APOPTOSIS= Programmed cell death

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”; 1^st 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 1^st 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.

Increase in cytoplasmic calcium concentration and activation of protease known as “Caspase” also one or more endonucleases that degrade DNA at their inter-nucleosomal (Linker) section are also very characteristic events of apoptosis. Thus the product of DNA degradation are discontinuous and of the size of nucleosomal and oligonucleosomal DNA sections that generate a very characteristic “ladder” pattern during gel electrophoresis, also some typical gene activation also leads to apoptosis. Even during advance stage of apoptosis, the structural & functional integrity of the plasma membrane, mitochondria and lysosome are generally preserved. These considering all the changes that occur in cell undergoing apoptosis, the most characteristic feature of this process is active participation of the cell in its demise. The cell activates a cascade of molecular events which result in orderly degradation of the cell constituents with minimal impact on the neighboring tissue.
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.

The genetic and biological experiments explain the membrane of c.elegnas mitochondria, a complex of CED-9, PROCED-3 and dimer of CED-4 remains inactive. When apoptosis is when apoptosis is triggered, EGL-1 protein binds to CED-9, releasing CED-4 and ProCED-3. Tetramers of CED-4 are formed and cleaved the inhibitory domain from proCEd-3. Activated CED-3 is the central enzymes that activates the remaining caspases and executes the apoptosis program.During development, c. elegnas generates more cells than are found in adult worm, extras die via apoptosis. The CED and EGL proteins of c. elegnas work in a cascade that initial cell death. CED-3 and CED-4 are special protease called “caspases” which are key regulator to apoptosis.

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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