| Summary: | 碩士 === 國立臺灣海洋大學 === 生物科技研究所 === 96 === Activated Ras is one of the most common abnormalities in hematological malignancies. Ras plays an important role in receptor signaling in regulating a diverse cellular process such as proliferation, survival, differentiation and transformation. In contrast to H-, N-, and K-Ras which require ligand-receptor binding for their activation, ERas is constitutively activated and has been shown to be the culprit for the in vivo tumorigenic behavior of ES cells. In addition, ERas specifically interacts with PI3K and activates the downstream effector AKT but not with Raf of the MAPK cascade.
In this research proposal, we plan to investigate whether ERas, like HRasG12V can induce factor-independent growth of a GM-CSF/IL-3 dependent myeloid cell line, FDC-P1. In this study, a retrovirus infection system was used to express either ERas or HRasG12V in the target cells.
We first confirmed previous observation that both ERas and HRasG12V can transform 3T3-L1 fibroblasts using the anchorage- independent growth assay. We observed that ERas and HRasG12V had similar transformation efficiency. However we also observed that the colony size is smaller in the ERas transformed 3T3-L1 cells than those transformed by HRasG12V suggesting a relative lower transforming capability of the ERas protein.
In short-term survival assay after IL-3 withdrawal, both ERas and HRasG12V transduced FDC-P1 cells exhibit increased survival in the absence of IL-3. In this assay, control FDC-P1 cells died within 24 hr after growth factor withdrawal whereas it takes 48 hr for the ERas expressing cells. At the same time point, most of the HRasG12V expressing FDC-P1 cells are viable and the population continues to expand, suggestion factor-independent FDC-P1 cells are present. In addition, ERas and HRasG12V transduced FDC-P1 cells exhibit a moderate increase in the sensitivity of IL-3 as demonstrated by a shift in the dose response curve to IL-3.
Lastly direct abrogation of growth factor dependence has been shown in the HRasG12V transduced FDC-P1 cells. That a 3~4 fold increase in the frequency of factor independent clonogenic cells when the FDC-P1 was transduced with undiluted viral supernatant compared with those treated with 1:16 diluted viral supernatant implicate that additional events may also be involved in the process.
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