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extensive double-stranded fragmentation (LMW DNA) may easily be separated from very large, chromosomal length DNA (HMW DNA), e.g., by centrifugation and filtration. For the quantification of DNA fragmentation, most methods involve a step in which the DNA of the cells has to be labeled: Prior to the addition of the cell death-inducing agent or of the effector cells, the (target) cells are incubated either with the [3H]thymidine ([3H]-dT) isotope or the nucleotide analog 5-bromo-2’-deoxyuridine (BrdU). During DNA synthesis (DNA replication) these modified nucleotides are incorporated into the genomic DNA. Subsequently, those labeled cells are incubated with cell death-inducing agents or effector cells and the labeled DNA is either fragmented or retained in the cell nucleus. Further, researchers discovered that proteases were involved in the early stages of apoptosis. The appearance of these caspases sets off a cascade of events that disable a multitude of cell functions. Caspase activation can be analyzed in different ways: - By an in vitro enzyme assay. Activity of a specific caspase, for instance caspase 3, can be determined in cellular lysates by capturing of the caspase and measuring proteolytic cleavage of a suitable substrate (Sgonc et al., 1994). - By detection of cleavage of an in vivo caspase substrate. For instance caspase 3 is activated during early stages. Its substrate PARP (Poly- ADP-Ribose-Polymerase) and the cleaved fragments can be detected with the anti PARP antibody. TUNEL assay Extensive DNA degradation is a characteristic event which often occurs in the early stages of apoptosis. Cleavage of the DNA may yield double-stranded, LMW DNA fragments (mono- and oligonucleosomes) as well as single strand breaks (“nicks”) in HMW- DNA. Those DNA strand breaks can be detected by enzymatic labeling of the free 3’-OH termini with modified nucleotides (X-dUTP, X = biotin, DIG or fluorescein). Suitable labeling enzymes include DNA polymerase (nick translation) and terminal deoxynucleotidyl transferase (end labeling). DNA polymerase I catalyzes the template dependent addition of nucleotides when one strand of Apoptosis in plants 19 a double-stranded DNA molecule is nicked. Theoretically, this reaction (In Situ Nick Translation, ISNT) should detect not only apoptotic DNA, but also the random fragmentation of DNA by multiple endonucleases occurring in cellular necrosis. Terminal deoxynucleotidyl transferases (TdT) is able to label blunt ends of doublestranded DNA breaks independent of a template. The end-labeling method has also been termed TUNEL (TdT-mediated XdUTP nick end labeling). The TUNEL method is more sensitive and faster than the ISNT method. In addition, in early stages cells undergoing apoptosis were preferentially labeled by the TUNEL reaction, whereas necrotic cells were identified by ISNT. Thus, experiments suggest the TUNEL reaction is more specific for apoptosis and the combined use of the TUNEL and nick translation techniques may be helpful to differentiate cellular apoptosis and necrosis (Gold et al., 1994). To allow exogenous enzymes to enter the cell, the plasma membrane has to be permeabilized prior to the enzymatic reaction. To avoid loss of LMW DNA from the permeabilized cells, the cells have to be fixed with formaldehyde or glutaraldehyde before permeabilization. This fixation crosslinks LMW DNA to other cellular constituents and precludes its extraction during the permeabilization step. If free 3’ ends in DNA are labeled with biotin- dUTP or DIGdUTP, the incorporated nucleotides may be detected in a second incubation step with (strept)avidin or an anti- DIG antibody. The immunocomplex is easily visible if the (strept)avidin or an anti- DIG antibody is conjugated with a reporter molecule (e.g., fluorescein, AP, POD). In contrast, the use of fluorescein-dUTP to label the DNA strand breaks allows the detection of the incorporated nucleotides directly with a fluorescence microscope or a flow cytometer. Direct labeling with fluorescein-dUTP offers several other advantages. Direct labeling produces less nonspecific background with sensitivity equal to indirect labeling and, thus, is as powerful as the indirect method in detecting apoptosis. Furthermore, the fluorescence may be converted into a colorimetric signal if an antifluorescein antibody conjugated with a reporter enzyme is added to the sample. Annexin V usage in plant PCD determination (Membran alteration) It has been shown that a number of changes in the cell
20 Narcin Palavan-Unsal et al. surface (membrane) markers occur during apoptosis, and any one of which may signal “remove now” to the phagocytes in the animal system. These membrane changes include: - Loss of terminal sialic acid residues from the side chains of cell surface glycoproteins, exposing new sugar residues. - Emergence of surface - Loss of asymmetry in cell membrane phospholipids, altering both the hydrophobicity and charge of the membrane surface In theory, any of these membrane changes could provide an assay for apoptotic cells. In fact, one of them has the alteration in phospholipid distribution. In normal cells, the distribution of phospholipids is asymmetric, with the inner membrane containing anionic phospholipids (such as phosphatidylserine) and the outer membrane having mostly neutral phospholipids. In apoptotic cells, however, the amount of phosphatidylserine (PS) on the outer surface of the membrane increases, exposing PS to the surrounding liquid. Annexin V, a calcium-dependent phospholipidbinding protein, has a high affinity for PS. Although it will not bind to normal living cells, Annexin V will bind to the PS exposed on the surface of apoptotic cells. Thus, Annexin V has proved suitable for detecting apoptotic in animal system. There are many studies which use conjugated Annexin V for plant early PCD detection (O’Brien et al., 1997). When we compare the flow cytometry techniques, propidum iodide (PI) is the most common dye to detect apoptosis with cell cycle status in one cell. The PI, which can only enter into, the nucleus of dead cells and intercalate with nuclear DNA, resulting in red fluorescence under ultraviolet light. It also intercalates into the major groove of double-stranded DNA and produces a highly fluorescent adducts that can be excited at 488 nm with a broad emission centred around 600 nm. Since PI can also bind to doublestranded RNA, it is necessary to treat the cells with RNase for optimal DNA resolution. The excitation of PI at 488 nm facilitates its use on the benchtop cytometers [PI can also be excited in the U.V. (351- 364 nm line from the argon laser) which should be considered when performing multicolour analysis on the multibeam cell sorters]. Hoechst33342 (HO342) is another DNA fluorochrome which can enter into both live and dead cells (Darzynkiewicz et al., 1992). Other flow cytometric based methods include the TUNEL assay, which measures DNA strand breaks and Annexin V binding, which detects relocation of membrane phosphatidyl serine from the intracellular surface to the extracellular surface. More recently, one mechanism, which has consistently been implicated in apoptosis, is CASPASE activity (cysteine proteases), typically caspase-3, which can be detected using fluorogenic substrates. Although there are many choices to determine PCD in plants, still there are some unknowns for plant PCD approaches. Well-determined animal system is key way to understand plant PCD but researchers need to investigate details about molecular basis of PCD. Therefore, new genomic and proteomic techniques to understand this question are remarkable. 2D gel electrophoresis or Yeast 2 hybrid techniques especially try to find other related proteins that are still unknown. Microarray technology is another new approach to understand gene expressions in different conditions. We believe that in a short time, new molecules which identify different stages of cell death will be clarified and begin to use for determination. References Bayly AC, Roberts RA and Dive C. Mechanism of apoptosis. In: Advences in Molecular and Cell Biology, Vol 20, Mechanism of Cell Toxicity. Bittar EE and Chipman JK (eds), Jain Press, Greenwich. 183-229, 1997. Beers EP and Freeman TB. Proteinase activity during tracheary element differentiation in zinnia mesophyll cultures. Plant Physiol. 113: 873-880, 1997. Bell PR. Megaspore abortion: a consequence of selective apoptosis? Int. J. Plant Sci. 157: 1-7, 1996. Blank A and McKeon T.A. Sinlge-strand-preferring nuclease activity in wheat leaves is increased in senescence and is negatively photoregulated. Proc. Natl Acad. Sci. USA, 86: 3169-3173, 1989. Bozhkov PV, Filonova LH, Suarez MF, Helmersson A, Smertenko AP, Zhivotovsky B and von Arnold S. VEIDase is a principal caspase-like activity involved in plant programmed cell death and essential for embryonic pattern formation. Cell Death and Differatiation. 11: 175-182, 2004. Brown PH and Ho TD. Biochemical properties and hormonal regulation of barley nuclease. Eur J. Biochem. 168: 357-364, 1987. Danon A, Delorme VG, Mailhac N and Gallois P. Plant programmed cell death: A common way to die. Plant Physiology and Biochemistry. 38: 647–655, 2000.
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