Microsoft word - r_200919016.doc

つくば生物ジャーナル Tsukuba Journal of Biology (2013) 12, 51 ERK activation in murine fibroblasts under various growth conditions 森島仁美（筑波大学 生物学類） 指導教員：林 純一（筑波大学 生命環境系） Raf/MEK/ERK pathway is known to regulate numerous Using the fibroblasts NIH3T3 and 3T3BXB-ER, ERK cellular events such as cellular proliferation, senescence, activation for the growth induction was analyzed. It was differentiation, apoptosis, and transformation. It is known that tested to see if the cellular outcomes could be controlled by differences in duration and intensity of ERK activation signals changing intensity of ERK activation, investigating effect these decisions of different biological responses in parameters which may stimulate accelerated cell growth by fibroblasts (1). For intensity, it is known that strong prolonged BXB-ER activation. Using Western blots and cell counting, activation of ERK leads the cells to go in senescence (2). On the differences of ERK signals between activation by serum and other hand, weaker ERK signal leads cellular proliferation. by BXB-ER were compared. As expected, the induction of Even though it remains unclear how the activation of one ERK seemed to be optimal in fibroblasts at various signaling molecule ERK leads to specific cellular responses (3), concentrations of serum, but BXB-ER activation by 100nM accurate regulation of ERK signal could be the key to balance tamoxifen induced robust ERK activation signal which led these responses (1). By regulating the ERK activation signals, cells to go in senescence. To change the cellular outcome, the cells could have totally different cellular outcomes such as activation levels of ERK were adjusted using different senescence or cell growth. For further research of the pathway, concentrations of tamoxifen/estrogen which acutely regulate analysis of ERK activation for the growth induction of murine BXB-ER activation. First, tamoxifen/estrogen concentrations fibroblasts was done. The aim of the research was to see if the were decreased to see the signal intensity and cell growth. By cellular outcomes activated by ERK could be controlled by lowering, it produced the similar growth rates as the control, conditional Raf using different activation kinetics, and to adjust but BXB-ER activation could not induce additional growth. parameters to induce accelerated cell growth in 3T3BXB-ER Second, MEK inhibitor, PD184352, was used to regulate the intensity of ERK activation with various serum concentrations. When ERK activation was inhibited by PD184352 (0.2µM-4µM), the cells grew slower than the control. Then, BXB-ER activation was induced by tamoxifen Murine immortalized fibroblasts (NIH3T3) and engineered 100nM in addition to PD184352.Under the condition, the cells (3T3BXB-ER) were used. 3T3BXBER cells are NIH3T3 additional BXB-ER activation could restore ERK cells, expressing Raf-1 kinase domain-estrogen receptor fusion phosphorylation levels, and also growth rates to the levels proteins, which could be regulated by tamoxifen or estrogen (4). they were before MEK inhibition, but not more. Therefore, The cells were kept in a humidified incubator at 37°C with the cells seemed to control the level of ERK activity very 5.0% carbon dioxide in the DMEM medium supplemented tightly, to be optimal for growth. It is possible that additional with 10% fetal bovine serum (SIGMA), 1% 200mM research should have been completed with smaller intervals L-glutamine (SIGMA), and 1% penicillin streptomycin. for PD184352 concentrations. Although the parameter, which induces accelerated cell growth, was not found with the The cells were counted on the 4th to 5th day of the experiments. research, I showed the trends of how cells react with different Images were taken with Canon IXY 14.1 mega pixels with an activation kinetics. For future research, I wish to find out about the mechanism of how ERK activation gives the specific cellular outcomes by the precise investigation of ERK Samples were loaded to 12% acrylamide gels for sodium dodecyl sulfate polyacrylamide gel electrophoresis. The following antibodies were used.［Primary antibody: P-p44/42 MAPK (T202/Y204) (Cell Signaling) / Anti-MAP Kinase (1) Wellbrock C, et al. Nat Rev Mol Cell Biol. 2004;5:875-886. (ERK-1, 2) (SIGMA), Secondary antibody: antibody rabbit (2) Martin, C, et al. PLoS ONE 2010;5(10):e13322. (Stabilized Peroxidase Conjugated Goat Rabbit (Thermo)) ］ (3) Ebisuya, M, et al. J Cell Sci 2005;118:2997-3002. Luminata Crescendo/Classico Western HRP Substrate (4) Lovric, J., et al. J Biol Chem 1998;273:22848-22855. (Milipore) was used for protein detection.

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