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Objective & Hypotheses
Programmed cell “suicide”—apoptosis—occurs in normal
cells that turn cancerous (Böttger et al, 2008). The primary
mechanism of apoptosis occurs in the nucleus using a special
protein: p53, but secondary action may occur in the
mitochondria, mediated by a certain enzyme: HAUSP (Figure
4.) (Böttger et al, 2008; Vaseva & Moll, 2008). Understanding
this pathway can further the development of cancer treatments
in diseases such as human neuroblastoma. and can enhance
our current knowledge of the cell cycle.


Discussion & Conclusion
Results
Figure 1. Morphology stain: visible membrane disruption
at time 6, and 24 hrs compared to 0 hrs.
 Localization of p53 to the nucleus indicated cellular stress
and efficacy of etoposide (Figure 2).
t = 0 hrs
t = 6 hrs
t = 24 hrs
Figure 2. Immunocytochemistry: Localization of p53 at
the nucleus at time 12 hrs compared to time 0 hrs
 At present, experimentation is being done to evaluate
hypothesis #2
t = 0 hrs
t = 12 hrs
Figure 3. Fluorometric Analysis: tagged fragments of
DNA at t = 24 hrs.
Materials & Methods
Detecting p53 and Apoptosis
1) Morphology stain: Romanovski stain
2) Immunocytochemistry: Vectastain
3) Fluorometric Analysis: Flourometric TUNEL assay
References
Böttger, S., Jerszyk, E., Low, B., &Walker, C. (2008). Genotoxic stressinduced expression of p53 and apoptosis in leukemic clam hemocytes
with cytoplasmically sequestered p53. Cancer Research, 68(3), 777-782.
Schwartzman, R.A. and Cidlowski, J.A. (1993). Apoptosis: The
biochemistry and molecular biology of programmed cell death.
Endocrine Review. 14, 133-151.
Induce Cellular Stress
IMR-32 Human Neuroblastoma cells were cultured to
a concentration of 1.4 x 105 cells/ml and were
exposed to 1.0 mM/L of the cancer drug, etoposide, for
0, 6, 12 & 24 hours.
 Fluorescent tagging of the 3’ ends of the DNA fragments
confirmed apoptosis (Figure 3).
 This evidence supports etoposide’s efficacy in inducing
apoptosis in IMR-32 cells
Hypothesis 1: Treating cells with cancer drug—etoposide—
will induce “cell suicide” via the nucleus
Hypothesis 2: Preventing p53 from entering the nucleus and
inhibiting the HAUSP enzyme will prevent apoptosis
 Visible “blebbing” in morphology stain with increased
time of etoposide exposure (Figure 1).
DAPI
Figure 4. p53 entry into the mitochondria
(Vaseva & Moll, 2009)
FITC
Vaseva, A .V. & Moll, U. M. (2009). The mitochondrial p53 pathway.
Biochem Biophys Acta,1787(5), 414-412.
doi:10.1016/j.bbabio.2008.10.005.
Acknowledgments
I would like to thank Dr. Charles Walker Ph.D., Cameron Vergato, Hannah Eldred, and
Sarah Yunes from the Walker Lab; Dr. Patricia Halpin Ph.D. and Dr. Stephen Pugh Ph.D. from
the UNH Manchester Biology Program; and the UNH URA grant donors and committee members.
Funding provided by the UNH URA donors and the Walker Lab.