Biomedical subjects

S K Durum

Publications and source records attributed to S K Durum.

At least 19 recordsLinked to original sources

PubMed2001

Generation of macrophages from early T progenitors in vitro.

Early T progenitors in the thymus have been reported to have the capacity to develop into B cells, thymic dendritic cells, and NK cells. Here we describe conditions that induce early T progenitors to develop into macrophages. Initially, we observed that early T progenitors could be induced to develop into macrophages by cytokines produced from a thymic stromal cell line, TFGD, and later we found that the cytokine mixture of M-CSF plus IL-6 plus IL-7 also induced macrophage differentiation from pro-T cells. M-CSF by itself was unable to induce macrophage differentiation from early T progenitors. To correlate this observation with the developmental potential of early T progenitors, mouse embryonic thymocytes were sorted into four populations, pro-T1 to pro-T4, based on the expression of CD44 and CD25, and then cultured with TFGD culture supernatant. We found that pro-T1 and pro-T2 cells, but not pro-T3 and pro-T4 cells, generate macrophages. Limiting dilution analysis of the differentiation capability of sorted pro-T2 cells also confirmed that pro-T2 cells could generate macrophages. These results suggest that T cells and thymic macrophages could originate from a common intrathymic precursor.

Animals↗

PubMed2001

Thymic emigrants isolated by a new method possess unique phenotypic and functional properties.

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.

Abatacept↗

PubMed2001

Trophic factor withdrawal: p38 mitogen-activated protein kinase activates NHE1, which induces intracellular alkalinization.

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.

Activating Transcription Factor 2↗

PubMed2001

Interferon-alpha suppresses the antiapoptotic effect of NF-kB and sensitizes renal cell carcinoma cells in vitro to chemotherapeutic drugs.

BACKGROUND: Immunochemotherapy (ICT) with interleukin-2 (IL-2) and interferon-alpha (IFNalpha) with a secondary effector (5-fluorouracil, 5 FU) is the only promising treatment for advanced renal cell carcinoma (RCC). With IFNalpha, besides the activation mechanisms of the immunosystem, a direct antitumor effect on tumor cells is expected. MATERIALS AND METHODS: NF-kB activity in three permanent cell lines (Hep2, HepG2, HT29) and in primary RCC cell lines was measured after incubation with tumor necrosis factor-alpha (TNFalpha), IFNalpha, IFN-gamma, TNFalpha+IFNalpha, and IFNgamma+TNFalpha, respectively. NF-kB activity and induction of apoptosis by chemotherapeutic drugs (5FU and doxorubicin) were determined in cells transfected with a constitutively active NF-kB p65 or a dominant negative IkB. RESULTS: NF-kB signaling induced by TNFalpha is suppressed by IFNalpha and IFNgamma in the permanent cell lines and in the primary RCC tumor cell cultures. In an in vitro ICT model we show that pretreatment of RCC with IL-2 and IFNalpha leads to a diminished NF-kB response to TNFalpha. In certain tumors, this correlates with increased susceptibility to investigated chemotherapeutic drugs as shown by annexin stain and cell elimination. Modulation of the cellular NF-kB state by a constitutively active p65 or a dominant negative IkB mimics this effect. The IkB construct leads to the same effects as IL-2/IFNalpha pretreatment as shown by predominant elimination of the transfected cells from the overall population, while introduction of p65 leads to a partial rescue from the effect of IL-2 and IFNalpha. The described effect, however, applies only to a selection of primary cell cultures. CONCLUSIONS: Besides the immunomodulation effects, treatment of RCC with IL-2/IFNalpha leads to a proapoptotic state in certain tumors. The relevant mediator seems to be IFNalpha by suppression of the antiapoptotic effect of NF-kB. These data can provide an experimental base for correlation with real patient outcome after ICT.

Antineoplastic Agents↗

PubMed2000

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.

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).

Adenosine Diphosphate↗

PubMed2000

Interleukin (IL)-7 induces rapid activation of Pyk2, which is bound to Janus kinase 1 and IL-7Ralpha.

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.

Animals↗

PubMed2000

Induction of oxidative DNA damage in u937 cells by TNF or anti-Fas stimulation.

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.

Adenosine↗

PubMed1999

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

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.

Animals↗

PubMed1999

Defining functional domains of Ku80: DNA end binding and survival after radiation.

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.

Animals↗

PubMed1999

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.

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).

Animals↗

PubMed1999

Interleukin-7: physiological roles and mechanisms of action.

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.

Animals↗

PubMed1998

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

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.

Animals↗

PubMed1998

Characterization of gene expression, genomic structure, and chromosomal localization of Hells (Lsh).

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.

Animals↗

PubMed1998

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.

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.

Animals↗

PubMed1998

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.

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.

Animals↗

PubMed1997

Defining the minimal domain of Ku80 for interaction with Ku70.

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.

Antigens, Nuclear↗

PubMed1997

IL-7 activates alpha4beta1 integrin in murine thymocytes.

IL-7, a cytokine produced by thymic epithelium, was shown to induce adhesion of murine thymocytes to gelatin-coated membranes. A major binding component of gelatin was identified as fibronectin. IL-7-induced adhesion was observed for all of the major thymocyte subsets, including double-negative, double-positive, and single-positive cells, and specific IL-7R were verified on each subset. Fibronectin binding was mediated via alpha4beta1 integrin (VLA-4), which is expressed at high levels on thymocytes. VLA-4 surface expression was not increased following IL-7 treatment, but was shown to undergo rapid tyrosine phosphorylation on the beta1 subunit. This tyrosine phosphorylation was blocked by genistein, which also blocked IL-7-induced adhesion. IL-7 was detected on the extracellular matrix of the thymus, suggesting that it could promote matrix association through an integrin pathway.

Animals↗

PubMed1997

Defective T-cell receptor gamma gene rearrangement in interleukin-7 receptor knockout mice.

T-cell receptor (TCR) genes need to be rearranged by a site specific-VDJ recombinase before they are expressed. This process, initiated in CD44+25+ thymocytes, takes place during the early stage of T-cell differentiation in the thymus. Interleukin-7 receptor alpha chain knockout (IL-7R-/-) mice are severely deficient in B-lymphocytes and alpha beta T-cells and completely lack the gamma delta T-cell lineage. Thymocyte development is arrested at a very early stage (DN CD44+CD25-). Because this arrest is earlier than in mice with a block in VDJ recombination, we examined the rearrangement status of TCR genes in thymocytes from IL-7R-/- mice. The TCR beta locus showed a nearly normal pattern of VDJ rearrangements, consistent with the presence of alpha beta T-cells in these mice. However, TCR gamma locus rearrangement was absent or severely reduced for all the V gamma genes analyzed (V gamma 3, V gamma 4, V gamma 1.1, V gamma 1.2 and V gamma 2). In contrast, the delta locus showed little reduction in rearrangement. The defect in gamma rearrangements in IL-7R-/- thymocytes is not simply due to an absence of mature gamma delta T-cells, since TCR delta-/- mice, which also have only alpha beta T-cells, had normal levels of gamma and delta rearrangements. These findings indicate that one or both of the two known ligands of IL-7R, IL-7 and thymic stromal lymphopoietin (TSLP) serves as an extrinsic signal to specifically rearrange the TCR gamma locus.

Animals↗