Accelerated cellular senescence (ACS) has been described for tumor cells treated with chemotherapy and radiation. Following exposure to genotoxins, tumor cells undergo terminal growth arrest and adopt morphologic and marker features suggestive of cellular senescence. ACS is elicited by a variety of chemotherapeutic agents in the p53-null, p16-deficient human non-small cell H1299 carcinoma cells. After 10 to 21 days, infrequent ACS cells (1 in 10 6 ) can bypass replicative arrest and reenter cell cycle. These cells express senescence markers and resemble the parental cells in their transcription profile. We show that these escaped H1299 cells overexpress the cyclindependent kinase Cdc2/ /Cdk1. The escape from ACS can be disrupted by Cdc2/ /Cdk1 kinase inhibitors or by knockdown of Cdc2/ /Cdk1 with small interfering RNA and can be promoted by expression of exogenous Cdc2/ /Cdk1. We also present evidence that ACS occurs in vivo in human lung cancer following induction chemotherapy. Viable tumors following chemotherapy also overexpress Cdc2/ /Cdk1. We propose that ACS is a mechanism of in vivo tumor response and that mechanisms aberrantly up-regulate Cdc2/ /Cdk1 promotes escape from the senescence pathway may be involved in a subset of tumors and likely accounts for tumor recurrence/ /progression. (Cancer Res 2005; 65(7): 2795-803)

Therapy‐induced accelerated cellular senescence (ACS) is a reversible tumor response to chemotherapy that is likely detrimental to the overall therapeutic efficacy of cancer treatment. To further understand the mechanism by which cancer cells can escape the sustained cell cycle arrest in ACS, we established a tissue culture model, in which the p53‐null NCI‐H1299 cells can be induced into senescence by an abbreviated exposure to a chemotherapeutic agent. Previously, we have reported that senescent cells overexpress Cdc2/Cdk1 when they bypassed the prolonged arrest and their viability is dependent on Cdc2/Cdk1 kinase activity. In our study, we show that human survivin is the immediate downstream effector of the Cdc2/Cdk1 mediated survival signal. Survivin cooperates with Cdc2/Cdk1 to inhibit apoptosis following chemotherapy and promote senescence escape. Using HIV‐1 TAT peptides to disrupt survivin phosphorylation by Cdc2/Cdk1, we also found that phosphorylated survivin is necessary both for the escape of senescent cells and for maintenance of subsequent viability after bypassing senescence. These results further propose survivin as an important determinant of senescence reversibility and as a putative molecular target to enforce cell death in ACS.

The growth arrest and DNA damage-inducible protein (GADD34) mediates growth arrest and apoptosis in response to DNA damage, negative growth signals, and protein malfolding. GADD34 binds to protein phosphatase-1 (PP1) and can attenuate translational elongation of key transcriptional factors through dephosphorylation of eukaryotic initiation factor-2␣. We reported previously that the human trithorax leukemia fusion protein (HRX) can bind to GADD34 and abrogate GADD34-mediated apoptosis in response to UV irradiation. We found that hSNF5/INI1, a component of the hSWI/SNF chromatin remodeling complex, also binds to GADD34 and can coexist with GADD34 and HRX fusion proteins as a trimolecular complexes in vivo. In the present report, we demonstrate that hSNF5/INI1 binds to GADD34 in part through the PP1 docking site within a domain homologous to herpes simplex virus-1 ICP34.5. We found that hSNF5/INI1 can bind PP1 independently and weakly stimulate its phosphatase activity in solution and in complex with GADD34. hSNF5/INI1 and PP1 do not compete for binding to GADD34 but rather form a stable heterotrimeric complex with GADD34. We also show that Epstein-Barr nuclear protein 2, which binds hSNF5/INI1, can disrupt hSNF5/INI1 binding to GADD34 and partially reverse the GADD34-mediated growth suppression function in Ha-ras expressing HIH-3T3 (3T3-ras) cells. These results implicate hSNF5/INI1 in the function of GADD34 and suggest that hSNF5/INI1 may regulate PP1 activity in vivo. GADD341 is a growth arrest and DNA damage-inducible gene (1), whose level of transcript is increased in response to a variety of agents that elicit genomic damage and apoptosis (2, 3) as well as by amino acid deprivation and by agents that lead to protein malfolding in the endoplasmic reticulum (4). The transcriptional regulation of GADD34 has been shown to be independent of functional p53 (2). At its carboxyl terminus, GADD34 harbors a highly conserved domain homologous to ICP34.5 of HSV1, a virulence factor that blocks the premature shut off of protein synthesis in HSV1-infected neuroblastoma cells and may interfere with apoptosis (5, 6). Although the homologous domain of murine GADD34 (MyD116) can supply the anti-apoptotic functions of viral ICP34.5 (7), mammalian GADD34 likely functions to facilitate both growth arrest and apoptosis in response to DNA damage and other cellular stresses (3,8,9).Recently both viral ICP34.5 and mammalian GADD34 proteins have been shown to regulate the activity of protein phosphatase 1 (PP1) in vitro (10, 11). ICP34.5 coexists in a high molecular weight complex with PP1 in cellular extracts derived from HSV1-infected HeLa cells (10). Mutations in either ICP34.5 or GADD34 which disrupt their binding to PP1 also impair PP1 activity and the protein malfolding response (4, 12). Recent evidence suggests that the translational elongation factor eIF2␣ is a likely biological substrate for PP1 (10 -12). eIF2␣ can be phosphorylated by several serine/threonine kinases, including the double-stranded RNA-activated kinase that i...

3The cellular stress response protein GADD34 mediates growth arrest and apoptosis in response to DNA damage, negative growth signals, and protein malfolding. GADD34 binds to protein phosphatase PP1 and can attenuate the translational elongation of key transcriptional factors through dephosphorylation of eukaryotic initiation factor 2␣ (eIF2␣). Recently, we reported the involvement of human SNF5/INI1 (hSNF5/INI1) protein in the functions of GADD34 and showed that hSNF5/INI1 binds GADD34 and stimulates the bound PP1 phosphatase activity. To better understand the regulatory and functional mechanisms of GADD34, we undertook a yeast two-hybrid screen with full-length GADD34 as bait in order to identify additional protein partners of GADD34. We report here that human cochaperone protein BAG-1 interacts with GADD34 in vitro and in SW480 cells treated with the proteasome inhibitor z-LLL-B to induce apoptosis. Two other proteins, Hsp70/ Hsc70 and PP1, associate reversibly with the GADD34-BAG-1 complex, and their dissociation is promoted by ATP. BAG-1 negatively modulates GADD34-bound PP1 activity, and the expression of BAG-1 isoforms can also mask GADD34-mediated inhibition of colony formation and suppression of transcription. Our findings suggest that BAG-1 may function to suppress the GADD34-mediated cellular stress response and support a role for BAG-1 in the survival of cells undergoing stress.The eventual fate of cells following exposure to genotoxic stress is determined by signaling from competing pathways favoring either death or survival. The subsequent down-regulation of both p53-dependent and -independent apoptosis pathways may be key to both cell survival and oncogenesis. The growth arrest and DNA damage-inducible protein GADD34 mediates growth arrest and apoptosis in response to stress signals elicited by genotoxic stress, amino acid deprivation, and protein malfolding at the endoplasmic reticulum (21,22,35). The GADD34 transcript is stabilized under conditions of cellular stress, leading to the observed rise in its mRNA level independent of cellular p53 status (24).The GADD34 protein harbors a highly conserved carboxyterminal peptide domain homologous to herpes simplex virus type 1 (HSV-1) ICP34.5, a virulence factor that blocks the premature shutoff of protein synthesis in HSV-1-infected neuroblastoma cells and allows HSV-1-infected cells to circumvent apoptosis (6,10,11,19). Both ICP34.5 and mammalian GADD34 engage protein phosphatase-1 (PP1) and target dephosphorylation of the eukaryotic translation elongation factor eIF2␣ (13,20). In HSV-1-infected cells, this molecular event enables protein synthesis (19). In mammalian cells, the dephosphorylation attenuates eIF2␣-mediated induction of stress-responsive genes and has been proposed in feedback regulation of cellular stress response initiated by protein malfolding at the endoplasmic reticulum (35).The mechanism and regulation of GADD34-mediated growth suppression and apoptosis remain poorly understood.Recently we reported the association of the human SNF5...