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Apoptosis Cell Death, And Proliferation

Apoptosis Cell Death, And Proliferation

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http://www.roche-applied-science.com/sis/apoptosis/docs/manual_apoptosis.pdf - ACESSO em 13/09/2010.
 Roche Applied Science
Apoptosis, Cell Death, andCell Proliferation3rd edition
 
Ov
er 
v
iew of this Guide
01
How this guide can help you study cell death and cell proliferation?
02
When and why do cells die? Does the concentration of environmental
03
pollutants exert cytotoxic or cytostatic effects on cells? What factors
04
influence the rate and timing of cell proliferation? Researchers in basic,
05
industrial, and medical research are asking these questions and looking for
06
answers. Understanding the normal regulation of cell death and cell
07
proliferation will be critical
e.g.,
for the development of new and more
08
successful therapies for preventing and treating cancer and for the
09
screening of new anti-cancer compounds.
10
Many assays exist to measure cell death and cell proliferation. However, if
11
you have only recently become interested in cell death or cel
12
proliferation, you may find the diversity of such assays bewildering. You
13
may not be able to determine what each assay measures nor decide which
14
assays are best for your purposes. This guide is designed to help you make
15
such decisions. It presents a brief overview of cell death and cell
16
proliferation, along with the major assays currently available to measure
17
each. In addition, it clearly lists the advantages and the disadvantages of
18
these assays.
19
For those who want to eliminate radioactivity from their laboratories, this
20
review also describes a number of non-radioactive assays that can serve as
21
alternatives to radioactive assays. Wherever possible, the review will
22
compare the sensitivity of the radioactive and non-radioactive assays.
Cell Death
 VI
 
by Andrew H. Wyllie
01
Over the past five or six years there has been a near-exponential increase
02
in publications on apoptosis. Around 30 new molecules have been discovered
03
whose known functions are exclusively to do with the initiation or
04
regulation of apoptosis. A further 20 molecules at least, although already
05
associated with important roles in signalling or DNA replication,
06
transcription or repair, have been recognised as affecting the regulation
07
of apoptosis.
08
This article is dedicated to young scientists thinking of entering this
09
exploding area of biology, and to those more mature ones who happened to be
10
looking elsewhere when the blast reached them, and consequently are in need
11
of a rapid introduction to the present state of affairs.
12
The term apoptosis first appeared in the biomedical literature in 1972, to
13
delineate a structurally-distinctive mode of cell death responsible for
14
cell loss within living tissues1.
 
15
The cardinal morphological features are cell shrinkage, accompanied by
16
transient but violent bubbling and blebbing from the surface, and
17
culminating in separation of the cell into a cluster of membrane-bounded
18
bodies. Organellar structure is usually preserved intact, but the nucleus
19
undergoes a characteristic condensation of chromatin, initiated at
20
sublamellar foci and often extending to generate toroidal or cap-like,
21
densely heterochromatic regions. Changes in several cell surface molecules
22
also ensure that, in tissues, apoptotic cells are immediately recognised
23
and phagocytosed by their neighbours. The result is that many cells can be
24
deleted from tissues in a relatively short time with little to show for it
25
in conventional microscopic sections.
26
This remarkable process is responsible for cell death in development,
27
normal tissue turnover, atrophy induced by endocrine and other stimuli,
28
negative selection in the immune system, and a substantial proportion of T-
29
cell killing. It also accounts for many cell deaths following exposure to
30
cytotoxic compounds, hypoxia or viral infection. It is a major factor in
31
the cell kinetics of tumors, both growing and regressing. Many cancer
32
therapeutic agents exert their effects through initiation of apoptosis, and
33
even the process of carcinogenesis itself seems sometimes to depend upon a
34
selective, critical failure of apoptosis that permits the survival of cells
35
after mutagenic DNA damage. Apoptosis probably contributes to many chronic
36
degenerative processes, including Alzheimer¶s disease, Parkinson¶s disease
37
and heart failure. So how does it work?
38
Molecular genetic studies on the hard-wired developmental cell death
39
programme of the nematode Caenorhabditis elegans led to discovery of a set
40
of proteins, widely represented by homologues in other species, and
41
responsible for turning on or off the final commitment to death2. In the
42
nematode these proteins include the products of the
c
ed3
and
c
ed4
genes
43
(
which initiate cell suicide),
c
ed9
(
which prevents it) and a series of
44
some seven genes involved in recognition and phagocytosis of the doomed
45
cell.
46
CED3 is the prototype of a family of around a dozen mammalian proteases,
47
called caspases because of the obligatory cysteine in their active site and
48
their predilection for cutting adjacent to aspartate residues. Mammalian
49
caspases appear to constitute an autocatalytic cascade, some members
50
(
notably caspase 8 or FLICE) being ³apical´ and more susceptible to
51
modification by endogenous regulatory proteins, whilst others
(
notably
52
caspase 3 ± also called CPP32, Yama and apopain) enact the final,
53
irreversible commitment to death. Study of caspase substrates is providing
54
interesting insights into the ways in which cells dismantle their structure
55
and function. Such substrates include ± not surprisingly ± cytoskeletal
56
proteins such as actin and fodrin and the nuclear lamins, but also an array
57
of regulatory and chaperone-like proteins whose function is altered by
58
cleavage in subtle and suggestive ways3. A recent example is the nuclease
59
chaperone ICAD, whose cleavage permits nuclear entry by a distinctive
60
apoptosis nuclease responsible for chromatin cleavage to oligonucleosome
61
fragments4.VII
61
Caspases appear to be present in most if not all cells in inactive pro-
62
enzyme form, awaiting activation by cleavage. One of the killing mechanisms
63
of cytotoxic T cells is a protease, granzyme B, that is delivered to the
64
target cell by the T cell granules and triggers these latent pro-enzymes.
65
There are endogenous triggers also, and the first to be discovered ± the
.
66
elegans
CED4 protein and its mammalian homologue ± is particularly
 
67
intriguing because of its mitochondrial origin5. Thus CED4 could be the
68
signal that initiates apoptosis under conditions of shut-down of cellular
69
energy metabolism, or when there is a critical level of cell injury
70
affecting mitochondrial respiration. In this way CED4 may act as the link
71
between agents long known to be associated with mitochondrial injury, such
72
as calcium and reactive oxygen species, and the initiation of apoptosis.
73
A second mitochondrial protein of enormous significance in apoptosis is
74
BCL2, a mammalian homologue of the nematode CED9 protein. BCL2 has the
75
tertiary structure of a bacterial pore-forming protein, and inserts into
76
the outer membrane of mitochondria. It abrogates apoptosis, probably
77
through binding CED4 and another protein BAX, with which it forms
78
heterodimers and which, like CED4, is also a ³killer´ protein6. Both BCL2
79
and BAX have several structurally and functionally similar homologues and
80
some of this family at least also tap into other cell membranes such as the
81
outer nuclear membrane and the endoplasmic reticulum.
82
So are there other sources of death transducers, activating the caspase
83
cascade because of injury to or signals arising in other parts of the cell
84
than mitochondria? There are already examples that show that the answer is
85
yes. Thus, the oncosuppressor protein p53 is activated following some types
86
of DNA damage and can trigger apoptosis. One way ± but only one of several
87
± whereby this happens is through transcriptional activation of BAX7.
88
The second messenger ceramide, a product of membrane-linked acid
89
sphingomyelinase activation, may act as a signal for plasma membrane
90
damage8. And a powerful caspase activating system is mediated by cytokine
91
receptors of the tumor necrosis factor family, notably fas/apo-1/CD95, TNF
92
receptor I, and others. These receptors, on receiving a death stimulus from
93
binding their ligand, initiate a series of protein-protein interactions,
94
building a complex
(
the death initiating signalling complex or DISC) which
95
eventually recruits and activates caspase 8
9
.
96
These mechanisms for coupling cell injury to apoptosis have mostly depended
97
on activation of pre-formed proteins. Apoptosis can also be initiated
(
and
98
forestalled) by transcriptional mechanisms, although rather little is known
99
about most of them. An outstanding example is the Drosophila gene
eape
,
100
transcriptionally activated around two hours prior to developmental and
101
injury-induced deaths in this organism. Drosophila apoptosis can occur
102
without
eape
 
transactivation, but requires very substantially enhanced
103
stimuli, suggesting that
eape
 
adjusts a threshold for apoptosis
104
initiation10.
105
Another gene whose transcription can initiate death is the familiar
106
immediate early gene
c-myc
11
. Transcriptional activation of
c-myc
 
107
initiates entry into DNA synthesis and is required for sustained re-entry
108
in repeated cell cycles, but
c-myc
 
activation in the absence of concurrent
109
cytokine support triggers apoptosis. This can also be interpreted as a
110
³threshold regulatory´ effect: ±
c-myc
 
expression increases the cellular
111
requirement for survival factors such as IGF-1.
112
Impressive confirmation of the significance of these pathways to apoptosis
113
is available from study of transforming viruses. These are hardened
114
survivors in the labyrinth of cell regulation, and have found keys to
115
allow escape from cell death in a variety of ways. Thus the transforming
116
papovavirus SV40, adenovirus type 12, Human Papilloma Virus type 16 and
117
Epstein-Barr Virus all have proteins that inactivate apoptosis through
118
inactivation of p53 or binding of BAX
12
. Even lytic viruses possess

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