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.

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IntroductionDendritic cells (DC) are professional antigen-presenting cells that are critically involved in the initiation of T cell-dependent immune responses as a consequence of their high expression of major histocompatibility complex (MHC) and costimulatory molecules. 1 DC are sparsely distributed throughout the body and, in most tissues, are present in an immature state, showing a high capacity for antigen uptake and processing but unable to stimulate T cells. 1,2 Once activated by inflammatory stimuli or infectious agents, DC undergo a maturation process whose hallmarks are up-regulated expression of costimulatory (CD40, CD80, and CD86) and adhesion (CD54 and CD58) molecules, migration into lymphoid organs, and subsequent acquisition of the capacity to activate quiescent, naïve, and memory lymphocytes. [1][2][3][4] In vitro, DC can be derived from either precursor cells or peripheral blood monocytes 5-8 when the appropriate cytokine signals are provided. Immature monocytederived DC (MDDC) can be obtained from peripheral blood monocytes in the presence of granulocyte-macrophage colonystimulating factor (GM-CSF) and interleukin-4 (IL-4). Addition of lipopolysaccharide (LPS) or tumor necrosis factor ␣ (TNF-␣) leads to the appearance of MDDC with all the morphologic, phenotypic, and functional characteristics of mature DC, 5,6 including de novo expression of CD83, 9 up-regulated expression of adhesion and costimulatory molecules, 1-4,10 loss of mannose-receptor-mediated endocytosis, synthesis and release of IL-12, and enhanced antigen presentation capacity. 11 Thus, in vitro maturation of MDDC represents a useful system for analyzing the molecular and functional changes that take place during acquisition of optimal T-cell-stimulating activity by DC.At least 3 distinct mitogen-activated protein kinase (MAPK) signaling cascades exist in mammals, including the extracellular signal-regulated kinase (ERK), the c-Jun N-terminal kinase (JNK), and the p38 MAPK pathways. 12 These kinases are activated by phosphorylation by distinct upstream MAPK kinases. The ERK signaling cascade regulates cell proliferation and differentiation in response to mitogens and growth factors, whereas the JNK and p38 MAPK pathways are preferentially activated by stress-inducing agents. 12 The availability of specific inhibitors for the ERK and p38 MAPK pathways allows evaluation of their respective involvement in cellular responses to extracellular stimuli. The ERK pathway inhibitors PD98059 13 and U0126 14 prevent activation of mitogenactivated protein kinase kinase (MEK) 1/2, 15 upstream activators of ERK 1/2, whereas the pyridinyl imidazole SB203580 inhibits p38 MAPK activity. 15,16 The intracellular signaling pathways implicated in maturation of MDDC are just beginning to be explored. TNF-␣ stimulation of immature MDDC initiates activation of several MAPKs, including For personal use only. on May 11, 2018. by guest www.bloodjournal.org From ERK 2, stress-activated protein kinase-JNK, and p38 MAPK. 10,17,18 Several reports have suggested t...

DNA repair by homologous recombination is involved in maintaining genome stability. Previous data report that wild-type p53 suppresses homologous recombination and physically interacts with Rad51. Here, we show the in vivo binding of wild-type p53 to a p53 response element in the promoter of Rad51 and the downregulation of Rad51 messenger RNA and protein by wild-type p53, favoured by DNA damage. Moreover, wild-type p53 inhibits Rad51 foci formation in response to double-strand breaks, whereas p53 contact mutant R280K fails to repress Rad51 mRNA and protein expression and Rad51 foci formation. We propose that transcriptional repression of Rad51 by p53 participates in regulating homologous recombination, and impaired Rad51 repression by p53 mutants may contribute to malignant transformation.

The gene PTTG1 (encoding the pituitary tumor-transforming 1 protein) is overexpressed in several different tumor types, is tumorigenic in vivo and shows transcriptional activity. The PTTG1 protein is cell-cycle regulated and was identified as the human securin (a category of proteins involved in the regulation of sister-chromatid separation) on the basis of biochemical similarities with the Pds1p protein of budding yeast and the Cut2p protein of fission yeast. To unravel the function of human securin in oncogenesis, we carried out a phage-display screening to identify proteins that interact with securin. Notably, we isolated the p53 tumor suppressor. Pull-down and co-immunoprecipitation assays demonstrated that p53 interacts specifically with securin both in vitro and in vivo. This interaction blocks the specific binding of p53 to DNA and inhibits its transcriptional activity. Securin also inhibits the ability of p53 to induce cell death. Moreover, we observed that transfection of H1299 cells with securin induced an accumulation of G2 cells that compensated for the loss of G2 cells caused by transfection with p53. We demonstrated the physiological relevance of this interaction in PTTG1-deficient human tumor cells (PTTG1(-/-)): both apoptotic and transactivating functions of p53 were potentiated in these cells compared to parental cells. We propose that the oncogenic effect of increased expression of securin may result from modulation of p53 functions.

Quantitative PCR technology may have a major role in the near future as an adjunctive diagnostic tool in both epidemiological and clinical studies in periodontology. However, culture techniques still hold some inherent capabilities, which makes this diagnostic tool the current reference standard in periodontal microbiology.

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These data suggest that not only IL1beta, IFNgamma, and TNFalpha but also IL10, IL12, and IL17 are involved in the OA-TMJ pathogenesis. Furthermore, an inflammatory response characterised by the predominant expression of IL12 mRNA and down-regulated expression of IL10 mRNA is associated with the degenerative changes observed in OA-TMJ.