mTORC2/AKT/HSF1/HuR constitute a feed-forward loop regulating Rictor expression and tumor growth in glioblastoma.

Overexpression of Rictor has been demonstrated to result in increased mechanistic target of rapamycin C2 (mTORC2) nucleation and activity leading to tumor growth and increased invasive characteristics in glioblastoma multiforme (GBM). However, the mechanisms regulating Rictor expression in these tumors is not clearly understood. In this report, we demonstrate that Rictor is regulated at the level of mRNA translation via heat-shock transcription factor 1 (HSF1)-induced HuR activity. HuR is shown to directly bind the 3' untranslated region of the Rictor transcript and enhance translational efficiency. Moreover, we demonstrate that mTORC2/AKT signaling activates HSF1 resulting in a feed-forward cascade in which continued mTORC2 activity is able to drive Rictor expression. RNAi-mediated blockade of AKT, HSF1 or HuR is sufficient to downregulate Rictor and inhibit GBM growth and invasive characteristics in vitro and suppress xenograft growth in mice. Modulation of AKT or HSF1 activity via the ectopic expression of mutant alleles support the ability of AKT to activate HSF1 and demonstrate continued HSF1/HuR/Rictor signaling in the context of AKT knockdown. We further show that constitutive overexpression of HuR is able to maintain Rictor expression under conditions of AKT or HSF1 loss. The expression of these components is also examined in patient GBM samples and correlative associations between the relative expression of these factors support the presence of these signaling relationships in GBM. These data support a role for a feed-forward loop mechanism by which mTORC2 activity stimulates Rictor translational efficiency via an AKT/HSF1/HuR signaling cascade resulting in enhanced mTORC2 activity in these tumors.

Delayed adjustable sutures: a multicentred clinical review.

PURPOSE: To characterise the results of the largest patient series to date undergoing closed conjunctival delayed adjustable suture techniques. METHODS: A multicentre retrospective review of 440 operations (patients aged 10-91 years) by five surgeons at four centres were evaluated for surgical outcomes associated with the delayed adjustable suture technique. RESULTS: 26% (116 of 440) of all patients required postoperative manipulation, with individual surgeon rates ranging from 13% to 56%. The majority of these patients did not complain of diplopia in target gaze and/or had satisfactory cosmetic improvement as evaluated at the 1-3 months follow-up visit (84%). Transient complications included dellen, poor conjunctival appearance, filamentary keratitis, infection, granuloma, exposed suture and corneal abrasion. Serious complications were rare. CONCLUSIONS: This large multicentred series characterises the closed conjunctival delayed adjustable suture technique for the correction of strabismus. It may present some significant advantages to more traditional adjustable suture techniques.

The von Hippel-Lindau protein sensitizes renal carcinoma cells to apoptotic stimuli through stabilization of BIM(EL).

von Hippel-Lindau (VHL) disease is caused by germ-line mutations in the VHL tumor suppressor gene and is the most common cause of inherited renal cell carcinoma (RCC). Mutations in the VHL gene also occur in a large majority of sporadic cases of clear-cell RCC, which have high intrinsic resistance to chemotherapy and radiotherapy. Here we show that VHL-deficient RCC cells express lower levels of the proapoptotic Bcl-2 family protein BIM(EL) and are more resistant to etoposide and UV radiation-induced death compared to the same cells stably expressing the wild-type VHL protein (pVHL). Reintroducing pVHL into VHL-null cells increased the half-life of BIM(EL) protein without affecting its mRNA expression, and overexpressing pVHL inhibited BIM(EL) polyubiquitination. Suppressing pVHL expression with RNA interference resulted in a decrease in BIM(EL) protein and a corresponding decrease in the sensitivity of RCC cells to apoptotic stimuli. Directly inhibiting BIM(EL) expression in pVHL-expressing RCC cells caused a similar decrease in cell death. These results demonstrate that pVHL acts to promote BIM(EL) protein stability in RCC cells, and that destabilization of BIM(EL) in the absence of pVHL contributes to the increased resistance of VHL-null RCC cells to certain apoptotic stimuli.

Cytosine arabinoside rapidly activates Bax-dependent apoptosis and a delayed Bax-independent death pathway in sympathetic neurons.

Cytosine arabinoside (ara-C) is a nucleoside analog used in the treatment of hematologic malignancies. One of the major side effects of ara-C chemotherapy is neurotoxicity. In this study, we have further characterized the cell death induced by ara-C in sympathetic neurons. Similar to neurons undergoing trophic factor deprivation-induced apoptosis, ara-C-exposed neurons became hypometabolic before death and upregulated c-myb, c-fos, and Bim. Bax deletion delayed, but did not prevent, ara-C toxicity. Neurons died by apoptosis, indicated by the release of mitochondrial cytochrome-c and caspase-3 activation. p53-deficient neurons demonstrated decreased sensitivity to ara-C, but neither p53 nor multiple p53-regulated genes were induced. Mature neurons showed increased ara-C resistance. These results demonstrate that molecular mechanisms underlying ara-C-induced death are similar to those responsible for trophic factor deprivation-induced apoptosis. However, substantial differences in neuronal death after these two distinct stress stimuli exist since ara-C toxicity, unlike the developmental death, can proceed in the absence of Bax.

Analysis of the NF-kappa B and PI 3-kinase/Akt survival pathways in nerve growth factor-dependent neurons.

Nerve growth factor (NGF) readdition to NGF-deprived neurons can halt Jun N-terminal kinase (JNK) activation, cytochrome c release, and cell death through mechanisms that may involve phosphatidylinositol (PI) 3-kinase, Akt, and nuclear factor kappa B (NF-kappaB). We found that expression of the NF-kappaB protein c-Rel in NGF-deprived neurons blocks cytochrome c release but does not inhibit c-Jun phosphorylation. Conversely, inhibition of NF-kappaB in NGF-maintained neurons promotes cytochrome c release and cell death. In contrast to c-Rel, activated PI 3-kinase and Akt inhibit c-Jun phosphorylation but have only a small effect on cytochrome c release. Finally, although c-Rel can protect neurons from death caused by inhibitors of PI 3-kinase or Akt, NF-kappaB function is not critical for Akt-promoted survival. These results suggest that the PI 3-kinase/Akt and NF-kappaB survival pathways target distinct cell death events in neurons.

SM-20 is a novel mitochondrial protein that causes caspase-dependent cell death in nerve growth factor-dependent neurons.

Sympathetic neurons undergo protein synthesis-dependent apoptosis when deprived of nerve growth factor (NGF). Expression of SM-20 is up-regulated in NGF-deprived sympathetic neurons, and ectopic SM-20 is sufficient to promote neuronal death in the presence of NGF. We now report that SM-20 is a mitochondrial protein that promotes cell death through a caspase-dependent mechanism. SM-20 immunofluorescence was present in the cytoplasm in a punctate pattern that colocalized with cytochrome oxidase I and with mitochondria-selective dyes. Analysis of SM-20/dihydrofolate reductase fusion proteins revealed that the first 25 amino acids of SM-20 contain a functional mitochondrial targeting sequence. An amino-terminal truncated form of SM-20 was not restricted to mitochondria but instead localized throughout the cytosol and nucleus. Nevertheless, the truncated SM-20 retained the ability to induce neuronal death, similar to the wild type protein. SM-20-induced death was accompanied by caspase-3 activation and was blocked by a general caspase inhibitor. Additionally, overexpression of SM-20, under conditions where cell death is blocked by a general caspase inhibitor, did not result in widespread release of cytochrome c from mitochondria. These results indicate that SM-20 is a novel mitochondrial protein that may be an important mediator of neurotrophin-withdrawal-mediated cell death.

Glycogen synthase kinase-3 beta activity is critical for neuronal death caused by inhibiting phosphatidylinositol 3-kinase or Akt but not for death caused by nerve growth factor withdrawal.

Numerous studies reveal that phosphatidylinositol (PI) 3-kinase and Akt protein kinase are important mediators of cell survival. However, the survival-promoting mechanisms downstream of these enzymes remain uncharacterized. Glycogen synthase kinase-3 beta (GSK-3 beta), which is inhibited upon phosphorylation by Akt, was recently shown to function during cell death induced by PI 3-kinase inhibitors. In this study, we tested whether GSK-3 beta is critical for the death of sympathetic neurons caused by the withdrawal of their physiological survival factor, the nerve growth factor (NGF). Stimulation with NGF resulted in PI 3-kinase-dependent phosphorylation of GSK-3 beta and inhibition of its protein kinase activity, indicating that GSK-3 beta is targeted by PI 3-kinase/Akt in these neurons. Expression of the GSK-3 beta inhibitor Frat1, but not a mutant Frat1 protein that does not bind GSK-3 beta, rescued neurons from death caused by inhibiting PI 3-kinase. Similarly, expression of Frat1 or kinase-deficient GSK-3 beta reduced death caused by inhibiting Akt. In NGF-maintained neurons, overexpression of GSK-3 beta caused a small but significant decrease in survival. However, expression of neither Frat1, kinase-deficient GSK-3 beta, nor GSK-3-binding protein inhibited NGF withdrawal-induced death. Thus, although GSK-3 beta function is required for death caused by inactivation of PI 3-kinase and Akt, neuronal death caused by NGF withdrawal can proceed through GSK-3 beta-independent pathways.

The survival of sympathetic neurons promoted by potassium depolarization, but not by cyclic AMP, requires phosphatidylinositol 3-kinase and Akt.

Phosphatidylinositol (PI) 3-kinase and Akt protein kinase mediate trophic factor-dependent survival in certain neurons. However, a role for these enzymes in neuronal survival promoted by other agents is unclear. We have tested PI 3-kinase and Akt for their role in survival promoted by membrane-depolarizing concentrations of extracellular potassium and the cell-permeable cyclic AMP analogue 8-(4-chlorophenylthio)cyclic AMP (cpt-cAMP). Depolarization of sympathetic neurons resulted in an increase in the activities of both PI 3-kinase and Akt. In addition, the PI 3-kinase inhibitor LY294002 was a potent inducer of cell death in depolarized neurons. Stimulation with cpt-cAMP resulted in relatively small increases in PI 3-kinase and Akt activities, and neurons maintained with cpt-cAMP were more resistant to LY294002-induced death than were depolarized neurons. Expression of either dominant-negative PI 3-kinase or dominant-negative Akt blocked survival promoted by depolarization but not by cpt-cAMP. These results indicate that a PI 3-kinase/Akt pathway is required for survival of sympathetic neurons mediated by depolarization but not by cpt-cAMP. Thus, the survival of sympathetic neurons can be maintained through PI 3-kinase/Akt-dependent and -independent pathways.

Expression of the SM-20 gene promotes death in nerve growth factor-dependent sympathetic neurons.

Sympathetic neurons undergo apoptosis when deprived of nerve growth factor (NGF). Inhibitors of RNA or protein synthesis block this death, suggesting that gene expression is important for apoptosis in this system. We have identified SM-20 as a new gene that increases in expression in sympathetic neurons after NGF withdrawal. Expression of SM-20 also increases during neuronal death caused by cytosine arabinoside or the phosphatidylinositol 3-kinase inhibitor LY294002. In addition, SM-20 protein synthesis is elevated in NGF-deprived neurons compared with neurons maintained with NGF. Importantly, expression of SM-20 in sympathetic neurons causes cell death in the presence of NGF. These results suggest that SM-20 may function to regulate cell death in neurons.

Nerve growth factor-dependent activation of NF-kappaB contributes to survival of sympathetic neurons.

Neurotrophins activate multiple signaling pathways in neurons. However, the precise roles of these signaling molecules in cell survival are not well understood. In this report, we show that nerve growth factor (NGF) activates the transcription factors NF-kappaB and AP-1 in cultured sympathetic neurons. Activated NF-kappaB complexes were shown to consist of heterodimers of p50 and Rel proteins (RelA, as well as c-Rel), and NF-kappaB activation was found to occur independently of de novo protein synthesis but in a manner that required the action of the proteasome complex. Treatment with the NF-kappaB inhibitory peptide SN50 in the continuous presence of NGF resulted in dose-dependent induction of cell death. Under the conditions used, SN50 was shown to selectively inhibit NF-kappaB activation but not the activation of other cellular transcription factors such as AP-1 and cAMP response element-binding protein. Cells treated with SN50 exhibited morphological and biochemical hallmarks of apoptosis, and the kinetics of cell killing were accelerated relative to death induced by NGF withdrawal. Finally, experiments were conducted to test directly whether NF-kappaB could act as a survival factor for NGF-deprived neurons. Microinjection of cells with an expression plasmid encoding NF-kappaB (c-Rel) resulted in enhanced neuronal survival after withdrawal of NGF, whereas cells that were transfected with a vector encoding a mutated derivative of c-Rel lacking the transactivation domain underwent cell death to the same extent as control cells. Together, these findings suggest that the activation of NF-kappaB/Rel transcription factors may contribute to the survival of NGF-dependent sympathetic neurons.

Phosphatidylinositol 3-kinase and Akt protein kinase are necessary and sufficient for the survival of nerve growth factor-dependent sympathetic neurons.

Recent studies have suggested a role for phosphatidylinositol (PI) 3-kinase in cell survival, including the survival of neurons. We used rat sympathetic neurons maintained in vitro to characterize the potential survival signals mediated by PI 3-kinase and to test whether the Akt protein kinase, a putative effector of PI 3-kinase, functions during nerve growth factor (NGF)-mediated survival. Two PI 3-kinase inhibitors, LY294002 and wortmannin, block NGF-mediated survival of sympathetic neurons. Cell death caused by LY294002 resembles death caused by NGF deprivation in that it is blocked by a caspase inhibitor or a cAMP analog and that it is accompanied by the induction of c-jun, c-fos, and cyclin D1 mRNAs. Treatment of neurons with NGF activates endogenous Akt protein kinase, and LY294002 or wortmannin blocks this activation. Expression of constitutively active Akt or PI 3-kinase in neurons efficiently prevents death after NGF withdrawal. Conversely, expression of dominant negative forms of PI 3-kinase or Akt induces apoptosis in the presence of NGF. These results demonstrate that PI 3-kinase and Akt are both necessary and sufficient for the survival of NGF-dependent sympathetic neurons.

Cloudy lenses and issues: a pedigree of unoperated congenital cataracts.

1. Although there are many strong recommendations regarding congenital cataracts in the literature, little has been written about the initial decision of whether to operate. 2. Because this condition does not often occur and a large number of patients is necessary to determine the predictive value, a large, collaborative study is required to make significant headway in this area. 3. If we can identify the factors that have strong predictive value for good vision after cataract surgery, we may start to define the profile of children who will have good vision, with or without surgery.

Altered gene expression in neurons during programmed cell death: identification of c-jun as necessary for neuronal apoptosis.

We have examined the hypothesis that neuronal programmed cell death requires a genetic program; we used a model wherein rat sympathetic neurons maintained in vitro are deprived of NGF and subsequently undergo apoptosis. To evaluate gene expression potentially necessary for this process, we used a PCR-based technique and in situ hybridization; patterns of general gene repression and selective gene induction were identified in NGF-deprived neurons. A temporal cascade of induced genes included "immediate early genes," which were remarkable in that their induction occurred hours after the initial stimulus of NGF removal and the synthesis of some required ongoing protein synthesis. The cascade also included the cell cycle gene c-myb and the genes encoding the extracellular matrix proteases transin and collagenase. Concurrent in situ hybridization and nuclear staining revealed that while c-jun was induced in most neurons, c-fos induction was restricted to neurons undergoing chromatin condensation, a hallmark of apoptosis. To evaluate the functional role of the proteins encoded by these genes, neutralizing antibodies were injected into neurons. Antibodies specific for either c-Jun or the Fos family (c-Fos, Fos B, Fra-1, and Fra-2) protected NGF-deprived neurons from apoptosis, whereas antibodies specific for Jun B, Jun D, or three nonimmune antibody preparations had no protective effect. Because these induced genes encode proteins ranging from a transcription factor necessary for death to proteases likely involved in tissue remodeling concurrent with death, these data may outline a genetic program responsible for neuronal programmed cell death.

Adult treatment with removal of all four permanent canines.

The permanent canines-especially in the maxillary arch-have always been considered of prime importance, even before the "cuspid protection" hypothesis became well known to most orthodontists in the 1960s. In the adult case presented, periodontal considerations and other factors led to the unconventional (and likely controversial) extraction of all four canines.

Cloudy lenses and issues: a pedigree of unoperated congenital cataracts.

The issue of when congenital cataracts should be removed seems to be well resolved, with most recommendations stating that all significant central lens opacities should be removed by 4 months of age, with removal most commonly occurring the first month. Although there are many strong recommendations regarding congenital cataracts in the literature, little has been written about the initial decision of whether or not to operate. A pedigree is presented of multiple generations with inherited bilateral central cataracts. Two affected children, who did not undergo surgery, were followed from birth. These two children developed essentially normal vision, despite seemingly significant central opacities. This pedigree emphasizes the need to further examine prognostic factors regarding bilateral infantile cataracts and to analyze the initial surgical decision. Because this condition does not often occur and a large number of patients is necessary to determine the predictive value, a large, collaborative study is required to make significant headway in this area. If we can identify the factors that have strong predictive value for good vision after cataract surgery, we may start to define the profile of children who will have good vision, with or without surgery.

Analysis of cell cycle-related gene expression in postmitotic neurons: selective induction of Cyclin D1 during programmed cell death.

Sympathetic neurons undergo RNA and protein synthesis-dependent programmed cell death when deprived of nerve growth factor. To test the hypothesis that neuronal programmed cell death is a consequence of conflicting growth signals which cause the inappropriate activation of cell cycle genes, we have analyzed cell cycle-related genes for their expression in postmitotic neurons. Surprisingly, many of these genes are expressed in neurons, although cdc2, cdk2, and cyclin A are not. During programmed cell death, the expression of most of these genes, including several cyclins and the Rb and p53 tumor suppressor genes, decreases similar to that of neuronal genes. In contrast, cyclin D1 expression is selectively induced in dying neurons. Cyclin D1 mRNA levels peak 15-20 hr after nerve growth factor withdrawal, concurrent with the time that neurons become committed to die. These results provide an extensive characterization of cell cycle gene expression in postmitotic neurons and provide the evidence for a gene induced during neuronal programmed cell death.

NGF deprivation and neuronal degeneration trigger altered beta-amyloid precursor protein gene expression in the rat superior cervical ganglia in vivo and in vitro.

In order to study the expression of beta-amyloid precursor protein (APP) isoforms during neuronal degeneration we have used the rat superior cervical ganglia (SCG) as an experimental model. In the neonate these sympathetic ganglia are nerve growth factor (NGF) dependent and in vivo administration of anti-NGF antiserum results in exaggerated neuronal degeneration. Analysis of APP mRNA transcripts in the SCG, following NGF deprivation, revealed a coincident decrease in APP695 and augmentation of APP751/770. These changes were specific to the SCG and were not seen in sensory ganglia. Subsequent in vitro studies, using primary dissociated cultures of sympathetic or cortical neurones, confirmed these changes in APP gene expression during neuronal degeneration. These observations may have important implications for the generation of beta-amyloid in Alzheimer's disease.

Cell death genes in invertebrates and (maybe) vertebrates.

That naturally occurring cell death in the nervous and other systems is an active and physiologically appropriate process has received much attention recently and has gained a significant degree of acceptance. The identification of cell death genes in invertebrates, the characterization of gene products that function as cell death suppressors, and the demonstration that some proto-oncogenes elicit cell death, as well as proliferation, in certain cell types have heightened interest in the mechanism of programmed cell death. Yet, evidence for a genetic program for cell death in vertebrates remains circumstantial and, so far, vertebrate 'cell death' genes exist only in theory.

Phosphorylation of conserved serine residues does not regulate the ability of mosxe protein kinase to induce oocyte maturation or function as cytostatic factor.

Expression of the mosxe protein kinase is required for the normal meiotic maturation of Xenopus oocytes and overexpression induces maturation in the absence of other stimuli. In addition, mosxe functions as a component of cytostatic factor (CSF), an activity responsible for arrest of the mature egg at metaphase II. After microinjection of Xenopus oocytes with in vitro synthesized RNA encoding either wild-type mosxe or kinase-inactive mosxe(R90), both proteins are phosphorylated exclusively on serine residues and exhibit essentially identical chymotryptic maps. Since the phosphorylated kinase-inactive mosxe(R90) protein was recovered from resting oocytes that have not yet begun to translate endogenous mosxe, this indicates that the major phosphopeptides of mosxe(R90) are phosphorylated by a preexisting protein kinase present in resting oocytes, and are not the result of autophosphorylation. The results presented here also indicate that the mosxe protein does not undergo significant phosphorylation at unique sites during oocyte maturation. If the biological activity of mosxe were regulated by phosphorylation, a site of regulatory phosphorylation would most likely be conserved among mos proteins of different species. Site-directed mutagenesis was used to construct 13 individual serine----alanine mutations at conserved residues (3, 16, 18, 25, 26, 57, 71, 76, 102, 105, 127, 211, and 258). These 13 mutants were analyzed for their abilities to induce oocyte maturation and to function as CSF. Results obtained with the mosxe(A105) mutant revealed that serine-105 is required for both maturation induction and CSF activity, even though serine-105 does not represent a major site of phosphorylation. All of the remaining serine----alanine mosxe mutants induced oocyte maturation and exhibited CSF activity comparable with the wild type. These results demonstrate that none of the conserved serines examined in this study function as regulatory phosphorylation sites for these biological activities. Peptide mapping of the remaining mosxe mutants identified serine-3 as a major phosphorylation site in vivo, which is contained within the chymotryptic peptide MPSPIPVERF.