Boc-D-FMK

The cyclin-dependent kinase inhibitor p27KIP1 is a crucial component of the mammalian restriction point, and as such is subject to multiple regulatory mechanisms. It has recently been shown that the abundance of p27KIP1 is also regulated during apoptosis; p27KIP1 is cleaved by a Z-VAD-fmk-sensitive caspase during apoptosis induced by growth factor deprivation in endothelial cells, and also following exposure of myeloid leukaemia cells to etoposide. Here, we investigate p27KIP1 regulation in B- and T-lymphoid cells undergoing apoptosis. We observe that p27KIP1 is down-regulated following exposure to a variety of apoptotic stimuli including an agonistic anti-Fas antibody, cycloheximide and etoposide. Further investigation revealed the existence of two different routes of p27KIP1 regulation in lymphoid cells undergoing apoptosis. The first pathway is utilized by lymphoid cells stimulated through Fas, is abrogated in a caspase-8-deficient T-cell line, and is blocked by the caspase inhibitors Z-VAD-fmk and Boc-D-fmk. In contrast, the loss of p27KIP1 in cells exposed to cycloheximide and etoposide occurs in the absence of caspase-8 or any Z-VAD-fmk- or Boc-D-fmk-sensitive caspase activities.

The role of p38 mitogen-activated protein kinase (MAPK) in apoptosis is a matter of debate. Here, we investigated the involvement of p38 MAPK in endothelial apoptosis induced by tumor necrosis factor alpha (TNF). We found that activation of p38 MAPK preceded activation of caspase-3, and the early phase of p38 MAPK stimulation did not depend on caspase activity, as shown by pretreatment with the caspase inhibitors z-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk) and Boc-Asp(OMe)-fluoromethylketone (BAF). The p38 MAPK inhibitor SB203580 significantly attenuated TNF-induced apoptosis in endothelial cells, suggesting that p38 MAPK is essential for apoptotic signaling. Furthermore, we observed a time-dependent increase in active p38 MAPK in the mitochondrial subfraction of cells exposed to TNF. Notably, the level of Bcl-x(L) protein was reduced in cells undergoing TNF-induced apoptosis, and this reduction was prevented by treatment with SB203580. Immunoprecipitation experiments revealed p38 MAPK-dependent serine-threonine phosphorylation of Bcl-x(L) in TNF-treated cells. Exposure to lactacystin prevented both the downregulation of Bcl-x(L) and activation of caspase-3. Taken together, our results suggest that TNF-induced p38 MAPK-mediated phosphorylation of Bcl-x(L) in endothelial cells leads to degradation of Bcl-x(L) in proteasomes and subsequent induction of apoptosis.

Caspases are implicated in apoptotic cell death after spinal cord injury (SCI), but the relative contribution of these proteases to the secondary injury process has been only partially described. We examined the activation of caspases 1, 2, 3, 6, 8, and 9 from 1 hr to 7 days after moderate contusion injury induced by a weight-drop method in the rat. Tissue homogenates from a 1-cm segment of cord that contained the site of impact were processed by fluorometric enzymatic activity assays and/or immunoblotting methods. Caspases 3, 8, and 9 were activated from 1 to 72 hr after injury, whereas caspases 1, 2, and 6 were not. Double-label immunohistochemistry utilizing antibodies for CNS cell-type-specific markers and active subunits of caspases 3, 8, or 9 showed that, at 4 and 72 hr after injury, these caspases were primarily activated in neurons and oligodendrocytes, rather than in astrocytes. Active caspase subunits were present in neurons within the necrotic lesion core at 4 hr after injury and in cells more than several segments away at 4 or 72 hr after injury. Intrathecal injection of the pan-caspase inhibitor Boc-Asp (OMe)-fluoromethylketone (Boc-d-fmk) at 15 min after injury improved locomotor function 21 and 28 days later.