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J C Reed

Publications and source records attributed to J C Reed.

At least 451 records · Page 25Linked to original sources
PubMed1993

Coupling of GTP-binding to the T cell receptor (TCR) zeta-chain with TCR-mediated signal transduction.

The zeta-subunit of the TCR binds GTP and is a well characterized substrate for a TCR-activated tyrosine kinase. To examine the possible coupling of GTP-binding to zeta with TCR-mediated signal transduction, a mutant (termed J32-3.2) of the T cell line Jurkat (J32) was used. Anti-TCR/CD3 stimulation of the TCR/CD3+ J32-3.2 cells resulted in a weak stimulation of both the phosphatidyl inositol and tyrosine kinase signal transduction pathways, as measured by changes in the level of free intracellular calcium, tyrosine phosphorylation of TCR-zeta, CD3-epsilon and ZAP-70, p56lck, or p59fyn tyrosine kinase activity and IL-2 gene activation. The impaired responsiveness of J32-3.2 cells to anti-TCR/CD3 mAb correlated with a low basal level of GTP-binding to zeta. Furthermore, in J32-3.2 cells TCR activation by antibody ligation caused a weaker increase in GTP-binding to the zeta-chain, as compared with that of wild-type J32 cells, which indicates for the first time that GTP-binding to zeta can be modulated by extracellular signals and suggest that the role of GTP-binding to zeta is to couple the TCR to intracellular signal transduction mechanisms.

Amino Acid Sequence↗
PubMed1993

Apoptosis induced by withdrawal of interleukin-3 (IL-3) from an IL-3-dependent hematopoietic cell line is associated with repartitioning of intracellular calcium and is blocked by enforced Bcl-2 oncoprotein production.

The regulation of intracellular pools of Ca2+ was investigated in an interleukin-3 (IL-3)-dependent hematopoietic cell line 32D that undergoes programmed cell death ("apoptosis") when deprived of lymphokine. Comparisons were made with 32D cells that had been stably transfected with a bcl-2 expression plasmid that encodes a 26-kDa intracellular integral-membrane protein known to abrogate apoptosis resulting from IL-3 withdrawal. Removal of IL-3 from cultures of 32D cells or control-transfected 32D-NEO cells for 1-2 days led to cell cycle arrest and oligonucleosomal DNA fragmentation and was associated with lower cytosolic free Ca2+ concentrations ([Ca2+]i), as measured by Indo-1 fluorescence of viable cells in Ca(2+)-containing media. In bcl-2-expressing 32D-BCL2 cells, IL-3 withdrawal also resulted in cessation of proliferation, but [Ca2+]i levels were not decreased and DNA fragmentation was markedly suppressed. Nonmitochondrial stores of Ca2+ were also significantly diminished in IL-3-deprived 32D-NEO but not in 32D-BCL2 cells, based on measurements of Ca2+ release into the cytosol following exposure of cells to thapsigargin (an inhibitor of endoplasmic reticulum Ca(2+)-ATPases) under Ca(2+)-free conditions. In contrast, estimates of mitochondrial Ca2+ stores using an uncoupler of oxidative phosphorylation 1799 (2,6-dihydroxy-1,1,1,7,7,7-he xafluoro-2,6-bis(trifluoromethyl)heptan-4-one[bis(he xafluoroacetonyl)]acetone) suggested that IL-3 deprivation leads to an increase in this intracellular pool of Ca2+ in 32D-NEO but not in 32D-BCL2 cells. Re-addition of IL-3 to factor-deprived 32D-NEO cells reversed the changes in thapsigargin- and 1799-releasable Ca2+ pools and rescued many of the cells from death. Measurements of total cellular Ca2+ revealed no difference in 32D-NEO cells before and after IL-3 withdrawal, suggesting that the observed alterations in mitochondrial and nonmitochondrial Ca2+ pools result from intracellular repartitioning of Ca2+. Treatment of IL-3-deprived 32D-NEO cells with Ca2+ ionophores blocked DNA fragmentation and prolonged cell survival, whereas addition of Ca2+ chelators to IL-3-stimulated 32D cells resulted in oligonucleosomal DNA fragmentation and cell death, suggesting that diminutions in the concentrations of Ca2+ in cytosol, endoplasmic reticulum, or other intracellular compartments either directly or indirectly regulate apoptosis in these lymphokine-dependent hematopoietic cells.

Animals↗
PubMed1993

Conversion of lytic to persistent alphavirus infection by the bcl-2 cellular oncogene.

Little is known about virus-host cell interactions that regulate the lytic potential of viruses during productive replication. Sindbis virus (SV), a single-stranded positive-sense RNA virus in the alphavirus genus (family Togaviridae), results in lytic infection in most vertebrate cell lines, but persistent productive infection in post-mitotic neurons. The cellular oncogene bcl-2, which encodes an inner mitochondrial membrane protein of M(r) 26,000 (ref. 2), blocks programmed cell death (apoptosis) in neurons. We therefore investigated whether SV infection induces programmed cell death in non-neuronal cells, and if so, whether virus-induced programmed cell death can be blocked by transfection with bcl-2. We demonstrate that SV infection of baby hamster kidney (BHK-2), mouse neuroblastoma (N18), and rat prostatic adenocarcinoma (AT-3) cells results in programmed cell death, whereas SV infection of bcl-2-transfected AT-3 cells results in long-term persistent productive infection. Thus cellular bcl-2 oncogene expression plays a role in the establishment of persistent viral infection by blocking virus-induced programmed cell death.

Adenocarcinoma↗
PubMed1993

Bcl-2 blocks apoptosis in cells lacking mitochondrial DNA.

When the mammalian proto-oncogene bcl-2 is overexpressed it can protect various types of cells both from normal and from experimentally induced apoptosis, but the molecular mechanisms involved are unknown. Although the Bcl-2 protein is membrane-associated, its subcellular location is controversial: two studies have suggested that it is mainly associated with the nuclear envelope and endoplasmic reticulum, whereas another study has suggested that it is mainly located in the inner mitochondrial membrane. The latter study has suggested that Bcl-2 might protect cells from apoptosis by altering mitochondrial function and that mitochondria may be involved in apoptosis. Here we report that human mutant cell lines that lack mitochondrial DNA (mtDNA), and therefore do not have a functional respiratory chain, can still be induced to die by apoptosis, and that they can be protected from apoptosis by the overexpression of bcl-2, suggesting that neither apoptosis nor the protective effect of bcl-2 depends on mitochondrial respiration. We also show that the Bcl-2 protein in overexpressing cells is associated with the nuclear envelope and endoplasmic reticulum, as well as with mitochondria.

Alkaloids↗
PubMed1993

Bcl-2 oncoprotein blocks chemotherapy-induced apoptosis in a human leukemia cell line.

Previous studies have shown that the bcl-2 gene encodes a mitochondrial protein that contributes to neoplastic cell expansion primarily by promoting cell survival through interference with "programmed cell death" (PCD), also termed "apoptosis." Because many chemotherapeutic drugs are capable of initiating pathways leading to apoptosis, we determined whether deregulated bcl-2 expression could render cells resistant to several drugs commonly used in the treatment of non-Hodgkin's lymphomas, including dexamethasone (DEX), methotrexate (MTX), 1-beta-D-arabinofuranosyl-cytosine (Ara-C), etoposide (VP-16), vincristine (VC), cisplatin (CP), and hydroperoxycyclophosphamide (4-HC). For these experiments, we achieved high levels of p26-Bcl-2 protein production in a human pre-B-cell leukemia line 697 by stable infection with a recombinant bcl-2-containing retrovirus and then compared these cells with control virus-infected 697 cells. Control 697 cells were induced to undergo apoptosis by all drugs tested as defined by DNA degradation into oligonucleosomal-length fragments, cell shrinkage, and subsequent cell death. In contrast, 697 cells with elevated Bcl-2 protein levels exhibited strikingly prolonged cell survival and markedly reduced DNA fragmentation when cultured in the presence of these antineoplastic agents. Although high levels of Bcl-2 protein protected 697 cells from the acute cytotoxic effects of DEX and the other drugs tested, Bcl-2 did not prevent these drugs from suppressing the proliferation of 697 cells. However, when 697 cells were treated with DEX or MTX for 3 days, then washed and cultured in semisolid media without drugs, bcl-2-virus-infected cells gave rise to colonies at much higher frequencies than 697 cells stably infected with control virus. These results indicate that by protecting 697 leukemic cells from the acute cytotoxicity of DEX and some other chemotherapeutic drugs, high levels of p26-Bcl-2 can create the opportunity for re-initiation of cell growth when drugs are withdrawn. The findings may be relevant to clinical correlative studies of non-Hodgkin's lymphoma patients that have found an association between worse prognosis and bcl-2 gene rearrangements or t[14;18] translocations.

Antineoplastic Agents↗
PubMed1993

Phosphorylation of calmodulin by the catalytic subunit of casein kinase II is inhibited by the regulatory subunit.

Casein kinase II (CKII) is composed of a catalytic subunit (alpha) and a regulatory subunit (beta) that combine to form an alpha 2 beta 2 holoenzyme. The alpha-subunit monomer is enzymatically active, albeit kinetically attenuated relative to the holoenzyme, and the addition of purified beta subunit stimulates its activity against casein (C. Cochet and E. M. Chambaz, 1983, J. Biol. Chem. 258, 1403-1406). Here we report a kinetic analysis of the phosphorylation of various protein and peptide substrates by the alpha subunit and the holoenzyme of Drosophila melanogaster CKII. We demonstrate that the alpha subunit, like the holoenzyme, is competent to phosphorylate typical physiological substrates such as the regulatory (RII) subunit of cAMP-dependent protein kinase (cAMPdPK), as well as artificial substrates such as alpha-casein and the synthetic peptide RRREEETEEE. The Km of the alpha subunit in each case is similar to that of the holoenzyme, whereas the Vmax is 5- to 60-fold lower. In contrast, calmodulin, a protein that is significantly phosphorylated by the holoenzyme only in the presence of polybasic compounds, is readily phosphorylated by the alpha subunit alone. While the Km values of the alpha subunit and the holoenzyme for calmodulin are similar, the Vmax of the alpha subunit is at least 10-fold higher than that of the holoenzyme. These results suggest that while the alpha subunit contains the necessary determinants for CKII substrate specificity, the beta subunit can either inhibit or activate it, in a substrate-dependent manner. Finally, we also demonstrate that polybasic compounds stimulate not only the holoenzyme but, to a lesser extent, the alpha subunit as well.

Amino Acid Sequence↗
PubMed1993

Protooncogene-encoded protein kinases in interleukin-2 signal transduction.

Protooncogenes are the normal forms of cellular genes that when altered in their expression or coding sequences can contribute to neoplastic transformation. As these genes often are important for normal cellular growth control, we explored the possibility that protein kinases encoded by particular protooncogenes could participate in signal transduction pathways regulated by the T cell growth factor, interleukin-2 (IL-2). In this review we summarize our findings to date regarding Raf-1, a serine/threonine-specific kinase that becomes phosphorylated on tyrosine residues and enzymatically activated in response to IL-2 stimulation. In addition, we describe our investigations of Lck and Lyn, two closely related protein tyrosine kinases of the src gene family that physically associate with the IL-2 receptor complex and whose activities are regulated by IL-2 in at least some T cells and B cells, respectively.

Animals↗
PubMed1993

Investigations of antisense oligonucleotides targeted against bcl-2 RNAs.

Expression of the bcl-2 gene becomes deregulated in many non-Hodgkin lymphomas as the result of t(14;18) chromosomal translocations. Because bcl-2 regulates the survival of cells, and because its over-expression is associated with cellular resistance to killing by chemotherapeutic drugs and gamma-irradiation, this gene and its mRNA and protein products represent ideal targets for designing novel therapeutic strategies for the treatment of cancer. Here we describe the effects of an 18-mer phosphodiester oligonucleotide that is complementary to the first 6 codons of the bcl-2 mRNA's open reading frame. When tested for inhibition of in vitro protein synthesis using RNAse-H-supplemented reticulocyte lysates and RNA prepared by in vitro transcription of a human bcl-2 cDNA, the bcl-2 antisense (AS) oligomer completely abolished Bcl-2 protein production at 10 microM, but had no effect on the in vitro translation of a chicken bcl-2 RNA that contained three mismatches relative to the oligomer binding site on the human bcl-2 RNA. A control 18-mer having the same base composition as the AS oligomer but with scrambled order (SC) was not inhibitory. Addition of AS and SC oligomers to cultures of a NIH-3T3 fibroblast cell line that had been stably infected with a recombinant retrovirus containing the same human bcl-2 cDNA used for in vitro transcription/translation experiments revealed concentration-dependent reductions in the relative levels of the 26-kD human Bcl-2 protein (as determined by immunoblotting) by the AS but not by the SC oligomer. Similar results were obtained when AS and SC oligomers were applied to a t(14;18)-containing lymphoma cell line SU-DHL-4 that was cultured in low-serum media. When used at 200 microM, the bcl-2 AS oligomer produced 84-95% reductions in Bcl-2 protein levels in SU-DHL-4 cells but had relatively little effect on the levels of other mitochondrial control proteins, suggesting that the inhibitory effects were specific. Treatment of SU-DHL-4 cells with AS oligomer lead to essentially complete loss of bcl-2 mRNA from cells within 1 day of addition to cultures, but presumably because of the long half-life of the Bcl-2 protein (approximately 14 h), commensurate reductions in Bcl-2 protein levels did not occur until 3 days.(ABSTRACT TRUNCATED AT 400 WORDS)

3T3 Cells↗
PubMed1993

Somatic point mutations in the translocated bcl-2 genes of non-Hodgkin's lymphomas and lymphocytic leukemias: implications for mechanisms of tumor progression.

The t[14;18] chromosomal translocation is the most common cytogenetic abnormality found in hematolymphoid malignancies. The t[14;18] fuses the bcl-2 gene at 18q21 with the immunoglobulin heavy-chain locus at 14q32, resulting in deregulated expression of bcl-2 and production of high levels of its encoded 26-kD protein in the majority of non-Hodgkin lymphomas. Recent data indicate that somatic point mutations frequently occur in translocated bcl-2 alleles, possibly because of the somatic hypermutation mechanism that is associated with the immunoglobulin gene loci and that normally contributes to antibody diversity. In some cases, these mutations can affect the open reading frame of the bcl-2 gene and thereby alter Bcl-2 proteins. Here, we review the currently available data about the incidence, biological effects, and possible clinical importance of somatic mutations within the translocated bcl-2 genes of human lymphomas and leukemias.

Alleles↗
PubMed1993

Magnetic resonance imaging of the mediastinum.

Magnetic resonance (MR) imaging is becoming a primary modality for evaluating the mediastinum. MR affords multiplanar imaging capabilities without exposing patients to ionizing radiation. The inherent contrast effect of different mediastinal tissues sharply delineates anatomic structures on MR images without contrast enhancement. Gradient-echo and phase-mapping techniques permit noninvasive qualitative and quantitative assessment of mediastinal blood flow. High soft tissue contrast and flow analysis capabilities make MR imaging a valuable modality for evaluating mediastinal vascular disorders. Various mediastinal tumors and their extent are best identified by the use of T1-weighted, T2-weighted, and gadolinium-enhanced images. Both primary and secondary chest wall lesions may be assessed with standard spin-echo MR images. Complex pleuroparenchymal lesions may be evaluated by means of a multiplanar approach and modified pulse sequences. This article addresses the technical parameters governing MR imaging of the mediastinum and describes MR characteristics of various pathologic conditions.

Humans↗
PubMed1993

Effect of bcl-2 proto-oncogene expression on cellular sensitivity to tumor necrosis factor-mediated cytotoxicity.

Introduction and expression of the proto-oncogene bcl-2 (B-cell lymphoma/leukemia 2) has been shown to extend the survival of certain hematopoietic cell lines after growth factor deprivation, by blocking apoptosis or programmed cell death. We investigated the effect of bcl-2 expression on cellular sensitivity to lysis by tumor necrosis factor (TNF), a cytokine capable of inducing apoptosis in several tumor cell lines. Introduction of the human bcl-2 gene in the highly TNF-sensitive L929 mouse fibrosarcoma cell line did not result in altered TNF sensitivity. Likewise, NIH3T3 and REF cells, which are resistant to TNF cytotoxicity but become TNF sensitive upon cotreatment with actinomycin D or upon expression of the adenovirus E1A gene, did not show altered TNF sensitivity upon bcl-2 transfection. Despite constitutive expression of the endogenous bcl-2 gene, human MCF7 breast carcinoma cells, as well as HL60 promyelocytic leukemia and U937 histiocytic lymphoma cell lines were found to be TNF sensitive. bcl-2-overexpressing derivatives of these cell lines did not acquire reduced TNF sensitivity and still exhibited the characteristic pattern of internucleosomal DNA fragmentation of TNF-induced apoptosis. Moreover, bcl-2 expression in the interleukin 3 (IL-3)-dependent myeloid cell line 32D protected these cells from apoptosis resulting from growth factor deprivation, but not from apoptosis induced by TNF. These data clearly establish the absence of a correlation between bcl-2 gene expression and cellular sensitivity to TNF-induced cell lysis. These findings are discussed in the context of the hypothesis of different pathways for induction of apoptosis, only some of which are affected by bcl-2 expression.

3T3 Cells↗
PubMed1992

Differential effects of Bcl-2 on T and B cells in transgenic mice.

We have produced bcl-2 transgenic mice by using a construct which mimics the t(14;18) translocation in human follicular lymphomas. Although lymphoid tissues from all transgenic mice contained high levels of human Bcl-2 protein, transgene expression was differentially regulated within the B- and T-cell compartments of lines derived from various founder mice. We have characterized the phenotypes of two lines of bcl-2 transgenic mice (line 2 and line 6) in which bcl-2 transgene expression was restricted primarily to the T- or B-cell lineages, respectively. Analysis of line 6 lymphocytes revealed a polyclonal expansion of B cells, and these B cells exhibited prolonged survival in vitro. In line 2 mice, numbers of T cells in the peripheral lymphoid tissues were more moderately elevated despite enhanced T-cell survival in vitro. Line 2 transgenic mice also showed significantly increased proportions of thymocytes with a mature phenotype. Taken together, these findings suggest different roles for bcl-2 in the in vivo regulation of B- and T-cell development and homeostasis.

Animals↗
PubMed1992

Anergic Th1 cells express altered levels of the protein tyrosine kinases p56lck and p59fyn.

Tolerance in T lymphocytes can result from clonal anergy, or paralysis, of Ag-specific T cells. To investigate the molecular mechanisms responsible for anergy, a system in which tolerance can be induced in vitro was employed. Anergy, as defined by long-lived nonresponsiveness to normal antigenic stimulation for IL-2 production, was produced in cloned murine CD4+ Th1 cells. Here we report that such anergic Th1 cells express constitutively reduced amounts of the protein tyrosine kinase p56lck and constitutively elevated levels of the protein tyrosine kinase p59fyn. Because protein tyrosine phosphorylation is known to be important for the normal induction of IL-2 synthesis, these results suggest that T cell anergy may be maintained, at least in part, by alterations in tyrosine phosphorylation signaling events.

Animals↗
PubMed1992

bcl-2 gene transfer increases relative resistance of S49.1 and WEHI7.2 lymphoid cells to cell death and DNA fragmentation induced by glucocorticoids and multiple chemotherapeutic drugs.

The S49.1 and WEHI7.2 murine lymphoid cell lines have been used extensively as models for investigations of programmed cell death ("apoptosis") induced by glucocorticoids such as dexamethasone. Infection of these thymus-derived T-cell lines with a recombinant retrovirus encoding the human M(r) 26,000 Bcl-2 oncoprotein resulted in marked resistance to DEX-mediated cell death and DNA degradation into oligonucleosomal fragments, without interfering with the ability of dexamethasone to suppress cellular proliferation and without lowering levels of glucocorticoid receptors. In contrast, high levels of p26-Bcl-2 production did not block cell killing and DNA fragmentation induced by H2O2, suggesting that the Bcl-2 impairs some but not all pathways for cell death in S49.1 and WEHI7.2 cells that are associated with the DNA fragmentation pattern typical of apoptosis. S49.1 and WEHI7.2 cells infected with bcl-2 but not control retrovirus also exhibited increased resistance to cell killing and DNA fragmentation induced by a wide variety of reagents, including the calcium ionophore ionomycin, the phorbol ester tetradecanoylphorbol acetate, the dihydrofolate reductase inhibitor methotrexate, the antimetabolite 1-beta-D-arabinofuranosylcytosine, and the microtubule inhibitor vincristine. These findings provide evidence that p26-Bcl-2 interferes with a pathway for cell death that is activated by multiple drugs used for the treatment of cancer.

Animals↗
PubMed1992

Molecular cloning and DNA sequence analysis of cDNA encoding chicken homologue of the Bcl-2 oncoprotein.

We have isolated a 2228 bp cDNA clone encoding a chicken homologue of the human Bcl-2 oncoprotein by low-stringency hybridization screening of a lambda gt10 cDNA library derived from a chicken B-cell lymphoma. DNA sequence analysis of this cDNA revealed an open reading frame predicting a polypeptide of 232 amino acids and an M(r) of 25,839. The predicted protein is highly homologous to the human (73%) and mouse (70%) Bcl-2 proteins, and contains a hydrophobic stretch of amino acids within its carboxyl-end (213-229) consistent with an integral membrane protein. Areas of very high sequence homology shared by all three Bcl-2 proteins at the NH2-terminus (amino acids 1-33) and within the last 150 amino acids of these proteins suggest the presence of at least two evolutionarily conserved domains within the family of Bcl-2 proteins that may be important either for their targeting to mitochondria or their ability to block programmed cell death.

Amino Acid Sequence↗
PubMed1992

A strategy for generating monoclonal antibodies against recombinant baculovirus-produced proteins: application to the Bcl-2 oncoprotein.

A strategy is described for production of monoclonal antibodies against recombinant proteins that are produced using the baculovirus expression system and that requires no prior purification of the protein of interest. Crude lysates prepared from cultured Sf9 insect cells infected with recombinant or control baculoviruses are absorbed to nitrocellulose filters and used in a dot-immunobinding assay for screening hybridomas. The monoclonal antibody-producing hybridomas are derived by immunization of mice with a synthetic peptide corresponding to a hydrophilic region in the recombinant protein of interest. By using the baculovirus-produced recombinant protein as the screening antigen and by comparing antibody binding to filters containing control Sf9 lysates, hybridomas are identified that produce monoclonal antibodies with specific reactivity for the recombinant protein of interest and that can then subsequently be used to assist in the large-scale purification of the recombinant protein from baculovirus-infected cells. We applied this method to recombinant 26-kDa human Bcl-2 (B-cell lymphoma/leukemia-2), an integral membrane oncoprotein that regulates programmed cell death ("apoptosis") in hematolymphoid cells through unknown mechanisms. Two mouse monoclonal antibodies were produced that specifically bound the recombinant Bcl-2 baculoprotein in both solution and solid-phase assays.

Amino Acid Sequence↗