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Cisplatin is one of the major chemotherapeutic weapons used against different human cancers, although its mechanism to induce apoptosis is not fully understood. The presence of wild type p53 has been suggested to be important for cisplatin cytotoxicity, hence we found that cisplatin induced apoptosis in cell lines with functional p53. Using the HCT116 colon carcinoma derived cell line we have established that the apoptotic activity of cisplatin requires the onset of a p53-mediated p38alpha MAPK pathway through generation of reactive oxygen species (ROS). HCT116 p53-deficient cells were much less sensitive to apoptosis by cisplatin than their p53wt counterparts, where apoptosis was strongly inhibited by antioxidants. Moreover, the presence of pifithrin-alpha, an inhibitor of p53 transcriptional activity, blocked cisplatin-induced apoptosis, reduced the generation of ROS produced upon cisplatin treatment. In addition, we have identified p38alpha as the isoform necessary for cisplatin-induced apoptosis, upon activation by p53-mediated ROS production. p38alpha MAPK contributes to further activation of p53, which leads to a positive feedback loop, p38alpha MAPK/p53. We conclude that the p53/ROS/p38alpha MAPK cascade is essential for cisplatin-induced cell death in HCT116 cells and the subsequent p38alpha/p53 positive feedback loop strongly enhances the initial p53 activation.

p38␣ mitogen-activated protein (MAP) kinase is a broadly expressed signaling molecule that participates in the regulation of cellular responses to stress as well as in the control of proliferation and survival of many cell types. We have used cell lines derived from p38␣ knockout mice to study the role of this signaling pathway in the regulation of apoptosis. Here, we show that cardiomyocytes and fibroblasts lacking p38␣ are more resistant to apoptosis induced by different stimuli. The reduced apoptosis of p38␣-deficient cells correlates with decreased expression of the mitochondrial proapoptotic protein Bax and the apoptosis-inducing receptor Fas/CD-95. Cells lacking p38␣ also have increased extracellular signalregulated kinase (ERKs) MAP kinase activity, and the up-regulation of this survival pathway seems to be at least partially responsible for the reduced levels of apoptosis in the absence of p38␣. Phosphorylation of the transcription factor STAT3 on Ser-727, mediated by the extracellular signal-regulated kinase MAP kinase pathway, may contribute to the decrease in both Bax and Fas expression in p38␣؊/؊ cells. Thus, p38␣ seems to sensitize cells to apoptosis via both up-regulation of proapoptotic proteins and down-regulation of survival pathways. INTRODUCTIONThe family of p38 mitogen-activated protein kinases (MAPKs) are strongly activated by stress and inflammatory cytokines, but nonstressful stimuli can also activate p38 MAPKs, leading to the regulation of cellular functions such as proliferation, differentiation, and survival. Four different p38 MAPK family members have been identified, p38␣, ␤, ␥, and ␦, also known as stress-activated kinase (SAPK)2a, SAPK2b, SAPK3, and SAPK4, respectively, which may have both overlapping and specific functions (reviewed by Cohen, 1997;Nebreda and Porras, 2000;Ono and Han, 2000;Kyriakis and Avruch, 2001). p38␣ is broadly expressed and is also the most abundant p38 family member present in most cell types. Targeted inactivation of the mouse p38␣ gene results in embryonic death due to a placental defect (Adams et al., 2000;Mudgett et al., 2000;Tamura et al., 2000). Studies using cell lines have suggested an important role for p38 MAPKs in the regulation of cardiomyocyte differentiation, hypertrophy, and apoptosis (Wang et al., 1998;Zechner et al., 1998;Mackay and Mochly-Rosen, 1999;Saurin et al., 2000; Stephanou et al., 2000). However, p38␣ does not seem to play a critical role in the development of the embryonic heart in mice (Adams et al., 2000), although heterozygous p38␣ϩ/Ϫ mice have been reported to be less sensitive to myocardial cell death caused by ischemia-reperfusion (Otsu et al., 2003). In addition, cardiac-specific expression of dominant negative forms of p38␣ and their activators MKK3 and MKK6 enhances cardiac hypertrophy in transgenic mice and/or makes the mice resistant to cardiac fibrosis (Braz et al., 2003;Zhang et al., 2003b).The role of p38 MAPKs in apoptosis depends on the cell type and the stimuli (reviewed by Nebreda and Porras, 2000). Most of the d...

p38alpha MAP kinase is activated in response to many cellular stresses and also regulates the differentiation and/or survival of various cell types in vitro, including skeletal muscle cells and cardiomyocytes. Here we show that targeted inactivation of the mouse p38alpha gene results in embryonic lethality at midgestation correlating with a massive reduction of the myocardium and malformation of blood vessels in the head region. However, this defect appears to be secondary to insufficient oxygen and nutrient transfer across the placenta. When the placental defect was rescued, p38alpha(-/-) embryos developed to term and were normal in appearance. Our results indicate that p38alpha is required for placental organogenesis but is not essential for other aspects of mammalian embryonic development.

Notch, a transmembrane receptor member of the homeotic epidermal growth factor-like family of proteins, participates in cell-to-cell signaling to control cell fate during development. Activated Notch-1 constructs lacking the extracellular region prevent differentiation of several mammalian cells in vitro. This effect, however, bypasses the normal mechanisms of cell-to-cell interactions in which Notch-1 participates. We investigated the role of Notch-1 in the hormone-induced adipocyte differentiation of 3T3-L1 fibroblasts, a paradigmatic model of adipogenesis that requires cell-to-cell contact. Unlike other differentiation models, Notch-1 expression and function were necessary conditions for adipogenesis. Impaired Notch-1 expression by antisense Notch-1 constructs prevented adipocyte differentiation. Strategies aimed at blocking putative Notch/ligand interactions also blocked adipogenesis, implicating Notch as a critical molecule in cell-to-cell signaling necessary for differentiation. Inhibition of Notch-1 expression or function decreased the expression of peroxisomal proliferatoractivated receptors ␦ and ␥, transcription factors that control adipocyte differentiation and that are up-regulated at cell confluence. These results implicate Notch in the commitment of 3T3-L1 cells to undergo adipogenesis by controlling the expression of the principal regulators of this process.

Mammalian 3T3-L1 cells differentiate into adipocytes after continuous exposure to pharmacological doses of insulin or physiological doses of insulin-like growth factor I (IGF-1). Expression of transfected ras oncogenes led to differentiation of these cells into adipocytes in the absence of externally added insulin or IGF-I. Cells transfected with normal ras genes or the tyrosine kinase trk oncogene did not differentiate. Transfection with a dominant inhibitory ras mutant resulted in inhibition of differentiation. Exposure of untransfected 3T3-L1 cells to insulin stimulated formation of the active Ras.GTP complex. These observations indicate that Ras proteins participate in signal transduction pathways initiated by insulin and IGF-I in these cells.

In 3T3-L1 fibroblasts, Ras proteins mediate both insulin-induced differentiation to adipocytes and its activation of cytosolic serine/threonine kinases, including Raf-1 kinase, mitogen-activated protein kinase (MAPK), and Rsk. Here, we report that insulin-and Ras-induced activation of MAPK is not required for the differentiation process and in fact antagonizes it. The treatment of 3T3-L1 preadipocytes with MEK-specific inhibitor PD98059 blocked insulin-and Ras-induced MAPK activation but had no effect on or slightly enhanced adipocytic differentiation. Tumor necrosis factor alpha (TNF-␣), an inhibitor of insulin-stimulated adipogenesis, activated MAPK in 3T3-L1 cells. PD98059 treatment blocked MAPK activation by TNF-␣ and reversed the blockade of adipogenesis mediated by low (1 ng/ml) TNF-␣ concentrations. 3T3-L1 transfectants containing hyperactivated MEK1 or overexpressed MAPK displayed impaired adipocytic differentiation. PD98059 treatment also reversed the blockade of differentiation in MEK1 transfectants. These results indicate that MAPK does not promote but can contribute to inhibition of the process of adipocytic differentiation of 3T3-L1 cells.Adipocytic differentiation is a complex process regulated by many hormones, growth factors, and cytokines. Whereas some of those signals stimulate differentiation to adipocytes, others inhibit this event (13,20), presumably based on the ability to ultimately alter gene expression. Preadipocytic cell lines, such as 3T3-L1, undergo differentiation after continuous exposure to pharmacological doses of insulin or physiologic doses of insulin-like growth factor 1 and are additionally induced by glucocorticoids and fatty acids (27,37,41,43). In contrast, tumor necrosis factor alpha (TNF-␣), a cytokine secreted by macrophages and adipose tissue, inhibits adipocytic differentiation (44, 45). However, the mechanisms that mediate the actions of insulin and other ligands on differentiation have yet to be clearly defined.We demonstrated previously that overexpression of Ras induces adipocytic differentiation of 3T3-L1 cells (3, 34), implicating Ras proteins as obligatory signaling intermediates in insulin-stimulated differentiation pathways. Our prior studies also showed that Ras proteins are necessary and sufficient for insulin activation of the cytosolic kinases Raf-1, mitogen-activated protein kinase (MAPK), and Rsk (35, 36); furthermore, Raf-1 participates downstream of Ras in the signaling cascade resulting in adipocytic differentiation. However, in contrast to its role in proliferating cells (39, 51, 54), Raf-1 kinase activation in differentiating 3T3-L1 cells is completely dissociated from activation of MAPK by insulin (35,36), suggesting that at least two separate signals emerge from Ras after insulin stimulation, regulating either growth or differentiation depending on the state of the cell or the presence of other environmental factors. Dissociation between Raf-1 kinase and MAPK has also previously been described for other cell types (15,39,(55)(56)(57).To further de...

Background: p38␣ MAPK is activated by stress stimuli, which can regulate cell death. Results: In response to H 2 O 2 , p38␣ MAPK increases SOD and catalase levels, impairs ROS accumulation, and leads to cell survival. Conclusion: p38␣ MAPK signals survival under moderate oxidative stress through up-regulation of antioxidant defenses. Significance: To know how p38␣ regulates ROS levels is important for cell homeostasis.