Transcript Inducing Apoptosis of Glioblastoma Multiforme Cells Using a Bcl

Inducing Apoptosis of Glioblastoma Multiforme Cells Using a Bcl-2 Specific siRNA Sequence
Valerie P. Capozziello* and Jeffrey P. Thompson
York College of Pennsylvania, Department of Biological Sciences
Glioblastoma multiforme (GBM) is the most common
brain cancer of middle aged Americans. Unfortunately,
survival rates are typically less than 1 year from time of
diagnosis. Following surgery, GBM patients typically
endure chemical and radiotherapies to slow tumor
regrowth. Research suggests that under these
treatments GBM cells regress by entering apoptosis, or
programmed cell death. We investigated the use of
small interfering RNAs (siRNAs) to induce apoptosis in
human GBM cells in vitro. Current research suggests
that siRNAs eliminate a particular target protein from a
cell by degrading the target’s mRNA cellular pool.
Bcl-2 is an anti-apoptotic protein in cells. Using Bcl-2
targeted siRNAs, we attempted to eliminate Bcl-2 from
U87mg (human GBM) cells, cultured in vitro, inducing
them into apoptosis. Preliminary data suggests that
U87mg cell number is reduced (cell death enhanced)
when treated with Bcl-2 siRNA compared to U87mg
control cells, treated with a scrambled siRNA
sequence.
This research aims to investigate the use of siRNAs to
downregulate the expression of the Bcl-2 gene and
thereby induce apoptosis of U87MG Human GBM cells
(Figure 1).
Materials and Methods
The suppression of genes by siRNA is a pathway that
begins with double-stranded RNA (dsRNA) and is a
two-step process (Figure 2).
•RNA interference (RNAi) machinery detects dsRNA
and breaks it into approximately 21-23 nucleotide long
segments.
• These segments are bound in an RNA-protein
complex that mediates the detection and destruction of
mRNAs containing the same sequence as the dsRNA
(Pierce 2002).
Figure 2.
Introduction
Glioblastoma multiforme (GBM) is the most common
brain cancer among middle aged Americans and has a
mean survival rate of approximately one-year (Julien et
al. 2000). Cancer is the unregulated growth and
proliferation of cells. Cell growth is a process normally
controlled by apoptosis. Apoptosis is programmed cell
death brought on by a variety of stimuli. This process
involves decreased cell volume, modification of the
cytoskeleton and degradation of DNA. After this occurs
the cell releases tiny membrane-bound apoptotic
bodies containing intact organelles that are quickly
phagocitized. A new set of oncogenes is being defined
that affect this apoptotic pathway (Julien et al. 2000).
One of these is the Bcl-2 gene. Bcl-2 does not
necessarily affect proliferation but it does inhibit
apoptosis (Reed 1994). Bcl-2 has been found to be
overexpressed in GBM (Julien et al. 2000). Not only
does Bcl-2 affect apoptosis but also increased
production of the gene has been found to increase
resistance to chemotherapies and radiation treatment,
therapies aimed at inducing apoptosis (Reed 1994).
Figure 1.
Figure 1. U87MG Human GBM cells.
Experiments
Aseptically transferred 4x105 dilution of U87MG cells
into a 96-well cell culture plate
Transfected cells with siRNA and Transmessenger
Transfection reagent complex
Optimization reaction was performed to access the
correct ratio of siRNA to TransMessenger to allow
the most transfection
Target sequence experimental siRNA and scrambled
sequence control siRNA were used along with cells
untreated with siRNA
Efficiency of transfection was measured by reading
the fluorescence of the cells after incubation with
siRNA complexes
Cells were transfected with siRNA and
Transmessenger Transfection Reagent complexes
at optimal ratio with a larger sample size
Transfection was accessed in target sequence and
scrambled sequence cells
2500
Fluorescence (485nm/535nm,
0.1s)
Objective
2250
2000
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1000
750
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250
0
1:3
1:6
1:12
Target Sequence
Figure 3. Mean fluorescence of U87MG cells after
transfection. Three ratios of siRNA to Transmessenger
Transfection reagent were studied for both the target
and scrambled sequence. The 1:12 ratio had the
highest transfection rate in both cases.
Figure 4.
1.0×10 -04
9.0×10 -05
8.0×10 -05
7.0×10 -05
6.0×10 -05
5.0×10 -05
4.0×10 -05
3.0×10 -05
2.0×10 -05
1.0×10 -05
0.0×10 -00
Cell viability was accessed by incubating cells with
cell proliferation reagent PMS-MTS
Change in absorbance of the reagent quantified
amount of cell viability
Standardized cell viability to amount of transfection to
calculate cell death using the following calculation
Absorbance of control– Absorbance of experimental
Fluorescent units
Figure 2. siRNA mechanism of gene suppression.
•Sequence specific siRNAs can now be readily
ordered to match a particular part of a gene sequence.
•Instructions for picking a fragment were obtained
from xeragon.com, the same company from which the
siRNAs were ordered.
•The target sequence was verified to be found in the
Bcl-2 gene and not in any other gene sequence.
•The gene fragment of the Bcl-2 gene
5’-AACATCGGCCCTGTGGATGACTG-3’
was chosen according to those guidelines.
•In addition, siRNA was tagged with flourescein on the
3’-end, during synthesis by the company, for
identification and quantification of transfection.
Results
1. Optimal concentration of siRNA to Transmessenger
Transfection reagent was 1:12 for both the target
sequence and the scrambled sequence (Figure 3).
2. Transfection efficiency was greater in the scrambled
versus target sequence.
3. Target sequence caused approximately three times
the amount of cell death compared to scrambled
sequence (Figure 4).
4. Standardization was required because of differing
amounts of transfection between target and
scrambled sequence.
1:3
1:6
1:12
Scrambled Sequence
Ratio of siRNA duplex to Transmessenger Reagent
 Absorbance/Fluorescent Unit
Abstract
Figure 3.
Target Sequence
Scrambled Sequence
siRNA
Figure 4. Calculated amount of cell death in U87MG
cells after transfection and cell viability assay.
Discussion
This data suggests that the siRNA mechanism is a
possible method to induce cell death in human GBM
cells. Efficiency of transfection is affected by ratio of
siRNA to Transmessenger Transfection reagent and
this must be optimized. Amount of transfection was also
affected by differing uptake by cells even though they
were presented with the complexes in the same
manner. This is why standardization of cell death was
required. Further study is needed to determine whether
the observed death is due to apoptosis.
Future Directions
1. Perform Western blot to determine if there is a
decrease in the Bcl-2 protein level.
2. Determine if death is through apoptosis by looking
for apoptotic markers.
Literature Cited
http://www.xeragon.com/siRNA_review.html Accessed 9/9/2002
http://www.xeragon.com/siRNA_support.html Accessed 9/9/2002
Julien, T., Frankel, B., Longo, S., Kyle, M., Gibson, S., Shillitoe, E., and
Ryken, T. 2000. Antisense-mediated inhibition of the Bcl-2 gene induces
apoptosis in the human malignant glioma. Surgical Neurology. 53:360-9.
Pierce, J.L. 2002. RNAi turns the message off.The Scientist. 16:44-6.
Reed, J.C. 1994. Bcl-2 and the regulation of programmed cell death. The
Journal of Cell Biology. 124:1-6.
Acknowledgments
The Pennsylvania Academy of Science for funding