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J C Reed

Publications and source records attributed to J C Reed.

At least 415 records · Page 23Linked to original sources
PubMed1994

Immunohistochemical analysis of in vivo patterns of Bcl-X expression.

The in vivo patterns of bcl-X gene expression were assessed in human and mouse tissues using an immunohistochemical approach. Polyclonal antisera were raised against synthetic peptides corresponding to amino acids 46-66 and 61-79 of the human Bcl-X protein and were shown to be specific for detection of human and mouse Bcl-X-L and Bcl-X-S proteins by immunoblotting. Bcl-X immunoreactivity was detected in a wide variety of cell types and was typically present in the cytosol in a punctate pattern suggestive of association with intracellular organelles. Among the cell types with prominent Bcl-X immunostaining were: (a) a variety of neuronal populations in the brain as well as sensory neurons in dorsal root ganglia; (b) cortical (but not medullary) thymocytes and activated lymphocytes and plasma cells in lymph nodes; (c) several types of cells in the bone marrow, including megakaryocytes, red cell precursors, and some types of differentiating myeloid cells; (d) reproductive tissues, including the spermatocytes and spermatids in the testes and germinal epithelium of the ovary; and (e) a variety of epithelial cells including mammary epithelium, the secretory epithelial and basal cells of the prostate, uterine endometrium, gastric and intestinal epithelial cells, renal tubule epithelium, and differentiated keratinocytes in the upper layers of the epidermis but not in the basal cells. In many cases, these patterns of Bcl-X expression were strikingly different from those reported previously for Bcl-2, suggesting that Bcl-X and Bcl-2 regulate cell life and death at different stages of cell differentiation through tissue-specific control of their expression.

Amino Acid Sequence↗
PubMed1994

Cell-free apoptosis in Xenopus egg extracts: inhibition by Bcl-2 and requirement for an organelle fraction enriched in mitochondria.

Apoptotic cell death involves a ritual series of morphological changes, presumably reflecting a conserved molecular pathway. We now report that the nuclear events typical of apoptosis can be reproduced in "apoptotic" Xenopus egg extracts. In this cell-free system, nuclear assembly and protein import are initially normal; after 2-4 hr, however, a process of nuclear destruction ensues involving chromatin condensation and the shrinkage and fragmentation of the nuclei. This apoptotic process, which also occurs in nuclei added exogenously, is blocked by the addition of baculovirus-expressed Bcl-2 protein. To block the disintegration of nuclei that are added later, Bcl-2 must be present during this latent period. "Apoptosis" in these extracts requires a dense organelle fraction enriched in mitochondria. The cell-free system described here provides a novel tool for understanding intracellular events in apoptosis and the inhibitory function of Bcl-2.

Animals↗
PubMed1994

Bcl-2 inhibits T-cell-mediated cytolysis of a leukemia cell line.

The bcl-2 gene becomes dysregulated in its expression in a wide variety of human cancers and has been shown to block both spontaneous and drug-induced cell death, thus conferring a selective survival advantage on malignant cells. The biochemical mechanism by which bcl-2 promotes cell survival remains enigmatic but appears to involve a downstream event in an evolutionarily conserved cell death pathway. Here we report that gene transfer-mediated increases in Bcl-2 protein levels in the human leukemia line Jurkat render these cells more resistant to induction of DNA fragmentation and cytolysis by a cloned T-cell. The killing mechanism used by these particular T-cells was consistent with apoptosis, as opposed to necrosis, in that DNA degradation occurred as a prelysis event. The findings raise the possibility that dysregulation of bcl-2 gene expression could play a role in the avoidance of immune surveillance mechanisms by cancer cells.

Animals↗
PubMed1994

Protein tyrosine kinase p56-Lck regulates lymphocyte function-associated 1 adhesion molecule expression, granule exocytosis, and cytolytic effector function in a cloned T cell.

Elevated levels of p56-Lck kinase activity were achieved in an interleukin 2 (IL-2)-dependent cloned cytolytic T cell CTLL-2 through gene transfer approaches. CTLL-2-Lck cells remained dependent on IL-2 for growth and survival in culture but exhibited markedly elevated, IL-2-independent cytolytic activity against a variety of tumor targets. This immune cell effector function was similar to the non-major histocompatibility complex-restricted cytolytic activity previously described for lymphokine activated killer (LAK) cells, and involved a cytolytic mechanism that was independent of protein synthesis in either the T cells or the tumor targets. Characterization of CTLL-2-Lck cells revealed markedly elevated levels of both the alpha (CD11a) and beta (CD18) chains of the cell adhesion molecule lymphocyte function-associated 1 (LFA-1) and increased binding of these T cells to a recombinant protein representing the extracellular domain of the LFA-ligand, intercellular adhesion molecule 1 (ICAM-1). Antibodies to CD11a partially abrogated cytolytic killing of tumor target cells by CTLL-2-Lck cells, suggesting that the upregulation in LFA protein levels potentially accounts at least in part for the phenotype of these T cells. Gene transfer-mediated elevations in p56-Lck kinase activity in an IL-3-dependent myeloid cell clone 32D.3 also resulted in increased LFA-1 expression, demonstrating that the findings are not unique to CTLL-2 cells. In addition to upregulation of LFA-1 expression, CTLL-Lck cells also exhibited more efficient exocytosis of cytotoxic granules upon activation with Ca(2+)-ionophore and phorbol ester, relative to control transfected and untransfected CTLL-2 cells. The findings functionally link the Lck kinase to a T cell effector pathway involved in cell-mediated cytotoxicity.

Cell Adhesion↗
PubMed1994

Progression of the myeloid differentiation program is dominant to transforming growth factor-beta 1-induced apoptosis in M1 myeloid leukemic cells.

Hematopoiesis is a profound example of cell homeostasis that is regulated throughout life. This process requires the participation of many factors, including positive and negative regulators of growth and differentiation, that determine survival, growth stimulation, differentiation, functional activation, and programmed cell death. Understanding the effects of multiple stimuli on specific cells at the molecular and cellular level is crucial towards understanding how the population of blood cells maintains a homeostatic state. Two appropriate stimuli for analysis, both of which are found in bone marrow, are differentiation-inducing cytokines, which induce terminal differentiation associated with growth arrest, ultimately culminating in programmed cell death, and transforming growth factor-beta 1 (TGF-beta 1), which induces rapid growth arrest and apoptosis of hematopoietic cells. Previously, we have shown, using M1 myeloblastic leukemic cells as a model system, that differentiation-inducing cytokines induce terminal differentiation associated with growth arrest and, only after 5 to 7 days, apoptosis, whereas TGF-beta 1 induces rapid growth arrest and apoptosis. In this report, we show that M1 myeloid leukemic cells treated concomitantly with the differentiation inducer interleukin-6 and TGF-beta 1 undergo terminal differentiation, in which modulators of the MyD118 gene product, previously shown to be a positive regulator of TGF-beta 1-induced apoptosis, are implicated to play a role in protecting the cells from TGF-beta 1-induced apoptosis. Furthermore, using M1 cell variants blocked at different stages after induction of differentiation, including M1myb and M1myc, as well as conditionally blocked M1mycer, it has been shown that the dominance of interleukin-6 to TGF-beta 1-induced apoptosis is dependent on the progression of the differentiation program. Further studies with M1 and the genetically engineered M1 cell variants will be instrumental towards molecularly dissecting the interaction of hematopoietic differentiation with a variety of apoptotic pathways.

Animals↗
PubMed1994

Cloning and disruption of CKB2, the gene encoding the 32-kDa regulatory beta'-subunit of Saccharomyces cerevisiae casein kinase II.

Casein kinase II of Saccharomyces cerevisiae is composed of two distinct catalytic subunits, alpha and alpha', and two distinct regulatory subunits, beta and beta' (Padmanabha, R. and Glover, C. V. C. (1987) J. Biol. Chem. 262, 1829-1835; Bidwai, A. P., Reed, J. C., and Glover, C. V. C. (1994) Arch. Biochem. Biophys. 309, 348-355). We report here the cloning, sequencing, and disruption of the CKB2 gene encoding the beta'-subunit. The deduced amino acid sequence of Ckb2 displays only 40-45% identity to other beta-subunit sequences reported to date, allowing a better definition of conserved features of this protein. Most notable is the conservation of a cysteine-containing sequence, CPX3C-X22-CPXC, which may constitute a novel metal-binding motif. The degree of sequence divergence of Ckb2 is comparable to that of the Drosophila Stellate protein, a testis-specific protein of unknown function, suggesting that the latter may function as a second beta-subunit in Drosophila. CKB2 is located on the right arm of chromosome XV between the HIR2 and WHI2 loci and has not been previously identified genetically. Haploid and homozygous diploid cells harboring a ckb2 null allele are viable, demonstrating that the beta'-subunit does not have an essential function distinct from that of beta. Strains lacking a functional CKB2 gene appear to grow normally on both fermentable and non-fermentable carbon sources, mate and sporulate normally, and display normal resistance to nitrogen starvation and heat shock. However, haploid strains harboring disruptions of both the beta' gene and either of the catalytic subunit genes exhibit a synthetic phenotype consisting of slow growth and flocculation in rich glucose medium. The occurrence of this synthetic phenotype implies that the beta'-subunit interacts physically and/or functionally with both the alpha- and alpha'-subunits in vivo.

Amino Acid Sequence↗
PubMed1994

Identification of a p53-dependent negative response element in the bcl-2 gene.

Recently, we have shown that the p53 tumor suppressor gene product can inhibit expression of the bcl-2 gene. In this report, we explored the molecular basis for p53-mediated down-regulation of bcl-2 gene expression using a cotransfection approach involving p53 expression plasmids and chloramphenicol acetyltransferase (CAT) reporter gene constructs containing regions from the bcl-2 gene. When transfected into a p53-deficient human lung cancer cell line H358, reporter gene constructs containing only the promoter region of bcl-2 and upstream sequences were not suppressed by p53. Inclusion of bcl-2 gene sequences corresponding to the 5' untranslated region in bcl-2/CAT constructs, however, resulted in p53-dependent down-regulation. A 195-base pair segment from the bcl-2 gene 5' untranslated region was found to be capable of conferring p53-dependent repression on a heterologous expression plasmid containing CAT under the control of an SV40 immediate early-region promoter. This p53-negative response element functioned in an orientation-independent manner when placed either upstream or downstream of the SV40-CAT transcription unit. The results demonstrate the existence of a negative response element in the bcl-2 gene through which p53 may either directly or indirectly transcriptionally down-regulate expression of this gene involved in the regulation of programmed cell death.

Base Sequence↗
PubMed1994

Interleukin 7-dependent B lymphocyte precursor cells are ultrasensitive to apoptosis.

We have compared the sensitivity of clonogenic interleukin 7 (IL-7)-dependent murine B cell precursors with that of clonogenic mature B cells and myeloid precursors to alpha-particles from plutonium-238 and X radiation. All three populations are relatively sensitive, but B cell precursors are ultrasensitive. This differential sensitivity is also observed with corticosteroid, etoposide, and cisplatin, all apoptosis-inducing drugs used in the treatment of leukemia and other cancers. Further, we show that x-rays and drugs induce the bulk of the B cell precursor population to undergo rapid apoptosis, despite the continued presence of IL-7. B cell precursors were found to express very low levels of BCL-2 protein compared with mature splenic B cells and their resistance to x-rays and corticosteroid could be enhanced by expression of a BCL-2 transgene. These data have important implications for normal lymphopoiesis and for the behavior of leukemic lymphoid precursor cells.

Alpha Particles↗
PubMed1994

Role of bcl-2 and IL-5 in the regulation of anti-IgM-induced growth arrest and apoptosis in immature B cell lines. A cooperative regulation model for B cell clonal deletion.

Recent studies of transgenic mice have confirmed that clonal deletion is involved in the development of B cells. However, little is known about intercellular and intracellular molecular events regulating B cell clonal deletion. We investigated the role of bcl-2 and cytokines in the regulation of B cell clonal deletion using anti-IgM-induced growth arrest and apoptosis in immature B cell lines as a model. We show here that overexpression of Bcl-2 protein in stably transfected immature B cells partially inhibits anti-Ig M-induced apoptosis but does not affect growth arrest. Similarly, IL-5 has a strong inhibitory effect on anti-IgM-mediated apoptosis but has a weak inhibitory effect on growth arrest. Finally, although both bcl-2 overexpression and exogenous IL-5 cooperate with bacterial LPS to block apoptosis, bcl-2 overexpression and exogenous IL-5 have no additive inhibitory effect on anti-Ig induced apoptosis. These findings indicate that anti-IgM-induced apoptosis is independently regulated from growth arrest and is controlled by at least two independent pathways: One is regulated by either Bcl-2 protein or IL-5 and the other is regulated by LPS. Activation of both the bcl-2/IL-5 and LPS pathways is necessary for complete inhibition of apoptosis, and presumably, clonal selection of the immature B cells.

Animals↗
PubMed1994

Cloning and sequencing of a cDNA encoding the rat Bcl-2 protein.

A rat cDNA encoding the Bcl-2 protein was cloned and sequenced. The primary amino-acid sequence deduced from the nucleotide sequence reveals a 236-aa protein having extensive homology with the mouse (95%), human (87%) and chicken (71%) Bcl-2 proteins.

Amino Acid Sequence↗
PubMed1994

Prolactin induces rapid phosphorylation and activation of prolactin receptor-associated RAF-1 kinase in a T-cell line.

The receptor for prolactin (PRL) is a member of the hematopoietic receptor family that also includes the receptors for interleukins 2-7. PRL is synthesized and is secreted by human T lymphocytes and acts as a lymphokine necessary, but not sufficient, for T lymphocyte progression through the G1 phase of the cell cycle. Although data now indicate that PRL serves an immunomodulatory role in vitro and in vivo, the mechanisms of PRL receptor signal transduction in T cells have not been defined. We demonstrate here that PRL induced the phosphorylation of the p72-74 serine/threonine kinase c-Raf-1 in the PRL-dependent rat T-cell line Nb2. Associated with this inducible phosphorylation of Raf-1 was a concentration- and time-dependent activation of in vitro Raf-1 autokinase and substrate kinase activities, which correlated with the PRL-induced proliferation of Nb2 cells. Co-immunoprecipitation studies revealed association of Raf-1 with PRL receptors in Nb2 cells. These results revealed that all isoforms of the PRL receptor (short, intermediate, and long) are expressed in Nb2 cells and associate with Raf-1. In contrast to the PRL-dependent Nb2 cells, phosphorylation and activation of Raf-1 were constitutive in the Nb2-derived, PRL-independent, T-cell line Sp. These studies demonstrate for the first time an association between the PRL receptor and a serine/threonine kinase affiliated with signal transduction.

Animals↗
PubMed1994

Subcellular localization of the bcl-2 protein in malignant and normal lymphoid cells.

The bcl-2 oncogene is expressed in lymphoid and myeloid cells as well as in neurons and several types of epithelial cells and inhibits programmed cell death (apoptosis). Deregulation by the t(14;18) translocation in lymphoid malignancies induces inappropriate cell survival and serves as one of the steps toward a fully malignant behavior. Using pre- and postembedding immunoelectron microscopy in normal and neoplastic lymphocytes, we demonstrate bcl-2 immunoreactivity to the mitochondrial outer circumference and the nuclear envelope and to a lesser degree to the cell membrane. Mitochondrial staining was patchy, reminiscent of mitochondrial contact zones. Additionally, there was a suggestion of association with nuclear pores. In these regions, transmembrane transport is mediated. This may suggest that bcl-2 exerts its function in this process.

3,3'-Diaminobenzidine↗
PubMed1994

Casein kinase II of Saccharomyces cerevisiae contains two distinct regulatory subunits, beta and beta'.

The subunit composition of casein kinase II (CKII) from S. cerevisiae has been difficult to define, particularly with respect to the existence and number of regulatory (beta) subunits. A single, integral beta subunit, a loosely associated beta subunit, two distinct beta subunits, and a complete absence of beta subunits have all been proposed. Our laboratory reported yeast CKII to be composed of four polypeptides of 42, 41, 35, and 32 kDa (R. Padmanabha and C. V. C. Glover, 1987, J. Biol. Chem. 262, 1829-1835). The 42- and 35-kDa polypeptides were identified as distinct catalytic subunits, alpha and alpha', on the basis of N-terminal sequencing and subsequent molecular cloning. The 41- and 32-kDa polypeptides were found to undergo autophosphorylation, a characteristic of the beta subunit in other species, but antibodies raised against the beta subunit of Drosophila CKII crossreacted only with the 41-kDa polypeptide. In order to clarify the subunit composition of yeast CKII, particularly with regard to the 32-kDa polypeptide, we have purified the enzyme to homogeneity using a modified procedure. Based on the results of autophosphorylation studies, Western blotting, peptide mapping of the 41- and 32-kDa peptides, and sequencing of subunit-specific peptides, we demonstrate that the 32-kDa polypeptide is an additional beta subunit (beta') distinct from the 41-kDa beta subunit. This represents the first demonstration of beta subunit heterogeneity in purified CKII from any species.

Amino Acid Sequence↗
PubMed1994

Purification and characterization of the bcl-2 protein.

The oncogene product bcl-2 functions as a repressor of programmed cell death and is a 26-kDa protein with a single predicted transmembrane segment located at the carboxyl terminus. The bcl-2 protein seems to function in different subcellular compartments, as evidenced by several biochemical and ultrastructural studies. The present study was performed to purify bcl-2 protein in significant quantities necessary for structural and functional studies. For this purpose, the bcl-2 gene was over-expressed in either baculovirus system or lymphocytes. Initially, attempts were undertaken to purify bcl-2 protein using conventional methods such as ion exchange or gel filtration chromatography. During these purification attempts we determined that bcl-2 protein is highly hydrophobic and prone to aggregation as might be expected for an integral membrane protein. By ion exchange and gel filtration chromatography, this protein could be partially purified. In order to purify bcl-2 to apparent homogeneity and avoid the aggregation problem, we prepared immunoaffinity columns using a monoclonal antibody developed against a synthetic peptide chosen from residues 61-76 of the amino acid sequence of human bcl-2. The antibody was either coupled to CNBr-activated Sepharose 4B or cross-linked into protein A-Sepharose by dimethylpimelimidate dihydrochloride. Cellular extract equivalent to 10(8) bcl-2-overexpressing insect cells or lymphocytes was applied to immunoaffinity columns. Approximately 500 micrograms purified bcl-2 protein could be recovered as estimated by silver staining and immunoblotting. Furthermore, purified bcl-2 protein was electroporated into Pre-B lymphocytes which do not express this protein in sufficient quantity to delay the onset of glucocorticoid-induced apoptosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Reversal of chemoresistance of lymphoma cells by antisense-mediated reduction of bcl-2 gene expression.

The bcl-2 gene is expressed in many types of human tumours and becomes transcriptionally deregulated in the majority of non-Hodgkin's lymphomas as the result of t(14;18) chromosomal translocations. The 26-kDa Bcl-2 protein has been shown to block programmed cell death (apoptosis) induced by many types of stimuli, including a wide variety of chemotherapeutic drugs and radiation. The presence of bcl-2 in tumor cells has been correlated with poor responses to therapy in patients with some types of cancer. To explore further the relevance of bcl-2 to drug resistance, we used antisense (As) approaches to achieve reductions in the levels of steady state Bcl-2 protein levels in t(14;18)-containing human lymphoma cell lines. Both synthetic bcl-2-As oligonucleotides and inducible expression plasmids that produce bcl-2-As transcripts induced reductions in bcl-2 expression, resulting in a marked enhancement in the sensitivity of neoplastic cells to conventional chemotherapeutic drugs such as cytosine arabinoside (ara-C) and methotrexate (MTX). These results suggest that novel therapeutics targeted against bcl-2 could provide the means for improved treatment of cancer by affecting physiological pathways distal to the targets of cytotoxic drugs.

Animals↗
PubMed1994

Regulation of chemoresistance by the bcl-2 oncoprotein in non-Hodgkin's lymphoma and lymphocytic leukemia cell lines.

BACKGROUND: The bcl-2 gene becomes activated by 14;18 chromosomal translocations in the majority of low-grade non-Hodgkin's lymphomas (NHLs) and is expressed at high levels in the absence of gene rearrangements in a high proportion of B-cell chronic lymphocytic leukemias (B-CLLs). The protein encoded by bcl-2 contributes to neoplastic cell expansion by prolonging cell survival through its ability to block programmed cell death (apoptosis). Because many chemotherapeutic drugs have been shown ultimately to kill tumor cells through mechanisms consistent with programmed cell death, we tested whether the relative levels of bcl-2 oncoprotein influence the sensitivity of lymphoma and leukemia cell lines to killing by conventional cytotoxic drugs commonly used in the treatment of cancer. METHODS: Leukemia cell lines with low levels of bcl-2 expression were stably infected with recombinant bcl-2 retroviruses to achieve elevations in bcl-2 protein levels. Lymphoma cell lines with high levels of bcl-2 expression as the result of 14;18 translocations were either stably transfected with inducible bcl-2 antisense expression plasmids or treated with bcl-2 antisense oligonucleotides to achieve reductions in bcl-2 protein levels. The sensitivity of these genetically modified cells to killing by various antineoplastic drugs was then determined. RESULTS: Gene transfer-mediated elevations in bcl-2 protein levels in lymphocytic leukemia cell lines was correlated with markedly elevated resistance to killing by all cytotoxic drugs tested. Conversely, antisense-mediated reductions in bcl-2 protein levels in t(14;18)-containing NHL cell lines resulted in enhanced sensitivity to all anticancer drugs. CONCLUSIONS: The relative levels of bcl-2 oncoprotein represent one of the key determinants of the sensitivity of lymphocytic cells to killing by essentially all drugs currently available for the treatment of cancer.

Animals↗