Biomedical subjects

J G Monroe

Publications and source records attributed to J G Monroe.

At least 55 records · Page 3Linked to original sources

PubMed1995

HIV-mediated B-lymphocyte activation and lymphomagenesis.

Non-Hodgkin's (1ii)lymphoma is an AIDS-defining event in a significant percent of U.S. patients infected with the human immunodeficiency virus (HIV). Advances in anti-retroviral treatment and management of opportunistic infection have been accompanied by an increase in the incidence of these lymphomas. In the immunocompromised state of patients late in the course of HIV infection, these lymphomas represent a complication of HIV infection that is associated with an extremely poor prognosis. Currently, there is little understanding of the pathogenesis of HIV-associated lymphomas, which are nearly exclusively of B-cell origin. Experimental data do not support HIV infection in these lymphomas. While some lymphomas show evidence of EBV infection, the majority do not. Polyclonal B-cell hyperactivity during the early phases of HIV infection argues that chronic B-cell stimulation may be the major process predisposing B-cells in the HIV-infected individual to malignant transformation. The mechanism of this stimulation of normal B cells and its relationship to AIDS-associated lymphomas remain poorly understood. In this review, we will summarize current information on HIV-associated B lymphoma and then discuss our views on the association and regulation of HIV-related hyperactivity on the pathogenesis of this lymphoma.

B-Lymphocytes↗

PubMed1995

The EGR1 protein contains a discrete transcriptional regulatory domain whose deletion results in a truncated protein that blocks EGR1-induced transcription.

Egr-1 is a ubiquitous immediate-early gene whose expression is induced by a wide range of different stimuli. A requirement for egr-1 expression has been demonstrated in pathways leading to both proliferation and differentiation, suggesting that egr-1 is a critical intermediary in determining the long-term cellular response to a stimulus. To determine how egr-1 coordinates a cellular response to receptor-mediated stimulation, we have developed a transient cotransfection assay to map functional domains in the EGR1 protein. We localized an activation domain to a serine/threonine/proline-rich region between amino acids 174 and 270. Using this information, we designed a mutant that lacks this activation domain, but retains the DNA-binding domain. When cotransfected into fibroblasts with an EGR1-dependent reporter, this mutant inhibited the transcriptional activity of both endogenous EGR1, as well as exogenously expressed, wild-type EGR1 protein. These data demonstrate that the activation domain of EGR1 is critical for the activity of the protein, and that a mutant lacking this domain can dominantly inhibit wild-type EGR1 function.

3T3 Cells↗

PubMed1995

A ternary complex factor-dependent mechanism mediates induction of egr-1 through selective serum response elements following antigen receptor cross-linking in B lymphocytes.

Induction of the primary response gene egr-1 occurs rapidly following antigen receptor cross-linking in B lymphocytes. Antisense studies have demonstrated that this induction is necessary for their subsequent activation to this signal. The present study examines the molecular mechanism whereby the receptor-generated signals interact with the egr-1 promoter to elicit transcription. Deletion mapping and point mutations have indicated that two of the five serum response elements (SREs) in the egr-1 promoter can mediate induction. Of the two critical SREs, both are capable of mediating maximal induction even in the absence of the other SRE. Our results also indicate that adjacent Ets motifs are necessary for induction. Like the c-fos SRE, the egr-1 SRE/Ets sites are occupied by a multiprotein (ternary) complex containing a homodimer of serum response factor and an unidentified member of the Ets family of transcription factors. The identification of a ternary complex-dependent mechanism of egr-1 induction, along with selective utilization of SREs in B lymphocytes, suggests that a complicated array of signaling cascades interacts with unique combinations of regulatory elements in the egr-1 promoter in different cell types.

Animals↗

PubMed1994

Surface IgM-stimulated proliferation, inositol phospholipid hydrolysis, Ca2+ flux, and tyrosine phosphorylation are not altered in B cells from p59fyn-1- mice.

The surface immunoglobulin M (sIgM)-associated src family protein tyrosine kinases (PTKs) p55blk, p59fyn, and p53/56lyn become activated in B cells within seconds following sIgM cross-linking. Studies using protein tyrosine kinase (PTK) inhibitors have demonstrated that PTK activity is crucial for downstream events such as calcium flux, inositol phospholipid hydrolysis, and cell cycle entry. The roles that the individual src family PTKs play in sIgM signaling are largely unknown, however. In order to determine whether p59fyn plays a distinct role in sIgM signal transduction, the signaling capabilities of B cells isolated from fyn "knockout" mice were evaluated. We observed that in the absence of p59fyn, there was no demonstrable compromise of the sIgM-coupled signaling events measured (tyrosine phosphorylation, inositol phospholipid hydrolysis, and Ca2+ flux). We propose that either p59fyn is not involved in coupling sIgM to these specific signaling pathways or that other PTKs are able to compensate for the absence of p59fyn, indicating redundancy in the sIgM signaling pathways.

Animals↗

PubMed1994

A mutant of the WEHI-231 B lymphocyte line that is resistant to phorbol esters is still sensitive to antigen receptor-mediated growth inhibition.

In order to gain a better understanding of the pathways linking receptor immunoglobulin (sIg) crosslinking to downstream B lymphocyte responses, mutants were generated that were defective in sIg-associated signaling pathways. The murine B lymphoma WEHI-231 has proven to be a useful model for studies of sIg-mediated signal transduction. Signaling through sIgM using anti-receptor antibodies (anti-mu) leads to growth arrest and apoptosis of this continuously proliferating cell line. Direct activation of protein kinase C (PKC) with phorbol esters also can mediate this response in WEHI-231. This negative growth response is a useful characteristic that can be exploited to generate signaling mutants that are resistant to the growth-inhibiting effects of anti-mu or phorbol ester. Using this approach, we selected a mutant, PR30-3, in which signaling was blocked downstream of the phorbol diester response element, presumably PKC. Although no longer responsive to phorbol ester stimulation, PR30-3 is not defective in PKC expression or function. Western blot analyses of cellular lysates shows the mutant to express the PKC isoforms alpha, beta, and delta, at levels not markedly different from wild-type WEHI-231. PR30-3 expresses active PKC as shown by its ability to phosphorylate a PKC-specific peptide in vitro. PR30-3 and WEHI-231 express equivalent levels of sIgM expression and nearly indistinguishable second messenger responses in the form of increases in [Ca2+]i and inositol phospholipid hydrolysis. Both PR30-3 and WEHI-231 demonstrate rapid induction of tyrosine kinase activation following sIgM signaling, although there is a reproducible difference in the ability to phosphorylate a 40-kDa substrate in PR30-3. Interestingly, tyrosine phosphorylation of this substrate is induced by phorbol ester stimulation in the wild-type but not the mutant PR30-3. We observed that phorbol ester stimulation of PR30-3 induces the expression of the early response gene c-fos, previously shown to be PKC dependent in this cell line. These results indicate that the signaling component(s) defective in PR30 lie downstream of PKC but upstream of the commitment point for growth inhibition and cell death. Finally, although PR30-3 is resistant to the inhibitory effects of phorbol ester, proliferation is nonetheless still inhibited in response to anti-mu stimulation. These results suggest that the growth inhibitory response of WEHI-231 to anti-mu and phorbol ester involves different pathways.

Animals↗

PubMed1993

Endogenous expression of delta on the surface of WEHI-231. Characterization of its expression and signaling properties.

WEHI-231 is a murine B cell lymphoma that has been used extensively as a model for the immature stage B cell and its functional response to Ag receptor cross-linking as a model for immature B cell tolerance. This cell line expresses sIgM, CD5, and FcR gamma, but lacks the B cell-specific isoform of CD45 (B220). This study demonstrates for the first time that WEHI-231, in contrast to classically defined immature B cells, expresses delta on its surface. Analysis of delta on WEHI-231 revealed structural differences with respect to that on BAL-17 or primary splenic B cells. Although the m.w. of delta on the latter two B cell populations was similar, delta on WEHI-231 manifested a marked increase in its apparent m.w. deduced by SDS-PAGE. This difference was found to be due primarily to differential N-linked glycosylation. Signal transduction through the endogenous sIgD on WEHI-231 was investigated. In contrast to cross-linking of sIgM, cross-linking of the endogenous surface IgD on WEHI-231 was unable to generate a negative growth response in these cells. This inability may be due to uncoupling from normal surface Ig signaling pathways. The signaling properties of the endogenous sIgD on WEHI-231 differ from that on primary B cells and other sIgD-expressing cell lines. Whereas sIgD on splenic B lymphocytes or the mature B cell line BAL-17 is coupled to inositol phospholipid hydrolysis and calcium mobilization, cross-linking of sIgD on WEHI-231 failed to elicit these events, although induced changes in tyrosine phosphorylation were observed. Thus, endogenous expression of surface IgD on WEHI-231 is inconsistent with its representing the classically defined immature stage B cell. The structural and signaling differences associated with delta on these cells suggest the potential for developmentally regulated delta function and model for study of sIgD signal transduction.

Animals↗

PubMed1992

Developmentally regulated association of a 56-kD member of the surface immunoglobulin M receptor complex.

Immature and mature B cells differ in the signals generated and transduced through their antigen receptor, surface immunoglobulin M (sIgM). Whereas signals generated through sIgM on mature B cells initiate a program leading to the positive activation of these cells, signaling through this receptor at the immature stage of development leads to a state of induced unresponsiveness or tolerance. Our previous studies have described developmental differences in sIgM transmembrane signaling that are independent of ligand-receptor affinity. In an attempt to understand the molecular basis for signaling differences between immature and mature B cells, we have analyzed the sIgM receptor complex in neonatal and adult mouse splenic B cells. While previously described components of this complex do not exhibit marked developmentally regulated differences in their association with sIgM, we have identified a 56-kD protein that associates with sIgM in mature (antigen-responsive), but not immature (tolerance-sensitive) B cells. This protein (p56) associates with sIgM as a homodimer, is constitutively phosphorylated on tyrosine, and is coimmunoprecipitated with IgM but not IgD. The observed inability to iodinate p56 suggests it is an intracellular component of the receptor complex. Based upon its migration in one- and two-dimensional gel electrophoresis we show, however, that p56 is distinct from the blk, lyn, or fyn src family kinases that have been shown to be associated with sIgM in mature B cells. The developmentally regulated participation of p56 in the B cell antigen receptor complex suggests a role in the differential signaling mediated via sIgM on immature and mature B cells.

Animals↗

PubMed1992

Differential responsiveness of immature- and mature-stage murine B cells to anti-IgM reflects both FcR-dependent and -independent mechanisms.

Mature and immature B cells differ in their responses to antigen receptor crosslinking. Whereas mature B cells enter cell cycle in response to such stimulation, immature B cells exhibit proliferative unresponsiveness and undergo induced tolerance following surface immunoglobulin (sIg) engagement. Previous studies evaluating antigen receptor-mediated negative signaling have utilized intact goat anti-immunoglobulin (anti-Ig) antibodies as polyclonal ligands based upon observations that the Fc portion of these reagents does not interact with and mediate negative signaling through the FcR on mature B cells. Thus, the negative effects of goat anti-Ig on immature B cells have been attributed solely to signals mediated via their antigen receptors. In the studies reported here we show that the activation unresponsiveness inherent to immature B cells is FcR independent. However, we also show that immature B cells are sensitive to FcR-mediated inhibition and that these effects can be mediated by intact goat antibodies at concentrations that promote positive activation signals in mature B cells. Our results demonstrate that inhibition of immature B cell LPS responses by anti-Ig antibodies, used in previous studies as an in vitro model for B cell tolerance induction, is an FcR-mediated phenomenon. We show that developmentally associated anti-Ig-mediated inhibition of LPS requires the use of intact antibodies, and that this inhibition can be blocked by the anti-FcR monoclonal antibody 2.4G2. Flow cytometric analysis of FcR-positive B cells indicates that both mature and immature B cells express equivalent levels of FcR gamma. Therefore, the sensitivity of immature, but not mature, cells to intact goat anti-mu antibodies suggests that either FcRs or their associated inhibitory pathways change during B cell development.

Animals↗

PubMed1992

Role of primary response genes in generating cellular responses to growth factors.

Cellular responses to growth and differentiation factors involve a complex cascade of signals that begins at the cell surface, traverses the cytoplasm, and eventually enters the nucleus. Although a great deal is known about the surface and cytoplasmic stages of this cascade, the nuclear events involved in transducing and translating growth and differentiation signals are less well understood. Recent work has implicated a set of genes known as primary response genes as critical for this process. To propagate the activation signal, these genes possess the ability not only to directly respond to upstream biochemical events, but also to transmit the signal downstream by modulating the unique changes in gene expression necessary for a particular cellular response. In this review we discuss how transcription factors encoded by primary response genes may be responsible for regulating tissue- or stimulus-specific responses associated with cellular activation.

Base Sequence↗

PubMed1991

Signaling through surface IgM in tolerance-susceptible immature murine B lymphocytes. Developmentally regulated differences in transmembrane signaling in splenic B cells from adult and neonatal mice.

During the course of B lymphocyte development, newly emerging surface Ig+ B cells pass through a stage when Ag-Ag receptor interactions lead not to immune responsiveness but to a state of functional tolerance. We have explored the molecular basis of antigenic nonresponsiveness and tolerance susceptibility using tolerance-susceptible surface Ig+ splenic B lymphocytes from neonatal mice and anti-mu chain antibodies as a polyclonal ligand. In this population of cells, surface IgM is uncoupled from the inositol phospholipid (PI)-hydrolysis pathway at a point proximal to the receptor; anti-mu antibodies did not stimulate inositol phosphate generation despite the fact that PI-hydrolysis was observed after treatment with A1F4, implicating the existence of a functional G protein and phospholipase C. Further evidence for a difference early in the signal transduction pathway stems from the finding that anti-mu stimulation does not induce the expression of two immediate/early PKC-linked genes egr-1 and c-fos. This appears to be the primary signaling difference between the mature and immature B cells from the neonatal mouse splenic population, as these cells undergo a G0-G1 cell cycle phase transition when surface IgM is bypassed using phorbol diester and calcium ionophore. Interestingly, despite undetectable levels of PI-hydrolysis, we observed equivalent receptor-mediated changes in intracellular calcium when comparing the immature and mature populations. These results indicate incomplete coupling of surface IgM to the signal transduction machinery operative in mature, immunocompetent B cells and suggests a molecular mechanism accounting for the differential processing of surface IgM signals into activation vs tolerogenic responses observed in these two stages of B cell development.

Aging↗

PubMed1990

Egr-1 expression in surface Ig-mediated B cell activation. Kinetics and association with protein kinase C activation.

We have studied the expression of an immediate/early type gene, Egr-1, in murine B lymphocyte responses to Ag receptor-generated signals. The Egr-1 gene encodes a zinc finger protein with sequence-specific DNA binding activity and is believed to act as an intracellular "third messenger," to couple receptor-generated signals to activation-associated changes in gene expression. We show here that Egr-1 mRNA expression is rapidly and transiently (returning to basal levels by 6 h) induced after receptor crosslinking with anti-receptor antibodies. Egr-1 protein expression is more prolonged, maintaining detectable levels through 12 h. The induction of Egr-1 is a primary response to Ag receptor signaling, as it is independent of new protein synthesis and is inhibited by actinomycin D. We have also examined the linkage of Egr-1 to known signaling pathways associated with G0 to G1 transition by these cells in response to signals generated through the B cell Ag receptor. Egr-1 mRNA was not induced after elevation of intracellular free Ca2+. In contrast, the pharmacologic agents PMA and SC-9, which directly activate protein kinase C, both cause marked increases in Egr-1 mRNA levels with the same kinetics as observed after anti-receptor antibody stimulation. Further, the protein kinase C inhibitors H7, sangivamycin, and staurosporin block anti-receptor antibody-induced expression of Egr-1, thus, B cell Ag receptor-linked Egr-1 expression is likely coupled to the protein kinase C component of transmembrane signaling. Preliminary promoter mapping studies are consistent with this conclusion, because both PMA and anti-receptor antibody act through the same or overlapping cis-regulatory elements.

Animals↗

PubMed1990

Methylation of an immediate-early inducible gene as a mechanism for B cell tolerance induction.

Stage-specific gene regulation is important in determining cell function during development. Immature B cells expressing membrane-bound immunoglobulin M (mIgM) are sensitive to antigen-induced tolerance, whereas mature B cells are activated by antigen. Previous studies have established an association between Egr-1 gene induction and antigen receptor (mIgM)-mediated activation of mature B cells. Here it is shown that the immature B cell line WEHI-231 and tolerance-sensitive bone marrow-derived B cells do not express Egr-1. It is further shown that lack of inducible expression in these cells is due to specific methylation of the Egr-1 gene. Thus, covalent inactivation of an activation-associated gene may explain tolerance sensitivity at specific stages of B cell development.

Animals↗

PubMed1989

Involvement of a specific guanine nucleotide binding protein in receptor immunoglobulin stimulated inositol phospholipid hydrolysis.

The role of a specific guanine nucleotide binding (G protein) protein in coupling murine B lymphocyte receptor immunoglobulin to inositol phospholipid hydrolysis was investigated. Using an in vitro system with isolated membranes, we have observed specific enhancement of GTP binding subsequent to ligand-induced receptor crosslinking. Induced increases were inhibited by pretreatment with pertussis toxin which catalyzed ADP-ribosylation of a 43 kDa substrate. Involvement of this G protein with receptor immunoglobulin-induced inositol phospholipid hydrolysis was evidenced by the ability of pertussis toxin to block this response. This report, then, indicates that the B lymphocyte antigen receptor belongs to a family of receptors which are linked to inositol phospholipid hydrolysis through a G protein.

Adenosine Diphosphate Ribose↗

PubMed1989

Isolation and characterization of a B lymphocyte mutant with altered signal transduction through its antigen receptor.

A receptor surface Ig (sIg) signaling variant of WEHI-231 was constructed to investigate components and linkages between various signaling events associated with signal transduction through sIg. Unlike the wildtype, crosslinking of sIgM on VS2.12-cl.2 did not result in downregulation of proliferation. Similarly, receptor crosslinking was uncoupled from inositol phospholipid (PI) hydrolysis and upregulation of c-fos expression in the variant. The signaling defect in VS2.12-cl.2 appears to be proximal to phospholipase C activation as direct G protein activation by A1F4- triggers PI hydrolysis and bypassing PI hydrolysis using phorbol diester stimulation of protein kinase C restores the inhibitable phenotype and the ability to upregulate c-fos. Even more interesting, sIg-linked Ca2+ responses by VS2.12-cl.2 are equivalent to these observed in the wildtype WEHI-231. These latter results suggest that contrary to current thought, sIg-generated signals may not be coupled to Ca2+ fluxes entirely via inositol phospholipid hydrolysis. Thus, VS2.12-cl.2 is a new and powerful tool with which to analyze signaling through sIg at the molecular level.

Antibodies↗

PubMed1989

Signalling through sIgA on a novel murine B lymphoma.

A novel murine B lymphoma expressing membrane-associated IgA was isolated and used to compare mechanisms of signal transduction by sIgM and sIgA. Like other isotypes so far studied, crosslinking of sIgA by anti-immunoglobulin antibodies stimulates hydrolysis of inositol phospholipids and causes elevation of intracellular free calcium. Furthermore, signals generated through sIgA are coupled to elevation of c-fos proto-oncogene expression. Coupling appears to be through the protein kinase C rather than through the Ca2+ component of sIg signalling as phorbol diester, but the Ca2+ ionophore cannot mediate this effect. Thus these results, coupled with those from earlier studies, show that early signal transduction through surface immunoglobulin appears to be similar regardless of the particular isotype involved in binding ligand.

Animals↗

PubMed1989

Differential expression of a zinc finger-encoding gene in response to positive versus negative signaling through receptor immunoglobulin in murine B lymphocytes.

Egr-1 is a murine early growth factor-inducible gene that encodes a protein with zinc fingers and that is believed to be involved in transcriptional regulation. Expression of this gene was investigated in murine B lymphocytes stimulated through their receptor for antigen (surface immunoglobulin [sIg]) with antireceptor antibodies (anti-sIg). Rapid (by 15 min) up-regulation of Egr-1 mRNA expression was observed after sIg cross-linking at a dose of anti-sIg sufficient to drive the majority of cells into cell cycle. Interestingly, signaling through sIg on the murine B-lymphoma cell line WEHI-231 did not up-regulate Egr-1 expression even though similar signaling pathways, including up-regulation of c-fos expression, are associated with this receptor in these cells. Importantly, cell growth and proliferation of WEHI-231 cells were inhibited by anti-sIg stimulation, which suggested a relationship between Egr-1 expression and differential processing of receptor immunoglobulin signals with respect to cellular growth responses. This notion was further supported by the finding that murine B lymphomas whose proliferation was not inhibited by anti-sIg showed receptor immunoglobulin-coupled Egr-1 expression. In further support of this association are results showing that under conditions in which Egr-1 expression was induced in WEHI-231 cells in response to stimulation by anti-sIg, a concomitant change was observed in the growth response of these cells. These results, then, indicate a potential role for Egr-1 expression in the translation of receptor-generated signals into cellular activation or induced unresponsiveness.

Animals↗

PubMed1988

Up-regulation of c-fos expression is a component of the mIg signal transduction mechanism but is not indicative of competence for proliferation.

Control of entry into and progression through the early phases of cell cycle in B lymphocytes is poorly understood at the molecular level. Products of the c-fos proto-oncogene have been implicated in regulation of G0 to G1 cell cycle phase transition and cell proliferation in other systems. In view of these observations, the relationship between signals generated through receptor Ig which alter the B cells position in cell cycle and relative level of c-fos expression was investigated. Not unexpectantly, anti-Ig under conditions which promote G0-G1 and G1-S phase transition was observed to selectively up-regulate expression of c-fos. More interestingly, however, anti-Ig-induced cross-linking of surface Ig on the WEHI-231 B lymphoma also caused rapid and transient up-regulation of c-fos mRNA levels although it was associated with inhibition of proliferation of these cells. These results are important because they show that 1) c-fos expression is inducible in both normal and transformed B lymphocytes as a consequence of signals generated through receptor Ig, and 2) up-regulation of c-fos expression is not positively linked to B cell proliferation but rather appears to be a component of the surface Ig signal transduction mechanism. Finally, studies utilizing phorbol diesters suggest that pathways leading through protein kinase C are involved in both the growth inhibition and c-fos expression WEHI-231 following membrane-associated Ig cross-linking.

Animals↗

PubMed1988

Studies of surface immunoglobulin-dependent B cell activation.

Studies from a number of laboratories have firmly established the potential of surface immunoglobulin-generated signals in B lymphocyte activation. While clearly there are multiple ways of activating B lymphocytes, some of which may not involve surface immunoglobulin, it is clear that crosslinking of surface immunoglobulin whether by antigen or antireceptor antibody can generate signals relevant to B cell activation. Although considerable insight into the mechanism of transduction of mIg-generated signals across the plasma membrane has been realized, a molecular explanation for linking inositol phospholipid hydrolysis to changes within the cytoplasm and nucleus of the B cell is still speculative. A more rigorous definition of the PKC and calcium components of the mIg signal transduction pathway are critical for a thorough understanding of the mechanism of signal transduction by this receptor. The use of tumor cell models allowing selection of mutants within the signalling pathway(s) will be invaluable to fully defining the critical molecular and biochemical events involved in B cell activation.

Animals↗