Apoptosis DNA Fragmentation
Encyclopedia
Apoptosis
DNA
fragmentation is a key feature of programmed cell
death and also occurs in certain stages of necrosis
. Apoptosis is characterized by the activation of endogenous endonucleases with subsequent cleavage of chromatin DNA into internucleosomal fragments of 180 BP
and multiples thereof.
DNA cleavage during apoptosis occurs at sites between nucleosomes, protein-containing structures that occur in chromatin
at ~200-BP intervals. This DNA fragmentation is often analyzed using agarose gel electrophoresis
to demonstrate a "ladder" pattern at ~200-BP intervals
. Necrosis
, on the other hand, is characterized by random DNA fragmentation which forms a "smear" on agarose gels.
DNA fragmentation is a secondary consequence, rather than an integral cause, of apoptosis. Endonuclease involved might be similar to DNAse I, a potential indication that the DNA fragmentation might occur after the release of enzymes from cytoplasmic membrane lysis
, an event that would potentially occur only after the final lytic event in the apoptotic sequence. More recently, data have shown that specific proteases residing in the cytoplasm mediate the terminal events of apoptosis, including those of nuclear morphology. Even so, the detection of DNA fragmentation and the presence of single strand ends of DNA has continued to be an assay used in many studies to detect apoptotic cells, particularly in intact tissues. This is in spite of the fact that necrosis also produces single-strand DNA ends in cell nuclei. Therefore, the interpretation of these in situ assays of DNA fragmentation [in situ nick-translation (ISNT); terminal transferase (TUNEL
) must be carefully assessed together with morphological features of apoptotic cells.
Even though much work has been performed on the analysis of apoptotic events, little information is available to link the timing of morphological features at the cell surface and in the nucleus to the biochemical degradation of DNA in the same cells. Apoptosis can be initiated by a myriad of different mechanisms in different cell types, and the kinetics of these events vary widely, from only a few minutes to several days depending on the cell system.
There are many methods to assess the DNA fragmentation caused by apoptosis and also many commercial kits are available. Some new methods do quantification assays for DNA Fragmentation by flow cytometry and fluorescent assays.
The discovery of the stereotypical internucleosomal degradation of genomic DNA (lymphocytes, thymocytes etc.) to a regular repeating oligonucleosomal fragments generated by Ca/Mg-dependent endonuclease is accepted as one of the best-characterized biochemical marker of the process known in molecular and cellular biology as apoptosis
or programmed cell death.
In 1951 Glucksmann A. described on morphological grounds evidence characterizing selective death as a normal process of embryonic development. In 1964-1965 Lokshin R.A. And Williams C.M. referred to this process as a programmed cell death in recognition of “timing and synchrony involved”. In 1970 Williamson R. described that cytoplasmic DNA isolated from mouse liver cells after culture is characterized with a fragments of a series of multiples of a unit molecular weight of 135000 Dalton, consistent with a hypothesis that these DNA fragments are specific degradation product of nuclear DNA. In 1972 Kerr J., Wyllie A. and Currie A. described on morphological grounds the features of a programmed cell death/apoptosis which exhibits specific morphological characteristics distinctly different from necrosis. In 1973 Hewish D.R. And Burgoyne L.A. in the context of study of subchromatin structure found that chromatin is accessible to the Ca++/Mg++ endonuclease with formation of digest product with a regular series of molecular weight similar to the previously described by Williamson R. In 1974 Williams J.R., Little J.B.,Shipley W. U. using cells exposed to widely differing type of trauma found, that during cell death degraded DNA in “every case had a modal value of between 10(x6) and10(x7) Dalton and cellular metabolism is required to produce degradation of DNA”. However this observation was without indication “whether the incision attack on the DNA molecule was a random or rather at particular site,that have structural or functional meaning”. In 1976 Scalka M., Matyasova M, Cejkova M. described internucleosomal fragmentation of irradiated lymphoid chromatin DNA in vivo. Six year passed from 1972 to 1978/1980 until discovery and evaluation of internucleosomal fragmentation of DNA during apoptotic cell death as a hallmark of apoptosis. Since 1972 (Kerr J., Wyllie A., and Currie A.) it is accepted that glucocorticoids induced death of lymphocytes belongs to the apoptosis. In 1978 Zakharyan Robert A., Pogosian R. originally presented paper revealing that glucocorticoids induced DNA degradation in rat lymphoid tissue in thymus and spleen occurred in a specific pattern producing fragments of DNA electrophoretically similar to those observed after treatment of chromatin with microccoccal nuclease,which indicated internucleosomal cleavege pattern of DNA degradation occurred during apoptosis. Thus, the first link between programmed cell death/apoptosis and internucleosomal fragmentation of chromatin DNA was discovered and soon became as a specific feature of apoptosis.
In 1980 Wyllie A. reported additional evidence for occurring internucleosomal DNA cleavage pattern as a specific feature in glucocorticoid treated thymocytes undergoing apoptosis. Internucleosomal DNA cleavage pattern was observed as a specific feature of apoptosis in 1978/1980 and became as a hallmark of programmed cell death and ERA of apoptosis in molecular and cellular biology was started.
Degradation of nuclear DNA into nucleosomal units is one of the hallmarks of apoptotic cell death. It occurs in response to various apoptotic stimuli in a wide variety of cell types. Molecular characterization of this process identified a specific DNase (CAD, caspase-activated DNase) that cleaves chromosomal DNA in a caspase-dependent manner. CAD is synthesized with the help of ICAD (inhibitor of CAD), which works as a specific chaperone for CAD and is found complexed with ICAD in proliferating cells. When cells are induced to undergo apoptosis, caspases-in particular caspase 3-cleave ICAD to dissociate the CAD:ICAD complex, allowing CAD to cleave chromosomal DNA. Cells that lack ICAD or that express caspase-resistant mutant ICAD thus do not show DNA fragmentation during apoptosis, although they do exhibit some other features of apoptosis and die.
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Apoptosis
Apoptosis is the process of programmed cell death that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation...
DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
fragmentation is a key feature of programmed cell
Cell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
death and also occurs in certain stages of necrosis
Necrosis
Necrosis is the premature death of cells in living tissue. Necrosis is caused by factors external to the cell or tissue, such as infection, toxins, or trauma. This is in contrast to apoptosis, which is a naturally occurring cause of cellular death...
. Apoptosis is characterized by the activation of endogenous endonucleases with subsequent cleavage of chromatin DNA into internucleosomal fragments of 180 BP
Base pair
In molecular biology and genetics, the linking between two nitrogenous bases on opposite complementary DNA or certain types of RNA strands that are connected via hydrogen bonds is called a base pair...
and multiples thereof.
DNA cleavage during apoptosis occurs at sites between nucleosomes, protein-containing structures that occur in chromatin
Chromatin
Chromatin is the combination of DNA and proteins that make up the contents of the nucleus of a cell. The primary functions of chromatin are; to package DNA into a smaller volume to fit in the cell, to strengthen the DNA to allow mitosis and meiosis and prevent DNA damage, and to control gene...
at ~200-BP intervals. This DNA fragmentation is often analyzed using agarose gel electrophoresis
Electrophoresis
Electrophoresis, also called cataphoresis, is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field. This electrokinetic phenomenon was observed for the first time in 1807 by Reuss , who noticed that the application of a constant electric...
to demonstrate a "ladder" pattern at ~200-BP intervals
DNA laddering
DNA laddering is a phenomenon seen in laboratory tests; it is a sensitive indicator of programmed cell death, specifically of apoptosis. It was first described in 1980 by A. H. Wyllie at the University of Edinburgh Medical School....
. Necrosis
Necrosis
Necrosis is the premature death of cells in living tissue. Necrosis is caused by factors external to the cell or tissue, such as infection, toxins, or trauma. This is in contrast to apoptosis, which is a naturally occurring cause of cellular death...
, on the other hand, is characterized by random DNA fragmentation which forms a "smear" on agarose gels.
DNA fragmentation is a secondary consequence, rather than an integral cause, of apoptosis. Endonuclease involved might be similar to DNAse I, a potential indication that the DNA fragmentation might occur after the release of enzymes from cytoplasmic membrane lysis
Lysis
Lysis refers to the breaking down of a cell, often by viral, enzymic, or osmotic mechanisms that compromise its integrity. A fluid containing the contents of lysed cells is called a "lysate"....
, an event that would potentially occur only after the final lytic event in the apoptotic sequence. More recently, data have shown that specific proteases residing in the cytoplasm mediate the terminal events of apoptosis, including those of nuclear morphology. Even so, the detection of DNA fragmentation and the presence of single strand ends of DNA has continued to be an assay used in many studies to detect apoptotic cells, particularly in intact tissues. This is in spite of the fact that necrosis also produces single-strand DNA ends in cell nuclei. Therefore, the interpretation of these in situ assays of DNA fragmentation [in situ nick-translation (ISNT); terminal transferase (TUNEL
TUNEL assay
Terminal deoxynucleotidyl transferase dUTP nick end labeling is a method for detecting DNA fragmentation by labeling the terminal end of nucleic acids.- Method :...
) must be carefully assessed together with morphological features of apoptotic cells.
Even though much work has been performed on the analysis of apoptotic events, little information is available to link the timing of morphological features at the cell surface and in the nucleus to the biochemical degradation of DNA in the same cells. Apoptosis can be initiated by a myriad of different mechanisms in different cell types, and the kinetics of these events vary widely, from only a few minutes to several days depending on the cell system.
There are many methods to assess the DNA fragmentation caused by apoptosis and also many commercial kits are available. Some new methods do quantification assays for DNA Fragmentation by flow cytometry and fluorescent assays.
Programmed Cell Death/Apoptosis
Historical background: The discovery of stereotypical internucleosomal fragmentation of chromatine DNA as a hallmark of apoptosis.The discovery of the stereotypical internucleosomal degradation of genomic DNA (lymphocytes, thymocytes etc.) to a regular repeating oligonucleosomal fragments generated by Ca/Mg-dependent endonuclease is accepted as one of the best-characterized biochemical marker of the process known in molecular and cellular biology as apoptosis
Apoptosis
Apoptosis is the process of programmed cell death that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation...
or programmed cell death.
In 1951 Glucksmann A. described on morphological grounds evidence characterizing selective death as a normal process of embryonic development. In 1964-1965 Lokshin R.A. And Williams C.M. referred to this process as a programmed cell death in recognition of “timing and synchrony involved”. In 1970 Williamson R. described that cytoplasmic DNA isolated from mouse liver cells after culture is characterized with a fragments of a series of multiples of a unit molecular weight of 135000 Dalton, consistent with a hypothesis that these DNA fragments are specific degradation product of nuclear DNA. In 1972 Kerr J., Wyllie A. and Currie A. described on morphological grounds the features of a programmed cell death/apoptosis which exhibits specific morphological characteristics distinctly different from necrosis. In 1973 Hewish D.R. And Burgoyne L.A. in the context of study of subchromatin structure found that chromatin is accessible to the Ca++/Mg++ endonuclease with formation of digest product with a regular series of molecular weight similar to the previously described by Williamson R. In 1974 Williams J.R., Little J.B.,Shipley W. U. using cells exposed to widely differing type of trauma found, that during cell death degraded DNA in “every case had a modal value of between 10(x6) and10(x7) Dalton and cellular metabolism is required to produce degradation of DNA”. However this observation was without indication “whether the incision attack on the DNA molecule was a random or rather at particular site,that have structural or functional meaning”. In 1976 Scalka M., Matyasova M, Cejkova M. described internucleosomal fragmentation of irradiated lymphoid chromatin DNA in vivo. Six year passed from 1972 to 1978/1980 until discovery and evaluation of internucleosomal fragmentation of DNA during apoptotic cell death as a hallmark of apoptosis. Since 1972 (Kerr J., Wyllie A., and Currie A.) it is accepted that glucocorticoids induced death of lymphocytes belongs to the apoptosis. In 1978 Zakharyan Robert A., Pogosian R. originally presented paper revealing that glucocorticoids induced DNA degradation in rat lymphoid tissue in thymus and spleen occurred in a specific pattern producing fragments of DNA electrophoretically similar to those observed after treatment of chromatin with microccoccal nuclease,which indicated internucleosomal cleavege pattern of DNA degradation occurred during apoptosis. Thus, the first link between programmed cell death/apoptosis and internucleosomal fragmentation of chromatin DNA was discovered and soon became as a specific feature of apoptosis.
In 1980 Wyllie A. reported additional evidence for occurring internucleosomal DNA cleavage pattern as a specific feature in glucocorticoid treated thymocytes undergoing apoptosis. Internucleosomal DNA cleavage pattern was observed as a specific feature of apoptosis in 1978/1980 and became as a hallmark of programmed cell death and ERA of apoptosis in molecular and cellular biology was started.
Degradation of nuclear DNA into nucleosomal units is one of the hallmarks of apoptotic cell death. It occurs in response to various apoptotic stimuli in a wide variety of cell types. Molecular characterization of this process identified a specific DNase (CAD, caspase-activated DNase) that cleaves chromosomal DNA in a caspase-dependent manner. CAD is synthesized with the help of ICAD (inhibitor of CAD), which works as a specific chaperone for CAD and is found complexed with ICAD in proliferating cells. When cells are induced to undergo apoptosis, caspases-in particular caspase 3-cleave ICAD to dissociate the CAD:ICAD complex, allowing CAD to cleave chromosomal DNA. Cells that lack ICAD or that express caspase-resistant mutant ICAD thus do not show DNA fragmentation during apoptosis, although they do exhibit some other features of apoptosis and die.
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