Mouse Lung Fibroblast Cell Survival and p53 Expression After Exposure to Acetophenone
Max Eismann, Alley Masocco, Eric Sprys
Division of Natural and Health Sciences, Seton Hill University, Greensburg, PA
Introduction:
Tobacco products such as cigarettes contain upwards of
7,000 added chemicals and these chemicals can cause serious
health risks to humans who inhale them (FDA, 2017). There are
20 carcinogens in tobacco smoke that cause lung tumors in
humans (Hecht, 1999). One of the many chemicals found in
cigarettes is acetophenone. This chemical is a severe skin and
eye irritant (HSDB, 2017). This irritation can cause severe stress
on cells that have had the chemical exposure. Another form of
stress to cells can come from changes in pH. This stress can
cause the protein, p53, to be expressed inside of the cell. This
protein is known as the tumor suppressor gene, and can
activate cell apoptosis upon cellular exposure to stress
(Alberoni, et al. 2002). The MIC-1 gene is a target protein for
the p53 gene (Alberoni, et al. 2002). MIC-1 is a downstream
regulator for p53 gene expression (Alberoni, et al. 2002). In our
experiment we test different concentrations of acetophenone
on mouse lung fibroblast cells. The group predicted that as
concentration of acetophenone is increased, the expression of
p53 would increase and cell survival would decrease.
Methods:
Cell Culture: Mouse fibroblast cells were incubated at 37OC. The cells were
passaged using aseptic technique every 2-3 days and using a media containing
10% fetal bovine serum. The amount of new cells being added to a dish was
determined by using a hemocytometer to count the cells. The cells were dosed
with different concentrations of acetophenone 16 hours before they were
studied. The concentrations were negative control, 10 uM, 40 uM, and 85 uM.
Trypan blue was used on the cells under a hemocytometer to determine the
number of alive and dead cells. This data was used to calculate percent
survival and percent attachment which gave a quantitative look at the impact
of acetophenone.
LIVE/DEAD Viability/Cytotoxicity Assay: Cells were grown in a 4-well chamber
and dosed with the different concentrations of acetophenone. The cells were
dyed with a solution containing EthD1 and Calcein AM and incubated at 37OC.
The cells were then observed using fluorescence microscopy.
qRT-PCR: TaqMan and cDNA were resuspended by gently vortexing. 2 master
mixes were made, 1 for GAPDH and another for p53. PCR reaction mix was
placed in 8 wells of the reaction plate. cDNA was placed in the appropriate
wells. Plate was centrifuged and loaded into the qRT-PCR instrument for
testing.
SDS-PAGE: Biorad Protean II Mini gel was cleaned and assembled and resolving
gel (12.5%) and stacking gel (6%) were poured into the assembly. Frame was
placed inside electrophoresis chamber, which included running buffer.
Samples were boiled for 5 minutes and centrifuged at 16,000 rpm. Wells were
loaded and samples were run.
Western Blotting: Membranes were cut and presoaked in transfer buffer.
Transfer cell was assembled then placed into chamber with transfer buffer and
ice pack. Immunoblotting was conducted to yield results of protein
identification.
Results:
Figure 2: The percent survival of lung fibroblast cell treated with different
concentrations of acetophenone. This graphs depicts slight upward trend of survival.
The cells treated with 0 uM and 10 uM concentrations underwent three dosages of
acetophenone. Whereas, the cells treated with the 40uM and 85 uM concentrations
were exposed acetophenone twice. The standard deviation bars correlate with the
number of dosage trials.
A B
Figure 3: a-b The percent attachment of lung fibroblast cell treated with different
concentrations of acetophenone. a This graph depicts a downward trend in attachment
amongst the acetophenone dosages. The cells treated with 0 uM and 10 uM
concentrations underwent three dosages of acetophenone. Whereas, the cells treated
with the 40uM and 85 uM concentrations were exposed acetophenone twice. The
standard deviation bars correlate with the number of dosage trials. b
Figure 4: LIVE/DEAD Assay of mouse lung fibroblast cell treated with different
concentrations of 2.6 mM acetophenone compared to the control at 100X
magnification showing a qualitative survival analysis. The 2 mM EthD-1 stain (red)
illuminates the mouse fibroblast cells with broken cell membranes (dead cells) a,c, e,
and g. The 4mM calcein AM stain (green) demonstrates cells that are undergoing
intracellular esterase activity and have intact membranes b, d,f, and h. All imagines
were captured at 100X magnification. Columns from left to right indicate increasing
concentration for acetophenone 0 uM to 85 uM.
Figure 5: Ponceau stain protein analysis of mouse lung fibroblast cell treated with
acetophenone using nitrocellulose membrane. The Ponceau stain indicates general
protein expression (darker toned red) in the mouse lung fibroblast cells, it can not be
determined whether or not p53 was expressed. The protein expression (darker toned
red) appears to be contained in each dosage well of acetophenone (0uM - 85uM) which
indicates that the protein failed to run.
A
Figure 6: a qRT-PCR baseline showing raw data of p53 expression in mouse lung
fibroblast cell treated with acetophenone. The red horizontal line shows the
threshold value for GAPDH expression. The blue horizontal line shows the threshold
value for p53 expression. b Demonstrates the fold increase of p53 expression in the
cells. The 40uM dosage of acetophenone had the largest fold increase value.
Conclusions:
● The percent attachment results show acetophenone did
not have detached many cells from the pates
● The high percent survival could mean that the
concentrations of acetophenone were too low to kill
cells
○ Acetophenone could also not be toxic to mouse lung
fibroblast cells
● Proteins were too large to travel through gel in western
blot
● 40 uM concentration having the highest protein
expression could be because of errors during protein
isolation
● The qRT-PCR data does not show a clear connection
between acetophenone concentration and p53
expression
● Errors in cell plates could have been caused by
inexperience in cell passaging
○ Could explain why 85 uM plate had a larger amount of
live cells than the other plates
○ Also explains low number of cells in control plate
when calculating percent attachment
Future Studies:
● Use higher concentrations of acetophenone
○ May get more p53 expression
● Do more assays to determine percent attachment and
percent survival
○ Will give us more data to see if the current data
collected is accurate
● Test how other ketone chemicals impact the cell survival
and p53 expression in Mouse Lung Fibroblast cells.
References:
Acetophenone. (2017, September 14). Retrieved from https://toxnet.nlm.nih.gov/cgi-bin/sis/search/a?dbs hsdb:@term @DOCNO 969
Albertoni, M., Shaw, P. H., Nozaki, M., Godard, S., Tenan, M., Hamou, M., . . . Hegi, M. E. (2002). Anoxia induces macrophage inhibitory cytokine-1
(MIC-1) in glioblastoma cells independently of p53 and HIF-1. Oncogene,21(27), 4212-4219. doi:10.1038/sj.onc.1205610
Center for Tobacco Products. (n.d.). Products, Ingredients & Components - How Cigarettes Are Made and How You Can Make a Plan to Quit. Retrieved
from https://www.fda.gov/TobaccoProducts/Labeling/ProductsIngredientsComponents/ucm529397.htm
Fornsaglio, J., & Sheffler, Z. (2018, May 17). Cell Biology Laboratory. Retrieved from
https://itunes.apple.com/us/book/cell-biology-laboratory/id842833018?mt=11
Hecht, S. S. (1999). Tobacco Smoke Carcinogens and Lung Cancer. JNCI Journal of the National Cancer Institute,91(14), 1194-1210.
doi:10.1093/jnci/91.14.1194