| Popis: |
Neuroblastoma (NB) is the most common solid tumour in paediatrics, arising from primitive neural crest cells. The tumour suppressor protein, p53 is inactivated in NB through aberrant cytoplasmic localization, thus contributing to its tumourigenicity and multidrug resistance. Regulation of the p53 response pathway occurs through phosphorylation, however there may be dysregulation of p53 as NB contains abnormally high expression of PKCs. We investigated the role of PKC-mediated phosphorylation as a mechanism responsible for the p53 cytoplasmic sequestration in NB cell lines, [MR-32 and SHSY5Y. A pharmacological approach utilizing protein kinase inhibitors including H7, Bisindolyamide I (BisI), and Go6976 were tested on their ability to relocalize p53 and reintroduce function. All the inhibitors were able to relocalize p53, however, only the general kinase and PKC inhibitors H7 and BisI, respectively were able to induce a decrease in cell proliferation and cell cycle accumulation as demonstrated by flow cytometric analysis. The conventional PKC isoform inhibitor Go6976 had no effect on p53 function albeit able to relocalize p53. To further substantiate the effects with H7 and BisI as being p53-dependant, increased expression of Bax and p21 proteins served as a hallmark of p53 function. Antibody-mediated inhibition of PKC allowed us to identify the PKC isozymes involved in p53 regulation. Inhibition of PKCalpha resulted in accumulation and nuclear relocalization of p53. Furthermore substantiating p53 as a PKC substrate in vivo, serine residue phosphorylation of p53 decreased upon PKCalpha inhibition. We found that two independent PKC phosphorylation events and isoforms are responsible for regulation of p53 relocalization and activation. Specifically for NB, PKC inhibition can initiate and active the p53 response pathway. Electrospray ionization was used to monitor p53 peptide phosphorylation in vitro. Spectra revealed that phosphorylation variants ranging from mono- to tri-phosphorylated peptides can be detected. Thus, ESI was verified as an effective method of monitoring in vitro phosphorylation of p53 peptides. |
