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J Imbert

Publications and source records attributed to J Imbert.

At least 37 records · Page 2Linked to original sources
PubMed1996

Elf-1 and Stat5 bind to a critical element in a new enhancer of the human interleukin-2 receptor alpha gene.

The interleukin 2 receptor alpha-chain (IL-2R alpha) gene is a key regulator of lymphocyte proliferation. IL-2R alpha is rapidly and potently induced in T cells in response to mitogenic stimuli. Interleukin 2 (IL-2) stimulates IL-2R alpha. transcription, thereby amplifying expression of its own high-affinity receptor. IL-2R alpha transcription is at least in part controlled by two positive regulatory regions, PRRI and PRRII. PRRI is an inducible proximal enhancer, located between nucleotides -276 and -244, which contains NF-kappaB and SRE/CArG motifs. PRRII is a T-cell-specific enhancer, located between nucleotides -137 and -64, which binds the T-cell-specific Ets protein Elf-1 and HMG-I(Y) proteins. However, none of these proximal regions account for the induction of IL-2R alpha transcription by IL-2. To find new regulatory regions of the IL-2R alpha gene, 8.5 kb of the 5' end noncoding sequence of the IL-2R alpha gene have been sequenced. We identified an 86-nucleotide fragment that is 90% identical to the recently characterized murine IL-2-responsive element (mIL-2rE). This putative human IL-2rE, designated PRRIII, confers IL-2 responsiveness on a heterologous promoter. PRRIII contains a Stat protein binding site that overlaps with an EBS motif (GASd/EBSd). These are essential for IL-2 inducibility of PRRIII/CAT reporter constructs. IL-2 induced the binding of Stat5a and b proteins to the human GASd element. To confirm the physiological relevance of these findings, we carried out in vivo footprinting experiments which showed that stimulation of IL-2R alpha expression correlated with occupancy of the GASd element. Our data demonstrate a major role of the GASd/EBSd element in IL-2R alpha regulation and suggest that the T-cell-specific Elf-1 factor can serve as a transcriptional repressor.

Animals↗
PubMed1996

Glutamine synthetase induction by glucocorticoids is preserved in skeletal muscle of aged rats.

Glutamine synthetase (GS) is a glucocorticoid-inducible enzyme that has a key role for glutamine synthesis in muscle. We hypothesized that the glucocorticoid induction of GS could be altered in aged rats, because alterations in the responsiveness of some genes to glucocorticoids were reported in aging. We compared the glucocorticoid-induced GS in fast-twitch and slow-twitch skeletal muscles (tibialis anterior and soleus, respectively) and heart from adult (age 6-8 mo) and aged (age 22 mo) female rats. All animals received dexamethasone (Dex) in their drinking water (0.77 +/- 0.10 and 0.80 +/- 0.08 mg/day per adult and aged rat, respectively) for 5 days. Dex caused an increase in both GS activity and GS mRNA in fast-twitch and slow-twitch skeletal muscles from adult and aged rats. In contrast, Dex increased GS activity in heart of adult rats, without any concomitant change in GS mRNA levels. Furthermore, Dex did not affect GS activity in aged heart. Thus the responsiveness of GS to an excess of glucocorticoids is preserved in skeletal muscle but not in heart from aged animals.

Aging↗
PubMed1996

Activation of the transcription from the human immunodeficiency virus type 1 (HIV-1) long terminal repeat by autologous and heterologous cell-to-cell contact.

Recent studies have demonstrated that the activation of HIV-1 provirus and of the Long Terminal Repeat of HIV-1 (HIV-1-LTR) may occur upon cell-to-cell contact between peripheral blood lymphocytes (PBLs) and infected or transfected (HT29) human colonic carcinoma cells. Using transient or stable transfections, we ascertained that the HIV-1 LTR was up-regulated by cell-to-cell contact in various cell lines. The degree of cell-to-cell contact responsiveness was cell type dependent. In contrast, in transient transfection, the HIV-1-LTR was strongly induced by Tat expression in all cell types tested. This indicates that there are differences in the induction mechanism for these two stimuli, even though Tat protein has been previously reported to induce cell adhesion. Except for the PBLs/transfected cells interactions, the results also demonstrate that the cell-to-cell contact-induced HIV-1-LTR activation was highest after contact between autologous as compared to heterologous cells. Previous experiments have shown that, in HT29 cells, cell-to-cell contact activation of the HIV-1-LTR involved the NF-kappa B tandem binding site and activates DNA binding of the nuclear factor NF-kappa B. In this work, we show that in a stably transfected cell line, the principal enhancer element was also involved in the HIV-1-LTR cell-to-cell contact activation. On the other hand, in the HT29 cell line, the NF-kappa B binding appeared to involve the RGD motif of the cell-binding domain, which indicates that a post-integrin receptor event could be implicated.

Cell Communication↗
PubMed1995

In vivo regulation of interleukin-2 receptor alpha gene transcription by the coordinated binding of constitutive and inducible factors in human primary T cells.

IL-2R alpha transcription is developmentally restricted to T cells and physiologically dependent on specific stimuli such as antigen recognition. To analyse the mechanisms used to activate IL-2R alpha transcription as well as those used to block it in non-expressing cells, we determined the protein-DNA interactions at the IL-2R alpha locus in three different cell types using the DMS/LMPCR genomic footprinting method. CD25/IL-2R alpha can be efficiently induced in primary human T cells since approximately 100% express this gene when receiving an appropriate combination of mitogenic stimuli. To understand why IL-2R alpha is not expressed in other haematopoietic cell types, we analysed BJAB B lymphoma cells which do not express the IL-2R alpha gene and contain constitutively active nuclear NF-kappa B. Primary fibroblasts from embryo and adult skin were selected to examine the mechanisms that may be used to keep the IL-2R alpha gene inactive in non-haematopoietic cells. The three main results are: (i) the stable in vivo occupancy of IL-2R alpha kappa B element in resting T cells, most probably by constitutive NF-kappa B p50 homodimer that could impair SRF binding to the flanking SRE/CArG box; (ii) its inducible occupancy by NF-kappa B p50-p65 associated with the binding of an SRE/CArG box DNA-binding factor upon mitogenic stimulation; and (iii) a correlation between the precommitment of T cells to activation and the presence of stable preassembled protein-DNA complexes in contrast with the bare IL-2R alpha locus in non-T cells.

Antineoplastic Agents↗
PubMed1995

Hematological reconstitution and gene therapy: retroviral transfer of the bacterial beta-galactosidase activity into human hematopoietic CD34+ cell populations and into T lymphocytes derived from the peripheral blood.

We report the possibility to transfer marker genes coding for beta-galactosidase activity using retroviral vectors into human peripheral blood CD34+ cells, peripheral blood T-lymphocytes and into the growth factor-dependent human hematopoietic cell line TF-1. Using the MFG-nisLacZ and the FLac vector and various packaging cell lines, we demonstrated retroviral transfer and high expression of a bacterial beta-galactosidase activity induced by the nisLacZ gene or the Sh-ble/LacZ gene. Kinetics of expression of the transgenes were analyzed both in primary cells and cell lines. Absence of cytotoxicity related to the expression of the bacterial beta-galactosidase was assessed in both cell types. These results open interesting prospectives for the use of the beta-galactosidase activity to mark and follow the fate of genetically modified cells isolated from patients prior to reimplantation.

Antigens, CD↗
PubMed1994

Binding of phosphatidylinositol-3-OH kinase to CD28 is required for T-cell signalling.

The engagement of CD28 with its ligand B7.1/CD80 results in potent costimulation of T-cell activation initiated through the CD3/T-cell receptor complex. The biochemical basis of CD28 costimulatory function is poorly understood. The signalling pathways used by CD28 are unlike those used by the CD3/T-cell receptor in that they are resistant to cyclosporin A and independent of changes in cyclic AMP concentrations. These differences suggest that each pathway provides unique biochemical information which is required for T-cell activation. We report here that CD28 becomes tyrosine-phosphorylated following interaction with B7.1/CD80, which induces formation of a complex with phosphatidylinositol-3-OH kinase, mediated by the SH2 domains of the p85 subunit of the kinase. Phosphatidylinositol-3-OH kinase is a heterodimer of this 85K regulatory subunit and a 110K catalytic subunit, and is a common substrate for most receptor tyrosine kinases and some cytokine receptors, binding through its SH2 domain to phosphotyrosine in the motif Tyr-X-X-Met in the CD28 sequence, which is highly conserved between human, mouse and rat and lies in the intracellular domain. We show that CD28 mutants that have their kinase-binding site deleted or the tyrosine at position 173 substituted by phenylalanine do not associate with the kinase after CD28 stimulation and cannot stimulate production of interleukin-2. Our results suggest that phosphatidylinositol-3-OH kinase is critical for signalling by CD28.

Animals↗
PubMed1994

Regions of the CD8 molecule involved in the regulation of CD2-mediated activation.

The CD8 molecule regulates T cell activation mediated via the CD3 T cell receptor and the adhesion molecule CD2. CD8 mAbs have been found to inhibit early (Ca2+ rise) as well as late events (cytotoxicity, proliferation, and lymphokine secretion) mediated via the CD2 pathway. A panel of eight anti-human CD8 mAbs was tested for inhibition of CD2-mediated Ca2+ rise in a cytotoxic T cell clone. The inhibition ranged from 5 to 53% independently of mAb isotype and affinity measured by half saturation binding. We then characterized these mAbs for their reactivity toward three mutants of the human CD8 alpha carrying amino acid sequence changes in the surface-exposed loops homologous to the immunoglobulin CDR1, 2, and 3. The mutations included replacement of the human CD8 alpha CDR1- and CDR2-like loops by the homologous mouse sequences and the insertion of a glycine in the middle of the CDR3-like loop. Thus, five mAbs were found to be affected by the mutation in the CDR2-like loop but not by alterations in the other CDR-like loops. Conversely, the other two mAbs (8E1.7 and B9.8) were affected only by mutations in the CDR1- and CDR3-like loops, respectively. Cross-inhibition experiments were essentially in agreement with these results. Interestingly, all the mAbs directed against the CDR2-like loop were potent inhibitors of CD2-mediated Ca2+ rise, with one exception probably due to poor affinity. Thus, in addition to being a site of interaction with major histocompatibility complex Class I as recent data have indicated, this region of the CD8 alpha subunit may play a role in regulating T cell activation.

Antibodies, Monoclonal↗
PubMed1994

Chromosomal location and expression of the genes coding for Ku p70 and p80 in human cell lines and normal tissues.

Ku protein is a relatively abundant DNA-binding nuclear protein complex composed of two polypeptide subunits, p70 and p80. Ku has been recently identified as the regulatory component of the DNA-dependent protein kinase that phosphorylates RNA polymerase II. To further characterize in vivo regulation of Ku protein, we studied the expression of the transcripts coding for the Ku p70 and p80 subunits in different human cell lines and normal tissues by Northern blot hybridization, using specific cDNA probes. The expression level of both genes was approximately 10-fold higher in established cell lines than in normal tissues. However, mRNA expression levels in permanent cell lines correlated more strongly with their proliferative state than with their level of malignant transformation. In purified T lymphocytes induced to proliferate by the combined action of monoclonal antibodies directed against the CD2 and CD28 adhesion molecules, Ku p70 and p80 mRNA steady-state levels increased as soon as 6 h after activation and lasted at least 72 h. The human genes coding for the Ku p70 and p80 subunits were localized by cytogenetic mapping, using fluorescence in situ hybridization, to 22q13 and 2q33-->q35, respectively.

Antigens, Nuclear↗
PubMed1994

Highly efficient retroviral gene transfer into human primary T lymphocytes derived from peripheral blood.

T lymphocytes are a promising cell vehicle for gene therapy purposes. By cocultivating retroviral vector producing cells and target cells, highly efficient gene transfer was achieved with activated human T lymphocytes derived from peripheral blood with vectors carrying different forms of the bacterial beta-galactosidase gene including the regular LacZ gene, the Sh-ble::LacZ gene and the nlsLacZ gene. Infection kinetics of T cells activated by a combination of monoclonal antibodies directed against CD2 and CD28 indicated that the highest efficiencies of transduction were obtained when the cocultivation began 4 days after stimulation. In fact, with the FLac vector, a new retroviral vector which expresses the Sh-ble::LacZ gene, we observed up to 78% transduction efficiency assessed by X-gal staining performed 2 days after the end of the cocultivation. Expression of the transduced genes was observed throughout the period of culture. Neither the cocultivation step nor the expression of the transduced Sh-ble::LacZ gene altered cell culture proliferation or the expression of selected cell surface antigens. In addition, we showed that CD4+ and CD8+ T cells were equally transduced.

Antibodies, Monoclonal↗
PubMed1993

The role of NF-kappa B1 (p50/p105) gene expression in activation of human blood T-lymphocytes via CD2 and CD28 adhesion molecules.

Stimulation of primary human T-lymphocytes via CD2 and CD28 adhesion molecules induces a long-lasting proliferation (> 3 weeks). This potent activation does not require accessory cells, such as monocytes, but depends on persistent interleukin 2 (IL-2) secretion and receptivity, which is associated with high and prolonged expression of the inducible CD25/IL-2 receptor alpha (IL-2R alpha) chain gene. The transcription factor NF-kappa B participates in the regulation of both IL-2 and IL-2R alpha genes, as well as multiple cellular genes involved in T-cell proliferation. To evaluate the role of NF-kappa B in human peripheral blood T-lymphocytes, we previously analyzed the activation of NF-kappa B-related complexes in response to CD2+CD28 costimulation. We demonstrated a long-term induction of p50/p65 heterodimer, a putative p65/c-Rel heterodimer, and a constitutive nuclear expression of KBF1/p50 homodimers. As the role of p50 remains unclear, we focused our present study on NF-kappa B1 (p50/p105) gene regulation. Using electrophoretic mobility shift assays and Western and Northern blot analyses, we studied NF-kappa B1 gene expression during T-cell stimulation via CD2+CD28. We observed a transient 4- to 5-fold increase of NF-kappa B1 gene expression at both the mRNA and protein levels, lasting for at least 24 h. p50 DNA-binding activity apparently stays highly controlled when p105 expression is enhanced by a physiological stimulus of peripheral blood T-cells. Partial inhibition of p50 and p105 expression by NF-kappa B1 antisense oligonucleotides significantly reduced T-cell proliferation and CD25/IL-2R alpha cell surface expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens, CD↗
PubMed1993

Activation of primary human T-lymphocytes through CD2 plus CD28 adhesion molecules induces long-term nuclear expression of NF-kappa B.

Stimulation of highly purified human T-cells via CD2 and CD28 adhesion molecules induces and maintains proliferation for more than 3 weeks. This potent interleukin 2 (IL-2)-dependent activation does not require monocytes or accessory cells. Long-lasting IL-2 receptivity is associated with high-level expression of the inducible IL-2 receptor alpha chain (IL-2R alpha) gene that is regulated at both transcriptional and posttranscriptional levels. Increase of IL-2R alpha gene transcription involves the enhanced binding of the transcription factor NF-kappa B to its consensus sequence in the 5'-regulatory region of the IL-2R alpha gene. To dissect the molecular basis for the unusually persistent transcription of the IL-2R alpha gene, we analyzed nuclear NF-kappa B binding to a radiolabeled IL-2R alpha kappa B-specific oligonucleotide probe during the time course of CD2 + CD28 activation. Resting T-cell nuclear extracts contained KBF1/p50 homodimer. After stimulation, two new kappa B-specific complexes were identified as NF-kappa B p50-p65 heterodimer and putative c-Rel homodimer or c-Rel-p65 heterodimer. Both inducible complexes persisted for at least 3 weeks. Their relative levels were very similar for the duration of proliferation. In parallel, CD2 + CD28 activation triggered a significant intracellular thiol decrease, suggesting that oxygen radicals are involved in the signaling pathway of adhesion molecules. Finally, micromolar amounts of pyrrolidine dithiocarbamate, an oxygen radical scavenger that efficiently blocked the nuclear appearance of NF-kappa B in T-lymphocytes, also inhibited IL-2 secretion, IL-2R alpha cell surface expression, and T-cell proliferation. Together, these results suggest that NF-kappa B plays an important role in long-term activation of human primary T-lymphocytes via CD2 + CD28.

Antibodies, Monoclonal↗
PubMed1989

Transcription factor MBF-I interacts with metal regulatory elements of higher eucaryotic metallothionein genes.

Metallothionein (MT) gene promoters in higher eucaryotes contain multiple metal regulatory elements (MREs) that are responsible for the metal induction of MT gene transcription. We identified and purified to near homogeneity a 74-kilodalton mouse nuclear protein that specifically binds to certain MRE sequences. This protein, MBF-I, was purified employing as an affinity reagent a trout MRE that is shown to be functional in mouse cells but which lacks the G+C-rich and SP1-like sequences found in many mammalian MT gene promoters. Using point-mutated MREs, we showed that there is a strong correlation between DNA binding in vitro and MT gene regulation in vivo, suggesting a direct role of MBF-I in MT gene transcription. We also showed that MBF-I can induce MT gene transcription in vitro in a mouse extract and that this stimulation requires zinc.

Animals↗
PubMed1989

[Not Available].

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France↗
PubMed1988

[Not Available].

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France↗
PubMed1987

Localization of the mcf.2 transforming sequence to the X chromosome.

A transforming sequence was identified using co-transfection of DNA from the human mammary carcinoma cell line MCF-7 and of a G418 resistance gene into NIH 3T3 cells, followed by tumor formation in athymic mice. This sequence, named mcf.2, was molecularly cloned. A transforming activity resides in a cosmid clone of 42 kb. mcf.2 did not cross-hybridize with the known oncogenes tested. In situ hybridization localized it on the X chromosome, probably at q27. This localization was confirmed by hybridization to a panel of human--rodent cell line DNAs.

Animals↗