Control of apoptosis by cytokines.
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Animals↗
Biomedical subjects
W S May
Publications and source records attributed to W S May.
Regulation of CD34 expression in differentiating M1 cells.
CD34 is a cell surface glycoprotein expressed on hematopoietic stem and progenitor cells, but not on mature blood cells. In the present study we found that CD34 downregulation during hematopoiesis occured at the level of transcriptional initiation. Two transcription initiation sites (TISs) were identified in each of three different CD34+ cell lines; these TISs were located at 120 and 80 bp 5' of the translation start site, respectively. The promoter lacks TATA elements and, like other TATA-less promoters, the TISs conform to the consensus sequence for an INR (PyPyCAPyPyPyPy). An additional 3000 bp of upstream genomic DNA were sequenced and found to contain consensus sites for transcription factors, suggesting their potential role in gene regulation. Transient transfection assays using CD34 promoter-luciferase reporter constructs, containing sequences up to 3 kb upstream and inclusive of the TIS, indicate that this promoter drives transcription in hematopoietic CD34+ cells but not CD34+ nonhematopoietic cells. Both cell type specific expression and full promoter activity are maintained in constructs that contain as little as 454 bp upstream of the TISs. Optimal promoter activity requires the 5' untranslated region of exon 1, which contains a 51-bp element that has the potential to form an extensive secondary structure. In the plasmid DNA, however, this secondary structure was not detectable by P1 nuclease digestion. At least three proteins present in uninduced M1 nuclear extracts bind to this element. Two of the three proteins were identified as Sp 1 and Sp 3 based on supershift experiments. These data suggest that CD34 expression by hematopoietic stem and progenitor cells involves hematopoietic cell-specific factors that interact with regulatory elements within the first 230 bp of the promoter and that optimal expression requires a 60-bp segment of the 5' untranslated region.
3T3 Cells↗
Characterization of mouse lymphohematopoietic stem cells lacking spleen colony-forming activity.
The classical definition of lymphohematopoietic stem cells (LHSC), the most primitive progenitors of all blood cells, requires that they have the capacity for self-renewal and for the long-term production of all blood cell lineages. However, other characteristics of LHSC have been debated. Our previous data suggested that mouse LHSC are very slowly proliferating cells that generate delayed multilineage engraftment, while "radioprotection" (rapid engraftment that will prevent early death from radiation-induced marrow aplasia) results from more committed progenitors. Alternatively, some groups have reported that mouse LHSC are responsible for both radioprotection and long-term repopulation of all blood cell lineages. A possible explanation for this difference is that cells with the capacity for long-term production of all blood cell lineages are biologically heterogeneous. We now show that 10 LHSC can generate all blood cell lineages for the lifetime of the animal. However, these cells lacked radioprotection and spleen colony-forming activity. LHSC were identified and isolated by their small size, their lack of expression of antigens characteristic of mature blood cell lineages, and their high expression of aldehyde dehydrogenase. In addition, these cells were found to express undetectable or low levels of many antigens presumed to mark LHSC, including Thy-1, Ly-6A/E (Sca-1), c-kit, and CD34. There appears to be at least two classes of LHSC with the capacity for long-term production of all blood cell lineages: one that generates both radioprotection and long-term engraftment and one that produces delayed but durable engraftment. Our data suggest that this latter class may represent a very primitive class of LHSC.
Aldehyde Dehydrogenase↗
CD34: structure, biology, and clinical utility.
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Acute Disease↗
Drug development train gathering steam.
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Drug Design↗
Full-length but not truncated CD34 inhibits hematopoietic cell differentiation of M1 cells.
CD34 is expressed on human and murine hematopoietic stem and progenitor cells and its clinical usefulness for isolation of stem/progenitor cells has been well established. Although expression of CD34 is regulated in a developmental stage-specific manner, the function of CD34 is not known. Recently we have shown that both a full-length and truncated form of CD34 protein is expressed by hematopoietic cells (Blood 84:691, 1994). To test whether failure to suppress either form of CD34 could affect terminal myeloid differentiation, we constitutively expressed these CD34 proteins in murine M1 myeloid leukemia cells, which can be terminally differentiated to macrophages by treatment with interleukin-6 of leukemia inhibitory factor. Surprisingly our results show that forced expression of the full-length but not the truncated form of CD34 impedes terminal differentiation by these agents. Because the difference between the two forms of CD34 protein resides in the length of their respective cytoplasmic tail domains, our findings strongly suggest that the cytoplasmic domain region of full-length CD34 is responsible for the observed maturation arrest phenotype. These findings suggest a potential negative regulatory role for full-length CD34 in hematopoietic cell differentiation and may explain, at least in part, the block in maturation observed in CD34+ acute myeloid leukemia.
Acute Disease↗
Interleukin-3 and bryostatin-1 mediate hyperphosphorylation of BCL2 alpha in association with suppression of apoptosis.
Using murine myeloid factor-dependent FDC-P1/ER cells, we demonstrate that the hematopoietic growth factors interleukin-3 and erythropoietin and bryostatin-1, a macrocyclic lactone natural product and potent activator of protein kinase C (PKC), suppress apoptosis and induce the rapid serine phosphorylation of Bc12 alpha. Expression of recombinant wild type Bc12 alpha in NFS/N1.H-7 cells confirms that murine Bc12 alpha is phosphorylated following PKC activation. The PKC inhibitors H-7 and staurosporine, but not the protein kinase A inhibitor HA1004, block not only interleukin-3- and bryostatin-1-induced hyperphosphorylation of Bc12 alpha but also their anti-apoptotic effect on growth factor-dependent cells, suggesting a role for activated PKC in both processes. A potential direct role for a classic isoform of PKC is indicated by the Ca(2+)-dependent nature of phosphorylation of Bc12 alpha mediated by purified PKC in vitro. Comparative phosphopeptide maps confirm that Bc12 alpha phosphorylation occurs on identical serine site(s) whether phosphorylation occurs in cells following agonist treatment or directly by PKC in vitro. These findings strongly support a role for activated PKC in growth factor-induced Bc12 alpha phosphorylation as well as suppression of apoptosis.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
Interleukin 3 stimulates protein synthesis by regulating double-stranded RNA-dependent protein kinase.
In a murine interleukin 3 (IL-3)-dependent cell line, IL-3 deprivation resulted in increased autophosphorylation of double-stranded RNA-dependent protein kinase (PKR) that has been reported to inhibit protein synthesis by phosphorylating the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha). Autophosphorylation was characterized by a shift up in mobility of PKR on SDS/PAGE gels from a 60- to a 64-kDa form. In vitro kinase studies comparing the autophosphorylated 64-kDa PKR with the nonphosphorylated 60-kDa PKR confirmed that only the 64-kDa form was active for eIF-2 alpha phosphorylation. PKR activation in vivo was associated with phosphorylation of eIF-2 alpha and inhibition of protein synthesis. Addition of IL-3 to deprived cells elicited a reciprocal response characterized by the rapid dephosphorylation of PKR and eIF-2 alpha, indicating inactivation of PKR. This was rapidly followed by the full recovery of protein synthesis. Furthermore, upon IL-3 addition, a 97-kDa phosphotyrosine-containing protein becomes rapidly and transiently associated with PKR prior to dephosphorylation of PKR and eIF-2 alpha. Genistein, a tyrosine kinase inhibitor, blocks both phosphorylation of the 97-kDa phosphoprotein and protein synthesis after IL-3 addition, suggesting a role for the 97-kDa phosphoprotein in the mechanism of inactivation of PKR and stimulation of protein synthesis. Thus, IL-3 appears to positively regulate protein synthesis by inducing the inactivation of PKR in a growth factor signaling pathway.
Animals↗
Characterization of murine CD34, a marker for hematopoietic progenitor and stem cells.
CD34 is expressed on human hematopoietic stem and progenitor cells, and its clinical usefulness for the purification of stem cells has been well established. However, a similar pattern of expression for murine CD34 (mCD34) has not yet been determined. Two polyclonal anti-mCD34 antibodies that specifically recognize both endogenous and recombinant murine CD34 were developed to characterize the mCD34 protein and to determine its pattern of expression on murine cell lines and hematopoietic progenitor cells. Fluorescence-activated cell sorter analysis showed that mCD34 is expressed on NIH/3T3 embryonic fibroblasts, PA6 stromal cells, embryonic stem cells, M1 leukemia cells, and a subpopulation of normal bone marrow cells. Murine CD34 was found to be a glycoprotein expressed on the cell surface as either a full-length (approximately 100 kD) or truncated (approximately 90 kD) protein in NIH/3T3 and PA6 cells. Recombinant full-length CD34, when expressed in the CHO-K1 cell line, had a molecular weight of approximately 105 kD. Full-length CD34 expressed on M1 leukemia cells, had a higher apparent molecular weight (110 kD). These results suggest that there are glycosylation differences between CD34 expressed by different cell types. The full-length form, but not the truncated form, is a phosphoprotein that is hyperphosphorylated in response to 12-0-Tetradecanoyl phorbol 13-acetate treatment, suggesting potential functional differences between the two forms. Selection of the 3% highest-expressing CD34+ bone marrow cells enriched for the hematopoietic precursors that form colony-forming unit-spleen (CFU-S), CFU-granulocyte-macrophage, and burst-forming unit-erythroid. Transplantation of lethally irradiated mice with these cells demonstrated both short- and long-term repopulating ability, indicating that this population contains both functional hematopoietic progenitors and the putative stem cell. These antibodies should be useful to select for murine hematopoietic stem cells.
3T3 Cells↗
Protein kinase C-mediated serine phosphorylation directly activates Raf-1 in murine hematopoietic cells.
We have previously found that Raf-1, which is activated by hematopoietic growth factors in association with phosphorylation, is required for hematopoietic cell proliferation. Recently, 12-O-tetradecanoylphorbol 13-acetate has been found to mediate Raf-1 phosphorylation, suggesting that protein kinase C (PKC) may be involved in the Raf-1 activation mechanism(s). Since PKC can be activated by hematopoietic growth factors, it was investigated as a potential "Raf-1 kinase-kinase." Results demonstrate that bryostatin 1, a pharmacologic activator of PKC, induces activation of Raf-1 in FDC-P1 cells. PKC inhibitors H7 and staurosporine block both bryostatin 1- and interleukin-3-mediated Raf-1 phosphorylation and FDC-P1 cell proliferation. Additionally, an antisense c-raf oligodeoxyribonucleotide specifically inhibits bryostatin 1-mediated proliferation, indicating a necessary role for Raf-1 in PKC signaling. Purified PKC can phosphorylate Raf-1 serine residues to high stoichiometry in vitro. Comparative phosphopeptide maps localize two PKC phosphorylation sites to Raf-1 phosphopeptides isolated from hematopoietic growth factor- or bryostatin 1-stimulated cells. The sites of PKC-mediated Raf-1 phosphorylation are deduced to be Ser497 and Ser619. Furthermore, PKC-mediated serine phosphorylation is sufficient to activate the enzymatic function of Raf-1 in vitro. These findings demonstrate that activated PKC can promote hematopoietic cell growth by regulating the enzymatic activity of Raf-1 through direct serine phosphorylation.
Amino Acid Sequence↗
Gö 6976, a selective inhibitor of protein kinase C, is a potent antagonist of human immunodeficiency virus 1 induction from latent/low-level-producing reservoir cells in vitro.
Human immunodeficiency virus (HIV-1) infection is followed by a period of latency or a low-level-persistent (LLP) state that results in an asymptomatic infection of the host. Productive viral expression may be triggered by a variety of activators including mitogens, antigens, and cytokines. Protein kinase C (PKC) has been shown to be important in the intracellular cascade of signals induced by such activators. With U1 and ACH-2 cell lines representative of an HIV-1 postintegration state, the effect of Gö 6976, a synthetic inhibitor of PKC was tested. Gö 6976 is a nonglycosidic indolocarbazole found to potently inhibit HIV-1 induction by Bryostatin 1, tumor necrosis factor alpha, and interleukin 6. Gö 6976 effectively blocks viral transcription induced by Bryostatin 1 or tumor necrosis factor alpha that leads to the inhibition of intracellular viral protein synthesis and viral shedding. Gö 6976 also blocks interleukin 6-mediated posttranscriptional induction of viral proteins. The IC50 of Gö 6976 shows a 12- to 60-fold more potent effect than for H-7, another PKC inhibitor with a similar mechanism. The inhibitory effect is reduced when Gö 6976 is not added before or within 1 hr of induction by the potent PKC activator Bryostatin 1. However, U1 cells can be grown for long periods in a nontoxic concentration of Gö 6976 (300 nM), which confers virtual inhibition of HIV-1 induction without the development of resistance. Results indicate that inhibition of HIV-1 proviral induction from latent/low-level-producing infectious states with potent PKC inhibitors like Gö 6976 may represent an additional and promising antiviral approach.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
BMT for severe aplastic anemia using cyclosporine.
Between 1984 and 1991 24 patients with severe aplastic anemia (SAA) were transplanted with HLA identical sibling donor BM. The overall long-term survival was 79 +/- 8%. The average age was 21 years (range 4-53 years) and the median pre-transplant disease duration was 35 days (range 12-2998 days). Over one-half (15 of 24) of the patients had received > 10 units of blood product transfusions prior to BMT. The pre-transplant conditioning regimen consisted of 200 mg/kg cyclophosphamide (CY). Cyclosporine (CYA) was administered from 2 days prior to BMT and continued for 6-12 months. Two of the 24 patients failed to achieve primary engraftment (FTE). One of these patients had autologous recovery of BM function and is alive and well. Five of the 22 patients who engrafted failed to sustain engraftment (FTSE). Of these, three are alive and well following a second BMT or marrow boost. Only 1 of the 22 patients who engrafted had clinically significant (i.e. Stage II-IV) acute GVHD. No patient developed chronic GVHD. Our results indicate that BMT following a regimen consisting of CY with the continuous use of CYA in the post-transplant period is well tolerated and associated with excellent long-term survival. The high incidence of secondary graft instability (i.e. FTSE), however, suggests that future studies should focus on post-transplantation immunomodulation.
Adolescent↗
Up-regulation of surface CD34 is associated with protein kinase C-mediated hyperphosphorylation of CD34.
CD34 is a transmembrane sialoglycoprotein expressed by early hematopoietic progenitor cells as well as endothelial cells. Previously we found that CD34 is rapidly and stoichiometrically phosphorylated by activated protein kinase C (PKC) (Fackler, M.J., Civin, C.I., Sutherland, D.R., Baker, M.A., and May, W.S. (1990) J. Biol. Chem. 265, 11056-11061). In the present study, we find dose-dependent up-regulation of CD34 surface expression following treatment of normal human CD34+ bone marrow progenitor cells, cord blood-derived KMT-2, or KG1 a myeloid leukemia cells with the PKC activator 12-O-tetradecanoylphorbol-13-acetate. Up-regulation begins within 1 min of treatment, is maximal by 30 min, is maintained for at least 3 h, and is associated with CD34 hyperphosphorylation. A specific inhibitor of PKC, 2,6-diamino-N-(1[1-(1-oxotridecyl)-2-piperadinyl]methyl)h exan-amide (NPC 15437), blocks both up-regulation and hyperphosphorylation of CD34. CD34 up-regulation is independent of transcription and/or translation and results from the recruitment of preformed intracellular CD34. The endocytosis rate of surface CD34 is unaltered by 12-O-tetradecanoylphorbol-13-acetate. Thus, activation of PKC mediates increased surface expression of the CD34 molecule possibly as a result of phosphorylation of CD34.
Antigens, CD↗
Differential effects of bryostatin 1 and phorbol ester on human breast cancer cell lines.
The effects of the protein kinase C (PKC) activators, phorbol ester 12-O-tetradecanoyl-13-phorbol acetate (TPA) and the marine natural product, bryostatin 1, on the growth and morphology of human breast cancer cell lines were examined. TPA (1 to 100 nM) inhibited growth of four of six cell lines by up to 75% in 5-day cultures. Bryostatin 1 inhibited growth of only MCF-7 cells and only at a high dose (100 nM). However, bryostatin 1 completely antagonized the growth inhibition and morphological changes induced by TPA in MCF-7 cells. The divergent effects of these two agents are associated with differing effects on PKC activity and isoform expression in MCF-7 cells. TPA induced rapid translocation of the PKC-alpha isozyme and PKC activity to the membrane fraction of MCF-7 cells. In contrast, bryostatin 1 treatment resulted in the loss of the PKC-alpha isozyme and PKC activity from both cytosolic and membrane compartments within 10 min of treatment. In coincubation assays the bryostatin 1 effect was dominant over that of TPA. Similar effects on PKC-alpha isozyme and PKC activity were seen in a second cell line whose growth was inhibited by TPA but not by bryostatin 1, MDA-MB-468. In contrast, in the T47D cell line, where TPA was not growth inhibitory, TPA failed to induce translocation of PKC-alpha to the cell membrane. Bryostatin, however, still caused loss of PKC-alpha isozyme and PKC activity from cytosolic and membrane fractions. Thus, differential actions of bryostatin 1 and TPA on PKC activity and alpha-isoform level in the membrane-associated fraction of MCF-7 and MDA-MB-468 cells may account for the divergent effects of these two agents on cell growth and morphology. These results suggest that the PKC-alpha isoform may specifically play a role in inhibiting growth of human breast cancer cells.
Breast Neoplasms↗
Activated protein kinase C directly phosphorylates the CD34 antigen in acute lymphoblastic leukemia cells.
The precursors of all blood cell lineages are contained within the 1-3% of bone marrow cells which express the CD34 antigen, and this population can reconstitute the hematopoietic system of lethally irradiated animals and humans. A potential regulatory role for the CD34 antigen in progenitor cell function and differentiation was indicated by our recent findings that the CD34 antigen can be phosphorylated in vivo to high stoichiometry in primitive CD34+ cell-lines by activated protein kinase C. To exclude the possibility that these effects were restricted to cell-lines, we have performed similar experiments on fresh cells from a patient with drug-resistant acute lymphoblastic leukemia. Similar to our previous findings, we found the CD34 antigen to be hyperphosphorylated in lymphoblasts labeled in the presence of active phorbols. The same peptides which were hyperphosphorylated in phorbol-stimulated cell-lines were also phosphorylated in phorbol-stimulated lymphoblasts. These data indicate that CD34 is a substrate molecule for PKC in fresh CD34+ lymphoblasts and underline the role of modulators of PKC activity in the biology of primitive leucocytes.
Adult↗
Four interleukin-2 surface binding proteins detected in rat spleen cells.
Four specific interleukin-2 (IL-2) surface binding proteins can be detected by covalent cross-linking of [125I]IL-2 to rat spleen cells that have been activated with various stimuli including concanavalin A (Con A), phytohaemagglutinin (PHA), calcium ionophore, and phorbol dibutyrate (PDB) with or without calcium ionophore. These four cross-linked proteins could not be demonstrated in either unstimulated T cells or in activated T cells when binding was performed in the presence of a 20-100-fold excess of unlabelled IL-2. The molecular weights of the four cross-linked proteins, after subtraction of the molecular weight contribution of IL-2 are: 53,000, 70,000, 90,000 and 118,000. The 53,000 MW protein was identified as the rat IL-2 receptor (IL-2R) alpha-chain by immune precipitation. Additionally, results suggest that the rat IL-2R alpha-chain is tightly complexed to both the 118,000 and 90,000 MW IL-2 binding proteins. Purification of surface labelled proteins from activated cells using IL-2 affinity chromatography yields four proteins with similar molecular weight to those identified by cross-linking plus an additional non-ligand cross-linked protein of 46,000 MW. The 46,000 MW band may be a non-binding associated protein since it was not seen following [125I]IL-2 binding cross-linking. Tryptic digests and two-dimensional separation of the affinity-isolated proteins indicate that unique peptide maps are generated for the 46,000, 53,000 and 70,000 MW proteins and excludes the possibility that the bands identified by cross-linking represents cross-linking of multiple ligands to the 53,000 MW subunit. However, the 90,000 and 118,000 MW bands yield peptide maps that closely resemble each other suggesting that these binding proteins may be related. These results suggest that at least four IL-2 surface binding proteins may constitute the rat IL-2R system.
Animals↗
Response of Jurkat T cells to phorbol ester and bryostatin. Development of sublines with distinct functional responses and changes in protein kinase C activity.
T lymphocyte activation is initiated as a result of the interaction between the TCR complex and Ag as seen in the framework of a membrane-bound MHC molecule. Receptor stimulation results in a rise in free intracellular Ca2+ and the activation of protein kinase C (PKC). Bryostatin (Bryo) and phorbol esters (e.g., 12-O-tetradecanoylphorbol 13-acetate (TPA] are PKC activators with somewhat different immunologic effects. We compared the effect of Bryo and TPA on the T cell tumor line Jurkat and derivatives of Jurkat cells grown in media supplemented with 100 nM Bryo ("BR100" cells) or 100 nM TPA ("TP100" cells). In untreated Jurkat cells, there is a dose- and time-dependent decrease in proliferation, compared to media controls, after the administration of as little as 10 nM TPA. This can be reversed in a dose- and time-dependent manner by Bryo. Interestingly, the expression of the transferrin receptor parallelled this effect on proliferation. Furthermore, Jurkat cells grown continuously in 100 nM TPA regained full proliferative capacity after several weeks in culture and transferrin receptor expression returned to near the level seen in untreated Jurkat cells. The chromatographic separation of PKC activity in these three cell lines showed that total PKC activity was dramatically decreased in both the TP100 and BR100 cells when compared to untreated Jurkat cells. However, in the TP100 cells there exists a peak of activity that is activated by Bryo, but not TPA. Western blots of whole cell lysates of the three cell lines showed that PKC-alpha and PKC-beta II were both down-regulated in BR100 and TP100 cells compared to untreated Jurkat cells. PKC-gamma was not detected in any of the cell lines. Therefore, the Bryo-specific peak seen in TP100 cells may be PKC-delta, -epsilon, -zeta, -eta, or a novel PKC isoform. This could provide the basis for a molecular characterization of the differences in PKC activation between phorbol esters and Bryo.
Antigens, CD↗
c-myc gene-induced alterations in protein kinase C expression: a possible mechanism facilitating myc-ras gene complementation.
The mechanism(s) by which the c-myc nuclear protein and the membrane-associated ras protein interact to mediate phenotypic changes is unknown. We now find that c-mcy gene expression is associated with alterations in the principal signal transduction pathway through which the ras protein is thought to function. We studied the transcript and protein expression of protein kinase C (PKC) isoforms in a culture line of human small cell lung cancer cells (NCI H209) in which expression of inserted c-myc and Ha-ras genes together, but not alone, causes a transition to a large cell phenotype. In control H209 cells, at the transcript and cell membrane protein levels, PKC-alpha is the dominant PKC species. In this cell line, the expression of an exogenous c-myc gene, but not of a viral Ha-ras gene, causes a 5- to 10-fold increase in the PKC-beta isoform transcript and protein. The insertion of ras into the exogenous myc-expressing 209 cells, in addition to causing phenotypic transition, results in the translocation of the PKC-beta protein from the cytosol to the membrane fraction and a decrease in membrane-associated PKC-alpha. Concomitant with these changes, the increased PKC isoform transcript levels induced by myc alone are completely reversed. These observations suggest that a complex set of PKC transcript and protein alterations, most prominently involving an increased PKC-beta protein level in the cell membrane, a decrease in PKC-alpha protein, and a decrease in all PKC isoform transcripts, may represent a fundamental event(s) for c-myc collaboration with Ha-ras to alter cell phenotype.
Carcinoma, Small Cell↗