Ras Proteins Induce Senescence by Altering the Intracellular Levels of Reactive Oxygen Species

doi:10.1074/jbc.274.12.7936

Ras Proteins Induce Senescence by Altering the Intracellular Levels of Reactive Oxygen Species*

From the ^‡Cardiology Branch and the ^¶Pathology Section, NHLBI and the ^‖Laboratory of Molecular Growth Regulation, NICHD, National Institutes of Health, Bethesda, Maryland 20892

Abstract

Human diploid fibroblasts eventually lose the capacity to replicate in culture and enter a viable but nonproliferative state of senescence. Recently, it has been demonstrated that retroviral-mediated gene transfer into primary fibroblasts of an activated ras gene (V12ras) rapidly accelerates development of the senescent phenotype. Using thisin vitro system, we have sought to define the mediators of Ras-induced senescence. We demonstrate that expression of V12Ras results in an increase in intracellular and in particular, mitochondrial reactive oxygen species. The ability of V12Ras to induce growth arrest and senescence is shown to be partially inhibited by coexpression of an activated rac1 gene. A more dramatic rescue of V12Ras-expressing cells is demonstrated when the cells are placed in a low oxygen environment, a condition in which reactive oxygen species production is inhibited. In addition, in a 1% oxygen environment, Ras is unable to trigger an increase in the level of the cyclin-dependent kinase inhibitor p21 or to activate the senescent program. Under normoxic (20% O₂) conditions, the V12Ras senescent phenotype is demonstrated to be unaffected by scavengers of superoxide but rescued by scavengers of hydrogen peroxide. These results suggest that in normal diploid cells, Ras proteins regulate oxidant production and that a rise in intracellular H₂O₂ represents a critical signal mediating replicative senescence.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵§ Supported by the Howard Hughes Medical Institute National Institutes of Health Research Scholars program.
↵** To whom correspondence should be addressed: Cardiology Branch, NHLBI, National Institutes of Health, 10 Center Dr., Bethesda, MD 20892-1650. Tel.: 301-402-4081; Fax: 301-402-0888; E-mail:finkelt{at}gwgate.nhlbi.nih.gov.
↵2 A. C. Lee and T. Finkel, unpublished observations.
Abbreviations:

ROS

reactive oxygen species

β-gal

β-galactosidase

DCF

dichlorodihydrofluorescin diacetate

FACS

fluorescence-activated cell sorter

MnTMPyP

Mn(111) tetrakis 1-methyl 4-pyridyl porphyrin pentachloride

NAC

N-acetylcysteine

SOD

superoxide dismutase

DHR123

dihydrorhodamine 123

IL

interleukin
- Received September 1, 1998.
- Revision received November 16, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.