S K Durum

Publications (68)420.96 Total impact

• Article: Cell cycling through Cdc25A: transducer of cytokine proliferative signals.

  C Kittipatarin, W Q Li, D V Bulavin, S K Durum, A R Khaled
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  ABSTRACT: A balance between survival and proliferative signals maintains a constant number of T lymphocytes that populate the mammalian immune system, a process termed "homeostasis". Central to this process is the availability of a stromal cell product--the cytokine interleukin-7 (IL-7). We recently showed that IL-7, in addition to protecting cells from apoptosis, drives the cell cycling of lymphocytes through regulation of the stability of the phosphatase, Cdc25A, a key activator of cyclin-dependent kinases (cdks). IL-7 achieves this by controlling the activity of p38 MAP kinase (MAPK), which can phosphorylate Cdc25A, triggering its degradation. Sustained expression of Cdc25A had diverse effects: it promoted cell cycling, even in presence of cell cycle inhibitors such p27Kip1, and prevented cell shrinkage in response to cytokine deprivation. Herein we show a role for Cdc25A as a transducer of cytokine-driven proliferation and discuss novel implications for cell growth from the perspective of the requirements for maintenance of lymphocyte homeostasis.
  Cell cycle (Georgetown, Tex.) 06/2006; 5(9):907-12. · 5.36 Impact Factor
• Article: Retroviral transduction of IL-7Ralpha into IL-7Ralpha-/- bone marrow progenitors: correction of lymphoid deficiency and induction of neutrophilia.

  Q Jiang, W-Q Li, F B Aiello, K D Klarmann, J R Keller, S K Durum
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  ABSTRACT: Defects in the gene for the IL-7 receptor (R) alpha chain are one cause of severe combined immunodeficiency disease (SCID) based on a strict requirement for IL-7 in T lymphoid development and survival. We tested the feasibility and potentially undesirable consequences of IL-7Ralpha gene transfer as a therapy for this genetic defect. The murine IL-7Ralpha gene was introduced into IL-7Ralpha(-/-) bone marrow progenitors using retrovirus and transplanted into Rag(-/-) recipient mice. Both alphabeta and gammadelta T cells were reconstituted in thymus and spleen showing proof of principle. B-cell development was also restored in some mice, but their numbers were much lower than in the T-cell compartment. Splenomegaly was observed due to an increase in neutrophils. We showed that hematopoietic progenitors, after transfection with IL-7Ralpha, could respond to IL-7 in vitro by a striking production of neutrophils and other myeloid cells. These data indicate that although IL-7 is a critical lymphopoietin, ectopic expression of its receptor on multipotential progenitors can also induce production of myeloid cells, presumably through survival and proliferation signals that are not restricted to lymphoid cells. This supports the stochastic model of progenitor differentiation, in which cytokines give permissive and not instructive signals.
  Gene Therapy 01/2006; 12(24):1761-8. · 3.71 Impact Factor
• Article: Bax translocation and mitochondrial fragmentation induced by Helicobacter pylori.

  H Ashktorab, S Frank, A R Khaled, S K Durum, B Kifle, D T Smoot
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  ABSTRACT: Previous in vitro and in vivo studies have revealed an association between Helicobacter pylori infection and apoptosis in gastric epithelial cells. Although involvement of the Bcl-2 family of proteins as well as cytochrome c release has been demonstrated in H pylori induced cell death, the exact role of the mitochondria during this type of programmed cell death has not been fully elucidated. Therefore, we sought to determine whether or not Bax translocation and mitochondrial fragmentation occur on exposure of gastric epithelial cells to H pylori, resulting in cell death. Experiments were performed with human gastric adenocarcinoma (AGS) cells, AGS cells transfected with the HPV-E6 gene (which inactivates p53 function), AGS-neo cells (transfected with the backbone construct), mouse embryonic fibroblasts (MEFs), and p19(ARF) null (ARF(-/-)) MEFs. Cells were incubated with a cag positive H pylori strain for up to 24 hours, lysed, and cytoplasmic and mitochondrial membrane fractions were analysed by western blot for Bax translocation. Bax translocation was detected in AGS, AGS-neo, and normal MEF cells after exposure to H pylori for three hours, but not in ARF(-/-) MEFs cells. Translocation of Bax after H pylori incubation was also detected in AGS-E6 cells (inactive p53 gene) but to a lesser degree than in AGS-neo cells. In parallel studies, the mitochondrial morphology of living cells infected with H pylori was assessed by confocal microscopy. Mitochondrial fragmentation was detectable after 10 hours of H pylori incubation with AGS cells and after seven hours with MEF cells. In wild-type MEFs, mitochondrial fragmentation was significantly increased in comparison with ARF null MEFs (43% v 10.4%, respectively). Furthermore, mitochondrial depolarisation and caspase-3 activity were initiated within four hours in cells incubated with H pylori, and these events were inhibited by forced expression of Bcl-2. These data suggest that during H pylori induced apoptosis, Bax translocates to the mitochondria which subsequently undergo depolarisation and profound fragmentation. Functional ARF and p53 proteins may play an important role in H pylori induced mitochondrial modification.
  Gut 07/2004; 53(6):805-13. · 10.11 Impact Factor
• Article: Cutting edge: histone acetylation and recombination at the TCR gamma locus follows IL-7 induction.

  J Huang, S K Durum, K Muegge
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  ABSTRACT: IL-7 signaling is required for V(D)J recombination at the TCRgamma locus. We have recently reported that IL-7 controls chromatin accessibility for RAG-mediated cleavage. Inhibition of histone deacetylase substituted for the IL-7 signal, indicating a role for histone acetylation in altering chromatin accessibility. We found a greatly reduced histone 3 and histone 4 acetylation level in IL-7Ralpha(-/-) thymocytes in comparison with RAG(-/-) thymocytes or fetal thymocytes. Sterile transcripts, indicating an open chromatin configuration, were suppressed in IL-7Ralpha(-/-) and IL-7(-/-)RAG(-/-) thymocytes. Moreover, exogenously added IL-7 induced sterile transcripts from the TCRgamma constant region in cultured thymocytes from IL-7(-/-)RAG(-/-) mice. This induction correlated with increased histone acetylation at the J-promoter and C-enhancer regulatory elements at the TCRgamma locus. These results suggest that IL-7 regulates chromatin accessibility for V(D)J recombination by specifically altering histone acetylation within the TCRgamma locus.
  The Journal of Immunology 01/2002; 167(11):6073-7. · 5.79 Impact Factor
• Article: Trophic factor withdrawal: p38 mitogen-activated protein kinase activates NHE1, which induces intracellular alkalinization.

  A R Khaled, A N Moor, A Li, K Kim, D K Ferris, K Muegge, R J Fisher, L Fliegel, S K Durum
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  ABSTRACT: Trophic factor withdrawal induces cell death by mechanisms that are incompletely understood. Previously we reported that withdrawal of interleukin-7 (IL-7) or IL-3 produced a rapid intracellular alkalinization, disrupting mitochondrial metabolism and activating the death protein Bax. We now observe that this novel alkalinization pathway is mediated by the pH regulator NHE1, as shown by the requirement for sodium, blocking by pharmacological inhibitors or use of an NHE1-deficient cell line, and the altered phosphorylation of NHE1. Alkalinization also required the stress-activated p38 mitogen-activated protein kinase (MAPK). Inhibition of p38 MAPK activity with pharmacological inhibitors or expression of a dominant negative kinase prevented alkalinization. Activated p38 MAPK directly phosphorylated the C terminus of NHE1 within a 40-amino-acid region. Analysis by mass spectroscopy identified four phosphorylation sites on NHE1, Thr 717, Ser 722, Ser 725, and Ser 728. Thus, loss of trophic cytokine signaling induced the p38 MAPK pathway, which phosphorylated NHE1 at specific sites, inducing intracellular alkalinization.
  Molecular and Cellular Biology 12/2001; 21(22):7545-57. · 5.53 Impact Factor
• Source

  Available from: nshtvn.org

  Article: Measurement of soluble and membrane-bound interleukin 1 using a fibroblast bioassay.

  C A Dinarello, K Muegge, S K Durum
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  ABSTRACT: Interleukin 1 (IL-1) is both a major mediator of inflammation and an important signal for activation and differentiation of lymphoid cells. IL-1 is produced by a large variety of cell types, but in most immunologic systems, activated macrophages are its major cellular source. This unit describes a bioassay for testing biological fluids based on the ability of IL-1 to induce IL-8 or IL-6 in fibroblasts. Also listed are sources of commercially available kits (ELISAs, immunoassays) to measure IL-1, and antibodies useful in setting up these assays.
  Current protocols in immunology / edited by John E. Coligan ... [et al.] 06/2001; Chapter 6:Unit 6.2.
• Article: Thymic emigrants isolated by a new method possess unique phenotypic and functional properties.

  C K Lee, K Kim, L A Welniak, W J Murphy, K Muegge, S K Durum
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  ABSTRACT: T cells that emigrate from the thymus have primarily been studied in vivo using fluorescent dye injection of the thymus. This study examined the properties of thymocytes that emigrate from cultured thymic lobes in organ culture. Under these conditions, thymic emigrants displayed the expected phenotype, that of mature thymocytes expressing high levels of T-cell receptor (TCR-alphabeta) and either CD4 or CD8, and were observed to emigrate within 24 hours of positive selection. Emigration was inhibited by cytochalasin D, pertussis toxin, or Clostridium difficile toxin B, implicating an active motility process. Most of the surface markers on alphabeta-thymic emigrants (Thy1, CD44, CD69, CD25, leukocyte functional antigen-1, intercellular adhesion molecule-1, alpha(4)-integrin, alpha(5)-integrin, CD45, and CD28) were expressed at a surface density similar to that on mature intrathymic cells and peripheral splenic T cells. Heterogeneous expression of L-selectin and heat-stable antigen (HSA) suggested that subsets emerge from the thymus with a commitment to different migration patterns. The only marker on emigrants not found on either intrathymic cells or mature spleen T cells was CTLA-4, which could dampen the response of emigrants to peripheral antigens. Antigen responsivenes measured in vitro against allogeneic dendritic cells showed a proliferative response comparable to that of splenic T cells. In vivo, however, thymic emigrants failed to induce an acute graft-versus-host reaction in allogeneic severe combined immunodeficiency recipients. This suggests that a mechanism operating in vivo, perhaps tolerance or migration pattern, attenuates the response of emigrants against antigens that did not induce their deletion in the thymus.
  Blood 04/2001; 97(5):1360-9. · 9.90 Impact Factor
• Article: Interleukin-3 withdrawal induces an early increase in mitochondrial membrane potential unrelated to the Bcl-2 family. Roles of intracellular pH, ADP transport, and F(0)F(1)-ATPase.

  A R Khaled, D A Reynolds, H A Young, C B Thompson, K Muegge, S K Durum
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  ABSTRACT: Cytokines such as interleukin-3 (IL-3) promote the survival of hematopoietic cells through mechanisms that are not well characterized. Withdrawal of IL-3 from an IL-3-dependent pro-B cell line induced early stress-related events that preceded cell death by more than 40 h. Intracellular pH rose above pH 7.8, peaking 2-3 h post-IL-3 withdrawal, and induced a transient increase in mitochondrial membrane potential (Delta Psi(m)) detected using several different dyes. Similar events were observed following IL-7 withdrawal from a different dependent cell line. Bcl-2, Bax, and caspases were unrelated to these early events. Intracellular alkaline pH inhibited the mitochondrial import of ADP, which would limit ATP synthesis. Total cellular ATP sharply declined during this early period, presumably as a consequence of suppressed ADP import. This was followed by an increase in reduced pyridine nucleotides. The transient increase in Delta Psi(m) was blocked by oligomycin, an inhibitor of F(0)F(1-)ATPase that may have undergone reversal caused by the abnormal ADP-ATP balance within mitochondria. These findings suggest a novel sequence of early events following trophic factor withdrawal in which alkaline pH inhibits ADP import into mitochondria, reversing the F(0)F(1-)ATPase, which in turn consumes ATP and pumps out protons, raising Delta Psi(m).
  Journal of Biological Chemistry 04/2001; 276(9):6453-62. · 4.77 Impact Factor
• Article: Induction of oxidative DNA damage in u937 cells by TNF or anti-Fas stimulation.

  I Nathan, M Dizdaroglu, L Bernstein, U Junker, C Lee, K Muegge, S K Durum
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  ABSTRACT: TNF and Fas signaling pathways are reported to induce mitochondrial damage associated with production of oxygen radicals. We examined whether such radical production elicited detectable nuclear DNA damage in U937 cells following treatment with TNF or with anti-Fas antibodies. Using GC-mass spectroscopy for analysing base oxidation, several oxidized species increased significantly following TNF treatment, whereas anti-Fas resulted in less detectable oxidative damage using this assay. Cytogenetic analysis showed that, in the presence of aphidicolin, which blocks several types of DNA repair, TNF induced extensive chromosomal damage. Aphidicolin also synergized with TNF and anti-Fas in inducing cell death which was prevented by reducing atmospheric oxygen or addition of n -acetyl cysteine, a scavenger of oxygen radicals. Thus, several lines of evidence point to the TNF and Fas pathways inducing extensive oxidative DNA damage and repair, and suggest potential roles for these pathways in mutagenesis and aging.
  Cytokine 08/2000; 12(7):881-7. · 3.02 Impact Factor
• Article: Interleukin (IL)-7 induces rapid activation of Pyk2, which is bound to Janus kinase 1 and IL-7Ralpha.

  N Benbernou, K Muegge, S K Durum
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  ABSTRACT: Interleukin-7 (IL-7) receptor signaling begins with activation of the Janus tyrosine kinases Jak1 and Jak3, which are associated with the receptor complex. To identify potential targets of these kinases, we examined Pyk2 (a member of the focal adhesion kinase family) using an IL-7-dependent murine thymocyte line, D1. We demonstrate that stimulation of D1 (or normal pro-T) cells by IL-7 rapidly increased tyrosine phosphorylation and enzymatic activity of Pyk2, with kinetics slightly lagging that of Jak1 and Jak3 phosphorylation. Conversely, IL-7 withdrawal resulted in a marked decrease of Pyk2 phosphorylation. Pyk2 was found to be physically associated with Jak1 prior to IL-7 stimulation and to increase its association with IL-7Ralpha chain following IL-7 stimulation. Pyk2 appeared to be involved in cell survival, because antisense Pyk2 accelerated the cell death process. Activation of Pyk2 via the muscarinic and nicotinic receptors using carbachol or via intracellular Ca(2+) rise using ionomycin/phorbol myristate acetate promoted survival in the absence of IL-7. These data support a role for Pyk2 in coupling Jak signaling to the trophic response.
  Journal of Biological Chemistry 04/2000; 275(10):7060-5. · 4.77 Impact Factor
• Source

  Available from: pnas.org

  Article: Withdrawal of IL-7 induces Bax translocation from cytosol to mitochondria through a rise in intracellular pH.

  A R Khaled, K Kim, R Hofmeister, K Muegge, S K Durum
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  ABSTRACT: IL-7 functions as a trophic factor during T lymphocyte development by a mechanism that is partly based on the induction of Bcl-2, which protects cells from apoptosis. Here we report a mechanism by which cytokine withdrawal activates the prodeath protein Bax. On loss of IL-7 in a dependent cell line, Bax protein translocated from the cytosol to the mitochondria, where it integrated into the mitochondrial membrane. This translocation was attributable to a conformational change in the Bax protein itself. We show that a rise in intracellular pH preceded mitochondrial translocation and triggered the change in Bax conformation. Intracellular pH in the IL-7-dependent cells rose steadily to peak over pH 7.8 by 6 hr after cytokine withdrawal, paralleling the time point of Bax translocation (a similar alkalinization and Bax translocation was also observed after IL-3 withdrawal from a dependent cell line). The conformation of Bax was directly altered by pH of 7.8 or higher and was demonstrated by increased protease sensitivity, exposure of N terminus epitopes, and exposure of a hydrophobic domain in the C terminus. Eliminating charged amino acids at the C or N termini of Bax induced a conformational change similar to that induced by raising pH, implicating these residues in the pH effect. Therefore, we have shown that by either cytokine withdrawal, experimental manipulation of pH, or site-directed mutagenesis, Bax protein changes conformation, exposing membrane-seeking domains, thereby inducing mitochondrial translocation and initiating the cascade of events leading to apoptotic death.
  Proceedings of the National Academy of Sciences 01/2000; 96(25):14476-81. · 9.68 Impact Factor
• Article: Defining functional domains of Ku80: DNA end binding and survival after radiation.

  O Osipovich, R J Duhe, P Hasty, S K Durum, K Muegge
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  ABSTRACT: The Ku heterodimeric protein (Ku80/Ku70) is an essential component of the double-strand break DNA repair pathway in mammalian cells. We have recently defined a central region within Ku80 that is required for heterodimerization with Ku70. We now identified a core region within Ku80 (amino acids 210 to 531) that is necessary for binding of Ku to DNA ends. Interaction with Ku70 and DNA end binding are important for Ku80 function in vivo, since Ku80 mutants lacking DNA end binding activity were unable to restore radiation resistance in Ku80 deficient fibroblast cell lines. However, Ku80 mutants were identified that retained DNA end binding activity but were unable to restore radiation survival, thus pointing to additional functional properties of Ku80. An N-terminal deletional mutant of Ku80 was able to suppress wild type Ku80 function for radiation survival in several cell lines, thus demonstrating dominant negative function.
  Biochemical and Biophysical Research Communications 09/1999; 261(3):802-7. · 2.48 Impact Factor
• Article: Interleukin-7: physiological roles and mechanisms of action.

  R Hofmeister, A R Khaled, N Benbernou, E Rajnavolgyi, K Muegge, S K Durum
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  ABSTRACT: Interleukin-7 (IL-7), a product of stromal cells, provides critical signals to lymphoid cells at early stages in their development. Two types of cellular responses to IL-7 have been identified in lymphoid progenitors: (1) a trophic effect and (2) an effect supporting V(D)J recombination. The IL-7 receptor is comprised of two chains, IL-7R alpha and gamma(c). Following receptor crosslinking, rapid activation of several classes of kinases occurs, including members of the Janus and Src families and PI3-kinase. A number of transcription factors are subsequently activated including STATs, c-myc, NFAT and AP-1. However, it remains to be determined which, if any, previously identified pathway leads to the trophic or V(D)J endpoints. The trophic response to IL-7 involves protecting lymphoid progenitors from a death process that resembles apoptosis. This protection is partly mediated by IL-7 induction of Bcl-2, however other IL-7-induced events are probably also involved in the trophic response. The V(D)J response to IL-7 is partly mediated through increased production of Rag proteins (which cleave the target locus) and partly by increasing the accessibility of a target locus to cleavage through chromatin remodeling.
  Cytokine & Growth Factor Reviews 04/1999; 10(1):41-60. · 7.81 Impact Factor
• Article: Cloning thymic precursor cells: demonstration that individual pro-T1 cells have dual T-NK potential and individual pro-T2 cells have dual alphabeta-gammadelta T cell potential.

  C K Lee, K Kim, T M Geiman, W J Murphy, K Muegge, S K Durum
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  ABSTRACT: Thymic progenitors have the capacity to generate alphabeta T cells, gammadelta T cells, and NK cells. To determine whether these three lineages derive from a single precursor cell or from different precursors, a procedure was developed for cloning precursor cells from mouse embryonic thymus. The progeny of each pro-T cell clone were then tested for the potential to generate alphabeta, gammadelta, and NK cells. Of these precursor clones, about half displayed dual potential, developing into either T cells or NK cells, demonstrating the existence of a common T/NK precursor cell in the thymus. The other half of the clones were restricted to T cell development. No precursor clones were restricted to NK development. The common T/NK precursors were shown to be of the pro-T1 (CD25(-)) stage whereas the T-restricted precursors were shown to be of the later pro-T2 (CD25(+)) stage. Both alphabeta and gammadelta T cells were generated from all clones derived from either pro-T1 or -T2 precursors. This shows that commitment of a cell to the alphabeta versus gammadelta lineages does not precede rearrangement of the TCR genes (which occurs immediately after the pro-T2 stage).
  Cellular Immunology 03/1999; 191(2):139-44. · 1.97 Impact Factor
• Article: Characterization of gene expression, genomic structure, and chromosomal localization of Hells (Lsh).

  T M Geiman, S K Durum, K Muegge
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  ABSTRACT: Hells (Lsh) is a lymphoid-specific presumptive helicase with highest expression in lymphoid precursor cells. Other members of the helicase family participate in maintenance of genome stability, DNA repair, and transcriptional control. Here we report the structure and chromosomal location of the Hells gene. The open reading frame of the murine Hells gene spans at least 26.6 kb of chromosomal DNA and is composed of 18 exons. The genomic structure of the seven helicase domains closely resembles that of mammalian Rad54, a gene whose product appears to be involved in recombination and double-strand break repair. The human homologue, the HELLS gene, has a mRNA expression pattern that is similar to murine Hells expression. Low-stringency hybridization in a Southern analysis reveals homologous Hells genes in a variety of species including Saccharomyces cerevisiae. FISH analysis maps the murine Hells gene to region C3-D1 on chromosome 19. The human homologue maps to a region of synteny on chromosome 10q23-q24, a breakpoint region frequently involved in human leukemia.
  Genomics 01/1999; 54(3):477-83. · 3.02 Impact Factor
• Source

  Available from: Leslie J Berg

  Article: Interleukin 7 receptor control of T cell receptor gamma gene rearrangement: role of receptor-associated chains and locus accessibility.

  S K Durum, S Candèias, H Nakajima, W J Leonard, A M Baird, L J Berg, K Muegge
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  ABSTRACT: VDJ recombination of T cell receptor and immunoglobulin loci occurs in immature lymphoid cells. Although the molecular mechanisms of DNA cleavage and ligation have become more clear, it is not understood what controls which target loci undergo rearrangement. In interleukin 7 receptor (IL-7R)alpha-/- murine thymocytes, it has been shown that rearrangement of the T cell receptor (TCR)-gamma locus is virtually abrogated, whereas other rearranging loci are less severely affected. By examining different strains of mice with targeted mutations, we now observe that the signaling pathway leading from IL-7Ralpha to rearrangement of the TCR-gamma locus requires the gammac receptor chain and the gammac-associated Janus kinase Jak3. Production of sterile transcripts from the TCR-gamma locus, a process that generally precedes rearrangement of a locus, was greatly repressed in IL-7Ralpha-/- thymocytes. The repressed transcription was not due to a lack in transcription factors since the three transcription factors known to regulate this locus were readily detected in IL-7Ralpha-/- thymocytes. Instead, the TCR-gamma locus was shown to be methylated in IL-7Ralpha-/- thymocytes. Treatment of IL-7Ralpha-/- precursor T cells with the specific histone deacetylase inhibitor trichostatin A released the block of TCR-gamma gene rearrangement. This data supports the model that IL-7R promotes TCR-gamma gene rearrangement by regulating accessibility of the locus via demethylation and histone acetylation of the locus.
  Journal of Experimental Medicine 01/1999; 188(12):2233-41. · 13.85 Impact Factor
• Article: CD16 cross-linking blocks rearrangement of the TCR beta locus and development of alpha beta T cells and induces development of NK cells from thymic progenitors.

  S K Durum, C K Lee, T M Geiman, W J Murphy, K Muegge
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  ABSTRACT: Mouse thymocytes normally develop into T lymphocytes, but the embryonic thymus also contains precursor cells capable of developing into NK cells. Here, we describe conditions that induce pro-T cells to develop into NK cells. CD16 is expressed on thymic pro-T cells. We observed that CD16 cross-linking during culture of embryonic thymic organs suppressed rearrangement of the TCR beta locus (but did not inhibit TCR gamma locus rearrangement). Rearrangement of the TCR beta locus is normally required for development to the CD4+CD8+, and this development was also suppressed by CD16 cross-linking. The ability of CD16 cross-linking to block alpha beta T cell development was not attributable to toxic effects, but rather was accompanied by promotion of development into NK cells, identified based on molecular and functional criteria. These results suggest that common lymphoid precursors can respond to environmental signals to commit to the alpha beta T vs NK developmental pathways.
  The Journal of Immunology 11/1998; 161(7):3325-9. · 5.79 Impact Factor
• Article: The trophic action of IL-7 on pro-T cells: inhibition of apoptosis of pro-T1, -T2, and -T3 cells correlates with Bcl-2 and Bax levels and is independent of Fas and p53 pathways.

  K Kim, C K Lee, T J Sayers, K Muegge, S K Durum
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  ABSTRACT: Signals from the IL-7R are essential for normal thymocyte development. We isolated thymocytes from early developmental stages and observed that suspensions of pro-T1, -T2, and -T3 cells rapidly died in culture. Addition of IL-7 promoted their survival, but did not induce cell division. Pro-T4 cells did not undergo rapid cell death, and their survival was therefore independent of IL-7. Death in the absence of IL-7 showed the hallmarks of apoptosis, including DNA fragmentation and annexin V binding; however, caspase inhibitors blocked DNA fragmentation, but did not block cell death. The trophic effect of IL-7 was partially inhibited by blocking protein synthesis. The p53 pathway was not involved in this death pathway, since pro-T cells from p53-/- mice also underwent cell death in the absence of IL-7. The Fas/Fas ligand pathway was not involved in cell death, since Fas-deficient pro-T cells died normally in the absence of IL-7, anti-Fas Abs did not protect cells from death in the absence of IL-7, and Fas expression was undetectable on cells at these stages. The IL-7 trophic affect correlated with increased intracellular levels of Bcl-2 and decreased levels of Bax, whereas no Bcl-X(L), Bcl-w, or Bad was detectable. Thus, maintaining a favorable Bcl-2/Bax ratio may account for the trophic action of IL-7.
  The Journal of Immunology 07/1998; 160(12):5735-41. · 5.79 Impact Factor
• Article: p53-dependent apoptosis and transcription of p21waf/cip1/sdi1 in SCID mice following gamma-irradiation.

  S M Candéias, S K Durum, K Muegge
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  ABSTRACT: The recruitment and activation of DNA-repair mechanisms at the sites of DNA-damage after exposure of cells to genotoxic stress are poorly understood. The DNA-dependent kinase (DNA-PK) was considered to be a likely candidate for initiating these events because of the conditions required for its activation, its phosphorylation of p53 in vitro and the extreme radiosensitivity induced by its inactivation in vivo. We analyzed irradiation-induced p53-activation in SCID mice, which lack DNA-PK activity, and found that p53-dependent apoptosis and p21waf/cip1/sdi1 transcription in these animals are at least as efficient as in wild-type mice. Thus, our results show that DNA-PK is not the main sensor for genotoxic stress and is not required for p53 activation. In fact, they rather suggest that DNA-PK may play a role in p53 down-regulation.
  Biochimie 11/1997; 79(9-10):607-12. · 3.02 Impact Factor
• Article: Defining the minimal domain of Ku80 for interaction with Ku70.

  O Osipovich, S K Durum, K Muegge
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  ABSTRACT: The Ku protein has a critical function in the repair of double-strand DNA breaks induced for example by ionizing radiation or during VDJ recombination. Ku serves as the DNA-binding subunit of the DNA-dependent kinase and is a heterodimeric protein composed of 80- and 70-kDa subunits. We used the two-hybrid system to analyze the interaction domains of the Ku subunits and to identify possible additional partners for Ku. Screening a human cDNA library with the Ku heterodimer did not reveal any novel partners. Screening with the individual subunits, we detected only Ku70 clones interacting with Ku80 and only Ku80 clones interacting with Ku70, indicating that these are the primary partners for one another. Ku80 and Ku70 formed only heterodimers and did not homodimerize. Ku80 was restricted to interacting with just one Ku70 molecule at a time. The minimal functional interaction domain of Ku80 that interacted with Ku70 was defined. It consisted of a 28-amino acid region extending from amino acid 449 to 477. This region was crucial for interaction with Ku70, since mutation within this critical site at amino acids 453 and 454 abrogated the ability to interact with Ku70. We furthermore verified that the same region is crucial for interaction with Ku70 using in vitro co-translation of both subunits followed by an immunoprecipitation with anti-Ku70 antibodies. This interaction domain of Ku80 does not contain any motif previously recognized in protein-protein interactions.
  Journal of Biological Chemistry 11/1997; 272(43):27259-65. · 4.77 Impact Factor