Expression of p53 and cell survival at different concentrations of

acetanilide
Introduction:

Jesse A. Cooper and Tyler P. Bowling

Division of Natural and Health Sciences, Seton Hill University, Greensburg, PA
Results:

B.

A.

Air pollutants contained in cigarette smoke propose public health concerns.

Propaganda throughout the world is focused on raising awareness on the
toxic chemicals that harm the lungs. While there is research linking lung
cancer and cigarette smoke, there is more to be known on how air toxins
affect adenoma lung cells. Mouse lung adenoma cells were grown in culture
and treated with three increasing doses of the cigarette toxin acetanilide.
Acetanilide is known to have a very intense toxicity that is harmful if inhaled
and, it can even cause skin and eye irritation (Acetanilide, n.d.). After
chemical treatment, quantitative analysis with trypan blue dye and
qualitative inquiry by fluorescent microscopy aided in counting and
observing living and dead cells. Cells were lysed to retrieve mRNA, and
reverse transcribed to cDNA to isolate the p53 protein. A qRT-PCR analysis
including an RNA normalizing control, GAPDH, was run to generate
quantitative values and CT values representing levels of gene expression.
To confirm gene expression from the qRT-PCR, a qualitative analysis SDSPAGE and Western blot was performed. Specific antibodies including p53
and actin were added to the western blot, and secondary antibodies like
mouse IgG were tagged with enzymes to detect for p53 and actin. During
qRT-PCR, we hypothesized that gene expression with increased doses of
acetanilide would decrease, but there was a spike in expression at the
lowest dose, 50uM. Western blot results came up inconclusive. Longer
incubation with antibodies could clear up ambiguities at this stage of the
project to compare the results with the qRT-PCR data.

Figure 2. Fold analysis of p53 expression showing the largest dose

expressing the largest fold increase in gene expression compared to
the control. Error bars express ambiguity in the qRT-PCR amplification
plot for the 50uM dose.

Figure 5: Part A is shows the decrease in survival rate

of cells with increased dose of acetanilide. Part B shows
the amount of attached cells with increased dosage of
acetanilide.

Conclusions:
With higher doses of acetanilide, cell survival rate
decreased.

Apoptosis occurs in cells with higher doses of

acetanilide with heightened p53 expression.

B
Methods:
Cell Culture:
A sample of adenoma mouse lung cells were passaged by standard
culture methods.The cells were washed in a 1X PBS solution and
Trypsin-EDTA and incubated at 37. Cells were then counted and the
appropriate amount of media was added depending on the cell
count.
Trypan Blue:
Quantitative cell survival rate was determined by the use of trypan
blue. Dead cells were indicated by blue dye.
LIVE/DEAD (Viability/Cytotoxicity) Assay:
Qualitative data collected to view toxicity assay of cells. Cells were
prepared by incubating and then by adding calcein-AM and ethidium
homodimer-1. The cells were then viewed under the fluorescent
microscope. Living cells were identified by green-fluorescent calceinAM. Dead cells were identified by red-fluorescent ethidium
homodimer-1.
qRT-PCR:
Quantitative analysis was done for p53 expression normalized to the
GAPDH control.. The samples were pelleted in microcentrifuge tubes
and the cDNA was added to the wells. The samples were then
analyzed by the qRT-PCR computer program for data analysis.
Western Blotting:
use thiswas
fontidentified
and style
p53Please
gene expression
through membrane staining of
the nitrocellulose. The primary antibodies used were p53 and actin.
The secondary antibody used for p53 was anti-mouse IgG HRP-linked
antibody, and the secondary antibody for actin was rabbit antibody.
SDS-PAGE ran the gel in order to perform the Western blot.These
were both added to a milk-TBST buffer.

Figure 1: Chemical Structure of Acetanilide.

The molecular formula is C6H5NH(COCH3).

Since Western blot data was inconclusive, our hypothesis

was declined. There was not enough data collected to
conclude if p53 expression levels decreased.

Figure 3. Fluorescent microscopy LIVE/DEAD cytotoxicity

assay. Figures A-C represent the living green cells with the
Calcein AM on the left and the dead cells with ethidium
bromide dye on the right.

Future Studies:
Collect qualitative data that represents protein
expression of p53 by Western blot.
This study should be repeated with additional
chemicals from cigarette smoke as possible.
This will allow scientists to find out which
chemicals are toxic.
Further research on mutagenesis to observe
mutations that occur in genetic DNA and its
correlation with cancer development.
By further studying each new isolated
chemical, the cellular mechanisms, and
proteins it may utilize, future researchers could
discover exactly which chemicals in cigarette
smoke, lead to particular cancer types.
Acknowledgements:
We would like to thank our lab assistant
Zachary Sheffler for his assistance in teaching
lab techniques.
We would also like to thank the Biochemistry
students: Jack Wardale, Jasmine Johnson, Logan
Telford, Gage Bollinger, Harry Jawanda, and
Patrick
Smith for their research and insight on
References:
the acetanilide.
Acetanilide. (n.d.). Retrieved April 21, 2016, from
http://chemistry.about.com/od/factsstructures/ig/Chemical-Structures--A/Acetanilide.htm

Figure 4: qRT-PCR graph for p53 gene expression

in adenoma cells at concentrations of 50 um,
150 um, and 250 um.