Mojgan Djavaheri-Mergny

Several polycations possessing substantial buffering capacity below physiological pH, such as lipopolyamines and polyamidoamine polymers, are efficient transfection agents per se--i.e., without the addition of cell targeting or membrane-disruption agents. This observation led us to test the cationic polymer polyethylenimine (PEI) for its gene-delivery potential. Indeed, every third atom of PEI is a protonable amino nitrogen atom, which makes the polymeric network an effective "proton sponge" at virtually any pH. Luciferase reporter gene transfer with this polycation into a variety of cell lines and primary cells gave results comparable to, or even better than, lipopolyamines. Cytotoxicity was low and seen only at concentrations well above those required for optimal transfection. Delivery of oligonucleotides into embryonic neurons was followed by using a fluorescent probe. Virtually all neurons showed nuclear labeling, with no toxic effects. The optimal PEI cation/anion balance for in vitro transfection is only slightly on the cationic side, which is advantageous for in vivo delivery. Indeed, intracerebral luciferase gene transfer into newborn mice gave results comparable (for a given amount of DNA) to the in vitro transfection of primary rat brain endothelial cells or chicken embryonic neurons. Together, these properties make PEI a promising vector for gene therapy and an outstanding core for the design of more sophisticated devices. Our hypothesis is that its efficiency relies on extensive lysosome buffering that protects DNA from nuclease degradation, and consequent lysosomal swelling and rupture that provide an escape mechanism for the PEI/DNA particles.

Multiple cellular stressors, including activation of the tumour suppressor p53, can stimulate autophagy. Here we show that deletion, depletion or inhibition of p53 can induce autophagy in human, mouse and nematode cells subjected to knockout, knockdown or pharmacological inhibition of p53. Enhanced autophagy improved the survival of p53-deficient cancer cells under conditions of hypoxia and nutrient depletion, allowing them to maintain high ATP levels. Inhibition of p53 led to autophagy in enucleated cells, and cytoplasmic, not nuclear, p53 was able to repress the enhanced autophagy of p53(-/-) cells. Many different inducers of autophagy (for example, starvation, rapamycin and toxins affecting the endoplasmic reticulum) stimulated proteasome-mediated degradation of p53 through a pathway relying on the E3 ubiquitin ligase HDM2. Inhibition of p53 degradation prevented the activation of autophagy in several cell lines, in response to several distinct stimuli. These results provide evidence of a key signalling pathway that links autophagy to the cancer-associated dysregulation of p53.

Activation of NF-κB and autophagy are two processes involved in the regulation of cell death, but the possible cross-talk between these two signaling pathways is largely unknown. Here, we show that NF-κB activation mediates repression of autophagy in tumor necrosis factor-α (TNFα)-treated Ewing sarcoma cells. This repression is associated with an NF-κB-dependent activation of the autophagy inhibitor mTOR. In contrast, in cells lacking NF-κB activation, TNFα treatment up-regulates the expression of the autophagy-promoting protein Beclin 1 and subsequently induces the accumulation of autophagic vacuoles. Both of these responses are dependent on reactive oxygen species (ROS) production and can be mimicked in NF-κB-competent cells by the addition of H₂O₂. Small interfering RNA-mediated knockdown of <i>beclin 1</i> and <i>atg7</i> expression, two autophagy-related genes, reduced TNFα- and reactive oxygen species-induced apoptosis in cells lacking NF-κB activation and in NF-κB-competent cells, respectively. These findings demonstrate that autophagy may amplify apoptosis when associated with a death signaling pathway. They are also evidence that inhibition of autophagy is a novel mechanism of the antiapoptotic function of NF-κB activation. We suggest that stimulation of autophagy may be a potential way bypassing the resistance of cancer cells to anti-cancer agents that activate NF-κB.

Beclin 1 has a key role in the initiation of autophagy, a process of self-cannibalism in which cytoplasmic constituents are sequestered and targeted for lysosomal degradation. In a recent issue of Cell Death & Disease, Wirawan et al. report the significant finding that caspases can cleave Beclin 1, thereby destroying its pro-autophagic activity. Moreover, the C-terminal fragment of Beclin 1 that results from this cleavage acquires a new function and can amplify mitochondrion-mediated apoptosis. Of note, the BH3 domain of Beclin 1 remains within the N-terminal fragment, which has no detectable pro-apoptotic activity. These findings provide important insights into the molecular cross talk between autophagy and apoptosis.

Proton pump inhibitors (PPI) target tumour acidic pH and have an antineoplastic effect in melanoma. The PPI esomeprazole (ESOM) kills melanoma cells through a caspase-dependent pathway involving cytosolic acidification and alkalinization of tumour pH. In this paper, we further investigated the mechanisms of ESOM-induced cell death in melanoma. ESOM rapidly induced accumulation of reactive oxygen species (ROS) through mitochondrial dysfunctions and involvement of NADPH oxidase. The ROS scavenger N-acetyl-L-cysteine (NAC) and inhibition of NADPH oxidase significantly reduced ESOM-induced cell death, consistent with inhibition of cytosolic acidification. Autophagy, a cellular catabolic pathway leading to lysosomal degradation and recycling of proteins and organelles, represents a defence mechanism in cancer cells under metabolic stress. ESOM induced the early accumulation of autophagosomes, at the same time reducing the autophagic flux, as observed by WB analysis of LC3-II accumulation and by fluorescence microscopy. Moreover, ESOM treatment decreased mammalian target of rapamycin signalling, as reduced phosphorylation of p70-S6K and 4-EBP1 was observed. Inhibition of autophagy by knockdown of Atg5 and Beclin-1 expression significantly increased ESOM cytotoxicity, suggesting a protective role for autophagy in ESOM-treated cells. The data presented suggest that autophagy represents an adaptive survival mechanism to overcome drug-induced cellular stress and cytotoxicity, including alteration of pH homeostasis mediated by proton pump inhibition.

Cyclic hypoxia and alterations in oncogenic signaling contribute to switch cancer cell metabolism from oxidative phosphorylation to aerobic glycolysis. A major consequence of up-regulated glycolysis is the increased production of metabolic acids responsible for the presence of acidic areas within solid tumors. Tumor acidosis is an important determinant of tumor progression and tumor pH regulation is being investigated as a therapeutic target. Autophagy is a cellular catabolic pathway leading to lysosomal degradation and recycling of proteins and organelles, currently considered an important survival mechanism in cancer cells under metabolic stress or subjected to chemotherapy. We investigated the response of human melanoma cells cultured in acidic conditions in terms of survival and autophagy regulation. Melanoma cells exposed to acidic culture conditions (7.0 < pH < 6.2) promptly accumulated LC3+ autophagic vesicles. Immunoblot analysis showed a consistent increase of LC3-II in acidic culture conditions as compared with cells at normal pH. Inhibition of lysosomal acidification by bafilomycin A1 further increased LC3-II accumulation, suggesting an active autophagic flux in cells under acidic stress. Acute exposure to acidic stress induced rapid inhibition of the mammalian target of rapamycin signaling pathway detected by decreased phosphorylation of p70S6K and increased phosphorylation of AMP-activated protein kinase, associated with decreased ATP content and reduced glucose and leucine uptake. Inhibition of autophagy by knockdown of the autophagic gene ATG5 consistently reduced melanoma cell survival in low pH conditions. These observations indicate that induction of autophagy may represent an adaptation mechanism for cancer cells exposed to an acidic environment. Our data strengthen the validity of therapeutic strategies targeting tumor pH regulation and autophagy in progressive malignancies.

p53 and JNK are two apoptosis-regulatory factors frequently deregulated in cancer cells and also involved in the modulation of autophagy. We have recently investigated the links between these two signalling pathways in terms of the regulation of autophagy. We showed that 2-methoxyestradiol (2-ME), an antitumoral compound, enhances autophagy and apoptosis in Ewing sarcoma cells through the activation of both p53 and JNK pathways. In this context, p53 regulates, at least partially, JNK activation which in turn modulates autophagy through two distinct mechanisms: on the one hand it promotes Bcl-2 phosphorylation resulting in the dissociation of the Beclin 1-Bcl-2 complex and on the other hand it leads to the upregulation of DRAM (Damage-Regulated Autophagy Modulator), a p53 target gene. The critical role of DRAM in 2-ME-mediated autophagy and apoptosis is underlined by the fact that its silencing efficiently prevents the induction of both processes. These findings not only report the interplay between JNK and p53 in the regulation of autophagy but also uncover the role of JNK activation in the regulation of DRAM, a pro-autophagic and proapoptotic protein.

The remarkable metabolic differences between cancer cells and normal cells result in the potential for targeted cancer therapy. The upregulation of glutaminolysis provides energetic advantages to cancer cells. The recently described link between glutaminolysis and autophagy, mediated by MTORC1, may constitute an attractive target for therapeutic strategies. A combination of therapies targeting simultane-ously cell signaling, cancer metabolism, and autophagy can solve therapy resistance and tumor relapse problems, commonly observed in patients treated with most of the current targeted therapies. In this review we summarize the mechanistic link between glutaminolysis and autophagy, and discuss the impacts of these processes on cancer progression and the potential for therapeutic intervention.

Repression of activation of c‐Jun N‐terminal kinase (JNK) participates in the anti‐apoptotic effect of nuclear factor‐κB (NF‐κB) in TNFα‐treated Ewing sarcoma cells. As oxidative stress is one of the most prominent activators of JNK, we investigated the relationship between TNFα‐induced NF‐κB activation and the control of oxidative stress. Inhibition of NF‐κB activation resulted in an increase in TNFα‐induced ROS production, lipid peroxidation and protein oxidation. Those ROS and lipid peroxides were both involved in TNFα‐induced apoptosis, whereas only ROS elevation triggered sustained JNK activation. TNFα increased the level of two antioxidant enzymes, thioredoxin and manganese superoxide dismutase by an NF‐κB‐dependent mechanism. Inhibition of expression or activity of these enzymes sensitized cells to TNFα‐induced apoptosis, indicating their functional role in protection from cell death. Thus, agents that inhibit activities of these enzymes may prove helpful in the treatment of Ewing tumors.

Abstract Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid metabolite involved in cancer development through stimulation of cell survival, proliferation, migration, and angiogenesis. Irreversible degradation of S1P is catalyzed by S1P lyase (SPL). The human SGPL1 gene that encodes SPL maps to a region often mutated in cancers. To investigate the effect of SPL deficiency on cell survival and transformation, the susceptibility to anticancer drugs of fibroblasts generated from SPL-deficient mouse embryos (Sgpl1−/−) was compared with that of cells from heterozygous (Sgpl1+/−) or wild-type (Sgpl1+/+) embryos. First, loss of SPL caused resistance to the toxic effects of etoposide and doxorubicin. Interestingly, heterozygosity for the Sgpl1 gene resulted in partial resistance to apoptosis. Secondly, doxorubicin-induced apoptotic signaling was strongly inhibited in Sgpl1−/− cells (phosphatidylserine externalization, caspase activation, and cytochrome c release). This was accompanied by a strong increase in Bcl-2 and Bcl-xL protein content. Whereas correction of SPL deficiency in Sgpl1−/− cells led to downregulation of antiapoptotic proteins, Bcl-2 and Bcl-xL small interfering RNA–mediated knockdown in SPL-deficient cells resulted in increased sensitivity to doxorubicin, suggesting that Bcl-2 upregulation mediates SPL protective effects. Moreover, SPL deficiency led to increased cell proliferation, anchorage-independent cell growth, and formation of tumors in nude mice. Finally, transcriptomic studies showed that SPL expression is downregulated in human melanoma cell lines. Thus, by affecting S1P metabolism and the expression of Bcl-2 members, the loss of SPL enhances cell resistance to anticancer regimens and results in an increased ability of cells to acquire a transformed phenotype and become malignant. [Cancer Res 2009;69(24):9346–53]

Fabry disease is a lysosomal storage disorder (LSD) caused by a deficiency in α-galactosidase A. The disease is characterized by severe major organ involvement, but the pathologic mechanisms responsible have not been elucidated. Disruptions of autophagic processes have been reported for other LSDs, but have not yet been investigated in Fabry disease. Renal biopsies were obtained from 5 adult male Fabry disease patients before and after 3 years of enzyme replacement therapy (ERT) with agalsidase alfa. Vacuole accumulation was seen in renal biopsies from all patients compared with control biopsies. Decreases in the number of vacuoles were seen after 3 years of ERT primarily in renal endothelial cells and mesangial cells. Measurement of the levels of LC3, a specific autophagy marker, in cultured cells from Fabry patients revealed increased basal levels compared to cells from non-Fabry subjects and a larger increase in response to starvation than seen in non-Fabry cells. Starvation in the presence of protease inhibitors did not result in a significant increase in LC3 in Fabry cells, whereas a further increase in LC3 was observed in non-Fabry cells, an observation that is consistent with impaired autophagic flux in Fabry disease. Overexpression of LC3 mRNA in Fabry fibroblasts compared to control cells is consistent with an upregulation of autophagy. Furthermore, LC3 and p62/SQSTM1 (that binds to LC3) staining in renal tissues and in cultured fibroblasts from Fabry patients supports impairment of autophagic flux. These findings suggest that Fabry disease is linked to a deregulation of autophagy.

Imatinib, the anti-Abl tyrosine kinase inhibitor used as first-line therapy in chronic myeloid leukemia (CML), eliminates CML cells mainly by apoptosis and induces autophagy. Analysis of imatinib-treated K562 cells reveals a cell population with cell cycle arrest, p27 increase and senescence-associated beta galactosidase (SA-β-Gal) staining. Preventing apoptosis by caspase inhibition decreases annexin V-positive cells, caspase-3 cleavage and increases the SA-β-Gal-positive cell population. In addition, a concomitant increase of the cell cycle inhibitors p21 and p27 is detected emphasizing the senescent phenotype. Inhibition of apoptosis by targeting Bim expression or overexpression of Bcl2 potentiates senescence. The inhibition of autophagy by silencing the expression of the proteins ATG7 or Beclin-1 prevents the increase of SA-β-Gal staining in response to imatinib plus Z-Vad. In contrast, in apoptotic-deficient cells (Bim expression or overexpression of Bcl2), the inhibition of autophagy did not significantly modify the SA-β-Gal-positive cell population. Surprisingly, targeting autophagy by inhibiting ATG5 is accompanied by a strong SA-β-Gal staining, suggesting a specific inhibitory role on senescence. These results demonstrate that in addition to apoptosis and autophagy, imatinib induced senescence in K562 CML cells. Moreover, apoptosis is limiting the senescent response to imatinib, whereas autophagy seems to have an opposite role.

G-quadruplex ligands exert their antiproliferative effects through telomere-dependent and telomere-independent mechanisms, but the inter-relationships among autophagy, cell growth arrest and cell death induced by these ligands remain largely unexplored. Here, we demonstrate that the G-quadruplex ligand 20A causes growth arrest of cancer cells in culture and in a HeLa cell xenografted mouse model. This response is associated with the induction of senescence and apoptosis. Transcriptomic analysis of 20A treated cells reveals a significant functional enrichment of biological pathways related to growth arrest, DNA damage response and the lysosomal pathway. 20A elicits global DNA damage but not telomeric damage and activates the ATM and autophagy pathways. Loss of ATM following 20A treatment inhibits both autophagy and senescence and sensitizes cells to death. Moreover, disruption of autophagy by deletion of two essential autophagy genes ATG5 and ATG7 leads to failure of CHK1 activation by 20A and subsequently increased cell death. Our results, therefore, identify the activation of ATM by 20A as a critical player in the balance between senescence and apoptosis and autophagy as one of the key mediators of such regulation. Thus, targeting the ATM/autophagy pathway might be a promising strategy to achieve the maximal anticancer effect of this compound.

Guanine-quadruplex structures (G4) are unusual nucleic acid conformations formed by guanine-rich DNA and RNA sequences and known to control gene expression mechanisms, from transcription to protein synthesis. So far, a number of molecules that recognize G4 have been developed for potential therapeutic applications in human pathologies, including cancer and infectious diseases. These molecules are called G4 ligands. When the biological effects of G4 ligands are studied, the analysis is often limited to nucleic acid targets. However, recent evidence indicates that G4 ligands may target other cellular components and compartments such as lysosomes and mitochondria. Here, we summarize our current knowledge of the regulation of lysosome by G4 ligands, underlying their potential functional impact on lysosome biology and autophagic flux, as well as on the transcriptional regulation of lysosomal genes. We outline the consequences of these effects on cell fate decisions and we systematically analyzed G4-prone sequences within the promoter of 435 lysosome-related genes. Finally, we propose some hypotheses about the mechanisms involved in the regulation of lysosomes by G4 ligands.

Most studies on the structure of DNA in telomeres have been dedicated to the double-stranded region or the guanosine-rich strand and consequently little is known about the factors that may bind to the telomere cytosine-rich (C-rich) strand. This led us to investigate whether proteins exist that can recognise C-rich sequences. We have isolated several nuclear factors from human cell extracts that specifically bind the C-rich strand of vertebrate telomeres [namely a d(CCCTAA)(n)repeat] with high affinity and bind double-stranded telomeric DNA with a 100xreduced affinity. A biochemical assay allowed us to characterise four proteins of apparent molecular weights 66-64, 45 and 35 kDa, respectively. To identify these polypeptides we screened alambdagt11-based cDNA expression library, obtained from human HeLa cells using a radiolabelled telomeric oligonucleotide as a probe. Two clones were purified and sequenced: the first corresponded to the hnRNP K protein and the second to the ASF/SF2 splicing factor. Confirmation of the screening results was obtained with recombinant proteins, both of which bind to the human telomeric C-rich strand in vitro.

UV‐A irradiation induces a time‐dependent activation of AP‐1 in NCTC 2544 human keratinocytes. 4 h after irradiation, a 2–3‐fold increase in AP‐1 activity is observed in human keratinocytes and fibroblasts. Activation is still detectable 24 h later. The UV‐A induced AP‐1 binding complex is shown to contain c‐Fos and c‐Jun proteins. Lipophilic vitamin E impedes UV‐A induced lipid peroxidation but does not prevent AP‐1 activation which is inhibited by N ‐acetylcysteine, a hydrophilic antioxidant. This finding suggests that UV‐A‐dependent AP‐1 activation is sensitive to the cellular redox state but is not related to membrane lipid peroxidation.

1-(2-Chlorophenyl-N-methylpropyl)-3-isoquinolinecarboxamide (PK11195) is a prototypic ligand of the peripheral benzodiazepine receptor (PBR), a mitochondrial outer membrane protein. PK11195 can be used to chemosensitize tumor cells to a variety of chemotherapeutic agents, both in vitro and in vivo. PK11195 has been suggested to exert this effect via inhibition of the multiple drug resistance (MDR) pump and by direct mitochondrial effects which could be mediated by the PBR. Here, we established a model system in which PK11195 and another PBR ligand, 7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin-2-one (Ro5-4864), sensitize to nutrient depletion-induced cell death. In this MDR-independent model, PK11195 and Ro5-4864 are fully active even when the PBR is knocked down by small interfering RNA. Cells that lack PBR possess low-affinity binding sites for PK11195 and Ro5-4864. The starvation-sensitizing effects of PK11195 are not due to a modulation of the adaptive response of starved cells, namely autophagy and NF-kappaB activation. Rather, it appears that the combination of PK11195 with autophagy or NF-kappaB inhibitors has a potent synergistic death-inducing effect. Starved cells treated with PK11195 exhibit characteristics of apoptosis, including loss of the mitochondrial transmembrane potential, mitochondrial cytochrome c release, caspase activation and chromatin condensation. Accordingly, stabilization of mitochondria by overexpression of Bcl-2 or expression of the viral mitochondrial inhibitor (vMIA) from cytomegalovirus inhibits cell death induced by PK11195 plus starvation. Thus, PK11195 potently sensitizes to apoptosis via a pathway that involves mitochondria, yet does not involve the PBR.

Abstract Ewing sarcoma and osteosarcoma are two aggressive cancers that affect bones and soft tissues in children and adolescents. Despite multimodal therapy, patients with metastatic sarcoma have a poor prognosis, emphasizing a need for more effective treatment. We have shown previously that 2-methoxyestradiol (2-ME), an antitumoral compound, induces apoptosis in Ewing sarcoma cells through c-Jun NH2-terminal kinase (JNK) activation. In the present study, we provide evidence that 2-ME elicits macroautophagy, a process that participates in apoptotic responses, in a JNK-dependent manner, in Ewing sarcoma and osteosarcoma cells. We also found that the enhanced activation of JNK by 2-ME is partially regulated by p53, highlighting the relationship of JNK and autophagy to p53 signaling pathway. Furthermore, we showed that 2-ME up-regulates damage-regulated autophagy modulator (DRAM), a p53 target gene, in Ewing sarcoma cells through a mechanism that involves JNK activation. The silencing of DRAM expression reduced both apoptosis and autophagy triggered by 2-ME in Ewing sarcoma and osteosarcoma cells. Our results therefore identify JNK as a novel mediator of DRAM regulation. These findings suggest that 2-ME or other anticancer therapies that increase DRAM expression or function could be used to effectively treat sarcoma patients. [Cancer Res 2009;69(17):6924–31]

The p62/SQSTM1 adapter protein has an important role in the regulation of several key signaling pathways and helps transport ubiquitinated proteins to the autophagosomes and proteasome for degradation. Here, we investigate the regulation and roles of p62/SQSTM1 during acute myeloid leukemia (AML) cell maturation into granulocytes. Levels of p62/SQSTM1 mRNA and protein were both significantly increased during all-trans retinoic acid (ATRA)-induced differentiation of AML cells through a mechanism that depends on NF-κB activation. We show that this response constitutes a survival mechanism that prolongs the life span of mature AML cells and mitigates the effects of accumulation of aggregated proteins that occurs during granulocytic differentiation. Interestingly, ATRA-induced p62/SQSTM1 upregulation was impaired in maturation-resistant AML cells but was reactivated when differentiation was restored in these cells. Primary blast cells of AML patients and CD34+ progenitors exhibited significantly lower p62/SQSTM1 mRNA levels than did mature granulocytes from healthy donors. Our results demonstrate that p62/SQSTM1 expression is upregulated in mature compared with immature myeloid cells and reveal a pro-survival function of the NF-κB/SQSTM1 signaling axis during granulocytic differentiation of AML cells. These findings may help our understanding of neutrophil/granulocyte development and will guide the development of novel therapeutic strategies for refractory and relapsed AML patients with previous exposure to ATRA.

Haematopoiesis is a tightly orchestrated process where a pool of hematopoietic stem and progenitor cells (HSPCs) with high self-renewal potential can give rise to both lymphoid and myeloid lineages. The HSPCs pool is reduced with ageing resulting in few HSPC clones maintaining haematopoiesis thereby reducing blood cell diversity, a phenomenon called clonal haematopoiesis. Clonal expansion of HSPCs carrying specific genetic mutations leads to increased risk for haematological malignancies. Therefore, it comes as no surprise that hematopoietic tumours develop in higher frequency in elderly people. Unfortunately, elderly patients with leukaemia or lymphoma still have an unsatisfactory prognosis compared to younger ones highlighting the need to develop more efficient therapies for this group of patients. Growing evidence indicates that macroautophagy (hereafter referred to as autophagy) is essential for health and longevity. This review is focusing on the role of autophagy in normal haematopoiesis as well as in leukaemia and lymphoma development. Attenuated autophagy may support early hematopoietic neoplasia whereas activation of autophagy in later stages of tumour development and in response to a variety of therapies rather triggers a pro-tumoral response. Novel insights into the role of autophagy in haematopoiesis will be discussed in light of designing new autophagy modulating therapies in hematopoietic cancers.

Autophagy is one of the main cellular catabolic pathways controlling a variety of physiological processes, including those involved in self-renewal, differentiation and death. While acute promyelocytic leukemia (APL) cells manifest low levels of expression of autophagy genes associated with reduced autophagy activity, the introduction of all-trans retinoid acid (ATRA)-a differentiating agent currently used in clinical settings-restores autophagy in these cells. ATRA-induced autophagy is involved in granulocytes differentiation through a mechanism that involves among others the degradation of the PML-RARα oncoprotein. Arsenic trioxide (ATO) is another anti-cancer agent that promotes autophagy-dependent clearance of promyelocytic leukemia retinoic acid receptor alpha gene (PML-RARα) in APL cells. Hence, enhancing autophagy may have therapeutic benefits in maturation-resistant APL cells. However, the role of autophagy in response to APL therapy is not so simple, because some autophagy proteins have been shown to play a pro-survival role upon ATRA and ATO treatment, and both agents can activate ETosis, a type of cell death mediated by the release of neutrophil extracellular traps (ETs). This review highlights recent findings on the impact of autophagy on the mechanisms of action of ATRA and ATO in APL cells. We also discuss the potential role of autophagy in the development of resistance to treatment, and of differentiation syndrome in APL.

Acyl-coenzyme A (CoA)–binding protein (ACBP), also known as diazepam-binding inhibitor (DBI), is an extracellular feedback regulator of autophagy. Here, we report that injection of a monoclonal antibody neutralizing ACBP/DBI (α-DBI) protects the murine liver against ischemia/reperfusion damage, intoxication by acetaminophen and concanavalin A, and nonalcoholic steatohepatitis caused by methionine/choline-deficient diet as well as against liver fibrosis induced by bile duct ligation or carbon tetrachloride. α-DBI downregulated proinflammatory and profibrotic genes and upregulated antioxidant defenses and fatty acid oxidation in the liver. The hepatoprotective effects of α-DBI were mimicked by the induction of ACBP/DBI-specific autoantibodies, an inducible Acbp/Dbi knockout or a constitutive Gabrg2 F77I mutation that abolishes ACBP/DBI binding to the GABA A receptor. Liver-protective α-DBI effects were lost when autophagy was pharmacologically blocked or genetically inhibited by knockout of Atg4b . Of note, α-DBI also reduced myocardium infarction and lung fibrosis, supporting the contention that it mediates broad organ-protective effects against multiple insults.

Acute promyelocytic leukemia (APL) results from a blockade of granulocyte differentiation at the promyelocytic stage. All-trans retinoic acid (ATRA) induces clinical remission in APL patients by enhancing the rapid differentiation of APL cells and the clearance of PML-RARα, APL's hallmark oncoprotein. In the present study, we demonstrated that both autophagy and Beclin 1, an autophagic protein, are upregulated during the course of ATRA-induced neutrophil/granulocyte differentiation of an APL-derived cell line named NB4 cells. This induction of autophagy is associated with downregulation of Bcl-2 and inhibition of mTOR activity. Small interfering RNA-mediated knockdown of BECN1 expression enhances apoptosis triggered by ATRA in NB4 cells but does not affect the differentiation process. These results provide evidence that the upregulation of Beclin 1 by ATRA constitutes an anti-apoptotic signal for maintaining the viability of mature APL cells, but has no crucial effect on the granulocytic differentiation. This finding may help to elucidate the mechanisms involved in ATRA resistance of APL patients, and in the ATRA syndrome caused by an accumulation of mature APL cells.

A series of novel 2,4,6-triarylpyridines have been synthesized and their interactions with intramolecular G-quadruplexes have been measured by Förster Resonance Energy Transfer (FRET) melting and Fluorescent Intercalator Displacement (FID) assays. A few of these compounds exhibit stabilization of G4-DNA that is comparable to other benchmark G4-DNA ligands with fair to excellent G4-DNA vs. duplex selectivity and significant cytotoxicity towards HeLa cells. The nature of the 4-aryl substituents along with side chain length governs the G4-DNA stabilization ability of the compounds. In addition, we demonstrate that there is a strong correlation between the ability of the compounds to stabilize the same G4-DNA sequence in K+ and Na+ conditions and a strong correlation between the ability of the compounds to stabilize different G4-DNA sequences in K+ or Na+ buffer.

Interferons (IFNs α, β and γ) and all trans retinoic acid (RA) have the ability to activate genes with GAS sites. We have found that the promoter of CD26/dipeptidylpeptidase IV (DPPIV) contains a consensus GAS site TTCnnnGAA located at bp-35 to -27, and computer analysis confirmed this sequence to be a putative Stat binding site. Consistent with this finding, we show that IFNs and RA rapidly enhanced CD26 gene and protein expression in chronic B lymphocytic leukemia (B-CLL) cells. Immunoblot analyses revealed that unstimulated B-CLL cells expressed detectable levels of serine/tyrosine-phosphorylated Stat1α, and RA and IFN-γ treatment led to increased levels of tyrosine phosphorylation of Stat1α and its nuclear accumulation. As shown by electrophoretic mobility shift assay, RA and IFN-γ increased the binding of a nuclear protein to the GAS-CD26 element. Shift-Western blotting identified Stat1α as the GAS-CD26 binding factor. Augmented levels of CD26 protein in malignant B cells cultured with IFNs or RA coincided with the enhancement of DPPIV activity. Taken together, our results are in favor of the IFN-/RA-mediated upregulation of CD26/DPPIV in B-CLL through the signaling pathway involving Stat1α and the GAS response element of CD26 promoter.

gamma-glutamyltranspeptidase (GGT) is a key enzyme implicated in the homeostasis of intracellular reduced glutathione (GSH) and hence in the regulation of the cellular redox state. Besides, the extracellular cleavage of GSH by GGT leads to reactive oxygen species (ROS) production, depending on the generation and enhanced reactivity of cysteinylglycine (CysGly). Using a model cell line, the V79 GGT, which highly expresses a human GGT transgene, we examined whether the GGT induced oxidant stress could modulate intracellular transcription factors. For the first time, we show that GGT-dependent ROS production induces the NF-kB-binding and transactivation activities. This induction mimicked the one observed by H(2)O(2) and was inhibited by catalase, suggesting the involvement of H(2)O(2) in the NF-kB activation.

The effect of cupric ion- or endothelial cell-oxidized low-density lipoproteins (LDL) on transcription factor AP1 activation was investigated by electrophoretic mobility shift assay. Both oxidized LDL induced AP1 activation in fibroblasts, endothelial and smooth muscle cells. This phenomenon was also observed in the presence of cycloheximide. alpha-Tocopherol, a lipophilic free radical scavenger, and N-acetylcysteine, an hydrophilic antioxidant, partially inhibited the stimulatory effect of Cu2+-oxidized LDL. LDL modified by the mixture of the oxygen radicals OH. and O2.-, which generated lipid peroxidation products, also initiated AP1 activation, whereas LDL modified by OH. alone, which did not lead to marked LDL lipid peroxidation, was ineffective. Thus, lipid peroxidation products seem at least partially involved in the activation mechanism. Since AP1 activity is essential for the regulation of genes involved in cell growth and differentiation, our study suggests that the oxidative stress induced by oxidized LDL might be related to the fibroproliferative response observed in the atherosclerotic plaque.

Stem cells isolated from the apical papilla of wisdom teeth (SCAPs) are an attractive model for tissue repair due to their availability, high proliferation rate and potential to differentiate in vitro towards mesodermal and neurogenic lineages. Adult stem cells, such as SCAPs, develop in stem cell niches in which the oxygen concentration [O2] is low (3–8% compared with 21% of ambient air). In this work, we evaluate the impact of low [O2] on the physiology of SCAPs isolated and processed in parallel at 21% or 3% O2 without any hyperoxic shock in ambient air during the experiment performed at 3% O2. We demonstrate that SCAPs display a higher proliferation capacity at 3% O2 than in ambient air with elevated expression levels of two cell surface antigens: the alpha-6 integrin subunit (CD49f) and the embryonic stem cell marker (SSEA4). We show that the mesodermal differentiation potential of SCAPs is conserved at early passage in both [O2], but is partly lost at late passage and low [O2], conditions in which SCAPs proliferate efficiently without any sign of apoptosis. Unexpectedly, we show that autophagic flux is active in SCAPs irrespective of [O2] and that this process remains high in cells even after prolonged exposure to 3% O2.

Abstract Acyl-CoA binding protein (ACBP) encoded by diazepam binding inhibitor (DBI) is an extracellular inhibitor of autophagy acting on the gamma-aminobutyric acid A receptor (GABA A R) γ2 subunit (GABA A Rγ2). Here, we show that lipoanabolic diets cause an upregulation of GABA A Rγ2 protein in liver hepatocytes but not in other major organs. ACBP/DBI inhibition by systemically injected antibodies has been demonstrated to mediate anorexigenic and organ-protective, autophagy-dependent effects. Here, we set out to develop a new strategy for developing ACBP/DBI antagonists. For this, we built a molecular model of the interaction of ACBP/DBI with peptides derived from GABA A Rγ2. We then validated the interaction between recombinant and native ACBP/DBI protein and a GABA A Rγ2-derived eicosapeptide (but not its F77I mutant) by pull down experiments or surface plasmon resonance. The GABA A Rγ2-derived eicosapeptide inhibited the metabolic activation of hepatocytes by recombinant ACBP/DBI protein in vitro. Moreover, the GABA A Rγ2-derived eicosapeptide (but not its F77I-mutated control) blocked appetite stimulation by recombinant ACBP/DBI in vivo, induced autophagy in the liver, and protected mice against the hepatotoxin concanavalin A. We conclude that peptidomimetics disrupting the interaction between ACBP/DBI and GABA A Rγ2 might be used as ACBP/DBI antagonists. This strategy might lead to the future development of clinically relevant small molecules of the ACBP/DBI system.

Humans are frequently exposed to bacterial genotoxins of the gut microbiota, such as colibactin and cytolethal distending toxin (CDT). In the present study, whole genome microarray-based identification of differentially expressed genes was performed in vitro on HT29 intestinal cells while following the ectopic expression of the active CdtB subunit of Helicobacter hepaticus CDT. Microarray data showed a CdtB-dependent upregulation of transcripts involved in positive regulation of autophagy concomitant with the downregulation of transcripts involved in negative regulation of autophagy. CdtB promotes the activation of autophagy in intestinal and hepatic cell lines. Experiments with cells lacking autophagy related genes, ATG5 and ATG7 infected with CDT- and colibactin-producing bacteria revealed that autophagy protects cells against the genotoxin-induced apoptotic cell death. Autophagy induction could also be associated with nucleoplasmic reticulum (NR) formation following DNA damage induced by these bacterial genotoxins. In addition, both genotoxins promote the accumulation of the autophagic receptor P62/SQSTM1 aggregates, which colocalized with foci concentrating the RNA binding protein UNR/CSDE1. Some of these aggregates were deeply invaginated in NR in distended nuclei together or in the vicinity of UNR-rich foci. Interestingly, micronuclei-like structures and some vesicles containing chromatin and γH2AX foci were found surrounded with P62/SQSTM1 and/or the autophagosome marker LC3. This study suggests that autophagy and P62/SQSTM1 regulate the abundance of micronuclei-like structures and are involved in cell survival following the DNA damage induced by CDT and colibactin. Similar effects were observed in response to DNA damaging chemotherapeutic agents, offering new insights into the context of resistance of cancer cells to therapies inducing DNA damage.

> L'autophagie est un processus d'autodigestion essentiel au maintien de l'homéostasie cellulaire.Elle se produit à l'état normal mais est également activée en réponse à différents stress.C'est un frein à la formation de tumeurs, mais les cellules cancéreuses peuvent la détourner à leur propre avantage pour favoriser leur développement et/ou résister aux traitements thérapeutiques.Chercheurs et cliniciens sont capables, à l'aide de molécules qui restent à optimiser, de moduler positivement ou négativement ce processus afin d'améliorer l'efficacité des thérapies.L'autophagie est donc au centre d'un véritable « bras de fer » entre les cellules cancéreuses et les thérapies anti-cancéreuses.< La formation de l'autophagosome requiert principalement les complexes ULK1 (unc-51-like kinase 1) et PI3K class III-BECN1 (phosphoinositide 3-kinase-class III-beclin 1), ainsi que deux systèmes de conjugaison comprenant celui de ATG12 à ATG5 et celui de MAP1LC3 (ou LC3, microtubule associated protein 1 light chain 3) à la phosphatidyléthanolamine (PE).Ce dernier conjugué, appelé LC3-II, constitue un marqueur d'identification des autophagosomes.Lorsque l'autophagie est sélective, elle implique aussi des récepteurs autophagiques tels que p62/séquestosome, NBR1 (neighbour of BRCA1 [breast cancer 1]) ou OPTN (optineurin).Ils créent un lien physique entre le cargo et les protéines autophagiques ancrées dans la membrane autophagosomale, telle que la protéine LC3-II, et permettent ainsi l'adressage sélectif des cargos à dégrader vers l'autophagosome [3].Ainsi, si l'autophagie constitue une réponse adaptative mise en place par la cellule en réponse aux stress environnementaux (comme la carence) ou intracellulaires, elle assure également un contrôle qualité du cytoplasme en dégradant sélectivement les organites endommagés et les protéines en quantités excessives.L'importance de l'autophagie dans l'oncogenèse a largement été documentée, mais son rôle précis dans l'initiation et la progression tumorale, dans l'apparition de métastases, ou dans les phénomènes de résistance aux chimiothérapies reste sujet à controverse et dépend du contexte cellulaire.En effet, en réponse à divers traitements, l'autophagie a été impliquée dans la résistance ou, au contraire, dans

Lysosomes play a key role in regulating cell death in response to cancer therapies, yet little is known on the possible role of lysosomes in the therapeutic efficacy of G-quadruplex DNA ligands (G4L) in cancer cells. Here, we investigate the relationship between the modulation of lysosomal membrane damage and the degree to which cancer cells respond to the cytotoxic effects of G-quadruplex ligands belonging to the triarylpyridine family. Our results reveal that the lead compound of this family, 20A promotes the enlargement of the lysosome compartment as well as the induction of lysosome-relevant mRNAs. Interestingly, the combination of 20A and chloroquine (an inhibitor of lysosomal functions) led to a significant induction of lysosomal membrane permeabilization coupled to massive cell death. Similar effects were observed when chloroquine was added to three new triarylpyridine derivatives. Our findings thus uncover the lysosomal effects of triarylpyridines compounds and delineate a rationale for combining these compounds with chloroquine to increase their anticancer effects.

Calcium ions (Ca2+) play important and diverse roles in the regulation of autophagy, cell death and differentiation. Here, we investigated the impact of Ca2+ in regulating acute promyelocytic leukemia (APL) cell fate in response to the anti-cancer agent all-trans retinoic acid (ATRA). We observed that ATRA promotes calcium entry through store-operated calcium (SOC) channels into acute promyelocytic leukemia (APL) cells. This response is associated with changes in the expression profiles of ORAI1 and STIM1, two proteins involved in SOC channels activation, as well as with a significant upregulation of several key proteins associated to calcium signaling. Moreover, ATRA treatment of APL cells led to a significant activation of calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) and its downstream effector AMP-activated protein kinase (AMPK), linking Ca2+ signaling to autophagy. Pharmacological inhibition of SOC channels and CAMKK2 enhanced ATRA-induced cell differentiation and death. Altogether, our results unravel an ATRA-elicited signaling pathway that involves SOC channels/CAMKK2 activation, induction of autophagy, inhibition of cellular differentiation and suppression of cell death. We suggest that SOC channels and CAMKK2 may constitute novel drug targets for potentiating the anti-cancer effect of ATRA in APL patients.

To assess Pseudomonas exotoxin A (ETA) compartmentalization, processing and cytotoxicity in vivo , we have studied the fate of internalized ETA with the use of the in vivo rodent liver model following toxin administration, cell‐free hepatic endosomes, and pure in vitro protease assays. ETA taken up into rat liver in vivo was rapidly associated with plasma membranes (5–30 min), internalized within endosomes (15–60 min), and later translocated into the cytosolic compartment (30–90 min). Coincident with endocytosis of intact ETA, in vivo association of the catalytic ETA‐A subunit and low molecular mass ETA‐A fragments was observed in the endosomal apparatus. After an in vitro proteolytic assay with an endosomal lysate and pure proteases, the ETA‐degrading activity was attributed to the luminal species of endosomal acidic cathepsins B and D, with the major cleavages generated in vitro occurring mainly within domain III of ETA‐A. Cell‐free endosomes preloaded in vivo with ETA intraluminally processed and extraluminally released intact ETA and ETA‐A in vitro in a pH‐dependent and ATP‐dependent manner. Rat hepatic cells underwent in vivo intrinsic apoptosis at a late stage of ETA infection, as assessed by the mitochondrial release of cytochrome c , caspase‐9 and caspase‐3 activation, and DNA fragmentation. In an in vitro assay, intact ETA induced ADP‐ribosylation of EF‐2 and mitochondrial release of cytochrome c , with the former effect being efficiently increased by a cathepsin B/cathepsin D pretreatment. The data show a novel processing pathway for internalized ETA, involving cathepsins B and D, resulting in the production of ETA fragments that may participate in cytotoxicity and mitochondrial dysfunction.

p62 regulates key signaling pathways including those that control cell death and autophagy. Recently, we reported that p62 is upregulated during all-trans retinoic acid (ATRA)-induced terminal differentiation of acute myeloid leukemia (AML) cells. This response reduces levels of ubiquitinated protein aggregates in mature cells and protects these cells against ATRA treatment. Thus, p62 confers a survival advantage to mature AML cells.

The binding, uptake, and degradation of epidermal growth factor (EGF) has been studied in MRC5 human fibroblasts and NCTC 2544 human keratinocytes following ultraviolet A (UVA) irradiation at doses up to 18.9 J/cm2, which are not lethal to cells under our experimental conditions. A dose-dependent reduction in EGF binding was observed, with an approximately 75% decrease at the maximal studied UVA dose. At lower doses (6 to 12 J/cm2), EGF binding was more affected by ultraviolet A in fibroblasts than in keratinocytes. In both cell types, this effect of UVA appeared to be related to a reduction of the affinity of the EGF receptor for EGF. Kinetic studies by pulse-chase experiments indicated that EGF is more rapidly internalized by keratinocytes than by fibroblasts, and that UVA exposure resulted in a slower decay of EGF intracellular content. A 24-h pretreatment of cells with 5 x 10(-5) M vitamin E strongly reduced the appearance of light-induced lipid peroxidation products, measured via assay of thiobarbituric acid reactive substances formation, but only partially prevented the UVA-induced alterations of EGF processing by cells. Finally, UVA exposure almost completely abolished the EGF-induced increase in diacylglycerol production from 14C-arachidonic acid-labeled lipids in both cell types. These results demonstrate that UVA radiation induces important changes in EGF processing and could participate in the light-induced degenerative processes of the skin.

UV-A irradiation caused a dose-dependent decrease in cellular oxygen consumption (56%) and ATP content (65%) in human NCTC 2544 keratinocytes, one hour after treatment. This effect was partially reversed by maintaining the irradiated cells in normal culture conditions for 24 h. Using malate/glutamate or succinate as substrates for mitochondrial electron transport, the oxygen uptake of digitonin-permeabilised cells was greatly inhibited following UV-A exposure. These results strongly suggest that UV-A irradiation affects the state 3 respiration of the mitochondria. However, under identical conditions, UV-A exposure did not reduce the mitochondrial transmembrane potential. The antioxidant, vitamin E inhibited UV-A-induced lipid peroxidation, but did not significantly prevent the UV-A-mediated changes in cellular respiration nor the decrease in ATP content, suggesting that these effects were not the result of UV-A dependent lipid peroxidation. UV-A irradiation also led to an increase in MnSOD gene expression 24 hours after treatment, indicating that the mitochondrial protection system was enhanced in response to UV-A treatment. These findings provide evidence that impairment of mitochondrial respiratory activity is one of the early results of UV-A irradiation for light doses much lower than the minimal erythemal dose.

Cytotoxicity of many plant and bacterial toxins requires their endocytosis and retrograde transport from endosomes to the endoplasmic reticulum. Using cell fractionation and immunoblotting procedures, we have assessed the fate and action of the plant toxin ricin in rat liver in vivo, focusing on endosome-associated events and induction of apoptosis. Injected ricin rapidly accumulated in endosomes as an intact A/B heterodimer (5-90 min) and was later (15-90 min) partially translocated to cytosol as A- and B-chains. Unlike cholera and diphtheria toxins, which also undergo endocytosis in liver, neither in cell-free endosomes loaded by ricin in vivo nor upon incubation with endosomal lysates did ricin undergo degradation in vitro. A time-dependent translocation of ricin across the endosomal membrane occurred in cell-free endosomes. Endosome-located thioredoxin reductase-1 was required for translocation as shown by its physical association with ricin chains and effects of its removal and inhibition. Ricin induced in vivo intrinsic apoptosis as judged by increased cytochrome c content, activation of caspase-9 and caspase-3, and enrichment of DNA fragments in cytosol. Furthermore, reduced ricin and ricin B-chain caused cytochrome c release from mitochondria in vivo and in vitro, suggesting that the interaction of ricin B-chain with mitochondria is involved in ricin-induced apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively kills various cancer cell types, but also leads to the activation of signaling pathways that favor resistance to cell death. Here, we investigated the as yet unknown roles of calcium signaling and autophagy regulatory proteins during TRAIL-induced cell death in leukemia cells. Taking advantage of the Gene Expression Profiling Interactive Analysis (GEPIA) project, we first found that leukemia patients present a unique TRAIL receptor gene expression pattern that may reflect their resistance to TRAIL. The exposure of NB4 acute promyelocytic leukemia cells to TRAIL induces intracellular Ca2+ influx through a calcium release-activated channel (CRAC)-dependent mechanism, leading to an anti-apoptotic response. Mechanistically, we showed that upon TRAIL treatment, two autophagy proteins, ATG7 and p62/SQSTM1, are recruited to the death-inducing signaling complex (DISC) and are essential for TRAIL-induced Ca2+ influx and cell death. Importantly, the treatment of NB4 cells with all-trans retinoic acid (ATRA) led to the upregulation of p62/SQSTM1 and caspase-8 and, when added prior to TRAIL stimulation, significantly enhanced DISC formation and the apoptosis induced by TRAIL. In addition to uncovering new pleiotropic roles for autophagy proteins in controlling the calcium response and apoptosis triggered by TRAIL, our results point to novel therapeutic strategies for sensitizing leukemia cells to TRAIL.

Macroautophagy is a lysosomal catabolic process involved in recycling cell components and maintaining cellular homeostasis. Identifying some of the molecules involved in the control and execution steps of autophagy has shed light on the close link between autophagy and tumour progression. Several tumour-suppressor proteins -including Beclin 1, a protein involved in autophagosome formation- positively regulate autophagy. Conversely, some oncogenic proteins display inhibitory effects on autophagy. The antitumoral role of autophagy is supported by its involvement in reducing chromosome instability, proliferation and inflammation of tumour cells. However, autophagy can also be a protumoral mechanism which helps tumour cells to adapt to changes in their microenvironment (hypoxia, starvation...). Moreover, autophagy is induced in response to several anticancer treatments. This response can either be a mechanism allowing cell survival or a mechanism promoting cell death. The aim of this article is to summarize recent progress focusing on the dual role of autophagy in cancer.

G-quadruplex (G4) ligands have shown anti-tumor activity by stabilizing G4 structures. We recently reported that the G4 ligand 20A promotes senescence in cancer cells through Ataxia Telangiectasia Mutated (ATM) and autophagy activation. Inhibition of both pathways directs cells to apoptosis, positioning ATM/autophagy axis as a linchpin between senescence and apoptosis.

Previous reports have demonstrated an increase in nuclear factor-κB (NF-κB) activity in response to UV radiation. These studies have essentially focused on the DNA-damaging fraction of solar UV radiation (UV-B and UV-C). In contrast, the effects of UV-A radiation (320–400 nm) on NF-κB are not well known. In this study, we present evidence that UV-A radiation induces a marked decrease in NF-κB DNA-binding activity in NCTC 2544 human keratinocytes. In addition, NCTC 2544 keratinocytes pretreated with UV-A fail to respond to NF-κB inducers. Moreover, UV-A radiation induces a decrease in NF-κB-driven luciferase reporter gene expression in NCTC 2544 keratinocytes. The expression of the gene encoding IκBα (IκB is the NF-κB inhibitor), which is closely associated with NF-κB activity, is also reduced (3-fold) upon UV-A treatment. Our results indicate that the UV-A-induced decrease in NF-κB DNA-binding activity is associated with a decrease in the levels of the p50 and p65 protein subunits. This is the first evidence that an oxidative stress, such as UV-A radiation, may induce a specific decrease in NF-κB activity in mammalian cells, probably through degradation of NF-κB protein subunits. These findings suggest that UV-A could modulate the NF-κB-dependent gene expression.

Autophagy is a catabolic lysosomal-dependent pathway involved in the degradation of cellular materials, supplying precursor compounds and energy for macromolecule synthesis and metabolic needs [...].

Lung cancer (LC) remains a leading cause of mortality worldwide, and new therapeutic strategies are urgently needed. One such approach revolves around the utilization of four-stranded nucleic acid secondary structures, known as G-quadruplexes (G4), which are formed by G-rich sequences. Ligands that bind selectively to G4 structures present a promising strategy for regulating crucial cellular processes involved in the progression of LC, rendering them potent agents for lung cancer treatment. In this review, we offer a summary of recent advancements in the development of G4 ligands capable of targeting specific genes associated with the development and progression of lung cancer.

Abstract Autophagy is an evolutionary conserved process that degrades and recycles cellular components through the lysosomal pathway to maintain cellular homeostasis. It has long been considered as a pro-survival mechanism to maintain viability under stressful conditions. However, a growing body of evidence demonstrates that autophagy could also promote or act as a pro-death program. Autophagy plays an ambivalent role in cancer: it can operate either as a pro-tumoral mechanism or paradoxically as an anti-tumoral mechanism depending on the context and the stage of the tumours, making it an interesting field of investigation. p62/SQSTM1 is an adapter protein playing a key role during the autophagy process. It promotes the selective degradation of ubiquitinated cellular substrates (i.e. proteins and organelles). As a multidomain protein adapter, p62 is involved in the regulation of various signaling pathways including those that control cell death pathways. In this regard, p62 can directly interact with the death receptor DR5 and promotes death receptor induced-apoptosis through caspase 8 stabilization. Acute myeloid leukemia (AML) is a heterogeneous disease characterized by an abnormal proliferation of myeloblasts (precursors of myeloid white blood cells) without differentiation. Acute promyelocytic leukemia (APL) is a subtype of AML characterized by the translocation t(15;17)(q22;q12) which results to the expression of the chimeric protein PML-RAR alpha. All-trans retinoic acid (ATRA) induces cancer cells differentiation and results in complete clinical remission in APL patients. It is now used as a first-line therapy. Despite the success of this treatment, some patients are refractory to ATRA or relapse. Thus, new therapeutic strategies are needed. We previously showed that ATRA-induced APL cell differentiation was associated with autophagy induction and p62/SQSMT1 accumulation, a response that confers a survival advantage to mature APL cells. As recent findings showed several molecular links between p62 and extrinsic apoptosis pathway, we decided to investigate the link between p62/SQSMT1 and cell death in AML cells upon treatment by TRAIL, an inducer of extrinsic apoptosis. We found that p62/SQSTM1 is required for apoptotic responses in APL cells. Our results also reveal the synergic cooperation between TRAIL and ATRA to increase apoptosis. We are currently investigating the molecular mechanisms underlying the role of autophagy as well as the autophagy receptor p62/SQSM1 in TRAIL-induced apoptosis in APL cells. Citation Format: Kelly Airiau, Olivier Micheau, Anne-Marie Vacher, Faten Mehri, Pierre Vacher, Mojgan Djavaheri-Mergny. Regulation of TRAIL-induced apoptotic signaling by the autophagy receptor p62 in acute promyelocytic leukemia cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3723.

The authors wish to make the following change to their paper [...]

The aconitase activity of the cytoplasmic iron regulatory protein-1 of NCTC 2544 keratinocytes is effectively inhibited by physiological doses of UVA. The time course of the photoinactivation is biphasic. A fast step is first observed corresponding to about 50% inactivation after exposure to 5 J/cm2of UVA followed by a much slower photoinactivation at higher doses. The water-soluble antioxidant N-acetylcysteine only partially inhibits the pho-toinduced inactivation of the cytoplasmic aconitase function, whereas the lipophilic vitamin E, the iron chelator, desferrioxamine and the superoxide dismutase inhibitor, diethyldithiocarbamate do not protect at all. As a consequence, reactive oxygen species such as O2-, H2O2 and lipid peroxides and hydroperoxides seem to play a rather minor role in the inactivation induced by the UVA pho-tooxidative stress although an oxidative stress produced by O2- H2O2 is known to inhibit reversibly and effectively cytoplasmic aconitase activity in mammalian cells.