p53 includes a major role in the response and execution of apoptosis after chemotherapy in many cancers. augmented cisplatin-induced apoptosis and cell kill via the Fas 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 high levels in the majority of TCs.9 Despite the increasing knowledge about p53 as 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 maintain 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 opinions 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 MDM2-p53 interaction with small SAR131675 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 SAR131675 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 MDM2-p53 interaction as a mean to trigger 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 1a-c Supplementary Physique 1). With immunofluorescence we found that p53 is usually predominantly localised to the cytoplasm while MDM2 was mainly present in the nucleus in all four cell lines (Physique 1a and Supplementary Physique 1). After exposure of cells to 8?mutations in TC has led to the hypothesis that constitutively expressed p53 Rabbit Polyclonal to HXK1. is functionally inactive. 31 Surprisingly SAR131675 high levels of wild-type p53 have been frequently observed in TC. These levels correlate with SAR131675 expression levels of the p53 transcriptional target MDM2 suggesting that p53 is usually functional in TC.11 13 17 In this study we show that treatment with the selective MDM2 antagonist Nutlin-3 causes a high induction of both p53 and MDM2 a massive induction of..