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Suppression of Ras-Induced Apoptosis by the Rac GTPase
Abstract
Ras is an essential component of signal transduction pathways that control cell proliferation, differentiation, and survival. In this study we have examined the cellular responses to high-intensity Ras signaling. Expression of increasing amounts of the oncogenic form of human HRas, HRasV12, results in a dose-dependent induction of apoptosis in both primary and immortalized cells. The induction of apoptosis by HRasV12 is blocked by activated Rac and potentiated by dominant interfering Rac. The ability of Rac to suppress Ras-induced apoptosis is dependent on effector pathway(s) controlled by the insert region and is linked to the activation of NF-κB. The apoptotic effect of HRasV12 requires the activation of both the ERK and JNK mitogen-activated protein kinase cascade and is independent of p53. These results demonstrate a role for Rac in controlling signals that are necessary for cell survival, and suggest a mechanism by which Rac activity can confer growth advantage to cells transformed by the
ras
oncogene.
... 49 Oncogenic stress was proposed to be a safeguard mechanism that prevents tumor growth. 50 In those cells that succumb to malignant transformation, oncogene-triggered survival signals are believed to outbalance the death signals, 46 and the cells survive. Our data indicate that RAS-induced ATG12 loss triggers cell survival signals, which are critical for the viability of the respective cells. ...
... RAS is well known to trigger death signals in cells. 11,50 Induction of these signals represents an aspect of a phenomenon called oncogenic stress 49 that is thought to preclude tumorigenesis in response to the emergence of oncogenic mutations of RAS and other proto-oncogenes. 50 It is believed that the likely reason why cancer cells survive, despite the presence of the antisurvival signals, is because in addition, oncoproteins, such as RAS, trigger the prosurvival signals which outbalance the death-promoting events in the indicated cells. ...
... 11,50 Induction of these signals represents an aspect of a phenomenon called oncogenic stress 49 that is thought to preclude tumorigenesis in response to the emergence of oncogenic mutations of RAS and other proto-oncogenes. 50 It is believed that the likely reason why cancer cells survive, despite the presence of the antisurvival signals, is because in addition, oncoproteins, such as RAS, trigger the prosurvival signals which outbalance the death-promoting events in the indicated cells. 46 According to our data, RAS-dependent loss of ATG12 in cancer cells represents one prosurvival event that promotes viability of these cells. ...
Activating mutations of RAS GTPase contribute to the progression of many cancers, including colorectal carcinoma. So far, attempts to develop treatments of mutant RAS-carrying cancers have been unsuccessful owing to insufficient understanding of the salient mechanisms of RAS signalling. We found that RAS downregulates the protein ATG12 in colon cancer cells. ATG12 is a mediator of autophagy, a process of degradation and reutilization of cellular components. In addition, ATG12 can kill cells via autophagy-independent mechanisms. We established that RAS reduces ATG12 levels in cancer cells by accelerating its proteasomal degradation. We further observed that RAS-dependent ATG12 loss in these cells is mediated by protein kinases MAP2K/MEK and MAPK1/ERK2-MAPK3/ERK1, known effectors of RAS. We also demonstrated that the reversal of the effect of RAS on ATG12 achieved by the expression of exogenous ATG12 in cancer cells triggers both apoptotic and non-apoptotic signals and efficiently kills the cells. ATG12 is known to promote autophagy by forming covalent complexes with other autophagy mediators, such as ATG5. We found that the ability of ATG12 to kill oncogenic RAS-carrying malignant cells does not require covalent binding of ATG12 to other proteins. In summary, we have identified a novel mechanism by which oncogenic RAS promotes survival of malignant intestinal epithelial cells. This mechanism is driven by RAS-dependent loss of ATG12 in these cells.
... ROCK-Myosin II also sustain NF-κB signaling in melanoma through enhanced secretion of inflammatory cytokines reinforcing Myosin II and generating a positive feedback loop (288). During transformation of fibroblasts, high levels of Ras induce apoptosis (289), but this is counterbalanced by Rac1-mediated activation of NF-κB, which promotes survival (289). In line with this, activation of RAC1/PAK1 downstream of a6b4 integrin promotes NF-κB-mediated resistance to apoptosis of mammary epithelial cells in three-dimensional (3-D) cultures (290). ...
... ROCK-Myosin II also sustain NF-κB signaling in melanoma through enhanced secretion of inflammatory cytokines reinforcing Myosin II and generating a positive feedback loop (288). During transformation of fibroblasts, high levels of Ras induce apoptosis (289), but this is counterbalanced by Rac1-mediated activation of NF-κB, which promotes survival (289). In line with this, activation of RAC1/PAK1 downstream of a6b4 integrin promotes NF-κB-mediated resistance to apoptosis of mammary epithelial cells in three-dimensional (3-D) cultures (290). ...
Rho GTPases are a family of small G proteins that regulate a wide array of cellular processes related to their key roles controlling the cytoskeleton. On the other hand, cancer is a multi-step disease caused by the accumulation of genetic mutations and epigenetic alterations, from the initial stages of cancer development when cells in normal tissues undergo transformation, to the acquisition of invasive and metastatic traits, responsible for a large number of cancer related deaths. In this review, we discuss the role of Rho GTPase signalling in cancer in every step of disease progression. Rho GTPases contribute to tumour initiation and progression, by regulating proliferation and apoptosis, but also metabolism, senescence and cell stemness. Rho GTPases play a major role in cell migration, and in the metastatic process. They are also involved in interactions with the tumour microenvironment and regulate inflammation, contributing to cancer progression. After years of intensive research, we highlight the importance of relevant models in the Rho GTPase field, and we reflect on the therapeutic opportunities arising for cancer patients.
... Several studies assessing the impact of RAS mutations on cell behavior correlated the signaling pathways activated by a specific mutation with a particular outcome such as cell death or cell cycle redistribution (27,28,30,33). Joneson and Bar-Sagi reported that overexpression of HRAS G12V induced apoptosis in a panel of primary and immortalized cells (36). However, the co-transfection of REF-52 fibroblasts with HRAS G12V and activated RAC blocked HRAS G12V-induced apoptosis, indicating that RAC signaling pathway is sufficient to antagonize RAS proapoptotic signals (36). ...
... Joneson and Bar-Sagi reported that overexpression of HRAS G12V induced apoptosis in a panel of primary and immortalized cells (36). However, the co-transfection of REF-52 fibroblasts with HRAS G12V and activated RAC blocked HRAS G12V-induced apoptosis, indicating that RAC signaling pathway is sufficient to antagonize RAS proapoptotic signals (36). As KRAS4B G12V and HRAS G12V differentially activate the RAC signaling pathway (30), Walsh and Bar-Sagi hypothesized that these mutant variants may differ in their ability to induce apoptosis (30). ...
In human cells, three closely related RAS genes, termed HRAS, KRAS, and NRAS, encode four highly homologous proteins. RAS proteins are small GTPases involved in a broad spectrum of key molecular and cellular activities, including proliferation and survival among others. Gain-of-function missense mutations, mostly located at codons 12, 13, and 61, constitutively activate RAS proteins and can be detected in various types of human cancers. KRAS is the most frequently mutated, followed by NRAS and HRAS. However, each isoform exhibits distinctive mutation frequency at each codon, supporting the hypothesis that different RAS mutants may lead to distinct biologic manifestations. This review is focused on the differences in signaling and phenotype, as well as on transcriptomics, proteomics, and metabolomics profiles related to individual RAS-mutated variants. Additionally, association of these mutants with particular targeted outcomes and rare mutations at additional RAS codons are discussed.
... Giving the fact that Rac takes place in the regulation of both transcription and activation of NF-κB and that NF-κB activation can lead to the suppression of apoptosis, it is conceivable that Rac may have at least an indirect protective role against Ras-induced apoptosis [42]. ...
... Ras is implicated in Rac regulation in two different ways: Firstly, by the activation via PI3Kinase [40,42,43] or, secondly, by interaction with the Rac GEF Tiam [44]. The control of Rac activity, and thus, the impact on cellular Ros levels, might represent an additional way of how Ras affects the overall cellular viability [43,45]. ...
One of the most obvious hallmarks of cancer is uncontrolled proliferation of cells partly due to independence of growth factor supply. A major component of mitogenic signaling is Ras, a small GTPase. It was the first identified human protooncogene and is known since more than three decades to promote cellular proliferation and growth. Ras was shown to support growth factor-independent survival during development and to protect from chemical or mechanical lesion-induced neuronal degeneration in postmitotic neurons. In contrast, for specific patho-physiological cases and cellular systems it has been shown that Ras may also promote cell death. Proteins from the Ras association family (Rassf, especially Rassf1 and Rassf5) are tumor suppressors that are activated by Ras-GTP, triggering apoptosis via e.g., activation of mammalian sterile 20-like (MST1) kinase. In contrast to Ras, their expression is suppressed in many types of tumours, which makes Rassf proteins an exciting model for understanding the divergent effects of Ras activity. It seems likely that the outcome of Ras signaling depends on the balance between the activation of its various downstream effectors, thus determining cellular fate towards either proliferation or apoptosis. Ras homologue enriched in brain (Rheb) is a protein from the Ras superfamily that is also known to promote proliferation, growth, and regeneration through the mammalian target of rapamycin (mTor) pathway. However, recent evidences indicate that the Rheb-mTor pathway may switch its function from a pro-growth into a cell death pathway, depending on the cellular situation. In contrast to Ras signaling, for Rheb, the cellular context is likely to modulate the whole Rheb-mTor pathway towards cellular death or survival, respectively.
... NF-κB blocks the death of tumor cells induced by the activation of oncogenes Ras. 331 NF-κB-dependent Rac guanosine triphosphatase effectively inhibits the p53-independent apoptosis response induced by high levels of Ras activity. Rac mutants that could not activate NF-kB are defective in inhibiting Ras-induced apoptosis. ...
Since nuclear factor of κ-light chain of enhancer-activated B cells (NF-κB) was discovered in 1986, extraordinary efforts have been made to understand the function and regulating mechanism of NF-κB for 35 years, which lead to significant progress. Meanwhile, the molecular mechanisms regulating NF-κB activation have also been illuminated, the cascades of signaling events leading to NF-κB activity and key components of the NF-κB pathway are also identified. It has been suggested NF-κB plays an important role in human diseases, especially inflammation-related diseases. These studies make the NF-κB an attractive target for disease treatment. This review aims to summarize the knowledge of the family members of NF-κB, as well as the basic mechanisms of NF-κB signaling pathway activation. We will also review the effects of dysregulated NF-κB on inflammation, tumorigenesis, and tumor microenvironment. The progression of the translational study and drug development targeting NF-κB for inflammatory diseases and cancer treatment and the potential obstacles will be discussed. Further investigations on the precise functions of NF-κB in the physiological and pathological settings and underlying mechanisms are in the urgent need to develop drugs targeting NF-κB for inflammatory diseases and cancer treatment, with minimal side effects.
... This also underscores the strong induction of apoptosis in 2D cultured cells induced by oncogenic RAS itself, documented here and by many others. We can postulate that this is not generated by a RAS-RASSF-Hippo signaling axis but more likely through activation of p53 or alternative pathways (74)(75)(76)(77). ...
Small guanosine triphosphatases (GTPases) of the RAS superfamily signal by directly binding to multiple downstream effector proteins. Effectors are defined by a folded RAS-association (RA) domain that binds exclusively to GTP-loaded (activated) RAS, but the binding specificities of most RA domains toward more than 160 RAS superfamily GTPases have not been characterized. Ten RA domain family (RASSF) proteins comprise the largest group of related effectors and are proposed to couple RAS to the proapoptotic Hippo pathway. Here, we showed that RASSF1-6 formed complexes with the Hippo kinase ortholog MST1, whereas RASSF7-10 formed oligomers with the p53-regulating effectors ASPP1 and ASPP2. Moreover, only RASSF5 bound directly to activated HRAS and KRAS, and RASSFs did not augment apoptotic induction downstream of RAS oncoproteins. Structural modeling revealed that expansion of the RASSF effector family in vertebrates included amino acid substitutions to key residues that direct GTPase-binding specificity. We demonstrated that the tumor suppressor RASSF1A formed complexes with the RAS-related GTPases GEM, REM1, REM2, and the enigmatic RASL12. Furthermore, interactions between RASSFs and RAS GTPases blocked YAP1 nuclear localization. Thus, these simple scaffolds link the activation of diverse RAS family small G proteins to Hippo or p53 regulation.
... ElA-induced apoptosis (Joneson and Bar-Sagi, 1999;Kauffmann-Zeh et al., 1997;Lin et al., 1995;Nalca et al., 1999). Ras can also protect epithelial cells from apoptosis induced by their detachment from the ECM . ...
Multi-stage tumourigenesis is associated with the accumulation of co-operating genetic lesions. One of the best studied models of carcinogenesis is experimentally induced tumours in the skin of mice. In tumorigenesis of the skin, activated Ras co-operates with mutations that inactivate the tumour suppressor p53. The absence of the cyclin dependent kinase inhibitor p21Cip1, a p53 target gene, has also been shown to co-operate with oncogenic ras in the induction of aggressive and relatively undifferentiated tumours in vivo. However, the molecular basis for these co-operations remains unresolved. Activation of oncogenes, including Ras, Myc and E1A, have been reported to stabilise and activate p53 via induction of the tumour suppressor p19ARF. Therefore, it is thought that ARF might be the specific link between oncogene activation and induction of p53. Since most malignancies are epithelial in origin and ms and p53 mutations are most frequently associated with epithelial derived tumours, I investigated the molecular mechanisms involved in co-operation between Ras, p53 and p2l Cip1 in the skin and the potential involvement of p19ARF. Primary mouse keratinocytes provide an excellent system to study growth and differentiation in the skin. When cultured in low calcium medium they behave similarly to the cells found in the basal layer of the epidermis. Here I show that activation of the Raf/MAP kinase pathway in primary mouse keratinocytes leads to a G1/G2 cycle arrest and to terminal differentiation. This is preceded by an increase in p21 Cip1 and p53 protein levels while p16INK4A and p19ARF levels appear unaffected. Raf activation in keratinocytes lacking p53 or p21Cip1 genes leads to expression of differentiation markers, but the cells do not cease to proliferate. Thus, loss of p53 or p21Cip1 function is necessary to disable growth-inhibitory Raf/Map kinase signalling, but a cell cycle arrest is not obligatory for the onset of terminal differentiation in keratinocytes. However, the response to Raf in p19ARF-/- keratinocytes was indistinguishable from wild type controls. Thus, p19ARF is not essential for Raf-induced p53 induction and cell cycle arrest in keratinocytes, indicating that oncogenes engage p53 activity via multiple mechanisms.
... In addition to Rac1-dependent stimulation of proliferation and cell migration (see Section 4), Rac1-mediated anti-apoptotic signals also favor Ras-dependent oncogenicity [272]. Pak1 is considered the main Rac1 effector that influences cortical actin cytoskeleton rearrangements most relevant for cell motility (see 4.1). ...
Calmodulin is a ubiquitous signalling protein that controls many biological processes due to its capacity to interact and/or regulate a large number of cellular proteins and pathways, mostly in a Ca2+-dependent manner. This complex interactome of calmodulin can have pleiotropic molecular consequences, which over the years has made it often difficult to clearly define the contribution of calmodulin in the signal output of specific pathways and overall biological response. Most relevant for this review, the ability of calmodulin to influence the spatiotemporal signalling of several small GTPases, in particular KRas and Rac1, can modulate fundamental biological outcomes such as proliferation and migration. First, direct interaction of calmodulin with these GTPases can alter their subcellular localization and activation state, induce post-translational modifications as well as their ability to interact with effectors. Second, through interaction with a set of calmodulin binding proteins (CaMBPs), calmodulin can control the capacity of several guanine nucleotide exchange factors (GEFs) to promote the switch of inactive KRas and Rac1 to an active conformation. Moreover, Rac1 is also an effector of KRas and both proteins are interconnected as highlighted by the requirement for Rac1 activation in KRas-driven tumourigenesis. In this review, we attempt to summarize the multiple layers how calmodulin can regulate KRas and Rac1 GTPases in a variety of cellular events, with biological consequences and potential for therapeutic opportunities in disease settings, such as cancer.
... http://dx.doi.org/10.1101/2020.02.05.923433 doi: bioRxiv preprint first posted online Feb. 5, 2020; many others. We can now postulate this is not generated by a RAS-RASSF-Hippo signalling axis, but more likely via activation of p53 or alternative pathways (72)(73)(74)(75). ...
Activated RAS GTPases signal by directly binding effector proteins. Effectors have a folded RAS association (RA) domain that binds exclusively to GTP-loaded RAS, but the specificity of most RA domains for >150 RAS superfamily GTPases is unknown. Ten RAS-association domain family (RASSF) proteins comprise the largest group of effectors, proposed to couple RAS to the pro-apoptotic Hippo pathway. We show that RASSF1-6 complex with Hippo kinase, while RASSF7-10 are a separate family related to p53-regulatory ASPP effectors. Only RASSF5 directly binds activated HRAS and KRAS. Structural modelling reveals that expansion of RASSFs in vertebrates included amino acid substitutions that alter their GTPase binding specificity. We demonstrate that the tumour suppressor RASSF1A complexes with the GTPases GEM, REM1, REM2 and the enigmatic RASL12. Interplay between RASSFs and RAS GTPases can drastically restrict YAP1 nuclear localization. Thus, these simple scaffolds can link activation of diverse RAS proteins to Hippo or p53 regulation.
... Increased metastatic capacity of the Tigar null cells was mirrored by the acquisition of a more mesenchymal phenotype, activation of ERK signaling through the decreased expression of the phosphatase Dusp6/MKP-3, and increased migration and invasion in vitro. Activation of ERK, while associated with tumor proliferation, migration, and survival, can also limit growth through mechanisms such as senescence or increased sensitivity to other stresses, especially when hyperactivated in the context of an increase of ROS level (Cagnol and Chambard, 2010;Hong et al., 2018; (legend on next page) Joneson and Bar-Sagi, 1999;Woods et al., 1997). It seems likely that DUSP6 expression can be fine-tuned to modulate ERK at different stages of tumor progression and that this ability is lost in TIGAR null cells. ...
The TIGAR protein has antioxidant activity that supports intestinal tissue repair and adenoma development. Using a pancreatic ductal adenocarcinoma (PDAC) model, we show that reactive oxygen species (ROS) regulation by TIGAR supports premalignant tumor initiation while restricting metastasis. Increased ROS in PDAC cells drives a phenotypic switch that increases migration, invasion, and metastatic capacity. This switch is dependent on increased activation of MAPK signaling and can be reverted by antioxidant treatment. In mouse and human, TIGAR expression is modulated during PDAC development, with higher TIGAR levels in premalignant lesions and lower TIGAR levels in metastasizing tumors. Our study indicates that temporal, dynamic control of ROS underpins full malignant progression and helps to rationalize conflicting reports of pro- and anti-tumor effects of antioxidant treatment.
... Par ailleurs, Rac1 protègerait également les cellules tumorales de l'apoptose induite via la génération de radicaux libres oxygénés (Pervaiz et al. 2001). Des travaux réalisés sur différentes lignées cellulaires (HEK, Swiss-3T3, M14, ect) auraient d'ailleurs montrés que la capacité de Rac1 à supprimer l'apoptose serait liée à l'activation de NF-kB (Joneson et Bar-Sagi 1999). ...
La schistosomiase est la seconde endémie parasitaire mondiale après le paludisme puisqu’en 2016, environ 200 millions de personnes ont été traitées pour cette parasitose. Plusieurs espèces de schistosomes peuvent être la cause de cette maladie dont Schistosoma mansoni, responsable de la schistosomiase intestinale. Son cycle de vie est complexe et comprend deux hôtes : un hôte définitif vertébré, l’Humain et un hôte intermédiaire qui est un mollusque d’eau douce. Actuellement, un seul médicament, le praziquantel, est utilisé contre toutes les espèces de schistosomes, mais son utilisation de façon massive et répétée à favoriser l’émergence de souches parasitaire tolérantes et/ou résistantes. La nécessité de trouver de nouveaux médicaments et de nouveaux traitements est donc devenue impérative.Les lysines désacétylases ou KDAC(s) constituent des cibles thérapeutiques intéressantes, notamment parce que ce sont des enzymes impliquées dans des processus cellulaires essentiels tels que la régulation de l'expression des gènes et du cycle cellulaire, ou encore la différenciation cellulaire. À ce jour, des inhibiteurs de KDAC(s) sont déjà approuvés dans le traitement du cancer et d’autres sont en essais cliniques.Chez S. mansoni, trois KDAC(s) de classe I ont été identifiées: HDAC 1, 3 et 8. D'autre part, l'utilisation d'inhibiteurs de KDAC(s) a démontré qu'il était possible d'induire l'apoptose et la mort des parasites en culture. Des études réalisées sur la protéine HDAC8 humaine et SmHDAC8 ont montré qu'il existait des différences significatives au niveau de la poche catalytique entre ces deux protéines. Ces données soulignent l’intérêt de développer des inhibiteurs sélectifs de SmHDAC8. Il est devenu, néanmoins essentiel de déterminer le rôle de SmHDAC8 dans la biologie du parasite et notamment ses partenaires protéiques. De ce fait, la première partie de ce travail de thèse s’est focalisé sur la mise en évidence de l’interactome de l’enzyme parasitaire SmHDAC8. Par l’utilisation du système en double hybride chez la levure et de la co-immunoprécipitation couplée à la spectrométrie de masse, nous avons identifié plusieurs partenaires de SmHDAC8 qui sont impliqués dans des processus essentiels à la cellule tel que la régulation de la transcription et de la traduction, le cycle cellulaire, le métabolisme, la réparation de l’ADN, la protéolyse ou encore le transport des protéines. Parmi ces interactants, nous avons également retrouvé la GTPase SmRho1 suggérant que l’enzyme SmHDAC8 serait impliqué dans la modulation de l’organisation du cytosquelette.Dans une seconde partie, nous nous sommes donc intéresser à l’interaction entre SmHDAC8 et la SmRho1. Nous avons initialement démontré que cette interaction était bien présente chez le parasite et notamment chez les vers adultes et les schistosomules. L’acétylation de SmRho1 sur la lysine K136 a également été mise en évidence par spectrométrie de masse et nous avons aussi pu observer un effet de l’inhibition de SmHDAC8 sur l’organisation du cytosquelette d’actine chez le parasite.Deux isoformes de SmRho1 (SmRho1.1 et SmRho1.2) ont été identifiées et caractérisées. La technique du double hybride chez la levure et la co-immunoprécipitation en ovocytes de xénope, a permis de démontrer que seule SmRho1.1 pouvait interagir avec SmHDAC8. Enfin, la caractérisation des motifs d'interaction entre SmHDAC8 et SmRho1.1, par co-immunoprécipitation en ovocytes de xénope, suggère que le domaine C-terminal de SmRho1 serait impliqué dans cette interaction. Ces données sont en faveur d’un rôle potentiel de SmHDAC8 dans la modulation du cytosquelette d’actine via son interaction spécifique avec la GTPase SmRho1.1.
... NF-κB and p53 antagonistically regulate each other's activity, while IKKβ was shown to directly phosphorylate p53 and promote its polyubiquitylation and degradation [15]. In addition to p53 mediated apoptosis, studies have demonstrated that GTPase HRAS159 and c-MYC induced NF-κB activation inhibits cell death [31,32]. NF-κB and autophagy intricately regulate each other's activity. ...
Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers and is the third highest among cancer related deaths. Despite modest success with therapy such as gemcitabine, pancreatic cancer incidence remains virtually unchanged in the past 25 years. Among the several driver mutations for PDAC, Kras mutation contributes a central role for its development, progression and therapeutic resistance. In addition, inflammation is implicated in the development of most human cancer, including pancreatic cancer. Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is recognized as a key mediator of inflammation and has been frequently observed to be upregulated in PDAC. Several lines of evidence suggest that NF-κB pathways play a crucial role in PDAC development, progression and resistance. In this review, we focused on emphasizing the recent advancements in the involvement of NF-κB in PADC’s progression and resistance. We also highlighted the interaction of NF-κB with other signaling pathways. Lastly, we also aim to discuss how NF-κB could be an excellent target for PDAC prevention or therapy. This review could provide insight into the development of novel therapeutic strategies by considering NF-κB as a target to prevent or treat PDAC.
... The anti-apoptotic effect of Rac1 was AKT2/MCL1 dependent. Rac1 inhibited apoptosis of fibroblast induced by Ras over-activation, and its mechanism is dependent on the activation of NF-κB induced by Rac1 [26]. Shen et al. found that Rac1 played a crucial role in high glucose-induced myocardial apoptosis by regulating the activation of NADPH oxidase and increasing production of ROS. ...
Rac1, known as a “molecular switch”, plays a crucial role in plenty of cellular processes. Rac1 aggravates the damage of myocardial cells in the process of myocardial ischemia-reperfusion during myocardial infarction through activating the NADPH oxidase and bringing about the reactive oxygen species(ROS) generation. Myocardial ischemia and hypoxia are the basic pathogenesis of myocardial infarction and the underlying mechanisms are intricate and varied. Moreover, the regulatory effect of Rac1 on myocardial cells in the condition of serum starvation and the potential mechanisms are still incompletely undefined. Therefore, heart-derived H9c2 cells cultured in 0% serum were used to mimic ischemic myocardial cells and to clarify the role of Rac1 in H9c2 cells and the underlying mechanisms during serum deficiency. After Rac1 was knocked down using specific siRNA, cell apoptosis was assessed by flow cytometry assay and cell proliferation was detected by CCK-8 assay and EdU assay. In addition, the expression and activation of protein in related signaling pathway were detected by Western blot and siRNAs was used to testify the signaling pathways. Our results indicated that Rac1 inhibited apoptosis, promoted proliferation and cell cycle progression of H9c2 cells during serum deficiency. We concluded that Rac1 inhibited apoptosis in an AKT2/MCL1 dependent way and promoted cell proliferation through JNK/c-JUN/Cyclin-D1.
... In particular, AKT can phosphorylate Bad, a pro-apoptotic member of the Bcl-2 family, and this phosphorylation causes Bad to bind to 14-3-3 in an inactive complex, instead of sequestering the antiapoptotic proteins Bcl-2 and Bcl-XL [225]. In addition to AKT, PI3K can activate another important survival factor, NF-B, through the activation of Rac [226][227][228]. NF-B is a potent transcription factor that induces the transcription of several anti-apoptotic genes, such as inhibitors of apoptosis proteins (IAPs) [229]. ...
The exploitation of the yeastSaccharomyces cerevisiaeas a biological model for the investigation of complex molecular processes conserved in multicellular organisms, such as humans, has allowed fundamental biological discoveries. When comparing yeast and human proteins, it is clear that both amino acid sequences and protein functions are often very well conserved. One example of the high degree of conservation between human and yeast proteins is highlighted by the members of the RAS family. Indeed, the study of the signaling pathways regulated by RAS in yeast cells led to the discovery of properties that were often found interchangeable with RAS proto-oncogenes in human pathways, and vice versa. In this work, we performed an updated critical literature review on human and yeast RAS pathways, specifically highlighting the similarities and differences between them. Moreover, we emphasized the contribution of studying yeast RAS pathways for the understanding of human RAS and how this model organism can contribute to unveil the roles of RAS oncoproteins in the regulation of mechanisms important in the tumorigenic process, like autophagy.
... However, introduction of a constitutively active form of ras alone (i.e. HrasV12) showed its potential of sensitizing cells to apoptosis [24][25][26] and even induction of premature cell senescence through association of p53 accumulation [27]. In our proliferation assay with reprogrammed mESC, the lack of a significant enhancement of proliferation of the reprogrammed mESC with HrasV12 alone could be in part explained by the induction of either cellular senescence or apoptosis through the expression of a constitutively active form of HrasV12 alone. ...
Cancer stem cells (CSCs) are a small subset of cancer cells responsible for maintenance and progression of several types of cancer. Isolation, propagation, and the differentiation of CSCs in the proper stem niches expose the intrinsic difficulties for further studies. Here we show that induced cancer like stem cells (iCLSCs) can be generated by in vitro oncogenic manipulation of mouse embryonic stem cells (mESCs) with well-defined oncogenic elements; SV40 LTg and HrasV12 by using a mouse stem virus long terminal repeat (MSCV-LTR)-based retroviral system. The reprogrammed mESCs using both oncogenes were characterized through their oncogenic gene expression, the enhancement of proliferation, and unhampered maintenance of stem properties in vitro and in vivo. In addition, these transformed cells resulted in the formation of malignant, immature ovarian teratomas in vivo. To successfully further expand these properties to other organs and species, more research needs to be done to fully understand the role of a tumor- favorable microenvironment. Our current study has provided a novel approach to generate induced cancer like stem cells through in vitro oncogenic reprogramming and successfully initiated organ-specific malignant tumor formation in an orthotopic small animal cancer model.
... For example, the RAF pathway was linked to proliferation [10], apoptosis [11], energy metabolism [12] and angiogenesis. The PI3K pathway was associated with overlapping functions such as cell proliferation [13], and evasion of apoptosis [14] as well as with specific functions such as macrophage recruitment [15]. The RALGDS/RAL pathway composed of two small GTP-binding proteins RALA and RALB contributes to proliferation, anchorage independent growth [16], tumorigenicity [17], migration and metastasis [18,19]. ...
Our understanding of oncogenic signaling pathways has strongly fostered current concepts for targeted therapies in metastatic colorectal cancer. The RALA pathway is novel candidate due to its independent role in controlling expression of genes downstream of RAS.
We compared RALA GTPase activities in three colorectal cancer cell lines by GTPase pull-down assay and analyzed the transcriptional and phenotypic effects of transient RALA silencing. Knocking-down RALA expression strongly diminished the active GTP-bound form of the protein. Proliferation of KRAS mutated cell lines was significantly reduced, while BRAF mutated cells were mostly unaffected. By microarray analysis we identified common genes showing altered expression upon RALA silencing in all cell lines. None of these genes were affected when the RAF/MAPK or PI3K pathways were blocked.
To investigate the potential clinical relevance of the RALA pathway and its associated transcriptome, we performed a meta-analysis interrogating progression-free survival of colorectal cancer patients of five independent data sets using Cox regression. In each dataset, the RALA-responsive signature correlated with worse outcome.
In summary, we uncovered the impact of the RAL signal transduction on genetic program and growth control in KRAS- and BRAF-mutated colorectal cells and demonstrated prognostic potential of the pathway-responsive gene signature in cancer patients.
... 26 Oncogenic stress is thought to serve as a safeguard mechanism that blocks tumor growth. 27 In those cells that do become malignant, oncogene-induced antiapoptotic signals outbalance the proapoptotic signals. 24 As a result, cancer cells remain viable. ...
Detachment of non-malignant epithelial cells from the extracellular matrix causes their apoptosis, a phenomenon called anoikis. By contrast, carcinoma cells are anoikis-resistant, and this resistance is thought to be critical for tumor progression. Many oncogenes trigger not only anti- but also pr-apoptotic signals. The proapoptotic events represent an aspect of a phenomenon called oncogenic stress, which acts as a safeguard mechanism blocking tumor initiation. In cells that become malignant, oncogene-induced antiapoptotic signals outbalance the proapoptotic ones. It is now thought that treatments blocking the antiapoptotic events but preserving the proapoptotic signals can be particularly effective in killing tumor cells. Whether or not oncogenes induce any proanoikis signals that can be used for enhancing the efficiency of approaches aimed at triggering anoikis of cancer cells has never been explored. β-Catenin is a major oncoprotein that is often activated in colorectal cancer and promotes tumor progression via mechanisms that are understood only in part. We found here that β-catenin triggers both anti- and proanoikis signals in colon cancer cells. We observed that the antianoikis signals prevail and the cells become anoikis-resistant. We further established that one proanoikis signal in these cells is triggered by β-catenin-induced downregulation of an apoptosis inhibitor tumor necrosis factor receptor 1 (TNFR1) and subsequent reduction of the activity of a transcription factor NF-κB (nuclear factor-κB), a mediator of TNFR1 signaling. We also found that the effect of β-catenin on TNFR1 requires the presence of transcription factor TCF1, a β-catenin effector. We demonstrated that ablation of β-catenin in colon cancer cells triggers their anoikis and that this anoikis is enhanced even further if low TNFR1 or NF-κB activity is artificially preserved in the β-catenin-deprived cells. Thus, inhibition of TNFR1 or NF-κB activity can be expected to enhance the efficiency of approaches aimed at blocking β-catenin-driven anoikis resistance of colon carcinoma cells.Oncogene advance online publication, 22 December 2014; doi:10.1038/onc.2014.415.
... Rac1 has also been shown to suppress Ras-induced apoptosis by a mechanism linked with NF-kB activation contributing to Ras oncogenic transformation. 90 Activated Rac1 can cooperate with activated membrane-targeted Raf-1 (Raf-CAAX) 84 to promote malignant transformation, while Rac1 and Raf induce E1A-dependent transformation of primary BRK rat epithelial cells. 91 Activated Rac1 also cooperates with MEK1 to promote growth transformation of FRTL-5 rat thyroid epithelial cells. ...
Rho GTPases are involved in the acquisition of all the hallmarks of cancer, which comprise 6 biological capabilities acquired during the development of human tumors. The hallmarks include proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, and activating invasion and metastasis programs, as defined by Hanahan and Weinberg. (1) Controlling these hallmarks are genome instability and inflammation. Emerging hallmarks are reprogramming of energy metabolism and evading immune destruction. To give a different view to the readers, we will not be focusing on invasion, metastasis or cytoskeletal remodelling, but we will review here how Rho GTPases contribute to other hallmarks of cancer with a special emphasis on malignant transformation.
... Rac proteins are general modulators of cell growth/ survival and apoptosis, depending on the cell type and the extracellular stimuli. In fibroblasts and hematopoietic cells, constitutively active Rac protects cells from apoptosis induced by various stimuli, including Ras activation (Joneson and Bar-Sagi, 1999), anti-tumor drugs (Jeong et al., 2002), UV-radiation (Murga et al., 2002), death receptor activation (Deshpande et al., 2000), and factor deprivation Pervaiz et al., 2001;Ruggieri et al., 2001). Paradoxically, Rac can also sensitize cells to induction of apoptosis by a variety of stimuli. ...
Rac GTPases are crucial signaling regulators in eukaryotic cells, acting downstream of many cell surface receptors. They play essential roles in diverse cellular functions including cytoskeleton dynamics, cell motility, cell survival and apoptosis. Their activities are controlled by a tightly regulated GDP/GTP cycle coupled with an alternation between cytoplasm and membrane compartments. Aberrant Rac signaling is found in some human cancers as a result of changes in the GTPase itself or in its regulation loops. This review highlights recent findings regarding the molecular and functional aspects of Rac that mediate tumorigenic transformation and metastasis. It also describes the cellular mechanisms that potentially explain the complex role of Rac in tumorigenesis. Finally, it discusses approaches for modulating Rac function as a potential anticancer strategy.
... transformed fibroblasts from apoptosis [18][19][20][21] . However, the precise mechanisms by which the Rac pathway transduces survival signals that inhibit apoptosis remain to be elucidated. ...
Apoptosis is one mechanism by which cancer cells can be eliminated. Therefore, understanding the signaling pathways that transduce apoptotic signals in cancer cells is an indispensable component of cancer research. Rac, a member of the Rho family of proteins, has been implicated in the regulation of cell survival and apoptosis. However, the mechanisms underlying this process remain to be elucidated. To understand the role of Rac in oral squamous cancer, we inhibited its activity by a Rac-specific small molecule inhibitor, NSC23766, or transfection of dominant negative Rac (Rac-DN), and discovered that inhibition of Rac activity elicits apoptosis in highly malignant oral squamous carcinoma (OSC-19) cells. Upon suppression of Rac, we observed up-regulation of c-Jun N-terminal kinase (JNK), leading to caspase-dependent apoptosis. Furthermore, stimulation of protein phosphatase (PP5) rescued apoptosis caused by Rac inhibition by dephosphorylating JNK. Taken together, inhibition of Rac activity leads to the suppression of PP5 activity, which results in extensive activation of JNK and caspase-dependent apoptosis. In conclusion, Rac inhibition may represent a novel therapeutic approach for oral squamous carcinoma.
... Rho proteins have been implicated in both pro-and anti-apoptotic signaling, and in the apoptotic process itself (reviewed in (158). More significantly, they are involved in the decision to commit to apoptosis; ectopic expression of active Rac1 can provide a survival signal to protect tumor cell lines or transformed fibroblasts from apoptosis (159)(160)(161). Other mechanisms that link Rho proteins to cell survival have been described in non-transformed haematopoetic cells: Rac2 is required for the activation of the pro-survival kinase AKT in mast cells (162), and Rho function prevents p53-dependent apoptosis during T-cell development (163). ...
Ondanks een hoge mate van homologie verschillen de kleine Rho GTPases Rac1 en Rac3 wezenlijk in de functie die ze uitoefenen. De beschreven verschillen in effecten op cell-matrix adhesie berust op het feit dat Rac1 en Rac3 onafhankelijk van elkaar en op een andere manier aan het multifunctionele eiwit Git1 binden. De hierdoor veranderde Git1 downstream signalering beïnvloedt de cel matrix adhesie waardoor cellen of spreiden of afronden. De mate van cel-matrix interacties heeft een grote invloed heeft op de groei, polarisatie en differentiatie van cellen en deregulatie van cel-matrix adhesies speelt een belangrijke rol bij de progressie van kankercellen. Uit dit onderzoek blijkt dat Rac1 en Rac3 op een antagonistische manier de cel-matrix adhesies beïnvloeden.
... RalBP1 is a protein with GTPase activating protein (GAP) activity towards Rac1 and Cdc42 (Cantor et al., 1995;Jullien-Flores et al., 1995;Park & Weinberg, 1995). Activated Rac1 and Cdc42 have been implicated in signalling towards cell survival (Joneson & Bar-Sagi, 1999;Osada et al., 1999) and, because of its GAP activity, RalBP1 is capable of inhibiting these functions. The GAP activity of RalBP1, in turn, can be inhibited by activated Ral, which translocates RalPB1 to the cell membrane and physically away from Rac1 and Cdc42 (Matsubara et al., 1997). ...
Like normal prostate cells, prostate cancer cells are dependent on androgens for growth and survival, and prostate cancer can be treated by androgen ablation therapy. However, after a period of time some of the prostate cancer cells no longer respond to androgen ablation and survive the therapy. This transition of androgen-dependent prostate cancer (ADPC) to androgen-independent prostate cancer (AIPC) is critical, since no effective therapy is available for the androgen-independent stage of the disease. The molecular mechanisms that underlie the transition are largely unknown. REPS2, the protein that is studied in this PhD thesis project, might be involved in a molecular mechanism that contributes to AIPC development, since REPS2 mRNA is downregulated in AIPC compared to ADPC.
With specific antibodies it was shown that the REPS2 protein level in AIPC is decreased compared to ADPC. Transient overexpression of REPS2 in prostate cancer cell lines induced apoptosis within 48 h, which indicates that REPS2 may play a role in the life-death balance of the cell. To elucidate cellular functions of REPS2, proteins were identified that bind REPS2. A large fragment of the NF-κB subunit p65 (RELA) was found to bind REPS2. This protein p65 is inactive in ADPC but active in AIPC, and might cause cell survival through inhibition of apoptotic cell death. Two other protein sequences that were found to bind REPS2 represent parts of TRAF4 and STAT6. Interestingly, these two proteins, like p65, are implicated in control of some aspects of the NF-κB pathway. Taken together, the results point to a putative inhibitory effect of REPS2 on NF-kB signalling and prostate cancer cell survival.
... RALBP1 is a protein with GAP (GTPase activating protein) activity towards RAC1 and CDC42 Jullien-Flores et al., 1995;Park and Weinberg, 1995). Activated RAC1 and CDC42 have been implicated in signalling towards cell survival (Joneson and Bar-Sagi, 1999;Osada et al., 1999) and, because of its GAP activity, RALBP1 is capable of inhibiting these functions. The GAP activity of RALBP1, in turn, can be inhibited by activated RALA, which translocates RALPB1 to the cell membrane and physically away from RAC1 and CDC42 (Matsubara et al., 1997). ...
Prostate cancer has a high incidence in the western world. Early detection of the
disease is crucial for successful management, since late stages of the disease cannot
be cured. Unfortunately, however, conventional detection through rising PSA levels is
already too late in one-third of the cases. These patients have metastatic disease,
which can only be treated temporarily by androgen ablation therapy. The main
problem of metastatic prostate cancer is a transition from initially androgen-dependent
growth to androgen-independent growth. Androgen-independence of prostate cancer
cells implies resistance to androgen ablation therapy, eventually leading to death of
the patient.
Next to androgens, many other growth and differentiation inducing factors play a
role during development and homeostasis of the prostate and during progression of
prostate cancer. Peptide growth factors like EGF, TGF-a, FGF, IGF, NGF, PDGF, VEGF,
and TGF-ß have all been hypothesized to be involved in prostate cancer growth. The
proposed mechanism of androgen-independent prostate cancer progression is through
stimulation of proliferation via these factors as compensation for lack of proliferation
stimulation through androgens. Furthermore, crosstalk between androgen signalling
and growth factor signalling seems to play a role in prostate cancer growth. Growth
factors are reported to activate androgen receptors and androgens to induce growth
factor and growth factor receptor expression.
In this thesis we focussed mainly on EGF signalling. First, because REPS2, a protein
potentially involved in androgen-independent prostate cancer, acts through affecting
EGF signalling, and second, because gene profiling indicated that EGF and androgen
signalling seem to intertwine in androgen-independent prostate cancer.
... We have found that expression of the same proteins off different promoter-based expression vector plasmids can lead to dramatically different outcomes 53 . Low levels of activated Ras cause growth proliferation, whereas high levels of Ras cause growth inhibition, in primary and immortalized rodent fibroblasts 54 . Second, much of the current literature has been generated by the use of constitutively activated mutants that are assumed to mimic accurately the function of endogenously activated protein. ...
... This supports the notion that at least in Balb/C and MEF cells, both Rac1 and Cdc42Hs activities are normally involved in survival signalling pathways. The anti-apoptotic signalling of the activated Rac, but not Cdc42Hs, has also been observed in apoptosis induced by constitutive Ras signalling (Joneson and Bar-Sagi, 1999). On the other hand, previous reports have established that activation of distinct Rho family members can lead to similar phenotypes via a coordinated regulation of a cascade of activations. ...
... Alternatively, Rho-GDI· may block the antiapoptotic functions of certain Rho-GTPases, such as Rac2 and Rho (20,21). Also Rac1 has been reported to act as a survival factor (22) either by activating NF-κB (23) or by inhibiting the pro-apoptotic factor BAD (24). However, it cannot be excluded that Rho-GDI· is not causally linked to CMF sensitivity in breast cancer patients and instead is a surrogate marker for drug resistance. ...
Rho-GDI· is an inhibitor of Rho-GTPases, which is involved in cancer progression. Little is known about its role in breast cancer progression. There is evidence, that Rho-GDI· may modulate drug resistance of breast cancer cells. To assess the importance of Rho-GDI· as a risk factor in invasive ductal breast cancer, cancer specimens of three groups of patients were analyzed for Rho-GDI· RNA (group 1, N=72 and group 2, N=73) or protein expression (group 3, N=90). In group 1, patients did not receive any adjuvant treatment, whereas, in groups 2 and 3, patients were treated with anti-estrogens and/or with chemotherapeutical drugs. Rho-GDI· RNA levels, measured by RT-PCR from fresh-frozen material, did not correlate with relapse-free survival in Kaplan-Meier analysis, except in a subgroup of CMF-only treated patients. In this subgroup, higher Rho-GDI· RNA levels were significantly associated with more favorable prognosis. Immunohistochemical analysis (group 3) confirmed the link between higher Rho-GDI· expression and better outcome. This was again particularly true for the CMF-only treated patients. Cox regression analysis revealed that high Rho-GDI· protein expression reduced the risk for a relapse by ~3-fold, even if adjusted for grading, tumor size, nodal and estrogen receptor (ER) status. The data suggest that Rho-GDI· is beneficial to patients who received adjuvant chemotherapy. Rho-GDI· is possibly a useful biomarker to predict the response of breast cancer patients to CMF treatment.
... It is also found that downregulation of REPS2 is accompanied by upregulation of NF-kB activity during progression of prostate cancer from androgen-dependent to androgen-independent growth, and that NF-kB may promote cell proliferation through interacting with the EH domain of REPS2 (Penninkhof et al., 2004). In addition, REPS2 may function to keep RalBP1 cytosolic where it displays a GAP activity towards Rac1 and Cdc42 to signal cell survival so that overexpression of REPS2 may lead to a strong inhibition of Rac1 and Cdc42 signalling, which may consequently result in the attendant induction of apoptosis (Jullien-Flores et al., 1995;Joneson et al., 1999;Rincon et al., 2009). Above molecular mechanisms may provide the theoretical basis of REPS2 for participating in the the progression of ESCC, but that need further research to testify in future clinical practice. ...
Objective:
REPS2 plays important roles in inhibiting cell proliferation, migration and in inducing apoptosis of cancer cells, now being identified as a useful biomarker for favorable prognosis in prostate and breast cancers. The purpose of this study was to assess REPS2 expression and to explore its role in esophageal squamous cell carcinoma (ESCC).
Methods:
Protein expression of REPS2 in ESCCs and adjacent non-cancerous tissues from 120 patients was analyzed by immunohistochemistry and correlated with clinicopathological parameters and patient outcome. Additionally, thirty paired ESCC tissues and four ESCC cell lines and one normal human esophageal epithelial cell line were evaluated for REPS2 mRNA and protein expression levels by quantitative RT-PCR and Western blotting.
Results:
REPS2 mRNA and protein expression levels were down-regulated in ESCC tissues and cell lines. Low protein levels were significantly associated with primary tumour, TNM stage, lymph node metastasis and recurrence (all, P < 0.05). Survival analysis demonstrated that decreased REPS2 expression was significantly associated with shorter overall survival and disease-free survival (both, P < 0.001), especially in early stage ESCC patients. When REPS2 expression and lymph node metastasis status were combined, patients with low REPS2 expression/lymph node (+) had both poorer overall and disease-free survival than others (both, P < 0.001). Cox multivariate regression analysis further revealed REPS2 to be an independent prognostic factor for ESCC patients.
Conclusions:
Our findings demonstrate that downregulation of REPS2 may contribute to malignant progression of ESCC and represent a novel prognostic marker and a potential therapeutic target for ESCC patients.
... However, Ras-induced transformation is dependent on its capacity to activate further downstream signaling pathways that protect transformed cells from apoptosis 3,4 . Rac signaling has been shown essential for Ras-induced transformation [5][6][7][8] , involving the activation of the NF-kB transcription factor, which initiates an anti-apoptotic transcriptional response and further promotes cell cycle progression by an increase in cyclin D1 expression [9][10][11] . ...
BACKGROUND & AIMS: In colorectal tumors, activating BRAF mutations occur alternative to KRAS oncogenic mutations, but in cell culture possess a much lower transforming capacity. Rac1b, a hyperactive Rac1 spliced variant, is over expressed in some colorectal tumors and activates the transcription factor nuclear factor-kappaB, which initiates a transcriptional response that promotes cell cycle progression and inhibits apoptosis. The aim of this study was to determine whether Rac1b overexpression is associated with B-Raf(V600E) in primary colorectal tumors and whether a functional cooperation between these 2 proteins exists in colorectal cells with a wild-type KRAS genotype.
METHODS: Screening of BRAF and KRAS mutations by direct sequencing and Rac1b mRNA expression analysis by quantitative real-time polymerase chain reaction were conducted in 74 samples (13 normal colonic mucosa, 45 primary colorectal tumors, and 16 colorectal cancer [CRC] cell lines). RNA interference and focus formation assays were used to assess the cooperation between Rac1b and B-Raf(V600E) in cancer cell viability.
RESULTS: Rac1b overexpression and B-Raf(V600E) are significantly associated in primary colorectal tumors (P = .008) and colorectal cell lines. The simultaneous suppression of both proteins dramatically decreased CRC cell viability through impaired cell-cycle progression and increased apoptosis.
CONCLUSIONS: Our data demonstrate that Rac1b and B-Raf(V600E) functionally cooperate to sustain colorectal cell viability and suggest they constitute an alternative survival pathway to oncogenic K-Ras. These results reveal a novel molecular characteristic of colon tumors containing B-Raf mutations and should help in defining novel targets for cancer therapy.
... One study found Tiam1-Rac inhibition to be required for TJ assembly 8 , whilst other studies have shown that Tiam1-Rac activity promotes TJ assembly 9,10 , consistent with it promoting AJs [11][12][13] . Moreover, precisely how Tiam1 contributes to tumourigenesis remains unknown, although its regulation of cell-cell adhesions, cell cycle progression 12,[14][15][16] and survival 14,[17][18][19][20][21] are all believed to be important. ...
Although Rac and its activator Tiam1 are known to stimulate cell-cell adhesion, the mechanisms regulating their activity in cell-cell junction formation are poorly understood. Here, we identify β2-syntrophin as a Tiam1 interactor required for optimal cell-cell adhesion. We show that during tight-junction (TJ) assembly β2-syntrophin promotes Tiam1-Rac activity, in contrast to the function of the apical determinant Par-3 whose inhibition of Tiam1-Rac activity is necessary for TJ assembly. We further demonstrate that β2-syntrophin localizes more basally than Par-3 at cell-cell junctions, thus generating an apicobasal Rac activity gradient at developing cell-cell junctions. Targeting active Rac to TJs shows that this gradient is required for optimal TJ assembly and apical lumen formation. Consistently, β2-syntrophin depletion perturbs Tiam1 and Rac localization at cell-cell junctions and causes defects in apical lumen formation. We conclude that β2-syntrophin and Par-3 fine-tune Rac activity along cell-cell junctions controlling TJ assembly and the establishment of apicobasal polarity.
Pancreatic ductal adenocarcinoma (PDAC) has a rising incidence and is one of the most lethal human malignancies. Much is known regarding the biology and pathophysiology of PDAC, but translating this knowledge to the clinic to improve patient outcomes has been challenging. In this Review, we discuss advances and practice-changing trials for PDAC. We briefly review therapeutic failures as well as ongoing research to refine the standard of care, including novel biomarkers and clinical trial designs. In addition, we highlight contemporary areas of research, including poly(ADP-ribose) polymerase inhibitors, KRAS-targeted therapies and immunotherapies. Finally, we discuss the future of pancreatic cancer research and areas for improvement in the next decade.
The RAF-MEK-ERK mitogen-activated protein kinase (MAPK) cascade is aberrantly activated in a diverse set of human cancers and the RASopathy group of genetic developmental disorders. This protein kinase cascade is one of the most intensely studied cellular signaling networks and has been frequently targeted by the pharmaceutical industry, with more than 30 inhibitors either approved or under clinical evaluation. The ERK-MAPK cascade was originally depicted as a serial and linear, unidirectional pathway that relays extracellular signals, such as mitogenic stimuli, through the cytoplasm to the nucleus. However, we now appreciate that this three-tiered protein kinase cascade is a central core of a complex network with dynamic signaling inputs and outputs and autoregulatory loops. Despite our considerable advances in understanding the ERK-MAPK network, the ability of cancer cells to adapt to the inhibition of key nodes reveals a level of complexity that remains to be fully understood. In this review, we summarize important developments in our understanding of the ERK-MAPK network and identify unresolved issues for ongoing and future study.
We recently described the synthetic lethality that results when mutant KRAS and mutant EGFR are coexpressed in human lung adenocarcinoma (LUAD) cells, revealing the biological basis for the mutual exclusivity of KRAS and EGFR mutations in lung cancers. We have now further defined the biochemical events responsible for the toxic effects of signaling through the RAS pathway. By combining pharmacological and genetic approaches, we have developed multiple lines of evidence that signaling through extracellular signal-regulated kinases (ERK1/2) mediates the toxicity. These findings imply that tumors with mutant oncogenes that drive signaling through the RAS pathway must restrain the activity of ERK1/2 to avoid cell toxicities and enable tumor growth. In particular, a dual specificity phosphatase, DUSP6, regulates phosphorylated (P)-ERK levels in lung adenocarcinoma cells, providing negative feedback to the RAS signaling pathway. Accordingly, inhibition of DUSP6 is cytotoxic in LUAD cells driven by either mutant KRAS or mutant EGFR, phenocopying the effects of co-expression of mutant KRAS and EGFR. Together, these data suggest that targeting DUSP6 or other feedback regulators of the EGFR-KRAS-ERK pathway may offer a strategy for treating certain cancers by exceeding an upper threshold of RAS-mediated signaling.
The three human RAS proteins are mutated and constitutively activated in ∼20% of cancers leading to cell growth and proliferation. For the past three decades, many attempts have been made to inhibit these proteins with little success. Recently; however, multiple methods have emerged to inhibit KRAS, the most prevalently mutated isoform. These methods and the underlying biology will be discussed in this review with a special focus on KRAS-plasma membrane interactions. © 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Societ
RAS (H-ras, K-ras, and N-ras), as the second largest mutated gene driver in various human cancers, has long been a vital research target for cancer. Its function is to transform the extracellular environment into a cascade of intracellular signal transduction. RAS mutant protein regulates tumor cell proliferation, apoptosis, metabolism and angiogenesis through downstream MAPK, PI3K and other signaling pathways. In KRAS or other RAS driven cancers, current treatments include direct inhibitors and upstream/downstream signaling pathway inhibitors. However, the research on these inhibitors has been largely restricted due to their escape inhibition and off-target toxicity. In this paper, we started with the role of normal and mutant RAS genes in cancer, elucidated the relevant RAS regulating pathways, and highlighted the important research advancements in RAS inhibitor research. We concluded that for the crosstalk between RAS pathways, the effect of single regulation may be limited, and the multi-target drug combined compensation mechanism is becoming a research hotspot.
Synthetic lethality results when mutant KRAS and EGFR proteins are co-expressed in human lung adenocarcinoma (LUAD) cells, revealing the biological basis for mutual exclusivity of KRAS and EGFR mutations. We have now defined the biochemical events responsible for the toxic effects by combining pharmacological and genetic approaches and to show that signaling through extracellular signal-regulated kinases (ERK1/2) mediates the toxicity. These findings imply that tumors with mutant oncogenes in the RAS pathway must restrain the activity of ERK1/2 to avoid toxicities and enable tumor growth. A dual specificity phosphatase, DUSP6, that negatively regulates phosphorylation of (P)-ERK is up-regulated in EGFR- or KRAS-mutant LUAD, potentially protecting cells with mutations in the RAS signaling pathway, a proposal supported by experiments with DUSP6-specific siRNA and an inhibitory drug. Targeting DUSP6 or other negative regulators might offer a treatment strategy for certain cancers by inducing the toxic effects of RAS-mediated signaling.
Background and purpose:
SKLB-163 is a novel benzothiazole-2-thiol derivative with antitumor activities. This study investigated the effects of SKLB-163 on nasopharyngeal carcinoma (NPC) and its mechanisms.
Materials and methods:
Rho GDP-dissociation inhibitor (RhoGDI) expression was examined in NPC cell lines by western blot. Effects of SKLB-163 on proliferation, migration and radiosensitivity were assessed by MTT, wound healing and colony formation assays in vitro. Anti-tumor and anti-metastatic effects, and radiosensitizing effects of SKLB-163 were evaluated in a NPC lung metastatic nude mouse model and a subcutaneous xenograft mouse model. Effects of SKLB-163 on proliferation and apoptosis were assessed by PCNA immunohistochemistry and TUNEL assay in vivo. Key molecules in RhoGDI/c-Jun N-terminal kinases-1 (JNK-1) pathway were examined by western blot.
Results:
RhoGDI was up-regulated in NPC cell lines. SKLB-163 inhibited proliferation and migration, and increased radiosensitivity of NPC cells. SKLB-163 inhibited tumor growth and metastases, and sensitized tumor to irradiation. The radiosensitizing effects were correlated with induction of apoptosis and suppression of proliferation. The molecular mechanism was the down-regulation of RhoGDI and activation of JNK-1 signaling, and the subsequent activation of caspase-3 and the decrease in phosphorylated AKT.
Conclusions:
SKLB-163 shows strong anti-tumor activities against NPC and sensitizes NPC to irradiation by affecting the RhoGDI/JNK-1 pathway.
Inflammation has been recognized as a hallmark of cancer and is known to play an essential role in the development and progression of most cancers, even those without obvious signs of inflammation and infection.
Nuclear factor-κB (NF-κB), a transcription factor that is essential for inflammatory responses, is one of the most important molecules linking chronic inflammation to cancer, and its activity is tightly regulated by several mechanisms.
Activation of NF-κB is primarily initiated by bacterial endotoxins such as lipopolysaccharide and pro-inflammatory cytokines such as tumour necrosis factor and IL-1. NF-κB activation occurs in cancer cells and in the tumour microenvironments of most solid cancers and haematopoietic malignancies.
NF-κB activation induces various target genes, such as pro-proliferative and anti-apoptotic genes, and NF-κB signalling crosstalk affects many signalling pathways, including those involving STAT3, AP1, interferon regulatory factors, NRF2, Notch, WNT–β-catenin and p53.
All known hallmarks of cancer involve NF-κB activation. In addition to enhancing cancer cell proliferation and survival, NF-κB and inflammation promote genetic and epigenetic alterations, cellular metabolic changes, the acquisition of cancer stem cell properties, epithelial-to-mesenchymal transition, invasion, angiogenesis, metastasis, therapy resistance and the suppression of antitumour immunity.
The prevalence of NF-κB activation in cancer-related inflammation makes it an attractive therapeutic target with the potential for minimal side effects.
Hexavalent chromium [Cr(VI)] is a well-known environment carcinogen. The exposure of Cr(VI) through contaminated soil, air particles and drinking water is a strong concern for the public health worldwide. While many studies have been done, it remains unclear which intracellular molecules transduce Cr(VI)-mediated carcinogenic signaling in cells to promote cancer. In this study, we demonstrated that upon Cr(VI) treatment, the intracellular receptor src was activated, which further upregulated Ras activity, leading to the augmentation of ROS and onset of ER stress in human lung epithelial BEAS-2B or keratinocytes. These cells were formed colonies in soft agar cultures following the persistent Cr(VI) treatment. Furthermore, anti-apoptotic factor Bcl-2 was upregulated and activated in the colonies. Thus, our study suggests that Cr(VI), though activating the src and Ras signaling axis, perturbs redox state and invokes ER stress for the establishment of carcinogenic actions in the cells. In this process, Bcl-2 appears playing an important role. By uncovering these intracellular targets, our study may help developing novel strategies for better environmental protection, especially in areas contaminated or polluted by Cr(VI) as well as for effective cancer treatments. This article is protected by copyright. All rights reserved
TIPE1 (tumor necrosis factor-α-induced protein 8-like 1 or TNFAIP8L1) is a newly identified member of the TIPE (TNFAIP8) family, which play roles in regulating cell death. However, the biologic functions of TIPE1 in physiologic and pathologic conditions are largely unknown. Here, we report the roles of TIPE1 in hepatocellular carcinoma (HCC). Evaluated by immunohistochemical staining, HCC tissues showed significantly downregulated TIPE1 expression compared with adjacent non-tumor tissues, which positively correlated with tumor pathologic grades and patient survival. Using a homograft tumor model in Balb/c mice, we discovered that TIPE1 significantly diminished the growth and tumor weight of murine liver cancer homografts. Consistently, TIPE1 inhibited both cell growth and colony formation ability of cultured HCC cell lines, which was further identified to be due to TIPE1-inducing apoptosis in a caspase-independent, necrostatin-1 (Nec-1)-insensitive manner. Furthermore, mechanistic investigations revealed that TIPE1 interacted with Rac1, and inhibited the activation of Rac1 and its downstream p65 and c-Jun N-terminal kinase pathway. Moreover, overexpression of constitutively active Rac1 partially rescued the apoptosis induced by TIPE1, and Rac1 knockdown significantly restored the deregulated cell growth induced by TIPE1 small interfering RNA. Our findings revealed that TIPE1 induced apoptosis in HCC cells by negatively regulating Rac1 pathway, and loss of TIPE1 might be a new prognostic indicator for HCC patients.Oncogene advance online publication, 21 July 2014; doi:10.1038/onc.2014.208.
Taken together, in multicellular organisms, cell number is regulated spatially by extracellular signals through cell interactions controlling proliferation and survival in local neighbourhoods. Instructions from neighbouring cells can induce cell proliferation, differentiation or death. These stimuli include cell-cell and cell-ECM adhesion, growth factors, cytokines, neuropeptides and mechanical factors. Signals from G-protein-coupled receptors, tyrosine ki-nase receptors and integrins cooperate to integrate information from multiple stimuli that regulate cell cycle progression. To allow for a regulation of these processes a tight link is necessary between cell attachement mechanisms and control of proliferation and differentiation, i.e., the cell cycle machinery.
Contrary to most other cells that make up a multicellular organism, neurons use the molecular circuitry developed to sense their relationship to other cells to reorganize their connectivity according to the requirements for information processing within a cellular network. This puts neurons on the permanent risk to erroneously convert signals derived from plastic synaptic changes into positional cues that will activate the cell cycle. Maintaining neurons in a differentiated but still highly plastic phenotype will, thus, be the challenge to prevent neurodegeneration.
Although functional roles have been assigned to many genes - e.g., those involved in cell-cycle regulation, growth signaling, or cancer - considerably less is known about the quantitative relationship between their expression level and outcome. We devised an intra-population competition to study oncogene dosage. Cell populations were engineered to express a range of H-Ras oncogene levels. Cells with different levels of H-Ras then "competed" for an increased share of the total cell population. By using flow cytometry to track the population composition over time, we determined the relationship between different H-Ras oncogene expression levels and net proliferation rate. Under culture conditions where wild-type Ras activation was suppressed, we found that increased and maximal net proliferation occurred when H-Ras G12V oncogene was expressed at a level 1.2-fold that of wild-type Ras. As the H-Ras G12V expression levels increased above this optimal level, proliferation rates decreased. Our findings suggest that the tumor evolution process may optimize gene expression levels for maximal cell proliferation. In principle, engineered intra-population competitions can be used to determine proliferation rates associated with the level of any ectopically expressed gene. The approach also may be used to determine proliferation rates associated with different cell species in a heterogeneous population or improve the proliferation rate of a cell line. We also envision that the tracking of intra-population competitions could be utilized to investigate the evolution of tumors in the body.
A well-defined explanation of the progression of breast cancer to the metastatic state is still lacking at the molecular level. The involvement of Rac1, a member of the Rho GTPases, in cellular processes implicated in tumor progression, such as proliferation, adhesion and invasion, is manifest. To identify target genes of Rac1 which mediate its effects on invasion and metastasis, we applied cDNA-RDA and microarray analyses. This work resulted in the identification of 85 independent gene fragments (among them 23 novel genes) which showed altered expression levels as a result of Rac1V12 and Rac1N17 expression. The difference in mRNA abundance of twenty genes has been reconfirmed by northern blot analysis. Among the twenty genes are previously identified genes associated with tumorigenesis and/or invasion. We focused our efforts on the characterization of cyclin D1 and COX-2 (both genes' expression levels were upregulated as a result of Rac1V12 expression) with respect to mediating Rac1s effects on breast tumor progression, and found a role for COX-2 in Rac1V12-triggered increase in cell growth. In addition, we initiated experiments to obtain full-length cDNA's of the novel isolated gene fragments, which may result in the identification of novel components involved in conferring tumorigenic phenotypic changes.
A balance between cell survival and cell death is necessary for multicellular organisms to maintain homeostasis. The family of p21-activated kinases (PAKs 1, 2, and 3) are Rho GTPase-regulated serine/threonine kinases implicated in the regulation of a variety of cellular processes. The aim of this review is to describe two mechanisms by which PAKs can influence cellular homeostasis. PAK2 is proteolytically cleaved and activated by caspases after stimulation of death receptors; this activation is implicated in the regulation of biochemical and morphological changes during apoptosis. PAK1 is activated by cell survival factors (for example, interleukin 3) and promotes survival via phosphorylation and inactivation of the pro-apoptotic Bcl-2 family member Bad. Drug Dev. Res. 52:542–548, 2001. © 2001 Wiley-Liss, Inc.
The eukaryotic cell cycle is an evolutionary highly conserved process that results in cell division into two daughter cells.
Nondividing cells rest outside the cell cycle. The cell cycle consists of an ordered set of events that are tightly regulated
by defined temporal and spatial expression, subcellular localization, and degradation of cell cycle regulators. The transition
through the cell cycle is controlled by ‘stop’ and ‘go signals’ at defined checkpoints. These checkpoints allow alternate
decisions between further progression through the cell cycle and growth arrest, in order to provide potentials for DNA repair
or induction of apoptosis. Postmitotic neurons in the CNS are generated from neuroepithelial stem cells, which are multipotent
cells that differentiate into progenitor cells of neurons and glial cells. Differentiated neurons are postmitotic cells that
have permanently withdrawn from the cell cycle. Currently accumulating evidence, however, shows that differentiated neurons
express a number of cell cycle regulators which suggest a role of these proteins beyond cell cycle regulation. Under conditions
of neurodegeneration in Alzheimer’s disease, neurons leave their differentiated stage and re‐enter the cell cycle. This process
which is driven by an abnormal activation of mitogenic pathways eventually results in cell death. Alzheimer’s disease should,
thus, be added to the list of proliferative disorders such as cancer, cardiovascular disease, infections and autoimmune diseases,
where cell cycle regulators are recognized targets for treatment. Here we attempt to give an overview on the current state
of knowledge on the regulation of the cell cycle in neurons under physiological and pathophysiological conditions.
The important contributions of Ras to tumor progression and maintenance are now well established. These functions couple Ras
to a variety of downstream activities that are mediated by multiple effector pathways. Among the critical Ras targets are
the Rho GTPases, which have emerged as a family of proteins that plays an essential role in normal and neoplastic cellular
growth, particularly in Ras-induced transformation. How Rho family GTPases relay Ras signaling to downstream components and
what roles they serve in Ras-induced oncogenesis will be the subject of this chapter
Today, a new chapter is being written in the book of Alzheimer disease, one that is challenging the longstanding view that
adult neurons are incapable of division, remain nonproliferative, and are terminally differentiated. Here, we review the provocative
notion that, in Alzheimer disease, whole populations of nonstem cell neurons leave their quiescent state and re-enter into
the cell cycle. However, such neuronal re-entry into the cell cycle is futile and ultimately leads to the neurodegeneration
that typifies Alzheimer disease.
The understanding of the Raf, PI3-kinase and RalGDS mediated pathways that relay physiological signals from oncogenic Ras-proteins
has been consistently improved over the recent years. The proliferative, anti-apoptotic and some of the more cell-type and
tumor-idiosyncratic effects of Ras GTPases in various scenarios could be ascribed to one of these effectors. However, individual
tumor cells undergo drastic changes in their cell fates and differentiation states which likely require the activation of
other than Raf-, PI3-kinase- and RalGDS-initiated signaling mechanisms. In addition, Ras GTPases participate in a multitude
of developmental processes that entail growth, proliferative, differentiative and migratory programs. Proteins such as AF-6,
Nore1, certain protein kinase C (PLC) isoforms, Tiam1, Rin1 and a few others have been identified as candidate Ras-effectors
mostly by virtue of their physical interaction properties in various affinity-based protocols but also as a result of genetic
and computational approaches. This selection of alternative binding partners for oncogenic Ras-proteins can thus serve as
a source for more in depth investigations of particular Ras-related phenomena. The following chapter will scrutinize these
molecules with respect to their functions and biochemical properties
Aim: An overview on the evidence-based indications for an immunosuppressive treatment with azathioprine in chronic inflammatory bowel diseases is given.
Crohn's Disease: In Crohn's disease, steroid-dependent Crohn's disease, fistulizing Crohn's disease and the maintenance of remission in Crohn's disease. The optimal dose is 2.5 mg/kg body weight, treatment should be maintained for at least 4 years.
Ulcerative Colitis: In ulcerative colitis, these are steroid dependency, the maintenance of remission in chronic active ulcerative colitis and the maintenance of remission after induction of remission with cyclosporin or tacrolimus in acute attacks of disease.
The Pyst1 and Pyst2 mRNAs encode closely related proteins, which are novel members of a family of dual‐specificity MAP kinase phosphatases typified by CL100/MKP‐1. Pyst1 is expressed constitutively in human skin fibroblasts and, in contrast to other members of this family of enzymes, its mRNA is not inducible by either stress or mitogens. Furthermore, unlike the nuclear CL100 protein, Pyst1 is localized in the cytoplasm of transfected Cos‐1 cells. Like CL100/ MKP‐1, Pyst1 dephosphorylates and inactivates MAP kinase in vitro and in vivo. In addition, Pyst1 is able to form a physical complex with endogenous MAP kinase in Cos‐1 cells. However, unlike CL100, Pyst1 displays very low activity towards the stress‐activated protein kinases (SAPKs) or RK/p38 in vitro, indicating that these kinases are not physiological substrates for Pyst1. This specificity is underlined by the inability of Pyst1 to block either the stress‐mediated activation of the JNK‐1 SAP kinase or RK/p38 in vivo, or to inhibit nuclear signalling events mediated by the SAP kinases in response to UV radiation. Our results provide the first evidence that the members of the MAP kinase family of enzymes are differentially regulated by dual‐specificity phosphatases and also indicate that the MAP kinases may be regulated by different members of this family of enzymes depending on their subcellular location.
We report the cloning of a novel human activator of c-Jun N-terminal kinase (JNK), mitogen-activated protein kinase kinase 7 (MKK7). The mRNA for MKK7 is widely expressed in humans and mice and encodes a 47-kDa protein (419 amino acids), as determined by immunoblotting endogenous MKK7 with an antibody raised against its N terminus. The kinase domain of MKK7 is closely related to a Drosophila JNK kinase dHep (69% identity) and to a newly identified ortholog from Caenorhabditis elegans (54% identity), and was more distantly related to MKK4, MKK3, and MKK6. MKK7 phosphorylated and activated JNK1 but failed to activate p38 MAPK in co-expression studies. In hematopoietic cells, endogenous MKK7 was activated by treatment with the growth factor interleukin-3 (but not interleukin-4), or by ligation of CD40, the B-cell antigen receptor, or the receptor for the Fc fragment of immunoglobulin. MKK7 was also activated when cells were exposed to heat, UV irradiation, anisomycin, hyperosmolarity or the pro-inflammatory cytokine tumor necrosis factor-alpha. Co-expression of constitutively active mutants of RAS, RAC, or CDC42 in HeLa epithelial cells or of RAC or CDC42 in Ba/F3 factor-dependent hematopoietic cells also activated MKK7, suggesting that MKK7 will be involved in many physiological pathways.
Regulation of phosphoinositide 3-kinase (PI 3-kinase) can occur by binding of the regulatory p85 subunit to tyrosine-phosphorylated proteins and by binding of the p110 catalytic subunit to activated Ras. However, the way in which these regulatory mechanisms act to regulate PI 3-kinase in vivo is unclear. Here we show that several growth factors (basic fibroblast growth factor [bFGF], platelet-derived growth factor [PDGF], and epidermal growth factor [EGF; to activate an EGF receptor-Ret chimeric receptor]) all activate PI 3-kinase in vivo in the neuroectoderm-derived cell line SKF5. However, these growth factors differ in their ability to activate PI 3-kinase-dependent signaling. PDGF and EGF(Ret) treatment induced PI 3-kinase-dependent lamellipodium formation and protein kinase B (PKB) activation. In contrast, bFGF did not induce lamellipodium formation but activated PKB, albeit to a small extent. PDGF and EGF(Ret) stimulation resulted in binding of p85 to tyrosine-phosphorylated proteins and strong Ras activation. bFGF, however, induced only strong activation of Ras. In addition, while Ras
Asn17
abolished bFGF activation of PKB, PDGF- and EGF(Ret)-induced PKB activation was only partially inhibited and lamellipodium formation was unaffected. Interestingly, in contrast to activation of only endogenous Ras (bFGF), ectopic expression of activated Ras did result in lamellipodium formation. From this we conclude that, in vivo, p85 and Ras synergize to activate PI 3-kinase and that strong activation of only endogenous Ras exerts a small effect on PI 3-kinase activity, sufficient for PKB activation but not lamellipodium formation. This differential sensitivity to PI 3-kinase activation could be explained by our finding that PKB activation and lamellipodium formation are independent PI 3-kinase-induced events.
Although substantial evidence supports a critical role for the activation of Raf-1 and mitogen-activated protein kinases (MAPKs) in oncogenic Ras-mediated transformation, recent evidence suggests that Ras may activate a second signaling pathway which involves the Ras-related proteins Rac1 and RhoA. Consequently, we used three complementary approaches to determine the contribution of Rac1 and RhoA function to oncogenic Ras-mediated transformation. First, whereas constitutively activated mutants of Rac1 and RhoA showed very weak transforming activity when transfected alone, their coexpression with a weakly transforming Raf-1 mutant caused a greater than 35-fold enhancement of transforming activity. Second, we observed that coexpression of dominant negative mutants of Rac1 and RhoA reduced oncogenic Ras transforming activity. Third, activated Rac1 and RhoA further enhanced oncogenic Ras-triggered morphologic transformation, as well as growth in soft agar and cell motility. Finally, we also observed that kinase-deficient MAPKs inhibited Ras transformation. Taken together, these data support the possibility that oncogenic Ras activation of Rac1 and RhoA, coupled with activation of the Raf/MAPK pathway, is required to trigger the full morphogenic and mitogenic consequences of oncogenic Ras transformation.
The transcription factors NF-kappa B and AP-1 have been implicated in the inducible expression of a variety of genes involved in responses to oxidative stress and cellular defense mechanisms. Here, we report that thioredoxin, an important cellular protein oxidoreductase with antioxidant activity, exerts different effects on the activation of NF-kappa B and AP-1. Transient expression or exogenous application of thioredoxin resulted in a dose-dependent inhibition of NF-kappa B activity, as demonstrated in gel shift and transactivation experiments. AP-1-dependent transactivation, in contrast was strongly enhanced by thioredoxin. A similar increase of AP-1 activity was also observed with other, structurally unrelated antioxidants such as pyrrolidine dithiocarbamate and butylated hydroxyanisole, indicating that the thioredoxin-induced increase of AP-1 activation was indeed based on an antioxidant effect. Moreover, the stimulatory effect on AP-1 activity was found to involve de novo transcription of the c-jun and c-fos components but to be independent of protein kinase C activation. These results suggest that thioredoxin plays an important role in the regulation of transcriptional processes and oppositely affects NF-kappa B and AP-1 activation.
The mitogen-activated protein (MAP) kinases Erk-1 and Erk-2 are proline-directed kinases that are themselves activated through concomitant phosphorylation of tyrosine and threonine residues. The kinase p54 (M(r) 54,000), which was first isolated from cycloheximide-treated rats, is proline-directed like Erks-1/2, and requires both Tyr and Ser/Thr phosphorylation for activity. p54 is, however, distinct from Erks-1/2 in its substrate specificity, being unable to phosphorylate pp90rsk but more active in phosphorylating the c-Jun transactivation domain. Molecular cloning of p54 reveals a unique subfamily of extracellularly regulated kinases. Although they are 40-45% identical in sequence to Erks-1/2, unlike Erks-1/2 the p54s are only poorly activated in most cells by mitogens or phorbol esters. However, p54s are the principal c-Jun N-terminal kinases activated by cellular stress and tumour necrosis factor (TNF)-alpha, hence they are designated stress-activated protein kinases, or SAPKs. SAPKs are also activated by sphingomyelinase, which elicits a subset of cellular responses to TNF-alpha (ref. 9). SAPKs therefore define a new TNF-alpha and stress-activated signalling pathway, possibly initiated by sphingomyelin-based second messengers, which regulates the activity of c-Jun.
We show that AP-1 is an antioxidant-responsive transcription factor. DNA binding and transactivation by AP-1 were induced in HeLa cells upon treatment with the antioxidants pyrrolidine dithiocarbamate (PDTC) and N-acetyl-L-cysteine (NAC), and upon transient expression of the antioxidative enzyme thioredoxin. While PDTC and NAC enhanced DNA binding and transactivation of AP-1 in response to phorbol ester, the oxidant H2O2 suppressed phorbol ester activation of the factor. H2O2 on its own was only a weak inducer of AP-1. Activation of AP-1 by PDTC was dependent on protein synthesis and involved transcriptional induction of c-jun and c-fos genes. Transcriptional activation of c-fos by PDTC was conferred by the serum response element, suggesting that serum response factor and associated proteins function as primary antioxidant-responsive transcription factors. In the same cell line, the oxidative stress-responsive transcription factor NF-kappa B behaved in a manner strikingly opposite to AP-1. DNA binding and transactivation by NF-kappa B were strongly activated by H2O2, while the antioxidants alone were ineffective. H2O2 potentiated the activation of NF-kappa B by phorbol ester, while PDTC and NAC suppressed PMA activation of the factor. PDTC did not influence protein kinase C (PKC) activity and PKC activation by PMA, indicating that the antioxidant acted downstream of and independently from PKC.
Substantial evidence supports a critical role for the activation of the Raf-1/MEK/mitogen-activated protein kinase pathway in oncogenic Ras-mediated transformation. For example, dominant negative mutants of Raf-1, MEK, and mitogen-activated protein kinase all inhibit Ras transformation. Furthermore, the observation that plasma membrane-localized Raf-1 exhibits the same transforming potency as oncogenic Ras suggests that Raf-1 activation alone is sufficient to mediate full Ras transforming activity. However, the recent identification of other candidate Ras effectors (e.g., RalGDS and phosphatidylinositol-3 kinase) suggests that activation of other downstream effector-mediated signaling pathways may also mediate Ras transforming activity. In support of this, two H-Ras effector domain mutants, H-Ras(12V, 37G) and H-Ras(12V, 40C), which are defective for Raf binding and activation, induced potent tumorigenic transformation of some strains of NIH 3T3 fibroblasts. These Raf-binding defective mutants of H-Ras induced a transformed morphology that was indistinguishable from that induced by activated members of Rho family proteins. Furthermore, the transforming activities of both of these mutants were synergistically enhanced by activated Raf-1 and inhibited by the dominant negative RhoA(19N) mutant, indicating that Ras may cause transformation that occurs via coordinate activation of Raf-dependent and -independent pathways that involves Rho family proteins. Finally, cotransfection of H-Ras(12V, 37G) and H-Ras(12V, 40C) resulted in synergistic cooperation of their focus-forming activities, indicating that Ras activates at least two Raf-independent, Ras effector-mediated signaling events.
The Pyst1 and Pyst2 mRNAs encode closely related proteins, which are novel members of a family of dual-specificity MAP kinase phosphatases typified by CL100/MKP-1. Pyst1 is expressed constitutively in human skin fibroblasts and, in contrast to other members of this family of enzymes, its mRNA is not inducible by either stress or mitogens. Furthermore, unlike the nuclear CL100 protein, Pyst1 is localized in the cytoplasm of transfected Cos-1 cells. Like CL100/ MKP-1, Pyst1 dephosphorylates and inactivates MAP kinase in vitro and in vivo. In addition, Pyst1 is able to form a physical complex with endogenous MAP kinase in Cos-1 cells. However, unlike CL100, Pyst1 displays very low activity towards the stress-activated protein kinases (SAPKs) or RK/p38 in vitro, indicating that these kinases are not physiological substrates for Pyst1. This specificity is underlined by the inability of Pyst1 to block either the stress-mediated activation of the JNK-1 SAP kinase or RK/p38 in vivo, or to inhibit nuclear signalling events mediated by the SAP kinases in response to UV radiation. Our results provide the first evidence that the members of the MAP kinase family of enzymes are differentially regulated by dual-specificity phosphatases and also indicate that the MAP kinases may be regulated by different members of this family of enzymes depending on their subcellular location.
The small GTPase Rac assembles with the cytosolic p47(phox) and p67(phox) and the membrane-associated flavocytochrome b558 to form the multicomponent respiratory burst oxidase. Mutation of amino acids in a region of Rac (residues 26-45), homologous to an effector region in Ras, was previously shown to interfere with Rac binding to the oxidase. Herein we have elucidated an additional region in Rac involved in regulating oxidase activity. Rho family small GTPases contain a 12-amino acid "insert" region (residues 124-135) that is not present in Ras. Point mutations in and deletion of this region were constructed and used for in vitro studies of the activation of PAK65 and NADPH oxidase. Apparent binding constants (based on EC50 values) of the mutant Rac proteins for the oxidase are at least 13-25-fold higher than for wild-type Rac. Mutations in the insert region versus the 26-45 effector region resulted in distinct kinetic consequences, pointing to different roles for these two protein regions: mutations in the insert region but not the 26-45 effector region resulted in an increase in the EC50 for p67(phox). Although mutations in the 26-45 amino acid effector region showed markedly diminished activation of both PAK and the NADPH oxidase, insert region mutations did not affect activation of PAK. We propose that the combinatorial use of the 26-45 effector region and the insert region provides the Rho family GTPases with versatility in their specificity for several downstream targets.
Rac and Cdc42 regulate a variety of responses in mammalian cells including formation of lamellipodia and filopodia, activation of the JNK MAP kinase cascade, and induction of G1 cell cycle progression. Rac is also one of the downstream targets required for Ras-induced malignant transformation. Rac and Cdc42 containing a Y40C effector site substitution no longer intact with the Ser/Thr kinase p65PAK and are unable to activate the JNK MAP kinase pathway. However, they still induce cytoskeletal changes and G1 cell cycle progression. Rac containing an F37A effector site substitution, on the other hand, no longer interacts with the Ser/Thr kinase p160ROCK and is unable to induce lamellipodia or G1 progression. We conclude that Rac and Cdc42 control MAP kinase pathways and actin cytoskeleton organization independently through distinct downstream targets.
The mitogen-activated protein (MAP) kinase family includes extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38/RK/CSBP (p38) as structurally and functionally distinct
enzyme classes. Here we describe two new dual specificity phosphatases of the CL100/MKP-1 family that are selective for inactivating
ERK or JNK/SAPK and p38 MAP kinases when expressed in COS-7 cells. M3/6 is the first phosphatase of this family to display
highly specific inactivation of JNK/SAPK and p38 MAP kinases. Although stress-induced activation of p54 SAPKβ, p46 SAPKγ (JNK1)
or p38 MAP kinases is abolished upon co-transfection with increasing amounts of M3/6 plasmid, epidermal growth factor-stimulated
ERK1 is remarkably insensitive even to the highest levels of M3/6 expression obtained. In contrast to M3/6, the dual specificity
phosphatase MKP-3 is selective for inactivation of ERK family MAP kinases. Low level expression of MKP-3 blocks totally epidermal
growth factor-stimulated ERK1, whereas stress-induced activation of p54 SAPKβ and p38 MAP kinases is inhibited only partially
under identical conditions. Selective regulation by M3/6 and MKP-3 was also observed upon chronic MAP kinase activation by
constitutive p21ras GTPases. Hence, although M3/6 expression effectively blocked p54 SAPKβ activation by p21rac (G12V), ERK1 activated by p21ras (G12V) was insensitive to this phosphatase. ERK1 activation by oncogenic p21ras was, however, blocked totally by co-expression of MKP-3. This is the first report demonstrating reciprocally selective inhibition
of different MAP kinases by two distinct dual specificity phosphatases.
The c-Jun N-terminal protein kinases (JNKs), also called stress-activated protein kinases, are members of the growing family of serine/threonine kinases in the mitogen-activated protein (MAP) kinase superfamily. Like other MAP kinases, JNKs are activated via phosphorylation on adjacent threonine and tyrosine residues and can be inactivated by a unique family of dual specificity phosphatases, called MAP kinase phosphatases (MKPs). MKPs are encoded by immediate early genes and induced in response to environmental stressors and growth factor stimulation. Two prevalent isoforms of MKP, MKP1 and MKP2, are co-expressed in a wide variety of cell types. In this study, we examined the actions of MKP1 and MKP2 on JNK1 and JNK2. JNK1 phosphorylation and activation was inhibited by expression of both MKP1 and MKP2, although MKP1 selectivity toward JNK1 appeared significantly higher than that of MKP2. In contrast, JNK2 activity was inhibited by either phosphatase to similar degrees. Both MKP1 and MKP2 were highly effective at blocking the activation of the physiological target of JNK activation, the transcription factor c-Jun. In PC12 cells, MKP1 and MKP2 are transcriptionally induced following stimulation by nerve growth factor. In these cells, UV light-evoked JNK activation was reduced by pretreatment with nerve growth factor. Therefore, JNKs may be selective targets of MKP action in certain cells.
The Rho family of small GTPases are critical elements involved in the regulation of signal transduction cascades from extracellular stimuli to the cell nucleus, including the JNK/SAPK signaling pathway, the c-fos serum response factor, and the p70 S6 kinase. Here we report a novel signaling pathway activated by the Rho proteins that may be responsible for their biological activities, including cytoskeleton organization, transformation, apoptosis, and metastasis. The human RhoA, CDC42, and Rac-1 proteins efficiently induce the transcriptional activity of nuclear factor kappaB (NF-kappaB) by a mechanism that involves phosphorylation of Ikappa Balpha and translocation of p50/p50 and p50/p65 dimers to the nucleus, but independent of the Ras GTPase and the Raf-1 kinase. We also show that activation of NF-kappaB by TNFalpha depends on CDC42 and RhoA, but not Rac-1 proteins, because this activity is drastically inhibited by their respective dominant-negative mutants. In contrast, activation of NF-kappaB by UV light was not affected by Rho, CDC42, or Rac-1 dominant-negative mutants. Thus, members of the Rho family of GTPases are involved specifically in the regulation of NF-kappaB-dependent transcription.
We show that AP-1 is an antioxidant-responsive transcription factor. DNA binding and transactivation by AP-1 were induced in HeLa cells upon treatment with the antioxidants pyrrolidine dithiocarbamate (PDTC) and N-acetyl-L-cysteine (NAC), and upon transient expression of the antioxidative enzyme thioredoxin. While PDTC and NAC enhanced DNA binding and transactivation of AP-1 in response to phorbol ester, the oxidant H2O2 suppressed phorbol ester activation of the factor. H2O2 on its own was only a weak inducer of AP-1. Activation of AP-1 by PDTC was dependent on protein synthesis and involved transcriptional induction of c-jun and c-fos genes. Transcriptional activation of c-fos by PDTC was conferred by the serum response element, suggesting that serum response factor and associated proteins function as primary antioxidant-responsive transcription factors. In the same cell line, the oxidative stress-responsive transcription factor NF-kappa B behaved in a manner strikingly opposite to AP-1. DNA binding and transactivation by NF-kappa B were strongly activated by H2O2, while the antioxidants alone were ineffective. H2O2 potentiated the activation of NF-kappa B by phorbol ester, while PDTC and NAC suppressed PMA activation of the factor. PDTC did not influence protein kinase C (PKC) activity and PKC activation by PMA, indicating that the antioxidant acted downstream of and independently from PKC.
The ras proto-oncogene is frequently mutated in human tumors and functions to chronically stimulate signal transduction cascades resulting in the synthesis or activation of specific transcription factors, including Ets, c-Myc, c-Jun, and nuclear factor kappa B (NF-κB). These Ras-responsive transcription factors are required for transformation, but the mechanisms by which these proteins facilitate oncogenesis have not been fully established. Oncogenic Ras was shown to initiate a p53-independent apoptotic response that was suppressed through the activation of NF-κB. These results provide an explanation for the requirement of NF-κB for Ras-mediated oncogenesis and provide evidence that Ras-transformed cells are susceptible to apoptosis even if they do not express the p53 tumor-suppressor gene product.
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Activation of the transcription factor NF-κB has been linked to apoptosis, with the factor playing either an anti-apoptotic or a pro-apoptotic role, depending on the type of cell in which it is expressed.
Oncogenic ras can transform most immortal rodent cells to a tumorigenic state. However, transformation of primary cells by ras requires either a cooperating oncogene or the inactivation of tumor suppressors such as p53 or p16. Here we show that expression of oncogenic ras in primary human or rodent cells results in a permanent G1 arrest. The arrest induced by ras is accompanied by accumulation of p53 and p16, and is phenotypically indistinguishable from cellular senescence. Inactivation of either p53 or p16 prevents ras-induced arrest in rodent cells, and E1A achieves a similar effect in human cells. These observations suggest that the onset of cellular senescence does not simply reflect the accumulation of cell divisions, but can be prematurely activated in response to an oncogenic stimulus. Negation of ras-induced senescence may be relevant during multistep tumorigenesis.
Activation of the transcription factor NF-κB by tumor necrosis factor (TNF) and interleukin-1 (IL-1) requires the NF-κB-inducing kinase (NIK). In a yeast two-hybrid screen for NIK-interacting proteins, we have identified a protein kinase previously known as CHUK. Overexpression of CHUK activates a NF-κB-dependent reporter gene. A catalytically inactive mutant of CHUK is a dominant-negative inhibitor of TNF-, IL-1-, TRAF-, and NIK-induced NF-κB activation. CHUK associates with the NF-κB inhibitory protein, IκB-α, in mammalian cells. CHUK specifically phosphorylates IκB-α on both serine 32 and serine 36, modifications that are required for targeted degradation of IκB-α via the ubiquitin-proteasome pathway. This phosphorylation of IκB-α is greatly enhanced by NIK costimulation. Thus, CHUK is a NIK-activated IκB-α kinase that links TNF- and IL-1-induced kinase cascades to NF-κB activation.
Apoptosis plays an important role during neuronal development, and defects in apoptosis may underlie various neurodegenerative
disorders. To characterize molecular mechanisms that regulate neuronal apoptosis, the contributions to cell death of mitogen-activated
protein (MAP) kinase family members, including ERK (extracellular signal-regulated kinase), JNK (c-JUN NH2-terminal protein kinase), and p38, were examined after withdrawal of nerve growth factor (NGF) from rat PC-12 pheochromocytoma
cells. NGF withdrawal led to sustained activation of the JNK and p38 enzymes and inhibition of ERKs. The effects of dominant-interfering
or constitutively activated forms of various components of the JNK-p38 and ERK signaling pathways demonstrated that activation
of JNK and p38 and concurrent inhibition of ERK are critical for induction of apoptosis in these cells. Therefore, the dynamic
balance between growth factor-activated ERK and stress-activated JNK-p38 pathways may be important in determining whether
a cell survives or undergoes apoptosis.
Background:
Stimulation of phaeochromocytoma PC12 cells by nerve growth factor leads to growth arrest and neuronal differentiation, whereas insulin induces various metabolic responses such as metabolism of glucose and lipids. Moreover, both insulin and epidermal growth factor stimulate the proliferation of PC12 cells. In spite of their different biological effects, nerve growth factor, insulin and epidermal growth factor induce very similar early responses in PC12 cells. Stimulation with nerve growth factor leads to the sustained activation and nuclear translocation of mitogen-activated protein (MAP) kinase. By contrast, both insulin and epidermal growth factor induce the transient activation of MAP kinase, without pronounced nuclear translocation of the enzyme. We have investigated whether the differential activation of signaling pathway components can account for the distinct cellular responses to these different growth factors.
Results:
By overexpressing insulin receptors in PC12 cells, we observed insulin-dependent neurite outgrowth, similar to that induced by nerve growth factor in both non-transfected and overexpressing cells. Overexpression of insulin receptors in PC12 cells leads to a more pronounced, but similar pattern of insulin-induced tyrosine-phosphorylated proteins in PC12 cells, including enhanced recruitment of Grb2/Sos into a complex with either Shc or IRS1. MAP kinase activation in response to insulin stimulation of cells overexpressing the insulin receptor is similar to MAP kinase activation in response to NGF stimulation of parental or overexpressing PC12 cells: the activation is prolonged and nuclear translocation of the enzyme occurs.
Conclusion:
The differential subcellular localization and duration of MAP kinase activation induced by insulin and NGF may explain the difference in the biological actions of these two factors on PC12 cells. Our results show that the strength of the signal generated by a receptor with tyrosine kinase activity can influence the downstream signaling pathway, leading to cell differentiation instead of cell proliferation.
Activated Ras proteins have either positive or negative effects on the regulation of apoptosis depending on cell type and other factors. In part, this is due to the ability of Ras to control directly multiple effector pathways, including PI3-kinase, which provides a universal survival signal, and Raf, which can inhibit survival. The mechanisms remain partly unclear, however, especially with regard to Raf effects on apoptosis regulation. Recently Ras has been shown to be able to protect cells from apoptosis either through activation of PKB/Akt via PI3-kinase, or through activation of NF-kappa B.
The function of rac, a ras-related GTP-binding protein, was investigated in fibroblasts by microinjection. In confluent serum-starved Swiss 3T3 cells, rac1 rapidly stimulated actin filament accumulation at the plasma membrane, forming membrane ruffles. Several growth factors and activated H-ras also induced membrane ruffling, and this response was prevented by a dominant inhibitory mutant rac protein, N17rac1. This suggests that endogenous rac proteins are required for growth factor-induced membrane ruffling. In addition to membrane ruffling, a later response to both rac1 microinjection and some growth factors was the formation of actin stress fibers, a process requiring endogenous rho proteins. Using N17rac1 we have shown that these growth factors act through rac to stimulate this rho-dependent response. We propose that rac and rho are essential components of signal transduction pathways linking growth factors to the organization of polymerized actin.
The protein products (p21) of the ras cellular proto-oncogenes are thought to transduce membrane signals necessary for the induction of cell division. However, there is uncertainty as to the precise role of ras p21 in mediating ligand-membrane receptor signals leading to cell differentiation. Treatment of rat phaeochromocytoma cells (PC12) with nerve growth factor (NGF) results in the induction of a number of phenotypic characteristics of sympathetic neurones, including cessation of cell division and outgrowth of neuronal processes (neurites). Here we report that microinjection of antibody to ras p21 into PC12 cells inhibited neurite formation and resulted in temporary regression of partially extended neurites, an effect which was observed up to 36 h after initiation of NGF treatment. Neurite formation induced by cyclic AMP was unaffected by injection of anti-p21 antibody. These results indicate that p21 is involved in the initiation phase of NGF-induced neurite formation in PC12 cells and has a role in hormone-mediated cellular responses distinct from cell proliferation.
Human tumours often contain DNA sequences not found in normal tissues which are able to transform cultured NIH 3T3 cells. In some tumours the gene responsible for this transformation belongs to the cellular ras gene family. A specific type of mutation is responsible for converting the cellular proto-oncogene into a ras oncogene capable of inducing transformation. In a study of the function of a cellular ras gene, its protein product (produced in a bacterial cell) was microinjected into NIH 3T3 cells; the recipient cells became morphologically transformed and were induced to initiate DNA synthesis in the absence of added serum, but only when cellular ras protein was injected at much higher concentrations than required with protein of the transforming ras gene. To further analyse the function of the cellular ras gene, we have now injected monoclonal antibodies against ras proteins into NIH 3T3 cells. We report here that NIH 3T3 cells induced to divide by adding serum to the culture medium are unable to enter the S phase of the cell cycle after microinjection of anti-ras antibody, showing that the protein product of the ras proto-oncogene is required for initiation of the S-phase in NIH 3T3 cells.
The Harvey murine sarcoma virus (Ha-MuSV) transforming gene, v-rasH, encodes a 21,000 molecular weight protein (p21) that is closely related to the p21 proteins encoded by the cellular transforming genes of the ras gene family. The primary translation product (prop21), which is found in the cytosol, undergoes posttranslational modification and the mature protein subsequently becomes associated with the inner surface of the plasma membrane and binds lipid tightly. The p21 proteins have the capacity to bind guanine nucleotides non-covalently in vitro. To assess the biological relevance of these biochemical features of the protein, we have now studied a series of deletion mutants located at or near the C-terminus of the viral p21 protein. Our tissue culture studies indicate that amino acids located at or near the C-terminus are required for cellular transformation, membrane association and lipid binding.
Normal and activated Ras proteins are known to act as signal transducers, mediating mitogenic responses. Interactions of p21ras and protein kinase C (PKC) are required in a number of mitogenic or activation signaling pathways. The constitutive expression of activated v-Haras in Jurkat cells, a human T lymphoblastoid cell line, renders the cells susceptible to apoptosis during transient down-regulation or inhibition of PKC. Similarly, the expression of v-Ki-ras in murine fibroblasts induces apoptosis during suppression of PKC activity. This Ras-specific cell death is dependent upon suppression of cellular PKC activity, and can be blocked by the survival-promoting bcl-2 gene product. In vivo phosphorylation studies indicate that Bcl-2 is a phosphoprotein, and the phosphorylation state of Bcl-2 is modulated in the setting of activated p21Ha-ras in response to inhibition of PKC. These findings suggest an interactive regulation of growth or apoptosis in cells which involves at least three molecules: p21ras, PKC and Bcl-2.
The GTPase Rac1 is a key component in the reorganization of the actin cytoskeleton that is induced by growth factors or oncogenic Ras1. Here we investigate the role of Rac1 in cell transformation and show that Rat1 fibroblasts expressing activated Val-12 Rac1 (Rac1 with valine at residue 12) display all the hallmarks of malignant transformation. In a focus-forming assay in NIH3T3 fibroblasts to measure the efficiency of transformation, we found that dominant-negative Asn-17 Rac1 inhibited focus formation by oncogenic Ras, but not by RafCAAX, a Raf kinase targeted to the plasma membrane by virtue of the addition of a carboxyterminal localization signal from K-Ras. This indicates that Rac is essential for transformation by Ras. In addition, Val-12 Rac1 synergizes strongly with RafCAAX in focus-formation assays, indicating that oncogenic Ras drives both the Rac and MAP-kinase pathways, which cooperate to cause transformation.
The eukaryotic transcription factor system specifically activated by peroxides is NF-κB. Micromolar concentrations of H2O2 can mobilize the sequestered cytoplasmic form of NF-κB in cultured cells. This involves release of the regulatory subunit IκB from a heterodimer of DNA-binding p50 and p65 (also called Rel-A) subunits and nuclear translocation of p50-p65. Oxidants activate protein kinases that trigger dissociation of the NF-KB-IκB. Additional evidence that NF-κB is an oxidative stress-responsive transcription factor comes from the inhibitory effects of various structurally unrelated antioxidants on the activation of NF-κB in response to many diverse stimuli. This chapter describes how the activation of NF-κB by oxidants and the inhibitory effects of antioxidants on activation of NF-κB are investigated using intact cultured cells. The procedures described in the chapter are particularly useful in achieving two goals. The first goal is to find novel inhibitors of NF-κB that may be potent antioxidative drugs interfering with the release of IκB from the cytoplasmic complex of NF-κB. The second goal is to understand molecular mechanisms underlying the oxidative stress response in higher eukaryotes—that is, to determine what molecules sense a disturbance of the intracellular levels of reactive oxygen intermediates (ROIs) and how they transmit their signals to the nucleus where genes are newly transcribed.
When Swiss 3T3 cells are acutely infected with Moloney murine sarcoma virus containing the v-mos oncogene, 90% of the cells round up and detach from the monolayer (floating cells) and express high levels of v-Mos. The majority of the floating cells are generated between 30 and 70 h post infection when the cellular level of Mos reaches approximately 0.1% of the total protein. Seventy percent of the floating cells exclude trypan blue but are growth arrested with 2C or 4C DNA content, whereas the remaining floating cells with < 2C DNA content, are dead or dying, and show characteristic apoptotic phenotypes. The apoptotic cells are most likely generated from cells in S-phase since these cells are absent from the viable floating cell population and the percentage of cells with < 2C DNA approximated the expected S-phase fraction of logarithmically growing cells. In addition, 5'-bromo-2'-deoxyuridine-labeling studies showed that approximately 50% of the floating cells with typical apoptotic phenotypes were metabolically-labelled with the drug. These analyses show that cell populations in different stages of the cell cycle are differently affected by high levels of v-Mos expression and cells in S-phase appear to be uniquely sensitive and undergo apoptosis.
We have developed a generalized approach, using two hybrid interactions, to isolate Ha-Ras effector loop mutations that separate the ability of Ha-Ras to interact with different downstream effectors. These mutations attenuate or eliminate Ha-ras(G12V) transformation of mammalian cells, but retain complementary activity, as demonstrated by synergistic induction of foci of growth-transformed cells, and by the ability to activate different downstream components. The transformation defect of Ha-ras(G12V, E37G) is rescued by a mutant, raf1, that restores interaction. These results indicate that multiple cellular components, including Raf1, are activated by Ha-Ras and contribute to Ha-Ras-induced mammalian cell transformation.
Mitogen-activated protein (MAP) kinase is the central component of a signal transduction pathway that is activated by growth factors interacting with receptors that have protein tyrosine kinase activity. The stimulation of PC12 phaeochromocytoma cells with nerve growth factor leads to the sustained activation and nuclear translocation of the p42 and p44 isoforms of MAP kinase and induces the differentiation of these chromaffin cells to a sympathetic-neuron-like phenotype. In contrast, stimulation with epidermal growth factor induces a transient activation of p42 and p44 MAP kinases without pronounced nuclear translocation and does not trigger cell differentiation. We have examined whether the differential activation of MAP kinases forms the basis of the differential response of the cells to the two factors.
By overexpressing either wild-type or mutant receptors for epidermal growth factor in PC12 cells, we found that p42 and p44 MAP kinase activity remains elevated for longer in cells that overexpress receptors than in untransfected cells. Epidermal growth factor promotes both a striking nuclear translocation of p42 MAP kinase and the differentiation of the overexpressing cells.
Our results strongly suggest that the distinct effects of nerve growth factor and epidermal growth factor on PC12 cell differentiation can be explained by differences in the extent and duration of activation of p42 and p44 MAP kinases in response to the two factors, without invoking a signal transduction pathway specific to nerve growth factor.
The ultraviolet (UV) response of mammalian cells is characterized by a rapid and selective increase in gene expression mediated by AP-1 and NF-kappa B. The effect on AP-1 transcriptional activity results, in part, from enhanced phosphorylation of the c-Jun NH2-terminal activation domain. Here, we describe the molecular cloning and characterization of JNK1, a distant relative of the MAP kinase group that is activated by dual phosphorylation at Thr and Tyr during the UV response. Significantly, Ha-Ras partially activates JNK1 and potentiates the activation caused by UV. JNK1 binds to the c-Jun transactivation domain and phosphorylates it on Ser-63 and Ser-73. Thus, JNK1 is a component of a novel signal transduction pathway that is activated by oncoproteins and UV irradiation. These properties indicate that JNK1 activation may play an important role in tumor promotion.
The small GTP-binding proteins Rac and Rho are key elements in the signal-transduction pathways respectively controlling the formation of lamellipodia and stress fibers induced by growth factors or oncogenic Ras. We recently reported that Rac function is necessary for Ras transformation and that expression of constitutively activated Rac1 is sufficient to cause malignant transformation. We now show that, although expression of constitutively activated V14-RhoA in Rat 1 fibroblasts does not cause transformation on its own, it strongly cooperates with constitutively active RafCAAX in focus-formation assays in NIH 3T3 cells. Furthermore, dominant-negative N19-RhoA inhibits focus formation by V12-H-Ras and RafCAAX in NIH 3T3 cells, and stable coexpression of N19-RhoA and V12-H-Ras in Rat1 fibroblasts reverts Ras transformation. Interestingly, stress fiber formation is inhibited in V12-H-Ras lines and restored by coexpression of N19-RhoA. We conclude that Rho drives at least two separate pathways, one that induces stress fiber formation and another one that is important for transformation by oncogenic Ras.
The RAS guanine nucleotide binding proteins activate multiple signaling events that regulate cell growth and differentiation. In quiescent fibroblasts, ectopic expression of activated H-RAS (H-RASV12, where V12 indicates valine-12) induces membrane ruffling, mitogen-activated protein (MAP) kinase activation, and stimulation of DNA synthesis. A mutant of activated H-RAS, H-RASV12C40 (where C40 indicates cysteine-40), was identified that was defective for MAP kinase activation and stimulation of DNA synthesis, but retained the ability to induce membrane ruffling. Another mutant of activated H-RAS, H-RASV12S35 (where S35 indicates serine-35), which activates MAP kinase, was defective for stimulation of membrane ruffling and induction of DNA synthesis. Expression of both mutants resulted in a stimulation of DNA synthesis that was comparable to that induced by H-RASV12. These results indicate that membrane ruffling and activation of MAP kinase represent distinct RAS effector pathways and that input from both pathways is required for the mitogenic activity of RAS.
The transcription factor NF-kappa B has attracted widespread attention among researchers in many fields based on the following: its unusual and rapid regulation, the wide range of genes that it controls, its central role in immunological processes, the complexity of its subunits, and its apparent involvement in several diseases. A primary level of control for NF-kappa B is through interactions with an inhibitor protein called I kappa B. Recent evidence confirms the existence of multiple forms of I kappa B that appear to regulate NF-kappa B by distinct mechanisms. NF-kappa B can be activated by exposure of cells to LPS or inflammatory cytokines such as TNF or IL-1, viral infection or expression of certain viral gene products, UV irradiation, B or T cell activation, and by other physiological and nonphysiological stimuli. Activation of NF-kappa B to move into the nucleus is controlled by the targeted phosphorylation and subsequent degradation of I kappa B. Exciting new research has elaborated several important and unexpected findings that explain mechanisms involved in the activation of NF-kappa B. In the nucleus, NF-kappa B dimers bind to target DNA elements and activate transcription of genes encoding proteins involved with immune or inflammation responses and with cell growth control. Recent data provide evidence that NF-kappa B is constitutively active in several cell types, potentially playing unexpected roles in regulation of gene expression. In addition to advances in describing the mechanisms of NF-kappa B activation, excitement in NF-kappa B research has been generated by the first report of a crystal structure for one form of NF-kappa B, the first gene knockout studies for different forms of NF-kB and of I kappa B, and the implications for therapies of diseases thought to involve the inappropriate activation of NF-kappa B.
The RAC guanine nucleotide binding proteins regulate multiple biological activities, including actin polymerization, activation
of the Jun kinase (JNK) cascade, and cell proliferation. RAC effector loop mutants were identified that separate the ability
of RAC to interact with different downstream effectors. One mutant of activated human RAC protein, RACV12H40 (with valine and histidine substituted at position 12 and 40, respectively), was defective in binding to PAK3, a Ste20-related
p21-activated kinase (PAK), but bound to POR1, a RAC-binding protein. This mutant failed to stimulate PAK and JNK activity
but still induced membrane ruffling and mediated transformation. A second mutant, RACV12L37 (with leucine substituted at position 37), which bound PAK but not POR1, induced JNK activation but was defective in inducing
membrane ruffling and transformation. These results indicate that the effects of RAC on the JNK cascade and on actin polymerization
and cell proliferation are mediated by distinct effector pathways that diverge at the level of RAC itself.
The signal transduction pathway leading to the activation of the transcription factor NF-kappaB remains incompletely characterized. We demonstrate that in HeLa cells, transient expression of a constitutively active mutant of the small GTP-binding protein rac1 (V12rac1) leads to a significant increase in NF-kappaB transcriptional activity. In addition, expression of a dominant-negative rac1 mutant (N17rac1) inhibits basal and interleukin 1beta-stimulated NF-kappaB activity. Gel shift analysis using nuclear extract prepared from HeLa cells infected with a recombinant adenovirus encoding N17rac1 (Ad.N17racl) showed reduced levels of cytokine-stimulated DNA binding to a consensus NF-kappaB binding site. We demonstrate that rac proteins function downstream of ras proteins in the activation of NF-kappaB. In addition, V12rac1 stimulation of NF-kappaB activity is shown to be independent of the ability of rac proteins to activate the family of c-jun amino-terminal kinases. In an effort to further explore how rac proteins might regulate NF-kappaB activity, we demonstrate that expression of V12rac1 in HeLa cells or stimulation with cytokine results in a significant increase in intracellular reactive oxygen species (ROS). Treatment of cells with either of two chemically unrelated antioxidants inhibits the rise in ROS that occurs following V12rac1 expression as well as the ability of V12rac1 to stimulate NF-kappaB activity. These results suggest that in HeLa cells, rac1 regulates intracellular ROS production and that rac proteins function as part of a redox-dependent signal transduction pathway leading to NF-kappaB activation.
Constitutive activation of mitogen-activated protein kinase (MAPK) is a property common to many oncoproteins, including Mos, Ras, and Raf, and is essential for their transforming activities. We have shown that high levels of expression of the Mos/MAPK pathway in Swiss 3T3 fibroblast cause cells in S phase to undergo apoptosis, while cells in G1 irreversibly growth arrest. Interestingly, cells in G2 and M phases also arrest at a G1-like checkpoint after proceeding through mitosis. These cells fail to undergo cytokinesis and are binucleated. Thus, constitutive overexpression of Mos and MAPK cannot be tolerated, and fibroblasts transformed by Mos express only low levels of the mos oncogene product. Here, we show that p53 plays a key role in preventing oncogene-mediated activation of MAPK. In the absence of p53 (p53-/-), the growth arrest normally observed in wild-type p53 (p53+/+) mouse embryo fibroblasts (MEFs) is markedly reduced. The mos transformation efficiency in p53-/- MEFs is two to three orders of magnitude higher than that in p53+/+ cells, and p53-/- cells tolerate > 10-fold higher levels of both Mos and activated MAPK. Moreover, we show that, like Mos, both v-ras and v-raf oncogene products induce apoptosis in p53+/+ MEFs. These oncogenes also display a high transforming activity in p53-/- MEFs, as does a gain-of-function MAPK kinase mutant (MEK*). Thus, the p53-dependent checkpoint pathway is responsive to oncogene-mediated MAPK activation in inducing irreversible G1 growth arrest and apoptosis. Moreover, we show that the chromosome instability induced by the loss of p53 is greatly enhanced by the constitutive activation of the Mos/MAPK pathway.
The viability of vertebrate cells depends on survival factors which activate signal transduction pathways that suppress apoptosis. Defects in anti-apoptotic signalling pathways are implicated in many pathologies including cancer, in which apoptosis induced by deregulated oncogenes must be forestalled for a tumour to become established. Phosphatidylinositol-3-kinase (PI(3)K) is involved in the intracellular signal transduction of many receptors and has been implicated in the transduction of survival signals in neuronal cells. We therefore examined the role of PI(3)K, its upstream effector Ras, and its putative downstream protein kinase effectors PKB/Akt and p70S6K (ref. 5) in the modulation of apoptosis induced in fibroblasts by the oncoprotein c-Myc. Here we show that Ras activation of PI(3)K suppresses c-Myc-induced apoptosis through the activation of PKB/Akt but not p70S6K. However, we also found that Ras is an effective promoter of apoptosis, through the Raf pathway. Thus Ras activates contradictory intracellular pathways that modulate cell viability. Induction of apoptosis by Ras may be an important factor in limiting the expansion of somatic cells that sustain oncogenic ras mutations.
Serrano et al. are not the first to have noted that oncogenes paradoxically induce cells to stop growing. The seed of the idea has come from observations of Evan and his group that the myc oncogene can, under certain conditions, elicit another type of anti-proliferative response—apoptosis. (Evan et al. 1992xEvan, G.I, Wyllie, A.H, Gilbert, C.S, Littlewood, T.D, Land, H, Brooks, M, Waters, C.M, Penn, L.Z, and Hancock, D.C. Cell. 1992; 69: 119–128Abstract | Full Text PDF | PubMed | Scopus (2366)See all ReferencesEvan et al. 1992). Others subsequently showed that the E1A oncogene, which is myc-like in some of its actions, also induces apoptosis (3xDebbas, M and White, E. Genes Dev. 1993; 7: 546–554Crossref | PubMedSee all References, 12xLowe, S.W and Ruley, H.E. Genes Dev. 1993; 7: 535–545CrossrefSee all References). This apoptosis can be reversed by several means, among them, the introduction of ras oncogene (Lin et al. 1995xLin, H.J, Eviner, V, Prendergast, G.C, and White, E. Mol. Cell. Biol. 1995; 15: 4536–4544See all ReferencesLin et al. 1995).Now we begin to see the symmetry in the design of the circuitry that programs the cell's defense mechanisms. ras and perhaps other similarly acting oncogenes induce a senescence which E1A can block. E1A induces an apoptosis which can be blocked by ras; ras appears also able to block myc-induced apoptosis (McKenna et al. 1996xMcKenna, W.G, Bernhard, E.J, Markiewicz, D.A, Rudoltz, M.S, Maity, A, and Muschel, R.J. Oncogene. 1996; 12: 237–245See all ReferencesMcKenna et al. 1996). Hence, senescence and apoptosis represent the two alternative responses that cells mount in response to these two functionally complementary classes of oncogenes.Unexplained by this are the precise physiologic and biochemical mechanisms by which the myc participates in these collaborations. Ostensibly, myc intervenes in the cell cycle clock machinery to prevent ras-induced senescence, but that is not yet shown experimentally. An important molecular clue is likely provided by the recent observation that the Myc protein can induce expression of the Cdc25A phosphatase, an important activator of G1 CDKs (Galaktionov et al. 1996xGalaktionov, K, Chen, X, and Beach, D. Nature. 1996; 382: 511–517Crossref | PubMed | Scopus (593)See all ReferencesGalaktionov et al. 1996).All this brings us a large step closer to realizing one of the major goals of current cancer research: rationalizing the complex, multi-step process of tumor progression in terms of the workings of the molecular machinery that operates inside cells to regulate their proliferation.
Activation of the transcription factor NF-kappaB has been linked to apoptosis, with the factor playing either an anti-apoptotic or a pro-apoptotic role, depending on the type of cell in which it is expressed.
The pathways by which mammalian Ras proteins induce cortical actin rearrangement and cause cellular transformation are investigated using partial loss of function mutants of Ras and activated and inhibitory forms of various postulated target enzymes for Ras. Efficient transformation by Ras requires activation of other direct effectors in addition to the MAP kinase kinase kinase Raf and is inhibited by inactivation of the PI 3-kinase pathway. Actin rearrangement correlates with the ability of Ras mutants to activate PI 3-kinase. Inhibition of PI 3-kinase activity blocks Ras induction of membrane ruffling, while activated PI 3-kinase is sufficient to induce membrane ruffling, acting through Rac. The ability of activated Ras to stimulate PI 3-kinase in addition to Raf is therefore important in Ras transformation of mammalian cells and essential in Ras-induced cytoskeletal reorganization.
Nuclear transcription factors of the NF-kappaB/Rel family are inhibited by IkappaB proteins, which inactivate NF-kappaB by trapping it in the cell cytoplasm. Phosphorylation of IkappaBs marks them out for destruction, thereby relieving their inhibitory effect on NF-kappaB. A cytokine-activated protein kinase complex, IKK (for IkappaB kinase), has now been purified that phosphorylates IkappaBs on the sites that trigger their degradation. A component of IKK was molecularly cloned and identified as a serine kinase. IKK turns out to be the long-sought-after protein kinase that mediates the critical regulatory step in NF-kappaB activation.
Ras proteins are membrane-bound GTP-binding proteins that play a critical role in the control of cell growth. Through a large number of genetic and biochemical studies it is becoming increasingly evident that the biological activity of Ras proteins is mediated by multiple signaling pathways. This review provides an account of the target proteins that interact with Ras and the functional consequences of these interactions. The relative contribution of the different Ras effector pathways to the mitogenic and oncogenic effects of Ras are discussed.
PKB/Akt is a serine/threonine kinase that contains a pleckstrin-homology (PH) domain and is activated in response to growth-factor treatment of cells by a mechanism involving phosphoinositide 3-OH kinase. PKB/Akt provides a survival signal that protects cells from apoptosis induced by various stresses, perhaps explaining its discovery as a retroviral oncogene and its amplification in many human tumours.
Activation of the transcription factor nuclear factor kappa B (NF-kappaB) by inflammatory cytokines requires the successive action of NF-kappaB-inducing kinase (NIK) and IkappaB kinase-alpha (IKK-alpha). A widely expressed protein kinase was identified that is 52 percent identical to IKK-alpha. IkappaB kinase-beta (IKK-beta) activated NF-kappaB when overexpressed and phosphorylated serine residues 32 and 36 of IkappaB-alpha and serines 19 and 23 of IkappaB-beta. The activity of IKK-beta was stimulated by tumor necrosis factor and interleukin-1 treatment. IKK-alpha and IKK-beta formed heterodimers that interacted with NIK. Overexpression of a catalytically inactive form of IKK-beta blocked cytokine-induced NF-kappaB activation. Thus, an active IkappaB kinase complex may require three distinct protein kinases.