The Difference in Biological Properties between Parental and v-Ha-ras Transformed NIH3T3 Cells.
- Author:
Michael LEE
1
;
Jun Ho AHN
;
Ki Hwan EUM
Author Information
1. Department of Biology, College of Natural Sciences, University of Incheon, Incheon, Korea. mikelee@incheon.ac.kr
- Publication Type:Original Article
- Keywords:
v-Ha-ras;
Transformed cells;
Raf-1;
Negative feedback
- MeSH:
Cell Line;
Cell Proliferation;
Genes, ras;
Humans;
Hydroquinones;
Lipids;
NIH 3T3 Cells;
Parents;
Phenotype;
Proteins;
Proto-Oncogene Proteins c-raf;
RNA, Small Interfering;
src-Family Kinases;
Transfection;
Tyrosine
- From:Cancer Research and Treatment
2009;41(2):93-99
- CountryRepublic of Korea
- Language:English
-
Abstract:
PURPOSE: We performed experiments to investigate the change in cellular signaling that occurs during the transformation of a normal cell to a cell capable of cancerous growth, and we did so by using the NIH 3T3 cells that were transformed by transfection with the v-Ha-ras oncogene. MATERIALS AND METHODS: Parental and v-Ha-ras transfected NIH 3T3 cells were chosen as test systems. The siRNA transfections were performed using Lipofectamine 2000. The cell proliferation reagent WST-1 was used for the quantitative determination of cellular proliferation. Immunoblot analysis was performed using the ECL-Plus chemiluminescent system and a KODAK Image Station 4000R. RESULTS: The v-Ha-ras-transformed cells were found to be significantly more resistant to PP2 treatment, which is a potent inhibitor of the Src family tyrosine kinases, than were the parental cells at earlier times after treatment. However, PP2 induced growth arrest and the senescence-like phenotypes in both cell lines after longer treatment. Furthermore, the Raf-1 kinase of the v-Ha-ras-transformed cells was not affected by the expressed level of Sprouty proteins, which are negative regulators of the MAPK pathway, as evidenced by the failure of siRNA-mediated knockdown of Spry4 to activate Raf-1 kinase. Dephostatin (a tyrosine phosphatase inhibitor) effectively inhibited the proliferation of the v-Ha-ras transformed cells, whereas dephostatin had only a small effect on the parental cells' proliferation. This implied an inhibitory role for tyrosine phosphatase that is specific to the signaling pathway in the v-Ha-ras transformed cells. CONCLUSION: Taken together, our results show that the sustained activation of the oncogenic pathways through their resistance to negative feedback regulation might contribute to the transformation of NIH 3T3 cells.
