In endothelial cells, H2O2 induces the rapid formation of focal adhesion complexes at the ventral face of the cells and a major reorganization of the actin cytoskeleton into dense transcytoplasmic stress fibers. This change in actin dynamics results from the activation of the mitogen-activated protein (MAP) kinase stress-activated protein kinase-2/p38 (SAPK2/p38), which, via MAP kinase-activated protein (MAPKAP) kinase-2/3, leads to the phosphorylation of the actin polymerization modulator heat shock protein of 27 kD (HSP27). Here we show that the concomitant activation of the extracellular signal-regulated kinase (ERK) MAP kinase pathway by H2O2 accomplishes an essential survival function during this process. When the activation of ERK was blocked with PD098059, the focal adhesion complexes formed under the plasma membrane, and the actin polymerization activity led to a rapid and intense membrane blebbing. The blebs were delimited by a thin F-actin ring and contained enhanced levels of HSP27. Later, the cells displayed hallmarks of apoptosis, such as DEVD protease activities and internucleosomal DNA fragmentation. Bleb formation but not apoptosis was blocked by extremely low concentrations of the actin polymerization inhibitor cytochalasin D or by the SAPK2 inhibitor SB203580, indicating that the two processes are not in the same linear cascade. The role of HSP27 in mediating membrane blebbing was assessed in fibroblastic cells. In control fibroblasts expressing a low level of endogenous HSP27 or in fibroblasts expressing a high level of a nonphosphorylatable HSP27, H2O2 did not induce F-actin accumulation, nor did it generate membrane blebbing activity in the presence or absence of PD098059. In contrast, in fibroblasts that expressed wild-type HSP27 to a level similar to that found in endothelial cells, H2O2 induced accumulation of F-actin and caused bleb formation when the ERK pathway was inhibited. Cis-platinum, which activated SAPK2 but induced little ERK activity, also induced membrane blebbing that was dependent on the expression of HSP27. In these cells, membrane blebbing was not followed by caspase activation or DNA fragmentation. We conclude that the HSP27-dependent actin polymerization–generating activity of SAPK2 associated with a misassembly of the focal adhesions is responsible for induction of membrane blebbing by stressing agents.

The stress-activated protein kinase p38 is often induced by cytotoxic agents, but its contribution to cell death is ill defined. In Rat-1 cells, we found a strong correlation between activation of p38 and induction of c-Myc-dependent apoptosis. In cells with deregulated c-Myc expression but not in control cells, cis-diamminedichloroplatinum induced p38 activity and typical features of apoptosis, including internucleosomal DNA degradation, induction of caspase activities, and both nuclear (nuclear condensation and fragmentation) and extranuclear (cell blebbing) morphological alterations. The pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone did not block p38 activation and the p38 inhibitor SB203580 had no detectable effect on the activation of caspases or the in vivo cleavage of several caspase substrates, suggesting that p38 and caspase activation can contribute distinct features of apoptosis. Accordingly, we found that cell blebbing was independent of caspase activity and, rather, depended on p38-sensitive changes in microfilament dynamics likely mediated by heat shock protein 27 phosphorylation. Furthermore, p38 activity contributed to both caspase-dependent and caspase-independent nuclear condensation and fragmentation, suggesting a role in an early event triggering both mechanisms of apoptosis or sensitizing the cells to the action of both types of apoptosis executioners. Inhibiting p38 also resulted in a significant enhancement in cell survival estimated by colony formation. This capacity to modulate the sensitivity to apoptosis in cells with deregulated c-Myc expression suggests an important role for p38 in tumor cell killing by chemotherapeutic agents. INTRODUCTIONApoptosis is an active form of cell death that plays an essential role in physiological and pathological conditions throughout the development and adult life of multicellular organisms, eliminating damaged cells or cells with defects in key-regulated processes such as growth (Kerr et al., 1972;Wyllie et al., 1980;Ellis and Horvitz, 1986). Not surprisingly, several tumors emerge with mutations in genes conferring apoptosis resistance (Kerr et al., 1994;Reed, 1999) allowing them to continue uncontrolled growth under conditions that would be proapoptotic to normal cells. Resistance to apoptosis may also contribute to drug resistance, inasmuch as several anticancer drugs induce apoptosis (Kaufmann and Earnshaw, 2000).Apoptosis is highly regulated. At the morphological level, it is characterized by membrane blebbing, cell shrinkage, chromatin condensation, nuclear/cytoplasmic fragmentation, and formation of dense bodies that are quickly removed via phagocytosis by neighboring cells. Apoptosis can be induced through receptor-mediated mechanisms and as a consequence of stresses, such as growth factor withdrawal (Frisch and Francis, 1994;Xia et al., 1995) or exposures to cytotoxic drugs (Hale et al., 1996;Muschel et al., 1998). Once triggered, the apoptotic program involves activation of a series of biochemical events comprising ...

Microtubule disruption provokes cytoskeleton and cell adhesion changes whose importance for apoptosis induction remains unclear. The present study focuses on the functional and the molecular adhesion kinetics that are induced by microtubule disruption-mediated apoptosis. We showed that antimicrotubules induce a biphasic sequence of adhesion response that precedes the onset of apoptosis and focal adhesion kinase hydrolysis. Antimicrotubules first induced an increase of the cellular adhesion paralleled by the raise of focal adhesion sites and actin contractility, which was followed by a sharp decrease of cell adhesion and disorganization of focal adhesion and actin stress fibers. The latter sequence of events ends by cell rounding, detachment from the extracellular matrix, and cell death. Microtubule-disrupting agents induced a sustained paxillin phosphorylation, before the activation of apoptosis, that requires the prior activation of extracellular signal-regulated kinase and p38 but not c-Jun NH 2 -terminal kinase. Interestingly, integrin-linked kinase overexpression rescued the antimicrotubule-mediated loss of cell viability. Altogether, these results propound that antimicrotubule agents induce anoikis through the loss of focal adhesion structure integrity.Focal adhesions (FAs) are specialized complexes of structural and signaling proteins that assemble or disassemble as cell migrate or enter into mitosis. They form, following the integrin-mediated cell attachment to extracellular matrices (ECM), anchoring actin filaments and microtubules (MTs) to the membrane of the cell (Mitra et al., 2005). One of the cornerstone proteins of FA architecture is paxillin (Brown and Turner, 2004). Paxillin connects integrins to the actin cytoskeleton via vinculin and talin (Brown and Turner, 2004) and focal adhesion kinase (FAK), the integrin-linked kinase (ILK) (Nikolopoulos and Turner, 2001). After integrin ligation, FAK undergoes tyrosine autophosphorylation, which triggers interactions with Src-family tyrosine kinases for its maximal activation. The FAK-Src complex further activates several signaling pathways, notably the prosurvival phosphatidylinositol 3-kinase/Akt pathway (Khwaja et al., 1998)