Selection of novel mediators of E2F1-induced apoptosis through retroviral expression of an antisense cDNA library

The E2F1 transcription factor is an essential mediator of p53-dependent and p53-independent apoptosis as part of an anti-tumour safeguard mechanism. In this study, a functional so-called technical knockout (TKO) approach was applied to Saos-2ERE2F1 cells that conditionally activate E2F1 by the addition of 4-hydroxytamoxifen to search for p53-independent pro-apoptotic E2F1 targets. The approach was based on random inactivation of genes after retroviral transfer of an antisense cDNA library enriched of E2F1-induced genes, followed by the selection of Saos-2ERE2F1 cells that survive in the presence of the apoptotic stimulus. We identified 13 novel E2F1 target genes encoding proteins of known cellular function, including apoptosis and RNA binding. FACS analysis revealed that E2F1-induced apoptosis was significantly attenuated in cell clones containing the antisense cDNA fragments of these genes, demonstrating their participation in E2F1 death pathways. Moreover, inactivation of the target genes resulted in a clear increase of cell viability (>80%) in response to E2F1 activation compared with controls (∼30%). Four genes showed an increase in expression intensity in the presence of cycloheximide, suggesting a direct effect of E2F1 on gene transcription, whereas one gene was identified as an indirect target. Our data provide new insight in the regulation of E2F1-induced apoptosis

Insulin-induced Increase in Anabolic Capacity is Blunted by Autophagic Inhibition in L6 Myotubes

Insulin is an anabolic hormone that acts on skeletal muscle cells to stimulate protein synthesis, an effect that is enhanced by the availability of amino acids. While autophagy within the cell provides an intracellular source of amino acids to support anabolism, little is known about how this pathway impacts the insulin-induced increase in anabolic capacity within skeletal muscle cells. PURPOSE: The purpose of this study was to determine the impact of autophagic inhibition in cultured L6 myotubes in conjunction with insulin stimulation in vitro. METHODS: Differentiated, cultured L6 myotubes were treated for 24 hours with or without insulin (100 nM) and NSC 185058 (100 μM), a specialized inhibitor of the autophagic catabolic pathway, in media enriched with 4% deuterium. Cells were harvested from each treatment group (n=3/group) 24 hours post-deuterium enrichment and were processed for protein synthesis and western blot protein analysis. A one-way ANOVA was used to compare groups, and when significant F ratios were present, a Student’s Newman-Keuls post hoc procedure was used to test differences among group means. Alpha was set at p≤0.05 for all analyses. RESULTS: Cells treated with insulin (INS) had a higher ratio of phosphorylated to total P70S6K compared to untreated (CON) cells and those incubated with both insulin and NSC 185058 (INS+NSC; 1694% and 327%, respectively; p\u3c0.05). INS+NSC also decreased the ratio of phosphorylated to total 4EBP1 relative to CON (-51%) and INS (-49%), although these differences were not significant (p\u3e0.05). Myofibrillar protein synthesis was stimulated with INS compared to CON and INS+NSC (30.3% and 70.1% respectively; p\u3c0.05) but was lower in INS+NSC relative to CON (-23.4%; p\u3c0.05). CONCLUSION: Results from our study indicate that insulin (100 nM) stimulates anabolism in skeletal muscle cells, but that addition of the autophagic inhibitor NSC 185058 (100 μM) blunts this effect to a level similar to or less than control. Further, our data suggest that the reduction of protein synthesis is mediated through the downregulation of the mTORC1 signaling pathway. While it is widely recognized that insulin promotes anabolic activity through both the direct stimulation of mTOR signaling and extracellular amino acid uptake, our data strongly indicate that autophagic processes are necessary for full anabolic responses in muscle. This decrease in anabolic capacity supports previous literature indicating that the amino acid availability impacts the stimulatory impact of insulin on protein synthesis

Autophagy, but Not Proteolysis, May Aid in Muscle Protein Synthesis

For muscle growth to occur, protein synthesis must be greater than protein degradation. However, up to this point, anabolic pathways have garnered the brunt of investigations examining anabolic capacity with little investigation into the connectedness of catabolic signaling on these anabolic targets. PURPOSE: The purpose of this study was to elucidate the contributions of proteasomal-dependent and autophagic-dependent catabolic pathways on anabolism via analysis of fractional synthetic rates (FSR) in L6 myotubes. METHODS: Differentiated, cultured L6 myoblasts were treated with media containing 4% deuterium oxide (stable isotope label) and a corresponding pharmacological treatment (NSC 185058 [autophagic inhibitor; 100 μM], MG-262 [proteasomal inhibitor; 0.01 μM] or DMSO control; n=3/group) during the final 24-hours of the differentiation period prior to harvest. The myofibrillar pellet of the processed samples was used to determine FSR via mass-spectrometry analysis. DMSO-treated myotubes served as controls, with a one-way analysis of variance and Tukey’s post-hoc test used to test for any differences among groups. RESULTS: Our results indicate that MG-262 had no impact on myofibrillar FSR when compared to DMSO control (MG-262 1.0993 %/day vs. control 1.239 %/day). However, NSC 185058 lowered myofibrillar FSR (NSC 185058 0.9009 %/day vs. control 1.239 %/day; P=0.0282). CONCLUSION: These data suggest that inhibition of autophagic machinery can impair anabolism. This may be due to autophagy’s role in increasing the amino acid pool within the cell. Further, the lack of inhibition seen from MG-262 suggests that there is a delineation of roles within the catabolic pathways in regard to their influence on anabolism in healthy, metabolically unchallenged myotubes

Autophagy is Required for the Anabolic Response to Muscle Contraction

Exercise is a key stimulus in regulating the behavior and metabolism of skeletal muscle, with exercise inducing muscular growth through activation of the anabolic mechanistic target of rapamycin kinase (mTOR). Separately, there is mounting evidence that exercise increases autophagy (one of the main routes by which intracellular proteins are degraded) and that the autophagic process may indeed be required for adaptations to exercise training. PURPOSE: To investigate the effects of autophagy inhibition on mTOR signaling and cellular anabolism after muscular contraction. METHODS: Cultured L6 myotubes were to exposed to electrical pulse stimulation using a stimulator set to deliver bipolar pulses of 30V at 100 Hz for 200 ms every fifth second for 60 minutes. Subsequently, cells received either vehicle control, or 100 μM NSC-185058, an antagonist of the key autophagy protein ATG4B and known inhibitor of autophagy. All groups were also exposed to 4% deuterium oxide, a stable isotopic tracer for measurements of protein synthesis. 24 hours post “exercise” bout, cells were lysed in ice-cold Norris buffer, and prepared for Western immunoblot of protein expression, or determination of protein fractional synthesis rate (FSR) of the myofibrillar fraction via mass-spectrometry analysis. Non-stimulated cells receiving vehicle control treatment served as controls, with a one-way analysis of variance and Tukey’s post-hoc test used to test for any differences between groups. RESULTS: We found that phosphorylation of a key downstream target of mTOR, P70S6 kinase, was roughly seven times greater in cells subjected to EPS and vehicle control (710.3%) relative to control (p0.05). While there was a trend for EPS treatment to increase expression of ATG4B, along with a reduction of ATG4B content as a result of NSC-185058 treatment, this finding did not rise to the level of statistical significance. There were no differences in FSR between cells exposed to EPS; however, NSC-185058 treatment significantly reduced FSR in EPS treated cells relative to controls (0.8712 %/hr vs 1.193 %/hr). CONCLUSION: These findings present two conclusions: high-intensity EPS as an in vitro model of exercise elevates mTOR signaling through P70S6K 24 hours post exercise, and mTOR activation as a result of muscular contraction is reliant upon autophagy in skeletal muscle. Further work will be required to elucidate the dynamics of this relationship, and the interplay between skeletal muscle autophagy and anabolism

Prognostic Factors and Survival in Pediatric and Adolescent Liposarcoma

Purpose. Liposarcoma is extremely rare in the pediatric population. To identify prognostic factors and determine treatment outcomes, we reviewed our institutional experience with pediatric liposarcoma. Methods. We retrospectively reviewed all pediatric patients (age < 22 years) with confirmed liposarcoma treated at Memorial Sloan-Kettering Cancer Center. Histologic subtype, tumor location, margin status, recurrence, and adjuvant therapy were analyzed and correlated with overall survival. Results. Thirtyfour patients (56% male) with a median age of 18.1 years were identified. Twenty-two (65%) had peripheral tumors and 12 (35%) had centrally located tumors. Histologically, 29 (85%) tumors were low grade, and 5 (15%) were high grade pleomorphic. Eleven (32%) had recurrent disease, 9 patients with central tumors and 2 patients with peripheral lesions. Eight deaths occurred, all in patients with central disease. Five-year overall survival was 78%, with a median follow-up time of 5.4 years (range, 0.3-30.3 years). Tumor grade (P = .003), histologic subtype (P = .01), and primary location (P < .001) all correlated with survival, as did stage (P < .001) and margin status (P = .001). Conclusions. Central location of the primary tumor, high tumor grade, and positive surgical margins are strongly correlated with poor survival in pediatric patients with liposarcoma

Systematic mutation analysis of KIAA0767 and KIAA1646 in chromosome 22q-linked periodic catatonia

BACKGROUND: Periodic catatonia is a familial subtype of schizophrenia characterized by hyperkinetic and akinetic episodes, followed by a catatonic residual syndrome. The phenotype has been evaluated in two independent genome-wide linkage scans with evidence for a major locus on chromosome 15q15, and a second independent locus on chromosome 22q(tel). METHODS: In the positional and brain-expressed candidate genes KIAA0767 and KIAA1646, we searched for variants in the complete exons and adjacent splice-junctions as well as in parts of the 5'- and 3'-untranslated regions by means of a systematic mutation screening in individuals from chromosome 22q-linked pedigrees. RESULTS: The mutation scan revealed 24 single nucleotide polymorphisms, among them two rare codon variants (KIAA0767: S159I; KIAA1646: V338G). However, both were neither found segregating with the disease in the respective pedigree nor found at a significant frequency in a case-control association sample. CONCLUSION: Starting from linkage signals at chromosome22q(tel )in periodic catatonia, we screened two positional brain-expressed candidate genes for genetic variation. Our study excludes genetic variations in the coding and putative promoter regions of KIAA0767 and KIAA1646 as causative factors for periodic catatonia

ASPP: a new family of oncogenes and tumour suppressor genes

The apoptosis stimulating proteins of p53 (ASPP) family consists of three members, ASPP1, ASPP2 and iASPP. They bind to proteins that are key players in controlling apoptosis (p53, Bcl-2 and RelA/p65) and cell growth (APCL, PP1). So far, the best-known function of the ASPP family members is their ability to regulate the apoptotic function of p53 and its family members, p63 and p73. Biochemical and genetic evidence has shown that ASPP1 and ASPP2 activate, whereas iASPP inhibits, the apoptotic but not the cell-cycle arrest function of p53. The p53 tumour suppressor gene, one of the most frequently mutated genes in human cancer, is capable of suppressing tumour growth through its ability to induce apoptosis or cell-cycle arrest. Thus, the ASPP family of proteins helps to determine how cells choose to die and may therefore be a novel target for cancer therapy

E2F and p53 Induce Apoptosis Independently during Drosophila Development but Intersect in the Context of DNA Damage

In mammalian cells, RB/E2F and p53 are intimately connected, and crosstalk between these pathways is critical for the induction of cell cycle arrest or cell death in response to cellular stresses. Here we have investigated the genetic interactions between RBF/E2F and p53 pathways during Drosophila development. Unexpectedly, we find that the pro-apoptotic activities of E2F and p53 are independent of one another when examined in the context of Drosophila development: apoptosis induced by the deregulation of dE2F1, or by the overexpression of dE2F1, is unaffected by the elimination of dp53; conversely, dp53-induced phenotypes are unaffected by the elimination of dE2F activity. However, dE2F and dp53 converge in the context of a DNA damage response. Both dE2F1/dDP and dp53 are required for DNA damage-induced cell death, and the analysis of rbf1 mutant eye discs indicates that dE2F1/dDP and dp53 cooperatively promote cell death in irradiated discs. In this context, the further deregulation in the expression of pro-apoptotic genes generates an additional sensitivity to apoptosis that requires both dE2F/dDP and dp53 activity. This sensitivity differs from DNA damage-induced apoptosis in wild-type discs (and from dE2F/dDP-induced apoptosis in un-irradiated rbf1 mutant eye discs) by being dependent on both hid and reaper. These results show that pro-apoptotic activities of dE2F1 and dp53 are surprisingly separable: dp53 is required for dE2F-dependent apoptosis in the response to DNA damage, but it is not required for dE2F-dependent apoptosis caused simply by the inactivation of rbf1