<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.     

<Emphasis Type="Italic">Drosophila</Emphasis> CENP-C is essential for centromere identity

Centromeres are specialized chromosomal domains that direct mitotic kinetochore assembly and are defined by the presence of CENP-A (CID in Drosophila) and CENP-C. While the role of CENP-A appears to be highly conserved, functional studies in different organisms suggest that the precise role of CENP-C in kinetochore assembly is still under debate. Previous studies in vertebrate cells have shown that CENP-C inactivation causes mitotic delay, chromosome missegregation, and apoptosis; however, in Drosophila, the role of CENP-C is not well-defined. We have used RNA interference depletion in S2 cells to address this question and we find that depletion of CENP-C causes a kinetochore null phenotype, and consequently, the spindle checkpoint, kinetochore–microtubule interactions, and spindle size are severely misregulated. Importantly, we show that CENP-C is required for centromere identity as CID, MEI-S332, and chromosomal passenger proteins fail to localize in CENP-C depleted cells, suggesting a tight communication between the inner kinetochore proteins and centromeres. We suggest that CENP-C might fulfill the structural roles of the human centromere-associated proteins not identified in Drosophila.     

Drosophila Smad2 Opposes Mad Signaling during Wing Vein Development

In the vertebrates, the BMP/Smad1 and TGF-β/Smad2 signaling pathways execute antagonistic functions in different contexts of development. The differentiation of specific structures results from the balance between these two pathways. For example, the gastrula organizer/node of the vertebrates requires a region of low Smad1 and high Smad2 signaling. In Drosophila, Mad regulates tissue determination and growth in the wing, but the function of dSmad2 in wing patterning is largely unknown. In this study, we used an RNAi loss-of-function approach to investigate dSmad2 signaling during wing development. RNAi-mediated knockdown of dSmad2 caused formation of extra vein tissue, with phenotypes similar to those seen in Dpp/Mad gain-of-function. Clonal analyses revealed that the normal function of dSmad2 is to inhibit the response of wing intervein cells to the extracellular Dpp morphogen gradient that specifies vein formation, as measured by expression of the activated phospho-Mad protein. The effect of dSmad2 depletion in promoting vein differentiation was dependent on Medea, the co-factor shared by Mad and dSmad2. Furthermore, double RNAi experiments showed that Mad is epistatic to dSmad2. In other words, depletion of Smad2 had no effect in Mad-deficient wings. Our results demonstrate a novel role for dSmad2 in opposing Mad-mediated vein formation in the wing. We propose that the main function of dActivin/dSmad2 in Drosophila wing development is to antagonize Dpp/Mad signaling. Possible molecular mechanisms for the opposition between dSmad2 and Mad signaling are discussed.     

Regulatory system for the G1‐arrest during neuronal development in Drosophila

Neuronal network consists of many types of neuron and glial cells. This diversity is guaranteed by the constant cell proliferation of neuronal stem cells following stop cell cycle re‐entry, which leads to differentiation during development. Neuronal differentiation occurs mainly at the specific cell cycle phase, the G1 phase. Therefore, cell cycle exit at the G1 phase is quite an important issue in understanding the process of neuronal cell development. Recent studies have revealed that aberrant S phase re‐entry from the G1 phase often links cellular survival. In this review we discuss the different types of G1 arrest on the process of neuronal development in Drosophila. We also describe the issue that aberrant S phase entry often causes apoptosis, and the same mechanism might contribute to sensory organ defects, such as deafness.     

Normal cells, but not cancer cells, survive severe Plk1 depletion

We previously reported the phenotype of depletion of polo-like kinase 1 (Plk1) using RNA interference (RNAi) and showed that p53 is stabilized in Plk1-depleted cancer cells. In this study, we further analyzed the Plk1 depletion-induced phenotype in both cancer cells and primary cells. The vector-based RNAi approach was used to evaluate the role of the p53 pathway in Plk1 depletion-induced apoptosis in cancer cells with different p53 backgrounds. Although DNA damage and cell death can occur independently of p53, p53-deficient cancer cells were much more sensitive to Plk1 depletion than cancer cells with functional p53. Next, the lentivirus-based RNAi approach was used to generate a series of Plk1 hypomorphs. In HeLa cells, two weak hypomorphs showed only slight G2/M arrest, a medium hypomorph arrested with 4N DNA content, followed later by apoptosis, and a strong Plk1 hypomorph underwent serious mitotic catastrophe. In well-synchronized HeLa cells, a medium level of Plk1 depletion caused a 2-h delay of mitotic progression, and a high degree of Plk1 depletion significantly delayed mitotic entry and completely blocked cells at mitosis. In striking contrast, normal hTERT-RPE1 and MCF10A cells were much less sensitive to Plk1 depletion than HeLa cells; no apparent cell proliferation defect or cell cycle arrest was observed after Plk1 depletion in these cells. Therefore, these data further support suggestions that Plk1 may be a feasible cancer therapy target.     

PpV,acting via the JNK pathway,represses apoptosis during normal development of <Emphasis Type="Italic">Drosophila</Emphasis> wing

Apoptosis is one of the main fundamental biological processes required for development of multicellular organisms. Inappropriate regulation of apoptosis can lead to severe developmental abnormalities and diseases. Therefore, the control of apoptosis, not only for its activation but also for its inhibition, is critically important during development. In contrast to the extensive studies of apoptosis induction, its inhibitory mechanisms that are even more vital in certain populations of cells actually are very far from being well understood. Here we report an inhibitory role of protein phosphatase V (PpV), a serine/threonine protein phosphatase, in controlling the apoptosis during Drosophila wing development. We observed that inhibition of ppv by RNAi in wing imaginal discs induced ectopic cell death and caspase activation, thus, resulted in a defective adult wing. Moreover, knocking-down ppv triggered the activation of c-Jun N-terminal kinase (JNK) signal, an evolutionarily conserved intracellular signaling that has been implicated to modulate the apoptotic machinery in many biological and experimental systems. Disrupting the JNK signal transduction was adequate to suppress the ppv effects for wing development. Together, we provided the evidence to demonstrate that ppv is required for normal wing development in maintaining the silence of apoptotic signal possibly through JNK pathway.     

Coordination between cell proliferation and apoptosis after DNA damage in Drosophila

Exposure to genotoxic stress promotes cell cycle arrest and DNA repair or apoptosis. These “life” or “death” cell fate decisions often rely on the activity of the tumor suppressor gene p53. Therefore, the precise regulation of p53 is essential to maintain tissue homeostasis and to prevent cancer development. However, how cell cycle progression has an impact on p53 cell fate decision-making is mostly unknown. In this work, we demonstrate that Drosophila p53 proapoptotic activity can be impacted by the G2/M kinase Cdk1. We find that cell cycle arrested or endocycle-induced cells are refractory to ionizing radiation-induced apoptosis. We show that p53 binding to the regulatory elements of the proapoptotic genes and its ability to activate their expression is compromised in experimentally arrested cells. Our results indicate that p53 genetically and physically interacts with Cdk1 and that p53 proapoptotic role is regulated by the cell cycle status of the cell. We propose a model in which cell cycle progression and p53 proapoptotic activity are molecularly connected to coordinate the appropriate response after DNA damage.Subject terms: Cell biology, Development, Gene regulation, Molecular biology     

TGEV nucleocapsid protein induces cell cycle arrest and apoptosis through activation of p53 signaling

Our previous studies showed that TGEV infection could induce cell cycle arrest and apoptosis via activation of p53 signaling in cultured host cells. However, it is unclear which viral gene causes these effects. In this study, we investigated the effects of TGEV nucleocapsid (N) protein on PK-15 cells. We found that TGEV N protein suppressed cell proliferation by causing cell cycle arrest at the S and G2/M phases and apoptosis. Characterization of various cellular proteins that are involved in regulating cell cycle progression demonstrated that the expression of N gene resulted in an accumulation of p53 and p21, which suppressed cyclin B1, cdc2 and cdk2 expression. Moreover, the expression of TGEV N gene promoted translocation of Bax to mitochondria, which in turn caused the release of cytochrome c, followed by activation of caspase-3, resulting in cell apoptosis in the transfected PK-15 cells following cell cycle arrest. Further studies showed that p53 inhibitor attenuated TGEV N protein induced cell cycle arrest at S and G2/M phases and apoptosis through reversing the expression changes of cdc2, cdk2 and cyclin B1 and the translocation changes of Bax and cytochrome c induced by TGEV N protein. Taken together, these results demonstrated that TGEV N protein might play an important role in TGEV infection-induced p53 activation and cell cycle arrest at the S and G2/M phases and apoptosis occurrence.     

Overexpression of <Emphasis Type="Italic">Drosophila</Emphasis> Rad51 protein (DmRad51) disrupts cell cycle progression and leads to apoptosis

Among proteins involved in homologous recombination, Rad51 is an essential enzyme in DNA repair and recombination. However, little is known about its role in cell cycle regulation and apoptosis. To examine the function of Drosophila Rad51 (DmRad51) in cell cycle regulation and apoptosis, DmRad51 protein was overexpressed using a heat shock-inducible promoter or the UAS–GAL4 binary expression system. We observed that ubiquitous expression of DmRad51 protein in flies carrying hsp26–Rad51 or UAS–Rad51 transgenes was lethal. Induction of DmRad51—more specifically in eye or wing imaginal discs—caused tissue-specific cell death in the domains of DmRad51 expression. Cell death was due to apoptosis, as shown by staining with the TdT-mediated dUTP nick-end labeling assay. Immunocytochemistry revealed that cells expressing DmRad51 colocalized with apoptotic cells. In addition, the phenotypes caused by the overexpression of DmRad51 were similar to those caused by ectopic expression of Reaper, a proapoptotic protein, and were partially suppressed by the coexpression of p35, an antiapoptotic protein. Using an antiphosphohistone H3 antibody, we also observed that the overexpression of DmRad51 protein disrupted normal cell cycle progression in eye imaginal discs. Taken together, these results show that ectopically expressed DmRad51 disrupts cell cycle regulation and induces apoptosis.     

Genetic analysis of dTSPO,an outer mitochondrial membrane protein,reveals its functions in apoptosis,longevity, and Aβ42‐induced neurodegeneration

The outer mitochondrial membrane (OMM) protein, the translocator protein 18 kDa (TSPO), formerly named the peripheral benzodiazepine receptor (PBR), has been proposed to participate in the pathogenesis of neurodegenerative diseases. To clarify the TSPO function, we identified the Drosophila homolog, CG2789/dTSPO, and studied the effects of its inactivation by P‐element insertion, RNAi knockdown, and inhibition by ligands (PK11195, Ro5‐4864). Inhibition of dTSPO inhibited wing disk apoptosis in response to γ‐irradiation or H₂O₂ exposure, as well as extended male fly lifespan and inhibited Aβ42‐induced neurodegeneration in association with decreased caspase activation. Therefore, dTSPO is an essential mediator of apoptosis in Drosophila and plays a central role in controlling longevity and neurodegenerative disease, making it a promising drug target.     

Allicin induces cell cycle arrest and apoptosis of breast cancer cells in vitro via modulating the p53 pathway

Background

The tumor suppressor protein p53 is a most promising target for the development of anticancer drugs. Allicin (diallylthiosulfinate) is one of the most active components of garlic (Alliium sativum L.) and possesses a variety of health-promoting properties with pharmacological applications. However, whether allicin plays an anti-cancer role against breast cancer cells through the induction of p53-mediated apoptosis remains unknown.

Methods and results

In this study, we investigate the anti-breast cancer effect of allicin in vitro by using MCF-7 and MD-MBA-231 cells. We found that allicin reduces cell viability, induces apoptosis and cell cycle arrest in both cells. Allicin activated p53 and caspase 3 expressions in both cells but produced different effects on the expression of p53-related biomarkers. In MDA-MB-231 cells, allicin up-regulated the mRNA and protein expression of A1BG and THBS1 while down-regulated the expression of TPM4. Conversely, the mRNA and protein expression of A1BG, THBS1 and TPM4 were all reduced in MCF-7 cells. Hence, allicin induces cell cycle arrest and apoptosis in breast cancer cells through p53 activation but it effects on the expression of p53-related biomarkers were dependent upon the specific type of breast cancer involved.

Conclusions

These findings suggest that allicin induces apoptosis and regulates biomarker expression in breast cancer cell lines through modulating the p53 signaling pathway. Furthermore, our results promote the utility of allicin as compound for further studies as an anticancer drug targeting p53.

     

The RASSF6 Tumor Suppressor Protein Regulates Apoptosis and the Cell Cycle via MDM2 Protein and p53 Protein

Ras association domain family (RASSF) 6 is a member of the C-terminal RASSF proteins such as RASSF1A and RASSF3. RASSF6 is involved in apoptosis in various cells under miscellaneous conditions, but it remains to be clarified how RASSF6 exerts tumor-suppressive roles. We reported previously that RASSF3 facilitates the degradation of MDM2, a major E3 ligase of p53, and stabilizes p53 to function as a tumor suppressor. In this study, we demonstrate that RASSF6 overexpression induces G₁/S arrest in p53-positive cells. Its depletion prevents UV- and VP-16-induced apoptosis and G₁/S arrest in HCT116 and U2OS cells. RASSF6-induced apoptosis partially depends on p53. RASSF6 binds MDM2 and facilitates its ubiquitination. RASSF6 depletion blocks the increase of p53 in response to UV exposure and up-regulation of p53 target genes. RASSF6 depletion delays DNA repair in UV- and VP-16-treated cells and increases polyploid cells after VP-16 treatment. These findings indicate that RASSF6 stabilizes p53, regulates apoptosis and the cell cycle, and functions as a tumor suppressor. Together with the previous reports regarding RASSF1A and RASSF3, the stabilization of p53 may be the common function of the C-terminal RASSF proteins.     

CAF-1 is required for efficient replication of euchromatic DNA in <Emphasis Type="Italic">Drosophila</Emphasis> larval endocycling cells

The endocycle constitutes an effective strategy for cell growth during development. In contrast to the mitotic cycle, it consists of multiple S-phases with no intervening mitosis and lacks a checkpoint ensuring the replication of the entire genome. Here, we report an essential requirement of chromatin assembly factor-1 (CAF-1) for Drosophila larval endocycles. This complex promotes histone H3–H4 deposition onto newly synthesised DNA in vitro. In metazoans, the depletion of its large subunit leads to the rapid accumulation of cells in S-phase. However, whether this slower S-phase progression results from the activation of cell cycle checkpoints or whether it reflects a more direct requirement of CAF-1 for efficient replication in vivo is still debated. Here, we show that, strikingly, Drosophila larval endocycling cells depleted for the CAF-1 large subunit exhibit normal dynamics of progression through endocycles, although accumulating defects, such as perturbation of nucleosomal organisation, reduction of the replication efficiency of euchromatic DNA and accumulation of DNA damage. Given that the endocycle lacks a checkpoint ensuring the replication of the entire genome, the biological context of Drosophila larval development offered a unique opportunity to highlight the requirement of CAF-1 for chromatin organisation and efficient replication processes in vivo, independently of checkpoint activation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A delay prior to mitotic entry triggers caspase 8-dependent cell death in p53-deficient Hela and HCT-116 cells

Stathmin/Oncoprotein 18, a microtubule destabilizing protein, is required for survival of p53-deficient cells. Stathmin-depleted cells are slower to enter mitosis, but whether delayed mitotic entry triggers cell death or whether stathmin has a separate pro-survival function was unknown. To test these possibilities, we abrogated the cell cycle delay by inhibiting Wee1 in synchronized, stathmin-depleted cells and found that apoptosis was reduced to control levels. Synchronized cells treated with a 4 hour pulse of inhibitors to CDK1 or both Aurora A and PLK1 delayed mitotic entry and apoptosis was triggered only in p53-deficient cells. We did not detect mitotic defects downstream of the delayed mitotic entry, indicating that cell death is activated by a mechanism distinct from those activated by prolonged mitotic arrest. Cell death is triggered by initiator caspase 8, based on its cleavage to the active form and by rescue of viability after caspase 8 depletion or treatment with a caspase 8 inhibitor. In contrast, initiator caspase 9, activated by prolonged mitotic arrest, is not activated and is not required for apoptosis under our experimental conditions. P53 upregulates expression of cFLIP_L, a protein that blocks caspase 8 activation. cFLIP_L levels are lower in cells lacking p53 and these levels are reduced to a greater extent after stathmin depletion. Expression of FLAG-tagged cFLIP_L in p53-deficient cells rescues them from apoptosis triggered by stathmin depletion or CDK1 inhibition during G2. These data indicate that a cell cycle delay in G2 activates caspase 8 to initiate apoptosis specifically in p53-deficient cells.     

Function of <Emphasis Type="Italic">Drosophila mob2</Emphasis> in photoreceptor morphogenesis

The Drosophila photoreceptor is a highly polarized cell; a mature photoreceptor cell in Drosophila contains a photosensitive structure (the rhabdomere) and a supporting membrane (stalk) at its apical membrane. In a screen to isolate genes involved in determining stalk and rhabdomere formation, this study has identified the Drosophila mob2 (Dmob2) gene. Dmob2 belongs to a Mob1/phocein domain protein family whose functions are involved in polarized cell growth and asymmetric cell fate determination in yeast. To study the role of Dmob2 in photoreceptor development, we have raised an antibody against the Dmob2 protein. An immunocytochemical study has shown that Dmob2 is mainly localized in the apical membrane of photoreceptor cells during early development. As development proceeds, Dmob2 is gradually confined to the rhabdomere base of the photoreceptor cells. RNA interference (RNAi) for knockdown Dmob2 expression during eye development impairs rhabdomere formation. Our study further shows that the subcellular localization of phosphorylated Moesin and Crumbs in the developing photoreceptor cell is disrupted in Dmob2 RNAi flies. This work thus reports a novel function of Dmob2 in photoreceptor cell development.     

Rab11 regulates JNK and Raf/MAPK‐ERK signalling pathways during Drosophila wing development

Developmental signalling pathways are regulated by intracellular vesicle trafficking in multicellular organisms. In our earlier communication, we have shown that mutation in Rab11 (a subfamily of the Ypt/Rab gene family) results in the activation of JNK signalling pathways in Drosophila eye. Here, we report that Rab11 regulates JNK and Raf/MAPK‐ERK signalling pathways during Drosophila wing development. Using immunofluorescence and immunohistochemical analyses, we show that overexpression of Rab11 in mutant wing imaginal disc cells triggers the induction of apoptosis and activation of JNK and ERK. Further, using a genetic approach it has been shown that Rab11 interacts with the components of these pathways during Drosophila wing development. In addition to this, in Rab11 mutant wing imaginal discs JNK activity was monitored using puc^E69, a P‐lacZ enhancer‐trap line inserted in puckered (puc). A strong induction of puc in Rab11 mutant wing imaginal disc cells provided a strong support that Rab11 regulates the JNK signalling pathway during Drosophila wing development.