One of the key players in cancer development is p53,
a tumor suppressor. p53 is a
transcription factor encoded by a gene whose disruption is associated with
approximately 50 to 55 percent of human cancers. The p53 protein acts as a
checkpoint in the cell cycle, either preventing or initiating programmed cell
death (apoptosis). Since cancer is the unchecked proliferation of cells,
disruption of p53 activation allows diseased cells to multiply or to trigger a
tumor's progression from benign to malignant. Functional profiling of this
protein allows precise characterization of mechanisms of its regulation.
FIGURE: Functional profiling of
p53 protein in 5 different cell lines (1=Human Endothelial Cell Line, 2=C6
Glioma Cell Line, 3=A431 Ovarian Cancer Cell Line, 4=3T3 Fibroblasts, 5=HT29
Colon Cancer Cell Line). A.
Schematic representation of human p53
protein structure. Phosphorylation of residues within the activation domain
at the N-terminus of the p53 protein results in activation of the protein. Three
serine residues associated with this activation are shown.. B.
Detection of total p53 protein and
phosphorylated forms associated with p53 protein function.
Antibodies specific for total p53 and for p53 phosphorylated at three
different sites within the activation domain were used in parallel analyses.
Twenty micrograms of total protein lysate was separated by PAGE on a 10% gel and
transferred through a 5-membrane stack from the BIOMAX MultiBlot Kit for
Proteins according to the manufacturer’s recommendations. Primary antibodies
were purchased from Cell Signaling Technologies and used at a 1:200 dilution.
The presence of the proteins was detected by the ECL reaction (ECL Plus,
Amersham) and visualized on BIOMAX MR X-ray film.
Discussion
Cell lines investigated clearly showed not only differences in the amount of total p53 protein but also displayed distinct differences in the phosphorylated forms of the protein. All tumor-derived cell lines showed p53 expression; as expected, the non-cancerous fibroblast cell line (lane #4) showed no p53 expression.
These results clearly show that p53 activity is affected in different ways in different cancers. For example, the ovarian cancer cell line (lane #3) showed increased expression of p53 activated through phosphorylation of serine 6 while the colon cancer cell line (lane #5) shows up-regulation of p53 phosphorylated at serine 15. Use of the Multi-Blot Kit greatly facilitated this analysis as all forms of p53 shown here are of virtually the same molecular weight. The only way to study these proteins in the same sample using conventional Western blotting would be to strip and reprobe the blot multiple times. This is obviously cumbersome, but more importantly, protein bound to the membrane is removed with each cycle of stripping. The Multi-Blot Kit allows confident comparisons of same sized proteins within a sample.