Involvement of p38 in Apoptosis-associated Membrane Blebbing and Nuclear Condensation

Abstract

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 ...

c-Myc potentiates the mitochondrial pathway of apoptosis by acting upstream of apoptosis signal-regulating kinase 1 (Ask1) in the p38 signalling cascade

“…Similar results were obtained with other anticancer drugs, such as doxorubicin or daunorubicin, suggesting that this effect is not restricted to cisplatin (Figures 1B and 1C). At the concentration of SB203580 used in the present study, the in vitro phosphorylation of heat-shock protein 27 ('Hsp27'), a downstream target of p38 activity, was totally inhibited [21]. Higher concentrations of SB203580 were no more efficient in blocking apoptosis (results not shown).…”

“…We reported recently [21] that activation of p38 by many of these toxic agents was also dependent on the deregulated expression of c-Myc and played an important role in the pro-apoptotic effect of c-Myc. In the present study, we examined further the interplay between c-Myc-dependent apoptosis and p38 activation using c-Myc-transformed Rat-1 cells exposed to cisplatin and other anticancer agents.…”

c-Fos Is a Mediator of the c-Myc-induced Apoptotic Signaling in Serum-deprived Hepatoma Cells via the p38 Mitogen-activated Protein Kinase Pathway

“…7C). Activation of p38 in the rat fibroblasts is known to be associated with Myc-dependent apoptosis when exposed to cisplatin (34). Furthermore, a significantly lowered activity of p38 and MKK6 has been reported in hepatocellular carcinoma compared with normal tissue, suggesting the importance of this pathway in controlling unrestricted cellular proliferation (65).…”

“…It could also involve release of cytochrome c (30), loss of mitochondrial potential (31), down-regulation of survival signals such as Bcl-2 (32) and nuclear factor-B in tumor necrosis factor-␣-mediated apoptosis (33), and up-regulation of p38 in cisplatin-induced apoptosis (34). Although the involvement of immediate early response gene c-fos is well documented in a variety of apoptotic stress stimuli leading to apoptosis (35)(36)(37), its involvement in the c-Myc-mediated apoptosis is not known.…”

Cisplatin-induced activation of the EGF receptor

“…Recent studies have documented that CDDP triggers the activation of the JNK and p38 mitogenactivated protein kinase (MAPK) cascades in tumor cells or transformed cell lines (Benhar et al, 2001;Deschesnes et al, 2001;Gebauer et al, 2000;Pandey et al, 1996). Activation of JNK or p38 by CDDP was shown to promote apoptotic cell death (Benhar et al, 2001;Deschesnes et al, 2001).…”