We developed a clonal WI-38hTERT/GFP-RAF1-ER immortal cell line to study RAF-induced senescence of human fibroblasts. Activation of the GFP-RAF1-ER kinase by addition of 4-hydroxy-tamoxifen led to a robust induction of senescence within one population doubling, accompanied by the assembly of heterochromatic foci. At least two pathways contribute in parallel to this senescence leading to the accumulation of p15, p16, p21 and p27 inhibitors of cyclin-dependent kinases (CKIs). Cells that traversed S phase after RAF1 kinase activation experienced a replicative stress manifested by phosphorylation of H2AX and Chk2 and synthesis of p21. However, about half the cells in the population were blocked without passing through S phase and did not show activation of DNA-damage checkpoints. When the cells were cultivated in 5% oxygen, RAF1 activation generated minimal reactive oxygen species, but RAF-induced senescence occurred efficiently in these conditions even in the presence of anti-oxidants or inhibitors of DNA checkpoint pathways. Despite the presence of heterochromatic foci, simultaneous knockdown of p16 and p21 with inactivation of the GFP-RAF1-ER kinase led to rapid reversion of the senescent state with the majority of cells becoming competent for long-term proliferation. These results demonstrate that replicative and oxidative stresses are not required for RAF-induced senescence, and this senescence is readily reversed upon loss of CKIs.

The absence of Tsa1, a key peroxiredoxin that functions to scavenge H2O2 in Saccharomyces cerevisiae, causes the accumulation of a broad spectrum of mutations including gross chromosomal rearrangements (GCRs). Deletion of TSA1 also causes synthetic lethality in combination with mutations in RAD6 and several key genes involved in DNA double-strand break repair. In the present study we investigated the causes of GCRs and cell death in these mutants. tsa1-associated GCRs were independent of the activity of the translesion DNA polymerases , , and Rev1. Anaerobic growth reduced substantially GCR rates of WT and tsa1 mutants and restored the viability of tsa1 rad6, tsa1 rad51, and tsa1 mre11 double mutants. Anaerobic growth also reduced the GCR rate of rad27, pif1, and rad52 mutants, indicating a role of reactive oxygen species in GCR formation in these mutants. In addition, deletion of TSA1 or H 2O2 treatment of WT cells resulted in increased formation of Rad52 foci, sites of repair of multiple DNA lesions. H 2O2 treatment also induced the GCRs. Our results provide in vivo evidence that oxygen metabolism and reactive oxygen species are important sources of DNA damages that can lead to GCRs and lethal effects in S. cerevisiae.dsDNA break ͉ gross chromosomal rearrangement ͉ translesion DNA synthesis ͉ peroxiredoxin M aintaining genome stability is crucial for cell growth and cell survival. Different genetic disorders, including most human cancers, are associated with different forms of genome instability (1-3). One type of genomic instability observed frequently in many cancers is gross chromosomal rearrangements (GCRs), such as translocations, deletions of chromosome arms, interstitial deletions, inversions, amplifications, chromosome fusions, and aneuploidy. Multiple genes and pathways that suppress GCRs have been characterized by using the yeast Saccharomyces cerevisiae as a model system (4-6). These include genes involved in DNA replication, recombination, and repair, cell-cycle checkpoints that function during DNA replication and repair, and pathways involved in telomere maintenance, chromatin assembly, and detoxification of reactive oxygen species (ROS) (refs. 7 and 8 and references therein). The importance of these pathways for suppressing genome instability is further emphasized by their roles in preventing the development of human cancer (7, 9, 10).Although many pathways that function in the suppression of GCRs have been discovered, little is known about the causes and origin of GCRs. It is generally thought that a major cause of DNA damage that leads to mutations is ROS, which are generated as a normal part of oxygen metabolism but are also produced by ionizing radiation, metabolism of exogenous compounds, and pathological processes such as infection and inflammation (11,12). ROS, such as the superoxide radical, H 2 O 2 , and the hydroxyl radical, can attack almost all cell components and can induce many types of DNA damage, including single-stranded DNA breaks and dsDNA breaks (DSB), base and sugar modificat...