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Biomedical subjects

D R Green

Publications and source records attributed to D R Green.

At least 163 records · Page 9Linked to original sources
PubMed1994

Protein-tyrosine phosphorylation regulates apoptosis in human eosinophils and neutrophils.

Early signaling events that control the process of programmed cell death are largely unknown. Tyrosine phosphorylation plays a major role in transmembrane signal transduction through most cell surface receptors. Granulocyte/macrophage colony-stimulating factor (GM-CSF), a cytokine released by activated T cells, has been shown to increase tyrosine phosphorylation in several cells and to inhibit granulocyte cell death in vitro. In this study, we demonstrate that the effect of GM-CSF on granulocyte cell death can be blocked by the tyrosine kinase inhibitor genistein, suggesting that increases in tyrosine phosphorylation are essential to inhibit cell death. To analyze the role of tyrosine phosphorylation for the regulation of granulocyte cell death more precisely, we increased levels of tyrosine phosphorylation using the protein-tyrosine phosphatase inhibitor phenylarsine oxide (PAO). Similar to GM-CSF, treatment of the cells with PAO was followed by high increases in tyrosine phosphorylation and inhibition of programmed cell death in human eosinophils and neutrophils. Strikingly, at low concentrations of the inhibitor and low induction of tyrosine phosphorylation, acceleration of apoptosis was observed. Genistein and herbimycin A reversed the effects of PAO on tyrosine phosphorylation and granulocyte apoptosis. These results suggest that programmed eosinophil and neutrophil death is regulated by early events of signal transduction pathways such as tyrosine phosphorylation.

Apoptosis↗
PubMed1994

BCR-ABL maintains resistance of chronic myelogenous leukemia cells to apoptotic cell death.

Apoptosis is the major form of cell death associated with the action of chemotherapeutic agents on tumor cells, and therefore the expression of genes that interfere with apoptosis can have important consequences for the efficacy of therapeutic approaches. Here we show that K562, a chronic myelogenous leukemia (CML) cell line expressing the BCR-ABL fusion protein, are resistant to the induction of apoptosis by a number of agents and conditions. Antisense oligodeoxynucleotides corresponding to the translation start of bcr downregulate bcr-abl protein in these cells and render them susceptible to induction of apoptosis by chemotherapeutic agents or serum deprivation. Expression of a temperature sensitive v-Abl protein reverses the effects of the antisense oligonucleotides, such that the cells remain resistant to apoptosis at the permissive temperature. These data indicate that bcr-abl acts as an anti-apoptosis gene in CML cells and suggests that the effect is dependent on the abl kinase activity in this chimeric protein. Inhibition of bcr-abl to render CML cells susceptible to apoptosis can be combined with therapeutic drugs and/or treatment capable of inducing apoptosis to provide an effective strategy for elimination of these cells.

Apoptosis↗
PubMed1994

Role of DNA fragmentation in T cell activation-induced apoptosis in vitro and in vivo.

Apoptotic cell death, characterized by DNA fragmentation and morphologic changes, has previously been shown to occur in immature thymocytes and some T cell hybridomas after activation. Like some other forms of apoptosis, DNA fragmentation during activation-induced cell death precedes the morphologic events. For apoptosis to proceed, activation of the cells must persist at least to the time of DNA fragmentation, before which the cells can remain viable if the activation signal is removed. Aurintricarboxylic acid (ATA) blocks activation-induced apoptotic cell death in a T cell hybridoma, and kinetic studies show that this inhibition occurs at or near the time of DNA fragmentation in the cells. Taken together with the ability of ATA to inhibit DNA fragmentation in isolated nuclei exposed to Ca2+ and Mg2+, these data strongly suggest that ATA prevents apoptosis via its ability to inhibit endogenous endonuclease activity, and, conversely, that this activity is required for this form of cell death. In vivo, ATA inhibits thymocyte depletion and DNA fragmentation induced by anti-CD3 Ab. Further, specific loss of V beta 8+ thymocytes after administration of staphylococcal enterotoxin B is blocked by administration of ATA. These observations support an essential role for DNA fragmentation as an irreversible step in activation-induced apoptosis in T cell hybridomas and during T cell development. This is contrasted with heat shock-induced cell death, in which inhibition of DNA fragmentation does not prevent loss of cell viability.

Animals↗
PubMed1994

Lymphoma models for B cell activation and tolerance. X. Anti-mu-mediated growth arrest and apoptosis of murine B cell lymphomas is prevented by the stabilization of myc.

Treatment of the WEHI-2131 or CH31 B cell lymphomas with anti-mu or transforming growth factor (TGF)-beta leads to growth inhibition and subsequent cell death via apoptosis. Since anti-mu stimulates a transient increase in c-myc and c-fos transcription in these lymphomas, we examined the role of these proteins in growth regulation using antisense oligonucleotides. Herein, we demonstrate that antisense oligonucleotides for c-myc prevent both anti-mu- and TGF-beta-mediated growth inhibition in the CH31 and WEHI-231 B cell lymphomas, whereas antisense c-fos has no effect. Furthermore, antisense c-myc promotes the appearance of phosphorylated retinoblastoma protein in the presence of anti-mu and prevents the progression to apoptosis as measured by propidium iodide staining. Northern and Western analyses show that c-myc message and the levels of multiple myc proteins were maintained in the presence of antisense c-myc, results indicating that myc species are critical for the continuation of proliferation and the prevention of apoptosis. These data implicate c-myc in the negative signaling pathway of both TGF-beta and anti-mu.

Animals↗
PubMed1994

Activation-induced apoptosis in lymphocytes.

Activation-induced apoptosis has been proposed as a mechanism for purging the immune repertoire of anti-self specificities, not only in development but also during the generation of somatic mutation in germinal centers. The pathways involved in driving immature and mature T and B cells to programmed cell death are reviewed with respect to two hypotheses, the pre-emptive death model, in which certain signals are obligatory for programmed cell death, and the two signal: death/survival model. Depending on the system, some data support the former pathway, in which certain signals are obligatory for programmed cell death, whereas other data are consistent with the two signal hypothesis. Moreover, recent data suggests that the c-myc protein plays a pivotal role in controlling this process. Finally conflicting roles of protein kinases, bcl-2 and p53 are reviewed and contrasted for involvement in activation-induced cell death in T and B lymphocytes.

Animals↗
PubMed1994

Dicing with death: dissecting the components of the apoptosis machinery.

Apoptosis, a mode of cell death commonly observed when death is a desirable or programmed event, has several characteristic structural features. These features appear to be induced by a range of gene products which, together, supervise and participate in the controlled dismantling of the cell. In this article the molecular components of the apoptotic machinery and the proteins implicated in the regulation of this mechanism of cell death are discussed.

Apoptosis↗
PubMed1994

Apoptosis as a goal of cancer therapy.

The elimination of a tumor cell population by exposure of the tumor to lethal doses of drugs or radiation is a well-established strategy in cancer therapy. Although better cytotoxic chemo- and radiotherapy regimens, coupled with the discovery of new chemotherapeutics, will continue to improve the effectiveness of such treatments, recent discoveries in the field of cell death promise to have a major impact in this area. Because apoptosis is an active process under molecular control, information concerning the regulation and execution of this type of cell death should lead to the development of more effective means of eliminating malignant cell populations. In addition, discovering how normal controls on apoptosis can fail should also improve our understanding of how tumors develop.

Apoptosis↗
PubMed1994

Promotion and inhibition of activation-induced apoptosis in T-cell hybridomas by oncogenes and related signals.

The Two Signal: Death/Survival Model suggests that cellular proliferation and physiological cell death should be intimately associated such that, in the absence of external influences, a normal cell departing from rest will have an equal probability of undergoing either process. The c-Myc protooncogene product has been implicated in cell cycle progression and in the control of gene expression, and more recently c-Myc has also been seen to promote apoptotic cell death. As predicted from the model, c-Myc-induced apoptosis is inhibited by growth factors or other anti-apoptotic signals including those provided by some oncogenes. Here, we discuss experiments that test the Two Signal: Death/Survival Model in the phenomenon of activation-induced apoptosis in T-cell hybridomas. Ligation of the antigen receptor on these cells leads to activation, resulting in cytokine production and apoptosis. Inhibition of c-Myc expression by addition of antisense oligodeoxynucleotides or transforming growth factor beta inhibits this form of apoptosis. Because c-Myc is known to bind to several cellular proteins, including Max, we further examined the effects of expression of a dominant negative Max on activation-induced apoptosis. We found that this Max mutant, which interferes with the function of the Myc/Max heterodimer, inhibits the induction of apoptosis by antigen receptor ligation. Thus, both Myc and Max play roles in activation-induced apoptosis, presumably via control of gene expression. Further, as predicted, signals generated from growth factor receptors or the v-Abl oncogene interfere with activation-induced apoptosis. In contrast, the anti-apoptotic effects of Bcl-2 are not active in this form of apoptosis. Finally, a role for Fas/Fas-ligand interactions in activation-induced apoptosis is considered.

Animals↗
PubMed1994

1994 William J. Stickel Silver Award. Closing base wedge versus Austin bunionectomies for metatarsus primus adductus.

Hallux abducto valgus with concomitant metatarsus primus adductus was treated by either an isolated Austin bunionectomy or by a combination of a modified McBride bunionectomy, along with a closing base wedge osteotomy, on a total of 73 patients (88 feet) from 1983 to 1993. Both subjective and objective similarities and differences were compared between these two groups of patients. Long-term elevation or depression of the first ray was analyzed by using a technique termed sagittal plane displacement. The prevalent preoperative symptoms were significantly reduced postoperatively in both groups of patients. Initial postoperative elevation of the first ray occurred in approximately one third of the cases in both groups. Long-term elevation of the first ray was greater with the base wedge osteotomy and did not change appreciably with the Austin procedure. The sagittal plane displacement method is a helpful tool in analyzing changes in the position of the first ray perioperatively.

Adult↗
PubMed1994

The abl oncogene family and apoptosis.

The role of the abl oncogene family in cellular transformation has been well established, but knowledge of its role in apoptosis is limited. Recent studies demonstrate that it may act as a suppressor of apoptosis in certain circumstances. The growth factor independence conferred on IL-3 dependent myeloid progenitor cell lines following v-Abl transformation is due to the suppressive effects of this oncogene on apoptosis. Similarly, inhibition of the deregulated activity of the p210(bcr-abl) protein in both myeloid progenitor lines and CML granulocytes has proven effective in reversing resistance to apoptosis in such cells. The Bcr-Abl fusion protein might therefore promote myeloid expansion by suppression of apoptotic cell death rather than through promoting proliferation. While oncogenic forms of Abl appear to be anti-apoptotic, the function of c-Abl remains elusive. Through the elucidation of the roles in cell growth and survival of the Abl family members we may gain valuable insights into the regulation of apoptosis and the mechanisms of oncogenesis.

Journal Article↗
PubMed1993

DNA fragmentation induced by cytotoxic T lymphocytes can result in target cell death.

Cytotoxic T lymphocyte (CTL)-mediated lysis is accompanied by fragmentation of target cell DNA into an oligonucleosome ladder, a hallmark of apoptosis. Is this a fortuitous coincidence, or could CTL be inducing lysis by activation of the suicide signal? In this report we demonstrate that CTL-mediated target cell death can be blocked with the drug aurintricarboxylic acid (ATA). The abrogation of death correlates with the inhibition of DNA fragmentation. While ATA prevented DNA fragmentation, it failed to significantly alter protein, RNA, or DNA synthesis in the cell lines over the dose range used. In addition, there was no inhibition of cell-cell interaction or granule exocytosis during CTL-mediated killing. ATA also significantly inhibited the cytolysis and DNA fragmentation mediated by isolated cytolytic granules, as well as the granular protein fragmentin. We developed an assay in which target cells could be separated from CTL after binding and programming for lysis. Once they had received the "kiss of death," target cells could be rescued from lysis (as indicated by inhibition of DNA fragmentation and increased target cell viability) by treatment with ATA. These results suggest that ATA blocks target cell death by inhibition of DNA fragmentation, and further, that chromatin degradation is a cause rather than a result of cell death in CTL-mediated lysis.

Animals↗
PubMed1993

Saying the 'S' word in public.

The mechanism by which self-reactive, peripheral lymphocytes are prevented from causing overt autoimmune disease is the subject of much debate. A recent meeting examined the evidence to support a role for T-cell mediated suppression in maintenance of peripheral tolerance.

Animals↗
PubMed1992

Antisense oligodeoxynucleotides as probes of T-lymphocyte gene function.

Conventional and thiophosphonate-derivatized oligonucleotides were employed to specifically regulate functional gene expression in murine T-cell hybridomas. For example, induction of apoptotic cell death following activation of T-cell hybridomas was examined using antisense oligonucleotides corresponding to several protooncogenes. We found that antisense oligodeoxynucleotides corresponding to c-myc inhibited both the characteristic DNA fragmentation and the loss of cell viability following activation without affecting production of lymphokines. Functional antisense oligonucleotides corresponding to c-fos had no effect in this system. These results demonstrate the use of antisense oligonucleotides to regulate function in T-cell hybridomas and provide valuable insights into the molecular bases of this biological phenomenon. Antisense oligonucleotides were also used to study another problem, the relation of T-cell-derived antigen-specific immunoregulatory factors to the T-cell receptor (TCR). Because the translation start of each TCR gene usually varies from one T cell to another, antisense oligonucleotides corresponding to the TCR V alpha or V beta of different cells were shown to act in a cell-specific manner. Furthermore, this method was used to demonstrate that a soluble antigen-specific regulatory activity produced by one of the T-cell lines depends on expression of the specific TCRa, an observation that has since been confirmed by gene transfer experiments. Expression of the CD3-TCR complex on the cell surface was also blocked by antisense oligonucleotides corresponding to CD3 gamma and CD3 zeta; however, neither these nor TCR V beta antisense oligonucleotides had any effect on production of the soluble regulatory activity.

Animals↗
PubMed1992

Apoptotic cell death induced by c-myc is inhibited by bcl-2.

Apoptosis is a form of physiological cell death, characterized by chromatin condensation, cytoplasmic blebbing and DNA fragmentation, which often depends on RNA and protein synthesis by the dying cell. The c-myc proto-oncogene, usually implicated in cell transformation, differentiation and cell-cycle progression also has a central role in some forms of apoptosis. These opposing roles of myc in cell growth and death require that other gene products dictate the outcome of c-Myc expression on a cell. A candidate for such a modifying gene is bcl-2, whose product prolongs cell survival and blocks apoptosis in some systems. Here we demonstrate that Bcl-2 prevents apoptotic death induced by c-Myc, provide a mechanism whereby cells can express c-Myc without undergoing apoptosis, and give a possible explanation for the ability of Bcl-2 to synergize with c-Myc in cell transformation.

Animals↗
PubMed1992

Role for c-myc in activation-induced apoptotic cell death in T cell hybridomas.

Immature T cells and some T cell hybridomas undergo apoptotic cell death when activated through the T cell receptor complex, a phenomenon that is probably related to antigen induced negative selection of developing T cells. This activation-induced apoptosis depends on active protein and RNA synthesis in the dying cells, although none of the genes required for this process have previously been identified. Antisense oligonucleotides corresponding to c-myc block the constitutive expression of c-Myc protein in T cell hybridomas and interfere with all aspects of activation-induced apoptosis without affecting lymphokine production in these cells. These data indicate that c-myc expression is a necessary component of activation-induced apoptosis.

Animals↗
PubMed1992

Microfilament-disrupting agents prevent the formation of apoptotic bodies in tumor cells undergoing apoptosis.

Apoptosis is a form of cell death in which the cell "participates," such that metabolic energy and often protein synthesis are required for the death to occur. Once begun, the process of apoptosis proceeds in an ordered fashion. In the earliest phase DNA fragmentation occurs, accompanied by cell shrinkage and dilation of the endoplasmic reticulum. This is followed by cell fragmentation with the formation of sealed membrane vesicles, termed apoptotic bodies. In the present study we have demonstrated that the fungal metabolite cytochalasin B inhibits cell fragmentation and the formation of apoptotic bodies, probably by its ability to interfere with actin polymerization. This effect was seen when HL-60 cells were pretreated with cytochalasin B and then exposed to one of a number of apoptosis-inducing agents, including UV irradiation, camptothecin, aphidocholin, or PMA plus ionomycin. The observed effect was not peculiar to HL-60 cells, inasmuch as it was also seen for both Molt-4 and U-937 cell lines. Cytochalasin B had no effect on DNA fragmentation occurring in the earliest stage of apoptosis, and it appeared to have no inhibitory effects on nuclear fragmentation. Staurosporin had an effect similar to that seen with cytochalasin B, probably due to its ability to inhibit protein kinase C, which is a known potentiator of microfilament assembly. These data demonstrate that microfilament assembly is necessary for the formation of apoptotic bodies in the later stages of the apoptotic process.

Actin Cytoskeleton↗
PubMed1992

Purification and analysis of proteinase-resistant mutants of recombinant platelet-derived growth factor-BB exhibiting improved biological activity.

Recombinant platelet-derived growth factor (PDGF)-BB was expressed and secreted from yeast in order to study the structure-function relationships of this mitogen. A simple purification scheme has been developed which yields greater than 95% pure PDGF-BB. Analysis of this recombinant PDGF-BB shows partial proteolysis after arginine-32. Substitution of this arginine residue, or arginine-28 [a potential KEX2 (lysine-arginine endopeptidase) cleavage site], prevents or reduces cleavage of PDGF-BB respectively. These mutations result in a 5-fold increase in expression levels of PDGF-BB, and the resulting mutant proteins show higher activity in a number of biological assays than the cleaved wildtype PDGF-BB. These data are in accord with previous work by Giese, LaRochelle, May-Siroff, Robbins & Aaronson [(1990) Mol. Cell Biol. 10, 5496-5501] suggesting that the region isoleucine-25-phenylalanine-37 is involved in PDGF-receptor binding.

3T3 Cells↗