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Showing papers by "St. Jude Children's Research Hospital published in 1998"


Journal ArticleDOI
TL;DR: It is demonstrated that glycogen synthase kinase-3beta (GSK-3 beta) phosphorylates cyclin D1 specifically on Thr-286, thereby triggering rapid cyclinD1 turnover, which leads to proteasomal degradation of D1 and linked to phosphorylation and proteolytic turnover of cyclin L1 and its subcellular localization during the cell division cycle.
Abstract: A family of cyclin-dependent kinases (CDKs) cooperatively regulates mammalian cell cycle progression (for review, see Sherr 1993). During G1 phase, D-type cyclins (D1, D2, and D3) are synthesized and assemble with either CDK4 or CDK6 in response to growth factor stimulation, thereby generating active holoenzymes that help inactivate the growth-suppressive function of the retinoblastoma protein (Rb) through its phosphorylation (for review, see Weinberg 1995). Cyclin D holoenzyme complexes also titrate CDK inhibitors, such as p27Kip1 and p21Cip1, facilitating the activation of cyclin E-CDK2 and subsequent entry into the DNA synthetic phase of the cell cycle (for review, see Sherr and Roberts 1995). Ras-mediated pathways are important for cyclin D1 induction and its assembly with CDKs. Overexpression of activated oncogenic Ras alleles, but not wild-type Ras, initiates DNA synthesis independently of growth factor stimulation (Feramisco et al. 1984). Conversely, microinjection of antibodies that inactivate Ras or introduction of certain dominant-negative Ras alleles can block S-phase entry induced by mitogens (Mulcahy et al. 1985; Mittnacht et al. 1997; Peeper et al. 1997). Both cyclin D1 expression and assembly require the sequential activities of Raf1, mitogen-activated protein kinase-kinases (MEK1 and MEK2), and the sustained activation of extracellular signal-regulated protein kinases (ERKs; Albanese et al. 1995; Lavoie et al. 1996; Winston et al. 1996; Aktas et al. 1997; Kerkhoff and Rapp 1997; Weber et al. 1997; Cheng et al. 1998). In turn, cyclin D1 degradation is mediated by phosphorylation-triggered, ubiquitin-dependent proteolysis (Diehl et al. 1997). Polyubiquitination of protein substrates involves the sequential action of three distinct enzymes termed E1, E2 (UBC; ubiquitin-conjugating enzyme), and E3 (ubiquitin ligase; Ciechanover 1994; King et al. 1996). Specificity of substrate recognition is dependent on several factors including E2 and E3 selectivity (King et al. 1996; Skowyra et al. 1997; Renny-Feldman et al. 1997), recognition motifs within the target proteins themselves (Glotzer et al. 1991), and, in some cases, a requirement for phosphorylation of specific residues within the substrate (Deshaies et al. 1995; Clurman et al. 1996; Lanker et al. 1996; Won et al. 1996). Ubiquitin-dependent degradation of cyclin D1 requires phosphorylation of a specific threonine residue (Thr-286) located near the protein carboxyl terminus, and this phosphorylation is not mediated by cyclin D-dependent kinases themselves (Diehl et al. 1997). Because the kinase that phosphorylates this residue has not yet been identified, it remains unclear whether cyclin D1 proteolysis, like its synthesis and assembly, is subject to mitogen regulation. The subcellular distribution of D-type cyclins is also likely to be regulated by cell cycle-dependent events. Cyclin D1 accumulates in the nuclei of cells during G1 phase, but once DNA replication begins, it disappears from the nucleus (Baldin et al. 1993), despite the fact that its level of synthesis does not decrease markedly during S phase (Matsushime et al. 1991). The mechanisms that regulate the periodic subcellular redistribution of cyclin D1 during the cell division cycle have also not been defined. We now demonstrate that glycogen synthase kinase-3β (GSK-3β) catalyzes the phosphorylation of cyclin D1 on Thr-286, thereby regulating cyclin D1 turnover in response to mitogenic signals. In turn, GSK-3β-mediated phosphorylation of cyclin D1 redirects the protein from the nucleus to the cytoplasm. Our results support a model in which phosphorylation of cyclin D1 on Thr-286 by GSK-3β links processes governing cyclin D1 subcellular localization with its proteasomal degradation.

2,159 citations


Journal ArticleDOI
TL;DR: The results suggest transmission of the virus from infected chickens to the child without another intermediate mammalian host acting as a "mixing vessel" illustrates the importance of intensive global influenza surveillance.

1,366 citations


Journal ArticleDOI
29 May 1998-Cell
TL;DR: The phenotypes of the mice demonstrate an essential, and often redundant, role for the two Stat5 proteins in a spectrum of physiological responses associated with growth hormone and prolactin.

1,298 citations


Journal ArticleDOI
TL;DR: MEFs that survive myc overexpression sustain p53 mutation or ARF loss during the process of establishment and become immortal, and ARF regulates a p53-dependent checkpoint that safeguards cells against hyperproliferative, oncogenic signals.
Abstract: Establishment of primary mouse embryo fibroblasts (MEFs) as continuously growing cell lines is normally accompanied by loss of the p53 or p19ARF tumor suppressors, which act in a common biochemical pathway. myc rapidly activates ARF and p53 gene expression in primary MEFs and triggers replicative crisis by inducing apoptosis. MEFs that survive myc overexpression sustain p53 mutation or ARF loss during the process of establishment and become immortal. MEFs lacking ARF or p53 exhibit an attenuated apoptotic response to myc ab initio and rapidly give rise to cell lines that proliferate in chemically defined medium lacking serum. Therefore, ARF regulates a p53-dependent checkpoint that safeguards cells against hyperproliferative, oncogenic signals.

1,282 citations


Journal ArticleDOI
01 May 1998-Cell
TL;DR: Reconstitution experiments demonstrate that Jak2 is not required for the generation of lymphoid progenitors, their amplification, or functional differentiation, and plays a critical, nonredundant role in the function of a specific group of cytokines receptors.

1,110 citations


Journal ArticleDOI
01 Sep 1998-Blood
TL;DR: Polyclonal donor-derived T-cell lines specific for EBV proteins can be used safely to prevent EBV-related immunoblastic lymphoma after allogeneic marrow transplantation and may also be effective in the treatment of established disease.

1,061 citations


Journal ArticleDOI
TL;DR: A structural basis for the hypothesis that pigs may serve as “mixing vessels” for the generation of human-avian influenza A virus reassortants, similar to those responsible for the 1957 and 1968 pandemics is demonstrated.
Abstract: Genetic and biologic observations suggest that pigs may serve as “mixing vessels” for the generation of human-avian influenza A virus reassortants, similar to those responsible for the 1957 and 1968 pandemics. Here we demonstrate a structural basis for this hypothesis. Cell surface receptors for both human and avian influenza viruses were identified in the pig trachea, providing a milieu conducive to viral replication and genetic reassortment. Surprisingly, with continued replication, some avian-like swine viruses acquired the ability to recognize human virus receptors, raising the possibility of their direct transmission to human populations. These findings help to explain the emergence of pandemic influenza viruses and support the need for continued surveillance of swine for viruses carrying avian virus genes.

1,010 citations


Journal ArticleDOI
TL;DR: Overexpression of p19(ARF) in wild type or ARF-null mouse embryo fibroblasts increases the half-life of p53 from 15 to approximately 75 min, correlating with an increased p53-dependent transcriptional response and growth arrest.
Abstract: The INK4a-ARF locus encodes two proteins, p16INK4a and p19ARF, that restrain cell growth by affecting the functions of the retinoblastoma protein and p53, respectively. Disruption of this locus by deletions or point mutations is a common event in human cancer, perhaps second only to the loss of p53. Using insect cells infected with baculovirus vectors and NIH 3T3 fibroblasts infected with ARF retrovirus, we determined that mouse p19ARF can interact directly with p53, as well as with the p53 regulator mdm2. ARF can bind p53-DNA complexes, and it depends upon functional p53 to transcriptionally induce mdm2 and the cyclin-dependent kinase inhibitor p21Cip1, and to arrest cell proliferation. Binding of p19ARF to p53 requires the ARF N-terminal domain (amino acids 1–62) that is necessary and sufficient to induce cell cycle arrest. Overexpression of p19ARF in wild type or ARF-null mouse embryo fibroblasts increases the half-life of p53 from 15 to ≈75 min, correlating with an increased p53-dependent transcriptional response and growth arrest. Surprisingly, when overexpressed at supra-physiologic levels after introduction into ARF-null NIH 3T3 cells or mouse embryo fibroblasts, the p53 protein is handicapped in inducing this checkpoint response. In this setting, reintroduction of p19ARF restores p53’s ability to induce p21Cip1 and mdm2, implying that, in addition to stabilizing p53, ARF modulates p53-dependent function through an additional mechanism.

939 citations


Journal ArticleDOI
TL;DR: Emerging evidence is providing valuable insights into the molecular circuitry through which p19 modulates p53 activity as part of a checkpoint response to oncogenic, hyperproliferative signals.
Abstract: The retinoblastoma (Rb) and p53 genes are not essential for completion of the cell division cycle, but disruption of their functions is central to the life history of most, if not all, cancer cells (for review, see Weinberg 1995; Sherr 1996; Levine 1997). Surprisingly, Rb and p53 are themselves regulated by two proteins encoded by a single genetic locus, INK4a/ARF, the products of which, p16 and p19, are also potent tumor suppressors. The role of p16 as an inhibitor of cyclin D-dependent kinases has been appreciated since its discovery (Serrano et al. 1993). Now, emerging evidence is providing valuable insights into the molecular circuitry through which p19 modulates p53 activity as part of a checkpoint response to oncogenic, hyperproliferative signals.

721 citations


Journal ArticleDOI
01 Jun 1998-Immunity
TL;DR: Virus-specific CD8+ effector T cells are enriched in the lungs of mice with primary influenza pneumonia, though later detection of memory T cells (mCTL) in the mediastinal lymph nodes (MLN) or spleen by peptide-based staining protocols is at the limits of flow cytometric analysis.

707 citations


Journal ArticleDOI
20 Dec 1998-Virology
TL;DR: The analysis of the antigenic and biological properties of the H5N1 influenza viruses isolated from chickens, ducks, and geese from farms and poultry markets in Hong Kong during 1997 and compares them with those of virus isolated from the index human case revealed limited antigenic drift in 15 years.

Journal ArticleDOI
TL;DR: High-resolution in situ hybridization analyses to determine the pattern of reelin expression in the developing forebrain of the mouse revealed a complex pattern of cellular and regional expression that is consistent with Reelin having multiple roles in brain development and adult brain function.
Abstract: The reelin gene encodes an extracellular protein that is crucial for neuronal migration in laminated brain regions. To gain insights into the functions of Reelin, we performed high-resolution in situ hybridization analyses to determine the pattern of reelin expression in the developing forebrain of the mouse. We also performed double-labeling studies with several markers, including calcium-binding proteins, GAD65/67, and neuropeptides, to characterize the neuronal subsets that express reelin transcripts. reelin expression was detected at embryonic day 10 and later in the forebrain, with a distribution that is consistent with the prosomeric model of forebrain regionalization. In the diencephalon, expression was restricted to transverse and longitudinal domains that delineated boundaries between neuromeres. During embryogenesis, reelin was detected in the cerebral cortex in Cajal-Retzius cells but not in the GABAergic neurons of layer I. At prenatal stages, reelin was also expressed in the olfactory bulb, and striatum and in restricted nuclei in the ventral telencephalon, hypothalamus, thalamus, and pretectum. At postnatal stages, reelin transcripts gradually disappeared from Cajal-Retzius cells, at the same time as they appeared in subsets of GABAergic neurons distributed throughout neocortical and hippocampal layers. In other telencephalic and diencephalic regions, reelin expression decreased steadily during the postnatal period. In the adult, there was prominent expression in the olfactory bulb and cerebral cortex, where it was restricted to subsets of GABAergic interneurons that co-expressed calbindin, calretinin, neuropeptide Y, and somatostatin. This complex pattern of cellular and regional expression is consistent with Reelin having multiple roles in brain development and adult brain function.

Journal ArticleDOI
TL;DR: The MEK/ERK pathway not only acts transcriptionally to induce the cyclin D1 gene but functions posttranslationally to regulate cyclinD1 assembly with CDK4 and to thereby help cancel p27(Kip1)-mediated inhibition.
Abstract: A constitutively active form of mitogen-activated protein kinase kinase (MEK1) was synthesized under control of a zinc-inducible promoter in NIH 3T3 fibroblasts. Zinc treatment of serum-starved cells activated extracellular signal-regulated protein kinases (ERKs) and induced expression of cyclin D1. Newly synthesized cyclin D1 assembled with cyclin-dependent kinase-4 (CDK4) to form holoenzyme complexes that phosphorylated the retinoblastoma protein inefficiently. Activation of the MEK1/ERK pathway neither triggered degradation of the CDK inhibitor kinase inhibitory protein-1 (p27Kip1) nor led to activation of cyclin E- and A-dependent CDK2, and such cells did not enter the DNA synthetic (S) phase of the cell division cycle. In contrast, zinc induction of active MEK1 in cells also engineered to ectopically overexpress cyclin D1 and CDK4 subunits generated levels of cyclin D-dependent retinoblastoma protein kinase activity approximating those achieved in cells stimulated by serum. In this setting, p27Kip1 was mobilized into complexes containing cyclin D1; cyclin E- and A-dependent CDK2 complexes were activated; and serum-starved cells entered S phase. Thus, although the activity of p27Kip1 normally is canceled through a serum-dependent degradative process, overexpressed cyclin D1-CDK complexes sequestered p27Kip1 and reduced the effective inhibitory threshold through a stoichiometric mechanism. A fraction of these cells completed S phase and divided, but they were unable to continuously proliferate, indicating that other serum-responsive factors ultimately became rate limiting for cell cycle progression. Therefore, the MEK/ERK pathway not only acts transcriptionally to induce the cyclin D1 gene but functions posttranslationally to regulate cyclin D1 assembly with CDK4 and to thereby help cancel p27Kip1-mediated inhibition.

Journal ArticleDOI
TL;DR: Immunological detection of residual leukaemic cells at any point in the treatment course is a powerful predictor of relapse in children with ALL and should be considered for cases with persistent disease beyond the first 3 months of continuation therapy.

Journal ArticleDOI
20 Mar 1998-Cell
TL;DR: Extraction of soluble lumenal proteins from microsomes and reconstitution with purified proteins demonstrate, by fluorescence collisional quenching, that BiP seals the lumenAL end of this pore.

Journal ArticleDOI
TL;DR: Adjusting the dose of methotrexate to account for the patient's ability to clear the drug can improve the outcome in children with B-lineage acute lymphoblastic leukemia.
Abstract: Background The rate of clearance of antileukemic agents differs by a factor of 3 to 10 among children with acute lymphoblastic leukemia. We hypothesized that the outcome of treatment would be improved if doses were individualized to prevent low systemic exposure to the drugs in patients with fast drug clearance. Methods We stratified and randomly assigned 182 children with newly diagnosed acute lymphoblastic leukemia to postremission regimens that included high-dose methotrexate and teniposide plus cytarabine. The doses of these drugs were based on body-surface area (in the conventional-therapy group) or the rates of clearance of the three medications in each patient (in the individualized-treatment group). In the individualized-treatment group, doses were increased in patients with rapid clearance and decreased in patients with very slow clearance. Results Patients who received individualized doses had significantly fewer courses of treatment with systemic exposures below the target range than did patien...

Journal ArticleDOI
TL;DR: The data indicate that the current methods for monitoring resistant mutants are potentially flawed because no tissue culture system adequately reflects the receptor specificity of human respiratory tract epithelium.
Abstract: Zanamivir, a neuraminidase inhibitor, has shown promise as a drug to control influenza. During prolonged treatment with zanamivir, a mutant virus was isolated from an immunocompromised child infected with influenza B virus. A hemagglutinin mutation (198 Thr-->Ile) reduced the virus affinity for receptors found on susceptible human cells. A mutation in the neuraminidase active site (152 Arg-->Lys) led to a 1000-fold reduction in the enzyme sensitivity to zanamivir. When tested in ferrets, the mutant virus had less virulence than the parent; however, it had a growth preference over the parent in zanamivir-treated animals. Despite these changes, the sensitivity of the mutant virus to zanamivir assessed by a standard test in MDCK cells was unaffected. These data indicate that the current methods for monitoring resistant mutants are potentially flawed because no tissue culture system adequately reflects the receptor specificity of human respiratory tract epithelium.

Journal ArticleDOI
15 May 1998-Science
TL;DR: In wild-type, but not Atm-/- mice, up-regulation of p53 coincided with cell death, suggesting that Atn-dependent apoptosis in the CNS is mediated by p53, and p53 null mice showed a similar lack of radiation-induced cell death in the developing nervous system.
Abstract: Ataxia telangiectasia (AT) is characterized by progressive neurodegeneration that results from mutation of the ATM gene. However, neither the normal function of ATM in the nervous system nor the biological basis of the degeneration in AT is known. Resistance to apoptosis in the developing central nervous system (CNS) of Atm-/- mice was observed after ionizing radiation. This lack of death occurred in diverse regions of the CNS, including the cerebellum, which is markedly affected in AT. In wild-type, but not Atm-/- mice, up-regulation of p53 coincided with cell death, suggesting that Atm-dependent apoptosis in the CNS is mediated by p53. Further, p53 null mice showed a similar lack of radiation-induced cell death in the developing nervous system. Atm may function at a developmental survival checkpoint that serves to eliminate neurons with excessive DNA damage.

Journal ArticleDOI
TL;DR: The data suggest that interaction of pneumococci with the PAF receptor results in sorting so as to transcytose bacteria across the cell while non-PAF receptor entry shunts bacteria for exit and reentry on the apical surface in a novel recycling pathway.
Abstract: Although Streptococcus pneumoniae is a major cause of meningitis in humans, the mechanisms underlying its traversal from the circulation across the blood-brain barrier (BBB) into the subarachnoid space are poorly understood. One mechanism might involve transcytosis through microvascular endothelial cells. In this study we investigated the ability of pneumococci to invade and transmigrate through monolayers of rat and human brain microvascular endothelial cells (BMEC). Significant variability was found in the invasive capacity of clinical isolates. Phase variation to the transparent phenotype increased invasion as much as 6-fold and loss of capsule approximately 200-fold. Invasion of transparent pneumococci required choline in the pneumococcal cell wall, and invasion was partially inhibited by antagonists of the platelet-activating factor (PAF) receptor on the BMEC. Pneumococci that gained access to an intracellular vesicle from the apical side of the monolayer subsequently were subject to three fates. Most opaque variants were killed. In contrast, the transparent phase variants were able to transcytose to the basal surface of rat and human BMEC in a manner dependent on the PAF receptor and the presence of pneumococcal choline-binding protein A. The remaining transparent bacteria entering the cell underwent a previously unrecognized recycling to the apical surface. Transcytosis eventually becomes a dominating process accounting for up to 80% of intracellular bacteria. Our data suggest that interaction of pneumococci with the PAF receptor results in sorting so as to transcytose bacteria across the cell while non-PAF receptor entry shunts bacteria for exit and reentry on the apical surface in a novel recycling pathway.

Journal ArticleDOI
01 May 1998-Blood
TL;DR: AML1-ETO not only neutralizes the normal biologic activity of AML1 but also directly induces aberrant hematopoietic cell proliferation, which is similar to that seen following homozygous disruption of either AML 1 or CBFbeta.

Journal ArticleDOI
TL;DR: Results indicate that Dab1 functions downstream of Reln in a signaling pathway that controls cell positioning in the developing brain.
Abstract: Mutation of either reelin (Reln) or disabled-1 (Dab1) results in widespread abnormalities in laminar structures throughout the brain and ataxia in reeler and scrambler mice. Both exhibit the same neuroanatomical defects, including cerebellar hypoplasia with Purkinje cell ectopia and disruption of neuronal layers in the cerebral cortex and hippocampus. Despite these phenotypic similarities, Reln and Dab1 have distinct molecular properties. Reln is a large extracellular protein secreted by Cajal-Retzius cells in the forebrain and by granule neurons in the cerebellum. In contrast, Dab1 is a cytoplasmic protein which has properties of an adapter protein that functions in phosphorylation-dependent intracellular signal transduction. Here, we show that Dab1 participates in the same developmental process as Reln. In scrambler mice, neuronal precursors are unable to invade the preplate of the cerebral cortex and consequently, they do not align within the cortical plate. During development, cells expressing Dab1 are located next to those secreting Reln at critical stages of formation of the cerebral cortex, cerebellum and hippocampus, before the first abnormalities in cell position become apparent in either reeler or scrambler. In reeler, the major populations of displaced neurons contain elevated levels of Dab1 protein, although they express normal levels of Dab1 mRNA. This suggests that Dab1 accumulates in the absence of a Reln-evoked signal. Taken together, these results indicate that Dab1 functions downstream of Reln in a signaling pathway that controls cell positioning in the developing brain.

Journal ArticleDOI
TL;DR: Evidence to support the hypothesized ordering of groups in terms of neuropsychologic toxicity was obtained with regard to Performance Intelligence Quotient (IQ), Full Scale IQ, Attention, Reading, and Arithmetic.
Abstract: PURPOSEThe purpose of this study was to test the hypothesis that survivors of medulloblastoma who were younger at diagnosis and those who received standard-dose cranial irradiation (SRT) of 36 Gy would have a lower performance on standardized tests of cognitive function and achievement than children who were older and those treated with reduced-dose cranial irradiation (RRT) of 23.4 Gy.PATIENTS AND METHODSEligible patients had been treated on Pediatric Oncology Group (POG) study 8631 for low-risk medulloblastoma that randomized patients to receive RRT or SRT after surgical resection. Those who were alive and free of progressive disease 6.1 to 9.9 years from completion of treatment were eligible for this study. Of the 35 eligible patients, 22 patients (13 SRT, nine RRT) participated in a battery of tests that included intellectual and academic development as well as ratings of health-related quality of life.RESULTSPatients were stratified by treatment group (SRT v RRT) and into younger (Y) and older (O) gr...

Journal ArticleDOI
TL;DR: Results contradict the view that these compounds directly disrupt membranes and suggest that their widespread use will select for resistant bacterial populations.

Journal ArticleDOI
TL;DR: Long persistence of PMPApp, particularly in resting lymphocytes, may be unique to the nucleoside phosphonate analogs and indicates that effective levels of the active metabolite can be achieved and maintained with relatively infrequent administration of the parent drug.
Abstract: Bis(isopropyloxymethylcarbonyl) 9-R-(2-phosphonomethoxypropyl)adenine [bis(POC)PMPA] has been identified as a novel prodrug of PMPA. The anti-human immunodeficiency virus activity of bis(POC)PMPA was >100-fold greater than that of PMPA in both an established T-cell line and primary peripheral blood lymphocytes. This improved efficacy was shown to be due to a rapid intracellular uptake of the prodrug resulting in an increased intracellular accumulation of PMPA diphosphate (PMPApp), the pharmacologically active metabolite. PMPApp levels in bis(POC)PMPA-treated cells exceeded by >1,000-fold the levels seen in cells treated with unmodified PMPA in both resting and activated peripheral blood lymphocytes. Significant differences in the intracellular catabolism of PMPA metabolites were noted between the resting and activated lymphocytes. The half-life for the disappearance of PMPApp, derived from either bis(POC)PMPA or PMPA, was 12 to 15 h in the activated lymphocytes and 33 to 50 h in the resting lymphocytes. This long persistence of PMPApp, particularly in resting lymphocytes, may be unique to the nucleoside phosphonate analogs and indicates that effective levels of the active metabolite can be achieved and maintained with relatively infrequent administration of the parent drug.

Journal ArticleDOI
TL;DR: The application of new technologies for identifying interacting proteins has connected the studies on topoisomerases to other areas of human biology including genome stability and aging, and highlights the importance of understanding how topoisomersases participate in the normal processes of transcription, DNA replication, and genome stability.

Journal ArticleDOI
TL;DR: This review summarizes what is currently known about signaling pathways involved in apoptosis, with particular emphasis on the function of the cysteine proteases known as caspases.
Abstract: Since the discovery that cells can activate their own suicide program, investigators have attempted to determine whether the events that are associated with this form of cell death are genetically determined. The discovery that the ced-3 gene of Caenorhabditis elegans encodes a cysteine protease essential for developmentally regulated apoptosis ignited interest in this area of research. As a result, we now know that cell death is specified by a number of genes and that this biologic process contributes significantly to development, tumorigenesis, and autoimmune disease. In this review I summarize what is currently known about signaling pathways involved in apoptosis, with particular emphasis on the function of the cysteine proteases known as caspases. However, there is also evidence that protease-independent cell death pathways exist. Is there a relationship between these two distinct mechanisms? If so, how do they communicate? Finally, even though the involvement of tumor necrosis factor/nerve growth factor family of receptors and cysteine proteases has been elegantly established as a component of many apoptotic signaling pathways, what happens downstream of these initial events? Why are only a selected group of cellular proteins--many nuclear--the targets of these proteases? Are nuclear events essential for apoptosis in vivo? Are the cellular genes that encode products involved in apoptotic signaling frequent targets of mutation/alteration during tumorigenesis? These are only a few questions that may be answered in the next ten years.

Journal ArticleDOI
15 Apr 1998-Blood
TL;DR: The CTLs persisted for more than 13 weeks postinfusion and retained their potent antiviral effects in vivo, thereby enhancing the patient immune response to EBV, and may have value in the treatment of EBV-positive Hodgkin's disease.

Journal ArticleDOI
TL;DR: PLC-γ is an important downstream target of NPM-ALK that contributes to its mitogenic activity and is likely to be important in the molecular pathogenesis of large-cell anaplastic lymphomas.
Abstract: Large-cell anaplastic lymphoma is a subtype of non-Hodgkin’s lymphoma characterized by the expression of CD30. More than half of these lymphomas have a chromosomal translocation, t(2;5), that leads to the expression of a hybrid protein comprised of the nucleolar phosphoprotein nucleophosmin (NPM) and the anaplastic lymphoma kinase (ALK). Here we show that transfection of the constitutively active tyrosine kinase NPM-ALK into Ba/F3 and Rat-1 cells leads to a transformed phenotype. Oncogenic tyrosine kinases transform cells by activating the mitogenic signal transduction pathways, e.g., by binding and activating SH2-containing signaling molecules. We found that NPM-ALK binds most specifically to the SH2 domains of phospholipase C-γ (PLC-γ) in vitro. Furthermore, we showed complex formation of NPM-ALK and PLC-γ in vivo by coimmunoprecipitation experiments in large-cell anaplastic lymphoma cells. This complex formation leads to the tyrosine phosphorylation and activation of PLC-γ, which can be corroborated by enhanced production of inositol phosphates (IPs) in NPM-ALK-expressing cells. By phosphopeptide competition experiments, we were able to identify the tyrosine residue on NPM-ALK responsible for interaction with PLC-γ as Y664. Using site-directed mutagenesis, we constructed a comprehensive panel of tyrosine-to-phenylalanine NPM-ALK mutants, including NPM-ALK(Y664F). NPM-ALK(Y664F), when transfected into Ba/F3 cells, no longer forms complexes with PLC-γ or leads to PLC-γ phosphorylation and activation, as confirmed by low IP levels in these cells. Most interestingly, Ba/F3 and Rat-1 cells expressing NPM-ALK(Y664F) also show a biological phenotype in that they are not stably transformed. Overexpression of PLC-γ can partially rescue the proliferative response of Ba/F3 cells to the NPM-ALK(Y664F) mutant. Thus, PLC-γ is an important downstream target of NPM-ALK that contributes to its mitogenic activity and is likely to be important in the molecular pathogenesis of large-cell anaplastic lymphomas.

Journal ArticleDOI
TL;DR: AMS-1–ETO may contribute to leukemogenesis by specifically inhibiting C/EBP-α- and AML-1B-dependent activation of myeloid promoters and blocking differentiation.
Abstract: AML-1B is a hematopoietic transcription factor that is functionally inactivated by multiple chromosomal translocations in human acute myeloblastic and B-cell lymphocytic leukemias. The t(8;21)(q22;q22) translocation replaces the C terminus, including the transactivation domain of AML-1B, with ETO, a nuclear protein of unknown function. We previously showed that AML-1-ETO is a dominant inhibitor of AML-1B-dependent transcriptional activation. Here we demonstrate that AML-1-ETO also inhibits C/EBP-alpha-dependent activation of the myeloid cell-specific, rat defensin NP-3 promoter. AML-1B bound the core enhancer motifs present in the NP-3 promoter and activated transcription approximately sixfold. Similarly, C/EBP-alpha bound NP-3 promoter sequences and activated transcription approximately sixfold. Coexpression of C/EBP-alpha with AML-1B or its family members, AML-2 and murine AML-3, synergistically activated the NP-3 promoter up to 60-fold. The t(8;21) product, AML-1-ETO, repressed AML-1B-dependent activation of NP-3 and completely inhibited C/EBP-alpha-dependent activity as well as the synergistic activation. In contrast, the inv(16) product, which indirectly targets AML family members by fusing their heterodimeric DNA binding partner, CBF-beta, to the myosin heavy chain, inhibited AML-1B but not C/EBP-alpha activation or the synergistic activation. AML-1-ETO and C/EBP-alpha were coimmunoprecipitated and thus physically interact in vivo. Deletion mutants demonstrated that the C terminus of ETO was required for AML-1-ETO-mediated repression of the synergistic activation but not for association with C/EBP-alpha. Finally, overexpression of AML-1-ETO in myeloid progenitor cells prevented granulocyte colony-stimulating factor-induced differentiation. Thus, AML-1-ETO may contribute to leukemogenesis by specifically inhibiting C/EBP-alpha- and AML-1B-dependent activation of myeloid promoters and blocking differentiation.

Journal ArticleDOI
TL;DR: Findings indicate that expression of the functional λ5/14.1 is critical for B cell development in the human and that when expressed in COS cells, the allele carrying the pseudogene sequence resulted in defective folding and secretion of mutant λ 5/ 14.1.
Abstract: B cell precursors transiently express a pre–B cell receptor complex consisting of a rearranged mu heavy chain, a surrogate light chain composed of λ5/14.1 and VpreB, and the immunoglobulin (Ig)-associated signal transducing chains, Igα and Igβ. Mutations in the mu heavy chain are associated with a complete failure of B cell development in both humans and mice, whereas mutations in murine λ5 result in a leaky phenotype with detectable humoral responses. In evaluating patients with agammaglobulinemia and markedly reduced numbers of B cells, we identified a boy with mutations on both alleles of the gene for λ5/14.1. The maternal allele carried a premature stop codon in the first exon of λ5/14.1 and the paternal allele demonstrated three basepair substitutions in a 33-basepair sequence in exon 3. The three substitutions correspond to the sequence in the λ5/14.1 pseudogene 16.1 and result in an amino acid substitution at an invariant proline. When expressed in COS cells, the allele carrying the pseudogene sequence resulted in defective folding and secretion of mutant λ5/14.1. These findings indicate that expression of the functional λ5/14.1 is critical for B cell development in the human.