These total results suggest a good regulation of MDM2 getting together with p53

These total results suggest a good regulation of MDM2 getting together with p53. a solid induction of apoptosis in -resistant and cisplatin-sensitive TC cells. Suppression of wild-type p53 induced level of resistance to Nutlin-3 in TC cells, demonstrating the key role of p53 for Nutlin-3 sensitivity. More specifically, our results indicate that p53-dependent induction of Fas membrane expression (threefold) and enhanced Fas/FasL interactions at VHL the cell surface are important mechanisms of Nutlin-3-induced apoptosis in TC cells. Importantly, an analogous Fas-dependent mechanism of apoptosis upon Nutlin-3 treatment is usually executed in wild-type p53 expressing Hodgkin lymphoma and acute myeloid leukaemia cell lines. Finally, we demonstrate that Nutlin-3 strongly augmented cisplatin-induced apoptosis and cell kill via the Fas Medroxyprogesterone death receptor pathway. This effect is usually most pronounced in cisplatin-resistant TC cells. as well as genes that induce cell-cycle arrest, such as cyclin-dependent kinase inhibitor 1A gene mutations are found and wild-type p53 is usually expressed at Medroxyprogesterone high levels in the majority of TCs.9 Despite the increasing knowledge about p53 as Medroxyprogesterone a transactivator and cellular gatekeeper for cell growth and division, the effects of wild-type p53 (and mutated p53) on drug sensitivity of human tumours including TC are still not clear. We have previously shown that this response to cisplatin-induced DNA damage in TC cell lines is related to an induction of p53 expression and activation of the Fas death receptor pathway.2, 9 Several other studies have reported the effect of wild-type p53 expression on chemo-sensitivity of human TC cell lines with contrasting and sometimes conflicting results.3, 10, 11, 12, 13, 14, 15 Tumours that retain wild-type p53 are supposed to have other defects in the p53 pathway, such as the presence of microRNA (miR)-371-373, miR-106b-seed-family members or cytoplasmic p21, the lack of phosphatase and tensin homologue (PTEN) expression or the increased mouse double minute 2 (MDM2) expression.16, 17, 18, 19 MDM2, as transcriptional target of p53, is the main negative feedback regulator of p53. By binding to the transactivation domain name of p53, MDM2 is able to regulate p53 activity and stability via several mechanisms such as promoting p53 degradation through ubiquitination, stimulating p53 nuclear export, and inhibiting acetylation of p53.7 Interfering in the MDM2Cp53 interaction, with small molecules like RITA and Nutlin-3, provides an attractive strategy for (re)activating wild-type p53 in a non-genotoxic way. This (re)activation leads to cell-cycle arrest and or apoptosis in tumour cells with wild-type p53.20, 21, 22, 23 Restoration of p53 function by Nutlin-3 may thus have profound therapeutic effect on tumours that have retained wild-type p53, particularly if MDM2 activity is disproportionally increased.23 Recently, Nutlin-3-induced apoptosis was investigated in a small panel of TC cell lines, and only additive effects were seen in combination with cisplatin. However, no mechanistic insights in Nutlin-3-induced apoptosis were offered.24, 25 In this study, we explore the potential of disrupting the MDM2Cp53 conversation as a mean to activate p53 in TC. The role of p53 and MDM2 in cisplatin-induced apoptosis has been investigated using cisplatin-sensitive and -resistant human TC models. Finally, the importance of the Fas death receptor pathway in Nutlin-3 induced apoptosis has been studied. Results P53 and MDM2 cellular localisation and cisplatin response in TC Cells In the present study, we have used a panel of cisplatin-sensitive and -resistant wild-type p53 expressing TC cell lines to compare cisplatin responses (Table 1) with the cellular localisation of p53 and MDM2, and MDM2-p53 complex formation (Figures 1aCc, Supplementary Physique 1). With immunofluorescence, we found that p53 is usually predominantly localised to the cytoplasm, while MDM2 was mainly present Medroxyprogesterone in the nucleus in all four cell lines (Physique 1a and Supplementary Physique 1). After exposure of cells to 8?expression levels and lower levels of Oct4 and miR-106b family members, high levels of cytoplasmic p21, which is a key determinant of resistance to cisplatin-induced apoptosis.19 Cisplatin-induced apoptosis in TC cells also involves activation of the Fas death receptor pathway via elevated Fas membrane expression. High cytoplasmic p21 levels inhibit Fas death receptor-mediated.