Download Update from Erica Schenhals, February 2013: This semester began

Update from Erica Schenhals, February 2013:
This semester began by trying to identify any response to oxytocin in Jurkat T cells. Previous
experiments showed inconclusive results, so new experiments were conducted to answer several
hypotheses. First, I starved the Jurkat cells, which sensitizes the cells to a response that might
not normally be observed. I then treated these cells with varying concentrations of oxytocin
purchased from 3 different companies. The cells were then analyzed by Western blot using a
PKC antibody that will show a general, nonspecific increase in activation of intracellular proteins.
This antibody does not identify which proteins are being activated, but it will show if any
activation is occurring. Analysis showed no change in activation. Next, I used this new PKC
antibody to test proteins isolated from previous experiments conducted in Fall 2011. This
antibody confirmed that oxytocin was not inducing activation in the Jurkat cells. Next, I utilized
a new protocol to induce activation in the Jurkat cells. Certain intracellular proteins will only
become activated when the cells are first adhered. Utilizing poly-D-lysine will simulate
physiological adherence in the cells. Treatment with poly-D-lysine and then with oxytocin
showed no activation of PKC, MEK, p38, ERK, or JNK. Next, I utilized flow cytometry to
identify any apoptosis in the Jurkat cells. Initial experiments conducted in Spring 2011 indicated
that oxytocin was inducing apoptosis in Jurkat cells. Flow cytometry analysis showed no
increase in apoptosis compared to the control, suggesting that oxytocin does not induce apoptosis.
Finally, I utilized a new protocol for immunofluorescence to identify the oxytocin receptor in the
Jurkat cells. I used an anti-OTR antibody, which resulted in non-specific binding, and therefore
inconclusive results. My previous experiments have shown that the Jurkat cells have the mRNA
for the oxytocin receptor, but I have not had a successful method for determining expression of
the receptor.
Because I had not seen a significant, consistent response to oxytocin in the Jurkat cells, we
decided to purchase a cell line known to respond to oxytocin. A breast cancer cell line, Hs578T,
was used to determine if the oxytocin I have been using is effective. The Hs578T cells were
treated with oxytocin at varying concentrations and time. I received positive results for these
experiments that not only confirmed that the oxytocin is effective, but also provided novel results
that allowed me to submit an abstract to attend the American Society for Cell Biology
conference in San Francisco. My results indicated that exposure to oxytocin for 5 minutes
induces a dose-dependent increase in PKC and ERK activation. I also showed that 50 nM of
oxytocin induces a transient increase in PKC, ERK, and p38 activation. Lastly, results indicated
that 100 nM of oxytocin significantly increases ERK activation and induces a 10-fold increase in
ERK activation after 1 minute. I submitted these results in the abstract, and presented the results
at the ASCB conference in December.
After receiving positive results in the Hs578T breast cancer cells, we decided to continue with
this cell line. Moving on from the short-term experiments, I treated the cells for 12, 24, and 48
hours with oxytocin, dexamethasone, and a combination of both. Dexamethasone induces
apoptosis and is used as both a control and to determine if oxytocin could have a protective
effect. There was no significant change in PKC, ERK, or AKT activation. Oxytocin does not
appear to be causing any change after long-term exposure. Returning to short-term experiments,
I conducted an experiment treating the Hs578T cells with LiquidTrust. LiquidTrust is oxytocin
that is sold online and publicly advertised. It is not regulated by the FDA and the side effects
have not yet been determined. The results are currently inconclusive, but I will be continuing
with this line of inquiry.
Now that I have determined that the oxytocin is effective, we know that the results on the Jurkat
cells are likely true negative results. In order to return to investigating the immune effects of
oxytocin, we obtained a mouse thymus from UNC to isolate thymocytes. Thymocytes are
immature T cells and are more sensitive to environmental changes and therefore intracellular
responses. We isolated the thymocytes and tested them for viability after treatment with
oxytocin for 6 hours. These results are currently inconclusive, but will be repeated in the
upcoming semester. Thymocytes were also treated with oxytocin for 1 minute or 5 minutes at
either 100 nM or 10 uM. The limited number of thymocytes resulted in low concentrations of
protein, which did not allow for conclusive Western blot analysis. New thymuses will be
obtained and the experiment will be repeated.