Cycloheximide in a cell

Cycloheximide (CHX) can contribute to apoptotic processes, either in conjunction with another agent (e.g. tumor necrosis factor) or on its own. The apoptotic process is now known to involve the well orchestrated interactions of cell death receptors, death receptor adaptors, caspases, and Bcl-2 family members. Although a number of stimuli have been reported to result in the up-regulation of the Fas receptor and its ligand (e.g.UV, c-Myc, and certain chemotherapeutic drugs), there are many other stimuli for which the mechanism responsible for their action is still unknown. An example of the latter is the ability of cycloheximide (CHX) to either promote or inhibit apoptosis in divergent cell types and in response to varying death stimuli. A large body of evidence has shown that CHX can potentiate, and in some cases (e.g. TNFα stimulation and staurosporine) be necessary for, the apoptotic effects of certain death stimuli. The studies of Martin et al. and Tuschida et al. further indicated that CHX, independently of other stimuli, is also capable of promoting apoptosis in a number of transformed cell lines and normal cells. Jacobsonet al. has shown that staurosporine- and staurosporine/cycloheximide-induced death is mediated by a caspase-3-like activity that is blocked by Z-VAD-FMK, a synthetic tripeptide inhibitor that demonstrates broad caspase specificity. More recently, Woo et al. demonstrated that bone marrow neutrophils from caspase-3−/− mice no longer undergo CHX-induced apoptosis, indicating that caspase-3 expression is most likely required for this type of cell death.

Chemical structure of cycloheximide

The ability of CHX to induce cell death varies considerably from one cell line to another, suggesting that the continuous synthesis of a regulatory protein that blocks apoptosis is required for the normal growth of these CHX-sensitive cell lines. Sensitivity to CHX is not necessarily determined by cell type alone since cell lines from the same tissue and stage of development (e.g. Jurkat and CEM C7 T-cells) can be affected in very different ways. Furthermore, although cell death triggered by cell-surface receptors (e.g. Fas, DR3, and TNF receptor-1) requires an adaptor protein such as FADD to promote an apoptotic signal, cell death triggered by other stimuli (e.g. E1A, c-Myc, and Adriamycin) may not. Therefore, it is of interest to determine the basis of the cellular differences that result in cellular sensitivity or insensitivity to agents like CHX as well as the mechanism of CHX-induced apoptosis.

The transformed human T-cell lines Jurkat and CEM C7 are representative of a similar T-cell developmental stage, and they are equally sensitive to agonistic anti-Fas mAbs and TNFα. However, they exhibit disparate responses when exposed to CHX. We therefore set out to determine which apoptotic signaling pathway components are involved in CHX-induced Jurkat cell death as well as the basis for the CEM C7 cellular resistance to CHX. Here we demonstrate that disruption of normal FADD function inhibits apoptotic signaling in these cells. This may indicate that in addition to its role as an adaptor that links TNF-related receptors to caspase activation, FADD mediates certain apoptotic signals through a receptor-independent pathway(s). Finally, we demonstrate that FADD and caspase-8 (FLICE) coalesce into what appear to be perinuclear death effector filaments (DEFs) in wild-type Jurkat, Jurkat-FADD-DN, and CEM C7 cells treated with CHX, even though only the wild-type Jurkat cells apoptose. These results suggest that the redistribution of FADD and caspase-8 into these filament structures is necessary, but not sufficient, for cell death to occur.

Sources: Damu Tang,  M. Lahti, Jose Grenet and Vincent J. Kidd


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