Interferons are cytokines with potent antiviral and antiproliferative activities. We report that although a transient exposure to ␤-interferon induces a reversible cell cycle arrest, a sustained treatment triggers a p53-dependent senescence program. ␤-Interferon switched on p53 in two steps. First, it induced the acetylation of p53 at lysine 320 and its dephosphorylation at serine 392 but not p53 activity. Later on, it triggered a DNA signaling pathway, the phosphorylation of p53 at serine 15 and its transcriptional activity. In agreement, ␤-interferon-treated cells accumulated ␥-H2AX foci and phosphorylated forms of ATM and CHK2. The DNA damage signaling pathway was activated by an increase in reactive oxygen species (ROS) induced by interferon and was inhibited by the antioxidant N-acetyl cysteine. More important, RNA interference against ATM inhibited p53 phosphorylation at serine 15, p53 activity and senescence in response to ␤-interferon. ␤-Interferon-induced senescence was more efficient in cells expressing either, p53, or constitutive allele of ERK2 or RasV12. Hence, ␤-interferon-induced senescence targets preferentially cells with premalignant changes. INTRODUCTIONMany cell types are able to enter a stable and viable postmitotic state in response to oncogenic stresses such as DNA damage, short telomeres, and certain oncogenes. This condition, known as cellular senescence, is regulated by tumor suppressors such as p53, RB, p16INK4a , p19 ARF , and PML and engages a specific gene expression program (Ferbeyre, 2002;Lowe et al., 2004;Shay and Roninson, 2004). By preventing the expansion of potentially malignant cells, senescence may act as a barrier to tumor formation. Several signaling pathways must connect different stressors to the senescence program. For example, short telomeres or DNA damage activate the senescence program through the checkpoint proteins ATM, ATR, CHK1, and CHK2 (d'Adda di Fagagna et al., 2003;Herbig et al., 2004;Zglinicki et al., 2005). On the other hand, oncogenic stresses do not regulate senescence through telomere shortening and they may signal to senescence regulators through the production of reactive oxygen species (Lee et al., 1999;Wu et al., 2004;Catalano et al., 2005). In addition, it is established that some senescence regulators (p53, PML, and IFI16) are also targets of interferon-stimulated transcription factors (Lavau et al., 1995;Stadler et al., 1995;Takaoka et al., 2003;Xin et al., 2003Xin et al., , 2004de Stanchina et al., 2004). Accordingly, senescence prevented ras-transformation in wild-type fibroblasts, but not in fibroblasts from mice lacking the interferon regulated transcription factor IRF1 (Tanaka et al., 1994). Together, these studies suggest that signaling through the interferon pathway may play a role in senescence.Interferons comprise a family of cytokines with antiviral and antiproliferative activity. They include the type I interferon family (mainly ␣ and ␤-interferon) and type II or ␥-interferon (Taniguchi and Takaoka, 2002). Type-I interferon, usually pr...

SOCS1 is lost in many human tumors, but its tumor suppression activities are not well understood. We report that SOCS1 is required for transcriptional activity, DNA binding, and serine 15 phosphorylation of p53 in the context of STAT5 signaling. In agreement, inactivation of SOCS1 disabled p53-dependent senescence in response to oncogenic STAT5A and radiation-induced apoptosis in T cells. In addition, SOCS1 was sufficient to induce p53-dependent senescence in fibroblasts. The mechanism of activation of p53 by SOCS1 involved a direct interaction between the SH2 domain of SOCS1 and the N-terminal transactivation domain of p53, while the C-terminal domain of SOCS1 containing the SOCS Box mediated interaction with the DNA damage-regulated kinases ATM/ATR. Also, SOCS1 colocalized with ATM at DNA damage foci induced by oncogenic STAT5A. Collectively, these results add another component to the p53 and DNA damage networks and reveal a mechanism by which SOCS1 functions as a tumor suppressor.

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Saccharomyces cerevisiae transport protein particle (TRAPP) is a family of related multisubunit complexes required for endoplasmic reticulum-to-Golgi transport (TRAPP I), endosome-to-Golgi transport (TRAPP II) or cytosol to vacuole targeting (TRAPP III). To gain insight into the relationship between these complexes, we generated random and targeted mutations in the Trs23p core subunit. Remarkably, at physiological salt concentrations only two peaks (TRAPP I and a high molecular weight peak) are detected in wild-type cells. As the salt was raised, the high molecular weight peak resolved into TRAPP II and III peaks. Deletion of a Saccharomycotina-specific domain of Trs23p resulted in destabilization of TRAPP I but had no effect on TRAPP II or III. This mutation had no observable growth phenotype, normal levels of Ypt1p-directed guanine nucleotide exchange factor activity in vivo and did not display any in vivo nor in vitro blocks in membrane traffic. Biochemical analysis indicated that TRAPP I could be produced from the TRAPP II/III peak in vitro by increasing the salt concentration. Our data suggest that the SMS domain of Trs23p is responsible for the in vitro appearance of TRAPP I in S. cerevisiae. The implications of these findings are discussed.

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae exists in either a haploid or diploid state. The ability to combine alleles from two haploids and the ability to introduce modifications to the genome requires the production and dissection of asci. Asci production from haploid cells begins with the mating of two yeast haploid strains with compatible mating types to produce a diploid strain. This can be accomplished in a number of ways either on solid medium or in liquid. It is advantageous to select for the diploids in medium that selectively promotes their growth compared to either of the haploid strains. The diploids are then allowed to sporulate on nutrient-poor medium to form asci, a bundle of four haploid daughter cells resulting from meiotic reproduction of the diploid. A mixture of vegetative cells and asci is then treated with the enzyme zymolyase to digest away the membrane sac surrounding the ascospores of the asci. Using micromanipulation with a microneedle under a dissection microscope one can pick up individual asci and separate and relocate the four ascopores. Dissected asci are grown for several days and tested for the markers or alleles of interest by replica plating onto appropriate selective media.