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Papers by Maria Gagkou

Research paper thumbnail of Supplementary Figures 1-6 from Suppression of Apoptosis by PIF1 Helicase in Human Tumor Cells

Research paper thumbnail of Supplementary Figures 1-6 from Suppression of Apoptosis by PIF1 Helicase in Human Tumor Cells

Supplementary Figures 1-6 from Suppression of Apoptosis by PIF1 Helicase in Human Tumor Cells

Research paper thumbnail of DNA replication stress in CHK1-depleted tumour cells triggers premature (S-phase) mitosis through inappropriate activation of Aurora kinase B

Cell Death and Disease, 2014

The disruption of DNA replication in cells triggers checkpoint responses that slowdown S-phase pr... more The disruption of DNA replication in cells triggers checkpoint responses that slowdown S-phase progression and protect replication fork integrity. These checkpoints are also determinants of cell fate and can help maintain cell viability or trigger cell death pathways. CHK1 has a pivotal role in such S-phase responses. It helps maintain fork integrity during replication stress and protects cells from several catastrophic fates including premature mitosis, premature chromosome condensation and apoptosis. Here we investigated the role of CHK1 in protecting cancer cells from premature mitosis and apoptosis. We show that premature mitosis (characterized by the induction of histone H3 phosphorylation, aberrant chromatin condensation, and persistent RPA foci in arrested S-phase cells) is induced in p53-deficient tumour cells depleted of CHK1 when DNA synthesis is disrupted. These events are accompanied by an activation of Aurora kinase B in S-phase cells that is essential for histone H3 Ser10 phosphorylation. Histone H3 phosphorylation precedes the induction of apoptosis in p53 À / À tumour cell lines but does not appear to be required for this fate as an Aurora kinase inhibitor suppresses phosphorylation of both Aurora B and histone H3 but has little effect on cell death. In contrast, only a small fraction of p53 þ / þ tumour cells shows this premature mitotic response, although they undergo a more rapid and robust apoptotic response. Taken together, our results suggest a novel role for CHK1 in the control of Aurora B activation during DNA replication stress and support the idea that premature mitosis is a distinct cell fate triggered by the disruption of DNA replication when CHK1 function is suppressed.

Research paper thumbnail of Enhanced H2AX phosphorylation, DNA replication fork arrest, and cell death in the absence of Chk1

H2AX phosphorylation at serine 139 (H2AX) is a sensitive indicator of both DNA damage and DNA rep... more H2AX phosphorylation at serine 139 (H2AX) is a sensitive indicator of both DNA damage and DNA replication stress. Here we show that H2AX formation is greatly enhanced in response to replication inhibitors but not ionizing radiation in HCT116 or SW480 cells depleted of Chk1. Although H2AX phosphorylation precedes the induction of apoptosis in such cells, our results suggest that cells containing H2AX are not committed to death. H2AX foci in these cells largely colocalize with RPA foci and their formation is dependent upon the essential replication helicase cofactor Cdc45, suggesting that H2AX phosphorylation occurs at sites of stalled forks. However Chk1-depleted cells released from replication inhibitors retain H2AX foci and do not appear to resume replicative DNA synthesis. BrdU incorporation only occurs in a minority of Chk1-depleted cells containing H2AX foci after release from thymidine arrest and, in cells incorporating BrdU, DNA synthesis does not occur at sites of H2AX foci. ...

Research paper thumbnail of The Journal of Cell Biology

BubR1 performs several roles during mitosis, affecting the spindle assembly checkpoint (SAC), mit... more BubR1 performs several roles during mitosis, affecting the spindle assembly checkpoint (SAC), mitotic timing, and spindle function, but the interdependence of these functions is unclear. We have analyzed in Drosophila melanogaster the mitotic phenotypes of kinase-dead (KD) BubR1 and BubR1 lacking the N-terminal KEN box. bubR1-KD individuals have a robust SAC but abnormal spindles with thin kinetochore fibers, suggesting that the kinase activity modulates microtubule capture

Research paper thumbnail of Meuth M: ATR and Chk1 suppress a caspase-3-dependent apoptotic response following DNA replication stress. PLoS Genet 2009

The related PIK-like kinases Ataxia-Telangiectasia Mutated (ATM) and ATM- and Rad3-related (ATR) ... more The related PIK-like kinases Ataxia-Telangiectasia Mutated (ATM) and ATM- and Rad3-related (ATR) play major roles in the regulation of cellular responses to DNA damage or replication stress. The pro-apoptotic role of ATM and p53 in response to ionizing radiation (IR) has been widely investigated. Much less is known about the control of apoptosis following DNA replication stress. Recent work indicates that Chk1, the downstream phosphorylation target of ATR, protects cells from apoptosis induced by DNA replication inhibitors as well as IR. The aim of the work reported here was to determine the roles of ATM- and ATR-protein kinase cascades in the control of apoptosis following replication stress and the relationship between Chk1-suppressed apoptotic pathways responding to replication stress or IR. ATM and ATR/Chk1 signalling pathways were manipulated using siRNA-mediated depletions or specific inhibitors in two tumour cell lines or fibroblasts derived from patients with inherited mutat...

Research paper thumbnail of Abstract B56: Human PIF1 helicase depletion sensitizes RAS-transformed human fibroblasts to gemcitabine and suppresses DNA replication fork progression and initiation efficiency

Molecular Cancer Therapeutics, 2013

Pif1 helicase is implicated in a wide range of DNA transactions that are required for orderly rep... more Pif1 helicase is implicated in a wide range of DNA transactions that are required for orderly replication and genome stability in lower and higher eukaryotes. While mouse pif1-/- knockouts show no phenotype and PIF1 is dispensable in non-cancerous human cells, Pif1 knockdowns sensitize many tumor cell lines to replication inhibitors, slow S-phase progression, and trigger apoptosis. Here we investigated the effects of PIF1 depletion on a non-tumor cell line, MRC5-SV2, and HRAS-transformed derivatives. We show that PIF1 depletion reduces the growth and viability of RAS-transformed MRC5-SV2 cells relative to parental and increases sensitivity to the therapeutic DNA replication inhibitor gemcitabine. Since previous work suggested that PIF1 depletion affected S-phase entry and transition in cultured tumor cells, we used single fiber analysis of pulse-labeled DNA to investigate the potential role of PIF1 in these events. Here we show that PIF1 depletion triggers replication stress characterized by slower fork rates and increased fork arrest during normal cycling conditions in non-tumor MRC5-SV2 cells. These effects were enhanced in the RAS-transformed derivative. Impaired fork movement upon PIF1-depletion did not augment DNA double-stranded break formation or trigger DNA damage responses. However PIF1 depletion affected resumption of DNA synthesis after prolonged replication inhibitor exposure, accompanied by diminished new origin firing and S-phase entry. Taken together, our data establish a novel functional role for human PIF1 in DNA replication that becomes critical for cell growth during oncogenic stress. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B56. Citation Format: Mary E. Gagou, Anil Ganesh, Mark Meuth. Human PIF1 helicase depletion sensitizes RAS-transformed human fibroblasts to gemcitabine and suppresses DNA replication fork progression and initiation efficiency. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B56.

Research paper thumbnail of Separating the spindle, checkpoint, and timer functions of BubR1

The Journal of Cell Biology, 2009

The BubR1 kinase domain controls spindle attachment to the kinetochores, whereas the KEN domain r... more The BubR1 kinase domain controls spindle attachment to the kinetochores, whereas the KEN domain regulates activation of the spindle assembly checkpoint.

Research paper thumbnail of Human Embryonic Stem Cells Fail to Activate CHK1 and Commit to Apoptosis in Response to DNA Replication Stress

STEM CELLS, 2012

Pluripotent cells of the early embryo, to which embryonic stem cells (ESCs) correspond, give rise... more Pluripotent cells of the early embryo, to which embryonic stem cells (ESCs) correspond, give rise to all the somatic cells of the developing fetus. Any defects that occur in their genome or epigenome would have devastating consequences. Genetic and epigenetic change in human ESCs appear to be an inevitable consequence of long-term culture, driven by selection of variant cells that have a higher propensity for self-renewal rather than either differentiation or death. Mechanisms underlying the potentially separate events of mutation and subsequent selection of variants are poorly understood. Here, we show that human ESCs and their malignant counterpart, embryonal carcinoma (EC) cells, both fail to activate critical S-phase checkpoints when exposed to DNA replication inhibitors and commit to apoptosis instead. Human ESCs and EC cells also fail to form replication protein A, cH2AX, or RAD51 foci or load topoisomerase (DNA) II binding protein 1 onto chromatin in response to replication inhibitors. Furthermore, direct measurements of single-stranded DNA (ssDNA) show that these cells fail to generate the ssDNA regions in response to replication stress that are necessary for the activation of checkpoints and the initiation of homologous recombination repair to protect replication fork integrity and restart DNA replication. Taken together, our data suggest that pluripotent cells control genome integrity by the elimination of damaged cells through apoptosis rather than DNA repair, and therefore, mutations or epigenetic modifications resulting in an imbalance in cell death control could lead to genetic instability.

Research paper thumbnail of ATR and Chk1 Suppress a Caspase3–Dependent Apoptotic Response Following DNA Replication Stress

Plos Genetics, 2009

The related PIK-like kinases Ataxia-Telangiectasia Mutated (ATM) and ATM-and Rad3-related (ATR) p... more The related PIK-like kinases Ataxia-Telangiectasia Mutated (ATM) and ATM-and Rad3-related (ATR) play major roles in the regulation of cellular responses to DNA damage or replication stress. The pro-apoptotic role of ATM and p53 in response to ionizing radiation (IR) has been widely investigated. Much less is known about the control of apoptosis following DNA replication stress. Recent work indicates that Chk1, the downstream phosphorylation target of ATR, protects cells from apoptosis induced by DNA replication inhibitors as well as IR. The aim of the work reported here was to determine the roles of ATM-and ATR-protein kinase cascades in the control of apoptosis following replication stress and the relationship between Chk1-suppressed apoptotic pathways responding to replication stress or IR. ATM and ATR/Chk1 signalling pathways were manipulated using siRNA-mediated depletions or specific inhibitors in two tumour cell lines or fibroblasts derived from patients with inherited mutations. We show that depletion of ATM or its downstream phosphorylation targets, NBS1 and BID, has relatively little effect on apoptosis induced by DNA replication inhibitors, while ATR or Chk1 depletion strongly enhances cell death induced by such agents in all cells tested. Furthermore, early events occurring after the disruption of DNA replication (accumulation of RPA foci and RPA34 hyperphosphorylation) in ATR-or Chk1-depleted cells committed to apoptosis are not detected in ATM-depleted cells. Unlike the Chk1-suppressed pathway responding to IR, the replication stress-triggered apoptotic pathway did not require ATM and is characterized by activation of caspase 3 in both p53-proficient and-deficient cells. Taken together, our results show that the ATR-Chk1 signalling pathway plays a major role in the regulation of death in response to DNA replication stress and that the Chk1-suppressed pathway protecting cells from replication stress is clearly distinguishable from that protecting cells from IR.

Research paper thumbnail of Recruitment of Mad2 to the kinetochore requires the Rod/Zw10 complex

Current biology, 2005

cytoplasmic in interphase [7, 11, 12], whereas Mad2 is associated with the nucleoplasm and nuclea... more cytoplasmic in interphase [7, 11, 12], whereas Mad2 is associated with the nucleoplasm and nuclear envelope Avenue de la Terrasse 91198 Gif sur Yvette [11, 13, 14] (Figure 1A). In fly neuroblasts, as in Hela cells [15] but unlike in PtK cells [11], BubR1 is the first France 2 Institute of Cell and Molecular Biosciences to accumulate on kinetochores during prophase (at very low levels initially); it precedes Mad2 and Rod by The Medical School Framlington Place 2-5 min (Movie S1). Mad2 and Rod begin to label kinetochores only during nuclear-envelope breakdown (NEB), University of Newcastle Newcastle NE2 4HH easily recognized by the invasion of Rod into the nu-United Kingdom cleoplasm. The first kinetochore-associated Mad2 signals above the nucleoplasmic background are seen simultaneously with the first Rod signal (Figure 1A and Summary Movie S2). In prometaphase, the kinetochores brightly label with Compromising the activity of the spindle checkpoint all three proteins (Figure 1B). Because cytoplasmic permits mitotic exit in the presence of unattached ki-Mad2 signal is consistently higher than either BubR1 or netochores and, consequently, greatly increases the Rod, Mad2 kinetochore labeling appears relatively less rate of aneuploidy in the daughter cells [1-3]. The metaprominent. As the kinetochores capture MTs, Mad2 and zoan checkpoint mechanism is more complex than in Rod both are transported poleward (Figures 1B-1D; yeast in that it requires additional proteins and activi-Movies S3 and S4; and Figures S2 and S4), again conties besides the classical Mads and Bubs. Among sistent with previous reports [7, 9]. This process, called these are Rod, Zw10, and Zwilch, components of a "shedding," requires dynein/dynactin and may be im-700 Kdal complex (Rod/Zw10) [4-6] that is required portant for shutting off the checkpoint once MTs are for recruitment of dynein/dynactin to kinetochores [7, properly attached [7, 16-18]. 8] but whose role in the checkpoint is poorly under-These live images reveal a robustness that was not stood. The dynamics of Rod and Mad2, examined in evident for Mad2 transport in earlier studies in PtK cells different organisms, show intriguing similarities as and Drosophila cells [9, 16, 19], although it can be seen well as apparent differences [7, 9]. Here we simultasometimes even by immunostaining (Figure S5). It is neously follow GFP-Mad2 and RFP-Rod and find they difficult to quantify these signals, but the films clearly are in fact closely associated throughout early mitoshow that new cytosolic Mad2 is continuously recruited sis. They accumulate simultaneously on kinetochores to kinetochores even after MT capture and replaces and are shed together along microtubule fibers after that lost to shedding; the total Mad2 signal on KMTs attachment. Their behavior and position within atover the duration of prometaphase and metaphase is tached kinetochores is distinct from that of BubR1; far greater than the original kinetochore-associated sig-Mad2 and Rod colocalize to the outermost kinetnal. This is particularly evident in Movie S4, where ochore region (the corona), whereas BubR1 is slightly metaphase is prolonged. Thus Mad2, like Rod, estabmore interior. Moreover, Mad2, but not BubR1, Bub1, lishes a flux of recruitment to and shedding from at-Bub3, or Mps1, requires Rod/Zw10 for its accumulatached kinetochores. tion on unattached kinetochores. Rod/Zw10 thus con-GFP-Rod and RFP-Mad2 show a near-perfect coincitributes to checkpoint activation by promoting Mad2 dence of signal in prometaphase and early metaphase, recruitment and to checkpoint inactivation by recruitnot only on kinetochores but also along the KMTs (Figing dynein/dynactin that subsequently removes Mad2 ures 1C and 1D; Movies S3 and S4). The overall patfrom attached kinetochores. terns of the two proteins are superimposable (Figures 1C and 1D, Figure S4). Where discrete particles of GFP-Results and Discussion Mad2 could be followed, they always contained RFP-Rod (Figure 1D and Figure S4). These results suggest Rod and Mad2 Colocalize throughout Prometaphase that Mad2 and Rod/Zw10 remain associated as they and Early Metaphase, but BubR1 Does Not leave the kinetochore along the KMTs. To gain insight into the role of Rod/Zw10 relative to By late metaphase, Mad2 signal has essentially disother checkpoint proteins, we undertook a study of fluappeared from kinetochores and is only faintly visible orescently tagged (GFP and mRFP1 [10]) Rod on the spindle above the cytoplasmic Mad2 back-(CG1569), Mad2 (CG17498), and BubR1 (CG7838) in a ground, whereas Rod shedding continues robustly up single cell type, the Drosophila larval neuroblast. All to anaphase onset (Figure 1C and Movie S3). In larval three fusion proteins are controlled by their natural proneuroblasts, the timing of NEB to anaphase onset is typically 7-12 min, of which metaphase lasts 2-8 min (see also [20]). There does not appear to be much delay

Research paper thumbnail of BubR1-and Polo-coated DNA tethers facilitate poleward segregation of acentric chromatids

The mechanisms that safeguard cells against chromosomal instability (CIN) are of great interest, ... more The mechanisms that safeguard cells against chromosomal instability (CIN) are of great interest, as CIN contributes to tumorigenesis. To gain insight into these mechanisms, we studied the behavior of cells entering mitosis with damaged chromosomes. We used the endonuclease I-CreI to generate acentric chromosomes in Drosophila larvae. While I-CreI expression produces acentric chromosomes in the majority of neuronal stem cells, remarkably, it has no effect on adult survival. Our live studies reveal that acentric chromatids segregate efficiently to opposite poles. The acentric chromatid poleward movement is mediated through DNA tethers decorated with BubR1, Polo, INCENP, and Aurora-B. Reduced BubR1 or Polo function results in abnormal segregation of acentric chromatids, a decrease in acentric chromosome tethering, and a great reduction in adult survival. We propose that BubR1 and Polo facilitate the accurate segregation of acentric chromatids by maintaining the integrity of the tethers that connect acentric chromosomes to their centric partners.

Research paper thumbnail of Extensive RPA2 hyperphosphorylation promotes apoptosis in response to DNA replication stress in CHK1 inhibited cells

Nucleic Acids Research, 2015

The replication protein A (RPA)-ssDNA complex formed at arrested replication forks recruits key p... more The replication protein A (RPA)-ssDNA complex formed at arrested replication forks recruits key proteins to activate the ATR-CHK1 signalling cascade. When CHK1 is inhibited during DNA replication stress, RPA2 is extensively hyperphosphorylated. Here, we investigated the role of RPA2 hyperphosphorylation in the fate of cells when CHK1 is inhibited. We show that proteins normally involved in DNA repair (RAD51) or control of RPA phosphorylation (the PP4 protein phosphatase complex) are not recruited to the genome after treatment with CHK1 and DNA synthesis inhibitors. This is not due to RPA2 hyperphosphorylation as suppression of this response does not restore loading suggesting that recruitment requires active CHK1. To determine whether RPA2 hyperphosphorylation protects stalled forks from collapse or induction of apoptosis in CHK1 inhibited cells during replication stress, cells expressing RPA2 genes mutated at key phosphorylation sites were characterized. Mutant RPA2 rescued cells from RPA2 depletion and reduced the level of apoptosis induced by treatment with CHK1 and replication inhibitors however the incidence of double strand breaks was not affected. Our data indicate that RPA2 hyperphosphorylation promotes cell death during replication stress when CHK1 function is compromised but does not appear to be essential for replication fork integrity.

Research paper thumbnail of ATR (ataxia telangiectasia and Rad3 related)

Atlas of Genetics and Cytogenetics in Oncology and Haematology, 2011

This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 ... more This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence.

Research paper thumbnail of Human PIF1 helicase supports DNA replication and cell growth under oncogenic-stress

Oncotarget, Jan 30, 2014

Unwinding duplex DNA is a critical processing step during replication, repair and transcription. ... more Unwinding duplex DNA is a critical processing step during replication, repair and transcription. Pif1 are highly conserved non-processive 5'->3' DNA helicases with well-established roles in maintenance of yeast genome stability. However, the function of the sole member of Pif1 family in humans remains unclear. Human PIF1 is essential for tumour cell viability, particularly during replication stress, but is dispensable in non-cancerous cells and Pif1 deficient mice. Here we report that suppression of PIF1 function slows replication fork rates and increases arrested forks during normal cycling conditions. Importantly, PIF1-dependent replication impediments impair S-phase progression and reduce proliferation rates of RAS oncogene-transformed fibroblasts, where replication fork slowing is exacerbated, but not parental, non-cancerous cells. Disrupted fork movement upon PIF1-depletion does not enhance double-stranded break formation or DNA damage responses but affects resumptio...

Research paper thumbnail of The ribosomal P-proteins of the medfly Ceratitis capitata form a heterogeneous stalk structure interacting with the endogenous P-proteins, in conditional P0-null strains of the yeast Saccharomyces cerevisiae

Nucleic Acids Research, 2000

The genes encoding the ribosomal P-proteins CcP0, CcP1 and CcP2 of Ceratitis capitata were expres... more The genes encoding the ribosomal P-proteins CcP0, CcP1 and CcP2 of Ceratitis capitata were expressed in the conditional P0-null strains W303dGP0 and D67dGP0 of Saccharomyces cerevisiae, the ribosomes of which contain either standard amounts or are totally deprived of the P1/P2 proteins, respectively. The presence of the CcP0 protein restored cell viability but reduced the growth rate. In the W303CcP0 strain, all four acidic yeast proteins were found on the ribosomes, but in notably less quantity, while a preferable binding of the YP1α/YP2β pair was established. In the absence of the endogenous P1/P2 proteins in the D67CcP0 strain, the complementation capacity of the CcP0 protein was considerably reduced. The simultaneous expression of the three medfly genes resulted in alterations of the stalk composition: both the CcP1 and CcP2 proteins were found on the particles substituting the YP1α and YP2α proteins, respectively, but their presence did not alter the growth rate, except in the case of the YP1α/β defective strain, where a helping effect on the binding of the YP2α and YP2β proteins on the ribosomes was confirmed. Therefore, the medfly ribosomal P-proteins complement the yeast P-protein deficient strains forming an heterogeneous ribosomal stalk, which, however, is not functionally equivalent to the endogenous one.

Research paper thumbnail of Enhanced H2AX Phosphorylation, DNA Replication Fork Arrest, and Cell Death in the Absence of Chk1

Molecular Biology of the Cell, 2010

H2AX phosphorylation at serine 139 (γH2AX) is a sensitive indicator of both DNA damage and DNA re... more H2AX phosphorylation at serine 139 (γH2AX) is a sensitive indicator of both DNA damage and DNA replication stress. Here we show that γH2AX formation is greatly enhanced in response to replication inhibitors but not ionizing radiation in HCT116 or SW480 cells depleted of Chk1. Although H2AX phosphorylation precedes the induction of apoptosis in such cells, our results suggest that cells containing γH2AX are not committed to death. γH2AX foci in these cells largely colocalize with RPA foci and their formation is dependent upon the essential replication helicase cofactor Cdc45, suggesting that H2AX phosphorylation occurs at sites of stalled forks. However Chk1-depleted cells released from replication inhibitors retain γH2AX foci and do not appear to resume replicative DNA synthesis. BrdU incorporation only occurs in a minority of Chk1-depleted cells containing γH2AX foci after release from thymidine arrest and, in cells incorporating BrdU, DNA synthesis does not occur at sites of γH2AX...

Research paper thumbnail of Targeted Expression of the Class II Phosphoinositide 3-Kinase in Drosophila melanogaster Reveals Lipid Kinase-Dependent Effects on Patterning and Interactions with Receptor Signaling Pathways

Molecular and Cellular Biology, 2004

Phosphoinositide 3-kinases (PI3Ks) can be divided into three distinct classes (I, II, and III) on... more Phosphoinositide 3-kinases (PI3Ks) can be divided into three distinct classes (I, II, and III) on the basis of their domain structures and the lipid signals that they generate. Functions have been assigned to the class I and class III enzymes but have not been established for the class II PI3Ks. We have obtained the first evidence for a biological function for a class II PI3K by expressing this enzyme during Drosophila melanogaster development and by using deficiencies that remove the endogenous gene. Wild-type and catalytically inactive PI3K_68D transgenes have opposite effects on the number of sensory bristles and on wing venation phenotypes induced by modified epidermal growth factor (EGF) receptor signaling. These results indicate that the endogenous PI3K_68D may act antagonistically to the EGF receptor-stimulated Ras-mitogen-activated protein kinase pathway and downstream of, or parallel to, the Notch receptor. A class II polyproline motif in PI3K_68D can bind the Drk adaptor protein in vitro, primarily via the N-terminal SH3 domain of Drk. Drk may thus be important for the localization of PI3K_68D, allowing it to modify signaling pathways downstream of cell surface receptors. The phenotypes obtained are markedly distinct from those generated by expression of the Drosophila class I PI3K, which affects growth but not pattern formation.

Research paper thumbnail of Characterization of acidic ribosomal proteins from three developmental stages of the medfly Ceratitis capitata

IUBMB Life, 1998

Acidic proteins extracted with 0.5 M NH4Cl and 50% ethanol from ribosomes of the medfly Ceratitis... more Acidic proteins extracted with 0.5 M NH4Cl and 50% ethanol from ribosomes of the medfly Ceratitis capitata showed two major bands of MW 15 and 17 kD after SDS electrophoresis. Isoelectrofocusing of acidic proteins resolved two groups of bands at pH 4.5 and 3.5. Similar patterns were observed both from the acidic ribosomal protein fraction and from total ribosomes, treated with RNase. Treatment with alkaline phosphatase reduced the number of bands with a shift to a higher pI, indicating dephosphorylation. The phosphorylation pattern of the acidic proteins changed at three different stages of development, six day larvae, white pupae and 0-2 days old embryos. The two protein groups correspond to multi-phosphorylated forms of eucaryotic acidic ribosomal proteins P1 and P2. This was shown by immunoblotting with specific monoclonal antibodies.

Research paper thumbnail of Stage-specific expression of the chitin synthase DmeChSA and DmeChSB genes during the onset of Drosophila metamorphosis

Insect Biochemistry and Molecular Biology, 2002

Chitin, the major structural polysaccharide of arthropods, is an important constituent of the ins... more Chitin, the major structural polysaccharide of arthropods, is an important constituent of the insect extracellular structures, cuticle and gut peritrophic matrix. Synthesis of cuticular chitin is strictly coordinated with the ecdysone-regulated molting cycle of insect development (the term "ecdysone" is used in this paper instead of "ecdysteroids" since the exact ratio of various hormonal forms changes during metamorphosis). Based on observed similarities between the fungal chitin synthases and other processive β-glycosyltransferases, we have identified the first insect chitin synthase genes, DmeChSA and DmeChSB (Database accession numbers: EMBL/GenBank/DDBJ A83122, A83126, AJ309488, AJ309489), from Drosophila melanogaster. Chromosomal localization has identified these genes close to and on either side of the centromere of the third chromosome. Partial cDNA clones of both genes have been isolated from a pupal cDNA library. To obtain the first insight into the transcriptional regulation of chitin synthesis, we have monitored the expression of DmeChSA and DmeChSB during the periods of the late-larval and prepupal ecdysone pulses that direct metamorphosis. Transcripts of either gene are barely detected prior to and during the late-larval ecdysone pulse. Once the late-larval ecdysone pulse is ceased completely, both DmeChSA and DmeChSB genes show a remarkable up-regulation.

Research paper thumbnail of Supplementary Figures 1-6 from Suppression of Apoptosis by PIF1 Helicase in Human Tumor Cells

Research paper thumbnail of Supplementary Figures 1-6 from Suppression of Apoptosis by PIF1 Helicase in Human Tumor Cells

Supplementary Figures 1-6 from Suppression of Apoptosis by PIF1 Helicase in Human Tumor Cells

Research paper thumbnail of DNA replication stress in CHK1-depleted tumour cells triggers premature (S-phase) mitosis through inappropriate activation of Aurora kinase B

Cell Death and Disease, 2014

The disruption of DNA replication in cells triggers checkpoint responses that slowdown S-phase pr... more The disruption of DNA replication in cells triggers checkpoint responses that slowdown S-phase progression and protect replication fork integrity. These checkpoints are also determinants of cell fate and can help maintain cell viability or trigger cell death pathways. CHK1 has a pivotal role in such S-phase responses. It helps maintain fork integrity during replication stress and protects cells from several catastrophic fates including premature mitosis, premature chromosome condensation and apoptosis. Here we investigated the role of CHK1 in protecting cancer cells from premature mitosis and apoptosis. We show that premature mitosis (characterized by the induction of histone H3 phosphorylation, aberrant chromatin condensation, and persistent RPA foci in arrested S-phase cells) is induced in p53-deficient tumour cells depleted of CHK1 when DNA synthesis is disrupted. These events are accompanied by an activation of Aurora kinase B in S-phase cells that is essential for histone H3 Ser10 phosphorylation. Histone H3 phosphorylation precedes the induction of apoptosis in p53 À / À tumour cell lines but does not appear to be required for this fate as an Aurora kinase inhibitor suppresses phosphorylation of both Aurora B and histone H3 but has little effect on cell death. In contrast, only a small fraction of p53 þ / þ tumour cells shows this premature mitotic response, although they undergo a more rapid and robust apoptotic response. Taken together, our results suggest a novel role for CHK1 in the control of Aurora B activation during DNA replication stress and support the idea that premature mitosis is a distinct cell fate triggered by the disruption of DNA replication when CHK1 function is suppressed.

Research paper thumbnail of Enhanced H2AX phosphorylation, DNA replication fork arrest, and cell death in the absence of Chk1

H2AX phosphorylation at serine 139 (H2AX) is a sensitive indicator of both DNA damage and DNA rep... more H2AX phosphorylation at serine 139 (H2AX) is a sensitive indicator of both DNA damage and DNA replication stress. Here we show that H2AX formation is greatly enhanced in response to replication inhibitors but not ionizing radiation in HCT116 or SW480 cells depleted of Chk1. Although H2AX phosphorylation precedes the induction of apoptosis in such cells, our results suggest that cells containing H2AX are not committed to death. H2AX foci in these cells largely colocalize with RPA foci and their formation is dependent upon the essential replication helicase cofactor Cdc45, suggesting that H2AX phosphorylation occurs at sites of stalled forks. However Chk1-depleted cells released from replication inhibitors retain H2AX foci and do not appear to resume replicative DNA synthesis. BrdU incorporation only occurs in a minority of Chk1-depleted cells containing H2AX foci after release from thymidine arrest and, in cells incorporating BrdU, DNA synthesis does not occur at sites of H2AX foci. ...

Research paper thumbnail of The Journal of Cell Biology

BubR1 performs several roles during mitosis, affecting the spindle assembly checkpoint (SAC), mit... more BubR1 performs several roles during mitosis, affecting the spindle assembly checkpoint (SAC), mitotic timing, and spindle function, but the interdependence of these functions is unclear. We have analyzed in Drosophila melanogaster the mitotic phenotypes of kinase-dead (KD) BubR1 and BubR1 lacking the N-terminal KEN box. bubR1-KD individuals have a robust SAC but abnormal spindles with thin kinetochore fibers, suggesting that the kinase activity modulates microtubule capture

Research paper thumbnail of Meuth M: ATR and Chk1 suppress a caspase-3-dependent apoptotic response following DNA replication stress. PLoS Genet 2009

The related PIK-like kinases Ataxia-Telangiectasia Mutated (ATM) and ATM- and Rad3-related (ATR) ... more The related PIK-like kinases Ataxia-Telangiectasia Mutated (ATM) and ATM- and Rad3-related (ATR) play major roles in the regulation of cellular responses to DNA damage or replication stress. The pro-apoptotic role of ATM and p53 in response to ionizing radiation (IR) has been widely investigated. Much less is known about the control of apoptosis following DNA replication stress. Recent work indicates that Chk1, the downstream phosphorylation target of ATR, protects cells from apoptosis induced by DNA replication inhibitors as well as IR. The aim of the work reported here was to determine the roles of ATM- and ATR-protein kinase cascades in the control of apoptosis following replication stress and the relationship between Chk1-suppressed apoptotic pathways responding to replication stress or IR. ATM and ATR/Chk1 signalling pathways were manipulated using siRNA-mediated depletions or specific inhibitors in two tumour cell lines or fibroblasts derived from patients with inherited mutat...

Research paper thumbnail of Abstract B56: Human PIF1 helicase depletion sensitizes RAS-transformed human fibroblasts to gemcitabine and suppresses DNA replication fork progression and initiation efficiency

Molecular Cancer Therapeutics, 2013

Pif1 helicase is implicated in a wide range of DNA transactions that are required for orderly rep... more Pif1 helicase is implicated in a wide range of DNA transactions that are required for orderly replication and genome stability in lower and higher eukaryotes. While mouse pif1-/- knockouts show no phenotype and PIF1 is dispensable in non-cancerous human cells, Pif1 knockdowns sensitize many tumor cell lines to replication inhibitors, slow S-phase progression, and trigger apoptosis. Here we investigated the effects of PIF1 depletion on a non-tumor cell line, MRC5-SV2, and HRAS-transformed derivatives. We show that PIF1 depletion reduces the growth and viability of RAS-transformed MRC5-SV2 cells relative to parental and increases sensitivity to the therapeutic DNA replication inhibitor gemcitabine. Since previous work suggested that PIF1 depletion affected S-phase entry and transition in cultured tumor cells, we used single fiber analysis of pulse-labeled DNA to investigate the potential role of PIF1 in these events. Here we show that PIF1 depletion triggers replication stress characterized by slower fork rates and increased fork arrest during normal cycling conditions in non-tumor MRC5-SV2 cells. These effects were enhanced in the RAS-transformed derivative. Impaired fork movement upon PIF1-depletion did not augment DNA double-stranded break formation or trigger DNA damage responses. However PIF1 depletion affected resumption of DNA synthesis after prolonged replication inhibitor exposure, accompanied by diminished new origin firing and S-phase entry. Taken together, our data establish a novel functional role for human PIF1 in DNA replication that becomes critical for cell growth during oncogenic stress. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B56. Citation Format: Mary E. Gagou, Anil Ganesh, Mark Meuth. Human PIF1 helicase depletion sensitizes RAS-transformed human fibroblasts to gemcitabine and suppresses DNA replication fork progression and initiation efficiency. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B56.

Research paper thumbnail of Separating the spindle, checkpoint, and timer functions of BubR1

The Journal of Cell Biology, 2009

The BubR1 kinase domain controls spindle attachment to the kinetochores, whereas the KEN domain r... more The BubR1 kinase domain controls spindle attachment to the kinetochores, whereas the KEN domain regulates activation of the spindle assembly checkpoint.

Research paper thumbnail of Human Embryonic Stem Cells Fail to Activate CHK1 and Commit to Apoptosis in Response to DNA Replication Stress

STEM CELLS, 2012

Pluripotent cells of the early embryo, to which embryonic stem cells (ESCs) correspond, give rise... more Pluripotent cells of the early embryo, to which embryonic stem cells (ESCs) correspond, give rise to all the somatic cells of the developing fetus. Any defects that occur in their genome or epigenome would have devastating consequences. Genetic and epigenetic change in human ESCs appear to be an inevitable consequence of long-term culture, driven by selection of variant cells that have a higher propensity for self-renewal rather than either differentiation or death. Mechanisms underlying the potentially separate events of mutation and subsequent selection of variants are poorly understood. Here, we show that human ESCs and their malignant counterpart, embryonal carcinoma (EC) cells, both fail to activate critical S-phase checkpoints when exposed to DNA replication inhibitors and commit to apoptosis instead. Human ESCs and EC cells also fail to form replication protein A, cH2AX, or RAD51 foci or load topoisomerase (DNA) II binding protein 1 onto chromatin in response to replication inhibitors. Furthermore, direct measurements of single-stranded DNA (ssDNA) show that these cells fail to generate the ssDNA regions in response to replication stress that are necessary for the activation of checkpoints and the initiation of homologous recombination repair to protect replication fork integrity and restart DNA replication. Taken together, our data suggest that pluripotent cells control genome integrity by the elimination of damaged cells through apoptosis rather than DNA repair, and therefore, mutations or epigenetic modifications resulting in an imbalance in cell death control could lead to genetic instability.

Research paper thumbnail of ATR and Chk1 Suppress a Caspase3–Dependent Apoptotic Response Following DNA Replication Stress

Plos Genetics, 2009

The related PIK-like kinases Ataxia-Telangiectasia Mutated (ATM) and ATM-and Rad3-related (ATR) p... more The related PIK-like kinases Ataxia-Telangiectasia Mutated (ATM) and ATM-and Rad3-related (ATR) play major roles in the regulation of cellular responses to DNA damage or replication stress. The pro-apoptotic role of ATM and p53 in response to ionizing radiation (IR) has been widely investigated. Much less is known about the control of apoptosis following DNA replication stress. Recent work indicates that Chk1, the downstream phosphorylation target of ATR, protects cells from apoptosis induced by DNA replication inhibitors as well as IR. The aim of the work reported here was to determine the roles of ATM-and ATR-protein kinase cascades in the control of apoptosis following replication stress and the relationship between Chk1-suppressed apoptotic pathways responding to replication stress or IR. ATM and ATR/Chk1 signalling pathways were manipulated using siRNA-mediated depletions or specific inhibitors in two tumour cell lines or fibroblasts derived from patients with inherited mutations. We show that depletion of ATM or its downstream phosphorylation targets, NBS1 and BID, has relatively little effect on apoptosis induced by DNA replication inhibitors, while ATR or Chk1 depletion strongly enhances cell death induced by such agents in all cells tested. Furthermore, early events occurring after the disruption of DNA replication (accumulation of RPA foci and RPA34 hyperphosphorylation) in ATR-or Chk1-depleted cells committed to apoptosis are not detected in ATM-depleted cells. Unlike the Chk1-suppressed pathway responding to IR, the replication stress-triggered apoptotic pathway did not require ATM and is characterized by activation of caspase 3 in both p53-proficient and-deficient cells. Taken together, our results show that the ATR-Chk1 signalling pathway plays a major role in the regulation of death in response to DNA replication stress and that the Chk1-suppressed pathway protecting cells from replication stress is clearly distinguishable from that protecting cells from IR.

Research paper thumbnail of Recruitment of Mad2 to the kinetochore requires the Rod/Zw10 complex

Current biology, 2005

cytoplasmic in interphase [7, 11, 12], whereas Mad2 is associated with the nucleoplasm and nuclea... more cytoplasmic in interphase [7, 11, 12], whereas Mad2 is associated with the nucleoplasm and nuclear envelope Avenue de la Terrasse 91198 Gif sur Yvette [11, 13, 14] (Figure 1A). In fly neuroblasts, as in Hela cells [15] but unlike in PtK cells [11], BubR1 is the first France 2 Institute of Cell and Molecular Biosciences to accumulate on kinetochores during prophase (at very low levels initially); it precedes Mad2 and Rod by The Medical School Framlington Place 2-5 min (Movie S1). Mad2 and Rod begin to label kinetochores only during nuclear-envelope breakdown (NEB), University of Newcastle Newcastle NE2 4HH easily recognized by the invasion of Rod into the nu-United Kingdom cleoplasm. The first kinetochore-associated Mad2 signals above the nucleoplasmic background are seen simultaneously with the first Rod signal (Figure 1A and Summary Movie S2). In prometaphase, the kinetochores brightly label with Compromising the activity of the spindle checkpoint all three proteins (Figure 1B). Because cytoplasmic permits mitotic exit in the presence of unattached ki-Mad2 signal is consistently higher than either BubR1 or netochores and, consequently, greatly increases the Rod, Mad2 kinetochore labeling appears relatively less rate of aneuploidy in the daughter cells [1-3]. The metaprominent. As the kinetochores capture MTs, Mad2 and zoan checkpoint mechanism is more complex than in Rod both are transported poleward (Figures 1B-1D; yeast in that it requires additional proteins and activi-Movies S3 and S4; and Figures S2 and S4), again conties besides the classical Mads and Bubs. Among sistent with previous reports [7, 9]. This process, called these are Rod, Zw10, and Zwilch, components of a "shedding," requires dynein/dynactin and may be im-700 Kdal complex (Rod/Zw10) [4-6] that is required portant for shutting off the checkpoint once MTs are for recruitment of dynein/dynactin to kinetochores [7, properly attached [7, 16-18]. 8] but whose role in the checkpoint is poorly under-These live images reveal a robustness that was not stood. The dynamics of Rod and Mad2, examined in evident for Mad2 transport in earlier studies in PtK cells different organisms, show intriguing similarities as and Drosophila cells [9, 16, 19], although it can be seen well as apparent differences [7, 9]. Here we simultasometimes even by immunostaining (Figure S5). It is neously follow GFP-Mad2 and RFP-Rod and find they difficult to quantify these signals, but the films clearly are in fact closely associated throughout early mitoshow that new cytosolic Mad2 is continuously recruited sis. They accumulate simultaneously on kinetochores to kinetochores even after MT capture and replaces and are shed together along microtubule fibers after that lost to shedding; the total Mad2 signal on KMTs attachment. Their behavior and position within atover the duration of prometaphase and metaphase is tached kinetochores is distinct from that of BubR1; far greater than the original kinetochore-associated sig-Mad2 and Rod colocalize to the outermost kinetnal. This is particularly evident in Movie S4, where ochore region (the corona), whereas BubR1 is slightly metaphase is prolonged. Thus Mad2, like Rod, estabmore interior. Moreover, Mad2, but not BubR1, Bub1, lishes a flux of recruitment to and shedding from at-Bub3, or Mps1, requires Rod/Zw10 for its accumulatached kinetochores. tion on unattached kinetochores. Rod/Zw10 thus con-GFP-Rod and RFP-Mad2 show a near-perfect coincitributes to checkpoint activation by promoting Mad2 dence of signal in prometaphase and early metaphase, recruitment and to checkpoint inactivation by recruitnot only on kinetochores but also along the KMTs (Figing dynein/dynactin that subsequently removes Mad2 ures 1C and 1D; Movies S3 and S4). The overall patfrom attached kinetochores. terns of the two proteins are superimposable (Figures 1C and 1D, Figure S4). Where discrete particles of GFP-Results and Discussion Mad2 could be followed, they always contained RFP-Rod (Figure 1D and Figure S4). These results suggest Rod and Mad2 Colocalize throughout Prometaphase that Mad2 and Rod/Zw10 remain associated as they and Early Metaphase, but BubR1 Does Not leave the kinetochore along the KMTs. To gain insight into the role of Rod/Zw10 relative to By late metaphase, Mad2 signal has essentially disother checkpoint proteins, we undertook a study of fluappeared from kinetochores and is only faintly visible orescently tagged (GFP and mRFP1 [10]) Rod on the spindle above the cytoplasmic Mad2 back-(CG1569), Mad2 (CG17498), and BubR1 (CG7838) in a ground, whereas Rod shedding continues robustly up single cell type, the Drosophila larval neuroblast. All to anaphase onset (Figure 1C and Movie S3). In larval three fusion proteins are controlled by their natural proneuroblasts, the timing of NEB to anaphase onset is typically 7-12 min, of which metaphase lasts 2-8 min (see also [20]). There does not appear to be much delay

Research paper thumbnail of BubR1-and Polo-coated DNA tethers facilitate poleward segregation of acentric chromatids

The mechanisms that safeguard cells against chromosomal instability (CIN) are of great interest, ... more The mechanisms that safeguard cells against chromosomal instability (CIN) are of great interest, as CIN contributes to tumorigenesis. To gain insight into these mechanisms, we studied the behavior of cells entering mitosis with damaged chromosomes. We used the endonuclease I-CreI to generate acentric chromosomes in Drosophila larvae. While I-CreI expression produces acentric chromosomes in the majority of neuronal stem cells, remarkably, it has no effect on adult survival. Our live studies reveal that acentric chromatids segregate efficiently to opposite poles. The acentric chromatid poleward movement is mediated through DNA tethers decorated with BubR1, Polo, INCENP, and Aurora-B. Reduced BubR1 or Polo function results in abnormal segregation of acentric chromatids, a decrease in acentric chromosome tethering, and a great reduction in adult survival. We propose that BubR1 and Polo facilitate the accurate segregation of acentric chromatids by maintaining the integrity of the tethers that connect acentric chromosomes to their centric partners.

Research paper thumbnail of Extensive RPA2 hyperphosphorylation promotes apoptosis in response to DNA replication stress in CHK1 inhibited cells

Nucleic Acids Research, 2015

The replication protein A (RPA)-ssDNA complex formed at arrested replication forks recruits key p... more The replication protein A (RPA)-ssDNA complex formed at arrested replication forks recruits key proteins to activate the ATR-CHK1 signalling cascade. When CHK1 is inhibited during DNA replication stress, RPA2 is extensively hyperphosphorylated. Here, we investigated the role of RPA2 hyperphosphorylation in the fate of cells when CHK1 is inhibited. We show that proteins normally involved in DNA repair (RAD51) or control of RPA phosphorylation (the PP4 protein phosphatase complex) are not recruited to the genome after treatment with CHK1 and DNA synthesis inhibitors. This is not due to RPA2 hyperphosphorylation as suppression of this response does not restore loading suggesting that recruitment requires active CHK1. To determine whether RPA2 hyperphosphorylation protects stalled forks from collapse or induction of apoptosis in CHK1 inhibited cells during replication stress, cells expressing RPA2 genes mutated at key phosphorylation sites were characterized. Mutant RPA2 rescued cells from RPA2 depletion and reduced the level of apoptosis induced by treatment with CHK1 and replication inhibitors however the incidence of double strand breaks was not affected. Our data indicate that RPA2 hyperphosphorylation promotes cell death during replication stress when CHK1 function is compromised but does not appear to be essential for replication fork integrity.

Research paper thumbnail of ATR (ataxia telangiectasia and Rad3 related)

Atlas of Genetics and Cytogenetics in Oncology and Haematology, 2011

This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 ... more This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence.

Research paper thumbnail of Human PIF1 helicase supports DNA replication and cell growth under oncogenic-stress

Oncotarget, Jan 30, 2014

Unwinding duplex DNA is a critical processing step during replication, repair and transcription. ... more Unwinding duplex DNA is a critical processing step during replication, repair and transcription. Pif1 are highly conserved non-processive 5'->3' DNA helicases with well-established roles in maintenance of yeast genome stability. However, the function of the sole member of Pif1 family in humans remains unclear. Human PIF1 is essential for tumour cell viability, particularly during replication stress, but is dispensable in non-cancerous cells and Pif1 deficient mice. Here we report that suppression of PIF1 function slows replication fork rates and increases arrested forks during normal cycling conditions. Importantly, PIF1-dependent replication impediments impair S-phase progression and reduce proliferation rates of RAS oncogene-transformed fibroblasts, where replication fork slowing is exacerbated, but not parental, non-cancerous cells. Disrupted fork movement upon PIF1-depletion does not enhance double-stranded break formation or DNA damage responses but affects resumptio...

Research paper thumbnail of The ribosomal P-proteins of the medfly Ceratitis capitata form a heterogeneous stalk structure interacting with the endogenous P-proteins, in conditional P0-null strains of the yeast Saccharomyces cerevisiae

Nucleic Acids Research, 2000

The genes encoding the ribosomal P-proteins CcP0, CcP1 and CcP2 of Ceratitis capitata were expres... more The genes encoding the ribosomal P-proteins CcP0, CcP1 and CcP2 of Ceratitis capitata were expressed in the conditional P0-null strains W303dGP0 and D67dGP0 of Saccharomyces cerevisiae, the ribosomes of which contain either standard amounts or are totally deprived of the P1/P2 proteins, respectively. The presence of the CcP0 protein restored cell viability but reduced the growth rate. In the W303CcP0 strain, all four acidic yeast proteins were found on the ribosomes, but in notably less quantity, while a preferable binding of the YP1α/YP2β pair was established. In the absence of the endogenous P1/P2 proteins in the D67CcP0 strain, the complementation capacity of the CcP0 protein was considerably reduced. The simultaneous expression of the three medfly genes resulted in alterations of the stalk composition: both the CcP1 and CcP2 proteins were found on the particles substituting the YP1α and YP2α proteins, respectively, but their presence did not alter the growth rate, except in the case of the YP1α/β defective strain, where a helping effect on the binding of the YP2α and YP2β proteins on the ribosomes was confirmed. Therefore, the medfly ribosomal P-proteins complement the yeast P-protein deficient strains forming an heterogeneous ribosomal stalk, which, however, is not functionally equivalent to the endogenous one.

Research paper thumbnail of Enhanced H2AX Phosphorylation, DNA Replication Fork Arrest, and Cell Death in the Absence of Chk1

Molecular Biology of the Cell, 2010

H2AX phosphorylation at serine 139 (γH2AX) is a sensitive indicator of both DNA damage and DNA re... more H2AX phosphorylation at serine 139 (γH2AX) is a sensitive indicator of both DNA damage and DNA replication stress. Here we show that γH2AX formation is greatly enhanced in response to replication inhibitors but not ionizing radiation in HCT116 or SW480 cells depleted of Chk1. Although H2AX phosphorylation precedes the induction of apoptosis in such cells, our results suggest that cells containing γH2AX are not committed to death. γH2AX foci in these cells largely colocalize with RPA foci and their formation is dependent upon the essential replication helicase cofactor Cdc45, suggesting that H2AX phosphorylation occurs at sites of stalled forks. However Chk1-depleted cells released from replication inhibitors retain γH2AX foci and do not appear to resume replicative DNA synthesis. BrdU incorporation only occurs in a minority of Chk1-depleted cells containing γH2AX foci after release from thymidine arrest and, in cells incorporating BrdU, DNA synthesis does not occur at sites of γH2AX...

Research paper thumbnail of Targeted Expression of the Class II Phosphoinositide 3-Kinase in Drosophila melanogaster Reveals Lipid Kinase-Dependent Effects on Patterning and Interactions with Receptor Signaling Pathways

Molecular and Cellular Biology, 2004

Phosphoinositide 3-kinases (PI3Ks) can be divided into three distinct classes (I, II, and III) on... more Phosphoinositide 3-kinases (PI3Ks) can be divided into three distinct classes (I, II, and III) on the basis of their domain structures and the lipid signals that they generate. Functions have been assigned to the class I and class III enzymes but have not been established for the class II PI3Ks. We have obtained the first evidence for a biological function for a class II PI3K by expressing this enzyme during Drosophila melanogaster development and by using deficiencies that remove the endogenous gene. Wild-type and catalytically inactive PI3K_68D transgenes have opposite effects on the number of sensory bristles and on wing venation phenotypes induced by modified epidermal growth factor (EGF) receptor signaling. These results indicate that the endogenous PI3K_68D may act antagonistically to the EGF receptor-stimulated Ras-mitogen-activated protein kinase pathway and downstream of, or parallel to, the Notch receptor. A class II polyproline motif in PI3K_68D can bind the Drk adaptor protein in vitro, primarily via the N-terminal SH3 domain of Drk. Drk may thus be important for the localization of PI3K_68D, allowing it to modify signaling pathways downstream of cell surface receptors. The phenotypes obtained are markedly distinct from those generated by expression of the Drosophila class I PI3K, which affects growth but not pattern formation.

Research paper thumbnail of Characterization of acidic ribosomal proteins from three developmental stages of the medfly Ceratitis capitata

IUBMB Life, 1998

Acidic proteins extracted with 0.5 M NH4Cl and 50% ethanol from ribosomes of the medfly Ceratitis... more Acidic proteins extracted with 0.5 M NH4Cl and 50% ethanol from ribosomes of the medfly Ceratitis capitata showed two major bands of MW 15 and 17 kD after SDS electrophoresis. Isoelectrofocusing of acidic proteins resolved two groups of bands at pH 4.5 and 3.5. Similar patterns were observed both from the acidic ribosomal protein fraction and from total ribosomes, treated with RNase. Treatment with alkaline phosphatase reduced the number of bands with a shift to a higher pI, indicating dephosphorylation. The phosphorylation pattern of the acidic proteins changed at three different stages of development, six day larvae, white pupae and 0-2 days old embryos. The two protein groups correspond to multi-phosphorylated forms of eucaryotic acidic ribosomal proteins P1 and P2. This was shown by immunoblotting with specific monoclonal antibodies.

Research paper thumbnail of Stage-specific expression of the chitin synthase DmeChSA and DmeChSB genes during the onset of Drosophila metamorphosis

Insect Biochemistry and Molecular Biology, 2002

Chitin, the major structural polysaccharide of arthropods, is an important constituent of the ins... more Chitin, the major structural polysaccharide of arthropods, is an important constituent of the insect extracellular structures, cuticle and gut peritrophic matrix. Synthesis of cuticular chitin is strictly coordinated with the ecdysone-regulated molting cycle of insect development (the term "ecdysone" is used in this paper instead of "ecdysteroids" since the exact ratio of various hormonal forms changes during metamorphosis). Based on observed similarities between the fungal chitin synthases and other processive β-glycosyltransferases, we have identified the first insect chitin synthase genes, DmeChSA and DmeChSB (Database accession numbers: EMBL/GenBank/DDBJ A83122, A83126, AJ309488, AJ309489), from Drosophila melanogaster. Chromosomal localization has identified these genes close to and on either side of the centromere of the third chromosome. Partial cDNA clones of both genes have been isolated from a pupal cDNA library. To obtain the first insight into the transcriptional regulation of chitin synthesis, we have monitored the expression of DmeChSA and DmeChSB during the periods of the late-larval and prepupal ecdysone pulses that direct metamorphosis. Transcripts of either gene are barely detected prior to and during the late-larval ecdysone pulse. Once the late-larval ecdysone pulse is ceased completely, both DmeChSA and DmeChSB genes show a remarkable up-regulation.