MOLECULAR BIOLOGY ASSESMENT

1. By: Manuela Pabón Bernal
Molecular Biology
Third semester
Universidad Pontificia Bolivariana
2019-02

2. Diffuse Intrinsic Pontine
Glioma (DIPG)
• A rare, fast-growing, highly aggressive tumor that forms
in glial cells in the pons, that tend to spread to nearby
tissue and other parts of the brain stem, are hard to
treat, and have a poor prognosis.
• They usually occur in children (diagnosed 5-9 years),
equally in boys and girls.
• Makes up 10-15% of all brain tumors in children, with
about 100-300 new cases diagnosed each year in the
United States.
• Problems with eye movement; Facial weakness;
Sudden appearance of hearing problems; Trouble
chewing or swallowing; Limb weakness, difficulty
standing or walking, abnormal gaits, clumsiness,
unbalanced limb movements; Headache; and, Nausea
and vomiting from brain edema.

3. Wilm’s Tumor Protein
• Is encoded by the WT1 gene on chromosome 11p.
• Transcription factor that plays an important role in cellular
development and cell survival.
• Regulates the expression of numerous target genes,
including EPO.
• Plays an essential role for development of the urogenital
system.
• It has a suppressor as well as an oncogenic role in tumor
formation.
• Known as a protein highly associated with various
cancers including adult glioblastomas.
• WT1 has been ranked by the NCI as the Number 1 target
for cancer inmunotherapy.

4. General
objective
The differential expression of Wilms’ Tumor Protein in Diffuse
Intrinsic Pontine Glioma

5. Methods. Quantitative RT-
PCR
• The RNA amplification through its copy DNA synthesized a
priori, using a reverse transcriptase, followed by several
cycles of conventional PCR. This technique can determine
the expression of genes in various tissues.
• Performed qRT-PCR using cDNA from fresh frozen DIPG
specimens, which confirmed the significantly higher
expression of WT1 in tumor specimens compared with
adjacent healthy brain samples. Data showed higher
expression (6.4-fold) in the H3.3-mutant samples.

6. Methods.
Immunohistochemistry
• Uses antibodies to check for certain antigens (markers) in
a sample of tissue. The antibodies are usually linked to an
enzyme or a fluorescent dye. After the antibodies bind to
the antigen in the tissue sample, the enzyme or dye is
activated, and the antigen can then be seen under a
microscope.
• WT1 expression is associated with the mutation status of
oncohistone H3.
• They observed that WT1 expression, at both the mRNA
and protein level, was significantly higher in a number of
DIPG specimens. This led them to expand their
investigation to a cohort of 37 tumor specimens and 20
adjacent healthy specimens from 37 subjects diagnosed
with midline glioma and probed for WT1 expression by
IHC.

7. Methods. Western Blot
Assays
• Identify specific proteins from a complex mixture of proteins
extracted from cells. The technique uses three elements to
accomplish this task: (1) separation by size, (2) transfer to a
solid support, and (3) marking target protein using a proper
primary and secondary antibody to visualize.
• Using protein extracts from DIPG subjects for whom frozen
tumor and adjacent normal tissue was available. Western
blot analysis showed significant overexpression of WT1 in
DIPG specimens compared with adjacent normal samples.
• Western blot analysis using human DIPG primary cells
showed significantly higher level of WT1 in DIPG tumor cells
compared with healthy brain tissue specimens from pediatric
patients without CNS disease.

8. Methods.
Immunofluorescence
• Based on the identification of the reaction between an
specific antigen and antibody. The reaction is targeted
with a fluorescent reagent which evidences the reaction
itself. The intensity of the fluorescence indicates the
reaction intensity as well.
• Using antibodies against H3K27M, they probed for
mutant histones and found that 94.6% (35/37) of
subjects harbored the H3K27M mutation and
corroborated with the conclusion which stated that WT1
expression is associated with the mutation status of
oncohistone H3.
• When they performed IF using human DIPG primary
cells, WT1 was present in the cells but was localized to
the cytosol in both human DIPG primary cells tested.
Given the fact that WT1 is known to be a nuclear
protein, this finding was surprising.

9. Results

10. Results

11. Results

12. Author What it said
Match with the
article
Nakatsuka S, Oji Y, Horiuchi
T, et al.
Oji Y, Suzuki T, Nakano Y, et
al.
“Our cytosolic detection of WT1 is not
surprising as previous studies have
also shown cytosolic localization of
WT1
in glioblastoma cells and lung cancer
cells”.
NO
Chiba Y, Hashimoto N, Tsuboi
A, et al.
”WT1-directed immunotherapeutic trials
in adults with glioblastoma have
indicated better survival of patients with
higher WT1 expression levels receiving
WT1-specific immunotherapy”.
YES
O’Rourke D, Nasrallah M,
Desai A, et al.
“There has been a rapid increase in
studies investigating immunotherapy in
CNS tumors. For instance, vaccines YES
Discussio
n

13. • In my opinion this study was very well performed, used a wide
variety of methods and had coherent results with what they were
looking for and acknowledging now a days advances with genetic
therapies, it seems like a promising pathway in order to develop
health promoting tools, such as treatments or pharmaceutical aids
for patients with DIPG.
• Molecular biology’s importance stands up in every single conclusion
they had, and enhances the value of knowing the origin of diseases
from the simplest and smallest factors, which can fully help doctors
to improve not just these, but every patients’ quality of life.
Conclution
s

15. Bibliograph
y
https://www.cancer.gov/publications/dictionaries/cancer-
terms
https://www.uniprot.org/uniprot/P19544
Biologia Molecular, Sánchez. Lina. 2016. Universidad
Pontificia Bolivarian