Caspase cell culture

Sauerwald TM, Betenbaugh MJ, Oyler GA (2002), Inhibiting apoptosis in mammalian cell culture using the caspase inhibitor XIAP and deletion mutants, Biotechnol. Bioeng. 77 704-716. 

Sauerwald TM, Oyler GA, Betenbaugh MJ (2003), Study of caspase inhibitors for limiting death in mammalian cell culture, Biotechnol. Bioeng. 81 329-340. 

Caspase activation can occur downstream of cytochrome c release from mitochondria or after cell-surface death receptors are engaged. Apoptosis-inducing ligands can directly activate caspases by recruiting caspase-8 or caspase-10 to protein complexes formed on the cytoplasmic portions of the receptors (20). ELIS As for quantification of specific active caspases in cultured cells are used to quantify these events. By using the capture of the large subunit that contains the active site cysteine modified with the biotinylated inhibitor as an indicator of the active caspase state, it is now possible to specifically quantify active caspase-3 or caspase-7 without interference by other active caspases. 

Caspases are intracellular proteases and cleave their substrate proteins specifically behind an aspartate residue. Caspases are normally present as inactive proenzymes. However, during apoptosis, caspase activation is induced by cleavage at specific internal aspartate residues. Activation of the initiator caspases by pro-apoptotic signals leads to proteolytic activation of the execution caspases, which cleave a set of vital proteins and thus initiate and execute the apoptotic degradation phase. A self-contained on-chip cell culture and pretreatment microdevice system has been developed for the screening of caspase-3 expression in apoptotic cells. 

Apoptosis can be triggered by mitochondrial damage, which is followed by the release of cytochrome c and the caspase cascade [167], The BAX protein activates mitochondrial release of cytochrome c [168], while Bcl-2 is a mitochondrial protein inhibits the apoptotic process and promotes cell survival [169]. Ji et al. [163] research showed that occur a decrease in mitochondrial Bcl-2 and a markedly increase in mitochondrial level of Bax in the HepG2 cells treated with PA from 200 to 400 mM concentrations. The authors suggest that this mechanism can contribute to NASH and NAFLD installation, and especially may play an important role in the transition from steatosis to steatohepatitis in human. Other studies [170] on the effect of FAs-induced steatosis on cellular apoptosis have demonstrated that palmitic and oleic FA mixtures-induced steatosis is associated with apoptosis in hepatocyte cell cultures. Joshi-Barve s et al. [171] studies also showed that exposure to excess palmitic acid induces apoptosis and lL-8 production in hepatocytes in a relation of dose-dependent and time dependent manner, via activation of c-Jun amino terminal kinase (JNK/AP-1), and nuclear factor kappa B (NF-B) transcription factors for IL-8 expression. 

The release of cytochrome c from mitochondria to the cytosol is thought to be a key event for signal transduction in the apoptotic process. It has been suggested that it induces a series of biochemical reactions that result in caspase activation and subsequent cell death. Tamaki et al. [12] have developed a microchip-based system for direct monitoring of cytochrome c distribution during the apoptosis process. This system incorporated microscale cell culture, chemical stimulation, and a scanning-thermal-... 

Activated MAPKs have been found to modulate mitochondria-mediated apoptotic pathways, JNK in particular [71]. In fact, it appears that MAPK signaling may represent a potential cross-link between die different apoptotic pathways. JNK activation is not required for death-receptor-mediated apoptosis but is required for caspase-9 activation by the mitochondrial pathway, induced by a variety of proapoptotic stimuli [72,73]. JNK activation and c-Jun phosphorylation were found to be necessary to promote cytochrome c release from mitochondria, with the sequential assembly of the apoptosome and caspase-3 activation. The molecular mechanism of this effect was unclear, but it appeared to involve regulation of the expression and phosphorylation state of the Bcl-2 protein family and their recruitment to the mitochondrial outer membrane (Fig. 4). In a number of apoptotic models, JNK activation was associated with a downregulation of the antiapoptotic Bcl-2 and Bc1-Xl and upregulation of the proapoptotic Bax and Bad [74—77]. Two cell culture studies provided very strong evidence that JNK activation resulted in the phosphorylation of Bcl-2 and Bc1-Xl ex vivo, with the induction of apoptosis [76,77]. In other words, Bcl-2 and Bc1-Xl appear to be substrates of JNK, and phosphorylation results in their inactivation, thereby abolishing their ability to prevent cytochrome c release. 

---