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D R Green

Publications and source records attributed to D R Green.

At least 19 recordsLinked to original sources
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↗
PubMed1992

Characterization of the structure and conformation of platelet-derived growth factor-BB (PDGF-BB) and proteinase-resistant mutants of PDGF-BB expressed in Saccharomyces cerevisiae.

A detailed biophysical study of the secondary and tertiary structures of recombinant platelet-derived growth factor (PDGF)-BB produced in yeast has been carried out. The secondary structure of the molecule is composed of 54% beta-sheet with less than 5% ordered helix. The single tryptophan residue has been shown to be solvent-accessible; however, the ability of the side chain to rotate is severely restricted. The fluorescence emission is quenched at pH 7.0 and in the presence of high salt, but dequenched by titration to lower pH with a pK of 5.8. Two proteinase-resistant mutants of PDGF [( Ser28]- and [Pro32]-PDGF-BB) have also been characterized and shown to have secondary and tertiary structures indistinguishable from wild-type PDGF-BB. These are, therefore, suitable stable background molecules in which to carry out structure-activity-relationship studies on PDGF-BB.

Mutagenesis, Site-Directed↗
PubMed1992

Activation-induced apoptosis in lymphoid systems.

Lymphocytes become activated when antigen receptors on the cell surface are cross-linked, or when they are exposed to agents that mimic this signal. Although such activation is usually associated with the production of immune mediators (e.g. antibodies, cytokines) and entry into the cell cycle, it can alternatively lead to death via apoptosis. This activation-induced apoptosis was first observed in developing lymphocytes and has been proposed as a mechanism for negative selection, by which immature cells with potential for autoreactivity are eliminated from the maturation pathway. Activation-induced apoptosis has also been observed in normal, mature lymphocytes under some conditions, and this may account for the phenomena of peripheral deletion, in which mature T cells are eliminated upon exposure to high doses of antigen. It may also be an important mechanism whereby CD4+ T cells are depleted in HIV+ individuals. Although the phenomenon of activation-induced apoptosis is not understood, recent studies have begun to implicate specific signal transduction pathways and gene products in the process. Among the latter is the c-myc proto-oncogene, which paradoxically can play an essential role in several forms of apoptosis, including that induced by activation of lymphocytes.

Animals↗
PubMed1991

Immunoregulatory activity of the T-cell receptor alpha chain demonstrated by retroviral gene transfer.

We have previously described an antigen-specific I-Ad-restricted T-cell hybridoma, A1.1, that constitutively releases an antigen-specific immunoregulatory activity into supernatants. Using retrovirally mediated gene transfer, we have found that transfer of the T-cell receptor alpha chain (TCR alpha) gene from A1.1 to a number of other T-cell hybridomas effectively transferred the ability to produce the activity. Gene transfer of the TCR beta chain (TCR beta), however, did not transfer this ability. The regulatory activity from cells expressing the A1.1 TCR alpha bound to and was eluted from an anti-TCR alpha monoclonal antibody and displayed fine antigenic specificity identical to that of supernatants from A1.1. The possibility that this activity represents a secreted form of the TCR alpha (as opposed to shed cell-surface TCR) was examined in BW1100 cells, lacking TCR alpha and TCR beta, which produced the antigen-specific activity after gene transfer of the A1.1 TCR alpha gene. The expression of the immunoregulatory activity in supernatants correlated with a direct antigen-binding activity as detected by ELISA, thus raising the possibility that antigen binding is relevant to the mechanism of action of the soluble TCR alpha. We discuss these observations and our earlier studies suggesting an immunoregulatory role for soluble TCR alpha.

Amino Acid Sequence↗
PubMed1991

In vivo administration of monoclonal antibodies to the CD3 T cell receptor complex induces cell death (apoptosis) in immature thymocytes.

Some thymocytes, upon activation via the TCR complex in vitro, undergo apoptotic cell death. In this report, we examine the cell death induced in the thymus after administration of anti-CD3 or anti-TCR antibodies. We found that shortly after antibody injection, cortical thymocytes undergo apoptosis as characterized by morphologic changes and DNA fragmentation. Anti-CD3 administration led to depletion of nearly all CD4+CD8+ thymocytes, and approximately 50% of CD4+CD8- thymocytes. This depletion predominantly affected cells bearing low levels of CD3, although some depletion also occurred among cells expressing intermediate and high levels. Administration of an anti-TCR antibody also induced apoptosis, but affected significantly fewer thymocytes than anti-CD3. This effect was probably not due to different binding affinities for the two antibodies, because both antibodies show similar dose response effects in an in vitro model of activation-induced apoptosis. This work demonstrates that findings on activation-induced apoptosis in vitro can be extended to the in vivo situation, and further, that the activation of cortical thymocytes, in situ, results in apoptosis and removal of the activated cells. The possible relationships between this activation-induced cell death in immature thymocytes and the process of negative selection of autoreactive T cells is discussed.

Animals↗
PubMed1991

A T helper cell hybridoma produces an antigen-specific regulatory activity. Relationship to the T cell receptor by serology and antigenic fine specificity.

We have previously shown that a T cell hybridoma, A1.1, constitutively produces an Ag-specific regulatory factor with specificity for poly-18, a synthetic polypeptide. This cell also responds to poly-18 plus I-Ad by producing lymphokines. The antigenic specificity of the factor and the T cell appeared to be the same. This suggested the possibility that some part of the TCR, responsible for antigenic specificity of the cell, also imparts specificity to the A1.1-derived factor. This was supported by the observation that the factor was bound and eluted from a monospecific anti-TCR antiserum. Further, we demonstrated that antisense oligodeoxynucleotides corresponding to the TCR V alpha of A1.1 (but not TCR V beta) block production of the Ag-specific factor. Herein, we report recent findings that strengthen the proposed relationship between the TCR and the A1.1-derived factor. The factor was bound and eluted from a monoclonal anti-TCR C alpha antibody, but not from anti-TCR beta, anti-V beta 6, nor anti-CD3 epsilon. The anti-TCR C alpha antibody bound a Mr 46-kDa protein from A1.1 supernatants, which is the same apparent size at which activity could be eluted from an SDS-PAGE gel separation of concentrated factor. Antigenic fine-specificity analysis revealed that two amino acids in poly-18 are critical for the recognition of the antigen by the Ag-specific factor. These two amino acids appear to be those recognized by the TCR. The factor that was bound and eluted from the monoclonal anti-TCR C alpha showed this fine-specificity as well. This, combined with our earlier studies, supports the view that the A1.1-derived factor is encoded, at least in part, by TCR-alpha.

Amino Acid Sequence↗
PubMed1991

Two PDGF-B chain residues, arginine 27 and isoleucine 30, mediate receptor binding and activation.

PDGF may be involved in the pathogenesis of a variety of disorders including atherosclerosis and certain types of cancer. There is currently little understanding of the molecular structure of PDGF and of the critical amino acid residues involved in receptor binding and cell activation. Two such PDGF-B chain residues, arginine 27 and isoleucine 30, have been identified by a site-directed mutagenesis programme. Substitutions in these positions can lead to PDGF mutants defective in both receptor affinity and cell activation as judged by displacement of [125I]PDGF-BB, mitogenic assay and inositol lipid turnover. Circular dichroism and fluorescence spectroscopy show that such mutations do not disrupt the structure of PDGF.

3T3 Cells↗
PubMed1990

Regulatory function for murine intraepithelial lymphocytes. Two subsets of CD3+, T cell receptor-1+ intraepithelial lymphocyte T cells abrogate oral tolerance.

The murine intraepithelial lymphocyte (IEL) population is enriched in T cells that express the gamma delta-TCR, however, the biologic function served by these T cells remains obscure. IEL are considered to be major effector cells in mucosal immunity, and we have investigated whether IEL subsets could reverse orally induced systemic unresponsiveness (oral tolerance; OT) and support secondary type responses when adoptively transferred to mice orally tolerized with SRBC. When purified CD3+ IEL from mice orally primed with SRBC were transferred to adoptive hosts and challenged with SRBC, splenic IgM, IgG1, IgG2b, and IgA anti-SRBC plaque-forming cell responses were observed. However, CD3+ IEL from HRBC orally primed mice did not abrogate SRBC induced OT. Further, HRBC-primed CD3+, IEL converted HRBC-specific OT but not SRBC-specific OT. CD3+ IEL could be separated into four subsets based on expression of CD4 and CD8. CD3+, CD4-, 8+ T cells were the major subset (74.5%), with smaller numbers of CD4- and CD8- (double negatives, DN) (7.8%), CD4+, 8- (7.6%) and CD4+, CD8+ (double positives) (10.1%) T cells. Interestingly, both the CD3+, CD8+, and the CD3+, DN IEL subsets abrogated OT, resulting in significant IgM, IgG1, IgG2b, and IgA anti-SRBC plaque-forming cell responses when adoptively transferred to mice with OT. However, neither CD3+, CD4+, CD8-, nor double positive T cells affected OT when studied in this system. The CD3+, CD8+ IEL subset could be further separated into Thy-1+ (16.6%) and Thy-1- (83.4%) cells; adoptive transfer of Thy-1- cells abrogated oral tolerance whereas the Thy-1+ subset was without effect. When the expression of TCR on IEL with this biologic function was determined by use of monoclonal anti-alpha beta TCR (H57.597), TCR2-, CD3+ IEL possessed immunoregulatory function whereas the alpha beta-TCR+ (TCR2+) fraction did not abrogate OT. Immunoprecipitation of membrane fractions obtained from purified CD3+, CD4-, CD8+, Thy-1- IEL with polyclonal anti-delta peptide (Tyr-Ala-Asn-Ser-Phe-Asn-Asn-Glu-Lys-Leu) antibody revealed bands of 45 and 35 kDa, corresponding to the delta- and gamma-chains, respectively. These results suggest that gamma delta-TCR+ IEL possess a regulatory function, namely the restoration of immune responses in a state of oral tolerance. Further, both CD3+, CD4-, CD8+, Thy-1-, and CD3+, DN IEL T cells exhibit this effector contrasuppressor function.

Administration, Oral↗
PubMed1990

Activation-induced cell death in T cell hybridomas is due to apoptosis. Morphologic aspects and DNA fragmentation.

Some T cell hybridomas, upon activation via the TCR, rapidly undergo cell death. In this paper, we demonstrate that this activation-induced cell death (AICD) is accompanied by morphologic changes seen at the electron and light microscopy levels. The most striking changes are an extensive condensation of the chromatin and formation of membrane blebs. In addition to the morphologic changes, a significant portion of genomic DNA is broken at an interval of approximately 200 bp, producing a ladder of oligonucleosome-sized fragments after gel electrophoresis. Taken together, these observations indicate that AICD proceeds via apoptosis, or programmed cell death. This is additionally supported by the observation that AICD-associated phenomena are at least partially inhibited by cycloheximide or actinomycin D. Curiously, AICD and its associated DNA fragmentation are completely inhibited by aurintricarboxylic acid, a known nuclease inhibitor. The possible relationship between AICD in vitro, and the negative selection process (wherein selection may proceed via AICD of developing, autoreactive thymocytes) is discussed.

Aurintricarboxylic Acid↗
PubMed1990

Host natural suppressor activity regulates hemopoietic engraftment kinetics in antibody-conditioned recipient mice.

Resistance to semi-allogeneic or syngeneic hemopoietic stem cell engraftment can be reduced by treating the unirradiated host with anti-class I MHC antibody. In our previous studies we showed a direct correlation between such resistance and the level of natural suppressor (NS) activity in the host. Thus newborn mice that have high NS activity are very resistant to marrow engraftment, as are adults pretreated with CFA that increases NS activity in the bone marrow. We have now devised a method that allows us to follow hemopoietic engraftment kinetics within the marrow cavity itself by assaying individual CFU-granulocyte/macrophage progenitor cells for their host or donor origin over the immediate post-transplant period. By using this method, we find a close correlation between the rate of marrow engraftment and reduction in host NS activity. Marrow engraftment does not correlate with the reduction of either total host bone marrow cellular content or CFU-granulocyte/macrophage progenitor cell levels. NS activity is mediated by Thy-1-, partially radiosensitive, nylon wool nonadherent cells without NK activity. Adoptively transferred Thy-1-, irradiated spleen cells containing NS activity induced by pretreatment with CFA delayed engraftment kinetics in the marrow cavity. Thus hemopoietic engraftment in the marrow cavity appears to be controlled by an inhibitory regulatory activity that is reflected in the in vitro NS assay. These studies suggest new regulatory targets for selective host conditioning to eliminate resistance to marrow transplantation.

Animals↗
PubMed1990

Generation of a T-cell hybridoma producing a contrasuppressor factor for contact sensitivity.

C3H/HeN mice were immunized to induce contrasuppressor T-cell (Tcs) activity, splenic T cells from these mice were fused with the BW5147 thymoma, and the resulting hybridomas were tested for their ability to produce a contrasuppressor T-cell factor (TcsF). Nine TcsF-producing hybridomas were preliminarily identified by their ability to inhibit the effect of antigen-specific suppressor T-cell factor (TsF) on the adoptive transfer of contact sensitivity. One of these hybrids, AF5.C6, was cloned, the production of a contrasuppressor factor confirmed, and the high-titred TcsF produced by this cloned hybrid characterized. Hybridoma-derived TcsF is antigen-specific and specifically binds its antigen, but does not bear immunoglobulin (Ig) determinants. Thus, hybridoma-derived TcsF is serologically and functionally identical to an antigen-specific contrasuppressor factor for contact sensitivity, whose production from splenocyte cell cultures has previously been described. The generation of a hybridoma secreting a contrasuppressor factor identical to that produced by spleen cells significantly strengthens the hypothesis that the phenomenon of T-cell contrasuppression is mediated by a specific subset of cells whose activity is contrasuppressive. The further advantages of employing T-cell hybridomas for functional, biochemical and molecular genetic analyses of contrasuppression are also discussed.

Animals↗