Apoptosis caspase detection

The immunocytochemical staining of cytochrome C offers another alternative since, upon exposure to apoptotic stimuli, cytochrome C is rapidly released into the cytosol, an event that may be required for the completion of apoptosis in some systems (L2). The effect of cytosolic cytochrome C is thought to be the activation of caspases. The immunocytochemical staining of cytochrome C localized in mitochondria in healthy cells or diffused in the cell cytoplasm with monoclonal antibody (Promega) after induction of apoptosis, as detected by fluorescence microscopy, can be used for monitoring apoptosis (L2, M7, S8, T6). It is also a simple, rapid, specific mefhod for quanfifafive assessment of apoptotic cells. 

Fig. 6. Activation of caspases detected by the fluorochrome-labeled caspase (FLICA) inhibitors assay. FiL-60 cells were untreated (A), treated in culture with camptothecin to induce apoptosis (B) (ref. 26). The cells were then electrostatically attached to microscope slides, incubated with staining solution of FAM-VAD-FMK as described in the protocol, and their green fluorescence (integrated value and pixel of maximal intensity) measured by LSC. Note the appearance of apoptotic cell subpopulation characterized by the increased green fluorescence (above the marked threshold level of the maximal pixel) reflecting activation of caspases that bind FAM-VAD-FMK.

Apple juice phenolic compound extracts have the property to induce apoptosis in HT-29 cells [147]. Quercetin and phloretin dose-dependently induced both caspase-3 activity and DNA cleavage under serum-free conditions. Phloretin at 100 pM induced both the death receptor as well as the mitochondrial pathway of apoptosis induction, detected by activation of the initiator caspases-8 and -9 and the effector caspases-3 and -7 as well as by PARP cleavage. Activation of caspase-9 was accompanied by release of cytochrome c and the mitochondrial protein Smac/DIABLO from the mitochondria to the cytoplasm, and upregulation of proapoptotic Bax levels [43,44,148]. In general, berry extracts have the ability to stimulate apoptosis of the HT-29, COX-2-expressing colon cancer cells. Black raspberry and strawberry extracts showed the most significant proapoptotic effects against this cell line [43] (Tables 1 and 6). 

Gurtu, V., Kain, S. R. and Zhang, G. (1997). Fluorometric and colorimetric detection of caspase activity associated with apoptosis. Anal. Biochem. 251, 98-102. 

Furthermore, since the cell growth arrest is often linked to cell death. The annexin V staining positive cell or the amount of DNA fragmentation assessed by TUNEL and FACS analysis has been interpreted as indicative of apoptosis. The HDACI-induced apoptosis can also be determined by Western blotting of target proteins, detection of mitochondrial membrane potentials, activation of caspases and their substrate cleavages in a dose- and time-dependent manner. 

The group at Pharmacyclics developed a related phenyl hydroxamic add HDACi, PCI-34051 (27e) and demonstrated this to display greater than 200-fold selectivity for HDAC8 (IC5o = 10nM) over the other HDAC isoforms [40b]. Interestingly, this compound induced caspase-dependent apoptosis in T-cell lymphomas and leukemias at low micromolar concentrations, but not in other hematopoietic or solid tumor cell lines. Furthermore, PCI-34051 does not cause detectable tubulin or histone acetylation. 

On the other hand, there is strong evidence that the effects of flavonoids on cultured cells are not merely toxic but via induction of apoptosis or inhibition of proliferation. Thus signs of apoptosis, such as induction of caspase-3, fragmentation of DNA and chromatin condensation, are frequently detected upon cell exposure to flavonoids [213, 217-221]. In addition, the effects of flavonoids are generally reversible upon removal or addition of serum [222-224]. 

Yang, F., Sun, X., Beech, W., Teter, B., Wu, S., Sigel, J., Vinters, H. V., Frautschy, S. A., and Cole, G. M. 1998. Antibody to caspase-cleaved actin detects apoptosis in differentiated neuroblastoma and plaque-associated neurons and microglia in Alzheimer s disease. Am. J. Pathol. 152 379-389. 

Endonucleases. Endonucleases provide for DNA cleavage into small ( 200 base pairs) fragments, which is an essential step in apoptotic cascade (Wyllie 1980 Wyllie 1998). Endonucleases are stimulated by Ca2+ and their activation was detected in several cell types undergoing apoptosis (McConkey et al., 1988 Aw et al., 1990). The intimate mechanisms of endonucleases action remain not fully described at least in part they can be explained through the involvement of caspase-3 activated endonuclease (or caspase-activated DNAase - (Enari et al., 1998)). Nonetheless, the chromatin fragmentation was also observed in cell (and caspase)-free system, when isolated nuclei were treated with Ca2+ and ATP, suggesting the existence of caspase-independent DNA cleavage mechanism (Jones et al., 1989). 

Despite extensive research, identification of apoptotic cells remains an important unresolved issue. Apoptosis can be recognized by characteristic morphological features, which are difficult to be found in the heart. Furthermore, morphology alone does not enable recognition of cells early in the apoptotic pathway. Detection of activated caspases appears to be a reasonable way to detect apoptotic cells, given the central role of caspases in the process of apoptosis. It must be kept in mind, however, that caspases may contribute to necrotic cell death [80, 81] and caspase-independent apoptosis does occur [80]. 

Both techniques can be applied to most experimental models of apoptosis, including cells in culture and biopsies. In addition, techniques based on the cleavage of synthetic caspase substrates can be applied to caspases, activated in vitro and in vivo [82, 83], Whereas Western blotting is time consuming and mainly a qualitative assay, the detection of caspase activity by cleavage of synthetic substrates is a quantitative, relatively fast and sensitive method [82]. However, both techniques do not provide information about the type and distribution of the cells with activated caspases in the tissue examined. 

Immunohistochemistry, on the other hand, enables identification of activated caspases or their cleaved products in fixed archival tissue sections. This technique allows identification of cell(s) undergoing caspase activation, as well as analysis of the distribution of cell(s) in the tissue. Specific antibodies to various caspases are now commercially available, the most frequently studied being caspase-3. Studies in various human tissues and cells have shown that immunohistochemical detection of activated caspase-3 is a useful tool for identifying apoptotic cells in archival material, even before all of the morphological features of apoptosis occur [84-86]. Several target proteins cleaved by caspases can also be detected by immunohistochemistry for example PARP [87], actin [88, 89], and lamin B [90]. 

Apoptosis has been demonstrated to occur also in human MI. Studies of heart tissue obtained at autopsies from patients, who died of MI, suggest that apoptotic cardiomyocytes were most prominent in the border zones of MI (Fig. 1A and B) [111-123], whereas few were present in the remote myocardium [113, 114, 118-121]. The most commonly used method for detection of apoptosis in these studies was TUNEL. Using this method, a highly variable apoptotic rate was found, ranging from 0.8% [114] to 43% [118] in the border zone of MI, and from 0.05% [114] to 38% [118] in the remote myocardium. Using immunohistochemistry for activated (cleaved) caspase-3, a much lower apoptotic rate was reported in human MI [123]. 

Gorman AM, Hirt UA, Zhivotovsky B, Orrenius S, Ceccatelli S. Application of a fluoro-metric assay to detect caspase activity in thymus tissue undergoing apoptosis in vivo. J Immunol Methods 1999 226 43-48. 

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