Cancer Res Treat.  2009 Jun;41(2):93-99.

The Difference in Biological Properties between Parental and v-Ha-ras Transformed NIH3T3 Cells

Affiliations
  • 1Department of Biology, College of Natural Sciences, University of Incheon, Incheon, Korea. mikelee@incheon.ac.kr

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.

Keyword

v-Ha-ras; Transformed cells; Raf-1; Negative feedback

MeSH Terms

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
Hydroquinones
Lipids
Proteins
Proto-Oncogene Proteins c-raf
RNA, Small Interfering
Tyrosine
src-Family Kinases

Figure

  • Fig. 1 Cell morphology after long-term culture. Actively growing cells were seeded at a density of 104 cells/60-mm dish. The culture medium was replaced with fresh medium and then it was changed twice a week during the following 5 weeks. Morphological transformation was determined under a dissecting microscope. (A) Parental NIH3T3 cells, (B) v-Ha-ras transformed NIH3T3 cells.

  • Fig. 2 The differential inhibitory effect of PP2 on the parental and v-Ha-ras-transfected NIH 3T3 cells. (A) The observed phenotype of the parental and v-Ha-ras-transfected NIH 3T3 cells at 2 and 8-days after PP2 treatment. (B) Each cell line was treated with increasing concentrations of 10 µM of PP2 and then the cells were incubated in 96 well plates for 3 days. The absorbance at 450 nm is expressed as the mean±SD of quadruplicate determinants from one of three representative experiments. *p<0.01 as determined by two-tailed Student's T-test as compared to the vehicle control group.

  • Fig. 3 Differential Raf-1 kinase activation in the parental and v-Ha-ras-transfected NIH 3T3 cells. Subconfluent parental and v-Ha-ras-transfected NIH 3T3 cells were serum-deprived for 24 h. The cells were then treated with 10 µM PP2 for the final 30 min of serum deprivation before treatment with 100 nM PMA and/or 1 mM H2O2 for 5 min. (A) The cell lysates were isolated and Raf-1's electrophoretic mobility was detected by immunoblotting with anti-Raf-1 antibody. (B) The whole cell lysates were analyzed by Western blotting. The phosphorylated forms of MEK-1 were detected with immunoblotting using anti-phospho-MEK-1 antibody. The numbers listed below each band indicate the value quantified by Kodak Molecular Imaging software, and they are expressed as fold change in phosphorylation, and the value observed in the untreated cells was defined as 1.0. The results presented are representative of at least three independent experiments carried out under the described conditions. Inset, the v-Ha-ras-Transformed NIH 3T3 cells were transfected with Spry4 siRNA or a non-targeting control siRNA (mock) for 24 h, and this was followed by treatment with 10 µM PP2 for 30 min. The whole cell lysates were analyzed by Western blotting. The blot was probed with anti-Spry4 antibody to show the siRNA-mediated knockdown of Spry4.

  • Fig. 4 The differential effects of different kinase inhibitors on the cell proliferation of the parental and v-Ha-ras-transfected NIH 3T3 cells. The cells were exposed to the indicated inhibitors for 1 h [60 µM genistein (a tyrosine kinase inhibitor)], 30 min [10 µM PP2 (a Src inhibitor)], 16 h [50 µM AG490 (a EGFR kinase inhibitor)], 4 h [25 µM AG1296 (a PDGFR kinase inhibitor)] or 30 min [1 µM cypermethrin (a PP2B inhibitor), 20 µM dephostatin (a tyrosine phosphatase inhibitor), or 1 µM okadaic acid (a PP2A inhibitor)]. The cells were then washed and incubated in 96 well plates for the indicated days. The absorbance at 450 nm is expressed as the mean±SD of quadruplicate determinants from one of three representative experiments. *p<0.01 as determined by Dunnett's T-test as compared to the vehicle control group.


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