The GADD45 inhibition of Cdc2 kinase correlates with GADD45-mediated growth suppression

Cell cycle growth arrest is an important cellular response to genotoxic stress. Gadd45, a p53-regulated stress protein, plays an important role in the cell cycle G(2)-M checkpoint following exposure to certain types of DNA-damaging agents such as UV radiation and methylmethane sulfonate. Recent findings indicate that Gadd45 interacts with Cdc2 protein and inhibits Cdc2 kinase activity. In the present study, a series of Myc-tagged Gadd45 deletion mutants and a Gadd45 overlapping peptide library were used to define the Gadd45 domains that are involved in the interaction of Gadd45 with Cdc2. Both in vitro and in vivo studies indicate that the interaction of Gadd45 with Cdc2 involves a central region of the Gadd45 protein (amino acids 65-84). The Cdc2-binding domain of Gadd45 is also required for Gadd45 inhibition of Cdc2 kinase activity. Sequence analysis of the central Gadd45 region reveals no homology to inhibitory motifs of known cyclin-dependent kinase inhibitors, indicating that the Cdc2-binding and -inhibitory domains on Gadd45 are a novel motif. The peptide containing the Cdc2-binding domain (amino acids 65-84) disrupted the Cdc2-cyclin B1 protein complex, suggesting that dissociation of this complex results from a direct interaction between the Gadd45 and Cdc2 proteins. GADD45-induced cell cycle G(2)-M arrest was abolished when its Cdc2 binding motif was disrupted. Importantly, a short term survival assay demonstrated that GADD45-induced cell cycle G(2)-M arrest correlates with GADD45-mediated growth suppression. These findings indicate that the cell cycle G(2)-M growth arrest mediated by GADD45 is one of the major mechanisms by which GADD45 suppresses cell growth.     

B23 regulates GADD45a nuclear translocation and contributes to GADD45a-induced cell cycle G2-M arrest

Gadd45a is an important player in cell cycle G2-M arrest in response to genotoxic stress. However, the underlying mechanism(s) by which Gadd45a exerts its role in the control of cell cycle progression remains to be further defined. Gadd45a interacts with Cdc2, dissociates the Cdc2-cyclin B1 complex, alters cyclin B1 nuclear localization, and thus inhibits the activity of Cdc2/cyclin B1 kinase. These observations indicate that Gadd45a nuclear translocation is closely associated with its role in cell cycle G2-M arrest. Gadd45a has been characterized as a nuclear protein, but it does not contain a classical nuclear localization signal, suggesting that Gadd45a nuclear translocation might be mediated through different nuclear import machinery. Here we show that Gadd45a associates directly with B23 (nucleophosmin), and the B23-interacting domain is mapped at the central region (61-100 amino acids) of the Gadd45a protein using a series of Myc tag-Gadd45a deletion mutants. Deletion of this central region disrupts Gadd45a association with B23 and abolishes Gadd45a nuclear translocation. Suppression of endogenous B23 through a short interfering RNA approach disrupts Gadd45a nuclear translocation and results in impaired Gadd45a-induced cell cycle G2-M arrest. These findings demonstrate a novel association of B23 and Gadd45a and implicate B23 as an important regulator in Gadd45a nuclear import.     

Control of mitochondrial integrity by Bcl-2 family members and caspase-independent cell death

Programmed cell death (PCD) is essential for normal development and maintenance of tissue homeostasis in multicellular organisms. While it is now evident that PCD can take many different forms, apoptosis is probably the most well-defined cell death programme. The characteristic morphological and biochemical features associated with this highly regulated form of cell death have until recently been exclusively attributed to the caspase family of cysteine proteases. As a result, many investigators affiliate apoptosis with its pivotal execution system, i.e. caspase activation. However, it is becoming increasingly clear that PCD or apoptosis can also proceed in a caspase-independent manner and maintain key characteristics of apoptosis. Mitochondrial integrity is central to both caspase-dependent and-independent cell death. The release of pro-apoptotic factors from the mitochondrial intermembrane space is a key event in a cell's commitment to die and is under the tight regulation of the Bcl-2 family. However, the underlying mechanisms governing the efflux of these pro-death molecules are largely unknown. This review will focus on the regulation of mitochondrial integrity by Bcl-2 family members with particular attention to the controlled release of factors involved in caspase-independent cell death.     

Molecular cloning of rat GADD45gamma, gene induction and its role during neuronal cell death

To study the molecular mechanism of neuronal cell death, we carried out the screening of genes which were induced during the neuronal cell death of neuronal PC12. We cloned the cDNA of rat GADD45gamma, the third member of the GADD45 family. Induction of GADD45gamma mRNA was observed in the neuronal cell death caused by depletion of neurotrophic factor and Ca2+ ionophore treatment. Overexpression of GADD45gamma in superior cervical ganglion neurons caused cell death. These results suggest that GADD45gamma plays an important role in neuronal cell death.     

Bcl-2 family members localize to tobacco chloroplasts and inhibit programmed cell death induced by chloroplast-targeted herbicides

In mammalian cells, apoptosis is often mediated via organelles. While apoptotic-like cell death occurs in plants, the mechanistic details are unresolved. Transgenic tobacco plants have been generated that harbour selected animal anti-apoptotic genes. Subcellular fractionation followed by western blot analysis indicated that chloroplasts serve as a location for these animal anti-apoptotic proteins in addition to the established mitochondrial location. To explore the functional significance of this observation, tobacco plants were treated with three chloroplast-directed herbicides. Wild-type plants died and exhibited features associated with apoptosis. Transgenic plants survived and did not show any apoptotic-like characteristics. Moreover, the herbicide-induced apoptotic-like cell death was light requiring. It was concluded that chloroplasts may be involved in mediating certain types of plant programmed cell death.     

Amyloid fibrils formed by the programmed cell death regulator Bcl-xL

The accumulation of amyloid fibers due to protein misfolding is associated with numerous human diseases. For example, the formation of amyloid deposits in neurodegenerative pathologies is correlated with abnormal apoptosis. We report here the in vitro formation of various types of aggregates by Bcl-xL, a protein of the Bcl-2 family involved in the regulation of apoptosis. Bcl-xL forms aggregates in three states, micelles, native-like fibrils, and amyloid fibers, and their biophysical characterization has been performed in detail. Bcl-xL remains in its native state within micelles and native-like fibrils, and our results suggest that native-like fibrils are formed by the association of micelles. Formation of amyloid structures, that is, nonnative intermolecular β-sheets, is favored by the proximity of proteins within fibrils at the expense of the Bcl-xL native structure. Finally, we provide evidence of a direct relationship between the amyloid character of the fibers and the tertiary-structure stability of the native Bcl-xL. The potential causality between the accumulation of Bcl-xL into amyloid deposits and abnormal apoptosis during neurodegenerative diseases is discussed.     

Coenzyme depletion by members of the aerolysin family of pore-forming toxins leads to diminished ATP levels and cell death

Recent studies demonstrated that a variety of bacterial pore-forming toxins induce cell death through a process of programmed necrosis characterized by the rapid depletion of cellular ATP. However, events leading to the necrosis and depletion of ATP are not thoroughly understood. We demonstrate that ATP-depletion induced by two pore-forming toxins, the Clostridium perfringens epsilon-toxin and the Aeromonas hydrophila aerolysin toxin, is associated with decreased mitochondrial membrane potential and opening of the mitochondrial permeability transition pore. To gain further insight into the toxin-induced metabolic changes contributing to necrosis and depletion of ATP, we analyzed the biochemical profiles of 251 distinct compounds by GC/MS or LC/MS/MS following exposure of a human kidney cell line to the epsilon-toxin. As expected, numerous biochemicals were seen to increase or decrease in response to epsilon-toxin. However, the pattern of these changes was consistent with the toxin-induced disruption of major energy-producing pathways in the cell including disruptions to the beta-oxidation of lipids. In particular, treatment with epsilon-toxin led to decreased levels of key coenzymes required for energy production including carnitine, NAD (and NADH), and coenzyme A. Independent biochemical assays confirmed that epsilon-toxin and aerolysin induced the rapid decrease of these coenzymes or their synthetic precursors. Incubation of cells with NADH or carnitine-enriched medium helped protect cells from toxin-induced ATP depletion and cell death. Collectively, these results demonstrate that members of the aerolysin family of pore-forming toxins lead to decreased levels of essential coenzymes required for energy production. The resulting loss of energy substrates is expected to contribute to dissipation of the mitochondrial membrane potential, opening of the mitochondrial permeability transition pore, and ultimately cell death.     

Expression of cellular oncogene Bcl-xL prevents coronavirus-induced cell death and converts acute infection to persistent infection in progenitor rat oligodendrocytes

Murine coronavirus mouse hepatitis virus (MHV) causes persistent infection of the central nervous system (CNS) in rodents, which has been associated with demyelination. However, the precise mechanism of MHV persistence in the CNS remains elusive. Here we show that the progenitor oligodendrocytes (central glial 4 [CG-4] cells) derived from newborn rat brain were permissive to MHV infection, which resulted in cell death, although viral replication was restricted. Interestingly, treatment with fetal bovine serum or exogenous expression of cellular oncogene Bcl-xL prevented CG-4 cells from MHV-induced cell death. Significantly, overexpression of Bcl-xL alone was sufficient to convert acute to persistent, nonproductive infection in CG-4 cells. This finding indicates that intracellular factors rather than viral components play a critical role in establishing viral persistence in CNS cells. Although viral genomic RNAs continuously persisted in Bcl-xL-expressing CG-4 cells over 10 passages, infectious virus could no longer be isolated beyond 2 passages of the cell. Such a phenomenon resembles the persistent MHV infection in animal CNS. Thus, the establishment of a persistent, nonproductive infection in CG-4 cells may provide a useful in vitro model for studying viral persistence in animal CNS. The data also suggest that direct virus-host cell interaction is one of the underlying mechanisms that regulate viral persistence in CNS cells.     

GATA family members as inducers for cellular reprogramming to pluripotency

Members of the GATA protein family play important roles in lineage specification and transdifferentiation. Previous reports show that some members of the GATA protein family can also induce pluripotency in somatic cells by substituting for Oct4, a key pluripotency-associated factor. However, the mechanism linking lineage-specifying cues and the activation of pluripotency remains elusive. Here, we report that all GATA family members can substitute for Oct4 to induce pluripotency. We found that all members of the GATA family could inhibit the overrepresented ectodermal-lineage genes, which is consistent with previous reports indicating that a balance of different lineage-specifying forces is important for the restoration of pluripotency. A conserved zinc-finger DNA-binding domain in the C-terminus is critical for the GATA family to induce pluripotency. Using RNA-seq and ChIP-seq, we determined that the pluripotency-related gene Sall4 is a direct target of GATA family members during reprogramming and serves as a bridge linking the lineage-specifying GATA family to the pluripotency circuit. Thus, the GATA family is the first protein family of which all members can function as inducers of the reprogramming process and can substitute for Oct4. Our results suggest that the role of GATA family in reprogramming has been underestimated and that the GATA family may serve as an important mediator of cell fate conversion.     

p53-independent induction of GADD45 and GADD153 in mouse lungs exposed to hyperoxia

Previous studies have shown that lungs of adult mice exposed to >95% oxygen have increased terminal deoxyribonucleotidyltransferase dUTP nick end-label staining and accumulate p53, the expression of which increases in cells exposed to DNA-damaging agents. The present study was designed to determine whether hyperoxia also increased expression of the growth arrest and DNA damage (GADD) gene 45 and GADD153, which are induced by genotoxic stress through p53-dependent and -independent pathways. GADD proteins have been shown to inhibit proliferation and stimulate DNA repair and/or apoptosis. GADD45 and GADD153 mRNAs were not detected in lungs exposed to room air but were detected after 48 and 72 h of exposure to hyperoxia. In situ hybridization and immunohistochemistry revealed that hyperoxia increased GADD45 and GADD153 expression in the bronchiolar epithelium and GADD45 expression predominantly in alveolar cells that were morphologically consistent with type II cells. Hyperoxia also increased GADD expression in p53-deficient mice. Terminal deoxyribonucleotidyltransferase dUTP nick end-label staining of lung cells from p53 wild-type and p53-null mice exposed to hyperoxia for 48 h revealed that hyperoxia-induced DNA fragmentation was not modified by p53 deficiency. These studies are consistent with the hypothesis that hyperoxia-induced DNA fragmentation is associated with the expression of GADD genes that may participate in DNA repair and/or apoptosis.     

Bcl-2 family members inhibit oxidative stress-induced programmed cell death in Saccharomyces cerevisiae

Selected antiapoptotic genes were expressed in baker's yeast (Saccharomyces cerevisiae) to evaluate cytoprotective effects during oxidative stress. When exposed to treatments resulting in the generation of reactive oxygen species (ROS), including H(2)O(2), menadione, or heat shock, wild-type yeast died and exhibited apoptotic-like characteristics, consistent with previous studies. Yeast strains were generated expressing nematode ced-9, human bcl-2, or chicken bcl-xl genes. These transformants tolerated a range of oxidative stresses, did not display features associated with apoptosis, and remained viable under conditions that were lethal to wild-type yeast. Yeast strains expressing a mutant antiapoptotic gene (bcl-2 deltaalpha 5-6), known to be nonfunctional in mammalian cells, were unable to tolerate any of the ROS-generating insults. These data are the first report showing CED-9 has cytoprotective effects against oxidative stress, and add CED-9 to the list of Bcl-2 protein family members that modulate ROS-mediated programmed cell death. In addition, these data indicate that Bcl-2 family members protect wild-type yeast from physiological stresses. Taken together, these data support the concept of the broad evolutionary conservation and functional similarity of the apoptotic processes in eukaryotic organisms.     

Unknotting the roles of Bcl-2 and Bcl-xL in cell death

The antiapoptotic Bcl-2 family proteins Bcl-2 and Bcl-xL play important roles in inhibiting mitochondria-dependent extrinsic and intrinsic cell death pathways. It seems that these two proteins have distinct functions for inhibiting extrinsic and intrinsic cell death pathways. The overexpression of Bcl-2 is able to inhibit not only apoptotic cell death but also in part nonapoptotic cell death, which has the role of cell cycle arrest in the G1 phase, which may promote cellular senescence. The overexpression of Bcl-2 may also have the ability to enhance cell death in the interaction of Bcl-xL with other factors. The overexpression of Bcl-xL enhances autophagic cell death when apoptotic cell death is inhibited in Bax(-/-)/Bak(-/-) double knockout cells. This review discusses the previously unexplained aspects of Bcl-2 and Bcl-xL functions associated with cell death, for better understanding of their functions in the regulation.     

Perturbation of cell adhesion and microvilli formation by antisense oligonucleotides to ERM family members

To examine the functions of ERM family members (ezrin, radixin, and moesin), mouse epithelial cells (MTD-1A cells) and thymoma cells (L5178Y), which coexpress all of them, were cultured in the presence of antisense phosphorothioate oligonucleotides (PONs) complementary to ERM sequences. Immunoblotting revealed that the antisense PONs selectively suppressed the expression of each member. Immunofluorescence microscopy of these ezrin, radixin, or moesin "single-suppressed" MTD-1A cells revealed that the ERM family members are colocalized at cell-cell adhesion sites, microvilli, and cleavage furrows, where actin filaments are densely associated with plasma membranes. The ezrin/radixin/moesin antisense PONs mixture induced the destruction of both cell-cell and cell-substrate adhesion, as well as the disappearance of microvilli. Ezrin or radixin antisense PONs individually affected the initial step of the formation of both cell-cell and cell-substrate adhesion, but did not affect the microvilli structures. In sharp contrast, moesin antisense PONs did not singly affect cell-cell and cell-substrate adhesion, whereas it partly affected the microvilli structures. These data indicate that ezrin and radixin can be functionally substituted, that moesin has some synergetic functional interaction with ezrin and radixin, and that these ERM family members are involved in cell-cell and cell-substrate adhesion, as well as microvilli formation.     

GAPDH binds to active Akt,leading to Bcl-xL increase and escape from caspase-independent cell death

Increased glucose catabolism and resistance to cell death are hallmarks of cancers, but the link between them remains elusive. Remarkably, under conditions where caspases are inhibited, the process of cell death is delayed but rarely blocked, leading to the occurrence of caspase-independent cell death (CICD). Escape from CICD is particularly relevant in the context of cancer as apoptosis inhibition only is often not sufficient to allow oncogenic transformation. While most glycolytic enzymes are overexpressed in tumors, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is of particular interest as it can allow cells to recover from CICD. Here, we show that GAPDH, but no other glycolytic enzymes tested, when overexpressed could bind to active Akt and limit its dephosphorylation. Active Akt prevents FoxO nuclear localization, which precludes Bcl-6 expression and leads to Bcl-xL overexpression. The GAPDH-dependent Bcl-xL overexpression is able to protect a subset of mitochondria from permeabilization that are required for cellular survival from CICD. Thus, our work suggests that GAPDH overexpression could induce Bcl-xL overexpression and protect cells from CICD-induced chemotherapy through preservation of intact mitochondria that may facilitate tumor survival and chemotherapeutic resistance.     

Contribution of caveolin-1 alpha and Akt to TNF-alpha-induced cell death

We used retrovirus insertion-mediated random mutagenesis to generate tumor necrosis factor-alpha (TNF-alpha)-resistant lines from L929 cells. Using this approach, we discovered that caveolin-1 alpha is required for TNF-alpha-induced cell death in L929 cells. The need for caveolin-1 alpha in TNF-alpha-induced cell death was confirmed by the restoration of sensitivity to TNF-alpha after ectopic reconstitution of caveolin-1 alpha/beta expression. This caveolin-1 alpha-mutated line was also resistant to H(2)O(2) and staurosporine, but not to lonidamine. HepG2 cells are known to lack endogenous caveolins. HepG2 cells stably transfected with caveolin-1 alpha/beta were found to be much more sensitive to TNF-alpha than either parental cells transfected with caveolin-1 beta or parental cells transfected with an empty vector. In contrast to its extensively documented antiapoptotic effect, the elevated activity of Akt appears to be important in sensitizing caveolin-1-expressing cells to TNF-alpha, since pretreatment of cells with the phosphatidylinositide 3-kinase (PI3K) inhibitor LY-294002 or wortmannin completely blocked PI3K activation and markedly improved the survival of TNF-alpha-treated L929 cells. The survival rates of caveolin-1 alpha-normal and caveolin-1 alpha-deficient L929 cells were comparable after treatment with PI3K inhibitor and TNF-alpha. Similar results were obtained with HepG2 cells that stably expressed caveolin-1 alpha/beta or -beta and parental cells transfected with an empty vector. In summary, our results indicate that caveolin-1 alpha preferentially sensitizes L929 cells to TNF-alpha through the activation of a PI3K/Akt signaling pathway.