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Induction of cell proliferation in quiescent NIH 3T3 cells by oncogenic c-Raf-1
1Institut für Medizinische Strahlenkunde und Zellforschung, University of Würzburg, Germany.
Abstract:
The c-Raf-1 kinase is activated by different mitogenic stimuli and has been shown to be an important mediator of growth factor responses. Fusion of the catalytic domain of the c-Raf-1 kinase with the hormone binding domain of the estrogen receptor (deltaRaf-ER) provides a hormone-regulated form of oncogenic activated c-Raf-1. We have established NIH 3T3 cells stably expressing a c-Raf-1 deletion mutant-estrogen receptor fusion protein (c-Raf-1-BxB-ER) (N-BxB-ER cells). The transformed morphology of these cells is dependent on the presence of the estrogen antagonist 4-hydroxytamoxifen. Addition of 4-hydroxytamoxifen to N-BxB-ER cells arrested by density or serum starvation causes reentry of these cells into cell proliferation. Increases in the cell number are obvious by 24 h after activation of the oncogenic c-Raf-1 protein in confluent cells. The onset of proliferation in serum-starved cells is further delayed and takes about 48 h. In both cases, the proliferative response of the oncogenic c-Raf-1-induced cell proliferation is weaker than the one mediated by serum and does not lead to exponential growth. This is reflected in a markedly lower expression of the late-S- and G2/M-phase-specific cyclin B protein and a slightly lower expression of the cyclin A protein being induced at the G1/S transition. Oncogenic activation of c-Raf-1 induces the expression of the heparin binding epidermal growth factor. The Jnk1 kinase is putatively activated by the action of the autocrine growth factor. The kinetics of Jnk1 kinase activity is delayed and occurs by a time when we also detect DNA synthesis and the expression of the S-phase-specific cyclin A protein. This finding indicates that oncogenic activation of the c-Raf-1 protein can trigger the entry into the cell cycle without the action of the autocrine growth factor loop. The activation of the c-Raf-1-BxB-ER protein leads to an accumulation of high levels of cyclin D1 protein and a repression of the p27Kip1 cyclin-dependent kinase inhibitor under all culture conditions tested.
Insights
Oncogenic c-Raf-1 activation in NIH 3T3 cells drives cell proliferation and cyclin D1 expression. This oncogenic Raf protein can initiate cell cycle entry independently of autocrine growth factors.
Area of Science:
- Cell Biology
- Molecular Oncology
- Signal Transduction
Background:
- c-Raf-1 kinase is a key mediator of mitogenic stimuli and growth factor responses.
- Oncogenic activation of c-Raf-1 plays a crucial role in cell proliferation and transformation.
- Understanding the precise mechanisms of c-Raf-1-driven cell cycle regulation is vital for cancer research.
Purpose of the Study:
- To investigate the role of a hormone-regulated oncogenic c-Raf-1 fusion protein (c-Raf-1-BxB-ER) in NIH 3T3 cell proliferation.
- To elucidate the downstream signaling events and cell cycle kinetics induced by oncogenic c-Raf-1 activation.
- To determine if oncogenic c-Raf-1 can trigger cell cycle entry independently of autocrine growth factor loops.
Main Methods:
- Establishment of NIH 3T3 cells stably expressing the c-Raf-1-BxB-ER fusion protein (N-BxB-ER cells).
- Utilizing the estrogen antagonist 4-hydroxytamoxifen to activate the oncogenic c-Raf-1 protein.
- Monitoring cell proliferation, cell cycle progression, and expression of key cell cycle regulatory proteins (cyclins, p27Kip1) and growth factors (HB-EGF).
- Assessing Jnk1 kinase activity and its correlation with DNA synthesis.
Main Results:
- Activation of oncogenic c-Raf-1 by 4-hydroxytamoxifen induced cell proliferation in density-arrested and serum-starved N-BxB-ER cells.
- The proliferative response was weaker than serum-mediated growth, with delayed onset in serum-starved cells.
- Oncogenic c-Raf-1 activation led to increased cyclin D1, decreased cyclin A and B protein expression, and repressed p27Kip1.
- Heparin-binding epidermal growth factor (HB-EGF) expression was induced, and Jnk1 kinase activity correlated with DNA synthesis, suggesting c-Raf-1 can initiate cell cycle entry independently of autocrine loops.
Conclusions:
- Hormone-regulated oncogenic c-Raf-1 can induce cell cycle re-entry and proliferation in NIH 3T3 cells.
- Oncogenic c-Raf-1 activation influences the expression of cyclins and cyclin-dependent kinase inhibitors, modulating cell cycle progression.
- The findings demonstrate that oncogenic c-Raf-1 can initiate cell cycle entry without requiring an autocrine growth factor loop, highlighting its potent oncogenic potential.