PO-159 Expression profiles of P53/P73 isoforms in human melanoma cell lines

Abstract

IntroductionTP53 is the most frequently mutated gene in human cancer. However, in metastatic melanoma mutations of TP53 occur infrequently and p53 fails to function as a tumour suppressor. The altered expression of p53 family members, including p53/p73 isoforms, as well as of the interactions among them could affect normal function of p53. Furthermore, somatic BRAF mutations have been found in 37%–50% of all melanomas, of which almost 90% harbour the activating V600E mutation. Although initial response to BRAF inhibitors is highly effective, the resistant clones frequently develop, and, in treated patients disease progression is observed within 6 to 8 months. To address this, a better understanding of the genetic basis of melanoma initiation and progression is needed.Material and methodsThe expression profile of p53 and its potential interaction partners - p53 and p73 isoforms was determined in a panel of melanoma cell lines by western blot analysis and quantitative RT-PCR. We have determined the protein levels of p53/p73 isoforms in response to DNA damage treatment (γ-irradiation and etoposide) in cell lines with different TP53 mutational status using western blot analysis. Furthermore, vemurafenib resistant cells are generated and resistance was confirmed by MTT assay. Expression of p53/p73 isoforms was determined in these cells.Results and discussionsRelative expression analysis of metastatic melanoma cell lines revealed that the most expressed p53 isoforms are p53α and Δ133p53α, while Δ40p53ß, Δ40p53γ and Δ133p53γ are least expressed. Also, interestingly, relative expression of full length TAp73 was higher than ΔNp73. Furthermore, the most expressed proteins were p53α, Δ40p53α, Δ133p53γ and Δ160p53γ. Contrary to gene expression, the most expressed p73 isoform is oncogenic ΔNp73β. γ-irradiation induced accumulation of all p53α isoforms in p53 mutant melanoma cell lines, but not in p53 wild type cells. Levels of p53 beta isoforms remained the same, while gamma isoforms were undetectable. Upon γ-irradiation, accumulation of ΔNp73 isoforms was observed in p53 mutant cells. Treatment with etoposide induced expression of p53α isoform, and both TAp73 and ΔNp73 isoforms in p53 wild type cells. Furthermore, in vemurafenib resistant clones the changes in p53/p73 protein expression were observed.ConclusionTaken together, these analyses enabled us to detect p53/p73 isoforms in melanoma cell lines and gave us insight into their abundance in melanoma cell lines for further analyses of p53 interacting partners.