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1.  Dynamics of p53 and NF-κB regulation in response to DNA damage and identification of target proteins suitable for therapeutic intervention 
BMC Systems Biology  2012;6:125.
The genome is continuously attacked by a variety of agents that cause DNA damage. Recognition of DNA lesions activates the cellular DNA damage response (DDR), which comprises a network of signal transduction pathways to maintain genome integrity. In response to severe DNA damage, cells undergo apoptosis to avoid transformation into tumour cells, or alternatively, the cells enter permanent cell cycle arrest, called senescence. Most tumour cells have defects in pathways leading to DNA repair or apoptosis. In addition, apoptosis could be counteracted by nuclear factor kappa B (NF-κB), the main anti-apoptotic transcription factor in the DDR. Despite the high clinical relevance, the interplay of the DDR pathways is poorly understood. For therapeutic purposes DNA damage signalling processes are induced to induce apoptosis in tumour cells. However, the efficiency of radio- and chemotherapy is strongly hampered by cell survival pathways in tumour cells. In this study logical modelling was performed to facilitate understanding of the complexity of the signal transduction networks in the DDR and to provide cancer treatment options.
Our comprehensive discrete logical model provided new insights into the dynamics of the DDR in human epithelial tumours. We identified new mechanisms by which the cell regulates the dynamics of the activation of the tumour suppressor p53 and NF-κB. Simulating therapeutic intervention by agents causing DNA single-strand breaks (SSBs) or DNA double-strand breaks (DSBs) we identified candidate target proteins for sensitization of carcinomas to therapeutic intervention. Further, we enlightened the DDR in different genetic diseases, and by failure mode analysis we defined molecular defects putatively contributing to carcinogenesis.
By logic modelling we identified candidate target proteins that could be suitable for radio- and chemotherapy, and contributes to the design of more effective therapies.
PMCID: PMC3473366  PMID: 22979979
Topoisomerase inhibitors; Signal transduction; Cell cycle arrest; Apoptosis; Cancer; Logical model
2.  A methodology for the structural and functional analysis of signaling and regulatory networks 
BMC Bioinformatics  2006;7:56.
Structural analysis of cellular interaction networks contributes to a deeper understanding of network-wide interdependencies, causal relationships, and basic functional capabilities. While the structural analysis of metabolic networks is a well-established field, similar methodologies have been scarcely developed and applied to signaling and regulatory networks.
We propose formalisms and methods, relying on adapted and partially newly introduced approaches, which facilitate a structural analysis of signaling and regulatory networks with focus on functional aspects. We use two different formalisms to represent and analyze interaction networks: interaction graphs and (logical) interaction hypergraphs. We show that, in interaction graphs, the determination of feedback cycles and of all the signaling paths between any pair of species is equivalent to the computation of elementary modes known from metabolic networks. Knowledge on the set of signaling paths and feedback loops facilitates the computation of intervention strategies and the classification of compounds into activators, inhibitors, ambivalent factors, and non-affecting factors with respect to a certain species. In some cases, qualitative effects induced by perturbations can be unambiguously predicted from the network scheme. Interaction graphs however, are not able to capture AND relationships which do frequently occur in interaction networks. The consequent logical concatenation of all the arcs pointing into a species leads to Boolean networks. For a Boolean representation of cellular interaction networks we propose a formalism based on logical (or signed) interaction hypergraphs, which facilitates in particular a logical steady state analysis (LSSA). LSSA enables studies on the logical processing of signals and the identification of optimal intervention points (targets) in cellular networks. LSSA also reveals network regions whose parametrization and initial states are crucial for the dynamic behavior.
We have implemented these methods in our software tool CellNetAnalyzer (successor of FluxAnalyzer) and illustrate their applicability using a logical model of T-Cell receptor signaling providing non-intuitive results regarding feedback loops, essential elements, and (logical) signal processing upon different stimuli.
The methods and formalisms we propose herein are another step towards the comprehensive functional analysis of cellular interaction networks. Their potential, shown on a realistic T-cell signaling model, makes them a promising tool.
PMCID: PMC1458363  PMID: 16464248
3.  PIDDosome-independent tumor suppression by Caspase-2 
Cell Death and Differentiation  2012;19(10):1722-1732.
The PIDDosome, a multiprotein complex constituted of the ‘p53-induced protein with a death domain (PIDD), ‘receptor-interacting protein (RIP)-associated ICH-1/CED-3 homologous protein with a death domain' (RAIDD) and pro-Caspase-2 has been defined as an activating platform for this apoptosis-related protease. PIDD has been implicated in p53-mediated cell death in response to DNA damage but also in DNA repair and nuclear factor kappa-light-chain enhancer (NF-κB) activation upon genotoxic stress, together with RIP-1 kinase and Nemo/IKKγ. As all these cellular responses are critical for tumor suppression and deregulated expression of individual PIDDosome components has been noted in human cancer, we investigated their role in oncogenesis induced by DNA damage or oncogenic stress in gene-ablated mice. We observed that Pidd or Caspase-2 failed to suppress lymphoma formation triggered by γ-irradiation or 3-methylcholanthrene-driven fibrosarcoma development. In contrast, Caspase-2 showed tumor suppressive capacity in response to aberrant c-Myc expression, which did not rely on PIDD, the BH3-only protein Bid (BH3 interacting domain death agonist) or the death receptor ligand Trail (TNF-related apoptosis-inducing ligand), but associated with reduced rates of p53 loss and increased extranodal dissemination of tumor cells. In contrast, Pidd deficiency associated with abnormal M-phase progression and delayed disease onset, indicating that both proteins are differentially engaged upon oncogenic stress triggered by c-Myc, leading to opposing effects on tumor-free survival.
PMCID: PMC3438502  PMID: 22595758
apoptosis; Caspase-2; PIDDosome; cancer
4.  Host-pathogen systems biology: logical modelling of hepatocyte growth factor and Helicobacter pylori induced c-Met signal transduction 
The hepatocyte growth factor (HGF) stimulates mitogenesis, motogenesis, and morphogenesis in a wide range of tissues, including epithelial cells, on binding to the receptor tyrosine kinase c-Met. Abnormal c-Met signalling contributes to tumour genesis, in particular to the development of invasive and metastatic phenotypes. The human microbial pathogen Helicobacter pylori can induce chronic gastritis, peptic ulceration and more rarely, gastric adenocarcinoma. The H. pylori effector protein cytotoxin associated gene A (CagA), which is translocated via a type IV secretion system (T4SS) into epithelial cells, intracellularly modulates the c-Met receptor and promotes cellular processes leading to cell scattering, which could contribute to the invasiveness of tumour cells. Using a logical modelling framework, the presented work aims at analysing the c-Met signal transduction network and how it is interfered by H. pylori infection, which might be of importance for tumour development.
A logical model of HGF and H. pylori induced c-Met signal transduction is presented in this work. The formalism of logical interaction hypergraphs (LIH) was used to construct the network model. The molecular interactions included in the model were all assembled manually based on a careful meta-analysis of published experimental results. Our model reveals the differences and commonalities of the response of the network upon HGF and H. pylori induced c-Met signalling. As another important result, using the formalism of minimal intervention sets, phospholipase Cγ1 (PLCγ1) was identified as knockout target for repressing the activation of the extracellular signal regulated kinase 1/2 (ERK1/2), a signalling molecule directly linked to cell scattering in H. pylori infected cells. The model predicted only an effect on ERK1/2 for the H. pylori stimulus, but not for HGF treatment. This result could be confirmed experimentally in MDCK cells using a specific pharmacological inhibitor against PLCγ1. The in silico predictions for the knockout of two other network components were also verified experimentally.
This work represents one of the first approaches in the direction of host-pathogen systems biology aiming at deciphering signalling changes brought about by pathogenic bacteria. The suitability of our network model is demonstrated by an in silico prediction of a relevant target against pathogen infection.
PMCID: PMC2254585  PMID: 18194572
5.  A Cytosolic ATM/NEMO/RIP1 Complex Recruits TAK1 To Mediate the NF-κB and p38 Mitogen-Activated Protein Kinase (MAPK)/MAPK-Activated Protein 2 Responses to DNA Damage▿ 
Molecular and Cellular Biology  2011;31(14):2774-2786.
In multiple tumor types, activation of the transcription factor NF-κB increases the resistance of tumor cells to anticancer therapies and contributes to tumor progression. Genotoxic stress induced by chemotherapy or radiation therapy triggers the ATM-dependent translocation of NF-κB essential modifier (NEMO), also designated IκB kinase γ (IKKγ), from the nucleus to the cytosol, resulting in IκB kinase activation by mechanisms not yet fully understood. RIP1 has been implicated in this response and found to be modified in cells with damaged DNA; however, the nature of the RIP1 modification and its precise role in the pathway remain unclear. Here, we show that DNA damage stimulates the formation of a cytosolic complex containing ATM, NEMO (IKKγ), RIP1, and TAK1. We find that RIP1 is modified by SUMO-1 and ubiquitin in response to DNA damage and demonstrate that modified RIP1 is required for NF-κB activation and tumor cell survival. We show that ATM activates TAK1 in a manner dependent on RIP1 and NEMO. We also reveal TAK1 as a central mediator of the alternative DNA damage response pathway mediated by the p38 mitogen-activated protein kinase (MAPK)/MAPK-activated protein 2 (MAPKAP-2) kinases. These findings have translational implications and reveal RIP1 and TAK1 as potential therapeutic targets in chemoresistance.
PMCID: PMC3133388  PMID: 21606198
6.  PIDD Death-Domain Phosphorylation by ATM Controls Prodeath Versus Prosurvival PIDDosome Signaling 
Molecular cell  2012;47(5):681-693.
Biochemical evidence implicates the death-domain (DD) protein PIDD as a molecular switch capable of signaling cell survival or death in response to genotoxic stress. PIDD activity is determined by binding-partner selection at its DD: whereas recruitment of RIP1 triggers prosurvival NF-κB signaling, recruitment of RAIDD activates proapoptotic caspase-2 via PIDDosome formation. However, it remains unclear how interactor selection, and thus fate decision, are regulated at the PIDD platform. We show that the PIDDosome functions in the ‘Chk1-suppressed’ apoptotic response to DNA damage, a conserved ATM/ATR–caspase-2 pathway antagonized by Chk1. In this pathway, ATM phosphorylates PIDD on Thr788 within the DD. This phosphorylation is necessary and sufficient for RAIDD binding and caspase-2 activation. Conversely, nonphosphorylatable PIDD fails to bind RAIDD or activate caspase-2, and recruits prosurvival RIP1 instead. Thus, ATM phosphorylation of the PIDD DD enables a binary switch through which cells elect to survive or die upon DNA injury.
PMCID: PMC3444620  PMID: 22854598
7.  Chemotherapy Synergizes with Radioimmunotherapy Targeting La Autoantigen in Tumors 
PLoS ONE  2009;4(2):e4630.
To date, inefficient delivery of therapeutic doses of radionuclides to solid tumors limits the clinical utility of radioimmunotherapy. We aim to test the therapeutic utility of Yttrium-90 (90Y)-radio-conjugates of a monoclonal antibody, which we showed previously to bind specifically to the abundant intracellular La ribonucleoprotein revealed in dead tumor cells after DNA-damaging treatment.
Methodology/Principal Findings
Immunoconjugates of the DAB4 clone of the La-specific monoclonal antibody, APOMAB®, were prepared using the metal chelator, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and then radiolabeled with 90Y. Mice bearing established subcutaneous tumors were treated with 90Y-DOTA-DAB4 alone or after chemotherapy. Non-radiosensitizing cyclophosphamide/etoposide chemotherapy was used for the syngeneic EL4 lymphoma model. Radiosensitizing cisplatin/gemcitabine chemotherapy was used for the syngeneic Lewis Lung carcinoma (LL2) model, and for the xenograft models of LNCaP prostatic carcinoma and Panc-1 pancreatic carcinoma. We demonstrate the safety, specificity, and efficacy of 90Y-DOTA-DAB4-radioimmunotherapy alone or combined with chemotherapy. EL4 lymphoma-bearing mice either were cured at higher doses of radioimmunotherapy alone or lower doses of radioimmunotherapy in synergy with chemotherapy. Radioimmunotherapy alone was less effective in chemo- and radio-resistant carcinoma models. However, radioimmunotherapy synergized with radiosensitizing chemotherapy to retard significantly tumor regrowth and so prolong the survival of mice bearing LL2, LNCaP, or Panc-1 subcutaneous tumor implants.
We report proof-of-concept data supporting a unique form of radioimmunotherapy, which delivers bystander killing to viable cancer cells after targeting the universal cancer antigen, La, created by DNA-damaging treatment in neighboring dead cancer cells. Subsequently we propose that DAB4-targeted ionizing radiation induces additional cycles of tumor cell death, which further augments DAB4 binding to produce a tumor-lethal ‘genotoxic chain reaction’. Clinically, this approach may be useful as consolidation treatment after a drug-induced cell death among (small-volume) metastatic deposits, the commonest cause of cancer death.
This article is part II of a two-part series providing proof-of-concept for the diagnostic and therapeutic use of the DAB4 clone of the La-specific monoclonal antibody, APOMAB®.
PMCID: PMC2645682  PMID: 19247485
8.  Analysis of Gene Expression Using Gene Sets Discriminates Cancer Patients with and without Late Radiation Toxicity 
PLoS Medicine  2006;3(10):e422.
Radiation is an effective anti-cancer therapy but leads to severe late radiation toxicity in 5%–10% of patients. Assuming that genetic susceptibility impacts this risk, we hypothesized that the cellular response of normal tissue to X-rays could discriminate patients with and without late radiation toxicity.
Methods and Findings
Prostate carcinoma patients without evidence of cancer 2 y after curative radiotherapy were recruited in the study. Blood samples of 21 patients with severe late complications from radiation and 17 patients without symptoms were collected. Stimulated peripheral lymphocytes were mock-irradiated or irradiated with 2-Gy X-rays. The 24-h radiation response was analyzed by gene expression profiling and used for classification. Classification was performed either on the expression of separate genes or, to augment the classification power, on gene sets consisting of genes grouped together based on function or cellular colocalization.
X-ray irradiation altered the expression of radio-responsive genes in both groups. This response was variable across individuals, and the expression of the most significant radio-responsive genes was unlinked to radiation toxicity. The classifier based on the radiation response of separate genes correctly classified 63% of the patients. The classifier based on affected gene sets improved correct classification to 86%, although on the individual level only 21/38 (55%) patients were classified with high certainty. The majority of the discriminative genes and gene sets belonged to the ubiquitin, apoptosis, and stress signaling networks. The apoptotic response appeared more pronounced in patients that did not develop toxicity. In an independent set of 12 patients, the toxicity status of eight was predicted correctly by the gene set classifier.
Gene expression profiling succeeded to some extent in discriminating groups of patients with and without severe late radiotherapy toxicity. Moreover, the discriminative power was enhanced by assessment of functionally or structurally related gene sets. While prediction of individual response requires improvement, this study is a step forward in predicting susceptibility to late radiation toxicity.
Expression profiling can discriminate between groups of patients with and without severe late radiotherapy toxicity but not (yet) predict individual responses.
Editors' Summary
More than half the people who develop cancer receive radiotherapy as part of their treatment. That is, tumor cells are destroyed by exposing them to a source of ionizing radiation such as X-rays. Ionizing radiation damages the genetic material of cancer cells so that they can no longer divide. Unfortunately, it also damages nearby normal cells, although they are less sensitive to radiation than the cancer cells. Radiotherapists minimize how much radiation hits normal tissues by carefully aiming the X-rays at the tumor. Even so, patients often develop side effects such as sore skin or digestive problems during or soon after radiotherapy; the exact nature of the side effects depends on the part of the body exposed to the X-rays. In addition, a few patients develop severe late radiation toxicity, months or years after their treatment. Like early toxicity, late toxicity occurs in the normal tissues near the tumor site. For example, in prostate cancer—a tumor that forms in a gland in the male reproductive system that lies between the bladder and the end of the gut (the rectum)—late radiation toxicity affects rectal, bladder, and sexual function in 5%–10% of patients.
Why Was This Study Done?
It is not known why some patients develop late radiation toxicity, and it is impossible to predict before treatment which patients will have long-term health problems after radiotherapy. It would be useful to know this, because radiation levels might be reduced in those patients, while larger doses of radiation could be given to patients at low risk of late complications to ensure a complete eradication of their cancer. One theory is that some patients are genetically predisposed to develop severe late radiation toxicity. In other words, their genetic make-up makes it more likely that their tissues develop long-term complications after radiation damage. In this study, the researchers looked for markers of a genetic predisposition for late radiation toxicity by comparing radiation-induced changes in the pattern of cellular proteins in patients who had late radiation toxicity after radiotherapy with the changes seen in patients who did not develop such complications.
What Did the Researchers Do and Find?
The researchers recruited 38 patients who had been treated successfully with radiotherapy for prostate cancer two years previously. Of these, 21 had developed severe late radiation toxicity. They isolated lymphocytes (a type of immune system cell) from the patients' blood, stimulated the lymphocytes to divide, exposed them to X-rays, and analyzed the pattern of genes active in these cells—their gene expression profile—before and after irradiation. The researchers found that irradiation induced the expression of numerous genes in the lymphocytes, including many well-known radiation-responsive genes. They then used an analytical process called “random cross-validation” to look for a gene expression profile (or molecular signature) that was associated with late radiation toxicity. They report that a signature based on the radiation response of 50 individual genes correctly classified 63% of the patient population in terms of whether the patient had developed late radiation toxicity. A signature based on the radiation response of gene sets containing genes linked by function or cellular localization correctly classified 86% of the patient population.
What Do These Findings Mean?
Gene expression profiling identified groups of patients who had had severe late radiation toxicity pretty well, particularly when sets of related genes were used to classify the patients. The approach was not so good, however, at identifying individual patients who had had problems, being correct and certain only half the time. Additional studies are needed, therefore, before this promising approach can be used clinically to predict patient responses to radiotherapy. Overall, the study supports the idea that some patients are genetically predisposed to develop late radiation toxicity, and it also provides clues about which cellular pathways help to determine late radiation toxicity. Most of the genes and gene sets that discriminated between the patients with and without late radiation toxicity are involved in protein metabolism, apoptosis (a special sort of cell death), and stress signaling networks (pathways that protect cells from damage). This information, if confirmed, might help researchers to develop therapeutic interventions to minimize late radiation toxicity in vulnerable individuals.
Additional Information.
Please access these Web sites via the online version of this summary at
US National Cancer Institute patient information on radiotherapy and on prostate cancer
American Cancer Society information on radiation therapy
Cancer Research UK patient information on radiotherapy
Wikipedia pages on radiotherapy (note that Wikipedia is a free online encyclopedia that anyone can edit)
PMCID: PMC1626552  PMID: 17076557
9.  The Nuclear Guanine Nucleotide Exchange Factors Ect2 and Net1 Regulate RhoB-Mediated Cell Death after DNA Damage 
PLoS ONE  2011;6(2):e17108.
Commonly used antitumor treatments, including radiation and chemotherapy, function by damaging the DNA of rapidly proliferating cells. However, resistance to these agents is a predominant clinical problem. A member of the Rho family of small GTPases, RhoB has been shown to be integral in mediating cell death after ionizing radiation (IR) or other DNA damaging agents in Ras-transformed cell lines. In addition, RhoB protein expression increases after genotoxic stress, and loss of RhoB expression causes radio- and chemotherapeutic resistance. However, the signaling pathways that govern RhoB-induced cell death after DNA damage remain enigmatic. Here, we show that RhoB activity increases in human breast and cervical cancer cell lines after treatment with DNA damaging agents. Furthermore, RhoB activity is necessary for DNA damage-induced cell death, as the stable loss of RhoB protein expression using shRNA partially protects cells and prevents the phosphorylation of c-Jun N-terminal kinases (JNKs) and the induction of the pro-apoptotic protein Bim after IR. The increase in RhoB activity after genotoxic stress is associated with increased activity of the nuclear guanine nucleotide exchange factors (GEFs), Ect2 and Net1, but not the cytoplasmic GEFs p115 RhoGEF or Vav2. Importantly, loss of Ect2 and Net1 via siRNA-mediated protein knock-down inhibited IR-induced increases in RhoB activity, reduced apoptotic signaling events, and protected cells from IR-induced cell death. Collectively, these data suggest a mechanism involving the nuclear GEFs Ect2 and Net1 for activating RhoB after genotoxic stress, thereby facilitating cell death after treatment with DNA damaging agents.
PMCID: PMC3044157  PMID: 21373644
10.  Putting the brakes on p53-driven apoptosis 
Cell Cycle  2012;11(22):4122-4128.
Following genotoxic stress, cells activate a complex, kinase-based signaling network to arrest the cell cycle and initiate DNA repair or apoptosis. The tumor suppressor p53 lies at the heart of this DNA damage response. p53 mediates the transactivation of both cell cycle-regulating and pro-apoptotic clusters of target genes. However, it remains incompletely understood which signaling molecules dictate the choice between these two opposing p53-dependent cellular outcomes. Over recent years, numerous regulatory mechanisms impacting on the cellular outcome of p53 signaling have been described. However, no single dominant mechanism has thus far been identified to regulate the cellular choice between p53-driven apoptosis or senescence. The transcriptional regulator AATF has recently emerged as a novel factor impacting on the cellular outcome of the p53 response. Upon genotoxic stress, cytoplasmic pools of MRLC-bound AATF are phosphorylated through the p38MAPK/MK2 checkpoint kinase complex. This AATF phosphorylation results in the disruption of cytoplasmic MRLC3:AATF complexes followed by rapid nuclear localization of AATF. Once in the nucleus, AATF binds to the PUMA, BAX and BAK promoters to repress the DNA damage-induced expression of these pro-apoptotic p53 target genes. Depletion of AATF in tumor cells results in a dramatically enhanced response to DNA-damaging chemotherapeutics, both in vitro and in vivo. Furthermore, focal copy number gains at the AATF locus in neuroblastoma correlate with adverse prognosis and reduced overall survival in this typically p53-proficient malignancy. These data identify the p38/MK2/AATF signaling pathway as a critical repressor of p53-driven apoptosis in tumor cells and implicate this signaling cascade as a novel target for chemotherapy-sensitizing therapeutic efforts.
PMCID: PMC3524207  PMID: 22983126
AATF; DNA damage; MK2; apoptosis; checkpoint; kinase signaling; p53
11.  Evaluation of p21 promoter for interleukin 12 radiation induced transcriptional targeting in a mouse tumor model 
Molecular Cancer  2013;12:136.
Radiation induced transcriptional targeting is a gene therapy approach that takes advantage of the targeting abilities of radiotherapy by using radio inducible promoters to spatially and temporally limit the transgene expression. Cyclin dependent kinase inhibitor 1 (CDKN1A), also known as p21, is a crucial regulator of the cell cycle, mediating G1 phase arrest in response to a variety of stress stimuli, including DNA damaging agents like irradiation. The aim of the study was to evaluate the suitability of the p21 promoter for radiation induced transcriptional targeting with the objective to test the therapeutic effectiveness of the combined radio-gene therapy with p21 promoter driven therapeutic gene interleukin 12.
To test the inducibility of the p21 promoter, three reporter gene experimental models with green fluorescent protein (GFP) under the control of p21 promoter were established by gene electrotransfer of plasmid DNA: stably transfected cells, stably transfected tumors, and transiently transfected muscles. Induction of reporter gene expression after irradiation was determined using a fluorescence microplate reader in vitro and by non-invasive fluorescence imaging using fluorescence stereomicroscope in vivo. The antitumor effect of the plasmid encoding the p21 promoter driven interleukin 12 after radio-gene therapy was determined by tumor growth delay assay and by quantification of intratumoral and serum levels of interleukin 12 protein and intratumoral concentrations of interleukin 12 mRNA.
Using the reporter gene experimental models, p21 promoter was proven to be inducible with radiation, the induction was not dose dependent, and it could be re-induced. Furthermore radio-gene therapy with interleukin 12 under control of the p21 promoter had a good antitumor therapeutic effect with the statistically relevant tumor growth delay, which was comparable to that of the same therapy using a constitutive promoter.
In this study p21 promoter was proven to be a suitable candidate for radiation induced transcriptional targeting. As a proof of principle the therapeutic value was demonstrated with the radio-inducible interleukin 12 plasmid providing a synergistic antitumor effect to radiotherapy alone, which makes this approach feasible for the combined treatment with radiotherapy.
PMCID: PMC3832904  PMID: 24219565
Gene therapy; Transcriptional targeting; p21 promoter; Interleukin 12; Mouse tumor model; Radiotherapy; Plasmid DNA; Gene electrotransfer
12.  Chemotherapeutic Resistance: Surviving Stressful Situations 
Cancer research  2011;71(15):5062-5066.
Chemotherapeutic regimens involve the systemic administration of genotoxic compounds that induce cancer cell death via well-established DNA damage response signaling networks. Less understood is how the treatment of other cell types within the tumor microenvironment impacts therapeutic response. Here we discuss recent work that shows that tumor-adjacent cells can respond to genotoxic stress by engaging a paracrine secretory program. While this secretory response serves to protect progenitor cells and promote tissue regeneration in conditions of cellular stress, it can also be coopted by tumor cells to survive front-line chemotherapy. Thus, local pro-survival signaling may present a fundamental barrier to tumor clearance by genotoxic agents, suggesting that effective treatments need to target both cancer cells and the tumor microenvironment.
PMCID: PMC3148403  PMID: 21771909
13.  Senescence-Associated Secretory Phenotypes Reveal Cell-Nonautonomous Functions of Oncogenic RAS and the p53 Tumor Suppressor 
PLoS Biology  2008;6(12):e301.
Cellular senescence suppresses cancer by arresting cell proliferation, essentially permanently, in response to oncogenic stimuli, including genotoxic stress. We modified the use of antibody arrays to provide a quantitative assessment of factors secreted by senescent cells. We show that human cells induced to senesce by genotoxic stress secrete myriad factors associated with inflammation and malignancy. This senescence-associated secretory phenotype (SASP) developed slowly over several days and only after DNA damage of sufficient magnitude to induce senescence. Remarkably similar SASPs developed in normal fibroblasts, normal epithelial cells, and epithelial tumor cells after genotoxic stress in culture, and in epithelial tumor cells in vivo after treatment of prostate cancer patients with DNA-damaging chemotherapy. In cultured premalignant epithelial cells, SASPs induced an epithelial–mesenchyme transition and invasiveness, hallmarks of malignancy, by a paracrine mechanism that depended largely on the SASP factors interleukin (IL)-6 and IL-8. Strikingly, two manipulations markedly amplified, and accelerated development of, the SASPs: oncogenic RAS expression, which causes genotoxic stress and senescence in normal cells, and functional loss of the p53 tumor suppressor protein. Both loss of p53 and gain of oncogenic RAS also exacerbated the promalignant paracrine activities of the SASPs. Our findings define a central feature of genotoxic stress-induced senescence. Moreover, they suggest a cell-nonautonomous mechanism by which p53 can restrain, and oncogenic RAS can promote, the development of age-related cancer by altering the tissue microenvironment.
Author Summary
Cells with damaged DNA are at risk of becoming cancerous tumors. Although “cellular senescence” can suppress tumor formation from damaged cells by blocking the cell division that underlies cancer growth, it has also been implicated in promoting cancer and other age-related diseases. To understand how this might happen, we measured proteins that senescent human cells secrete into their local environment and found many factors associated with inflammation and cancer development. Different types of cells secrete a common set of proteins when they senesce. This senescence-associated secretory phenotype (SASP) occurs not only in cultured cells, but also in vivo in response to DNA-damaging chemotherapy. Normal cells that acquire a highly active mutant version of the RAS protein, which is known to contribute to tumor growth, undergo cellular senescence, and develop a very intense SASP, with higher levels of proteins secreted. Likewise, the SASP is more intense when cells lose the functions of the tumor suppressor p53. Senescent cells promote the growth and aggressiveness of nearby precancerous or cancer cells, and cells with a more intense SASP do so more efficiently. Our findings support the idea that cellular senescence can be both beneficial, in preventing damaged cells from dividing, and deleterious, by having effects on neighboring cells; this balance of effects is predicted by an evolutionary theory of aging.
By controlling how damaged cells modify their surrounding tissue environment, a tumor suppressor gene can restrain, and an oncogene can promote, the development of cancer.
PMCID: PMC2592359  PMID: 19053174
14.  ON/OFF and Beyond - A Boolean Model of Apoptosis 
PLoS Computational Biology  2009;5(12):e1000595.
Apoptosis is regulated by several signaling pathways which are extensively linked by crosstalks. Boolean or logical modeling has become a promising approach to capture the qualitative behavior of such complex networks. Here we built a large-scale literature-based Boolean model of the central intrinsic and extrinsic apoptosis pathways as well as pathways connected with them. The model responds to several external stimuli such as Fas ligand, TNF-α, UV-B irradiation, interleukin-1β and insulin. Timescales and multi-value node logic were used and turned out to be indispensable to reproduce the behavior of the apoptotic network. The coherence of the model was experimentally validated. Thereby an UV-B dose-effect is shown for the first time in mouse hepatocytes. Analysis of the model revealed a tight regulation emerging from high connectivity and spanning crosstalks and a particular importance of feedback loops. An unexpected feedback from Smac release to RIP could further increase complex II formation. The introduced Boolean model provides a comprehensive and coherent description of the apoptosis network behavior. It gives new insights into the complex interplay of pro- and antiapoptotic factors and can be easily expanded to other signaling pathways.
Author Summary
Apoptosis is one of the most investigated topics in the life sciences, especially as this kind of programmed cell death has been linked to several diseases. The strong desire to understand the function and regulation of apoptosis is unfortunately confronted with its complexity and its high degree of cross linking within the cell. Therefore we apply the so-called logical or Boolean mathematical modeling approach to comprehensively describe the numerous interactions in the apoptotic network. Classical Boolean modeling assumes that a certain cellular signal is either present (on) or absent (off). We use extensions of classical Boolean models, namely timescale constants and multi-value nodes, which allow the model to emulate typical apoptotic features. The mathematical model describes for the first time the numerous relevant interactions and signals that control apoptosis in a single and coherent framework. The logical model of apoptosis provides valuable information about the topology of the network including feedback loops and crosstalk effects. Proper investigation of the mutual interactions between species points towards hubs in the network with outstanding relevance. These species are of special interest concerning experimental intervention as well as drug target search. The model we present here is easy to use and freely available.
PMCID: PMC2781112  PMID: 20011108
15.  Feedback between p21 and reactive oxygen production is necessary for cell senescence 
The sustained activation of CDKN1A (p21/Waf1/Cip1) by a DNA damage response induces mitochondrial dysfunction and reactive oxygen species (ROS) production via signalling through CDKN1A-GADD45A-MAPK14- GRB2-TGFBR2-TGFbeta in senescing primary human and mouse cells in vitro and in vivo.Enhanced ROS production in senescing cells generates additional DNA damage. Although this damage is repairable and transient, it elevates the average levels of DNA damage response permanently, thus forming a positive feedback loop.This loop is necessary and sufficient to maintain the stability of growth arrest until a ‘point of no return' is reached during establishment of senescence.
The phenomenon of cellular ‘senescence'—the permanent arrest of division in normally proliferating mammalian cells such as fibroblasts—is thought to be a central component of the ageing process. Senescence contributes both to age-related loss of tissue homeostasis, as the loss of division capacity leads to impaired cell renewal, and also to protect against cancer, because it acts to block the uncontrolled proliferation of cells that may give rise to a malignant tumour. Replicative senescence is triggered by uncapped telomeres or by ‘unrepairable' non-telomeric DNA damage. Both lesions initiate the same canonical DNA damage response (DDR) (d'Adda di Fagagna, 2008). This response is characterized by activation of sensor kinases (ATM/ATR, DNA-PK), formation of DNA damage foci containing activated H2A.X (γH2A.X) and ultimately induction of cell cycle arrest through activation of checkpoint proteins, notably p53 (TP53) and the CDK inhibitor p21 (CDKN1A). This signalling pathway continues to contribute actively to the stability of the G0 arrest in fully senescent cells long after induction of senescence (d'Adda di Fagagna et al, 2003). However, senescence is more complex than mere CDKI-mediated growth arrest. Senescent cells alter their expression of literally hundreds of genes (Shelton et al, 1999), prominent among these being pro-inflammatory secretory genes (Coppe et al, 2008) and marker genes for a retrograde response induced by mitochondrial dysfunction (Passos et al, 2007a).
There is a growing evidence that multiple mechanisms interact to underpin ageing at the cellular level (Kirkwood, 2005; Passos et al, 2007b) necessitating a systems biology approach if the complex mechanisms of ageing are to be understood (Kirkwood, 2008). With respect to cell senescence, the two major unanswered questions are (i) How does a DNA lesion that can be repaired, at least in principle, induce and maintain irreversible growth arrest? and (ii) How does a growth arrest trigger a completely different cellular phenotype as soon as it becomes irreversible?
To understand those questions, we performed a kinetic analysis of the establishment phase of senescence initiated by DNA damage or telomere dysfunction, focussing on pathways downstream of the classical DDR. Using an approach that combined (i) in-silico interactome analysis, (ii) functional target gene inhibition, (iii) stochastic modelling, and (iv) live cell microscopy, we identified a positive feedback loop between DDR and mitochondrial production of reactive oxygen species (ROS) as necessary and sufficient for long-term maintenance of growth arrest. Using pathway log likelihood scores calculated by a quantitative in-silico interactome analysis to guide siRNA and small molecule inhibition experiments, and using results of sequential and combined inhibition experiments to refine the predictions from the interactome analysis, we found that DDR triggered mitochondrial dysfunction leading to enhanced ROS activation through a linear signal transduction through TP53, CDKN1A, GADD45A, p38 (MAPK14), GRB2, TGFBR2 and TGFβ(Figure 2D). We hypothesized that these ROS stochastically generate novel DNA damage in the nucleus, thus forming a positive feedback loop contributing to the long-term maintenance of DDR (Figure 3A). First confirmation came from static inhibitor experiments as before, showing that nuclear DNA damage foci frequencies in senescent cells were reduced if feedback signalling was suppressed. To formally establish the existence of a feedback loop and its relevance for senescence, we used live cell microscopy in combination with quantitative modelling.
We transformed the conceptual model shown in Figure 3A into a stochastic mechanistic model of the DDR feedback loop by extending the previously published model of the TP53/Mdm2 circuit (Proctor and Gray, 2008) to include reactions for synthesis/activation and degradation/deactivation/repair of CDKN1A, GADD45, MAPK14, ROS and DNA damage. The model replicated very precisely the kinetic behaviour of activated TP53, CDKN1A, ROS and DNA damage foci after initiation of senescence by irradiation. Having established its concordance with the experimental data, the model was then used to predict the effects of intervening in the feedback loop. The model predicted that any intervention reducing ROS levels by about half would decrease average DNA damage foci frequencies from six to four foci/nucleus within about 15 h. It further predicted that this would be sufficient to reduce CDKN1A to basal levels continuously for at least 6 h in about 20% of the treated cells, thus allowing a significant fraction of cells to escape from growth arrest and to resume proliferation. This should happen even if the intervention into the feedback loop was started at a late time point (e.g. 6 days) after induction of senescence.
To analyse DNA damage foci dynamics we used a reporter construct (AcGFP–53BP1c) that quantitatively reports single DNA damage foci kinetics in time-resolved live cell microscopy (Nelson et al, 2009). Foci frequency measurements quantitatively confirmed the prediction from the stochastic model. More importantly, we found that many individual foci in both telomere- and stress-dependent senescence had short lifespans with half-lives below 15 h. Feedback loop inhibition reduced only the frequencies of short-lived DNA damage foci in accordance with the hypothesis that ROS production contributed to DDR by constant replenishment of short-lived DNA damage foci.
Finally, we inhibited signalling through the loop at different time points after induction of senescence by ionizing radiation and measured ROS levels, DNA damage foci frequencies and proliferation markers. Treatments with the MAPK14 inhibitor SB203580 or the free radical scavenger PBN were used to block the loop. The results quantitatively confirmed the model prediction and indicated that the feedback loop between DDR and ROS production was both necessary and sufficient to maintain cell cycle arrest for at least 6–10 days after induction of senescence. Interestingly, the loop was still active at later time points and in deep senescence, but proliferation arrest was then stabilized by additional factor(s). This indicated that certain features of the senescent phenotype-like ROS production that might be responsible for the negative impact of senescent cells into their tissue environment can be successfully inhibited even in deep senescence. This may prove relevant for novel therapeutic studies aiming to modulate intracellular ROS levels in both aging and cancer.
Cellular senescence—the permanent arrest of cycling in normally proliferating cells such as fibroblasts—contributes both to age-related loss of mammalian tissue homeostasis and acts as a tumour suppressor mechanism. The pathways leading to establishment of senescence are proving to be more complex than was previously envisaged. Combining in-silico interactome analysis and functional target gene inhibition, stochastic modelling and live cell microscopy, we show here that there exists a dynamic feedback loop that is triggered by a DNA damage response (DDR) and, which after a delay of several days, locks the cell into an actively maintained state of ‘deep' cellular senescence. The essential feature of the loop is that long-term activation of the checkpoint gene CDKN1A (p21) induces mitochondrial dysfunction and production of reactive oxygen species (ROS) through serial signalling through GADD45-MAPK14(p38MAPK)-GRB2-TGFBR2-TGFβ. These ROS in turn replenish short-lived DNA damage foci and maintain an ongoing DDR. We show that this loop is both necessary and sufficient for the stability of growth arrest during the establishment of the senescent phenotype.
PMCID: PMC2835567  PMID: 20160708
aging; cell senescence; DNA damage foci; mitochondria; reactive oxygen
16.  Identification of nucleolin and nucleophosmin as genotoxic stress-responsive RNA-binding proteins 
Nucleic Acids Research  2002;30(10):2251-2260.
Genotoxic stress (DNA damage) can elicit multiple responses in mammalian cells, including the activation of numerous cascades of signal transduction that result in the activation of cellular genes involved in growth control, DNA repair and apoptosis. In an earlier report, we have shown that DNA-damaging agents can also induce the RNA-binding activity of several specific proteins that favor a double stem–loop RNA structure. Here we report the purification and identification of nucleophosmin (NPM) and nucleolin as two genotoxic stress-responsive RNA-binding proteins. UV radiation induces the protein expression levels and RNA-binding activity of NPM while nucleolin RNA-binding activity increases after UV or ionizing radiation exposure. Moreover, we have identified 40 mRNA ligands that are potentially regulated by nucleolin, several of which are stress-responsive transcripts. In addition, our data indicate that activation of nucleolin RNA-binding activity by genotoxic stress is mediated by stress-activated protein kinase p38. Our findings suggest that activation of the RNA-binding properties of nucleolin and NPM is part of the cellular response to genotoxic stress.
PMCID: PMC115285  PMID: 12000845
17.  Mitogen-activated protein kinase signal transduction and DNA repair network are involved in aluminum-induced DNA damage and adaptive response in root cells of Allium cepa L. 
In the current study, we studied the role of signal transduction in aluminum (Al3+)-induced DNA damage and adaptive response in root cells of Allium cepa L. The root cells in planta were treated with Al3+ (800 μM) for 3 h without or with 2 h pre-treatment of inhibitors of mitogen-activated protein kinase (MAPK), and protein phosphatase. Also, root cells in planta were conditioned with Al3+ (10 μM) for 2 h and then subjected to genotoxic challenge of ethyl methane sulfonate (EMS; 5 mM) for 3 h without or with the pre-treatment of the aforementioned inhibitors as well as the inhibitors of translation, transcription, DNA replication and repair. At the end of treatments, roots cells were assayed for cell death and/or DNA damage. The results revealed that Al3+ (800 μM)-induced significant DNA damage and cell death. On the other hand, conditioning with low dose of Al3+ induced adaptive response conferring protection of root cells from genotoxic stress caused by EMS-challenge. Pre-treatment of roots cells with the chosen inhibitors prior to Al3+-conditioning prevented or reduced the adaptive response to EMS genotoxicity. The results of this study suggested the involvement of MAPK and DNA repair network underlying Al-induced DNA damage and adaptive response to genotoxic stress in root cells of A. cepa.
PMCID: PMC4046574  PMID: 24926302
adaptive response; DNA damage; DNA repair; genome protection; MAP kinase; metabolic inhibitors; signal transduction
18.  Bystander signaling in C. elegans: proton microbeam studies 
Journal of Radiation Research  2014;55(Suppl 1):i118-i119.
Biological model: In this project, we investigated the control of radiation-induced genotoxic damage expression in somatic cells of the nematode Caenorhabditis elegans. We measured genotoxic damage in the C. elegans intestine by irradiating young larvae with 20 intestinal cells. Fourteen of these cells undergo exactly one nuclear division without cytoplasmic division leading to 14 binucleate cells. This nuclear division is synchronized and occurs at the first larval molt. Irradiation induces chromosome aberrations including dicentrics which we can quantify as stable anaphase bridges in the binucleate cells of young adult intestines. The endpoint is dose- and LET-dependent and we have demonstrated that individual intestinal cells have unique radiosensitivities.
Results: The project has two components, a genetic screen for genes that control cell sensitivity and a microbeam component to directly probe individual cells. The genetic screen has identified several genes in NHEJ repair and telomere metabolism that modulate overall bridge frequency. Knockout mutants of cku-70, cku-80 and lig-4 greatly sensitize animals for anaphase bridge induction. A statistical method was used to determine whether induction of bridges was strictly random and cell autonomous and we determined that expression of bridges in pairs of cells was, in fact, non-random which suggested that signaling between cells affected the pattern of bridge expression. This allowed us to conduct an RNAi and mutation screen for genes that control the signaling (block non-random distributions) and several candidates have been identified.
To directly test the notion that signaling of genotoxic damage occurs, we conducted experiments with alpha particles collimated through slits in metal foils and showed that genotoxic damage could be expressed many cell diameters away from a partial body exposure site. Thus, an in vivo bystander effect was demonstrated. Dose targeting was then improved to small regional exposures and eventually to individual cell targeting using 2 MeV protons from the microbeam facility at Texas A&M University. We now employ a green fluorescent protein (GFP)-expressing transgenic worm (rrIs1[elt-2::GFP]) to target GFP-positive gut cells via the gut-specific transcription factor elt-2. This allows alignment of the cell of interest over the microbeam aperture under appropriate fluorescence illumination.
Microbeam irradiation experiments for many pairwise combinations of cell signal transmission and reception (observed as expression of anaphase bridges) have been conducted and several interesting patterns emerge. (i) The signaling pattern is cell-specific and does not simply reflect cell–cell distance or require direct contact between cell pairs. (ii) The signal range can be as far as from cell pair 2 to cell pair 8 (>100 µm). (iii) There appears to be a functional compartment boundary at the pharynx/intestine valve as even high-dose exposures to the posterior pharyngeal bulb fail to induce bridges in nearby intestinal cells. (iv) The frequency of signal transmission and reception corresponds broadly to the overall frequency of bridges observed during whole-body irradiations which suggests that direct irradiation and ‘out-of-field’ effects may be additive. These patterns have been analyzed in terms of a cellular logic circuit map for signal transmission and reception.
A dose–response for a subset of microbeam-targeted cells was measured over the range of 5–20 Gy. Controlled cell pair targeting was used to test the potential additivity of signals and we found that effects were supra-additive. Finally, preliminary measurements were conducted on GFP-expressing transgenic strains that bore cku-70(tm1524) III and smk-1(mn156) V mutations which confer enhanced radiosensitivity. Cku-70 is a Ku-70 ortholog while smk-1 is orthologous to the mammalian and Dictyostelium discoideum SMEK (suppressor of MEK null) protein. In the cku-70(0/0) strain, the severity of the bridges in bystander cells was enhanced, suggesting that signal recipient cells employ NHEJ repair pathways in the expression of anaphase bridges.
Clinical trial registration number: Not applicable.
PMCID: PMC3941528
19.  Long-term Activation of c-Jun N-terminal Kinase through Receptor Interacting Protein is Associated with DNA Damage-induced Cell Death 
Activation of c-Jun N-terminal kinase (JNK), a member of the mitogen-activated protein kinase family, is an important cellular response that modulates the outcome of the cells which are exposed to the tumor necrosis factor (TNF) or the genotoxic stress including DNA damaging agents. Although it is known that JNK is activated in response to genotoxic stress, neither the pathways to transduce signals to activate JNK nor the primary sensors of the cells that trigger the stress response have been identified. Here, we report that the receptor interacting protein (RIP), a key adaptor protein of TNF signaling, was required to activate JNK in the cells treated with certain DNA damaging agents such as adriamycin (Adr) and 1-β-D-arabinofuranosylcytosine (Ara-C) that cause slow and sustained activation, but it was not required when treated with N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and short wavelength UV, which causes quick and transient activation. Our findings revealed that this sustained JNK activation was not mediated by the TNF (tumor necrosis factor) receptor signaling, but it required a functional ATM (ataxia telangiectasia) activity. In addition, JNK inhibitor SP-600125 significantly blocked the Adr-induced cell death, but it did not affect the cell death induced by MNNG. These findings suggest that the sustained activation of JNK mediated by RIP plays an important role in the DNA damage-induced cell death, and that the duration of JNK activation relays a different stress response to determine the cell fate.
PMCID: PMC2788634  PMID: 19967054
DNA damage; Cell death; Receptor interacting protein; c-Jun N-terminal kinase; Poly (ADP-ribose) polymerase
20.  Regulation of PIDD auto-proteolysis and activity by the molecular chaperone Hsp90 
Cell Death and Differentiation  2010;18(3):506-515.
In response to DNA damage, p53-induced protein with a death domain (PIDD) forms a complex called the PIDDosome, which either consists of PIDD, RIP-associated protein with a death domain and caspase-2, forming a platform for the activation of caspase-2, or contains PIDD, RIP1 and NEMO, important for NF-κB activation. PIDDosome activation is dependent on auto-processing of PIDD at two different sites, generating the fragments PIDD-C and PIDD-CC. Despite constitutive cleavage, endogenous PIDD remains inactive. In this study, we screened for novel PIDD regulators and identified heat shock protein 90 (Hsp90) as a major effector in both PIDD protein maturation and activation. Hsp90, together with p23, binds PIDD and inhibition of Hsp90 activity with geldanamycin efficiently disrupts this association and impairs PIDD auto-processing. Consequently, both PIDD-mediated NF-κB and caspase-2 activation are abrogated. Interestingly, PIDDosome formation itself is associated with Hsp90 release. Characterisation of cytoplasmic and nuclear pools of PIDD showed that active PIDD accumulates in the nucleus and that only cytoplasmic PIDD is bound to Hsp90. Finally, heat shock induces Hsp90 release from PIDD and PIDD nuclear translocation. Thus, Hsp90 has a major role in controlling PIDD functional activity.
PMCID: PMC3131991  PMID: 20966961
Caspase-2; Hsp90; NF-κB; PIDD
21.  Caspase-2-mediated cleavage of Mdm2 creates p53-induced positive feedback loop 
Molecular cell  2011;43(1):57-71.
Caspase-2 is an evolutionarily conserved caspase, yet its biological function and cleavage targets are poorly understood. Caspase-2 is activated by the p53 target gene product PIDD (also known as LRDD) in a complex called the Caspase-2-PIDDosome. We show that PIDD expression promotes growth arrest and chemotherapy resistance by a mechanism that depends on Caspase-2 and wild-type p53. PIDD-induced Caspase-2 directly cleaves the E3 ubiquitin ligase Mdm2 at Asp 367, leading to loss of the C-terminal RING domain responsible for p53 ubiquitination. As a consequence, N-terminally truncated Mdm2 binds p53 and promotes its stability. Upon DNA damage, p53 induction of the Caspase-2-PIDDosome creates a positive feedback loop that inhibits Mdm2 and reinforces p53 stability and activity, contributing to cell survival and drug resistance. These data establish Mdm2 as a cleavage target of Caspase-2 and provide insight into a mechanism of Mdm2 inhibition that impacts p53 dynamics upon genotoxic stress.
PMCID: PMC3160283  PMID: 21726810
p53; Mdm2; tumor suppressor; Caspase-2; DNA damage; Lrdd; PIDDosome; cancer
22.  Exquisite Sensitivity of TP53 Mutant and Basal Breast Cancers to a Dose-Dense Epirubicin−Cyclophosphamide Regimen 
PLoS Medicine  2007;4(3):e90.
In breast cancers, only a minority of patients fully benefit from the different chemotherapy regimens currently in use. Identification of markers that could predict the response to a particular regimen would thus be critically important for patient care. In cell lines or animal models, tumor protein p53 (TP53) plays a critical role in modulating the response to genotoxic drugs. TP53 is activated in response to DNA damage and triggers either apoptosis or cell-cycle arrest, which have opposite effects on cell fate. Yet, studies linking TP53 status and chemotherapy response have so far failed to unambiguously establish this paradigm in patients. Breast cancers with a TP53 mutation were repeatedly shown to have a poor outcome, but whether this reflects poor response to treatment or greater intrinsic aggressiveness of the tumor is unknown.
Methods and Findings
In this study we analyzed 80 noninflammatory breast cancers treated by frontline (neoadjuvant) chemotherapy. Tumor diagnoses were performed on pretreatment biopsies, and the patients then received six cycles of a dose-dense regimen of 75 mg/m2 epirubicin and 1,200 mg/m2 cyclophosphamide, given every 14 days. After completion of chemotherapy, all patients underwent mastectomies, thus allowing for a reliable assessment of chemotherapy response. The pretreatment biopsy samples were used to determine the TP53 status through a highly efficient yeast functional assay and to perform RNA profiling. All 15 complete responses occurred among the 28 TP53-mutant tumors. Furthermore, among the TP53-mutant tumors, nine out of ten of the highly aggressive basal subtypes (defined by basal cytokeratin [KRT] immunohistochemical staining) experienced complete pathological responses, and only TP53 status and basal subtype were independent predictors of a complete response. Expression analysis identified many mutant TP53-associated genes, including CDC20, TTK, CDKN2A, and the stem cell gene PROM1, but failed to identify a transcriptional profile associated with complete responses among TP53 mutant tumors. In patients with unresponsive tumors, mutant TP53 status predicted significantly shorter overall survival. The 15 patients with responsive TP53-mutant tumors, however, had a favorable outcome, suggesting that this chemotherapy regimen can overcome the poor prognosis generally associated with mutant TP53 status.
This study demonstrates that, in noninflammatory breast cancers, TP53 status is a key predictive factor for response to this dose-dense epirubicin–cyclophosphamide regimen and further suggests that the basal subtype is exquisitely sensitive to this association. Given the well-established predictive value of complete responses for long-term survival and the poor prognosis of basal and TP53-mutant tumors treated with other regimens, this chemotherapy could be particularly suited for breast cancer patients with a mutant TP53, particularly those with basal features.
Hugues de The and colleagues report thatTP53 status is a predictive factor for responsiveness in breast cancers to a dose-dense epirubicin-cyclophosphamide chemotherapy regimen, and suggests that this regimen might be well suited for patientsTP53 mutant tumors.
Editors' Summary
One woman in eight will develop breast cancer during her life. As with other cancers, breast cancer arises when cells accumulate genetic changes (mutations) that allow them to grow uncontrollably and to move around the body. These altered cells are called malignant cells. The normal human breast contains several types of cell, any of which can become malignant. In addition, there is more than one route to malignancy—different sets of genes can be mutated. As a result, breast cancer is a heterogeneous disease that cannot be cured with a single type of treatment. Ideally, oncologists would like to know before they start treating a patient which therapeutic approach is going to be successful for that individual. Recently, researchers have begun to identify molecular changes that might eventually allow oncologists to make such rational treatment decisions. For example, laboratory studies in cell lines or animals indicate that the status of a gene called TP53 determines the chemotherapy agents (drugs that preferentially kill rapidly dividing cancer cells) to which cells respond. p53, the protein encoded by TP53, is a tumor suppressor. That is, in normal cells it prevents unregulated growth by controlling the expression of proteins involved in cell division and cell death. Consequently, p53 is often inactivated during cancer development.
Why Was This Study Done?
Although laboratory studies have linked TP53 status to chemotherapy responses, little is known about this relationship in human breast cancers. The clinical studies that have investigated whether TP53 status affects chemotherapy responses have generally found that patients whose tumors contain mutant TP53 have a poorer response to therapy and/or a shorter survival time than those whose tumors contain normal TP53. In this study, the researchers have asked whether TP53 status affects tumor responses to a dose-intense chemotherapy regimen (frequent, high doses of drugs) given to women with advanced noninflammatory breast cancer before surgery. This type of treatment is called neoadjuvant chemotherapy and is used to shrink tumors before surgery.
What Did the Researchers Do and Find?
The researchers collected breast tumor samples from 80 women before starting six fortnightly cycles of chemotherapy with epirubicin and cyclophosphamide. After this, each woman had her affected breast removed and examined to see whether the chemotherapy had killed the tumor cells. The researchers determined which original tumor samples contained mutated TP53 and used a technique called microarray expression profiling to document gene expression patterns in them. Overall, 28 tumors contained mutated TP53. Strikingly, all 15 tumors that responded completely to neoadjuvant chemotherapy (no tumor cells detectable in the breast tissue after chemotherapy) contained mutated TP53. Nine of these responsive tumors were basal-cell–like breast tumors, a particularly aggressive type of breast cancer; only one basal-cell–like, TP53-mutated tumor did not respond to chemotherapy. Patients whose tumors were unresponsive to the neoadjuvant chemotherapy but contained mutated TP53 tended to die sooner than those whose tumors contained normal TP53 or those with chemotherapy-responsive TP53-mutated tumors. Finally, expression profiling identified changes in the expression of many p53-regulated genes, but did not identify an expression profile in the TP53-mutated tumors unique to those that responded to chemotherapy.
What Do These Findings Mean?
These findings indicate that noninflammatory breast tumors containing mutant TP53—in particular, basal-cell–like tumors—are very sensitive to dose-dense epirubicin and cyclophosphamide chemotherapy. Intensive regimens of this type have rarely been used in previous studies, which might explain the apparent contradiction between these results and the generally poor response to chemotherapy of TP53-mutated breast tumors. More tumors now need to be examined to confirm the association between complete response, TP53 status and basal-cell–like tumors. In addition, although complete tumor responses generally predict good overall survival, longer survival studies than those reported here are needed to show that the tumor response to this particular neoadjuvant chemotherapy regimen translates into improved overall survival. If the present results can be confirmed and extended, dose-dense neoadjuvant chemotherapy with epirubicin and cyclophosphamide could considerably improve the outlook for patients with aggressive TP53-mutant, basal-cell–like breast tumors.
Additional Information.
Please access these Web sites via the online version of this summary at
The US National Cancer Institute provides patient and physician information on breast cancer and general information on understanding cancer
Cancer Research UK offers patient information on cancer and breast cancer
The MedlinePlus encyclopedia has pages on breast cancer
Emory University's CancerQuest discusses the biology of cancer, including the role of tumor suppressor proteins
Wikipedia has pages on p53 (note that Wikipedia is a free online encyclopedia that anyone can edit)
PMCID: PMC1831731  PMID: 17388661
23.  A Logical Model Provides Insights into T Cell Receptor Signaling 
PLoS Computational Biology  2007;3(8):e163.
Cellular decisions are determined by complex molecular interaction networks. Large-scale signaling networks are currently being reconstructed, but the kinetic parameters and quantitative data that would allow for dynamic modeling are still scarce. Therefore, computational studies based upon the structure of these networks are of great interest. Here, a methodology relying on a logical formalism is applied to the functional analysis of the complex signaling network governing the activation of T cells via the T cell receptor, the CD4/CD8 co-receptors, and the accessory signaling receptor CD28. Our large-scale Boolean model, which comprises 94 nodes and 123 interactions and is based upon well-established qualitative knowledge from primary T cells, reveals important structural features (e.g., feedback loops and network-wide dependencies) and recapitulates the global behavior of this network for an array of published data on T cell activation in wild-type and knock-out conditions. More importantly, the model predicted unexpected signaling events after antibody-mediated perturbation of CD28 and after genetic knockout of the kinase Fyn that were subsequently experimentally validated. Finally, we show that the logical model reveals key elements and potential failure modes in network functioning and provides candidates for missing links. In summary, our large-scale logical model for T cell activation proved to be a promising in silico tool, and it inspires immunologists to ask new questions. We think that it holds valuable potential in foreseeing the effects of drugs and network modifications.
Author Summary
T-lymphocytes are central regulators of the adaptive immune response, and their inappropriate activation can cause autoimmune diseases or cancer. The understanding of the signaling mechanisms underlying T cell activation is a prerequisite to develop new strategies for pharmacological intervention and disease treatments. However, much of the existing literature on T cell signaling is related to T cell development or to activation processes in transformed T cell lines (e.g., Jurkat), whereas information on non-transformed primary T cells is limited. Here, immunologists and theoreticians have compiled data from the existing literature that stem from analysis of primary T cells. They used this information to establish a qualitative Boolean network that describes T cell activation mechanisms after engagement of the TCR, the CD4/CD8 co-receptors, and CD28. The network comprises 94 nodes and can be extended to facilitate interpretation of new data that emerge from experimental analysis of T cell activation. Newly developed tools and methods allow in silico analysis, and manipulation of the network and can uncover hidden/unforeseen signaling pathways. Indeed, by assessing signaling events controlled by CD28 and the protein tyrosine kinase Fyn, we show that computational analysis of even a qualitative network can provide new and non-obvious signaling pathways which can be validated experimentally.
PMCID: PMC1950951  PMID: 17722974
24.  Dynamical and Structural Analysis of a T Cell Survival Network Identifies Novel Candidate Therapeutic Targets for Large Granular Lymphocyte Leukemia 
PLoS Computational Biology  2011;7(11):e1002267.
The blood cancer T cell large granular lymphocyte (T-LGL) leukemia is a chronic disease characterized by a clonal proliferation of cytotoxic T cells. As no curative therapy is yet known for this disease, identification of potential therapeutic targets is of immense importance. In this paper, we perform a comprehensive dynamical and structural analysis of a network model of this disease. By employing a network reduction technique, we identify the stationary states (fixed points) of the system, representing normal and diseased (T-LGL) behavior, and analyze their precursor states (basins of attraction) using an asynchronous Boolean dynamic framework. This analysis identifies the T-LGL states of 54 components of the network, out of which 36 (67%) are corroborated by previous experimental evidence and the rest are novel predictions. We further test and validate one of these newly identified states experimentally. Specifically, we verify the prediction that the node SMAD is over-active in leukemic T-LGL by demonstrating the predominant phosphorylation of the SMAD family members Smad2 and Smad3. Our systematic perturbation analysis using dynamical and structural methods leads to the identification of 19 potential therapeutic targets, 68% of which are corroborated by experimental evidence. The novel therapeutic targets provide valuable guidance for wet-bench experiments. In addition, we successfully identify two new candidates for engineering long-lived T cells necessary for the delivery of virus and cancer vaccines. Overall, this study provides a bird's-eye-view of the avenues available for identification of therapeutic targets for similar diseases through perturbation of the underlying signal transduction network.
Author Summary
T-LGL leukemia is a blood cancer characterized by an abnormal increase in the abundance of a type of white blood cell called T cell. Since there is no known curative therapy for this disease, identification of potential therapeutic targets is of utmost importance. Experimental identification of manipulations capable of reversing the disease condition is usually a long, arduous process. Mathematical modeling can aid this process by identifying potential therapeutic interventions. In this work, we carry out a systematic analysis of a network model of T cell survival in T-LGL leukemia to get a deeper insight into the unknown facets of the disease. We identify the T-LGL status of 54 components of the system, out of which 36 (67%) are corroborated by previous experimental evidence and the rest are novel predictions, one of which we validate by follow-up experiments. By deciphering the structure and dynamics of the underlying network, we identify component perturbations that lead to programmed cell death, thereby suggesting several novel candidate therapeutic targets for future experiments.
PMCID: PMC3213185  PMID: 22102804
25.  Homeodomain Interacting Protein Kinase 2 Activation Compromises Endothelial Cell Response to Laminar Flow: Protective Role of p21waf1,cip1,sdi1 
PLoS ONE  2009;4(8):e6603.
In the cardiovascular system, laminar shear stress (SS) is one of the most important source of endothelial protecting signals. Physical and chemical agents, however, including ionising radiations and anticancer drugs, may injure endothelial cells determining an increase in oxidative stress and genotoxic damage. Whether the SS protective function remains intact in the presence of strong oxidants or DNA damage is currently unclear.
Methods and Results
To investigate this aspect a series of experiments were performed in which HUVEC were exposed to sub-lethal doses of the radio-mimetic compound Bleomycin (Bleo; 10 µg/ml) which generated free radicals (ROS) without significantly compromising cell survival. Remarkably, the application of a SS of 12 dyne/cm2 did not protect endothelial cells but markedly accelerated apoptosis compared to controls kept in static culture and in the presence of Bleo. Experiments with the inducible nitric oxide synthase (iNOS) inhibitor GW274150 significantly reduced the SS-dependent apoptosis indicating that the production of NO was relevant for this effect. At molecular level, the ataxia-telangectasia-mutated (ATM) kinase, the homeodomain-interacting protein kinase-2 (HIPK2) and p53 were found activated along a pro-apoptotic signalling pathway while p21waf1,cip1,sdi1 was prevented from its protective action. RNA interference experiments revealed that HIPK2 and p53 were both important for this process, however, only the forced expression p21waf1,cip1,sdi1 fully restored the SS-dependent pro-survival function.
This study provides the first evidence that, in the presence of genotoxic damage, laminar flow contributes to endothelial toxicity and death and identifies molecular targets potentially relevant in endothelial dysfunction and cardiovascular disease pathogenesis.
PMCID: PMC2719102  PMID: 19668373

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