Apoptosis marker

Translocation of the phosphatidylserine from the inner to the outer leaflet of the plasma membrane is an initial event related to the apoptotic process and possibly serves as a signal for the removal of apoptotic bodies by phagocytic cells (Martin et al., 1995). The exposure of this phospholipid has been largely used as a specific apoptosis marker. 

Many elements of complex intrinsic and extrinsic signaling pathways leading to apoptosis have been identified. During apoptosis, a cascade of proteases termed caspases is involved with upstream signaling events and downstream executioner events. Caspases are cysteine-dependent, aspartate-specific proteases that contain highly conserved cysteine residues in their active sites and cleave substrates leaving C terminal Asp residues. Caspase-3 is one of the main effector molecules of the apoptotic process. It cleaves several target proteins and serves as one of the executioner caspases that implement apoptosis. Despite reports of caspase-independent apoptosis (Broker et al. 2005), caspase-3 has become the most widely accepted and most frequently measured apoptosis marker for HTS. 

The mitomycins are a family of aziridine-containing antibiotics isolated from Streptomyces lavendulae. One of these antibiotics, mitomycin C (MMC), is an antitumor antibiotic frequently used for the treatment of breast, lung, stomach, intestinal, testicular, cholioepithelial, seminal and oral carcinoma [18-20]. When human oral squamous cell carcinoma cell lines (HSC-2, HSC-4) were treated with MMC, the viable cell number was dose-dependently reduced (CC50 of MMC against HSC-2, HSC-3 and HSC-4 cells 3.5, 9.7 and 18 xM, respectively, determined 24 hours after treatment). The rapid decline of polyamines (measured at 3 hours after MMC treatment) was observed prior to the expression of early apoptosis markers such as the production of annexin-positive cells and caspase activation (Table 1) [21]. The interactions... 

The other factor that may determine the type of cell death is the chemical structure of inducing agents [14]. We have recently found that u,(>-unsaluraled ketones such as 4,4-dimethyl-2-cyclopenten-l-one, a-methy-lene-y-butyrolactone, 5,6-dihydro-2H-pyran-2-one [15], codeinone [16], and morphinone [17] and a-hydroxylketones such as 3,3,3-trifluoro-2-hydroxy-1-phenyl-1-propanone induced caspase-independent cell death [18], induced vacuolization or autophagosome formation engulfing organelles, but without induction of apoptosis markers. 

We found that IM5 induced different types of cell death in HSC-2, HSC-4, and HL-60 cells. IM5 induced apoptotic characteristic in HL-60 cells. IM5 also induced DNA fragmentation, but no clear-cut laddering pattern in HSC-2 cells. On the other hand, IM5 induced the formation of secondary lysosomes without induction of apoptosis markers in HSC-4 cells, even though HSC-2... 

Smith, K.J., Graham, J.S., Hamilton, T.A., Skelton, H.G., Petrali, J.P., Hurst, C.G. (1997h). Immunohistochemical smdies of basement membrane proteins and proliferation and apoptosis markers in sulfur mustard induced cutaneous lesions in weanling pigs. J. Dermatol. Sci. 15 173-82. 

A second group of apoptosis markers that connect to activation of neuron apoptosis are cellular events that mediate mitochondrial release of the pro-apoptotic proteins. Permeabilization of the outer mitochondrial membrane (MOMP) is central to the apoptosis process and it is likely that we are close to an understanding of the specific mechanisms involved. Basically, a plethora of pro-apoptotic stimuh set into play increased expression, protein-protein interactions, and subcellular relocahza-tions of members of Bcl-2 family of proteins (Gross et al, 1999 Ryter et al, 2007). The founder of this group of proteins is the Bcl-2 oncogene, with the group... 

This class of lymphocytes differentiates from immuno-logically incompetent hematopoietic stem cells of the bone marrow within the thymus - hence, the name thymus-dependent (T-) lymphocytes. Two major subclasses develop simultaneously, T-helper lymphocytes (Th) and cytotoxic effector lymphocytes (Tc). The cytotoxic T-lymphocytes (carrying on the surface the differentiation marker CD8) destroy cells, which cany their cognate antigen bound to MHC class I molecules on the surface by inducing apoptosis. From an evolutionary point of view Tc cells appear to have developed predominantly to cope with vims infections. As vituses can only replicate within cells, Tc eliminate them by destroying their producers. 

Kinter AL, Umscheid CA, Arthos J, et al. HIV envelope induces virus expression from resting CD4+ T cells isolated from HIV-infected individuals in the absence of markers of cellular activation or apoptosis. J Immunol 2003 170(5) 2449-2455. 

Fig. 9.8 Apoptosis of HEK293 cells induced by SWCNTs. Bl DNA electrophoresis of cells cultured with 25g/ml SWCNTs for 1-5 days, M molecular marker, no. 1-5 denote the results of cells cultured for day 1-5, respectively B2 DNA electrophoresis results of control cells cultured for day 1-5 C the cell cycle distribution of HEK293 cells cultured with 25 lg/ml SWCNTs for 4 days, the percentage of sub-Gl cells (apoptosis cells) was 43.5%. (Grunlan et al., 2000. With permission from Elsevier) (See Color Plates)...

A key feature of apoptosis is that, similar to the cell cycle, the duration of apoptosis is variable and the process is asynchronous in most cell populations. Consequently, there are variable proportions of cells in distinct phases of apoptosis and a sample, collected from a culture in which apoptosis has been induced, will contain cells in all the phases of apoptosis. This could represent a major technical problem in the investigation of apoptosis and requires measurement of metabolic apoptotic changes concomitantly with established markers of apoptosis progression. Using the same cellular model described above, we investigated metabolic alterations induced by X-rays using flow cytometry and a panel of fluorochromes... 

The cells are cultured and in vitro restimulated with antigen (here PT) for 12 h to mimic the encounter with the specific antigen in the body. Earlier studies have shown that an in vitro restimulation of mouse spleen lymphocytes with antigen for 4 and 24 h, can be used to study immune response as well as other cellular processes, such as apoptosis, inflammation, and so on (see Chapter 16). Our experiments, however, showed that immune response markers are expressed at later time-points as compared to mouse, and that 12 h of in vitro restimulation is the ideal time-point for the assessment of immune response genes (see Table 17.1) (see Note 1). 

H. Garewal, H. Bernstein, C. Bernstein, R. Sampliner and C. Payne, Reduced bile-acid-induced apoptosis in normal colorectal mucosa a potential biological marker for cancer risk, Cancer Res., 1996,56(7), 1480. 

In aminoglycoside-treated animals, the cells can be led to canonical apop-totic death through activation of caspases. Caspase-9 forms an apoptosome complex with cytochrome c and APAF-1 and leads to apoptosis through activation of caspase-3. Aminoglycosides activate caspases in auditory structures conversely, inhibition of caspase activity successfully blocks neomycin-induced vestibulotoxicity. In contrast, apoptotic markers were essentially absent in a mouse model of chronic kanamycin ototoxicity where death of auditory sensory cells ensued via cathepsins. The activation of cathepsin D was accompanied by the nuclear translocation of endonuclease G, necrotic cleavage of PARP, and activation of p,-calpain, all facets of necrotic cell death. 

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