Biomedical subjects

J Marvel

Publications and source records attributed to J Marvel.

At least 19 recordsLinked to original sources

PubMed2006

Overexpression of Sp1 transcription factor induces apoptosis.

Transcription factor Sp1 has recently been shown to be overexpressed in a number of human cancers and its overexpression contributes to malignant transformation. Sp1 regulates the expression of a number of genes participating in multiple aspects of tumorigenesis such as angiogenesis, cell growth and apoptosis resistance. To better understand the role of increased Sp1 levels on apoptosis regulation we have used retroviruses to overexpress this protein in haematopoietic Baf-3 cells and in 3T3 fibroblasts. We have also used inducible expression systems to control ectopic Sp1 levels in different cell types. Surprisingly, Sp1 overexpression on its own induces apoptosis in all the cellular models tested. The apoptotic pathways induced by Sp1 overexpression are cell type specific. Finally, using a truncated form of Sp1, we show that Sp1-induced apoptosis requires its DNA-binding domain. Our results highlight that Sp1 levels in untransformed cells must be tightly regulated as Sp1 overexpression leads to the induction of apoptosis. Our results also suggest that cancer cells overexpressing Sp1 can avoid Sp1-induced apoptosis.

Animals↗

PubMed2003

A1/Bfl-1 expression is restricted to TCR engagement in T lymphocytes.

We analyzed regulation of the prosurvival Bcl-2 homologue A1, following T-cell receptor (TCR) or cytokine receptor engagement. Activation of CD4(+) or CD8(+) T cells by antigenic peptides induced an early but transient IL-2-independent expression of A1 and Bcl-xl mRNA and proteins, whereas expression of Bcl-2 was delayed and required IL-2. Cytokines such as IL-2, IL-4, IL-7 or IL-15 prevented apoptosis of activated T cells that effect being associated with the maintenance of Bcl-2, but not of A1 expression. However, restimulation of activated or posteffector T cells with antigenic peptide strongly upregulated A1 mRNA and maintained A1 protein expression. IL-4, IL-7 or IL-15 also prevented cell death of naive T cells. In those cells, cytokines upregulated Bcl-2, but not A1 expression. Therefore, in naive, activated and posteffector T cells, expression of A1 is dependent on TCR but not on cytokine receptor engagement, indicating that A1 is differently regulated from Bcl-xl and Bcl-2.

Animals↗

PubMed2002

Decreased glycolytic metabolism contributes to but is not the inducer of apoptosis following IL-3-starvation.

IL-3 regulates the glycolytic pathway. In Baf-3 cells IL-3 starvation leads to a decrease in glucose uptake and in lactate production. To determine if there is a link between the decreased metabolism induced by growth factor-starvation and the induction of cell death, we have compared the cell death characteristics and the metabolic modifications induced by IL-3-deprivation or glucose-deprivation in Baf-3 cells. We show that in both conditions cells die by an apoptotic process which involves the activation of similar Caspases. Different metabolic parameters (i.e. intracellular ATP levels and lactate accumulation in the culture medium) were measured. We show that IL-3 deprivation leads to a partial decrease in lactate production in contrast to glucose deprivation that completely inhibits lactate production. Similarly following IL-3-starvation a significant drop in the intracellular ATP levels in live cells is observed only after 16 h when a large fraction, more than 50 per cent of cells, is already apoptotic. On the contrary, glucose deprivation is followed by an abrupt decrease in ATP levels in the first 2 h of treatment. However, in the presence of IL-3, cells are able to survive for an extended time in these conditions since 70% of cells survived with low ATP levels for up to 16 h. This was not due to partial inhibition of the apoptotic process by the low level of ATP as glucose-deprivation in the absence of IL-3 led to faster death kinetics of Baf-3 cells compared with IL-3 starvation only. These results indicate that the drop in ATP levels and the triggering of apoptosis can be dissociated in time and that when the glycolytic pathway is strongly inhibited, cells are able to survive with relatively low ATP levels if IL-3 is present. Finally we show that induction of bcl-x by IL-3 protects cells from glucose-deprivation induced cell death.

Adenosine Triphosphate↗

PubMed2001

Activation of the phosphatidylinositol 3-kinase/Akt pathway protects against interleukin-3 starvation but not DNA damage-induced apoptosis.

Baf-3 cells are dependent on interleukin-3 (IL-3) for their survival and proliferation in culture. To identify anti-apoptotic pathways, we performed a retroviral-insertion mutagenesis on Baf-3 cells and selected mutants that have acquired a long term survival capacity. The phenotype of one mutant, which does not overexpress bcl-x and proliferates in the absence of IL-3, is described. We show that, in this mutant, Akt is constitutively activated leading to FKHRL1 phosphorylation and constitutive glycolytic activity. This pathway is necessary for the mutant to survive following IL-3 starvation but is not sufficient or necessary to protect cells from DNA damage-induced cell death. Indeed, inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in Baf-3 cells does not prevent the ability of IL-3 to protect cells against gamma-irradiation-induced DNA damage. This protective effect of IL-3 rather correlates with the expression of the anti-apoptotic Bcl-x protein. Taken together, these data demonstrate that the PI3K/Akt pathway is sufficient to protect cells from growth factor starvation-induced apoptosis but is not required for IL-3 inhibition of DNA damage-induced cell death.

Animals↗

PubMed2001

Involvement of inhibitory NKRs in the survival of a subset of memory-phenotype CD8+ T cells.

Inhibitory natural killer receptors (NKRs) such as killer cell immunoglobulin-like receptors (KIRs) in humans and Ly49 molecules in mice are expressed on NK cells and recognize multiple major histocompatibility (MHC) class I proteins. In humans and mice, a subset of CD8+ T cells also expresses NKRs and harbors a memory phenotype. Using mice that are transgenic for KIR2DL3 and its cognate HLA-Cw3 ligand, we show that engagement of inhibitory NKRs selectively drives the in vivo accumulation of a subset of memory-phenotype CD8+ T cells that express the beta chain of the interleukin 2 receptor. In vitro, recognition of MHC class I molecules by inhibitory NKRs on T cells down-regulated activation-induced cell death. These results unveil an MHC class I-dependent pathway that promotes the survival of a subset of memory-phenotype CD8+ T cells and also reveal an unexpected biological function for inhibitory NKRs on T cells.

Animals↗

PubMed2000

Characterization at the single-cell level of naive and primed CD8 T cell cytokine responses.

The aim of this study was to characterize differences between naive and primed CD8 T cells. Our results show that (i) naive and primed CD8 T cells display similar activation thresholds, with no direct evidence for a difference in their TCR signals, and (ii) primed cells differ mainly in their capacity to secrete IFN-gamma. A comparison of the two populations at the single-cell level demonstrated that the increased production of IFN-gamma by the primed cell subset is due to a larger proportion of single cells that are able to synthesize this cytokine early following activation. These results indicate that the intrinsic effector capabilities of individual CD8 T cells expressing the same TCR are heterogeneous and that cells with identical antigen specificity but increased effector capacities are generated or selected during the primary response.

Animals↗

PubMed2000

Effects of T3R alpha 1 and T3R alpha 2 gene deletion on T and B lymphocyte development.

Thyroid hormones bind to several nuclear receptors encoded by T3R alpha and T3R beta genes. There is now accumulating evidence that thyroid hormones act on the immune system. Indeed, mice deficient for thyroid hormones show a reduction in lymphocyte production. However, the mechanisms involved and, in particular, the role of the different thyroid hormone receptors in lymphocyte development have not been investigated. To address that question, we have studied lymphocyte development in mice deficient for the T3R alpha 1 and T3R alpha 2 gene products. A strong decrease in spleen cell numbers was found compared with wild-type littermates, B lymphocytes being more severely affected than T lymphocytes. A significant decrease in splenic macrophage and granulocyte numbers was also found. In bone marrow, a reduction in CD45+/IgM- pro/pre-B cell numbers was found in these mice compared with wild-type littermates. This decrease seems to result from a proliferation defect, as CD45+/IgM- cells incorporate less 5-bromo-2'-deoxyuridine in vivo. To define the origin of the bone marrow development defect, chimeric animals between T3R alpha-/- and Rag1-/- mice were generated. Results indicate that for B cells the control of the population size by T3R alpha 1 and T3R alpha 2 is intrinsic. Altogether, these results show that T3R alpha 1 or T3R alpha 2 gene products are implicated in the control of the B cell pool size.

Animals↗

PubMed1999

Role of PI3-kinase in Bcl-X induction and apoptosis inhibition mediated by IL-3 or IGF-1 in Baf-3 cells.

In Baf-3 cells, IL-3 and IGF-1 both inhibit cell death. These growth factors act at least on two different pathways involved in the inhibition of apoptosis. They both upregulate Bcl-X at the mRNA and protein levels and also activate a pathway which inhibits apoptosis in the absence of protein synthesis. Recently, these two growth factors have been shown to activate the PI3-kinase-AKT pathway which leads to the phosphorylation of the pro-apoptotic Bcl-XL regulator Bad. In this study, we have investigated the role of PI3-kinase in the regulation of Bcl-X expression and in the survival of Baf-3 cells. We show that PI3-kinase activation is involved in the upregulation of Bcl-X mRNA induced by both IL-3 and IGF-1. Moreover, PI3-kinase activity is also necessary for inhibition of apoptosis and caspase regulation by IGF-1 but not IL-3.

Animals↗

PubMed1999

Memory CD44(int) CD8 T cells show increased proliferative responses and IFN-gamma production following antigenic challenge in vitro.

F5 TCR transgenic mice challenged in vivo with peptide generate long-lived primed CD8 T cells that hyper-proliferate in response to peptide in vitro. These primed CD8 T cells can be subdivided into three distinct populations on the basis of CD44 cell surface expression. In this report, we show that among primed CD8 T cells, those expressing intermediate levels of CD44 appear to be true memory T cells by the measurement of a variety of characteristics. Indeed, these cells hyper-proliferate in response to peptide re-stimulation in vitro, and produce IFN-gamma with faster kinetics and at higher levels than naive populations in vitro. We also show that CD8 T cells expressing high levels of CD44 express several activation markers and cycle in vivo in the absence of antigen. However, this population is unable to respond to peptide stimulation in vitro as measured by both proliferation and IFN-gamma secretion. The origin and specificity of these cells is unknown. These results provide evidence that memory CD8 T cells are functionally different from naive CD8 T cells both in terms of proliferation and cytokine secretion. They identify the CD8/CD44(int) T cells as the population responsible for hyper-reactivity in vitro.

Animals↗

PubMed1999

Death by neglect as a deletional mechanism of peripheral tolerance.

In contrast to most organs, the anatomy of the liver may allow naive CD8(+) T cells to make direct contact with liver parenchymal cells. We have previously shown, using a combination of TCR transgenic T cells specific for H-2 K(b) and hepatocytes expressing a transgenic H-2 K(b) molecule, that hepatocytes can induce antigen-specific activation and proliferation of naive CD8(+) T cells independently of CD28 co-stimulation. However, T cell activation by hepatocytes leads to premature T cell death and tolerance, both in vivo and in vitro. In this study, we investigated the mechanisms of T cell death induced by hepatocytes in vitro using primary hepatocytes to activate purified CD8(+) T cells. Neither Fas nor tumor necrosis factor receptor were involved, indicating that hepatocyte- induced death was distinct from activation-induced cell death. Before they started to divide, T cells activated by hepatocytes expressed lower levels of the bcl-x(L) survival gene and 30 times less IL-2 mRNA than CD8(+) cells activated by splenic antigen-presenting cells. Since CD28 co-stimulation increases both IL-2 and bcl-x(L) expression, this suggests that hepatocyte-activated T cells die by neglect because they fail to receive effective co-stimulatory signals. In agreement with this model, premature death promoted by hepatocytes could be prevented by cross-linking CD28. Survival after CD28 cross-linking correlated with increased IL-2 and bcl-x(L) expression, and sustained T cell proliferation, while cytotoxic T lymphocyte activity was prolonged as compared with cells stimulated without CD28 co-stimulation. This study confirms that high- affinity TCR transgenic antigen-specific CD8(+) T cells can be activated to proliferate and differentiate into cytotoxic effector cells. However, prolonged T cell survival and cytotoxicity required CD28 co-stimulation as well. To our knowledge, this is the first report suggesting that tolerance in the context of lack of CD28 co-stimulation can result from Fas-independent peripheral deletion rather than from anergy.

Animals↗

PubMed1998

Tolerant CD8 T cells induced by multiple injections of peptide antigen show impaired TCR signaling and altered proliferative responses in vitro and in vivo.

The mechanisms responsible for peripheral CD8 T cell tolerance to foreign Ags remain poorly understood. In this study we have characterized the state of CD8 T cell tolerance induced in F5 TCR transgenic mice by multiple peptide injections in vivo. The tolerant state of CD8 T cells is characterized by impaired proliferative responses, increased sensitivity to cell death, and failure to acquire cytotoxic effector function after in vitro antigenic challenge. In vivo monitoring of CD8 T cell proliferation using 5-carboxyfluorescein diacetate succinimidyl ester showed that a large subset of the tolerant T cell population failed to divide in response to peptide. TCR down-regulation could not account for this loss of responsiveness to Ag since recombination-activating gene-1 (RAG-1)-/-F5 CD8 T cell responses were similar to those of RAG-1(-/-)F5 x RAG-1(-/-)F1 T lymphocytes, which express lower levels of the transgenic TCR. Analysis of early signal transduction in tolerant CD8 T cells revealed high basal levels of cytoplasmic calcium as well as impaired calcium mobilization and tyrosine phosphorylation after cross-linking of CD3epsilon and CD8alpha. Together these data indicate that repeated exposure to soluble antigenic peptide in vivo can induce a state of functional tolerance characterized by defective TCR signaling, impaired proliferation, and increased sensitivity to cell death.

Animals↗

PubMed1998

Bcl-X is the major pleiotropic anti-apoptotic gene activated by retroviral insertion mutagenesis in an IL-3 dependent bone marrow derived cell line.

In order to identify genes capable of inhibiting apoptosis induced by different pathways, without inducing proliferation we have performed retroviral insertion mutagenesis in the IL-3 dependent bone marrow derived Baf-3 cell line. Out of 200 mutants obtained in three separate mutagenesis experiments, four mutants were resistant to multiple apoptosis inducing pathways (including growth factor starvation, staurosporine, etoposide and cyclosporin A) and did not proliferate in the absence of IL-3. These four mutants overexpress the bcl-X gene following a retroviral insertion 5' of the translation initiation site. These results indicate that the bcl-X gene is a major pleiotropic anti-apoptotic gene in Baf-3 cells. They also suggest that the Bcl-2 family of genes might be the only one capable of inhibiting apoptosis induced by multiple pathways without inducing cell proliferation.

Alleles↗

PubMed1997

In bone marrow derived Baf-3 cells, inhibition of apoptosis by IL-3 is mediated by two independent pathways.

The inhibition of cell death by growth factors plays a key role in the maintenance of the haematopoietic system homeostasis. However the mechanisms involved in this inhibition are still poorly understood. In order to determine if inhibition of apoptosis by growth factors is dependent only on the expression of survival genes, we have studied that process in the bone marrow derived IL-3 dependent cell line Baf-3. We show that, following IL-3 starvation, mRNA and protein levels of Bcl-X but not Bcl-2 decrease rapidly preceeding the onset of death. The death of IL-3 starved cells is asynchronous, starting between 6 to 8 h with 50% death being reached after 10 to 12 h. At any time point, apoptosis can be rapidly inhibited by growth factor re-addition. This has allowed us to determine that the inhibition of apoptosis by growth factor takes place at two levels. The first one, which we have called short term inhibition, is independent of mRNA and protein synthesis i.e. it takes place in the absence of survival gene neosynthesis and can be demonstrated during the first 6 h following growth factor re-addition. The second one corresponds to long-term survival-more than 24 h survival-and is strongly correlated with the induction of Bcl-X but not Bcl-2 gene expression. This induction of Bcl-X by IL-3 is shown to be dependent on MAP-kinase activation.

Animals↗

PubMed1996

Resting memory CD8+ T cells are hyperreactive to antigenic challenge in vitro.

The characteristics of CD8+ T cells responsible for memory responses are still largely unknown. Particularly, it has not been determined whether different activation thresholds distinguish naive from memory CD8+ T cell populations. In most experimental systems, heterogeneous populations of primed CD8+ T cells can be identified in vivo after immunization. These cells differ in terms of cell cycle status, surface phenotype, and/or effector function. This heterogeneity has made it difficult to assess the activation threshold and the relative role of these subpopulations in memory responses. In this study we have used F5 T cell receptor transgenic mice to generate a homogeneous population of primed CD8+ T cells. In the F5 transgenic mice, peptide injection in vivo leads to activation of most peripheral CD8+ T cells. In vivo BrdU labeling has been used to follow primed T cells over time periods spanning several weeks after peptide immunization. Our results show that the majority of primed CD8+ T cells generated in this system are not cycling and express increased levels of CD44 and Ly6C. These cells remain responsive to secondary peptide challenge in vivo as evidenced by short term upregulation of activation markers such as CD69 and CD44. The activation thresholds of naive and primed CD8+ T cells were compared in vitro. We found that CD8+ T cells from primed mice are activated by peptide concentrations 10-50-fold lower than naive mice. In addition, the kinetics of interleukin 2R alpha chain upregulation by primed CD8+ T cells differ from naive CD8+ T cells. These primed hyperresponsive CD8+ T cells might play an important role in the memory response.

Amino Acid Sequence↗

PubMed1996

The product of the v-src-inducible gene nr-13 is a potent anti-apoptotic factor.

Tumorigenesis can be induced either by activating cell proliferation or by inhibiting metabolic pathways regulating programmed cell death (apoptosis). There is evidence suggesting that p60(v-src) and other tyrosine kinases protect cells against apoptosis. This effect could contribute to cell transformation by the Rous sarcoma virus. Mechanism of cell death inhibition by p60(v-src) remains largely unknown. We have recently reported that in avian cells p60(v-src) activates the expression of nr-13, a bcl-2-related gene. In this paper, we demonstrate, using the bone marrow derived cell line Baf-3 as an experimental model, that the product of this avian gene (nr-13) is a potent anti-apoptotic factor. In addition, we report that, in quail neuroretinal cells, nr-13 expression is activated upon infection by the Rous sarcoma virus (RSV) but not by other oncogenic retroviruses like FSV or MH2, suggesting that nr-13 is a specific target of v-src. Activation of nr-13 expression may be a key step in cellular transformation by v-src.

Animals↗

PubMed1996

Altered methional homoeostasis is associated with decreased apoptosis in BAF3 bcl2 murine lymphoid cells.

Methional is a potent inducer of apoptosis in an interleukin 3-dependent murine lymphoid cell line BAF3 b0 when it is added to the culture medium. In these cells transfected with the bcl2 gene, BAF3 bcl2, the apoptotic-inducing activity of methional is dramatically reduced. The addition of disulfiram (an inhibitor of aldehyde dehydrogenase) in order to reduce methional oxidation brought about an increase in apoptosis in BAF3 b0 but not in BAF3 bcl2 cells. In contrast, the addition of quercetin (an inhibitor of aldehyde reductase) in an attempt to diminish methional reduction increased apoptosis in both BAF3 b0 and BAF3 bcl2 cells. The extent of DNA fragmentation in BAF3 bcl2 cells approached that in BAF3 b0 cells in the presence of quercetin and exogenous methional, suggesting a defect in methional biosynthesis in BAF3 bcl2 cells. Direct evidence for this was obtained by measuring labelled methional in cells incubated with the sodium, salt of [U-14C]4-methylthio-2-oxobutanoic acid (MTOB), the precursor of methional. The 80% decrease in labelled methional in BAF3 bcl2 compared with BAF3 b0 cells was accompanied by a concomitant rise in the transamination of [14C]MTOB to [14C]methionine in BAF3 bcl2 cells. Inhibition of the transaminase, however, by a synthetic transition-state-type compound, pyridoxal-L-methionine ethyl ester, induced apoptosis in BAF3 b0 but not in BAF3 bcl2 cells, confirming that the defect in BAF3 bcl2 cells was not in the transaminase itself but rather in the oxidative decarboxylation step MTOB --> methional. In addition, no evidence was obtained for the synthesis of [14C]malondialdehyde from [14C]methional in BAF3 bcl2 cells. As these cells show no deficiency in their content of reactive oxygen species compared with that of BAF3 b0 cells, they may possess some other defect in the beta-hydroxylase enzyme system itself.

Aldehyde Dehydrogenase↗

PubMed1995

Expression in vivo of CD45RA, CD45RB and CD44 on T cell receptor-transgenic CD8+ T cells following immunization.

We used mice transgenic for a major histocompatibility complex class I-restricted T cell receptor to study the changes of phenotype in vivo which follow priming by antigen of CD8 T cells. We show that following priming with peptide, CD44 on CD8 T cells is up-regulated. The change of phenotype was relatively stable, as primed CD8 cells isolated from thymectomized mice 6 weeks after priming still expressed increased levels of CD44. CD8 T cells in these mice are still responsive to peptide and could represent long-lived primed cells. No down-regulation in vivo of the CD45RA or CD45RB isoforms was found, indicating that there is a differential regulation of the expression of CD44 and CD45RB by activated CD8 transgenic T cells. These results contradict earlier studies in vitro which showed that CD8 T cells which have been primed earlier belong to the CD45RA- or CD45RB- subset.

Animals↗

PubMed1994

Dominant interfering alleles define a role for c-Myb in T-cell development.

The transcription activator c-Myb is expressed at high levels in immature thymocytes and during T-cell activation and may be a regulator of T-cell differentiation. To investigate the role of c-Myb in T-cell development, we generated transgenic mice in which two dominant interfering Myb alleles, one a competitive inhibitor of DNA binding, and the other, an active repressor comprising the Myb DNA-binding domain linked to the Drosophila Engrailed transcription repressor domain, were expressed from early times onward in T cells. Both alleles partially blocked thymopoiesis and inhibited proliferation of mature T cells. The Myb-En chimera was the more efficient repressor and might serve as an archetype for the manufacture of other dominant interfering transcription factor alleles.

Alleles↗