structural fatty
Protein granules
Nucleus
Tumefacient
Disintegrated
Uncombined and vacuolated
Increased
Broken or pyknoted
Basophilic nuclear protein
Degenerated
Plasma
Eosinophilic
Dark eosin staining
Water rich cell
Water vesicle enlarged
Water rich cell
Cell structures are disappeared
Membrane and organelles
Broken
DNA
Degenerated
Content of cell
Flowed out
Inflammation
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The differences
between
necrosis and
apoptosis
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Apoptosis:
Apoptosis was named by Kerr in 1977.
The major features of apoptosis are as the follows:
① Chromatin is condensed and moved to nuclear membrane. The nucleus is
broken, and cell will form many apoptosomes by germination.
② Apoptosome contains organelles, pyknotic chromatin. Apoptosome can be
swallowed and digested by adjacent cells. Because apoptosome is always
enveloped by its membrane, it will not release out the content, so, it will not result
in inflammation. But necrosis always causes inflammation.
③ Apoptosis cell can synthesize some protein for itself. Necrosis cell can not
do so.
④ Endonuclease was activated, so, the chromatin is cut off at nucleosome
junction, and form many fragments with a 200bp length difference, so, a ladder
electrophoresis result can be obtained for apoptosis cell genome DNA.
⑤ Usually, apoptosis is a physiological procedure, but necrosis is a
pathological procedure.
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The differences between necrosis and apoptosis
Differences
Apoptosis
Necrosis
Causes
Physiological or pathological
Pathological
Field
Single distributed cells
Mass of tissue or cells
Membrane
No broken and form
apoptosome
Broken
Chromatin
Condensed under nuclear
membrane
Flocculent
Organelles
Almost no change
Tumefacient, disintegrated ER
Cell size
Shorten by condensation
Enlarged
Apoptosome
Yes. Swallowed by adjacent
macrophages
No. Autolysis. Swallowed by
macrophages
Genome DNA
Degenerated with regulation.
Ladder DNA electrophoresis
Disintegrated. Smeared
electrophoresis result
Synthesis of
protein
Yes
No
Regulation
By genes
Passively
Inflammation
No
Yes
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Left: Normal
thymocyte
Right: Apoptosis
thymocyte
(Apoptosome)
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There are many papers or books describe the apoptosis and programmed cell
death (PCD) as same concept. Actually, apoptosis is different from PCD. PCD
means that some cells must turn to apoptosis and death at some time points
following the spatiotemporal sequence for the individual development, and the
death of these cells are designated previously by genetic regulation system.
Apoptosis means the cell death regulated by genes, but it is not designated
previously by genetic system, and many effects from the cell environment can
regulate it. The final result for PCD is apoptosis, but, it is not true that all
apoptosises are programmed.
Robert Horvitz group of MIT found 14 genes are associated with the apoptosis
of C. elegans using a somatic mutation method. Ced-3 and Ced-4 can induce
apoptosis, Ced-9 can inhibit Ced-3 and Ced-4 and stop apoptosis. If Ced-9 is
deficient, the fetus will die from excessive apoptosis.
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On October 7, 2002, an American scientist and two British scientists won the
2002 Nobel prize because of their great contributions to the genetic regulation of
organ development and PCD.
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III. The molecular mechanism of apoptosis
In an embryo, apoptosis is one of the basic ways to maintain the cell quantity
in body for the normal development. In an adult, apoptosis can clean the old and
damaged cells for the body health.
Like cell proliferation, apoptosis is regulated by gene system exactly.
There are two major ways to apoptosis: 1. Activate the apoptotic enzyme,
caspase, by extracellular signals; 2. Activate caspase by the caspase activation
factor released from mitochondria.
Activated caspase can degenerate the important proteins in cell to cause
apoptosis.
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Apoptosis associated genes or proteins
1. Caspase family:
Caspase is a type of protease. These proteases are the key enzymes to cause
apoptosis. When they are activated by signals, the important proteins in cell will be
degenerated to cause the cell turn to apoptosis irreversibly. The caspases keep the
features as the follows: ① The enzyme activity depends on the nuclear affinity of
cysteine residue; ② The substrate is always cut off at the site post aspartate, that is
why it was named as caspase (cysteine aspartate-specific protease); ③ are the
tetramer composed of two large subunits and two small units. Large and small
subunits are encoded by same gene.
Interleukin-1 β-converting enzyme (ICE) is the homologous gene of the
nematode Ced-3 that was earliest found. Because ICE can cleave the precursor of
IL-1, it was so named. 11 ICE homologous proteins have been found in human cells.
They can be sorted as two types: ICE subgroup and Ced-3 family. The former
participates in inflammation. Ced-3 family participates in apoptosis and can be
sorted as two types: 1. Executioner or effector, such as caspase-3, 6, and 7, can
degenerate the structural and functional proteins in cell to cause apoptosis. But they
can not be activated by autocatalytic or self-splicing ways. 2. Initiators, such as
caspase-8, and 9 can be activated with signal by self-splicing way, and start
caspase cascade reactions. Caspase inhibitor, inhibitors of apoptosis proteins
(IAPs), is a big protein family in cell. IAPs, such as XIAP, can bind to caspase by
baculovirus IAP repeats domain (BIR domain) to inhibit caspase.
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The members of ICE family (A: 3 types of caspases, those in blue mean
caspases involved with inflammation, in red mean executioner, in green mean
initiators. B: Structural model of caspase-3. C: Activation of caspase-3) 24
(From Katja C. Zimmermann, et al. 2001)
2．Apaf-1:
Apaf-1 (apoptotic protease activating factor-1) is important to the apoptosis
in mitochondrion. If Apaf-1 is knockout, the mouse brain can not be developed
normally. Apaf-1 contains 3 domains: ① CARD (caspase recruitment domain) can
enrich caspase-9. ② Ced-4 homologous domain can bind to ATP/dATP. ③ C
terminal domain can bind to cytochrome c to activate Apaf-1.
Apaf-1/ cytochrome c complex can bind to ATP/dATP, enrich caspase-9 by
CARD domain, form apoptosome, activate caspase-3, and finally start caspase
cascade reactions.
3．Bcl-2 (B-cell lymphoma/Leukemia-2) family:
Bcl-2 is the apoptosis suppressor gene and integrin. 19 homologous genes
about Bcl-2 have been identified so far. Anti-apoptotic Bcl-2 members: Bcl-2, Bcl-xl,
Bcl-w, and Mcl-1. Pro-apoptotic Bcl-2 members: Bax, Bak, Bad, Bid, and Bim.
Bcl-2 proteins are mainly located on mitochondrion membrane. Most of proapoptotic proteins are located in plasma. When cell receive apoptosis signals, proapoptotic proteins will move to mitochondrion membrane to release out the
mitochondrion content, such as cytochrome c, to activate caspase, and result in
apoptosis.
The pro-apoptotic proteins can be activated by dephosphorating, modifying by
caspase, releasing from combined protein.
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Bcl-2 family (From Katja C. Zimmermann, et al. 2001)
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4. Fas:
Fas is also called as APO-1/CD95, and is the member of TNF receptor family.
Fas gene encodes a 45KD transmembrane protein that is distributed on thymocyte,
activated T and B cells, macrophages, the cells of liver, spleen, lungs, heart, brain,
intestine, testes, ovary, and others. The combination of Fas and Fas ligand can
activate caspase to cause apoptosis of target cells.
5．p53:
p53 is a cancer suppressor gene that can check the DNA replication at G
phase. If DNA was damaged, p53 will inhibit cell cycle till to the DNA is repaired.
Otherwise, the cell will be introduced to apoptosis.
6. c-myc:
c-myc is a proto-oncogene that is excessively expressed in many human
cancer tissue. c-myc can enhance cell proliferation and inhibit differentiation. cmyc is excessively expressed in apoptotic cells also. As a transcription regulator,
c-myc can activate proliferation genes and apoptotic genes. So, it presents two
choices to cells, proliferation or apoptosis. With GF and Bcl-2, c-myc promotes
proliferation, otherwise apoptosis.
7. ATM:
ATM (ataxia telangiectasia-mutated) gene is a DNA damage detector also.
1% of people are the zygotes with ATM absence. These persons are sensitive to
radiation rays and easy to suffer from cancers.
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Apoptosis intermediated by Fas:
The apoptosis receptors on cell surface are the TNF receptor (TNFR) including
Fas (Apo-1/CD95), TNFR1, DR3/WSL, DR4/TRAIL-R1, and DR5/TRAIL-R2.
Fas is transmembrane protein including extracellular part and the intracellular
part called death domain (DD). Fas ligand (FasL) combines to Fas and change
DD structure to link the DD of Fas associated death domains (FADD), then, the
DED (death effector domain) of FADD can bind to caspase-8 to form DISC
(death-inducing signaling complex). DISC can activate caspase-8, -10, and start
caspase cascade reactions to activate caspase-3, -6, and -7, the important proteins
in cell are degenerated, cell turns to apoptosis.
Caspase can activate CAD (caspase-activated Dnase). CAD can cut off DNA
at the links of nucleosome, and form DNA fragments with 200bp difference in length.
Fas/FasL is important immune system. By the Fas/FasL intermediation, the
activated T cells can clean the cell clones that take autoimmunity to the cells of self
body. This action can protect the body from damage. The abnormal apoptosis of
lymphocytes is the major pathogen to the autoimmune diseases. The cell toxic T
lymphocytes (CTL) can introduce apoptosis by FasL, but, some cancer cells can
introduce lymphocyte apoptosis to escape immunity attack.
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Apoptosis intermediated by Fas
(From Avi Ashkenazi and Vishva M. Dixit 1998)
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Mitochondria and apoptosis
As the apoptosis inducer, cytochrome c released from mitochondrion can
bind to Apaf-1, caspase-9 precursor, and ATP/dATP to form apoptosome, then,
activate caspase-3 to start caspases cascade reaction resulting in apoptosis.
Mitochondrion keeps always no damaged in apoptotic cell. So, how is the
cytochrome c released into plasma? Probably, it is released into plasma through
the permeability transition pore (PT pore) or the channel formed by the Bcl-2
family.
PT pore is composed of adenine nucleotide translocator (ANT) and
voltage dependent anion channel (VDAC). The members of Bcl-2 family
regulate the switch of PT pore. Pro-apoptotic members, such as Bax, can promote
PT pore opened by binding to ANT or VDAC. Anti-apoptotic members, such as
Bcl-2 and Bcl-xL, can bind to ANT or VDAC competently with pro-apoptotic
members, or block the pro-apoptotic member to bind to ANT and VDAC directly.
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The mutation of Ced-3 and Ced-4 can inhibit the cell death in each develop
stage in nematode. Caspase can not be activated in the mouse with Apaf-1
absence, but most of organs can be developed normally excepting excessive
nerve cells.
The proteins released out with cytochrome c include Smac (second
mitochondria-derived activator of caspase), AIF (apoptosis inducing factor)
and endonuclease G (Endo G). Smac can bind to the BIR domain of IAP
(inhibitor of apoptosis) to stop the inhibition of IAP to caspase. AIF causes
nucleus pyknosed and chromatin broken. Endo G cleaves DNA. So, if no caspase
involved, apoptosis can be still started by mitochondrion way.
In the cells that are responding to Fas, the cells of type I, such as
thymocytes, their caspase-8 is powerful to cause apoptosis after activated by Fas.
So, excessive expression of Bcl-2 can not inhibit the apoptosis introduced by Fas
in type I cells. The cells of type II, such as liver cells, the activation of caspase-8
intermediated by Fas is not enough to cause apoptosis. So, the apoptosis signals
must be enhanced by mitochondrion way in this type cells: activated caspase-8
can cleave Bid in plasma to form tBid (truncated Bid), then, tBid enters
mitochondrion and causes cytochrome c released out, finally, the apoptotic signal
is enhanced.
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Apoptosis caused by
cytochrome c
(From R. Chris Bleackley and Jeffrey A.
Heibein 2001)
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By the described as above, it is indicated that mitochondrion is both the
energy station and the center of apoptosis of regulation. Why is mitochondrion so
important to a cell? Each growth factor can promote glycose transported to
mitochondrion to enhance energy supply, so, when the GFs were inhibited, the cell
will turn to apoptosis. It is easy to understand that the GF inhibition causes
apoptosis, but, the detail about it is still kept unknown so far.
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