Cellular Biology

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Cellular Biology
School of Life Sciences Shaanxi Normal University
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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 Enlarged, Stained darkly, Inclusion contained in nucleus
Chromatin Condensed, Agglutinated, Broken, Dissolved
Plasma membrane Increased viscosity (mucosity), Decreased mobility
Plasma Accumulation of pigments, Formation of vesicles
Mitochondrion Decreased quantity, Enlarged size, Mutated and lost mtDNA
Golgi Body Broken
Nissl Body Disappeared
Inclusion Decreased glycogen, Accumulated fat
Nuclear Membrane 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, Alzheimers disease (AD) is caused by
the accumulation of ß- amyloid protein (ß-AP).
So, ß-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 extra-environments 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 nerve-endocrine 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 Hutchinson-Gilford
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, ß- amyloid
protein (ß-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 ?
necrosis ? 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.
Mitochondria
Tumefacient
ER
Other organelles
Disintegrated Primary stage of necrosis
Structural fatty Uncombined and
vacuolated
Protein granules Increased
Nucleus
Broken or pyknoted
Basophilic
nuclear protein Degenerated Later stage of
necrosis Plasma Eosinophilic
Dark eosin staining
Water rich cell Water vesicle
enlarged
Water rich cell Cell structures are
disappeared Latest stage of necrosis
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 ? Chromatin is
condensed and moved to nuclear membrane. The
nucleus is broken, and cell will form many
apoptosomes by germination. ? 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. ? Apoptosis cell can
synthesize some protein for itself. Necrosis cell
can not do so. ? 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. ? 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 ? The enzyme activity depends on the
nuclear affinity of cysteine residue ? The
substrate is always cut off at the site post
aspartate, that is why it was named as caspase
(cysteine aspartate-specific protease) ? are the
tetramer composed of two large subunits and two
small units. Large and small subunits are encoded
by same gene. Interleukin-1 ß-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) (From
Katja C. Zimmermann, et al. 2001)
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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 ? CARD
(caspase recruitment domain) can enrich
caspase-9. ? Ced-4 homologous domain can bind to
ATP/dATP. ? 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 pro-apoptotic
proteins are located in plasma. When cell receive
apoptosis signals, pro-apoptotic 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. c-myc
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.