Downstream gene expression of wild type p53 tumor suppressor gene versus mutated and null p53
ForfatterAljabri, Mohammad Belal
P53 is a key tumor suppressor and transcription factor protecting us from cancer. The wild type p53 protein functions as a regulatory protein, triggering a variety of cellular responses to different signals. Activation of p53 can lead to cell division arrest, DNA repair, or apoptosis. More than 60% of all human cancers contain p53 mutations. P53 is also reported in many studies to play a role in the control of other cellular important activities such as angiogenesis and glycolysis. In this study we aimed to to identify novel target genes of p53 by investigating the difference in down-stream gene expression of wt p53 in a the Saos-2 cell line which is devoid of p53 expression, in comparison to mutated form of p53 that has been reported to be associated with cancer and in relation to the lack of p53 expression. The aim was also to study protein-protein interactions between p53 and its protein partners in the different p53 variants. Two different p53 mutations (R249S and R273H), considered as hot mutations, were constructed by site-directed mutagenesis. The GeneSwitch system was used to make stable inducible p53 cell lines. This expression is controlled by mifepristone (inducer). Total proteins were isolated from the different cell lines and separated on 2D gels. The total protein expression in Saos-2 cells containing wild type p53, R249S or R273H mutants, in addition to cells with no p53 copy were compared. The expression patterns of the different samples were similar but not identical. Our results showed a different expression patterns in some vital proteins. Our results suggest a role of p53 in transcriptionally activating the β subunit of Prolyl 4-Hydroxylase which plays an important role in angiogenesis. Also our results show different patterns in expressing vimentin, which is the most abundant intermediate filament protein in various cell types, between the different cell lines. Also our results show a clear difference in the protein expression patterns of four proteins, which are essential in glycolysis (TIM, enolase 1, α-enolase and aldolase A.) suggesting a role of p53 in the metabolism of tumors particularly in glycolysis. Recent studies have implicated some of these proteins in cancer if not to p53 as well. Studies dealing with P53 and its partner proteins from the total protein using anti V5 antibody were attempted, but was not pursued further due to the short time. The way of doing this was proteomics.
ForlagUniversitetet i Tromsø
University of Tromsø
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