Jordi Carreras Puigvert

Diclofenac inhibits tumor necrosis factor-α-induced nuclear factor-κB activation causing synergistic hepatocyte apoptosis.

Drug‐induced liver injury (DILI) is an important clinical problem. It involves crosstalk between drug toxicity and the immune system, but the exact mechanism at the cellular hepatocyte level is not well understood. Here we studied the mechanism of crosstalk in hepatocyte apoptosis caused by diclofenac and the proinflammatory cytokine tumor necrosis factor α (TNF‐α). HepG2 cells were treated with diclofenac followed by TNF‐α challenge and subsequent evaluation of necrosis and apoptosis. Diclofenac caused a mild apoptosis of HepG2 cells, which was strongly potentiated by TNF‐α. A focused apoptosis machinery short interference RNA (siRNA) library screen identified that this TNF‐α‐mediated enhancement involved activation of caspase‐3 through a caspase‐8/Bid/APAF1 pathway. Diclofenac itself induced sustained activation of c‐Jun N‐terminal kinase (JNK) and inhibition of JNK decreased both diclofenac and diclofenac/TNF‐α‐induced apoptosis. Live cell imaging of GFPp65/RelA showed that diclofenac dampened the TNF‐α‐mediated nuclear factor kappaB (NF‐κB) translocation oscillation in association with reduced NF‐κB transcriptional activity. This was associated with inhibition by diclofenac of the TNF‐α‐induced phosphorylation of the inhibitor of NF‐κB alpha (IκBα). Finally, inhibition of IκB kinase β (IKKβ) with BMS‐345541 as well as stable lentiviral short hairpin RNA (shRNA)‐based knockdown of p65/RelA sensitized hepatocytes towards diclofenac/TNF‐α‐induced cytotoxicity. Conclusion: Together, our data suggest a model whereby diclofenac‐mediated stress signaling suppresses TNF‐α‐induced survival signaling routes and sensitizes cells to apoptosis. (HEPATOLOGY 2011;)

The cancer stem cell microenvironment and anti-cancer therapy

Purpose: Tumours are composed of a heterogeneous cell population. Cancer stem cells, which make up a minor fraction of a tumour, may be the cells that initiate and sustain tumour growth. Cancer stem cells are believed to share many properties with normal stem cells that render them relatively insensitive to classical radio- and chemotherapy. Conclusions: We discuss what those (cancer) stem cell properties are and how the interactions with the microenvironment – ‘the niche’ – control those aspects of (cancer) stem cell biology. We also describe possible strategies to target cancer stem cells in order to prevent cancers from escaping therapy.

Global phosphoproteome profiling reveals unanticipated networks responsive to cisplatin treatment of embryonic stem cells

ABSTRACT Cellular responses to DNA-damaging agents involve the activation of various DNA damage signaling and transduction pathways. Using quantitative and high-resolution tandem mass spectrometry, we determined global changes in protein level and phosphorylation site profiles following treatment of SILAC (stable isotope labeling by amino acids in cell culture)-labeled murine embryonic stem cells with the anticancer drug cisplatin. Network and pathway analyses indicated that processes related to the DNA damage response and cytoskeleton organization were significantly affected. Although the ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related) consensus sequence (S/T-Q motif) was significantly overrepresented among hyperphosphorylated peptides, about half of the >2-fold-upregulated phosphorylation sites based on the consensus sequence were not direct substrates of ATM and ATR. Eleven protein kinases mainly belonging to the mitogen-activated protein kinase (MAPK) family were identified as being regulated in their kinase domain activation loop. The biological importance of three of these kinases (cyclin-dependent kinase 7 [CDK7], Plk1, and KPCD1) in the protection against cisplatin-induced cytotoxicity was demonstrated by small interfering RNA (siRNA)-mediated knockdown. Our results indicate that the cellular response to cisplatin involves a variety of kinases and phosphatases not only acting in the nucleus but also regulating cytoplasmic targets, resulting in extensive cytoskeletal rearrangements. Integration of transcriptomic and proteomic data revealed a poor correlation between changes in the relative levels of transcripts and their corresponding proteins, but a large overlap in affected pathways at the levels of mRNA, protein, and phosphoprotein. This study provides an integrated view of pathways activated by genotoxic stress and deciphers kinases that play a pivotal role in regulating cellular processes other than the DNA damage response.

Silencing of the microtubule-associated proteins doublecortin-like and doublecortin-like kinase-long induces apoptosis in neuroblastoma cells.

Doublecortin-like kinase-long (DCLK-long) and doublecortin-like (DCL) are two splice variants of DCLK gene. DCL and DCLK-long are microtubule-associated proteins with specific expression in proliferative neural progenitor cells. We have tested the hypothesis that knockdown of DCL/DCLK-long by RNA interference technology will induce cell death in neuroblastoma (NB) cells. First, we analyzed the expression of DCL and DCLK-long in several human neuroblastic tumors, other tumors, and normal tissues, revealing high expression of both DCL and DCLK-long in NB and glioma. Secondly, gene expression profiling revealed numerous differentially expressed genes indicating apoptosis induction after DCL/DCLK-long knockdown in NB cells. Finally, apoptosis was confirmed by time-lapse imaging of phosphatidylserine translocation, caspase-3 activation, live/dead double staining assays, and fluorescence-activated cell sorting. Together, our results suggest that silencing DCL/DCLK-long induces apoptosis in NB cells.

High-throughput live cell imaging of apoptosis.

Apoptosis is important for embryonic development, tissue homeostasis, and removal of cells with (potentially transforming) DNA lesions or other types of injuries. Functional genomics screens performed to unravel apoptotic signaling cascades in the context of toxicant‐induced cell injury commonly use apoptosis as an end‐point. Here, a method to detect the accumulation of apoptotic cells in real time that is well suited for high‐throughput screens is described. The method uses automated microscopy in a 96‐well format setting to visualize binding of fluorescent annexin V to the outer membrane leaflet of apoptotic cells. The automated image acquisition is followed by quantitative analysis using bioinformatics software. A protocol for each of the steps in this kinetic method is described, which includes the caspase‐dependent apoptotic response to toxic compounds in multiple cell types and demonstrates that RNAi‐based gene silencing of candidate apoptotic regulators affects the apoptosis kinetics as expected. This protocol will be useful for functional genomics as well as chemical (drug) screens. Curr. Protoc. Cell Biol. 47:18.10.1‐18.10.13.

Cross-Talk between Integrins and Oncogenes Modulates Chemosensitivity

Chemotherapy often relies on cancer cell death resulting from DNA damage. The p53 tumor suppressor pathway that is an important player in DNA damage response is frequently inactivated in cancer. Genotoxicants also activate DNA damage-independent stress pathways and activity of oncogenic signaling and adhesive interactions with the cancer microenvironment can have a strong impact on chemosensitivity. Here, we have investigated how two different oncogenes modulate the response to genotoxicants in the context of two classes of integrin adhesion receptors. Epithelial cells expressing either β1 or β3 integrins, in which p53 activity is suppressed, undergo G2 arrest but show little apoptosis after treatment with cisplatin or other genotoxicants. The apoptotic response is strongly enhanced by the c-Src[Y530F] oncogene in cells expressing β1 integrins, whereas such sensitization is reduced when these cells are engineered to express β3 integrins instead. The H-Ras[G12V] oncogene fails to sensitize, regardless of the integrin expression profile. The enhanced sensitivity induced by c-Src[Y530F] in the context of β1 integrins does not rely on p53-mediated DNA damage signaling but instead involves increased endoplasmic reticulum stress and caspase-3 activation. Our data implicate that the expression profiles of oncogenes and integrins strongly affect the response to chemotherapeutics and may thus determine the efficacy of chemotherapy.

Systems Biology Approach Identifies the Kinase Csnk1a1 as a Regulator of the DNA Damage Response in Embryonic Stem Cells

Genotoxic stress induces Wnt signaling that attenuates apoptosis in embryonic stem cells. Stalling the Death Warrant The DNA damage response (DDR) involves an intricate network that regulates repair of the damage, cell cycle arrest, and apoptosis. In pluripotent embryonic stem (ES) cells, the DDR also regulates differentiation. With functional genomics, transcriptomics, and phosphoproteomics, Carreras Puigvert et al. investigated the DDR in ES cells treated with cisplatin, a DNA interstrand cross-linking drug. They found that the tumor suppressor and transcriptional regulator p53 promoted apoptosis but that Wnt promoted a competing, p53-independent survival signal. DNA damage suppressed the activity of casein kinase 1α (Csnk1a1 or CK1α), a kinase that phosphorylates β-catenin, promoting its degradation and thereby inhibiting Wnt signaling. These findings show that pro- and antiapoptotic signals from independent pathways compete to regulate cell survival in response to DNA damage. In pluripotent stem cells, DNA damage triggers loss of pluripotency and apoptosis as a safeguard to exclude damaged DNA from the lineage. An intricate DNA damage response (DDR) signaling network ensures that the response is proportional to the severity of the damage. We combined an RNA interference screen targeting all kinases, phosphatases, and transcription factors with global transcriptomics and phosphoproteomics to map the DDR in mouse embryonic stem cells treated with the DNA cross-linker cisplatin. Networks derived from canonical pathways shared in all three data sets were implicated in DNA damage repair, cell cycle and survival, and differentiation. Experimental probing of these networks identified a mode of DNA damage–induced Wnt signaling that limited apoptosis. Silencing or deleting the p53 gene demonstrated that genotoxic stress elicited Wnt signaling in a p53-independent manner. Instead, this response occurred through reduced abundance of Csnk1a1 (CK1α), a kinase that inhibits β-catenin. Together, our findings reveal a balance between p53-mediated elimination of stem cells (through loss of pluripotency and apoptosis) and Wnt signaling that attenuates this response to tune the outcome of the DDR.

Combining Doublecortin-Like Kinase Silencing and Vinca Alkaloids Results in a Synergistic Apoptotic Effect in Neuroblastoma Cells

Microtubule-destabilizing agents, such as vinca alkaloids (VAs), are part of the treatment currently applied in patients with high-risk neuroblastoma (NB). However, the development of drug resistance and toxicity make NB difficult to treat with these drugs. In this study we explore the combination of VAs (vincristine or vinblastine) with knockdown of the microtubule-associated proteins encoded by the doublecortin-like kinase (DCLK) gene by using short interference RNA (siRNA). We examined the effect of VAs and DCLK knockdown on the microtubule network by immunohistochemistry. We performed dose-response studies on cell viability and proliferation. By combining VA with DCLK knockdown we observed a strong reduction in the EC50 to induce cell death: up to 7.3-fold reduction of vincristine and 21.1-fold reduction of vinblastine. Using time-lapse imaging of phosphatidylserine translocation and a terminal deoxynucleotidyl transferase dUTP nick-end labeling-based assay, we found a significant increase of apoptosis by the combined treatment. Induction of caspase-3 activity, as detected via cleavage of N-acetyl-Asp-Glu-Val-Asp-7-amido-4-methylcoumarin, showed a 3.3- to 12.0-fold increase in the combined treatment. We detected significant increases in caspase-8 activity as well. Moreover, the multidrug dose effect calculated by using the median effect method showed a strong synergistic inhibition of proliferation and induction of apoptosis at most of the combined concentrations of siRNAs and VAs. Together, our data demonstrate that the silencing of DCLK sensitizes NB cells to VAs, resulting in a synergetic apoptotic effect.

The E3 Ubiquitin Ligase ARIH1 Protects against Genotoxic Stress by Initiating a 4EHP-Mediated mRNA Translation Arrest

ABSTRACT DNA damage response signaling is crucial for genome maintenance in all organisms and is corrupted in cancer. In an RNA interference (RNAi) screen for (de)ubiquitinases and sumoylases modulating the apoptotic response of embryonic stem (ES) cells to DNA damage, we identified the E3 ubiquitin ligase/ISGylase, ariadne homologue 1 (ARIH1). Silencing ARIH1 sensitized ES and cancer cells to genotoxic compounds and ionizing radiation, irrespective of their p53 or caspase-3 status. Expression of wild-type but not ubiquitinase-defective ARIH1 constructs prevented sensitization caused by ARIH1 knockdown. ARIH1 protein abundance increased after DNA damage through attenuation of proteasomal degradation that required ATM signaling. Accumulated ARIH1 associated with 4EHP, and in turn, this competitive inhibitor of the eukaryotic translation initiation factor 4E (eIF4E) underwent increased nondegradative ubiquitination upon DNA damage. Genotoxic stress led to an enrichment of ARIH1 in perinuclear, ribosome-containing regions and triggered 4EHP association with the mRNA 5′ cap as well as mRNA translation arrest in an ARIH1-dependent manner. Finally, restoration of DNA damage-induced translation arrest in ARIH1-depleted cells by means of an eIF2 inhibitor was sufficient to reinstate resistance to genotoxic stress. These findings identify ARIH1 as a potent mediator of DNA damage-induced translation arrest that protects stem and cancer cells against genotoxic stress.