The drosophila Bcl-2 family protein Debcl is targeted to the proteasome by the β-TrCP homologue slimb

The ubiquitin–proteasome system is one of the main proteolytic pathways. It inhibits apoptosis by degrading pro-apoptotic regulators, such as caspases or the tumor suppressor p53. However, it also stimulates cell death by degrading pro-survival regulators, including IAPs. In Drosophila, the control of apoptosis by Bcl-2 family members is poorly documented. Using a genetic modifier screen designed to identify regulators of mammalian bax-induced apoptosis in Drosophila, we identified the ubiquitin activating enzyme Uba1 as a suppressor of bax-induced cell death. We then demonstrated that Uba1 also regulates apoptosis induced by Debcl, the only counterpart of Bax in Drosophila. Furthermore, we show that these apoptotic processes involve the same multimeric E3 ligase—an SCF complex consisting of three common subunits and a substrate-recognition variable subunit identified in these processes as the Slimb F-box protein. Thus, Drosophila Slimb, the homologue of β-TrCP targets Bax and Debcl to the proteasome. These new results shed light on a new aspect of the regulation of apoptosis in fruitfly that identifies the first regulation of a Drosophila member of the Bcl-2 family.     

Drosophila Cyclin G Is a Regulator of the Notch Signalling Pathway during Wing Development

Notch signalling regulates a multitude of differentiation processes during Drosophila development. For example, Notch activity is required for proper wing vein differentiation which is hampered in mutants of either the receptor Notch, the ligand Delta or the antagonist Hairless. Moreover, the Notch pathway is involved in several aspects of Drosophila oogenesis as well. We have identified Drosophila Cyclin G (CycG) as a molecular interaction partner of Hairless, the major antagonist in the Notch signalling pathway, in vitro and in vivo. Loss of CycG was shown before to cause female sterility and to disturb the architecture of the egg shell. Nevertheless, Notch dependent processes during oogenesis appeared largely unaffected in cycG mutant egg chambers. Loss of CycG modified the dominant wing phenotypes of Notch, Delta and Hairless mutants. Whereas the Notch loss of function phenotype was ameliorated by a loss of CycG, the phenotypes of either Notch gain of function or of Delta or Hairless loss of function were enhanced. In contrast, loss of CycG had only a minor effect on the wing vein phenotype of mutants affecting the EGFR signalling pathway emphasizing the specificity of the interaction of CycG and Notch pathway members.     

The Unique Non-Catalytic C-Terminus of P37delta-PI3K Adds Proliferative Properties In Vitro and In Vivo

The PI3K/Akt pathway is central for numerous cellular functions and is frequently deregulated in human cancers. The catalytic subunits of PI3K, p110, are thought to have a potential oncogenic function, and the regulatory subunit p85 exerts tumor suppressor properties. The fruit fly, Drosophila melanogaster, is a highly suitable system to investigate PI3K signaling, expressing one catalytic, Dp110, and one regulatory subunit, Dp60, and both show strong homology with the human PI3K proteins p110 and p85. We recently showed that p37δ, an alternatively spliced product of human PI3K p110δ, displayed strong proliferation-promoting properties despite lacking the catalytic domain completely. Here we functionally evaluate the different domains of human p37δ in Drosophila. The N-terminal region of Dp110 alone promotes cell proliferation, and we show that the unique C-terminal region of human p37δ further enhances these proliferative properties, both when expressed in Drosophila, and in human HEK-293 cells. Surprisingly, although the N-terminal region of Dp110 and the C-terminal region of p37δ both display proliferative effects, over-expression of full length Dp110 or the N-terminal part of Dp110 decreases survival in Drosophila, whereas the unique C-terminal region of p37δ prevents this effect. Furthermore, we found that the N-terminal region of the catalytic subunit of PI3K p110, including only the Dp60 (p85)-binding domain and a minor part of the Ras binding domain, rescues phenotypes with severely impaired development caused by Dp60 over-expression in Drosophila, possibly by regulating the levels of Dp60, and also by increasing the levels of phosphorylated Akt. Our results indicate a novel kinase-independent function of the PI3K catalytic subunit.     

Multiple roles of the gene zinc finger homeodomain-2 in the development of the Drosophila wing

The gene zfh2 and its human homolog Atbf1 encode huge molecules with several homeo- and zinc finger domains. It has been reported that they play important roles in neural differentiation and promotion of apoptosis in several tissues of both humans and flies. In the Drosophila wing imaginal disc, Zfh2 is expressed in a dynamic pattern and previous results suggest that it is involved is proximal–distal patterning. In this report we go further in the analysis of the function of this gene in wing development, performing ectopic expression experiments and studying its effects in genes involved in wing development. Our results suggest that Zfh2 plays an important role controlling the expression of several wing genes and in the specification of those cellular properties that define the differences in cell proliferation between proximal and distal domains of the wing disc.     

P53 induction accompanying G2/M arrest upon knockdown of tumor suppressor HIC1 in U87MG glioma cells

Hypermethylated in cancer 1 (HIC1) is a novel tumor suppressor gene (tsg) frequently silenced by epigenetic modification, predominantly by methylation in different tumors. HIC1 functionally co-operates with p53 in cultured cells as well as in transgenic animals to suppress tumors and has binding site on its promoter. Its over expression often leads to cell cycle arrests. Although HIC1 proven to have role as tsg, its regulation to cell cycle and dependency upon p53 is grossly unknown. In this study, we investigated the role of HIC1 in cell cycle and proliferation of glioma cell line U87MG which has wild type p53, in both serum-containing and serum-deprived medium. Microscopic analysis and MTT assay showed reduced cell number and rate of proliferation upon HIC1 knock down compared to control siRNA (p = 0.025) and untreated cells (p = 0.03) in serum-containing medium and serum-free medium (p = 0.014 vs control siRNA; p = 0.018 vs untreated cells). Cell cycle analysis revealed an arrest at G2/M phase of cell cycle with no demonstrable increase in apoptosis with both medium. An increased expression of p53 concomitant with HIC1 knockdown was observed. Furthermore P21, a p53 responsive gene, along with p27 was significantly increased in comparison with controls. Our results demonstrated an important role of HIC1 for the normal progression of cell cycle, and at molecular level, it could affect the homeostasis of p53 as well as number of cell cycle-related genes, which may or may not be directly linked to p53.     

Functional analysis of Notch gene in the white-backed planthopper Sogatella furcifera (Hemiptera: Delphacidae)

Insect wing development is a complex process controlled by a series of genes. Notch is a key gene that regulates insect wing development. It is a receptor protein in the Notch signaling pathway that regulates the expression of downstream target genes after binding with ligands. In this study, we obtained the Notch gene from Sogatella furcifera (SfNotch) and analyzed its mRNA expression levels in different developmental stages and tissues. We also used RNA interference (RNAi) to investigate the biological function of SfNotch. Our results showed that SfNotch expression increased toward the end of the 3rd–5th instars, reaching its highest expression in the adult stage, and was especially high in the thorax and wing tissues. In addition, knockdown of SfNotch resulted in notched wings in 68.5% of S. furcifera. Our findings provide insight into the regulation of insect wing development and contribute to the molecular understanding of this process.     

XI-011 enhances cisplatin-induced apoptosis by functional restoration of p53 in head and neck cancer

Head and neck cancer (HNC), one of the most common cancers worldwide, frequently involves mutation of the TP53 gene and dysregulation of the p53 pathway. Overexpression of MDM2 or MDM4 inactivates the tumor-suppressive function of p53. Restoration of p53 function that counteracts these p53 repressors can lead to in vivo tumor regression. Therefore, the present study assessed the ability of the small molecule p53 activator XI-011 (NSC146109) to induce apoptosis in HNC by restoring p53 function. We tested the effects of XI-011 treatment in HNC cell lines, either individually or in combination with cisplatin and assessed growth suppression, cell cycle arrest, and apoptosis. The drug effects on in vivo growth of HNC cells were examined in mice xenograft model. XI-011 exerted the highest growth suppression in tumor cells that overexpress MDM4, in which p53 is degraded. XI-011 treatment downregulated MDM4 mRNA and protein levels, and upregulated expression of proapoptotic genes and promoted apoptosis, in a dose-dependent manner. The apoptotic response was blocked by inhibition of p53 or expression of MDM4, demonstrating that the effects of XI-011 depend on p53 and MDM4. In combination treatments, XI-011 acted synergistically with cisplatin to inhibit growth of HNC cells in vitro and in vivo. MDM4 inhibition and functional restoration of p53 by XI-011 effectively enhanced cisplatin-induced cytotoxicity in HNC cells, an activity that suggests a promising strategy for treating HNC.     

In the absence of Sonic hedgehog, p53 induces apoptosis and inhibits retinal cell proliferation, cell-cycle exit and differentiation in zebrafish

Background

Sonic hedgehog (Shh) signaling regulates cell proliferation during vertebrate development via induction of cell-cycle regulator gene expression or activation of other signalling pathways, prevents cell death by an as yet unclear mechanism and is required for differentiation of retinal cell types. Thus, an unsolved question is how the same signalling molecule can regulate such distinct cell processes as proliferation, cell survival and differentiation.

Methodology/Principal Findings

Analysis of the zebrafish shh ^−/− mutant revealed that in this context p53 mediates elevated apoptosis during nervous system and retina development and interferes with retinal proliferation and differentiation. While in shh ^−/− mutants there is activation of p53 target genes and p53-mediated apoptosis, an increase in Hedgehog (Hh) signalling by over-expression of dominant-negative Protein Kinase A strongly decreased p53 target gene expression and apoptosis levels in shh ^−/− mutants. Using a novel p53 reporter transgene, I confirm that p53 is active in tissues that require Shh for cell survival. Proliferation assays revealed that loss of p53 can rescue normal cell-cycle exit and the mitotic indices in the shh ^−/− mutant retina at 24, 36 and 48 hpf. Moreover, generation of amacrine cells and photoreceptors was strongly enhanced in the double p53 ^−/− shh ^−/− mutant retina suggesting the effect of p53 on retinal differentiation.

Conclusions

Loss of Shh signalling leads to the p53-dependent apoptosis in the developing nervous system and retina. Moreover, Shh-mediated control of p53 activity is required for proliferation and cell cycle exit of retinal cells as well as differentiation of amacrine cells and photoreceptors.     

Erythropoietin protects red blood cells from TRAIL1-induced cell death during red blood cell transition in Xenopus laevis

In anuran amphibians, larval red blood cells (RBCs) are replaced by adult-type RBCs during metamorphosis. We previously showed that tumor necrosis factor-related apoptosis-inducing ligand 1 (TRAIL1) induces apoptosis in larval-, but not adult-type RBCs in Xenopus laevis. We also found that protein kinase C (PKC) activation is involved in establishing resistance to TRAIL1-induced apoptosis in adult-type RBCs. Here, we investigated whether erythropoietin (EPO), which induces PKC activation in mammalian erythroblasts, is involved in the RBC transition in X. laevis. RT-PCR analysis revealed that epo mRNA was upregulated in the lung, from the metamorphic climax (stage 60) onward. In an RBC culture system, EPO pretreatment significantly attenuated the TRAIL1-induced death of larval- and adult-type RBCs isolated from tadpoles and adults, probably due partly to PKC activation. In samples from froglets undergoing RBC transition, which included both larval- and adult-type RBCs, EPO exhibited a stronger protective effect on the adult-type than the larval-type RBCs. Newly differentiated RBCs isolated from tadpoles treated with a hemolytic reagent were more resistant to TRAIL1-induced cell death than non-treated controls. These results suggest that EPO functions to protect adult-type RBCs from TRAIL1-induced cell death during RBC transition, and that the protective effect might decrease as RBCs age.     

A genetic screen for elements of the network that regulates neurogenesis in Drosophila

The development of external sensory organs on the notum of Drosophila is promoted by the proneural genes achaete and scute. Their activity defines proneural cell clusters in the wing imaginal disc. Ectopic expression, under control of the GAL4 system, of the proneural gene lethal of scute (l'sc) causes the development of ectopic bristles. Persistent ectopic expression of l'sc is not sufficient to impose a neural fate on any given cell. This implies that mutual inhibition, mediated by the Notch signaling pathway, occurs among the cells of the ectopic proneural cluster. Consequently, the dominant, quantifiable phenotype associated with ectopic expression of l'sc is modified by mutations in genes known to be involved in neurogenesis. This phenotype has been utilized to screen for dominant enhancers and suppressors that modify the number of ectopic bristles. In this way, about 100 000 progeny of EMS or X-ray-treated flies have been analyzed to identify autosomal genes involved in regulation of the neural fate. In addition 1200 chromosomes carrying lethal P-element insertions were screened for modifiers. Besides mutations in genes expected to modify the phenotype, we have isolated mutations in six genes not known so far to be involved in neurogenesis.     

Dis3L2 regulates cell proliferation and tissue growth through a conserved mechanism

Dis3L2 is a highly conserved 3’-5’ exoribonuclease which is mutated in the human overgrowth disorders Perlman syndrome and Wilms’ tumour of the kidney. Using Drosophila melanogaster as a model system, we have generated a new dis3L2 null mutant together with wild-type and nuclease-dead genetic lines in Drosophila to demonstrate that the catalytic activity of Dis3L2 is required to control cell proliferation. To understand the cellular pathways regulated by Dis3L2 to control proliferation, we used RNA-seq on dis3L2 mutant wing discs to show that the imaginal disc growth factor Idgf2 is responsible for driving the wing overgrowth. IDGFs are conserved proteins homologous to human chitinase-like proteins such as CHI3L1/YKL-40 which are implicated in tissue regeneration as well as cancers including colon cancer and non-small cell lung cancer. We also demonstrate that loss of DIS3L2 in human kidney HEK-293T cells results in cell proliferation, illustrating the conservation of this important cell proliferation pathway. Using these human cells, we show that loss of DIS3L2 results in an increase in the PI3-Kinase/AKT signalling pathway, which we subsequently show to contribute towards the proliferation phenotype in Drosophila. Our work therefore provides the first mechanistic explanation for DIS3L2-induced overgrowth in humans and flies and identifies an ancient proliferation pathway controlled by Dis3L2 to regulate cell proliferation and tissue growth.     

Notch signaling regulates growth and differentiation in the mammalian lens

The Notch signal transduction pathway regulates the decision to proliferate versus differentiate. Although there are a myriad of mouse models for the Notch pathway, surprisingly little is known about how these genes regulate early eye development, particularly in the anterior lens. We employed both gain-of-function and loss-of-function approaches to determine the role of Notch signaling in lens development. Here we analyzed mice containing conditional deletion of the Notch effector Rbpj or overexpression of the activated Notch1 intracellular domain during lens formation. We demonstrate distinct functions for Notch signaling in progenitor cell growth, fiber cell differentiation and maintenance of the transition zone. In particular, Notch signaling controls the timing of primary fiber cell differentiation and is essential for secondary fiber cell differentiation. Either gain or loss of Notch signaling leads to formation of a dysgenic lens, which in loss-of-function mice undergoes a profound postnatal degeneration. Our data suggest both Cyclin D1 and Cyclin D2, and the p27^Kip1 cyclin-dependent kinase inhibitor act downstream of Notch signaling, and define multiple critical functions for this pathway during lens development.     

Targeted cellular ionic calcium chelation by oxalates: Implications for the treatment of tumor cells

Background

The Notch signaling pathway is crucial in T-cell development, Notch1 mutations are frequently present in T-cell acute lymphoblastic leukemia (T-ALL). To investigate the feature of Notch1 mutation and its corresponding expression level in Chinese patients with T-ALL, detection of mutation and the expression level of Notch1 gene was preformed using RT-PCR, sequencing and real-time PCR respectively.

Results

Two Notch1 point mutations (V1578E and L1593P) located on HD-N domain were identified in three cases out of 13 T-ALL patients. The mutation on 4733 position (V1578E) found in two cases was a novel mutation. The overexpression of Notch1 was detected in all samples with T-ALL, moreover, significantly higher expression of Notch1 was detected in the T-ALL with Notch1 mutation group compared with T-ALL with WT Notch1 group (p = 0.0192).

Conclusions

Higher expression of Notch1 was associated with Notch1 mutation, more novel mutation of this gene might be identified in different populations and its contribution to the molecular pathogenesis of T-ALL is needed further research.     

<Emphasis Type="Italic">Drosophila</Emphasis> Chk2 and p53 proteins induce stage-specific cell death independently during oogenesis

In Drosophila, the checkpoint protein-2 kinase (DmChk2) and its downstream effector protein, Dmp53, are required for DNA damage-mediated cell cycle arrest, DNA repair and apoptosis. In this study we focus on understanding the function of these two apoptosis inducing factors during ovarian development. We found that expression of Dmp53, but not DmChk2, led to loss of ovarian stem cells. We demonstrate that expression of DmChk2, but not Dmp53, induced mid-oogenesis cell death. DmChk2 induced cell death was not suppressed by Dmp53 mutant, revealing for the first time that in Drosophila, over-expression of DmChk2 can induce cell death which is independent of Dmp53. We found that over-expression of caspase inhibitors such as DIAP1, p35 and p49 did not suppress DmChk2- and Dmp53-induced cell death. Thus, our study reveals stage-specific effects of Dmp53 and DmChk2 in oogenesis. Moreover, our results demonstrate that although DmChk2 and Dmp53 affect different stages of ovarian development, loss of ovarian stem cells by p53 expression and mid-oogenesis cell death induced by DmChk2 do not require caspase activity.     

The Transcription Factor Optomotor-Blind Antagonizes Drosophila Haltere Growth by Repressing Decapentaplegic and Hedgehog Targets

In Drosophila, decapentaplegic, which codes for a secreted signaling molecule, is activated by the Hedgehog signaling pathway at the anteroposterior compartment border of the two dorsal primordia; the wing and the haltere imaginal discs. In the wing disc, Decapentaplegic and Hedgehog signaling targets are implicated in cell proliferation and cell survival. However, most of their known targets in the wing disc are not expressed in the haltere disc due to their repression by the Hox gene Ultrabithorax. The T-box gene optomotor-blind escapes this repression in the haltere disc, and therefore is expressed in both the haltere and wing discs. Optomotor-blind is a major player during wing development and its function has been intensely investigated in this tissue, however, its role in haltere development has not been reported so far. Here we show that Optomotor-blind function in the haltere disc differs from that in the wing disc. Unlike its role in the wing, Optomotor-blind does not prevent apoptosis in the haltere but rather limits growth by repressing several Decapentaplegic and Hedgehog targets involved both in wing proliferation and in modulating the spread of morphogens similar to Ultrabithorax function but without disturbing Ultrabithorax expression.     

Puerarin Protects Against β-Amyloid-Induced Microglia Apoptosis Via a PI3K-Dependent Signaling Pathway

Puerarin extracted from Radix puerariae is well-known for its anti-oxidative and neuroprotective activities. In this study, we investigated the protective effect of puerarin on amyloid-β protein (Aβ)-induced cytotoxicity and its potential mechanisms in BV-2 and primary microglial cells. We found that pretreatment with puerarin afforded protection against Aβ-induced cytotoxicity through inhibiting apoptosis in BV-2 and primary microglial cells. This result was also confirmed by the activated caspase-3 assay. Phospho-Akt and Bcl-2 expression increased after pretreatment with puerarin in BV-2 and primary microglial cells exposed to Aβ, whereas Bax expression and cytochrome c release decreased. In addition, puerarin treatment prevented the loss of mitochondrial membrane potential and reactive oxygen species production. Interestingly, these effects of puerarin against Aβ insult were abolished by LY294002, an inhibitor of PI3K phosphorylation. Taken together, these findings suggest that puerarin prevents Aβ-induced microglial apoptosis via the activation of PI3K/Akt signaling pathway, and might be a potential preventive or therapeutic agent for Alzheimer’s disease.