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Tumour cells endure both oncogenic and environmental stresses during cancer progression. Transformed cells must meet increased demands for protein and lipid production needed for rapid proliferation and must adapt to exist in an oxygen- and nutrient-deprived environment. To overcome such challenges, cancer cells exploit intrinsic adaptive mechanisms such as the unfolded protein response (UPR). The UPR is a pro-survival mechanism triggered by accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER), a condition referred to as ER stress. IRE1, PERK and ATF6 are three ER anchored transmembrane receptors. Upon induction of ER stress, they signal in a coordinated fashion to re-establish ER homoeostasis, thus aiding cell survival. Over the past decade, evidence has emerged supporting a role for the UPR in the establishment and progression of several cancers, including breast cancer, prostate cancer and glioblastoma multiforme. This review discusses our current knowledge of the UPR during oncogenesis, tumour growth, metastasis and chemoresistance.

International audience; Cancer cells are exposed to intrinsic (oncogene) or extrinsic (microenvironmental) challenges, leading to activation of stress response pathways. The unfolded protein response (UPR) is the cellular response to endoplasmic reticulum (ER) stress and plays a pivotal role in tumor development. Depending on ER stress intensity and duration, the UPR is either pro-survival to preserve ER homeostasis or pro-death if the stress cannot be resolved. On one hand, the adaptive arm of the UPR is essential for cancer cells to survive the harsh conditions they are facing, and on the other hand, cancer cells have evolved mechanisms to bypass ER stress-induced cell death, thereby conferring them with a selective advantage for malignant transformation. Therefore, the mechanisms involved in the balance between survival and death outcomes of the UPR may be exploited as therapeutic tools to treat cancer

Death receptors are members of the tumour necrosis factor (TNF) receptor superfamily characterised by an ~80 amino acid long alpha-helical fold, termed the death domain (DD). Death receptors diversified during early vertebrate evolution indicating that the DD fold has plasticity and specificity that can be easily adjusted to attain additional functions. Eight members of the death receptor family have been identified in humans, which can be divided into four structurally homologous groups or clades, namely: the p75(NTR) clade (consisting of ectodysplasin A receptor, death receptor 6 (DR6) and p75 neurotrophin (NTR) receptor); the tumour necrosis factor receptor 1 clade (TNFR1 and DR3), the CD95 clade (CD95/FAS) and the TNF-related apoptosis-inducing ligand receptor (TRAILR) clade (TRAILR1 and TRAILR2). Receptors in the same clade participate in similar processes indicating that structural diversification enabled functional specialisation. On the surface of nearly all human cells multiple death receptors are expressed, enabling the cell to respond to a plethora of external signals. Activation of different death receptors converges on the activation of three main signal transduction pathways: nuclear factor-κB-mediated differentiation or inflammation, mitogen-associated protein kinase-mediated stress response and caspase-mediated apoptosis. While the ability to induce cell death is true for nearly all DRs, the FAS and TRAILR clades have specialised in inducing cell death. Here we summarise recent discoveries about the molecular regulation and structural requirements of apoptosis induction by death receptors and discuss how this information can be used to better explain the biological functions, similarities and distinguishing features of death receptors.

The endoplasmic reticulum (ER) is a highly dynamic organelle of fundamental importance present in all eukaryotic cells. The majority of synthesized structural and secreted proteins undergo post-translational modification, folding and oligomerization in the ER lumen, enabling proteins to carry out their physiological functions. Therefore, maintenance of ER homeostasis and function is imperative for proper cellular function. Physiological and pathological conditions can disturb ER homeostasis and thus negatively impact upon protein folding, resulting in an accumulation of unfolded proteins. Examples include hypoxia, hypo- and hyperglycemia, acidosis, and fluxes in calcium levels. Increased levels of unfolded/misfolded proteins within the ER lumen triggers a condition commonly referred to as ‘ER stress’. To combat ER stress, cells have evolved a highly conserved adaptive stress response referred to as the unfolded protein response (UPR). UPR signaling affords the cell a ‘window of opportunity’ for stress resolution however, if prolonged or excessive the UPR is insufficient and ER stress-induced cell death ensues. This review discusses the role of ER stress sensors IRE1, PERK and ATF6, describing their role in ER stress-induced death signaling with specific emphasis placed upon the importance of the intrinsic cell death pathway and Bcl-2 family regulation.

Microarray experiments are affected by several sources of variability. The paper demonstrates the major role of the day-to-day variability, it underlines the importance of a randomized block design when processing replicates over several days to avoid systematic biases and it proposes a simple algorithm that minimizes the day dependence.

The endoplasmic reticulum (ER) is the site of synthesis and folding of secretory and membrane bound proteins. The capacity of the ER to process proteins is limited and the accumulation of unfolded and misfolded proteins can lead to ER stress which has been associated with a wide range of diseases including cancer. In this review we initially provide an overview of our current understanding of how cells respond to ER stress at the molecular level and the key players involved in mediating the unfolded protein response (UPR). We review the evidence suggesting that the ER stress response could be important for the growth and development of tumors under stressful growth conditions such as hypoxia or glucose deprivation, which are commonly encountered by most solid tumors, and we analyse how it may be possible to exploit the unfolded protein response as an anticancer strategy. Two approaches to target the unfolded protein response are proposed-the first involves inhibiting components of the unfolded protein response so cells cannot adapt to stressful conditions and the second involves overloading the unfolded protein response so the cell is unable to cope, leading to cell death. We focused on proteins with an enzymatic activity that can be targeted by small molecule inhibitors as this is one of the most common approaches utilized by drug discovery companies. Finally, we review drugs currently in clinical development that affect the ER stress response and that may have potential as anti-tumor agents alone or in combination with other chemotherapeutics.

Cdc7 is an essential kinase that promotes DNA replication by activating origins of replication. Here, we characterized the potent Cdc7 inhibitor PHA-767491 (1) in biochemical and cell-based assays, and we tested its antitumor activity in rodents. We found that the compound blocks DNA synthesis and affects the phosphorylation of the replicative DNA helicase at Cdc7-dependent phosphorylation sites. Unlike current DNA synthesis inhibitors, PHA-767491 prevents the activation of replication origins but does not impede replication fork progression, and it does not trigger a sustained DNA damage response. Treatment with PHA-767491 results in apoptotic cell death in multiple cancer cell types and tumor growth inhibition in preclinical cancer models. To our knowledge, PHA-767491 is the first molecule that directly affects the mechanisms controlling initiation as opposed to elongation in DNA replication, and its activities suggest that Cdc7 kinase inhibition could be a new strategy for the development of anticancer therapeutics.

DNA replication is an essential process for cell division and as such it is a process that is directly targeted by several anticancer drugs. CDC7 plays an essential role in the activation of replication origins and has recently been proposed as a novel target for drug discovery. The MCM DNA helicase complex (MCM2-7) is a key target of the CDC7 kinase, and MCM phosphorylation status at specific sites is a reliable biomarker of CDC7 cellular activity. In this work we describe a cell-based assay that utilizes the "In Cell Western Technique'' (ICW) to identify compounds that affect cellular CDC7 activity. By screening a library of approved drugs and kinase inhibitors we found several compounds that can affect CDC7-dependent phosphorylation of MCM2 in HeLa cells. Among these, Mitoxantrone, a topoisomerase inhibitor, and Ryuvidine, previously described as a CDK4 inhibitor, cause a reduction in phosphorylated MCM2 levels and a sudden blockade of DNA synthesis that is accompanied by an ATM-dependent checkpoint response. This study sheds light on the previously observed cytotoxity of Ryuvidine, strongly suggesting that it is related to its effect of causing DNA damage.

Plants possess two major classes of cyclin-dependent kinases (CDK) with cyclin-binding motifs PSTAIRE (CDK-a) and PPTA/TLRE (CDK-b). Tobacco (Nicotiana tabacum L. cv Bright Yellow-2) cells are the most highly synchronizable plant culture, but no detailed analysis of CDK activities has been reported in this system. Here we describe isolation of new PPTALRE CDKs (Nicta;CdkB1) from Bright Yellow-2 cells and present detailed analysis of the mRNA, protein and kinase activity levels of CdkB1, and the PSTAIRE CDKA during the growth and cell cycles. CdkA andCdkB1 transcripts are more abundant in exponential than in stationary phase cells, but the two genes show strikingly different regulation during the cell cycle. CdkA mRNA and protein accumulate during G1 in cells re-entering the cell cycle, and immunoprecipitated histone H1 kinase activity increases at the G1/S boundary. Aphidicolin synchronized cells show the highest CDKA-associated histone H1 kinase activity during S-G2 phases, althoughCdkA mRNA and protein levels are not significantly regulated. In contrast, CdkB1 transcripts are present at very low levels until S phase and CDKB1 protein and kinase activity is almost undetectable in G1. CdkB1 mRNA accumulates through S until M phase and its associated kinase activity peaks at the G2/M boundary, confirming that transcription of PPTALRE CDKs is cell cycle regulated. We suggest that CDKA kinase activity likely plays roles at the G1/S phase boundary, during S phase, and at the G2/M phase transition, and that CDKB1 kinase activity is present only at G2/M.

In most plants, sucrose is the major transported carbon source. Carbon source availability in the form of sucrose is likely to be a major determinant of cell division, and mechanisms must exist for sensing sugar levels and mediating appropriate control of the cell cycle. We show that sugar availability plays a major role during the G(1) phase by controlling the expression of CycD cyclins in Arabidopsis. CycD2 mRNA levels increase within 30 min of the addition of sucrose; CycD3 is induced after 4 h. This corresponds to induction of CycD2 expression early in G(1) and CycD3 expression in late G(1) near the S-phase boundary. CycD2 and CycD3 induction is independent both of progression to a specific point in the cell cycle and of protein synthesis. Protein kinase activity of CycD2- and CycD3-containing cyclin-dependent kinases is consistent with the observed regulation of their mRNA levels. CycD2 and CycD3 therefore act as direct mediators of the presence of sugar in cell cycle commitment. CycD3, but not CycD2, expression responds to hormones, for which we show that the presence of sugars is required. Finally, protein phosphatases are shown to be involved in regulating CycD2 and CycD3 induction. We propose that control of CycD2 and CycD3 by sucrose forms part of cell cycle control in response to cellular carbohydrate status.

Recent discoveries of plant retinoblastoma (Rb) protein homologues and D-type cyclins suggest that control of the onset of cell division in plants may have stronger parallels with mammalian G1/S controls than with yeasts. In mammals, the Rb protein interacts specifically with D-type cyclins and regulates cell proliferation by binding and inhibiting E2F transcription factors. However, the developmental role of Rb in plants and its potential interaction with cell cycle regulators is unknown. We show that the maize Rb homologue ZmRb-1 is temporally and spatially regulated during maize leaf development. ZmRb-1 is highly expressed in differentiating cells, but almost undetectable in proliferating cells. In vitro, both ZmRb-1 and human Rb bind all classes of plant D-type cyclins with the involvement of a conserved N-terminal Leu-x-Cys-x-Glu (LxCxE) Rb-interaction motif. This binding is strongly reduced by mutation of the conserved Cys-470 of ZmRb-1. ZmRb-1 binds human and Drosophila E2F, and inhibits transcriptional activation of human E2F. We also show that ZmRb-1 is a good in vitro substrate for all human G1/S protein kinases. The functional conservation of proteins that control the G1/S transition in mammals and plants points to the existence of plant E2F homologues. We conclude that evolution of Rb and cyclin D proteins occurred after separation of the fungi from the higher eukaryotic lineage, but preceded the divergence of plant and animal kingdoms.

Since its discovery in 1945, our knowledge of the structure and many functions of the endoplasmic reticulum (ER) has advanced at a phenomenal rate. Early studies focused on the structure, which was then followed by biochemical and functional studies associated with calcium storage and release from the ER, protein folding and secretion, ER associated degradation (ERAD) and ER stress responses. Currently there is a significant interest in the role of ER in such cellular processes as cell death, autophagy and cross-talk with other organelles. In this chapter we give an overview of the structural characteristics and biochemical functioning of the ER and describe its manifold roles in cellular physiology. Finally, we explain how the sensitive nature of the protein folding process in the ER enables this organelle to act as a sensor of a broad range of cellular stresses. Signals emanating from the stressed ER play central roles in differentiation processes, cellular homeostasis and cell death.

The stimuli for neuronal cell death in neurodegenerative disorders are multi-factorial and may include genetic predisposition, environmental factors, cellular stressors such as oxidative stress and free radical production, bioenergy failure, glutamate-induced excitotoxicity, neuroinflammation, disruption of Ca2+-regulating systems, mitochondrial dysfunction and misfolded protein accumulation. Cellular stress disrupts functioning of the endoplasmic reticulum (ER), a critical organelle for protein quality control, leading to induction of the unfolded protein response (UPR). ER stress may contribute to neurodegeneration in a range of neurodegenerative disorders. This review summarizes the molecular events occurring during ER stress and the unfolded protein response and it specifically evaluates the evidence suggesting the ER stress response plays a role in neurodegenerative disorders.

Aurora kinases represent an appealing target for anticancer therapies and several Aurora inhibitors are in clinical development, including the potent pan-Aurora inhibitor Danusertib. Treatment with Aurora inhibitors has been shown to induce diverse biological responses in different tumor cells, in part depending on TP53 status. To characterize the effects of Danusertib at the transcriptional level we carried out gene expression profiling of wt and TP53 mutant tumor cells showing differential cell cycle response upon drug treatment. We found that treatment with Danusertib induces a strong transcriptional response only in TP53 wt cells, with an overlapping pattern of expression of TP53-dependent genes among the three cell lines tested, while a prevalent signature could not be identified in the two TP53 mutant cells, suggesting that TP53 status is a key determinant for the observed transcriptional effects. This work led to the identification of a number of genes consistently modulated by Aurora treatment in TP53 cells. One of these is GDF15, a secreted protein belonging to the TGF-β superfamily, for which we found a potential role in resistance to Danusertib, and which could represent a potential biomarker for Danusertib treatment in TP53 WT tumors and in surrogate tissues such as blood or skin.

During apoptosis, the process of mitochondrial outer membrane permeabilization (MOMP) represents a point-of-no-return as it commits the cell to death. Here we have assessed the role of caspases, Bcl-2 family members and the mitochondrial permeability transition pore on ER stress-induced MOMP and subsequent cell death. Induction of ER stress leads to upregulation of several genes such as Grp78, Edem1, Erp72, Atf4, Wars, Herp, p58ipk, and ERdj4 and leads to caspase activation, release of mitochondrial intermembrane proteins and dissipation of mitochondrial transmembrane potential (ΔΨm). Mouse embryonic fibroblasts (MEFs) from caspase-9, -2 and, -3 knock-out mice were resistant to ER stress-induced apoptosis which correlated with decreased processing of pro-caspase-3 and -9. Furthermore, pretreatment of cells with caspase inhibitors (Boc-D.fmk and DEVD.fmk) attenuated ER stress-induced loss ofΔΨm. However, only deficiency of caspase-9 and -2 could prevent ER stress-mediated loss ofΔΨm. Bcl-2 overexpression or pretreatment of cells with the cell permeable BH4 domain (BH4-Tat) or the mitochondrial permeability transition pore inhibitors, bongkrekic acid or cyclosporine A, attenuated the ER stress-induced loss ofΔΨm. These data suggest a role for caspase-9 and -2, Bcl-2 family members and the mitochondrial permeability transition pore in loss of mitochondrial membrane potential during ER stress-induced apoptosis.

Background Microarrays have been widely used for the analysis of gene expression and several commercial platforms are available. The combined use of multiple platforms can overcome the inherent biases of each approach, and may represent an alternative that is complementary to RT-PCR for identification of the more robust changes in gene expression profiles. In this paper, we combined statistical and functional analysis for the cross platform validation of two oligonucleotide-based technologies, Affymetrix (AFFX) and Applied Biosystems (ABI), and for the identification of differentially expressed genes. Results In this study, we analysed differentially expressed genes after treatment of an ovarian carcinoma cell line with a cell cycle inhibitor. Treated versus control RNA was analysed for expression of 16425 genes represented on both platforms. We assessed reproducibility between replicates for each platform using CAT plots, and we found it high for both, with better scores for AFFX. We then applied integrative correlation analysis to assess reproducibility of gene expression patterns across studies, bypassing the need for normalizing expression measurements across platforms. We identified 930 genes as differentially expressed on AFFX and 908 on ABI, with ~80% common to both platforms. Despite the different absolute values, the range of intensities of the differentially expressed genes detected by each platform was similar. ABI showed a slightly higher dynamic range in FC values, which might be associated with its detection system. 62/66 genes identified as differentially expressed by Microarray were confirmed by RT-PCR. Conclusion In this study we present a cross-platform validation of two oligonucleotide-based technologies, AFFX and ABI. We found good reproducibility between replicates, and showed that both platforms can be used to select differentially expressed genes with substantial agreement. Pathway analysis of the affected functions identified themes well in agreement with those expected for a cell cycle inhibitor, suggesting that this procedure is appropriate to facilitate the identification of biologically relevant signatures associated with compound treatment. The high rate of confirmation found for both common and platform-specific genes suggests that the combination of platforms may overcome biases related to probe design and technical features, thereby accelerating the identification of trustworthy differentially expressed genes.

CYCD3;1 expression in Arabidopsis is associated with proliferating tissues such as meristems and developing leaves but not with differentiated tissues. Constitutive overexpression of CYCD3;1 increases CYCD3;1-associated kinase activity and reduces the proportion of cells in the G1-phase of the cell cycle. Moreover, CYCD3;1 overexpression leads to striking alterations in development. Leaf architecture in overexpressing plants is altered radically, with a failure to develop distinct spongy and palisade mesophyll layers. Associated with this, we observe hyperproliferation of leaf cells; in particular, the epidermis consists of large numbers of small, incompletely differentiated polygonal cells. Endoreduplication, a marker for differentiated cells that have exited from the mitotic cell cycle, is inhibited strongly in CYCD3;1-overexpressing plants. Transcript analysis reveals an activation of putative compensatory mechanisms upon CYCD3;1 overexpression or subsequent cell cycle activation. These results demonstrate that cell cycle exit in the G1-phase is required for normal cellular differentiation processes during plant development and suggest a critical role for CYCD3 in the switch from cell proliferation to the final stages of differentiation.

D-type cyclins (CycD) play key roles in linking the Arabidopsis cell cycle to extracellular and developmental signals, but little is known of their regulation at the post-transcriptional level or of their cyclin-dependent kinase (CDK) partners. Using new antisera to CycD2 and CycD3, we demonstrate that the CDK partner of these Arabidopsis cyclins is the PSTAIRE-containing CDK Cdc2a. Previous analysis has shown that transcript levels of CycD2 and CycD3 are regulated in response to sucrose levels and that both their mRNA levels and kinase activity are induced with different kinetics during the G(1) phase of cells reentering the division cycle from quiescence. Here we analyze the protein levels and kinase activity of CycD2 and CycD3. We show that CycD3 protein and kinase activity parallel the abundance of its mRNA and that CycD3 protein is rapidly lost from cells in stationary phase or following sucrose removal. In contrast to both CycD3 and the regulation of its own mRNA levels, CycD2 protein is present at constant levels. CycD2 kinase activity is regulated by sequestration of CycD2 protein in a form inaccessible to immunoprecipitation and probably not complexed to Cdc2a.

OBJECTIVE--To define the region on human chromosome 19 carrying the gene for malignant hyperthermia susceptibility and to evaluate the use of flanking DNA markers in diagnosing susceptibility. DESIGN--Prospective molecular genetic linkage studies in a large malignant hyperthermia pedigree. SETTING--Irish malignant hyperthermia testing centre. SUBJECTS--A large Irish malignant hyperthermia pedigree. MAIN OUTCOME MEASURES--Routine diagnosis of susceptibility to malignant hyperthermia with in vitro contracture test on muscle biopsy specimens and genetic linkage between susceptibility and polymorphic DNA markers in a malignant hyperthermia family. RESULTS--Genetic typing of polymorphic DNA markers in a large Irish malignant hyperthermia pedigree generated a lod score of greater than 3 for the marker D19S9 and showed that the gene for susceptibility is flanked by the markers D19S9 and D19S16. These tightly linked flanking markers allowed non-invasive presymptomatic diagnosis of susceptibility in five untested subjects in the large pedigree with an accuracy of greater than 99.7%. CONCLUSIONS--DNA markers flanking the gene for susceptibility to malignant hyperthermia can be used with high accuracy to diagnose susceptibility in subjects in large known malignant hyperthermia pedigrees and may replace the previous in vitro contracture test for diagnosing this inherited disorder in large families with malignant hyperthermia.

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the TNF cytokine family and a selective inducer of apoptosis in a range of tumour cells, but not in healthy normal, untransformed cells. It is expressed by natural killer cells and natural killer-T cells when they encounter malignantly transformed cells and it is a key effector molecule in tumour immune surveillance. TRAIL has 5 receptors, which is the highest receptor promiscuity in the TNF ligand family. It binds to death receptor 4 (DR4) or DR5 on the surface of target cells [1] and initiates a conformational change which promotes association of the receptors with FADD facilitating pro-caspase-8 and/or pro-caspase-10 recruitment which then activates effector caspases to execute cell death [2]. Signalling through DR4 and DR5 can also activate pro-inflammatory intracellular molecules such as MAPK, PKB and NF-κB and overexpression of DR4 or DR5 has been shown to stimulate the release of inflammatory cytokines [3]. However, TRAIL also has three regulatory receptors. Two of these, decoy receptor 1(DcR1) and DcR2 are membrane bound and the third regulatory receptor, osteoprotegerin is a secreted protein. DcRs regulate TRAIL-induced apoptosis by either sequestering TRAIL from the death receptors or by forming inactive, heteromeric DcR1/2–DR4/5 complexes [1]. Indeed, DcRs have been shown to be highly expressed in a number of tumour tissues such as acute myeloid leukaemia, prostate cancer and breast cancer and their expression is linked with poor prognosis [4]. However DcR expression in tumour cells does not correlate with TRAIL sensitivity and non-transformed cells do not require DcRs to be protected from TRAIL-induced apoptosis, suggesting that the in vivo role of the DcRs may be more complex than originally thought [5] The tumor-specific cytotoxicity of TRAIL has been exploited as a therapeutic strategy by utilizing recombinant versions of TRAIL and agonistic antibodies against DR4 and DR5 [6]. While recombinant soluble human TRAIL was highly potent against a broad range of tumours in vitro and in pre-clinical studies, in clinical trials TRAIL has failed to exhibit the same potency [6]. One of the major shortcomings of the preclinical models was the lack of assessment of the contribution of the tumour microenvironment (TME). The TME consists of various cell types, soluble factors and signals from the extracellular matrix, and is in a reciprocal interaction with the tumour cells. It is thus important to understand the interplay of different cell types in the tumour microenvironment and the effect of the factors they express and secrete on tumour growth, development and resistance to therapy. Figure 1 Cell autonomous and supracellular regulation of TRAIL-sensitivity by decoy receptor 1 (DcR1) and -2 The study by O'Leary and colleagues explored the hypothesis that DcRs exerted a ‘supracellular level control’ of TRAIL-sensitivity rather than simply regulating TRAIL resistance at a cell-autonomous level [7]. They firstly examined the expression of the decoy receptors in tumour cells, tumour stroma and in non-malignant, tumour adjacent tissues. Interestingly they found that DcR1 and DcR2 are commonly expressed in tissues but tissue stroma only express DcR1. To determine whether DcR-expressing stromal cells influence the TRAIL sensitivity of tumour cells sharing the same microenvironment they generated and characterized DcR-insensitive TRAIL mutants and used a combination of mathematical modelling, cell based assays and a stroma/tumour co-culture system allowing them to model the effect of DcRs expressed by adjacent stromal cells on DR4/5 activation in tumour cells. They found that stromal DcRs profoundly reduced TRAIL-induced DR4/DR5 activation and protected tumour cells against TRAIL. The authors confirmed this finding in a 3D mixed-cell type (stroma-tumour) spheroid tumour model. Interestingly they also observed that TNF increased surface expression of the DcRs in fibroblasts raising the possibility that an inflammatory environment may induce DcR expression and promote TRAIL resistance. This study clearly demonstrates that stromal DcRs in the tumour microenvironment can exert trans-cellular regulation affecting tumour cells and it highlights the importance of developing therapeutic TRAIL variants that can selectively activate the two death inducing TRAIL receptors but are not mopped up by the decoy receptors present on stromal tissues. Future studies will hopefully establish a feed forward signalling loop that drives overall tissue sensitivity in order to achieve tumour eradication.