Christine Didier - Academia.edu (original) (raw)

Papers by Christine Didier

Leukemia

Resistance of acute myeloid leukemia (AML) to therapeutic agents is frequent. Consequently, the m... more Resistance of acute myeloid leukemia (AML) to therapeutic agents is frequent. Consequently, the mechanisms leading to this resistance must be understood and addressed. In this paper, we demonstrate that inhibition of deubiquitinylase USP7 significantly reduces cell proliferation in vitro and in vivo, blocks DNA replication progression and increases cell death in AML. Transcriptomic dataset analyses reveal that a USP7 gene signature is highly enriched in cells from AML patients at relapse, as well as in residual blasts from patient-derived xenograft (PDX) models treated with clinically relevant doses of cytarabine, which indicates a relationship between USP7 expression and resistance to therapy. Accordingly, singlecell analysis of AML patient samples at relapse versus at diagnosis showed that a gene signature of the pre-existing subpopulation responsible for relapse is enriched in transcriptomes of patients with a high USP7 level. Furthermore, we found that USP7 interacts and modulates CHK1 protein levels and functions in AML. Finally, we demonstrated that USP7 inhibition acts in synergy with cytarabine to kill AML cell lines and primary cells of patients with high USP7 levels. Altogether, these data demonstrate that USP7 is both a marker of resistance to chemotherapy and a potential therapeutic target in overcoming resistance to treatment.

Journal of cell science, Jan 4, 2018

Although the kinase CHK1 is a key player in the DNA damage response (DDR), several studies have r... more Although the kinase CHK1 is a key player in the DNA damage response (DDR), several studies have recently provided evidence of DDR-independent roles of CHK1, in particular following phosphorylation of its S280 residue. Here, we demonstrate that CHK1 S280 phosphorylation is cell cycle-dependent and peaks during mitosis. We found that this phosphorylation was catalyzed by the kinase PIM2, whose protein expression was also increased during mitosis. Importantly, we identified Polo-Like Kinase 1 (PLK1) as a direct target of CHK1 during mitosis. Genetic or pharmacological inhibition of CHK1 reduced the activating phosphorylation of PLK1 on T210 residue, and recombinant CHK1 was able to phosphorylate T210 of PLK1 Accordingly, phospho-S280 CHK1 and PLK1 exhibited similar specific mitotic localizations, and PLK1 was co-immunoprecipitated with phospho-S280 CHK1 from mitotic cell extracts. Moreover, CHK1-mediated phosphorylation of PLK1 was dependent on S280 phosphorylation by PIM2. PIM inhibit...

Molecular & Cellular Oncology

Resistance of acute myeloid leukemia to current therapies leads to frequent relapses. Identificat... more Resistance of acute myeloid leukemia to current therapies leads to frequent relapses. Identification of molecular mechanisms involved in chemoresistance constitutes a key challenge to define new therapeutic concepts. Here, we show that the ATR/CHK1 pathway, essential in maintaining genomic stability, is involved in resistance and proliferation characteristics of leukemic cells.

FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 1999

Human fibroblasts and keratinocytes possess nitric oxide synthases (NOS), which metabolize L-argi... more Human fibroblasts and keratinocytes possess nitric oxide synthases (NOS), which metabolize L-arginine (L-Arg) for producing nitric oxide (NO*). This report delineates the relations between NO* and UVA in the human keratinocyte cell line HaCaT. NOS activity was stimulated by exposure of cells to L-Arg just after irradiation. L-Arg (5 mM) supply led to an increase in UVA (25.3 J/cm(2)) cytotoxicity (% of viability 18 +/- 3%) whereas neither L-Arg itself nor UVA irradiation induced cell death at the doses used in this study. Cells were also treated either with L-thiocitrulline (L-Thio), an irreversible inhibitor of NOS, or with exogenous superoxide dismutase (SOD) and catalase. L-Thio and SOD prevented L-Arg-mediated deleterious effects in irradiated cells, whereas catalase was ineffective. Intracellular antioxidant enzyme activities were also determined. UVA/L-Arg stress altered catalase (66% decrease) and glutathione peroxidase (83% decrease). DNA damage was evaluated using the '...

Trace Elements in Man and Animals 10, 2002

The Journal of Cell Biology, 2004

ere, we describe a new muscle LIM domain protein, UNC-95, and identify it as a novel target for t... more ere, we describe a new muscle LIM domain protein, UNC-95, and identify it as a novel target for the RING finger protein RNF-5 in the Caenorhabditis elegans body wall muscle. unc-95(su33) animals have disorganized muscle actin and myosin-containing filaments as a result of a failure to assemble normal muscle adhesion structures. UNC-95 is active downstream of PAT-3/ ␤ -integrin in the assembly pathways of the muscle dense body and M-line attachments, and upstream of DEB-1/vinculin in the dense body assembly pathway. The translational UNC-95::GFP H fusion construct is expressed in dense bodies, M-lines, and muscle-muscle cell boundaries as well as in muscle cell bodies. UNC-95 is partially colocalized with RNF-5 in muscle dense bodies and its expression and localization are regulated by or a RING domain deleted mutant, rnf-5(tm794) , exhibit structural defects of the muscle attachment sites. Together, our data demonstrate that UNC-95 constitutes an essential component of muscle adhesion sites that is regulated by RNF-5.

Molecular and Cellular Biology, 2005

We report the identification and characterization of JAMP (JNK1 [Jun N-terminal kinase 1]-associa... more We report the identification and characterization of JAMP (JNK1 [Jun N-terminal kinase 1]-associated membrane protein), a predicted seven-transmembrane protein that is localized primarily within the plasma membrane and associates with JNK1 through its C-terminal domain. JAMP association with JNK1 outcompetes JNK1 association with mitogen-activated protein kinase phosphatase 5, resulting in increased and prolonged JNK1 activity following stress. Elevated expression of JAMP following UV or tunicamycin treatment results in sustained JNK activity and a higher level of JNK-dependent apoptosis. Inhibition of JAMP expression by RNA interference reduces the degree and duration of JNK activation and concomitantly the level of stress-induced apoptosis. Through its regulation of JNK1 activity, JAMP emerges as a membrane-anchored regulator of the duration of JNK1 activity in response to diverse stress stimuli.

Free Radical Biology and Medicine, 2001

The present study analyzes the expression of the thioredoxin/thioredoxin reductase (Trx/TR) syste... more The present study analyzes the expression of the thioredoxin/thioredoxin reductase (Trx/TR) system in UVA-irradiated human skin fibroblasts. Irradiation increases the intracellular level of Trx and a time-dependent increase of Trx mRNA is observed. Our data indicate that Trx translocates from the cytoplasm to the nucleus. In addition, UV exposure results in an increase in TR synthesis. In order to evaluate the function of Trx/TR system, we investigated the antioxidant role of Trx in transient transfected cells. The ROS accumulation in UVA irradiated cells was assessed using flow cytometry. A 3-fold decrease in ROS production was observed in transiently transfected fibroblasts. These results indicate that Trx acts as an antioxidant protein in UVA irradiated fibroblasts. As ROS are inducers of cell death, this raises the question as to whether Trx is able to protect cells from apoptosis and/or necrosis induced by UVA. Six hours after UVA-irradiation, 29.92% of cells were annexin-V positive. This population was significantly reduced in Trxtransfected cells (8.58%). Moreover, this work demonstrates that Trx prevents the loss of the membrane potential of the mitochondria, the depletion of cellular ATP content, and the loss of cell viability induced by irradiation.

Free Radical Biology and Medicine, 2001

Thioredoxin (Trx) plays important biological roles both intra-and extracellularly via thiol redox... more Thioredoxin (Trx) plays important biological roles both intra-and extracellularly via thiol redox control. We have previously demonstrated that Trx exhibited protective effects against UVA cytotoxicity in human skin fibroblasts. As an extension of the latter investigation, the present work is aimed at assessing ability of Trx to maintain genomic integrity in human skin fibroblasts upon exposure to UVA radiation. Indeed, UVA (320 -380 nm) is mutagenic and induces genomic damage to skin cells. The alkaline comet assay was used in association with DNA repair enzyme including formamido pyrimidine glycosylase (Fpg) and endonuclease III (endo III) to estimate the amount of modified bases together with the level of strand breaks and alkali-labile sites. The HPLC-EC assay was applied to assess 8-oxo-7,8-dihydro-2Ј-deoxyguanosine (8-oxodGuo) levels and to permit the calibration of comet assay as previously described. We reported that overexpression of human Trx (transient transfection) as well as exogenous human recombinant Trx added to the culture medium, decreased the level of DNA damage in UVA irradiated cells. Interestingly, transfection appeared to prevent UVA-induced 8-oxodGuo (3.06 au per Joules.cm Ϫ2 compared to 4.94 au per Joules.cm Ϫ2 for nontransfected cells). Moreover, Trx accumulates into nuclei in transfected cells. This finding supports the notion that Trx is important for the maintenance of the integrity of genetic information. This work demonstrated that under conditions of UVA oxidative stress, Trx prevented the UVA-induced DNA damage.

Science signaling, Jan 13, 2016

The nucleoside analog cytarabine, an inhibitor of DNA replication fork progression that results i... more The nucleoside analog cytarabine, an inhibitor of DNA replication fork progression that results in DNA damage, is currently used in the treatment of acute myeloid leukemia (AML). We explored the prognostic value of the expression of 72 genes involved in various aspects of DNA replication in a set of 198 AML patients treated by cytarabine-based chemotherapy. We unveiled that high expression of the DNA replication checkpoint gene CHEK1 is a prognostic marker associated with shorter overall, event-free, and relapse-free survivals and determined that the expression of CHEK1 can predict more frequent and earlier postremission relapse. CHEK1 encodes checkpoint kinase 1 (CHK1), which is activated by the kinase ATR when DNA replication is impaired by DNA damage. High abundance of CHK1 in AML patient cells correlated with higher clonogenic ability and more efficient DNA replication fork progression upon cytarabine treatment. Exposing the patient cells with the high abundance of CHK1 to SCH90...

Oncotarget, Jan 16, 2015

We investigated cell cycle regulation in acute myeloid leukemia cells expressing the FLT3-ITD mut... more We investigated cell cycle regulation in acute myeloid leukemia cells expressing the FLT3-ITD mutated tyrosine kinase receptor, an underexplored field in this disease. Upon FLT3 inhibition, CDC25A mRNA and protein were rapidly down-regulated, while levels of other cell cycle proteins remained unchanged. This regulation was dependent on STAT5, arguing for FLT3-ITD-dependent transcriptional regulation of CDC25A. CDC25 inhibitors triggered proliferation arrest and cell death of FLT3-ITD as well as FLT3-ITD/TKD AC-220 resistant cells, but not of FLT3-wt cells. Consistently, RNA interference-mediated knock-down of CDC25A reduced the proliferation of FLT3-ITD cell lines. Finally, the clonogenic capacity of primary FLT3-ITD AML cells was reduced by the CDC25 inhibitor IRC-083864, while FLT3-wt AML and normal CD34+ myeloid cells were unaffected. In good agreement, in a cohort of 100 samples from AML patients with intermediate-risk cytogenetics, high levels of CDC25A mRNA were predictive of ...

Leukemia Research, 2014

CHK1 Ser/Thr kinase, a well characterized regulator of DNA damage response, is also involved in n... more CHK1 Ser/Thr kinase, a well characterized regulator of DNA damage response, is also involved in normal cell cycle progression. In this study, we investigate how CHK1 participates to proliferation of acute myeloid leukemia cells expressing the mutated FLT3-ITD tyrosine kinase receptor. Pharmacological inhibition of CHK1 as well as its shRNA mediated down regulation reduced the proliferation rate of FLT-ITD expressing leukemic cell lines in a cytostatic manner. Flow cytometry analysis revealed no accumulation in a specific phase of the cell cycle upon CHK1 inhibition. Accordingly, lentiviral-mediated CHK1 overexpression increased the proliferation rate of FLT3-ITD expressing cells, as judged by cell viability and [3H] thymidine incorporation experiments. By contrast, expression of a ser280 mutant did not, suggesting that phosphorylation of this residue is an important determinant of CHK1 proliferative function. Clonogenic growth of primary leukemic cells from patients in semi-solid medium was reduced upon CHK1 inhibition, confirming the data obtained with leukemic established cell lines. Surprisingly, 3 out of 4 CHK1 inhibitory compounds tested in this study were also potent inhibitors of the FLT3-ITD tyrosine kinase receptor. Altogether, these data identify CHK1 as a regulator of FLT3-ITD-positive leukemic cells proliferation, and they open interesting perspectives in terms of new therapeutic strategies for these pathologies.

Trace Elements in Man and Animals 10, 2002

... Marie-Jeanne Richard, Nathalie Emonet-Piccardi, Christine Didier, Eric Jourdan, Marie-Thérèse... more ... Marie-Jeanne Richard, Nathalie Emonet-Piccardi, Christine Didier, Eric Jourdan, Marie-Thérèse Leccia, Marie ... Telephone: 476765147; Fax: 476765664; email: Marie-Jeanne.Richard@ujf-grenoble.fr ... Hanada, K., Sawamura, D., Tamai, K., Baba, T., Hashimoto, I., Muramatsu, T ...

RNF5 is a RING finger protein found to be important in the growth and development of Caenorhabdit... more RNF5 is a RING finger protein found to be important in the growth and development of Caenorhabditis elegans. The search for RNF5-associated proteins via a yeast two-hybrid screen identified a LIM-containing protein in C. elegans which shows homology with human paxillin. Here we demonstrate that the human homologue of RNF5 associates with the amino-terminal domain of paxillin, resulting in its ubiquitination. RNF5 requires intact RING and C-terminal domains to mediate paxillin ubiquitination. Whereas RNF5 mediates efficient ubiquitination of paxillin in vivo, protein extracts were required for in vitro ubiquitination, suggesting that additional modifications and/or an associated E3 ligase assist RNF5 targeting of paxillin ubiquitination. Mutant Ubc13 efficiently inhibits RNF5 ubiquitination, suggesting that RNF5 generates polychain ubiquitin of the K63 topology. Expression of RNF5 increases the cytoplasmic distribution of paxillin while decreasing its localization within focal adhesions, where it is primarily seen under normal growth. Concomitantly, RNF5 expression results in inhibition of cell motility. Via targeting of paxillin ubiquitination, which alters its localization, RNF5 emerges as a novel regulator of cell motility.

At the present time, the broad use of monoclonal antibodies (MAbs) in immunochemica l, structural... more At the present time, the broad use of monoclonal antibodies (MAbs) in immunochemica l, structural and functional studies of proteins, as well as in immunotherapy, and their capacity for monospecific interaction, reproducibility and production in large scale have made of these macromolecules a very important tool in bioengineering. The aim of this work was to develop an enzyme-linked immunosorbent assay

Oncogene, 2008

Acute myeloid leukemia (AML) cells exposed to genotoxic agents arrest their cell cycle at the G2/... more Acute myeloid leukemia (AML) cells exposed to genotoxic agents arrest their cell cycle at the G2/M checkpoint and are inherently chemoresistant. To understand the mechanism of this chemoresistance, we compared the ability of immature CD34 þ versus mature CD34À AML cell lines (KG1a and U937, respectively) to recover from a DNA damage-induced cell cycle checkpoint in G2. Here, we report that KG1a cells have a more stringent G2/M checkpoint response than U937 cells. We show that in both cell types, the CDC25B phosphatase participates in the G2/M checkpoint recovery and that its expression is upregulated. Furthermore, we show that CHK1 inhibition by UCN-01 in immature KG1a cells allows checkpoint exit and induces sensitivity to genotoxic agents. Similarly, UCN-01 treatment potentializes genotoxic-induced inhibition of colony formation efficiency of primary leukemic cells from AML patients. Altogether, our results demonstrate that checkpoint stringency varies during the maturation process and indicate that targeting checkpoint mechanisms might represent an attractive therapeutic opportunity for chemoresistant immature AML cells.

Molecular Biology of the Cell, 2007

Centrosomes are dynamic organelles that consist of a pair of cylindrical centrioles, surrounded b... more Centrosomes are dynamic organelles that consist of a pair of cylindrical centrioles, surrounded by pericentriolar material. The pericentriolar material contains factors that are involved in microtubule nucleation and organization, and its recruitment varies during the cell cycle. We report here that proteasome inhibition in HeLa cells induces the accumulation of several proteins at the pericentriolar material, including gamma-tubulin, GCP4, NEDD1, ninein, pericentrin, dynactin, and PCM-1. The effect of proteasome inhibition on centrosome proteins does not require intact microtubules and is reversed after removal of proteasome inhibitors. This accrual of centrosome proteins is paralleled by accumulation of ubiquitin in the same area and increased polyubiquitylation of nonsoluble gamma-tubulin. Cells that have accumulated centrosome proteins in response to proteasome inhibition are impaired in microtubule aster formation. Our data point toward a role of the proteasome in the turnover of centrosome proteins, to maintain proper centrosome function.

European Journal of Pharmacology, 2008

Polo-Kinase 1 (PLK1) is a key cell cycle regulator that is necessary for checkpoint recovery afte... more Polo-Kinase 1 (PLK1) is a key cell cycle regulator that is necessary for checkpoint recovery after DNA damageinduced G2 arrest. We have examined the effects of PLK inhibition in Acute Myelocytic Leukaemia (AML) cells, whose resistance to genotoxic agents is thought to be associated with checkpoint reinforcement. We report that in U937 AML cells, PLK1 participates in checkpoint recovery, and that inhibition of PLK by the GW843682X compound results in mitotic accumulation and apoptosis. We also found that when challenged with VP-16, inhibition of PLK1 prevented U937 cells from checkpoint exit. Finally, we found that treatment with GW843682X slightly reduced genotoxic-induced inhibition of colony formation efficiency of primary leukaemia cells (CFU-L) from AML patients.

Cancer Research, 2006

The effects of cell adhesion on leukemia cell proliferation remain poorly documented and somehow ... more The effects of cell adhesion on leukemia cell proliferation remain poorly documented and somehow controversial. In this work, we investigated the effect of adhesion to fibronectin on the proliferation of acute myeloid leukemia (AML) cell lines (U937 and KG1a) and CD34 + normal or leukemic primary cells. We observed an increased rate of proliferation of AML cells when adhered to fibronectin, concomitant with accelerated S-phase entry and accumulation of CDC25A. Conversely, normal CD34 + cell proliferation was decreased by adhesion to fibronectin with a concomitant drop in CDC25A expression. Importantly, we showed that both small interfering RNA (siRNA)-mediated CDC25A down-regulation and a recently developed CDC25 pharmacologic inhibitor impaired this adhesion-dependent proliferation, establishing a functional link between CDC25A accumulation and adhesion-dependent proliferation in leukemic cells. CDC25A accumulation was found only slightly dependent on transcriptional regulation and essentially due to modifications of the proteasomal degradation of the protein as shown using proteasome inhibitors and reverse transcription-PCR. Interestingly, CDC25A regulation was Chk1 dependent in these cells as suggested by siRNA-mediated down-regulation of this protein. Finally, we identified activation of the phosphatidylinositol 3-kinase/ Akt pathway as an adhesion-dependent regulation mechanism of CDC25A protein expression. Altogether, our data show that in leukemic cells adhesion to fibronectin increases CDC25A expression through proteasome-and Chk1-dependent mechanisms, resulting in enhanced proliferation. They also suggest that these adhesion-dependent proliferation properties of hematopoietic cells may be modified during leukemogenesis.

Leukemia

Resistance of acute myeloid leukemia (AML) to therapeutic agents is frequent. Consequently, the m... more Resistance of acute myeloid leukemia (AML) to therapeutic agents is frequent. Consequently, the mechanisms leading to this resistance must be understood and addressed. In this paper, we demonstrate that inhibition of deubiquitinylase USP7 significantly reduces cell proliferation in vitro and in vivo, blocks DNA replication progression and increases cell death in AML. Transcriptomic dataset analyses reveal that a USP7 gene signature is highly enriched in cells from AML patients at relapse, as well as in residual blasts from patient-derived xenograft (PDX) models treated with clinically relevant doses of cytarabine, which indicates a relationship between USP7 expression and resistance to therapy. Accordingly, singlecell analysis of AML patient samples at relapse versus at diagnosis showed that a gene signature of the pre-existing subpopulation responsible for relapse is enriched in transcriptomes of patients with a high USP7 level. Furthermore, we found that USP7 interacts and modulates CHK1 protein levels and functions in AML. Finally, we demonstrated that USP7 inhibition acts in synergy with cytarabine to kill AML cell lines and primary cells of patients with high USP7 levels. Altogether, these data demonstrate that USP7 is both a marker of resistance to chemotherapy and a potential therapeutic target in overcoming resistance to treatment.

Journal of cell science, Jan 4, 2018

Although the kinase CHK1 is a key player in the DNA damage response (DDR), several studies have r... more Although the kinase CHK1 is a key player in the DNA damage response (DDR), several studies have recently provided evidence of DDR-independent roles of CHK1, in particular following phosphorylation of its S280 residue. Here, we demonstrate that CHK1 S280 phosphorylation is cell cycle-dependent and peaks during mitosis. We found that this phosphorylation was catalyzed by the kinase PIM2, whose protein expression was also increased during mitosis. Importantly, we identified Polo-Like Kinase 1 (PLK1) as a direct target of CHK1 during mitosis. Genetic or pharmacological inhibition of CHK1 reduced the activating phosphorylation of PLK1 on T210 residue, and recombinant CHK1 was able to phosphorylate T210 of PLK1 Accordingly, phospho-S280 CHK1 and PLK1 exhibited similar specific mitotic localizations, and PLK1 was co-immunoprecipitated with phospho-S280 CHK1 from mitotic cell extracts. Moreover, CHK1-mediated phosphorylation of PLK1 was dependent on S280 phosphorylation by PIM2. PIM inhibit...

Molecular & Cellular Oncology

Resistance of acute myeloid leukemia to current therapies leads to frequent relapses. Identificat... more Resistance of acute myeloid leukemia to current therapies leads to frequent relapses. Identification of molecular mechanisms involved in chemoresistance constitutes a key challenge to define new therapeutic concepts. Here, we show that the ATR/CHK1 pathway, essential in maintaining genomic stability, is involved in resistance and proliferation characteristics of leukemic cells.

FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 1999

Human fibroblasts and keratinocytes possess nitric oxide synthases (NOS), which metabolize L-argi... more Human fibroblasts and keratinocytes possess nitric oxide synthases (NOS), which metabolize L-arginine (L-Arg) for producing nitric oxide (NO*). This report delineates the relations between NO* and UVA in the human keratinocyte cell line HaCaT. NOS activity was stimulated by exposure of cells to L-Arg just after irradiation. L-Arg (5 mM) supply led to an increase in UVA (25.3 J/cm(2)) cytotoxicity (% of viability 18 +/- 3%) whereas neither L-Arg itself nor UVA irradiation induced cell death at the doses used in this study. Cells were also treated either with L-thiocitrulline (L-Thio), an irreversible inhibitor of NOS, or with exogenous superoxide dismutase (SOD) and catalase. L-Thio and SOD prevented L-Arg-mediated deleterious effects in irradiated cells, whereas catalase was ineffective. Intracellular antioxidant enzyme activities were also determined. UVA/L-Arg stress altered catalase (66% decrease) and glutathione peroxidase (83% decrease). DNA damage was evaluated using the '...

Trace Elements in Man and Animals 10, 2002

The Journal of Cell Biology, 2004

ere, we describe a new muscle LIM domain protein, UNC-95, and identify it as a novel target for t... more ere, we describe a new muscle LIM domain protein, UNC-95, and identify it as a novel target for the RING finger protein RNF-5 in the Caenorhabditis elegans body wall muscle. unc-95(su33) animals have disorganized muscle actin and myosin-containing filaments as a result of a failure to assemble normal muscle adhesion structures. UNC-95 is active downstream of PAT-3/ ␤ -integrin in the assembly pathways of the muscle dense body and M-line attachments, and upstream of DEB-1/vinculin in the dense body assembly pathway. The translational UNC-95::GFP H fusion construct is expressed in dense bodies, M-lines, and muscle-muscle cell boundaries as well as in muscle cell bodies. UNC-95 is partially colocalized with RNF-5 in muscle dense bodies and its expression and localization are regulated by or a RING domain deleted mutant, rnf-5(tm794) , exhibit structural defects of the muscle attachment sites. Together, our data demonstrate that UNC-95 constitutes an essential component of muscle adhesion sites that is regulated by RNF-5.

Molecular and Cellular Biology, 2005

We report the identification and characterization of JAMP (JNK1 [Jun N-terminal kinase 1]-associa... more We report the identification and characterization of JAMP (JNK1 [Jun N-terminal kinase 1]-associated membrane protein), a predicted seven-transmembrane protein that is localized primarily within the plasma membrane and associates with JNK1 through its C-terminal domain. JAMP association with JNK1 outcompetes JNK1 association with mitogen-activated protein kinase phosphatase 5, resulting in increased and prolonged JNK1 activity following stress. Elevated expression of JAMP following UV or tunicamycin treatment results in sustained JNK activity and a higher level of JNK-dependent apoptosis. Inhibition of JAMP expression by RNA interference reduces the degree and duration of JNK activation and concomitantly the level of stress-induced apoptosis. Through its regulation of JNK1 activity, JAMP emerges as a membrane-anchored regulator of the duration of JNK1 activity in response to diverse stress stimuli.

Free Radical Biology and Medicine, 2001

The present study analyzes the expression of the thioredoxin/thioredoxin reductase (Trx/TR) syste... more The present study analyzes the expression of the thioredoxin/thioredoxin reductase (Trx/TR) system in UVA-irradiated human skin fibroblasts. Irradiation increases the intracellular level of Trx and a time-dependent increase of Trx mRNA is observed. Our data indicate that Trx translocates from the cytoplasm to the nucleus. In addition, UV exposure results in an increase in TR synthesis. In order to evaluate the function of Trx/TR system, we investigated the antioxidant role of Trx in transient transfected cells. The ROS accumulation in UVA irradiated cells was assessed using flow cytometry. A 3-fold decrease in ROS production was observed in transiently transfected fibroblasts. These results indicate that Trx acts as an antioxidant protein in UVA irradiated fibroblasts. As ROS are inducers of cell death, this raises the question as to whether Trx is able to protect cells from apoptosis and/or necrosis induced by UVA. Six hours after UVA-irradiation, 29.92% of cells were annexin-V positive. This population was significantly reduced in Trxtransfected cells (8.58%). Moreover, this work demonstrates that Trx prevents the loss of the membrane potential of the mitochondria, the depletion of cellular ATP content, and the loss of cell viability induced by irradiation.

Free Radical Biology and Medicine, 2001

Thioredoxin (Trx) plays important biological roles both intra-and extracellularly via thiol redox... more Thioredoxin (Trx) plays important biological roles both intra-and extracellularly via thiol redox control. We have previously demonstrated that Trx exhibited protective effects against UVA cytotoxicity in human skin fibroblasts. As an extension of the latter investigation, the present work is aimed at assessing ability of Trx to maintain genomic integrity in human skin fibroblasts upon exposure to UVA radiation. Indeed, UVA (320 -380 nm) is mutagenic and induces genomic damage to skin cells. The alkaline comet assay was used in association with DNA repair enzyme including formamido pyrimidine glycosylase (Fpg) and endonuclease III (endo III) to estimate the amount of modified bases together with the level of strand breaks and alkali-labile sites. The HPLC-EC assay was applied to assess 8-oxo-7,8-dihydro-2Ј-deoxyguanosine (8-oxodGuo) levels and to permit the calibration of comet assay as previously described. We reported that overexpression of human Trx (transient transfection) as well as exogenous human recombinant Trx added to the culture medium, decreased the level of DNA damage in UVA irradiated cells. Interestingly, transfection appeared to prevent UVA-induced 8-oxodGuo (3.06 au per Joules.cm Ϫ2 compared to 4.94 au per Joules.cm Ϫ2 for nontransfected cells). Moreover, Trx accumulates into nuclei in transfected cells. This finding supports the notion that Trx is important for the maintenance of the integrity of genetic information. This work demonstrated that under conditions of UVA oxidative stress, Trx prevented the UVA-induced DNA damage.

Science signaling, Jan 13, 2016

The nucleoside analog cytarabine, an inhibitor of DNA replication fork progression that results i... more The nucleoside analog cytarabine, an inhibitor of DNA replication fork progression that results in DNA damage, is currently used in the treatment of acute myeloid leukemia (AML). We explored the prognostic value of the expression of 72 genes involved in various aspects of DNA replication in a set of 198 AML patients treated by cytarabine-based chemotherapy. We unveiled that high expression of the DNA replication checkpoint gene CHEK1 is a prognostic marker associated with shorter overall, event-free, and relapse-free survivals and determined that the expression of CHEK1 can predict more frequent and earlier postremission relapse. CHEK1 encodes checkpoint kinase 1 (CHK1), which is activated by the kinase ATR when DNA replication is impaired by DNA damage. High abundance of CHK1 in AML patient cells correlated with higher clonogenic ability and more efficient DNA replication fork progression upon cytarabine treatment. Exposing the patient cells with the high abundance of CHK1 to SCH90...

Oncotarget, Jan 16, 2015

We investigated cell cycle regulation in acute myeloid leukemia cells expressing the FLT3-ITD mut... more We investigated cell cycle regulation in acute myeloid leukemia cells expressing the FLT3-ITD mutated tyrosine kinase receptor, an underexplored field in this disease. Upon FLT3 inhibition, CDC25A mRNA and protein were rapidly down-regulated, while levels of other cell cycle proteins remained unchanged. This regulation was dependent on STAT5, arguing for FLT3-ITD-dependent transcriptional regulation of CDC25A. CDC25 inhibitors triggered proliferation arrest and cell death of FLT3-ITD as well as FLT3-ITD/TKD AC-220 resistant cells, but not of FLT3-wt cells. Consistently, RNA interference-mediated knock-down of CDC25A reduced the proliferation of FLT3-ITD cell lines. Finally, the clonogenic capacity of primary FLT3-ITD AML cells was reduced by the CDC25 inhibitor IRC-083864, while FLT3-wt AML and normal CD34+ myeloid cells were unaffected. In good agreement, in a cohort of 100 samples from AML patients with intermediate-risk cytogenetics, high levels of CDC25A mRNA were predictive of ...

Leukemia Research, 2014

CHK1 Ser/Thr kinase, a well characterized regulator of DNA damage response, is also involved in n... more CHK1 Ser/Thr kinase, a well characterized regulator of DNA damage response, is also involved in normal cell cycle progression. In this study, we investigate how CHK1 participates to proliferation of acute myeloid leukemia cells expressing the mutated FLT3-ITD tyrosine kinase receptor. Pharmacological inhibition of CHK1 as well as its shRNA mediated down regulation reduced the proliferation rate of FLT-ITD expressing leukemic cell lines in a cytostatic manner. Flow cytometry analysis revealed no accumulation in a specific phase of the cell cycle upon CHK1 inhibition. Accordingly, lentiviral-mediated CHK1 overexpression increased the proliferation rate of FLT3-ITD expressing cells, as judged by cell viability and [3H] thymidine incorporation experiments. By contrast, expression of a ser280 mutant did not, suggesting that phosphorylation of this residue is an important determinant of CHK1 proliferative function. Clonogenic growth of primary leukemic cells from patients in semi-solid medium was reduced upon CHK1 inhibition, confirming the data obtained with leukemic established cell lines. Surprisingly, 3 out of 4 CHK1 inhibitory compounds tested in this study were also potent inhibitors of the FLT3-ITD tyrosine kinase receptor. Altogether, these data identify CHK1 as a regulator of FLT3-ITD-positive leukemic cells proliferation, and they open interesting perspectives in terms of new therapeutic strategies for these pathologies.

Trace Elements in Man and Animals 10, 2002

... Marie-Jeanne Richard, Nathalie Emonet-Piccardi, Christine Didier, Eric Jourdan, Marie-Thérèse... more ... Marie-Jeanne Richard, Nathalie Emonet-Piccardi, Christine Didier, Eric Jourdan, Marie-Thérèse Leccia, Marie ... Telephone: 476765147; Fax: 476765664; email: Marie-Jeanne.Richard@ujf-grenoble.fr ... Hanada, K., Sawamura, D., Tamai, K., Baba, T., Hashimoto, I., Muramatsu, T ...

RNF5 is a RING finger protein found to be important in the growth and development of Caenorhabdit... more RNF5 is a RING finger protein found to be important in the growth and development of Caenorhabditis elegans. The search for RNF5-associated proteins via a yeast two-hybrid screen identified a LIM-containing protein in C. elegans which shows homology with human paxillin. Here we demonstrate that the human homologue of RNF5 associates with the amino-terminal domain of paxillin, resulting in its ubiquitination. RNF5 requires intact RING and C-terminal domains to mediate paxillin ubiquitination. Whereas RNF5 mediates efficient ubiquitination of paxillin in vivo, protein extracts were required for in vitro ubiquitination, suggesting that additional modifications and/or an associated E3 ligase assist RNF5 targeting of paxillin ubiquitination. Mutant Ubc13 efficiently inhibits RNF5 ubiquitination, suggesting that RNF5 generates polychain ubiquitin of the K63 topology. Expression of RNF5 increases the cytoplasmic distribution of paxillin while decreasing its localization within focal adhesions, where it is primarily seen under normal growth. Concomitantly, RNF5 expression results in inhibition of cell motility. Via targeting of paxillin ubiquitination, which alters its localization, RNF5 emerges as a novel regulator of cell motility.

At the present time, the broad use of monoclonal antibodies (MAbs) in immunochemica l, structural... more At the present time, the broad use of monoclonal antibodies (MAbs) in immunochemica l, structural and functional studies of proteins, as well as in immunotherapy, and their capacity for monospecific interaction, reproducibility and production in large scale have made of these macromolecules a very important tool in bioengineering. The aim of this work was to develop an enzyme-linked immunosorbent assay

Oncogene, 2008

Acute myeloid leukemia (AML) cells exposed to genotoxic agents arrest their cell cycle at the G2/... more Acute myeloid leukemia (AML) cells exposed to genotoxic agents arrest their cell cycle at the G2/M checkpoint and are inherently chemoresistant. To understand the mechanism of this chemoresistance, we compared the ability of immature CD34 þ versus mature CD34À AML cell lines (KG1a and U937, respectively) to recover from a DNA damage-induced cell cycle checkpoint in G2. Here, we report that KG1a cells have a more stringent G2/M checkpoint response than U937 cells. We show that in both cell types, the CDC25B phosphatase participates in the G2/M checkpoint recovery and that its expression is upregulated. Furthermore, we show that CHK1 inhibition by UCN-01 in immature KG1a cells allows checkpoint exit and induces sensitivity to genotoxic agents. Similarly, UCN-01 treatment potentializes genotoxic-induced inhibition of colony formation efficiency of primary leukemic cells from AML patients. Altogether, our results demonstrate that checkpoint stringency varies during the maturation process and indicate that targeting checkpoint mechanisms might represent an attractive therapeutic opportunity for chemoresistant immature AML cells.

Molecular Biology of the Cell, 2007

Centrosomes are dynamic organelles that consist of a pair of cylindrical centrioles, surrounded b... more Centrosomes are dynamic organelles that consist of a pair of cylindrical centrioles, surrounded by pericentriolar material. The pericentriolar material contains factors that are involved in microtubule nucleation and organization, and its recruitment varies during the cell cycle. We report here that proteasome inhibition in HeLa cells induces the accumulation of several proteins at the pericentriolar material, including gamma-tubulin, GCP4, NEDD1, ninein, pericentrin, dynactin, and PCM-1. The effect of proteasome inhibition on centrosome proteins does not require intact microtubules and is reversed after removal of proteasome inhibitors. This accrual of centrosome proteins is paralleled by accumulation of ubiquitin in the same area and increased polyubiquitylation of nonsoluble gamma-tubulin. Cells that have accumulated centrosome proteins in response to proteasome inhibition are impaired in microtubule aster formation. Our data point toward a role of the proteasome in the turnover of centrosome proteins, to maintain proper centrosome function.

European Journal of Pharmacology, 2008

Polo-Kinase 1 (PLK1) is a key cell cycle regulator that is necessary for checkpoint recovery afte... more Polo-Kinase 1 (PLK1) is a key cell cycle regulator that is necessary for checkpoint recovery after DNA damageinduced G2 arrest. We have examined the effects of PLK inhibition in Acute Myelocytic Leukaemia (AML) cells, whose resistance to genotoxic agents is thought to be associated with checkpoint reinforcement. We report that in U937 AML cells, PLK1 participates in checkpoint recovery, and that inhibition of PLK by the GW843682X compound results in mitotic accumulation and apoptosis. We also found that when challenged with VP-16, inhibition of PLK1 prevented U937 cells from checkpoint exit. Finally, we found that treatment with GW843682X slightly reduced genotoxic-induced inhibition of colony formation efficiency of primary leukaemia cells (CFU-L) from AML patients.

Cancer Research, 2006

The effects of cell adhesion on leukemia cell proliferation remain poorly documented and somehow ... more The effects of cell adhesion on leukemia cell proliferation remain poorly documented and somehow controversial. In this work, we investigated the effect of adhesion to fibronectin on the proliferation of acute myeloid leukemia (AML) cell lines (U937 and KG1a) and CD34 + normal or leukemic primary cells. We observed an increased rate of proliferation of AML cells when adhered to fibronectin, concomitant with accelerated S-phase entry and accumulation of CDC25A. Conversely, normal CD34 + cell proliferation was decreased by adhesion to fibronectin with a concomitant drop in CDC25A expression. Importantly, we showed that both small interfering RNA (siRNA)-mediated CDC25A down-regulation and a recently developed CDC25 pharmacologic inhibitor impaired this adhesion-dependent proliferation, establishing a functional link between CDC25A accumulation and adhesion-dependent proliferation in leukemic cells. CDC25A accumulation was found only slightly dependent on transcriptional regulation and essentially due to modifications of the proteasomal degradation of the protein as shown using proteasome inhibitors and reverse transcription-PCR. Interestingly, CDC25A regulation was Chk1 dependent in these cells as suggested by siRNA-mediated down-regulation of this protein. Finally, we identified activation of the phosphatidylinositol 3-kinase/ Akt pathway as an adhesion-dependent regulation mechanism of CDC25A protein expression. Altogether, our data show that in leukemic cells adhesion to fibronectin increases CDC25A expression through proteasome-and Chk1-dependent mechanisms, resulting in enhanced proliferation. They also suggest that these adhesion-dependent proliferation properties of hematopoietic cells may be modified during leukemogenesis.