Fig 1.
RFLP analysis of genomic DNA to detect the 378C>G mutation in the p53 gene.
DNA, extracted from the four formalin-fixed paraffin-embedded tissue samples of colorectal cancer, was amplified using the primers annealing to intron 4 (forward) and exon 6 (reverse) and the PCR products were subjected to the Sca I restriction enzyme digestion. (A) Schematic presentation of amplified and the Sca I digested fragments on the p53 gene. Numbers indicate the sizes of DNA fragments in base pairs (bp). The band of 453 bp after the restriction enzyme digestion indicates the absence of Sca I site which is a characteristic of the 378C>G mutation. The band of 380 bp is a product of the Sca I restriction enzyme digestion and is synonymous to the wild type p53 gene. (B) Agarose gel electrophoresis of the amplified DNA fragments before (-) and after (+) the restriction enzyme digestion. Molecular weight markers from HyperLadder II (Bioline) are shown in bp on the right.
Fig 2.
Analysis of p53 expression in ECV-304 and EJ cells.
(A) Western blotting analysis of p53 protein in HeLa, GL-V, ECV-304 and EJ cells. (B) The full-length Western blot of EJ and GL-V cells probed with the p53 antibody. The molecular weight protein markers are indicated on the right. (C) Immunodetection of p21/Waf1 protein in ECV-304, EJ and GL-V cells before (-) and after exposure to 10 mM of sodium butyrate (NaBu) for 24 h (+). Immunodetection of GAPDH protein is used as an endogenous control.
Fig 3.
Sequence analysis of the 378C>G mutation in p53 protein.
(A) Schematic representation of the Exon 4-intron-Exon 5 boundaries demonstrating how the G for C substitution creates an alternative 3’splice site which, if used, eliminates the stop codon from the mutated mRNA. The three proteins are designated here as p53-WT, p53-aa125, p53ΔY126. (B) Sequence analysis of exon 4-exon 5 junction in the PCR product amplified from cDNA of EJ cells. (C, D) The representative chromatograms of the sequenced clones corresponding to p53-aa125 and p53ΔY126, respectively. The nucleotides of exon 4 are highlighted in blue. The chromatogram was generated using FinchTV software.
Fig 4.
p53-EGFP expression in NB1-T cells 24 hours after transfection with plasmids driving the expression of the wild type p53 (A, B), p53ΔY126 (C, D), p53-aa125 (E, F). Panels A, C, E show green fluorescence originated from the EGFP; panels B, D, F–blue fluorescence from counterstaining with DAPI.
Fig 5.
Functional properties of the p53ΔY126 and p53-aa125 proteins compared to the wild type protein.
(A) Induction of the p21/Waf1 expression by transient transfection of K562 cells with plasmids expressing different variants of p53 proteins. Mock corresponds to transfection with the vector pEGFP-N1. Cells were collected 24 hours after transfection, and the total cell extracts were analysed by Western blotting with the p21/Waf1 antibody and the GAPDH antibody used as an endogenous control. (B) Cell viability assay for the GL-V cells transfected with the corresponding plasmids. (C) Flow cytometry analysis of the p53-induced apoptosis in the K562 cells transfected with the corresponding plasmids. The paired t-Test was used for statistical analysis of the mock transfected cells and the cells transfected with p53-aa125 or p53ΔY126 or p53wt using data from at least three independent experiments. Asterisk (*) represents the statistical significant difference with P-value <0.05.