Measurement of HIF1a activity
Absorbance at 450 nm
1,2
1
0,8
0,6
0,4
0,2
0
Sample 1
Sample 2
Sample 3
Sample 4
Sample 5
Sample 6
Nuclear extracts amount (μg/ml)
Normal
IDH1-mutant
Figure 5 Measurement of HIF1a activity in normal brain tissue and IDH1-mutated
patient glioma tissue.
This figure is derived from plotting the absorbance of HIF1a assay at 450 nm from nuclear
extracts of normal brain tissue and glioma patients to measure the HIF1a activity. The IDH1mutant gene has significantly higher absorbance than the normal brain tissue, with its highest
value at 0.98 nm and the lowest value at 0.68 nm. The normal brain tissue values range from
0.38 nm to 0.21nm. Both normal brain tissue and IDH1-mutation show a constant fluctuation
in absorbance between the different samples but a gradual decrease over time. The IDH1mutation has a larger standard deviation bar indicating extensive spread from the mean.
However, the normal brain tissue has a smaller standard deviation bar denoting the less
widely dispersed the data is. Forced expression of IDH1-mutant in cells reduces the
formation of enzyme product α-ketoglutarate (α-KG) and increases the levels of HIF1a (Zhao
et al., 2009). The expression of HIF1a is higher in human gliomas containing an IDH1mutation than to tumors without a mutation. Therefore, IDH1-mutant gene acts as a tumor
suppressor that contributes to changes in cellular metabolism and a response to hypoxic and
oxidative stress. This leads to a hypermethylation phenotype which alters gene expression
and drives cancer progression.
Figure 6 Expression levels of cyclin D1, vascular endothelial growth factor (VEGF) and
platelet-derived growth factor A (PDGFA) in IDH1-mutated tumor lysates obtained
from glioma patient and normal brain.
This figure is derived from a western blot analysis that displays the expression levels of
cyclin D1, vascular endothelial growth factor and platelet-derived growth factor A protein in
tissue lysates obtained from normal brain tissue and glioma patient. Tubulin is used as an
internal standard loading control protein for the western blot to normalise the levels of
proteins detected by confirming that protein loading is across the same gel. Tubulin offers
advantages as loading controls as it exhibits stability under most experimental conditions,
ubiquitously expressed to high levels and is highly conserved. Gene expression from gliomas
with IDH1 mutation is higher than gliomas without IDH1 mutation. This is evident through
the thicker protein bands indicating a higher molecular weight. The intensity of the protein
bands appears to be similar for cyclin D1 and VEGF of normal brain tissue however there is a
higher intensity for PDGFA.