The proinflammatory cytokine tumour necrosis factor-alpha (TNF-alpha) regulates immune responses, inflammation and programmed cell death (apoptosis). The ultimate fate of a cell exposed to TNF-alpha is determined by signal integration between its different effectors, including IkappaB kinase (IKK), c-Jun N-terminal protein kinase (JNK) and caspases. Activation of caspases is required for apoptotic cell death, whereas IKK activation inhibits apoptosis through the transcription factor NF-kappaB, whose target genes include caspase inhibitors. JNK activates the transcription factor c-Jun/AP-1, as well as other targets. However, the role of JNK activation in apoptosis induced by TNF-alpha is less clear. It is unknown whether any crosstalk occurs between IKK and JNK, and, if so, how it affects TNF-alpha-induced apoptosis. We investigated this using murine embryonic fibroblasts that are deficient in either the IKKbeta catalytic subunit of the IKK complex or the RelA/p65 subunit of NF-kappaB. Here we show that in addition to inhibiting caspases, the IKK/NF-kappaB pathway negatively modulates TNF-alpha-mediated JNK activation, partly through NF-kappaB-induced X-chromosome-linked inhibitor of apoptosis (XIAP). This negative crosstalk, which is specific to TNF-alpha signalling and does not affect JNK activation by interleukin-1 (IL-1), contributes to inhibition of apoptosis.

Hypoxia and nutrient deprivation are environmental stresses governing the survival and adaptation of tumor cells in vivo. We have identified a novel role for the Rb tumor suppressor in protecting against nonapoptotic cell death in the developing mouse fetal liver, in primary mouse embryonic fibroblasts, and in tumor cell lines. Loss of pRb resulted in derepression of BNip3, a hypoxia-inducible member of the Bcl-2 superfamily of cell death regulators. We identified BNIP3 as a direct target of pRB/E2F-mediated transcriptional repression and showed that pRB attenuates the induction of BNIP3 by hypoxia-inducible factor to prevent autophagic cell death. BNIP3 was essential for hypoxia-induced autophagy, and its ability to promote autophagosome formation was enhanced under conditions of nutrient deprivation. Knockdown of BNIP3 reduced cell death, and remaining deaths were necrotic in nature. These studies identify BNIP3 as a key regulator of hypoxia-induced autophagy and suggest a novel role for the RB tumor suppressor in preventing nonapoptotic cell death by limiting the extent of BNIP3 induction in cells.Programmed cell death plays an important role in normal developmental processes and in eliminating potentially pathological cells from the organism, and increased resistance to apoptotic cell death is a hallmark of cancer (13). The role of nonapoptotic cell death, including autophagic cell death and programmed necrosis, in cancer is less clear (24,30,56). Autophagy is a well-conserved mechanism activated in response to nutrient deprivation, and it involves the catabolic degradation of macromolecules and organelles by autophagosomes to regenerate metabolites for energy and growth (24, 30). Excess or prolonged autophagy can lead to autophagic cell death (29,40), whereas inhibition of autophagy appears to promote necrotic cell death (7,12). In contrast to processes of apoptosis or autophagic cell death that have tumor suppressor functions, necrosis promotes tumor progression through induction of inflammatory responses that initiate regenerative proliferation and invasion (50). Thus, the factors determining whether a cell undergoes a specific type of cell death (apoptotic, autophagic, or necrotic) in response to a given stress are significant for understanding the progression of cancer.The RB tumor suppressor functions as a negative regulator of the cell cycle through inhibition of E2F transcription factors (49), but it plays other less well defined roles in promoting cell survival (5). In particular, loss of pRB has been shown to sensitize tumor cells to apoptosis induced by chemotherapeutic agents, and certain viral oncoproteins promote apoptosis through the inactivation of pRB (5). However, pRB previously has not been implicated in the regulation of nonapoptotic cell death.Mice lacking the function of pRb die in midgestation and exhibit various developmental defects, including increased cell death in the nervous system, lens, and liver (5). Although cell death in the lens and peripheral nervous system likely is due t...

The retinoblastoma tumor suppressor gene plays important roles in cell cycle control, differentiation and survival during development and is functionally inactivated in most human cancers. Early studies using gene targeting in mice suggested a critical role for pRb in erythropoiesis, while more recent experiments have suggested that many of the abnormal embryonic phenotypes in the Rb null mouse result from a defective placenta. To address this controversy and determine whether Rb has cell intrinsic functions in erythropoiesis, we examined the effects of Rb loss on red cell production following acute deletion of pRb in vitro and under different stress conditions in vivo. Under stress conditions, pRb was required to regulate erythroblast expansion and promote red cell enucleation. Acute deletion of Rb in vitro induced erythroid cell cycle and differentiation defects similar to those observed in vivo. These results demonstrate a cell intrinsic role for pRb in stress erythropoiesis and hematopoietic homeostasis that has relevance for human diseases.

SummaryAge-associated changes in apoptotic rates have been observed in a number of different tissues. While the implications of these findings remain unclear, a better understanding of how apoptosis is regulated may further our understanding of the aging process. The role of the JNK pathway in apoptosis has been highly controversial with studies suggesting that it plays a pro-apoptotic, antiapoptotic or no role in this process. Here we discuss what is currently known about JNK's role in apoptosis, highlighting recent findings regarding NF-κ κ κ κ B-mediated inhibition of JNK activation and its impact on TNF-α α α α induced apoptosis.

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Basic fibroblast growth factor (bFGF) induces cell death in cells of the Ewing's sarcoma family of tumors in vivo and in vitro.In this study we demonstrate that this is dependent on the rapid and sustained activation of (ESFT) 1 encompasses a group of malignancies, including Ewing's sarcoma, Askin's tumor of the chest wall, and peripheral primitive neuroectodermal tumor, which are thought to be of neural histogenesis (1-3). ESFT exhibit a common genetic rearrangement involving fusion of the 5Ј end of the EWS gene on chromosome 22 to the 3Ј portion of members of the Ets gene family of transcription factors. In over 90% of cases the Ets gene family member is fused to the Fli1 gene located on chromosome 11 (4). This results in the generation of a fusion gene, the protein product of which has been implicated in development of the transformed ESFT phenotype (5-7). ESFT typically arise in the bone or soft tissue of adolescents and young adults, ϳ15-30% of patients presenting with metastatic disease. The outcome for this group of patients is particularly poor despite the use of aggressive therapeutic regimes, emphasizing the need for new therapeutic strategies.A role for autocrine and/or paracrine growth factor survival loops in ESFT is well documented; the blockade of insulin-like growth factor/insulin-like growth factor receptor 1 (8 -11) or stem cell factor (SCF)/c-Kit (12, 13) circuits results in a decrease in ESFT cell number in both in vitro and in vivo models. Previous studies (14) have also suggested that a basic fibroblast growth factor (bFGF)/fibroblast growth factor receptor autocrine/paracrine survival loop may be important for the survival and proliferation of ESFT. However, we have found no evidence of such a survival loop, and we demonstrated recently that treatment of ESFT with bFGF results in the up-regulation of the death receptor p75NTR and induction of cell death (15, 16). The intracellular signaling pathways leading to the induction of cell death following exposure of ESFT cells to bFGF are unknown. Although our preliminary results have shown that incubation of ESFT cells with bFGF causes phosphorylation of fibroblast growth factor receptor 1 and activation of the downstream signaling molecules Ras and ERK (16), whether these events are important effectors of bFGF-induced cell death is not clear. Following receptor activation, phosphorylated tyrosines function as binding sites for a number of downstream adapter and signaling proteins, including the docking protein FRS2 that recruits several signal transduction molecules leading to activation of the mitogen-activated protein kinase (MAPK) cascade and the phosphatidylinositol 3-kinase-AKT anti-apoptotic pathway (17, 18). Recruitment of guanine nucleotide exchange factors (e.g. hSOS) leads to the conversion of the small GTPase Ras from an inactive GDP-bound state to an active GTP-bound state and activation of the extracellular signal-regulated kinase (ERK) pathway (19). Activation of the Ras-ERK pathway has been shown to mediate such diverse cellular...

The mechanism of bFGF-induced cell death in tumours of the Ewing's sarcoma family (ESFT) has been investigated. bFGF-induces phosphorylation of FGFr 1 and activation of Ras/ERK in ESFT cells that die when exposed to bFGF. Induction of cell death was associated with activation of both initiator (caspases-2, -8 and -10) and e ector (caspases-3, -6 and -7) caspases. Moreover, the general caspase inhibitor Z-VAD-FMK protected cells from bFGF-induced cell death. After treatment with bFGF, a loss of mitochondrial transmembrane potential was accompanied by down-regulation of Bcl-2. However, the observed cell death was not associated with release of cytochrome c from the mitochondria. Furthermore, expression of wild-type p53 was not required for bFGF-induced cell death. These observations suggest that bFGF-induced cell death may be mediated through a cell death receptor mechanism, supported by up-regulation of the p75 neurotrophin receptor. bFGF-induced cell death was associated with up-regulation of p21 and p53, down-regulation of PCNA and cyclin A and a decrease in active pRb1, changes consistent with accumulation of cells in G1. These data demonstrate that bFGF-induced cell death is e ected through a caspase-dependent and p53-independent mechanism, that may be mediated through a cell death receptor pathway.

Definitive erythropoiesis occurs in islands composed of a central macrophage in contact with differentiating erythroblasts. Erythroid maturation including enucleation can also occur in the absence of macrophages both in vivo and in vitro. We reported previously that loss of Rb induces cell-autonomous defects in red cell maturation under stress conditions, while other reports have suggested that the failure of Rb-null erythroblasts to enucleate is due to defects in associated macrophages. Here we show that erythropoietic islands are disrupted by hypoxic stress, such as occurs in the Rb-null fetal liver, that Rb ؊/؊ macrophages are competent for erythropoietic island formation in the absence of exogenous stress and that enucleation defects persist in Rb-null erythroblasts irrespective of macrophage function.