“Apoptotic DNA fragmentation”的意思、由来-开放百科全书

The enzyme responsible for apoptotic DNA fragmentation is the Caspase-Activated DNase (CAD). CAD is normally inhibited by another protein, the Inhibitor of Caspase Activated DNase (ICAD). During apoptosis, the apoptotic effector caspase, caspase-3, cleaves ICAD and thus causes CAD to become activated.^[7]

CAD cleaves DNA at internucleosomal linker sites between nucleosomes, protein-containing structures that occur in chromatin at ~180-bp intervals. This is because the DNA is normally tightly wrapped around histones, the core proteins of the nucleosomes. The linker sites are the only parts of the DNA strand that are exposed and thus accessible to CAD.

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, caspase 3 cleaves 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.

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. The presence or absence of particular apoptotic event(s), including DNA fragmentation, depends on the "time window" at which the kinetic process of apoptosis is being investigated. Often this may complicate identification of apoptotic cells if cell populations are analyzed only at a single time point e.g. after induction of apoptosis.

Historical background

The discovery of the internucleosomal fragmentation of genomic DNA to regular repeating oligonucleosomal fragments generated by Ca/Mg-dependent endonuclease is accepted as one of the best-characterized biochemical markers of apoptosis (programmed cell death).

In 1970, {{Smallcaps|Williamson}} described that cytoplasmic DNA isolated from mouse liver cells after culture was characterized by DNA fragments with a molecular weight consisting of multiples of 135 kDa. This finding was consistent with the hypothesis that these DNA fragments were a specific degradation product of nuclear DNA.^[8] In 1972, {{Smallcaps|Kerr}}, {{Smallcaps|Wyllie}}, and {{Smallcaps|Currie}} coined the term apoptosis and distinguished this type of cell death from necrosis based on morphological features.^[9] In 1973, {{Smallcaps|Hewish}} and {{Smallcaps|Burgoyne}}, during the study of subchromatin structure, found that chromatin is accessible to the Ca⁺⁺/Mg⁺⁺ endonuclease, resulting in the formation of a digestion product with a regular series of molecular weight similar to the one previously described by Williamson (1970).^[10] In 1974, {{Smallcaps|Williams}}, {{Smallcaps|Little}}, and {{Smallcaps|Shipley}}, using cells exposed to widely differing types of trauma, found that during cell death, degraded DNA in "every case had a modal value of between 10(x6) and 10(x7) Dalton and cellular metabolism is required to produce degradation of DNA". However, this observation was without indication of "whether the incision attack on the DNA molecule was a random or rather at a particular site, that have structural or functional meaning".^[11] In 1976, {{Smallcaps|Scalka}}, {{Smallcaps|Matyasova}}, and {{Smallcaps|Cejkova}} described internucleosomal fragmentation of irradiated lymphoid chromatin DNA in vivo.^[12]

Six years passed from 1972 to 1978/1980 until the discovery and evaluation of internucleosomal fragmentation of DNA during apoptotic cell death as a hallmark of apoptosis. Since 1972 ({{Smallcaps|Kerr}}, {{Smallcaps|Wyllie}}, and {{Smallcaps|Currie}}^[9]), it is accepted that glucocorticoid-induced death of lymphocytes is a form of apoptosis. In 1978, {{Smallcaps|Zakharyan}} and {{Smallcaps|Pogosyan}} presented a paper revealing that glucocorticoid-induced DNA degradation in rat lymphoid tissue, thymus, and spleen occurred in a specific pattern producing fragments of DNA that were electrophoretically similar to those observed after treatment of chromatin with microccoccal nuclease, which indicated internucleosomal cleavage pattern of DNA degradation occurred during apoptosis.^[13]^[14] 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, {{Smallcaps|Wyllie}} reported additional evidence for an internucleosomal DNA cleavage pattern as a specific feature of glucocorticoid-treated thymocytes undergoing apoptosis.^[2] The internucleosomal DNA cleavage pattern was observed as a specific feature of apoptosis in 1978/1980 and has become a recognised hallmark of programmed cell death since then. In 1992 Gorczyca et al. ^[3] and Gavrieli et al.^[4] independently described the DNA fragmentation assay based on the use of the terminal deoxynucleotidyl transferase (TUNEL) which become one of the standard methods to detect and identify apoptotic cells.

Detection assays

Treatment of cells with detergent, prior or concurrently with DNA fluorochrome, also reveals DNA fragmentation by virtue of the presence of the sub-G₁ cells or cell fragments, as defined by Nicoletti et al.^[5]

Apoptotic DNA fragmentation can also be detected by the TUNEL assay. The fluorochrome-based TUNEL assay applicable for flow cytometry, correlates the detection of DNA strand breaks with the cellular DNA content and thus with cell cycle-phase position. The avidin-peroxidase labeling TUNEL assay is applicable for light absorption microscopy. Many TUNEL-related kits are commercially available.

Apoptotic DNA fragmentation is also analyzed using agarose gel electrophoresis to demonstrate a "ladder" pattern at ~180-BP intervals.^[1] Necrosis, on the other hand, is usually characterized by random DNA fragmentation which forms a "smear" on agarose gels.

See also

References

1. ^Sakahira H, Enari M, NagataS. (1998) Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis. Nature.391(6662):96-9. Erratum in: Nature. 2015 Oct 29;526(7575):728. {{PMID|9422513}}
2. ^¹{{cite journal| author = Wyllie AH| date = 1980-04-10| title = Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation| journal = Nature| volume = 284| issue = 5756| pages = 555–556| issn = 0028-0836| doi = 10.1038/284555a0| pmid = 6245367}}
3. ^. Gorczyca W, Bruno S, Darzynkiewicz R, Gong J, Darzynkiewicz Z. DNA strand breaks occurring during apoptosis - their early insitu detection by the terminal deoxynucleotidyl transferase and nick translation assays and prevention by serine protease inhibitors. Int J Oncol. 1992 Nov;1(6):639-48. {{PMID|21584593}}
4. ^{{Cite journal | last1 = Gavrieli | first1 = Y. | last2 = Sherman | first2 = Y. | last3 = Ben-Sasson | first3 = S. A. | doi = 10.1083/jcb.119.3.493 | title = Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation | journal = The Journal of Cell Biology | volume = 119 | issue = 3 | pages = 493–501 | year = 1992 | pmid = 1400587| pmc =2289665 }}
5. ^{{cite journal |vauthors=Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C | date = 3 June 1991| title = A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry| journal = Journal of Immunological Methods| volume = 139| issue = 2| pages = 271–279| pmid = 1710634| doi = 10.1016/0022-1759(91)90198-O}}
6. ^{{cite journal|vauthors=Riccardi C, Nicoletti I | date = 9 November 2006| title = Analysis of apoptosis by propidium iodide staining and flow cytometry| journal = Nature Protocols| volume = 1| issue = 3| pages = 1458–1461| pmid = 17406435| doi = 10.1038/nprot.2006.238}}
7. ^{{Cite journal | last1 = Nagata | first1 = S. | last2 = Enari | first2 = M. | last3 = Sakahira | first3 = H. | last4 = Yokoyama | first4 = H. | last5 = Okawa | first5 = K. | last6 = Iwamatsu | first6 = A. | title = A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD | journal = Nature | volume = 391 | issue = 6662 | pages = 43–50 | year = 1998 | doi = 10.1038/34112 | pmid = 9422506| pmc = }}
8. ^{{cite journal| last = Williamson| first = Robert| date = 1970-07-14| title = Properties of Rapidly Labeled Deoxyribonucleic Acid Fragments Isolated from the Cytoplasm of Primary Cultures of Embryonic Mouse Liver Cells| journal = Journal of Molecular Biology| volume = 51| issue = 1| pages = 157–168| issn = 0022-2836| doi = 10.1016/0022-2836(70)90277-9| pmid = 5481278| url = http://linkinghub.elsevier.com/retrieve/pii/0022-2836(70)90277-9}}
9. ^¹{{cite journal| last1 = Kerr| first1 = John F. R.| last2 = Wyllie| first2 = Andrew| authorlink2 = Andrew Wyllie| last3 = Currie| first3 = Alastair|date=August 1972| title = Apoptosis: A Basic Biological Phenomenon with Wide-ranging Implications in Tissue Kinetics| journal = British Journal of Cancer| volume = 26| issue = 4| pages = 239–257| issn = 0007-0920| doi = 10.1038/bjc.1972.33| pmid = 4561027| pmc = 2008650}}
10. ^{{cite journal| last = Burgoyne| first = Leigh A.| date = 1973-05-15| title = Chromatin sub-structure. The digestion of chromatin DNA at regularly spaced sites by a nuclear deoxyribonuclease| journal = Biochemical and Biophysical Research Communications| volume = 52| issue = 2| pages = 504–510| issn = 0006-291X| doi = 10.1016/0006-291X(73)90740-7| pmid = 4711166| url = http://linkinghub.elsevier.com/retrieve/pii/0006-291X(73)90740-7| last2 = Burgoyne| first2 = Leigh A.}}
11. ^{{cite journal| last1 = Williams| first1 = Jerry R.| last2 = Little| first2 = John B.| last3 = Shipley| first3 = William U.| date = 1974-12-20| title = Association of mammalian cell death with a specific endonucleolytic degradation of DNA| journal = Nature| volume = 252| pages = 754–755| issn = 0028-0836| doi = 10.1038/252754a0| pmid = 4474604| issue = 5485}}
12. ^{{cite journal| last = Ceskova| first = M.| date = 1976-12-31| title = DNA in chromatin of irradiated lymphoid tissues degrades in vivo into regular fragments| journal = FEBS Letters| volume = 72| issue = 2| pages = 271–274| issn = 0014-5793| doi = 10.1016/0014-5793(76)80984-2| pmid = 16386038| last2 = Matyásová| first2 = J| last3 = Cejková| first3 = M}}
13. ^{{cite journal| last1 = Zakharyan| first1 = R. A.| last2 = Pogosyan| first2 = R. G.| year = 1978| title = Glucocorticoid induction of the degradation of lymphocyte chromatin DNA into regularly repeating fragments in vivo| journal = Doklady Akademii Nauk Armyanskoi SSR| volume = 67| issue = 2| pages = 110–114| issn = 0366-8606}} CODEN: DANAAW, CAN 90:115643 AN 1979:115643 CAPLUS (Copyright 2003 ACS)
14. ^Chemical Abstracts v.90,1979;90:115643n p.112.
15. ^Kajstura M, Halicka HD, Pryjma J, Darzynkiewicz Z. (2007) Discontinuous fragmentation of nuclear DNA during apoptosis revealed by discrete “sub-G1“ peaks on DNA content histograms. Cytometry A 71A:125-131. {{PMID|17252584}}
16. ^Wlodkowic D, Telford W, Skommer J, Darzynkiewicz Z. (2011) Apoptosis and beyond: Cytometry in studies of programmed cell death. Methods Cell Biol, 103:55-98. {{PMID|21722800}} {{PMC|3263828}}.

Mechanism

Historical background

Detection assays

See also

References

Further reading