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Senescence of human fibroblasts induced by oncogenic Raf
1Department of Microbiology and Immunology and G.W. Hooper Foundation, University of California at San Francisco (UCSF), California 94143-0552, USA.
Abstract:
The oncogenes RAS and RAF came to view as agents of neoplastic transformation. However, in normal cells, these genes can have effects that run counter to oncogenic transformation, such as arrest of the cell division cycle, induction of cell differentiation, and apoptosis. Recent work has demonstrated that RAS elicits proliferative arrest and senescence in normal mouse and human fibroblasts. Because the Raf/MEK/MAP kinase signaling cascade is a key effector of signaling from Ras proteins, we examined the ability of conditionally active forms of Raf-1 to elicit cell cycle arrest and senescence in human cells. Activation of Raf-1 in nonimmortalized human lung fibroblasts (IMR-90) led to the prompt and irreversible arrest of cellular proliferation and the premature onset of senescence. Concomitant with the onset of cell cycle arrest, we observed the induction of the cyclin-dependent kinase (CDK) inhibitors p21(Cip1) and p16(Ink4a). Ablation of p53 and p21(Cip1) expression by use of the E6 oncoprotein of HPV16 demonstrated that expression of these proteins was not required for Raf-induced cell cycle arrest or senescence. Furthermore, cell cycle arrest and senescence were elicited in IMR-90 cells by the ectopic expression of p16(Ink4a) alone. Pharmacological inhibition of the Raf/MEK/MAP kinase cascade prevented Raf from inducing p16(Ink4a) and also prevented Raf-induced senescence. We conclude that the kinase cascade initiated by Raf can regulate the expression of p16(Ink4a) and the proliferative arrest and senescence that follows. Induction of senescence may provide a defense against neoplastic transformation when the MAP kinase signaling cascade is inappropriately active.
Insights
Activating Raf-1 in human cells triggers irreversible cell cycle arrest and senescence, mediated by p16(Ink4a) but independent of p53. This MAP kinase pathway activation offers a defense against cancer.
Area of Science:
- Cell Biology
- Oncology
- Molecular Signaling
Background:
- Oncogenes RAS and RAF can induce neoplastic transformation.
- In normal cells, RAS and RAF can paradoxically inhibit proliferation, induce differentiation, and trigger apoptosis.
- RAS activation in fibroblasts causes proliferative arrest and senescence.
Purpose of the Study:
- To investigate the role of the Raf/MEK/MAP kinase cascade in inducing cell cycle arrest and senescence.
- To determine if activated Raf-1 can elicit these effects in human cells.
- To elucidate the specific molecular mechanisms, including the involvement of CDK inhibitors, underlying Raf-induced senescence.
Main Methods:
- Utilized conditionally active forms of Raf-1 in non-immortalized human lung fibroblasts (IMR-90).
- Assessed cell proliferation, senescence markers, and expression of cyclin-dependent kinase (CDK) inhibitors p21(Cip1) and p16(Ink4a).
- Employed HPV16 E6 oncoprotein to ablate p53 and p21(Cip1) expression; used ectopic p16(Ink4a) expression; and applied pharmacological inhibition of the Raf/MEK/MAP kinase cascade.
Main Results:
- Activation of Raf-1 led to prompt, irreversible cell proliferation arrest and premature senescence in IMR-90 cells.
- Cell cycle arrest and senescence were accompanied by the induction of p21(Cip1) and p16(Ink4a).
- Raf-induced cell cycle arrest and senescence occurred independently of p53 and p21(Cip1) but were dependent on p16(Ink4a) and the Raf/MEK/MAP kinase cascade.
Conclusions:
- The Raf/MEK/MAP kinase cascade regulates p16(Ink4a) expression, leading to proliferative arrest and senescence.
- Induction of senescence by this pathway acts as a potential defense mechanism against neoplastic transformation.
- Activated Raf-1 can induce senescence in normal human fibroblasts, highlighting a tumor-suppressive role for this signaling pathway under specific conditions.