Download Figure 6. Expression levels of IGF-1 variants in breast cancer cell

Expression levels of IGF-1 Variants in Breast Cancer Cell Lines
Jillian Hood (Clark Atlanta University),
Rupali Hire,PhD and Melissa Davis, PhD
Davison Life Science University of Georgia, Athens, GA 30602
METHODS Cont’d
ABSTRACT
Elevated levels of growth factors significantly
increase the risk of breast cancer. Insulin-like
Growth Factor 1 (IGF-1) is a protein that is encoded by
the IGF1 gene in humans and has a significant effect
on human growth and development. IGF-1 is a growth
factor that is released into the bloodstream and
adheres to IGF receptors within organs that can
eventually cause an increase in cell growth and
inhibit apoptosis in cancers. IGF-1 plays a role in
cancer progression. Lowering levels of IGF-1 can lead
to reduced growth of already initiated cancer cells.
Numerous transcript variants code for different IGF-1
isoforms of this gene.
IGF-1 is regulated by binding proteins. At this time;
there are six known IGF Binding Proteins (IGFBP-1 to
IGFBP-6) with varying functions related to IGF-1.
Data suggests that IGF binding proteins have
essential roles in their ability to control IGFs. Some
of these binding proteins bind to the growth factor
and seem to defuse the elevated levels and may
reduce the risk of cancer. Other IGF binding proteins
function as carrier proteins for IGF-1 and increase its
functionality. The purpose of this particular project is
to observe the interaction between IGF-1 and IGFBP-6
and how the binding protein is expressed in specific
cell lines.
INTRODUCTION
Variant 2
Variant 1 and 3
Figure3: Agarose gel of Reverse Transcription PCR reaction.
First well contains 100 Bp ladder. Lane 3 with cell line
CCD1059sk expressed variant 2. Lane 6 with cell line
CCD1059sk expressed variant 1 and 4 with the expected
product length of 233 base pairs.
Variant 1 and 3
Figure4: Agarose gel of Reverse Transcription PCR
reaction using primer IGFEXP_CG for each cell line. First
lane contains 100 Bp DNA ladder. Lane 4 with cell line
HCC 1599 expressed variant 1 and 3 . Lane 6 with cell line
CCD 1059sk expressed variant 1 and 3.
Variant 2
RPL
Figure 1: Co-staining of IGF-1 and IGFBP6 with cell lines HCC1806 (ER-), HCC70
(ER+), and CCD1059sk(ER+). The purpose of this fluorescence microscopy was to
observe the overlap or binding of IGF-1 and IGFBP6. IGFBP6 is expressed in all three cell
lines but is barely visible in cell lines HCC70 and CCD1059sk. In the first row, IGF-1 is
expressed in green for each cell line. The second row exhibits the binding protein (IGFBP6) which is strongly shown in cell line HCC1806 but not seen as much in cell line HCC 70
and CCD1059sk. The third row is a merge of IGF-1 and IGFBP-6.
Figure 5: Agarose gel of Reverse Transcription PCR reaction
using IGF-1 F and R primers in each cell line. First well contains
100 Bp DNA ladder. Lane 6 with cell line CCD1059sk expressed variant
2 with an expected product length of 232 Bp. The RPL (lanes 9-12)
sample was used as a positive control.
Triple negative breast cancer is the most frequently
detected subtype in African American women.
Between African Americans and Caucasians, the
incidence of triple negative breast cancer is more
likely to affect African Americans with a higher
mortality rate. Triple negative breast cancer refers to
any type of breast cancer that does not exhibit the
three most well-known receptors: estrogen,
progesterone, and hormone epidermal growth
factor receptor 2 (HER-2) genes. These receptors
normally provide instructions that aid in organ
development but overexpress in tumors, causing
them to over proliferate. These receptors are the
targets of drugs for cancer treatment. The absence
of these receptors, in triple negative tumors, makes
treatments such as hormone targeted therapies
unsuccessful. Therefore, we are in search of targets
that are specific to these triple negative tumor types.
CONCLUSIONS
It is evident that IGFBP6 is strongly expressed in HCC1806
compared to HCC70 and CC1059sk where is barely seen inside
the cell. The qPCR data in correlation to the agarose gels
exhibits that the higher the average dCT values the duller the
ban will appear on the gel and the lower the average dCT
values the brighter the band appears the gel.
ACKNOWLEDGEMENTS
I would like to thank Dr. Davis and Rupali Hire for their
advice, patience, support and encouragement
throughout this program.
REFERENCES
Variant 1 and 2
Variant 1 and 2
Variant 1 and 3
Variant 2
Variant 1 and 3
1.
Komen, Susan. "Facts for Life: Triple Negative Breast
Cancer." . N.p., 1 Oct. 2013. Web. .
<http://ww5.komen.org/uploadedFiles/Content_Binarie
s/KOMEED079100.pdf>.
2.
"IGF-1 (Insulin-Like Growth Factor 1)." MESORx. N.p., 1
Jan. 2014. Web. . <https://thinksteroids.com/steroidprofiles/igf-1/>.
3.
"Insulin-like growth factor binding proteins and their
role in controlling IGF actions.." National Center for
Biotechnology Information. U.S. National Library of
Medicine, n.d. Web. .
<http://www.ncbi.nlm.nih.gov/pubmed/9174662?report=
docsum>.
METHODS
Part 1: Co-staining of cell lines using IGF-1 and
IGFBP6 antibodies. All cell lines were fixed with 4 %
formaldehyde for 1 hour then washed with PBS 3
times. After being washed, the cells were incubated in
PBI and signal enhancer for 30 minutes at 37ºC then
washed three times with PBS. 100 uL of Primary
Antibody solution (IGFBP6 Antibody- Anti-IGFBP6 in
rabbit) by Sigma was added to the cells and incubated
at…cont’d
room temperature for 1 hour. 100 uL of secondary
antibody solution (Anti-hIGF-1 Affinity purified
GoatIgG) was added to the cells in the dark and
incubated at room temperature for one hour. 100 uL
of secondary antibody for IGFBP-6 and IGF-1
(Antibody for IGFBP6: AlexaFluor 350, donkey, antirabbit AlexaFluor 350 and Anti-body for IGF-1:
AlexaFluor 488, chicken, anti-goat ) was added to the
cells in the dark and incubated at room temperature
for one hour. After being incubated for one hour the
cells were rinsed with PBS twice and mounted on
mounting slide.
*note: in between adding antibody solutions cells
were washed three times with PBS.
* All dilutions were 1:100
Part 2: Reverse transcription polymerase chain
reaction. A master mix was prepared using platinum
blue, necessary forward and reverse primers, and
cDNA. Once reactions and 1µl of cDNA were prepared
and equally distributed into each PCR tube , the
reactions were ran in the PCR machine. Once the
reactions were ran through the PCR machine, agarose
gel was prepared and we ran gel electrophoresis.
*note: cDNA was not added to the control tubes
Part 3: Quantitative polymerase chain reaction. A
master mix containing SyBr Green, primers 1 and 2,
nuclease free water, and cDNA. After the master mix
was equally distributed into PCR tubes, they were ran
through the PCR machine and all values were
calculated and represented in chart.
Figure 2: Splicing Variants. This figure is general concept about the IGF1 gene structure and its expression using
alternative promoters and the alternative splicing which results the different transcript isoforms Exons 1-6 are a
representation of a gene. Exons 1 and 2 are the promoters and initiate where transcription begins to take place.
IGF-1 a-c are represented as isoforms of the gene. For IGF-1-a: Exons 1 or 2 is spliced into 3,4, and 6, but does not
express exon 5. For IGF-1-b:Exons 1 or 2 is spliced into 3, 4,and 5, but does not express exon 6. For IGF-1-c: Exons 1 or
2 is spliced into 3,4, partially 5, and 6. In this particular project, primers IGF-1 forward (F) and reverse (R) and
IGFEXP_CG1 F and R were used to detect variants 1 and 2, 1 and 4, and 2.
Figure 6. Expression levels of IGF-1 variants in breast cancer cell lines with RPL as
endogenous control. The higher the bar the less the expression levels of IGF-1 variants. The
intensity/ brightness corresponds inversely with the average dCT values.