Apoptosis
(Programmed
cell death)
1. Brief introduction
2. Mechanism
3. Histochemical detection
4. Mammalian Apoptosis
5. Bacteria Apoptosis
6. Macrophage digestion of apoptotic bodies
7. Alzheimer’s diseases
8. Cancer treatment
9. Morphological differences between Apoptosis
& Necrosis
10. Biochemical detection of specific DNA damage for
demonstration of apoptosis
11. References.
Brief
introduction
Apoptosis is shrinkage, chromatin condensation, nuclease fragmentation and other degradation of cellular components by enzymes (Caspases).
Its role in cell population control during growth
& development suggests that there is an inherent cellular mechanism which
leads the cell to distruction. Due to rapid dehydrations, cells that are round
in shape become convoluted/ elongated and diminished in size. Chromatin
structure which occurs simultaneous to cell shrinkage, starts at the nuclear
periphery and is followed by nuclear fragmentation.The process of programmed cell death may be initiated by a specific stimulus or as a result of several signals
received from the external environment. it occurs as a result of inherent
cellular mechanisms, which finally leads to self destruction. The cell
activates a series of molecular events that cause an orderly degradation of the
cellular constitutes with minimal impact on the neighboring tissues.
In vivo the apoptotic bodies are phagocytosed by
macrophages, hence evoking no inflammatory response. Also apoptosis is “an
organized & efficient process of cell death”; 1st described in
1972 but not analyzed yet past 10 years genetically.
Examples:
(I) Falling of tadpole’s tail, (II) Getting sick by infection
When cells are damaged from external injury, oxygen starvation, or energy depletion, leads to “Necrosis”. Necrotic cells swell because of their osmotic balance is perturbed; their proteins are denatures & damaged. Finally, the cell ruptures and dies. Necrosis, bursting cells can cause collateral damage to surrounding tissue making the solution worse
The 1st apoptosis phenomenon well
documented in the nematode “coenorhabditis elegnas” (this worm is normally
found in soil eating bacteria, a model organism for developmental genetics
because its easy to maintain, has four developmental stages & is
transparent). During development c. elegnas generates 1090 cells, But final
adult worm has only 959, other 131 cells die via apoptosis.
Through “Nomarski optics” transforms differences in density into differences in height, giving an image that looks like 3-D relief where visualizes tissues that are several cell deep.
In (c.
elegnas) many mutant worms were identified in which the number of cells in
the adult was more/less than 959, the wild type number. In which 4 Gene’s involved in aberrant
apoptosis:- ced5, ced4, ced9 (cell death) and egl-1(laying defective egg); Defectiveness were >959 cells in adult
worms, where ced-9 proteins inhibits apoptosis. The CED and EGL proteins work
in cascade that initiate cell death i.e. action of one protein activates next.
Reasons
for cell suicide:
·
For proper development of organelles,
the endometrium and synapses.
·
To destroy cells that represents a
threat to integrity of the organism.
·
Withdrawal of positive signals of cells
(Neurons)
·
Receipt of negative signals that
increases levels of oxidants within the cells and the damaged to DNA.
Senescence and
apoptosis
As the cell grows in
culture, they become old due to aging and they can’t proliferate any more. The
end of the proliferative life span of cell is referrer.
Cellular senescence:
The growth of the cells is usually measure as population doublings which’s the number
of times the cell population doubles in number during the period of culture and
is calculated by [No. of cells harvested- No. of cells seeded]/ log10².
The phenomenon of
senescence has been mostly studied with human fibroblast cultures. After 30-60 population
doublings, the culture is mainly composed of senescent fibroblasts. Thses
senescent fibroblasts are unable to divide in response to mitotic stimuli. It
must be noted that the cells do not appear suddenly, but they gradually
accumulate and increase in number during the life span of the culture.
Measurement
of senescence: The direct measurement of senescent
cells is rather difficult. Some of the indirect measure are:
1. Loss
of metabolic activity.
2. Lack
of labeled precursor incorporation in to DNA
3. Certain
histochemical techniques.
Senescence
associated ß-galactosidase activity assay: There occurs an
over expression of the lysosomal enzyme ß-galactosidase at senescence. This enzyme elevation is also associated with
an increase in the cell size as the cell enters a permanent non-dividing state.
The number of senescent cells in a
culture can be measured by senescence associated ß-galactosidase (SA- ß). The assay
consists of the following stages:
1. Wash
the calls and fix them using a fixative (Para formaldehyde) and wash again.
2. Add
the staining solution (X-gal powder in dimethy formamide dissolved in buffer)
to the fixed cells and incubate.
3. The
senescent cells display a dense blue colour which can be counted.
Reasons for insitu
apoptosis:
1. For
proper development- the formation of fingers and toes of the fetus requires the
removal of the tissue between them. This is usually carried out by apoptosis.
2. Destruction
of cells that pose threat to the integrity of the organism- programmed cell
death is needed to destroy and remove the cells that may otherwise dame the
organism. Some of which are: a) cells with damaged DNA during the course of
embryonic development. If they are not destroyed they may result in birth defects.
b) cells of the immune system, after their appropriate immune function, undergo
apoptosis. This is needed to prevent autoimmune diseases e.g. rheumatoid
arthritis. c) cell infected with viruses
are destroyed by apoptosis.
3. Cell
destruction due to negative signals- There are several negative signals within
the cells that promote apoptosis. These include accumulation of free radicals,
exposure to UV rays, X-rays and chemotherapeutic drugs.
Mechanism of apoptosis:
The
PCD may occur due to three different mechanisms
1. Internal
signals
2. External
signals
3.
Reactive oxygen species
Role of caspases in
apoptosis: a group of enzyme namely activated proteases play a
crucial role in the PCD. These proteases are actually Cysteinyl Aspartate
Specific Proteinases or Caspases. There are about 10 different type of caspases
acting on different substrates ultimately leading to cell death. For instance,
caspase I cleaves interleukin-1.
Inhibition of caspases activity:
since the caspases are closely involved in apoptosis. Its possible to prevent
cell death by inhibiting their activities. Certain specific peptides that can
inhibit caspases, and thus apoptosis have been identified.
Measurement of apoptosis
A simple and easy way detecting dead or dying cells
is direct microscopic observation. The dying cells are rounded with dense
bodied which can be identified under phase contrast microscope. The cells that
have undergone apoptosis contain fragmented chromatin which can be convention
staining techniques. In recent years more sensitive and reliable techniques
have been developed for measuring apoptosis some of them are:
A) Determination of ADP/ATP ratio: both
the growth and apoptosis of cells require ATP. But when there is growth arrest,
an elevation of ADP occurs. Thus measuring ADP/ATP ratios are commercially
available.
B) Tunel assay: a
significant biochemical event for the apoptosis the activation of endogenous
nuclease activity. This enzyme cleaves DNA into fragments with free
three-hydroxyl groups. The newly formed small DNA fragments can be extended by
employing the enzyme DNA polymerase. If labeled nucleotides are used for DNA
fragment extension they can be detected. TUNEL- TdT-mediated dUTI nick end
labeling assay. This labeled DNA can be visualized by fluorescence microscopy.
TUNEL is very fast and effective for the determination of DNA fragments formed
by endogenous nuclease activity. The apoptotic nuclei can be identified by a
fluorescent technique using fluoresceir isothiocynate and
4,6-diaminoplenylindole.
C) DNA Laddering test:
during the course of apoptosis, the genomic DNA is cleaved to mono- and oligo-
nucleosomal DNA fragments. These fragments can beseparated by agarose
electrophoresis, and detected. The nucleosomal fragments of apoptotic cells
give a characteristic ladder pattern or electrophoresis.
Limitation:
DNA laddering test is not very specific since several cell that have undergone
apoptosis may not show DNA ladders, for these reasons, DNA laddering test is
coupled with some other test for measurement of apoptosis
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