Synchronizing Mammalian Cells for Mitotic Analysis of the Localization of Survivin.

This paper describes an easy method to enrich the harvest of adherent mammalian cells at each stage of mitosis (from prometaphase to cytokinesis) by combining Eg5 inhibition using dimethylenastron (DMA) with mitotic shake-off, followed by timed release from the drug.

Mitochondrial survivin reduces oxidative phosphorylation in cancer cells by inhibiting mitophagy.

Survivin (also known as BIRC5) is a cancer-associated protein that is pivotal for cellular life and death - it is an essential mitotic protein and an inhibitor of apoptosis. In cancer cells, a small pool of survivin localises to the mitochondria, the function of which remains to be elucidated. Here, we report that mitochondrial survivin inhibits the selective form of autophagy called 'mitophagy', causing an accumulation of respiratory-defective mitochondria. Mechanistically, the data reveal that survivin prevents recruitment of the E3-ubiquitin ligase Parkin to mitochondria and their subsequent recognition by the autophagosome. The data also demonstrate that cells in which mitophagy has been blocked by survivin expression have an increased dependency on glycolysis. As these effects were found exclusively in cancer cells, they suggest that the primary act of mitochondrial survivin is to steer cells towards the implementation of the Warburg transition by inhibiting mitochondrial turnover, which enables them to adapt and survive.This article has an associated First Person interview with the first author of the paper.

Transporting cells over several days without dry-ice.

This paper describes a simple, hazard-free and inexpensive procedure that allows researchers to send cultured cells across the globe at ambient temperatures. The method enables transit of up to 2 weeks without compromising cell recovery. Its use will assist collaborators in distant laboratories to exchange cells without using dry-ice.

Survivin at a glance.

Survivin (also known as BIRC5) is an evolutionarily conserved eukaryotic protein that is essential for cell division and can inhibit cell death. Normally it is only expressed in actively proliferating cells, but is upregulated in most, if not all cancers; consequently, it has received significant attention as a potential oncotherapeutic target. In this Cell Science at a Glance article and accompanying poster, we summarise our knowledge of survivin 21 years on from its initial discovery. We describe the structure, expression and function of survivin, highlight its interactome and conclude by describing anti-survivin strategies being trialled.

Engulfment, persistence and fate of Bdellovibrio bacteriovorus predators inside human phagocytic cells informs their future therapeutic potential.

In assessing the potential of predatory bacteria, such as Bdellovibrio bacteriovorus, to become live therapeutic agents against bacterial infections, it is crucial to understand and quantify Bdellovibrio host cell interactions at a molecular level. Here, we quantify the interactions of live B. bacteriovorus with human phagocytic cells, determining the uptake mechanisms, persistence, associated cytokine responses and intracellular trafficking of the non-growing B. bacteriovorus in PMA-differentiated U937 cells. B. bacteriovorus are engulfed by U937 cells and persist for 24 h without affecting host cell viability and can be observed microscopically and recovered and cultured post-uptake. The uptake of predators is passive and depends on the dynamics of the host cell cytoskeleton; the engulfed predators are eventually trafficked through the phagolysosomal pathway of degradation. We have also studied the prevalence of B. bacteriovorus specific antibodies in the general human population. Together, these results quantify a period of viable persistence and the ultimate fate of B. bacteriovorus inside phagocytic cells. They provide new knowledge on predator availability inside hosts, plus potential longevity and therefore potential efficacy as a treatment in humans and open up future fields of work testing if predators can prey on host-engulfed pathogenic bacteria.

A cost-effective, analytical method for measuring metabolic load of mitochondria.

Analysis of cellular energetics is central to understanding metabolic diseases including diabetes and cancer. The two most commonly used methods to monitor cellular respiration are the Seahorse-XF system, and Glo™ assays, which are considered "gold standards". These commercial methods measure energetics indirectly and require considerable financial investment. Here we describe an alternative assay that enables accurate quantification of NADH turnover and that is affordable. This method measures resazurin reduction to resorufin at rising concentrations in the presence of purified mitochondrial extracts until NADH becomes a rate-limiting factor. This indicates the maximal level of NADH turnover in each sample and therefore infers metabolic activity. Here we compare MRC5, MCF7 and MDA231 cell lines which have differing metabolic profiles.

The use of decellularised animal tissue to study disseminating cancer cells.

Since the establishment of cell culture, common practice has been to grow adherent cells in 2D monolayers. Although cells behave completely differently when grown under these artificial conditions, the ease of 2D culturing has meant that this practice still prevails, and adopting conditions that more closely reflect the natural microenvironment has been met with substantial inertia. The alternative, animal models that mimic natural human physiology, are less accessible, strictly regulated and require licences and expensive facilities. Although transition from 2D to 3D cell culturing is gathering momentum, there is a clear need for alternative culturing methods that more closely resemble in vivo conditions. Here, we show that decellularised organs gleaned from discarded animal carcasses are ideal biomimetic scaffolds to support secondary tumour initiation in vitro Further, we describe how to decellularise tissue and perform basic histochemistry and immunofluorescence procedures for cell and matrix detection. Cancer cell behaviour on this matrix is followed by way of an example. Because integration into the traditional work flow is easy and inexpensive, we hope this article will encourage other researchers to adopt this approach.

Survivin inhibits excessive autophagy in cancer cells but does so independently of its interaction with LC3.

Survivin expression is pivotal to life and death at the cellular level. For the past decade its pro-survival activity has been attributed to its essential role in cell proliferation and its ability to inhibit apoptosis. However, a growing body of evidence suggests that it may also contribute to cell viability through an as yet undefined role in autophagy. We report that survivin overexpression in osteosarcoma (U2OS) cells is associated with increased LC3-II expression, smaller autophagosomes, enlarged lysosomes and reduced autophagic flux. We also demonstrate that survivin binds LC3 directly through a canonical LC3-interacting region (LIR) in its baculovirus inhibitors of apoptosis protein (IAP) repeat BIR domain, mutation of which inhibits the interaction, but does not abrogate its influence on autophagy. Collectively these data suggest that survivin expression restricts autophagic flux, thereby inhibiting late-stage autophagy and preventing cell death, but does so independently of LC3.

The N-terminus of survivin is a mitochondrial-targeting sequence and Src regulator.

Survivin (also known as BIRC5) is a cancer-associated protein that exists in several locations in the cell. Its cytoplasmic residence in interphase cells is governed by CRM1 (also known as XPO1)-mediated nuclear exportation, and its localisation during mitosis to the centromeres and midzone microtubules is that of a canonical chromosomal passenger protein. In addition to these well-established locations, survivin is also a mitochondrial protein, but how it gets there and its function therein is presently unclear. Here, we show that the first ten amino acids at the N-terminus of survivin are sufficient to target GFP to the mitochondria in vivo, and ectopic expression of this decapeptide decreases cell adhesion and accelerates proliferation. The data support a signalling mechanism in which this decapeptide regulates the tyrosine kinase Src, leading to reduced focal adhesion plaques and disruption of F-actin organisation. This strongly suggests that the N-terminus of survivin is a mitochondrial-targeting sequence that regulates Src, and that survivin acts in concert with Src to promote tumorigenesis.

Plasmodium P-Type Cyclin CYC3 Modulates Endomitotic Growth during Oocyst Development in Mosquitoes.

Cell-cycle progression and cell division in eukaryotes are governed in part by the cyclin family and their regulation of cyclin-dependent kinases (CDKs). Cyclins are very well characterised in model systems such as yeast and human cells, but surprisingly little is known about their number and role in Plasmodium, the unicellular protozoan parasite that causes malaria. Malaria parasite cell division and proliferation differs from that of many eukaryotes. During its life cycle it undergoes two types of mitosis: endomitosis in asexual stages and an extremely rapid mitotic process during male gametogenesis. Both schizogony (producing merozoites) in host liver and red blood cells, and sporogony (producing sporozoites) in the mosquito vector, are endomitotic with repeated nuclear replication, without chromosome condensation, before cell division. The role of specific cyclins during Plasmodium cell proliferation was unknown. We show here that the Plasmodium genome contains only three cyclin genes, representing an unusual repertoire of cyclin classes. Expression and reverse genetic analyses of the single Plant (P)-type cyclin, CYC3, in the rodent malaria parasite, Plasmodium berghei, revealed a cytoplasmic and nuclear location of the GFP-tagged protein throughout the lifecycle. Deletion of cyc3 resulted in defects in size, number and growth of oocysts, with abnormalities in budding and sporozoite formation. Furthermore, global transcript analysis of the cyc3-deleted and wild type parasites at gametocyte and ookinete stages identified differentially expressed genes required for signalling, invasion and oocyst development. Collectively these data suggest that cyc3 modulates oocyst endomitotic development in Plasmodium berghei.

Mitotic activity of survivin is regulated by acetylation at K129.

Survivin is a cancer-associated protein regulated by multiple factors, including acetylation at K129 within its C-terminal α-helical tail. Acetylation of survivin is being pursued as a potential prognostic marker in breast cancer. This modification at K129 may cause nuclear accumulation of survivin in interphase cells; however, whether this affects its essential role during mitosis has not been addressed. We posited whether mimicking acetylation of survivin at K129 alters its activity during mitosis. Fluorescence microscopy and time-lapse imaging showed that, mutating this site to an alanine to act as a constitutive acetyl mimetic, K129A, causes defects in chromosome segregation and cytokinesis. As a non-acetylatable version, K129R, also has difficulty during mitotic exit, we conclude that cyclical acetylation and deacetylation is required for fully functional survivin during mitosis.

The functional repertoire of survivin's tails.

Survivin is a multitasking protein that can inhibit cell death and that is essential for mitosis. Due to these prosurvival activities and the correlation of its expression with tumor resistance to conventional cancer treatments, survivin has received much attention as a potential oncotherapeutic target. Nevertheless, many questions regarding its exact role at the molecular level remain to be elucidated. In this study we ask whether the extreme C- and NH2 termini of survivin are required for it to carry out its cytoprotective and mitotic duties. When assayed for their ability to act as a cytoprotectant, both survivin1-120 and survivin11-142 were able to protect cells against TRAIL-mediated apoptosis, but when challenged with irradiation cells expressing survivin11-142 had no survival advantage. During mitosis, however, removing the NH2 terminal 10 amino acids (survivin11-142) had no apparent effect but truncating 22 amino acids from the C-terminus (survivin1-120) prevented survivin from transferring to the midzone microtubules during anaphase. Collectively the data herein presented suggest that the C-terminus is required for cell division, and that the NH2 terminus is dispensable for apoptosis and mitosis but required for protection from irradiation.

Sox2 acts as a transcriptional repressor in neural stem cells.

BACKGROUND: The transcription factor, Sox2, is central to the behaviour of neural stem cells. It is also one of the key embryonic stem cell factors that, when overexpressed can convert somatic cells into induced pluripotent cells. Although generally studied as a transcriptional activator, recent evidence suggests that it might also repress gene expression. RESULTS: We show that in neural stem cells Sox2 represses as many genes as it activates. We found that Sox2 interacts directly with members of the groucho family of corepressors and that repression of several target genes required this interaction. Strikingly, where many of the genes activated by Sox2 encode transcriptional regulators, no such genes were repressed. Finally, we found that a mutant form of Sox2 that was unable to bind groucho was no longer able to inhibit differentiation of neural stem cells to the same extent as the wild type protein. CONCLUSIONS: These data reveal a major new mechanism of action for this key transcription factor. In the context of our understanding of endogenous stem cells, this highlights the need to determine how such a central regulator can distinguish which genes to activate and which to repress.

Analysis of the functional repertoire of a mutant form of survivin, K129E, which has been linked to lung cancer.

BACKGROUND: Survivin is a protein that is normally present only in G2 and M-phases in somatic cells, however, in cancer cells, it is expressed throughout the cell cycle. A prosurvival factor, survivin is both an inhibitor of apoptosis and an essential mitotic protein, thus it has attracted much attention as a target for new oncotherapies. Despite its prevalence in cancer, reports of survivin mutations have mostly been restricted to loci within its promoter, which increase the abundance of the protein. To date the only published mutation within the coding sequence is an adenine > guanine substitution in exon 4. This polymorphism, which was found in a cohort of Korean lung cancer patients, causes a lysine > glutamic acid mutation (K129E) in the protein. However, whether it plays a causative role in cancer has not been addressed. METHODS: Using site directed mutagenesis we recapitulate K129E expression in cultured human cells and assess its anti-apoptotic and mitotic activities. RESULTS: K129E retains its anti-apoptotic activity, but causes errors in mitosis and cytokinesis, which may be linked to its reduced affinity for borealin. CONCLUSION: K129E expression can induce genomic instability by introducing mitotic aberrations, thus it may play a causative role in cancer.

The Plasmodium berghei Ca(2+)/H(+) exchanger, PbCAX, is essential for tolerance to environmental Ca(2+) during sexual development.

Ca(2+) contributes to a myriad of important cellular processes in all organisms, including the apicomplexans, Plasmodium and Toxoplasma. Due to its varied and essential roles, free Ca(2+) is tightly regulated by complex mechanisms. These mechanisms are therefore of interest as putative drug targets. One pathway in Ca(2+) homeostatic control in apicomplexans uses a Ca(2+)/H(+) exchanger (a member of the cation exchanger family, CAX). The P. falciparum CAX (PfCAX) has recently been characterised in asexual blood stage parasites. To determine the physiological importance of apicomplexan CAXs, tagging and knock-out strategies were undertaken in the genetically tractable T. gondii and P. berghei parasites. In addition, a yeast heterologous expression system was used to study the function of apicomplexan CAXs. Tagging of T. gondii and P. berghei CAXs (TgCAX and PbCAX) under control of their endogenous promoters could not demonstrate measureable expression of either CAX in tachyzoites and asexual blood stages, respectively. These results were consistent with the ability of parasites to tolerate knock-outs of the genes for TgCAX and PbCAX at these developmental stages. In contrast, PbCAX expression was detectable during sexual stages of development in female gametocytes/gametes, zygotes and ookinetes, where it was dispersed in membranous networks within the cytosol (with minimal mitochondrial localisation). Furthermore, genetically disrupted parasites failed to develop further from "round" form zygotes, suggesting that PbCAX is essential for ookinete development and differentiation. This impeded phenotype could be rescued by removal of extracellular Ca(2+). Therefore, PbCAX provides a mechanism for free living parasites to multiply within the ionic microenvironment of the mosquito midgut. Ca(2+) homeostasis mediated by PbCAX is critical and suggests plasmodial CAXs may be targeted in approaches designed to block parasite transmission.

Genomic DNA damage and ATR-Chk1 signaling determine oncolytic adenoviral efficacy in human ovarian cancer cells.

Oncolytic adenoviruses replicate selectively within and lyse malignant cells. As such, they are being developed as anticancer therapeutics. However, the sensitivity of ovarian cancers to adenovirus cytotoxicity varies greatly, even in cells of similar infectivity. Using both the adenovirus E1A-CR2 deletion mutant dl922-947 and WT adenovirus serotype 5 in a panel of human ovarian cancer cell lines that cover a 3-log range of sensitivity, we observed profound overreplication of genomic DNA only in highly sensitive cell lines. This was associated with the presence of extensive genomic DNA damage. Inhibition of ataxia telangiectasia and Rad3-related checkpoint kinase 1 (ATR-Chk1), but not ataxia telangiectasia mutated (ATM), promoted genomic DNA damage and overreplication in resistant and partially sensitive cells. This was accompanied by increased adenovirus cytotoxicity both in vitro and in vivo in tumor-bearing mice. We also demonstrated that Cdc25A was upregulated in highly sensitive ovarian cancer cell lines after adenovirus infection and was stabilized after loss of Chk1 activity. Knockdown of Cdc25A inhibited virus-induced DNA damage in highly sensitive cells and blocked the effects of Chk1 inhibition in resistant cells. Finally, inhibition of Chk1 decreased homologous recombination repair of virus-induced genomic DNA double-strand breaks. Thus, virus-induced host cell DNA damage signaling and repair are key determinants of oncolytic adenoviral activity, and promoting unscheduled DNA synthesis and/or impeding homologous recombination repair could potentiate the effects of oncolytic adenoviruses in the treatment of ovarian cancer.

Chromosome 'by-Aurora-ientation' during mitosis.

New evidence from three separate laboratories, published recently in Science, has shown that centromere positioning of the CPC (chromosomal passenger complex) during early mitosis is achieved through direct interaction between the CPP (chromosomal passenger protein) survivin and histone H3. In essence, an acidic pocket in the BIR (baculovirus inhibitor of apoptosis repeat) domain of survivin binds to the NH2 tail of histone H3 specifically when it is phosphorylated at threonine 3, a mark that is placed by the mitotic kinase, haspin. These data are significant, as they describe a fundamental mechanism, conserved throughout eukaryotes, which is essential for chromosome biorientation and the maintenance of genome stability during mitosis.

Threonine 48 in the BIR domain of survivin is critical to its mitotic and anti-apoptotic activities and can be phosphorylated by CK2 in vitro.

In this study we report that the protein kinase CK2 phosphorylates survivin specifically on threonine 48 (T48) within its BIR domain, and that T48 is critical to both the mitotic and anti-apoptotic roles of survivin. Interestingly, during mitosis T48 mutants localise normally, but are unable to support cell growth when endogenous survivin is removed by siRNA. In addition, while overexpression of survivin normally confers inhibition of TRAIL-mediated apoptosis, this protection is abolished by mutation of T48. Furthermore in interphase cells depletion of endogenous survivin causes redistribution of T48 mutants from the cytoplasm to the nucleus and treatment of cells expressing survivin-GFP with the CK2 inhibitor TBB phenocopies this nuclear redistribution. Finally, we show T48 mutants have increased affinity for borealin, and that this association and cell proliferation can be restored by introduction of a second mutation at T97. To our knowledge these data are the first to identify T48 as a key regulatory site on survivin, and CK2 as a mediator of its mitotic and anti-apoptotic functions.

The polo-like kinase inhibitor BI 2536 exhibits potent activity against malignant plasma cells and represents a novel therapy in multiple myeloma.

OBJECTIVE: Polo-like kinase 1 (Plk1) is a regulator of the cell cycle that has been implicated in the pathology of many cancers. We have investigated whether this kinase plays a role in multiple myeloma (MM) using the Plk1 inhibitor BI 2536. MATERIALS AND METHODS: We have used six MM cell lines and six patient-derived samples to determine the effects of the Plk1 inhibitor, BI 2536, on cell viability, apoptosis, and cytokinesis. We have also examined the effect of the microenvironment on these parameters and the effects of BI 2536 in combination with other antimyeloma agents. RESULTS: We show that MM cell lines and patient samples express PLK1 and that cell death by apoptosis occurs when Plk1 is inhibited. Cells treated with BI 2536 accumulate in the G(2)/M phase of the cell cycle causing endoduplication. The effects of BI 2536 are not abrogated when cells are cultured on extracellular matrix components, in the presence of interleukin-6, or with bone marrow stromal cells. CONCLUSIONS: Plk1 inhibition leads to cell death in MM cell lines and patient myeloma samples. Our data suggest that inhibition of Plk1 may have potential use as a therapeutic strategy in multiple myeloma.

p21 Promotes oncolytic adenoviral activity in ovarian cancer and is a potential biomarker.

The oncolytic adenovirus dl922-947 replicates selectively within and lyses cells with a dysregulated Rb pathway, a finding seen in > 90% human cancers. dl922-947 is more potent than wild type adenovirus and the E1B-deletion mutant dl1520 (Onyx-015). We wished to determine which host cell factors influence cytotoxicity. SV40 large T-transformed MRC5-VA cells are 3-logs more sensitive to dl922-947 than isogenic parental MRC5 cells, confirming that an abnormal G1/S checkpoint increases viral efficacy. The sensitivity of ovarian cancer cells to dl922-947 varied widely: IC50 values ranged from 51 (SKOV3ip1) to 0.03 pfu/cell (TOV21G). Cells sensitive to dl922-947 had higher S phase populations and supported earlier E1A expression. Cytotoxicity correlated poorly with both infectivity and replication, but well with expression of p21 by microarray and western blot analyses. Matched p21+/+ and -/- Hct116 cells confirmed that p21 influences dl922-947 activity in vitro and in vivo. siRNA-mediated p21 knockdown in sensitive TOV21G cells decreases E1A expression and viral cytotoxicity, whilst expression of p21 in resistant A2780CP cells increases virus activity in vitro and in intraperitoneal xenografts. These results highlight that host cell factors beyond simple infectivity can influence the efficacy of oncolytic adenoviruses. p21 expression may be an important biomarker of response in clinical trials.