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Dissertation/Thesis Abstract

Establishing optimal conditions for confirming successful inhibition of AKT1/2 phosphprylation in proliferating C2C12 myoblasts
by Fischler-Barraza, Maraliz, M.S., California State University, Los Angeles, 2013, 63; 1542772
Abstract (Summary)

It has been shown that MyoD-DNA binding at muscle specific genes can be inhibited in C2C12 differentiating cells by blocking the PI3K/AKT1/2 pathway. It is not known whether MyoD-DNA binding is regulated in the same manner during proliferation. We hypothesize that MyoD-DNA binding in proliferating cells is independent of the PI3K/AKT pathway. To test this hypothesis we set out to find the optimal conditions for blocking and detecting the phosphorylation of AKT1/2 (pAKT). C2C12 cells were cultured in growth medium and induced to differentiate in the presence or absence of AKT1/2 phosphorylation inhibitor (AKTi; 2μM, 3.5μM, 5μM, and 8μM). There was a dose-dependent difference in morphology after the cells had been induced to differentiate. Cells treated with higher doses of AKTi failed to reach 100% confluence, and differentiate into myotubes. There were no clear morphological changes in proliferating C2C12 myoblasts. Protein was collected from all conditions and western blots were performed. Membranes were probed for pAKT1/2, total AKT1/2, MyoD, and GAPDH. Blots showed bands for total AKT1/2, MyoD, and GAPDH, but pAKT detection was not reproducible. Although not detectable via protein blots, we did see that AKTi induces clear morphological disruptions that affects differentiating myoblasts similar to when other PI3K/AKT1/2 inhibitors are used. Protein was also used to perform an ELISA, which was unsuccessful. Apoptosis was also monitored +/-AKTi. The dye 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide, commonly known as JC-1, was used to determine the integrity of mitochondria in the early stages of apoptosis. The dye proved toxic to the cells and did not produce consistent results. In conclusion, we were unable to prove that phosphorylation of AKT1/2 was being inhibited, or if the inhibitor was inducing apoptosis. But we did see that AKTi interfered with the differentiation process. Proliferation also seemed to be reduced when comparing cells +/-AKTi at 24h and 48h. Despite the lack of corroboration by protein analysis, morphological changes in AKTi treated C2C12 cell suggest the inhibitor is causing an effect similar to other PI3K/AKT1/2 pathway inhibitors.

Indexing (document details)
Advisor: Sharp, Sandra
Commitee: McQueen, Nancy, Nissen, Robert M., Russo-Neustadt, Amelia
School: California State University, Los Angeles
Department: Biological Sciences
School Location: United States -- California
Source: MAI 52/01M(E), Masters Abstracts International
Subjects: Cellular biology
Keywords: Akt pathway, C2c12 myoblasts, Inhibtion, Myogenesis
Publication Number: 1542772
ISBN: 978-1-303-28260-7
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