Abstract
Wild-type p53 has a major role in the response and execution of apoptosis after chemotherapy in many cancers. Although high levels of wild-type p53 and hardly any TP53 mutations are found in testicular cancer (TC), chemotherapy resistance is still observed in a significant subgroup of TC patients. In the present study, we demonstrate that p53 resides in a complex with MDM2 at higher cisplatin concentrations in cisplatin-resistant human TC cells compared with cisplatin-sensitive TC cells. Inhibition of the MDM2–p53 interaction using either Nutlin-3 or MDM2 RNA interference resulted in hyperactivation of the p53 pathway and a strong induction of apoptosis in cisplatin-sensitive and -resistant TC cells. Suppression of wild-type p53 induced 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 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 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 death receptor pathway. This effect is most pronounced in cisplatin-resistant TC cells.
Highlights
FAS (TNFRSF6), as well as genes that induce cell-cycle arrest, such as cyclin-dependent kinase inhibitor 1A gene (CDKN1A), encoding p21cip1/waf[1], allowing time for DNA repair
Our results suggest that high sensitivity for cisplatin cytotoxicity and cisplatin-induced apoptosis is related to a reduction in mouse double minute 2 (MDM2)–p53 complex formation and a change in p53 cellular localisation
We demonstrate that wild-type p53 is sustained in complex with MDM2 in cisplatin-resistant testicular cancer (TC) cell lines following treatment with therapeutically relevant cisplatin concentrations
Summary
FAS (TNFRSF6), as well as genes that induce cell-cycle arrest, such as cyclin-dependent kinase inhibitor 1A gene (CDKN1A), encoding p21cip1/waf[1], allowing time for DNA repair. Interfering in the MDM2–p53 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 p53.20–23 Restoration of p53 function by Nutlin-3 may have profound therapeutic effect on tumours that have retained wild-type p53, 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. The importance of the Fas death receptor pathway in Nutlin-3 induced apoptosis has been studied
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