Administrative Information
‫المعلومات االدارية‬
:‫المرجع‬
Project Title - )‫عنوان المشروع (عربي وأجنبي‬
‫في استقرار المركز الحساس المشترك في الحمض النووي لإلنسان‬
‫تحديد دور الجينة‬
‫ جينة ذو دور‬:
‫محتمل في قمع الورم؟‬
Identification
of the role of MUCIN 15 gene in the stability of the Human Common Fragile Sites: a potential
Tumor Suppressor Gene?
Principal Investigator - ‫الباحث الرئيسي‬
‫رقم الهاتف‬
‫العنوان االلكتروني‬
‫العنوان‬
‫الوظيفية‬
‫المؤسسة‬
‫االسم‬
Telephone
e-mail
Address
Post
Institution Name
009616931952 eliane.elachkar@balamand.edu.lb
Deir El Assistant University Eliane
ext 3837
Balamand- Professor
of
El
El KouraBalamand Achkar
North
Lebanon
Co-Workers - ‫الباحثون المشاركون‬
‫العنوان االلكتروني‬
e-mail
2 years
‫المؤسسة‬
Institution
‫االسم‬
Name
: Duration -‫المدة التعاقدية للمشروع‬
Scientific Information
‫العلمية‬
‫المعلومات‬
ّ
Objectives - ‫الهدف‬
This project falls within CNRS research priorities as it deals with a multifactorial disease
causing a serious world-wide problem of public health, a disease named Cancer. The main
characteristics of this disease is the uncontrolled growth of a group of cells that can later invade
the adjacent tissues and sometimes reach other locations in the body through the lymph and the
blood fluids (Metastasis).
Cancer is triggered by several factors (genetic factor, age, obesity, environmental factors like
tobacco smoke, radiation, chemicals, alcohol consumption, diet, infectious agents,…..) and is
associated with genomic abnormalities that touch three categories of genes: the oncogenes
involved in the activation of the cell cycle and the inhibition of apoptosis, the tumor suppressor
genes responsible for the growth arrest and the induction of the programmed cell death, and the
genome integrity genes that control the repair of DNA damages due to spontaneous mutations
or induced by the effect of carcinogens.
Therefore, the isolation of new oncogenes or tumor suppressor genes represents a primordial
step in understanding the origin of genetic instability in cancerous cells and thus in targeting the
development of new drugs in Oncology.
Our main objective in this project is to determine the role played by the product of Mucin 15
gene (mapped to the fragile site FRA11D at the chromosomal band 11p14.2) in the stability of
the common fragile sites and to determine if this gene could be a potential tumor suppressor
gene. (Common fragile sites are site specific breaks, gaps or constrictions that constitute an
integral element of the human genome and other species. Breakpoints at common fragile sites
colocalize with 50% of the breaks observed in cancerous cells).
The normal expression level of mucin 15 gene will be measured by Western Blot in a
lymphoma cell line. Several small interfering RNA (siRNA) will be designed to shut down the
expression of this gene. The impact of this RNA degradation on the stability of fragile sites will
be determined by Fluorescent Microscopy analysis of metaphases. In brief, the breakpoints at
fragile sites after mucin 15 siRNA knockdown will be recorded and their frequency compared
to that of the breakpoints obtained at the same fragile sites in untreated cells. The protein level
variation expected will be detected by Western Blot.
Achievements -‫أالنجازات المحققة‬
Determination of the Optimal Concentration of Aphidicolin
The first objective of the project was to determine the optimal concentration of
aphidicolin (a partial inhibitor of DNA polymerases α, δ and ε) that can stimulate breakages at
fragile sites in the U937 lymphoma cells. In order to answer this question, six different
experimental conditions were set:

Untreated cells

Cells treated with aphidicolin with four different concentrations: 0.1, 0.2, 0.3 and
0.4 μg/ml, followed by nocodazole treatment at 5 μg/ml to accumulate cells in
metaphase for the microscopy analysis.

Cells treated with nocodazole only at 5 μg/ml
This experiment was done in triplicate. In each experiment 100 metaphases were
analyzed.
Microscopy analysis showed that untreated cells and cells treated with nocodazole only exhibit
between 0 to 2 breakages/cell which falls within the normal range. Cells treated with an
increasing concentration of aphidicolin showed an increase in the number of breakages (more
than 3 breakages per cell) associated with the partial inhibition of DNA polymerases α, δ and ε.
Cells treated with APC 0.1 μg/ml and 0.2 μg/ml showed an increase in the number of breakages
(17% and 41% of cells with more than 3 breakages per cell respectively) compared to normal
cells. Cells treated with APC 0.3 μg/ml and 0.4 μg/ml showed a higher increase in the number
of breakages (72% and 89% of cells with more than 3 breakages per cell respectively).
We selected the concentration of APC
0.3 μg/ml to be used for the transfection
experiment since they exhibit a large number of breakages compared to untreated cells and with
this concentration there is no drastic loss of the genetic material.
Cell transfection with siRNA against Mucin 15 mRNA
In this experiment 96 well plates were used with nine different conditions studied for two
time points: 48h and 72h after transfection. The nine different conditions are illustrated in the
table 1.
Table 1: The Nine Different Conditions of the Transfection Experiment.
1
2
3
4
5
6
7
8
9
Untreated U937 cells
Mock:U937 cells treated with transfection reagent
Negative Control: U937 cells treated with siRNA with no homology to any
mammalian gene
Cell death Control: U937 cells treated with siRNA targeting essential genes for
cell survival
U937 cells treated with siRNA targeting MUC15 gene
U937 cells treated with APC 0.3 μg/ml
U937 cells transfected with siRNA targeting MUC15 gene and treated with APC
0.3 μg/ml
APC Mock: U937 cells treated with transfection reagent and APC 0.3 μg/ml
U937 cells treated with nocodazole at 5 μg/ml
Cell Death Analysis by Flow Cytometry
Cell death was analyzed in order to monitor if the transfection with siRNA targeting the
MUC15 gene will affect the normal percentage of apoptosis of in vitro dividing cells.
Using SPSS, the statistical software, these data were analyzed with multiple ANOVA test to
find if there is a statistical significance. SPSS results showed that there is no statistical
difference between samples for the two time points.
Fluorescent Microscopy Breakages Analysis
Using the Zeiss epifluorescent microscope a breakage count per cell was done for each
experiment. Metaphases were divided for each time point into two parts: metaphases that fall in
the accepted normal range of breakages (between 0 and 2 breakages/cell) and metaphases that
exhibit a number of breakages equal or higher than 3 breakages per metaphase.
By comparing untreated cells and cells transfected with siRNA targeting MUC15 gene,
cells treated with APC alone and cells treated with APC and siRNA targeting MUC15 gene, we
can clearly notice an increase in the number of breakages per metaphase.
Microscopy analysis showed that after 48 hours of transfection approximately 9% of
metaphases in untreated cells have three or more breakages.
Mock samples containing lipid vesicles used for transfection and negative control samples
containing siRNA with no homology to any mammalian gene have respectively 6% and 15% of
their metaphases exhibiting three or more breakages.
Cells transfected with siRNA targeting essential genes for cell survival forming the cell
death control have 12% of their metaphases exhibiting three or more breakages.
Remarkably this low percentage of metaphases having three or more breakages in the
previous conditions will increase in the following conditions: cells transfected with siRNA
targeting MUC15 gene (33% of metaphases with ≥3 breakages); cells treated with APC alone
(34% of metaphases with ≥3 breakages); cells treated with APC then transfected with siRNA
targeting MUC15 gene (64% of metaphases with ≥3 breakages).
In the sample where the cells were treated with APC and lipid vesicle 18% of metaphases
exhibit three or more breakages. Cells treated with nocodazole alone have only 4% of their
metaphases exhibiting three or more breakages.
After 72 hours of transfection the results were identical to those found after 48 hours of
transfection.
Approximately 3% of metaphases in untreated cells have three or more breakages.
Mock samples containing lipid vesicles used for transfection and negative control samples
containing siRNA with no homology to any mammalian gene have both 13% of their
metaphases exhibiting three or more breakages.
Cells transfected with siRNA targeting essential genes for cell survival forming the cell
death control have 7% of their metaphases exhibiting three or more breakages.
Remarkably this low percentage of metaphases having three or more breakages in the
previous conditions will increase in the following conditions: cells transfected with siRNA
targeting MUC15 gene (30% of metaphases with ≥3 breakages); cells treated with APC alone
(34% of metaphases with ≥3 breakages); cells treated with APC then transfected with siRNA
targeting MUC15 gene (68% of metaphases with ≥3 breakages).
In the sample where the cells were treated with APC and lipid vesicle 11% of metaphases
exhibit three or more breakages. Cells treated with nocodazole alone have 4% of their
metaphases exhibiting three or more breakages.
A cooperative effect between APC and siRNA MUC15 on the increase of breakages
frequency is strikingly noticed for both time points 48h and 72h.
Hence, knocking down MUC15 gene has increased the number of breakages per
metaphase showing that this gene has a role in preventing breakages to occur which highlights
its potential tumor suppressor activity.
To investigate this increase in the frequency of breaks at FS, these results were analyzed
for statistical significance using the multiple ANOVA test.
Statistical analysis showed that after 48 hours of transfection cells treated with APC and
transfected with siRNA targeting MUC15 gene showed a significant difference in the number
of breakages compared to all other conditions except for those transfected with siRNA targeting
MUC15 gene alone and those treated with APC alone.
After 72 hours of transfection cells treated with APC and transfected with siRNA
targeting MUC15 gene showed a significant increase in the number of breakages compared to
all other conditions except for those transfected with siRNA targeting MUC15 gene alone.
These results also indicate that cells transfected with siRNA exhibit a high number of breakages
compared to all the other conditions even though if not treated with APC. Hence, the stability of
FS in this cell line is also affected by Mucin 15 knockdown.
Perspectives - ‫آفاق البحث‬
Our findings showed that the optimal APC concentration to induce common fragile sites
efficiently in U937 cell line is 0.3 μg/ml. Cell death percentage after treatment analyzed by flow
cytometry did not show any significance. Breakages microscopy analysis showed clearly that
by knocking down MUC15 gene, a significant increase in the number of breakages occurred
showing that MUC15 gene affect DNA instability and that MUC15 gene is a tumor suppressor
gene.
More experiments should be done in the future to confirm this finding since the discovery
of a new tumor suppressor gene is a critical issue in understanding the molecular mechanism
underlying the rearrangements in cancer.
The upcoming experiments should target the following points:
- Optimization of the western blot conditions to obtain a reproducible result
- Immunohistochemical staining: to compare the expression of this gene in normal tissues
and in tumor tissues.
Publications & Communications - ‫المنشورات والمساهمات في المؤتمرات‬
The results will be prepared for publication after the optimization of the western blot
conditions.
Abstract - ‫موجز عن نتائج البحث‬
Common fragile sites (CFS) are site specific breaks, gaps or constrictions that constitute
an integral element of the human genome and other species. These sites are induced by
aphidicolin (APC), a partial inhibitor of DNA polymerases α, δ and ε. According to previous
studies, breakpoints at CFS colocalize with 50% of the breaks observed in cancerous cells.
Some of the CFS are associated with tumor suppressor genes (TSG): FRA3B located at the
chromosome band 3p14.2 is associated with the TSG FHIT and FRA16D located at16q23
includes the TSG WWOX.
Our main objective in this study was to determine if the Mucin 15 gene located at the
fragile site FRA11D could be a potential tumor suppressor gene.
In order to reach our aim siRNAs were selected to knockdown Mucin 15 gene expression.
To determine the optimal aphidicolin concentration inducing fragile sites in the U937 cell line,
a breakage count was performed in triplicate after several treatments with APC. The APC
concentration of 0.3 μg/ml was shown to be the most efficient. Following siRNA MUC 15
transfection, western blot was performed to determine the expression level of Mucin 15 protein:
a decrease in the expression was noticed. Fluorescent microscopy was used to compare
breakage frequency in both control and siRNA MUC 15 transfected cells. A significant increase
in the number of breakages was observed in transfected cells and treated with aphidicolin.
This increased instability after Mucin 15 knockdown reflects the essential role of this
gene in the regulation of the stability at fragile sites and pinpoints its potential tumor suppressor
activity.
It is noteworthy to mention that the discovery of a new TSG at the level of fragile regions
is an important step in understanding the molecular mechanism underlying the genomic
abnormalities observed in cancer.