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H-ras and raf-1 cooperate in transformation of NIH3T3 fibroblasts
A Cuadrado1, J T Bruder, M A Heidaran
1Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892.
Abstract:
We examined the effect of overexpression of growth factor-regulated second messenger enzymes, alone and in combination, on transformation of NIH3T3 cells. Signal transducers included phospholipase C-gamma (PLC-gamma), protein kinase C-gamma (PKC-gamma), and two proto-oncogenes, c-H-ras and c-raf-1. Three of these proteins, PLC-gamma, PKC-gamma and Raf-1, did not transform NIH3T3 cells alone or in combination. c-H-ras, which under its own promoter control has low transforming activity, also did not cooperate with PLC-gamma or PKC-gamma. In contrast, the combination of normal or oncogenic p21 H-Ras with the Raf-1 kinase dramatically increased transformation efficiency. The level of Ras protein required for transformation was reduced in Raf-1 co-transfectants, implying that, at low levels of p21 Ras, p74 Raf-1 is rate limiting. As transformation by Ras depends on jun-mediated transcriptional events, we also examined H-ras and c-raf-1 cooperation in transcriptional transactivation of TPA-responsive element (TRE)-dependent reporters. Like the H-ras/c-raf-1 cooperation in transformation, we observed this synergistic stimulation of TRE-dependent transcription. This pathway for transformation and transcriptional activation by increased levels of normal Ras and Raf may be important in tumors that show overexpression but lack mutationally activated forms of these two proto-oncogenes.
Insights
The combination of Ras and Raf-1 proteins significantly enhances NIH3T3 cell transformation and gene transcription. This Ras-Raf interaction, crucial for cell growth, may play a role in certain cancers.
Area of Science:
- Molecular Biology
- Cell Biology
- Oncology
Background:
- Growth factor signaling pathways regulate cell growth and transformation.
- Proto-oncogenes like Ras and Raf are key players in cell signaling and cancer development.
- Understanding the interplay between signaling molecules is crucial for cancer research.
Purpose of the Study:
- To investigate the transforming potential of specific growth factor-regulated enzymes and proto-oncogenes.
- To determine the cooperative effects of phospholipase C-gamma (PLC-gamma), protein kinase C-gamma (PKC-gamma), c-H-ras, and c-raf-1 on NIH3T3 cell transformation.
- To explore the impact of Ras and Raf-1 on transcriptional activation.
Main Methods:
- NIH3T3 cells were transfected with genes encoding PLC-gamma, PKC-gamma, c-H-ras, and c-raf-1, alone and in combination.
- Cell transformation efficiency was assessed.
- Transcriptional transactivation of TPA-responsive element (TRE)-dependent reporters was examined to study H-ras and c-raf-1 cooperation.
Main Results:
- PLC-gamma, PKC-gamma, and Raf-1 alone or in combination did not induce cell transformation.
- c-H-ras showed low transforming activity and did not cooperate with PLC-gamma or PKC-gamma.
- Co-expression of Ras and Raf-1 synergistically increased NIH3T3 cell transformation and TRE-dependent transcriptional activation.
- Lower levels of Ras protein were required for transformation when co-expressed with Raf-1, indicating Raf-1 is rate-limiting at low Ras levels.
Conclusions:
- The combination of Ras and Raf-1 is a potent driver of cell transformation and transcriptional activation.
- This Ras-Raf pathway may be significant in tumors with overexpression but not necessarily activating mutations of these proto-oncogenes.
- Further research into the Ras-Raf interaction could offer insights into cancer mechanisms and therapeutic strategies.