[RadioTransNet, the French network for preclinical research in oncological radiotherapy]. / RadioTransNet, le réseau national de radiothérapie oncologique préclinique.

The ambition of the RADIOTRANSNET network, launched by the INCa at the end of 2018, is to create a French research consortium dedicated to preclinical radiotherapy to foster scientific and clinical interactions at the interface of radiotherapy and radiobiology, and to identify research priorities dedicated to innovation in radiotherapy. The activities of the network are organized around four major axes that are target definition, normal tissue, combined treatments and dose modelling. Under the supervision of the Scientific Council, headed by a coordinator designated by the SFRO and a co-coordinator designated by the SFPM, three leaders coordinate each axis: a radiation-oncologist, a medical physicist and a biologist, who are responsible for organizing a scientific meeting based on the consensus conference methodology to identify priority issues. The selected themes will be the basis for the establishment of a strategic research agenda and a roadmap to help coordinate national basic and translational research efforts in oncological radiotherapy. This work will be published and will be transmitted to the funding institutions and bodies with the aim of opening dedicated calls to finance the necessary human and technical resources. Structuration of a preclinical research network will allow coordinating the efforts of all the actors in the field and thus promoting innovation in radiotherapy.

Design and functionalities of the MADOR® software suite for dose-reduction management after DTPA therapy.

A software suite on biokinetics of radionuclides and internal dosimetry intended for the occupational health practitioners of nuclear industry and for expert opinions has been developed under Borland C++ Builder™. These computing tools allow physicians to improve the dosimetric follow-up of workers in agreement with the French regulations and to manage new internal contaminations by radionuclides such as Pu and/or Am after diethylene triamine penta-acetic acid treatments. In this paper, the concept and functionalities of the first two computing tools of this MADOR(®) suite are described. The release 0.0 is the forensic application, which allows calculating the derived recording levels for intake by inhalation or ingestion of the main radioisotopes encountered in occupational environment. Indeed, these reference values of activity are convenient to interpret rapidly the bioassay measurements and make decisions as part of medical monitoring. The release 1.0 addresses the effect of DTPA treatments on Pu/Am biokinetics and the dose benefit. The forensic results of the MADOR(®) suite were validated by comparison with reference data.

Expression of CD34 and CD7 on human T-cell acute lymphoblastic leukemia discriminates functionally heterogeneous cell populations.

Leukemia-initiating/repopulating cells (LICs), also named leukemic stem cells, are responsible for propagating human acute leukemia. Although they have been characterized in various leukemias, their role in T-cell acute lymphoblastic leukemia (T-ALL) is unclear. To identify and characterize LICs in T-ALL (T-LIC), we fractionated peripheral blood cell populations from patient samples by flow cytometry into three cell fractions by using two markers: CD34 (a marker of immature cells and LICs) and CD7 (a marker of early T-cell differentiation). We tested these populations in both in vitro culture assays and in vivo for growth and leukemia development in immune-deficient mice. We found LIC activity in CD7(+) cells only as CD34(+)CD7(-) cells contained normal human progenitors and hematopoietic stem cells that differentiated into T, B lymphoid and myeloid cells. In contrast, CD34(+)CD7(+) cells were enriched in LICs, when compared with CD34(-)CD7(+) cells. These CD34(+)CD7(+) cells also proliferated more upon NOTCH activation than CD34(-)CD7(+) cells and were sensitive to dexamethasone and NOTCH inhibitors. These data show that CD34 and CD7 expression in human T-ALL samples help in discriminating heterogeneous cell populations endowed with different LIC activity, proliferation capacity and responses to drugs.

Metabolomic analysis of normal and sickle cell erythrocytes.

Metabolic signatures of specialized circulating hematopoietic cells in physiological or human hematological diseases start to be described. We use a simple and highly reproductive extraction method of erythrocytes metabolites coupled with a liquid chromatography-mass spectrometry based metabolites profiling method to determine metabolomes of normal and sickle cell erythrocytes. Sickle cell erythrocytes and normal erythrocytes metabolomes display major differences in glycolysis, in glutathione, in ascorbate metabolisms and in metabolites associated to membranes turnover. In addition, the amounts of metabolites derived from urea cycle and NO metabolism that partly take place within erythrocyte were different between normal and sickle cell erythrocytes. These results show that metabolic profiling of red blood cell diseases can now be determined and might indicate new biomarkers that can be used for the follow-up of sickle cell patients.

Resveratrol, a natural dietary phytoalexin, possesses similar properties to hydroxyurea towards erythroid differentiation.

Resveratrol, a natural dietary polyphenol, has been postulated to be implicated in the cardioprotective effect of red wine and the low incidence of breast and prostate cancers among vegetarians and Orientals respectively. This compound inhibits ribonucleotide reductase as does hydroxyurea, the first therapeutic agent used in the treatment of sickle cell disease. Using the human erythroleukaemic K562 cell line as an in vitro model, we show here that 50 micromol/l of resveratrol induced a higher haemoglobin production (sevenfold) in K562 cells than 500 micromol/l of hydroxyurea (3.5-fold). This erythroid differentiation was linked to a dose- and time-dependent inhibition of cell proliferation associated with an equivalent increased expression of p21 mRNA, but with a higher increased level of p21 protein (sixfold) for cells treated with resveratrol than for those treated with hydroxyurea (1.5-fold). We also show that 50 micromol/l of resveratrol and 25 micromol/l of hydroxyurea induced variable but similar enhancements of fetal haemoglobin synthesis in cultured erythroid progenitors for the majority of the sickle cell patients studied. These inductions were linked to, but not correlated with, a variable decrease in erythroid burst-forming unit clone number. Taken together, these results show that resveratrol merits further investigations in sickle cell disease therapy.

Erythroblasts are a source of angiogenic factors.

In adult bone marrow, mature erythroblasts are produced within structures called erythroblastic islands and then cross the endothelial barrier to reach circulation. Erythroblastic islands are composed of a central macrophage surrounded by maturing erythroblasts. In this study, it is shown that erythroid cells, but not the other mature hematopoietic cells, coexpress 2 angiogenic factors, vascular endothelial growth factor A (VEGF-A) and placenta growth factor (PlGF). Secretion of both VEGF-A and PlGF increases during in vitro erythroid differentiation. Erythroblast-conditioned medium can induce both migration of monocytes and endothelial cells and the permeability of endothelial cells. These effects are inhibited by anti-PlGF and/or anti-VEGF antibodies. Finally, it is shown that VEGF-A and PlGF proteins are expressed by bone marrow erythroblasts in vivo. Angiogenic factors secreted by erythroblasts may promote interactions either with macrophages in erythroblastic islands or with endothelial cells that would facilitate the passage of erythroid cells through the endothelial barrier. (Blood. 2001;97:1968-1974)

Polydom: a secreted protein with pentraxin, complement control protein, epidermal growth factor and von Willebrand factor A domains.

To identify extracellular proteins with epidermal growth factor (EGF) domains that are potentially involved in the control of haemopoiesis, we performed degenerate reverse-transcriptase-mediated PCR on the murine bone-marrow stromal cell line MS-5 and isolated a new partial cDNA encoding EGF-like domains related to those in the Notch proteins. Cloning and sequencing of the full-length cDNA showed that it encoded a new extracellular multi-domain protein that we named polydom. This 387 kDa mosaic protein contained a signal peptide followed by a new association of eight different protein domains, including a pentraxin domain and a von Willebrand factor type A domain, ten EGF domains, and 34 complement control protein modules. The human polydom mRNA is strongly expressed in placenta, its expression in the other tissues being weak or undetectable. The particular multidomain structure of the encoded protein suggests an important biological role in cellular adhesion and/or in the immune system.

Impaired antibody affinity maturation process characterizes a subset of patients with common variable immunodeficiency.

Common variable immunodeficiency (CVID) is an heterogeneous syndrome characterized by decreased levels of serum Ig and recurrent bacterial infection. Here, we were interested to study whether a qualitative defect of the affinity Ab maturation process could be combined to the low level of serum Ig in a cohort of 38 CVID patients. For this, we designed a novel and rapid screening test for the detection of hypomutated V gene expressed by memory B cells. This test delineated a subset of 9/38 (23%) CVID patients with an abnormal pattern of Ig V gene mutation. The mean frequency of V gene mutation of this subset was significantly lower (1.74%) compared with other CVID patients (5.46%) and normal donors (6.5%) (p<0.0001). The mean age of this subgroup was significantly higher than other hypogammaglobulinemic patients with normal levels of V gene mutation (p<0.02), whereas no difference in the duration of symptoms was noted between the two groups. This suggests that hypomutation characterizes patients who began CVID late in life. Recently, it was shown that non-Ig sequences, such as the intronic BCL-6 gene, could be the target of the somatic hypermutation process in normal memory B cells. Our finding of a normal mutation frequency of the BCL-6 gene in two hypomutated CVID point to a defect of the Ig targeting of hypermutation machinery in these cases.

Molecular characterization of a novel gene family (PHTF) conserved from Drosophila to mammals.

PHTF1 (putative homeodomain transcriptional factor; HGMW-approved symbol PHTF1) is a putative homeobox gene located at band 1p11-p13 of the human genome. We report here the cloning and sequencing of its mouse and Drosophila orthologs. The conservation between mouse and human proteins extends over the entire protein and is localized at the putative homeodomain and at the N- and C-terminal regions of Drosophila protein sequence. Blast searches allowed us to identify another member of the PHTF family, PHTF2, located at 7q11.23-q21 of the human genome. The strongest homologies between human PHTF1 and PHTF2 are localized to the domains that we already described in Drosophila, i.e., the putative homeodomain and the N- and C-terminal regions. The human and mouse genes display 98% similarity to one another, 56% similarity with the Drosophila gene, and 67% similarity with PHTF2, suggesting that phtf might define a novel gene family of highly divergent homeobox genes. Finally, Northern blot analysis showed that while PHTF1 is expressed mainly in testis, PHTF2 is predominantly expressed in muscle, suggesting that these two genes may have acquired different functions after their duplication and divergence.

The MAL gene is expressed in primary mediastinal large B-cell lymphoma.

Primary mediastinal large B-cell lymphoma (PMBL) appears to be a distinct clinicopathologic entity among diffuse large B-cell lymphomas (DLBLs). To find molecular alterations associated with this disease, we compared the mRNAs expressed in 3 PMBLs and 3 peripheral DLBLs by differential display-reverse transcription (DDRT) and identified a mRNA specifically expressed in PMBLs. Sequence analysis showed that this mRNA is encoded by the MAL gene, the expression of which was shown to be restricted to the T-cell lineage during hematopoiesis. MAL gene expression was demonstrated by Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) in 8 of 12 PMBLs. However, there was little or no MAL gene expression in 8 peripheral DLBLs. Immunohistochemical analysis evidenced expression of MAL protein in tumoral B cells restricted to the PMBL subtype. Finally, Southern blot studies did not demonstrate rearrangement of the MAL gene. Altogether, our results indicate that MAL expression is recurrent in PMBLs, providing further evidence that PMBL represents a distinct entity among DLBLs. Because MAL protein is located in detergent-insoluble glycolipid-enriched membrane (GEM) domains involved in lymphocyte signal transduction, abnormal expression of MAL protein in the B-lymphoid lineage may have significant implications in PMBL lymphomagenesis.

Neuropilin-1 is expressed on bone marrow stromal cells: a novel interaction with hematopoietic cells?

In adult bone marrow, hematopoietic stem cells are found in close association with distinctive stromal cell elements. This association is necessary for maintenance of hematopoiesis, but the precise mechanisms underlying the cross-talk between stromal cells and hematopoietic stem cells are poorly understood. In this study, we used a bone marrow stromal cell line (MS-5) that is able to support human long-term hematopoiesis. This hematopoietic-promoting activity cannot be related to expression of known cytokines and is abolished by addition of hydrocortisone. Using a gene trap strategy that selects genes encoding transmembrane or secreted proteins expressed by MS-5 cells, we obtained several insertions that produced fusion proteins. In one clone, fusion protein activity was downregulated in the presence of hydrocortisone, and we show that insertion of the trap vector has occurred into the neuropilin-1 gene. Neuropilin-1 is expressed in MS-5 cells, in other hematopoietic-supporting cell lines, and in primary stromal cells but not in primitive hematopoietic cells. We show that neuropilin-1 acts as a functional cell-surface receptor in MS-5 cells. Two neuropilin-1 ligands, semaphorin III and VEGF 165, can bind to these cells, and the addition of VEGF 165 to MS-5 cells increases expression of 2 cytokines known to regulate early hematopoiesis, Tpo and Flt3-L. Finally, we show that stromal cells and immature hematopoietic cells express different neuropilin-1 ligands. We propose that neuropilin-1 may act as a novel receptor on stromal cells by mediating interactions between stroma and primitive hematopoietic cells.

Regulation of embryonic/fetal globin genes by nuclear hormone receptors: a novel perspective on hemoglobin switching.

The CCAAT box is one of the conserved motifs found in globin promoters. It binds the CP1 protein. We noticed that the CCAAT-box region of embryonic/fetal, but not adult, globin promoters also contains one or two direct repeats of a short motif analogous to DR-1 binding sites for non-steroid nuclear hormone receptors. We show that a complex previously named NF-E3 binds to these repeats. In transgenic mice, destruction of the CCAAT motif within the human epsilon-globin promoter leads to substantial reduction in epsilon expression in embryonic erythroid cells, indicating that CP1 activates epsilon expression; in contrast, destruction of the DR-1 elements yields striking epsilon expression in definitive erythropoiesis, indicating that the NF-E3 complex acts as a developmental repressor of the epsilon gene. We also show that NF-E3 is immunologically related to COUP-TF orphan nuclear receptors. One of these, COUP-TF II, is expressed in embryonic/fetal erythroid cell lines, murine yolk sac, intra-embryonic splanchnopleura and fetal liver. In addition, the structure and abundance of NF-E3/COUP-TF complexes vary during fetal liver development. These results elucidate the structure as well as the role of NF-E3 in globin gene expression and provide evidence that nuclear hormone receptors are involved in the control of globin gene switching.

T-cell expression of the human GATA-3 gene is regulated by a non-lineage-specific silencer.

The GATA-3 transcription factor is required for development of the T-cell lineage and Th2 cytokine gene expression in CD4 T-cells. We have mapped the DNase-I-hypersensitive (HS) regions of the human GATA-3 gene in T-cells and non-T-cells and studied their transcriptional activities. HS I-III, located 5' from the transcriptional initiation site, were found in hematopoietic and non-hematopoietic cells, whereas HS IV-VII, located 3' from the transcriptional start site, were exclusively observed in T-cells. Among these hypersensitive sites, two transcriptional control elements were found, one in the first intron of the GATA-3 gene and the other between 8.3 and 5.9 kilobases 5' from the GATA-3 transcriptional initiation site. The first intron acted as a strong transcriptional activator in a position-dependent manner and with no cell-type specificity. The upstream regulatory element could confer T-cell specificity to the GATA-3 promoter activity, and analysis of this region revealed a 707-base pair silencer that drastically inhibited GATA-3 promoter activity in non-T-cells. Two CAGGTG E-boxes, located at the 5'- and 3'-ends of the silencer, were necessary for this silencer activity. The 3'-CAGGTG E-box could bind USF proteins, the ubiquitous repressor ZEB, or the basic helix-loop-helix proteins E2A and HEB, and we showed that a competition between ZEB and E2A/HEB proteins is involved in the silencer activity.

Molecular identity of hematopoietic precursor cells emerging in the human embryo.

It is now accepted from studies in animal models that hematopoietic stem cells emerge in the para-aortic mesoderm-derived aorta-gonad-mesonephros region of the vertebrate embryo. We have previously identified the equivalent primitive hematogenous territory in the 4- to 6-week human embryo, under the form of CD34(+)CD45(+)Lin- high proliferative potential hematopoietic cells clustered on the ventral endothelium of the aorta. To characterize molecules involved in initial stem cell emergence, we first investigated the expression in that territory of known early hematopoietic regulators. We herein show that aorta-associated CD34(+) cells coexpress the tal-1/SCL, c-myb, GATA-2, GATA-3, c-kit, and flk-1/KDR genes, as do embryonic and fetal hematopoietic progenitors later present in the liver and bone marrow. Next, CD34(+)CD45(+) aorta-associated cells were sorted by flow cytometry from a 5-week embryo and a cDNA library was constructed therefrom. Differential screening of that library with total cDNA probes obtained from CD34(+) embryonic liver cells allowed the isolation of a kinase-related sequence previously identified in KG-1 cells. In addition to emerging blood stem cells, KG-1 kinase is also strikingly expressed in all developing endothelial cells in the yolk sac and embryo, which suggests its involvement in the genesis of both hematopoietic and vascular cell lineages in humans.

Identification of a novel cDNA, encoding a cytoskeletal associated protein, differentially expressed in diffuse large B cell lymphomas.

Diffuse large B-cell lymphomas (DLBL) constitute an heterogeneous clinico-pathological entity. To characterize molecular events related to histological subtypes, clinical presentation or outcome, we compared the mRNAs expressed in a limited series of DLBL by Differential display-reverse transcription (DDRT) and cloned a differential cDNA, that we called LB1. LB1 open reading frame encodes a 683 amino-acid polypeptide that does not show significant homology upon comparison to protein databases, nor any structural domain relating LB1 to an already known protein family. Immunofluorescence analysis of transfected COS cells showed a cytoplasmic filamentous staining, indicating that LB1 protein is tightly associated with cytoskeletal fibers. Two LB1 transcripts, a major 3.6-3.9 Kb and a minor 2.2 Kb transcripts, were detected among human haematopoietic and non-haematopoietic lines and tissues. LB1 transcripts were abundant in testis, thymus and in tumour derived cell lines, while barely detectable in liver, prostate and kidney. Concerning DLBL, LB1 expression was high in two cases of DLBL, and low or undetectable in four others, confirming the differential expression previously observed in the DDRT experiment. Furthermore, LB1 gene mapped to chromosome 13q14, a region that has been involved as a chromosomal breakpoint in DLBL. The cellular function of LB1 and its relationship with B cell maturation and/or oncogenesis remain to be established.

Chromatin immunoselection defines a TAL-1 target gene.

Despite the major functions of the basic helix-loop-helix transcription factor TAL-1 in hematopoiesis and T-cell leukemogenesis, no TAL-1 target gene has been identified. Using immunoprecipitation of genomic fragments bound to TAL-1 in the chromatin of murine erythro-leukemia (MEL) cells, we found that 10% of the immunoselected fragments contained a CAGATG or a CAGGTG E-box, followed by a GATA site. We studied one of these fragments containing two E-boxes, CAGATG and CAGGTC, followed by a GATA motif, and showed that TAL-1 binds to the CAGGTG E-box with an affinity modulated by the CAGATG or the GATA site, and that the CAGGTG-GATA motif exhibits positive transcriptional activity in MEL but not in HeLa cells. This immunoselected sequence is located within an intron of a new gene co-expressed with TAL-1 in endothelial and erythroid cells, but not expressed in fibroblasts or adult liver where no TAL-1 mRNA was detected. Finally, in vitro differentiation of embryonic stem cells towards the erythro/megakaryocytic pathways showed that the TAL-1 target gene expression followed TAL-1 and GATA-1 expression. These results establish that TAL-1 is likely to activate its target genes through a complex that binds an E-box-GATA motif and define the first gene regulated by TAL-1.

Transcription factor SCL is required for c-kit expression and c-Kit function in hemopoietic cells.

In normal hemopoietic cells that are dependent on specific growth factors for cell survival, the expression of the basic helix-loop-helix transcription factor SCL/Tal1 correlates with that of c-Kit, the receptor for Steel factor (SF) or stem cell factor. To address the possibility that SCL may function upstream of c-kit, we sought to modulate endogenous SCL function in the CD34(+) hemopoietic cell line TF-1, which requires SF, granulocyte/macrophage colony-stimulating factor, or interleukin 3 for survival. Ectopic expression of an antisense SCL cDNA (as-SCL) or a dominant negative SCL (dn-SCL) in these cells impaired SCL DNA binding activity, and prevented the suppression of apoptosis by SF only, indicating that SCL is required for c-Kit-dependent cell survival. Consistent with the lack of response to SF, the level of c-kit mRNA and c-Kit protein was significantly and specifically reduced in as-SCL- or dn-SCL- expressing cells. c-kit mRNA, c-kit promoter activity, and the response to SF were rescued by SCL overexpression in the antisense or dn-SCL transfectants. Furthermore, ectopic c-kit expression in as-SCL transfectants is sufficient to restore cell survival in response to SF. Finally, enforced SCL in the pro-B cell line Ba/F3, which is both SCL and c-kit negative is sufficient to induce c-Kit and SF responsiveness. Together, these results indicate that c-kit, a gene that is essential for the survival of primitive hemopoietic cells, is a downstream target of the transcription factor SCL.

p45 NF-E2 regulates expression of thromboxane synthase in megakaryocytes.

Transcription factor p45 NF-E2 is highly expressed in the erythroid and megakaryocytic lineages. Although p45 recognizes regulatory regions of several erythroid genes, mice deficient for this protein display only mild dyserythropoiesis but have abnormal megakaryocytes and lack circulating platelets. A number of megakaryocytic marker genes have been extensively studied, but none of them is regulated by NF-E2. To find target genes for p45 NF-E2 in megakaryopoiesis, we used an in vivo immunoselection assay: genomic fragments bound to p45 NF-E2 in the chromatin of a megakaryocytic cell line were immunoprecipitated with an anti-p45 antiserum and cloned. One of these fragments belongs to the second intron of the thromboxane synthase gene (TXS). We demonstrate that the TXS gene, which is mainly expressed in megakaryocytes, is indeed directly regulated by p45 NF-E2. First, its promoter contains a functional NF-E2 binding site; second, the intronic NF-E2 binding site is located within a chromatin-dependent enhancer element; third, p45-null murine megakaryocytes do not express detectable TXS mRNA, although TXS expression can be detected in other cells. These data, and the structure of the TXS promoter and enhancer, suggest that TXS belongs to a distinct subgroup of genes involved in platelet formation and function.

[Genetic control of hematopoiesis]. / Contrôle génétique de l'hématopoïèse.

Commitment and differentiation of hematopoietic stem cells are associated with the progressive restriction of cellular proliferation and the progressive expression of a subset of genes encoding the markers of mature cells. These two processes are genetically regulated and, in this paper, I review the expression and function of the GATA family of transcription factors as an example of this genetic regulation. GATA cis-acting elements are found in most of the regulatory regions of T-lymphoid, erythrocytic and megakaryocytic restricted genes. These GATA motifs are recognized by the members of a family of transcriptional regulators: the GATA family. Three members of this family, GATA-1, 2 and 3 are expressed in hematopoietic cells. They are necessary for the erythrocytic and megakaryocytic lineages (GATA-1), for the T-lymphoid lineage (GATA-3), and for the proliferation of uncommitted hematopoietic precursors (GATA-2). GATA-1 displays at least four functions: activation of the erythrocytic and megakaryocytic specific genes, regulation of the epsilon-->gamma globin switch and control of the cell cycle. These two last functions will be discussed to show the multiple facets of GATA-1 in the genetic regulation of hematopoiesis.