Functional analyses of the TEL-ARNT fusion protein underscores a role for oxygen tension in hematopoietic cellular differentiation.

The transcription factor hypoxia inducible factor 1 (HIF1), an HIF1alpha-aryl hydrocarbon receptor nuclear translocator (ARNT) dimeric factor, is essential to the cellular response to hypoxia. We described a t(1;12)(q21;p13) chromosomal translocation in human acute myeloblastic leukemia that involves the translocated Ets leukemia (TEL/ETV6) and the ARNT genes and results in the expression of a TEL-ARNT fusion protein. Functional studies show that TEL-ARNT interacts with HIF1alpha and the complex binds to consensus hypoxia response element. In low oxygen tension conditions, the HIF1alpha/TEL-ARNT complex does not activate transcription but exerts a dominant-negative effect on normal HIF1 activity. Differentiation of normal human CD34+ progenitors cells along all the erythrocytic, megakaryocytic and granulocytic pathways was accelerated in low versus high oxygen tension conditions. Murine 32Dcl3 myeloid cells also show accelerated granulocytic differentiation in low oxygen tension in response to granulocyte colony-stimulating factor. Interestingly, stable expression of the TEL-ARNT in 32Dcl3 subclones resulted in impaired HIF1-mediated transcriptional response and inhibition of differentiation enhancement in hypoxic conditions. Taken together, our results underscore the role of oxygen tension in the modulation of normal hematopoietic differentiation, whose targeting can participate in human malignancies.

Multistep virus-induced leukemogenesis in vitro: description of a model specifying three steps within the myeloblastic malignant process.

A helper-independent Friend leukemia virus was used to infect bone marrow cultures. This virus induces myeloblastic leukemia in mice after a long latency period. Infection of the bone marrow cultures resulted in the in vitro production of myeloblastic leukemogenesis after a long latency period. Three steps were observed in the evolution of the infected cultures, and permanent cell lines were derived at each step. This allowed us to individualize three successive events in the course of the myeloblastic transformation: (i) an abnormal responsiveness to the physiological hormone granulo-macrophagic colony-stimulating factor, (ii) the acquisition of growth autonomy, and (iii) the acquisition of in vivo tumorigenicity.

Monoclonal proliferation of Friend murine leukemia virus-transformed myeloblastic cells occurs early in the leukemogenic process.

Integrated Friend murine leukemia virus copies were analyzed by the Southern blotting procedure in myeloblastic cell lines obtained after in vitro infection of long-term mouse bone marrow cultures. Several steps leading to the generation of malignant myeloblastic cells after a long latency period were observed in the evolution of infected cultures. Shortly after infection, a random distribution of integrated provirus copies was observed in the DNA of normally differentiating myeloid cells. In contrast, a distinct pattern of integrated Friend murine leukemia virus copies was evident in the first non-differentiating immature myeloblastic cells appearing in cultures, suggesting a monoclonal origin of these cells. For each cell line, characteristic hybridizing fragments were conserved during the 1-year culture period necessary for the acquisition of tumorigenic properties and were also observed in tumors grafted in vivo. We can conclude that monoclonality is effective very early in the myeloid transformation process, as soon as the precursor cells are blocked in their differentiation.

Comparative effects of polycythemia-inducing Friend leukemia virus and Moloney leukemia virus on hematopoietic progenitors in murine long-term bone marrow cultures.

The specificity of murine leukemia virus-induced myelomonocytic phenotypic changes in long-term bone marrow culture have been examined by comparing the effects of polycythemia-inducing Friend leukemia virus (FVP) and Moloney murine leukemia virus (M-MuLV) known to have in vivo target cells in the erythroid and lymphoid lineage, respectively. Noninfected adn M-MuLV-infected cultures showed no modification in granulocyte macrophage colony-forming cell behavior and failed to generate cell line in WEHI-3-conditioned medium. In contrast, in FVP-infected cultures, granulocyte macrophage colony-forming cells became colony-stimulating factor independent, and the nonadherent cells gave rise to two cell lines in WEHI-3 conditioned medium with monocytic characteristics and no leukemogenic potential in vivo. These results confirm the ability of long-term bone marrow culture to unmask target cells for FVP within myelomonocytic progenitors, and the negative results in M-MuLV-infected cultures underline the specificity of the FVP-induced phenotypic changes. Despite a high level of virus production and the presence of T-cell precursors in the M-MuLV infected culture, T-cell transformation was not observed.

Alternative splicing of the Evi-1 zinc finger gene generates mRNAs which differ by the number of zinc finger motifs.

Evi-1 is a common integration site for endogenous ecotropic virus in myeloid leukemias of the AKXD mouse strain. This gene encodes a zinc finger protein with ten finger motifs. Myeloblastic leukemias obtained after infection with F-MuLV frequently exhibit proviral integration in the Fim-3 region which is genetically linked to Evi-1. This proviral integration always results in the expression of two mRNA transcripts, 5.7 kb and 4.7 kb long. We isolated two classes of cDNAs from a myeloblastic cell line with a F-MuLV provirus integrated in Fim-3. By sequence analysis, we found that one Evi-1 cDNA (E29) had an internal deletion of 972 nucleotides when compared to the full-length sequence previously published by Morishita et al. (1988). This deletion eliminated the 6th and 7th Evi-1 finger domains and was bordered by consensus donor and acceptor splice sequences. The E29 clone most likely resulted from alternative splicing of the Evi-1 gene. This was confirmed by Northern blot analysis and S1 mapping experiments. Therefore, the Evi-1 gene codes potentially for at least two proteins of 1042 and 718 amino acids differing in the numbers of their zinc-finger motifs.

Role of Gab proteins in phosphatidylinositol 3-kinase activation by thrombopoietin (Tpo).

In this study, we show that upon thrombopoietin (Tpo) stimulation the two adapter proteins Gab1 and Gab2 are strongly tyrosine phosphorylated and associated with Shc, SHP2, PI 3-kinase and Grb2 in mpl-expressing UT7 cells. Although Gab1 and Gab2 seem to mediate overlapping biological signals in many cells, only Gab1 is expressed and phosphorylated in response to Tpo in primary human megakaryocytic progenitors; furthermore, it associates with the same proteins. Although a low level of tyrosine phosphorylated IRS-2 protein is also detected in PI 3-kinase immunoprecipitates, Gab proteins are the essential proteins associated with PI 3-kinase after Tpo stimulation. We demonstrate that, albeit no association is detected between the Tpo receptor mpl and Gab proteins, Y112 located in the C-terminal cytoplasmic domain of mpl is required for Gab1/2 tyrosine phosphorylation. Gab proteins are not tyrosine phosphorylated after Tpo stimulation of UT-7 and Ba/F3 cells expressing a mpl mutant lacking Y112. Moreover, no activation of the PI 3-kinase/Akt pathway is observed in cells expressing this mpl mutant. Finally, we show that this mutant does not allow cell proliferation, thereby confirming that PI 3-kinase activation is required for Tpo-induced cell proliferation.

Characterization of the murine Mpl proto-oncogene, a member of the hematopoietic cytokine receptor family: molecular cloning, chromosomal location and evidence for a function in cell growth.

The v-mpl oncogene transduced in the myeloproliferative leukemia virus (MPLV) encodes a truncated form of a putative receptor protein that belongs to the cytokine receptor superfamily. We previously reported the cloning of complete human c-MPL cDNA. In the present report, we show that the murine Mpl proto-oncogene is located at the D-band of murine chromosome 4, in a region in synteny with human chromosome 1p34, where MPL was previously located. RNA blot analysis of murine hematopoietic tissues and cells lines indicated that Mpl is expressed in immature hematopoietic precursor cells. Molecular cloning of murine proto-oncogene c-Mpl cDNAs is also reported. Two cDNA species were isolated. One potentially encodes a transmembrane protein. The extracellular domain of this protein has two repeats of the cytokine receptor domain common to all members of this receptor family. The cytoplasmic domain has no protein kinase or phosphatase motifs, but does contain a sequence that has been shown to be essential for the transmission of a growth signal in several other members of the family. Comparison of murine and human putative proteins indicated that they shared 81% amino acid identity, the most conserved region being the cytoplasmic domain (91% identity). The other Mpl cDNA clones potentially encode a soluble form of this receptor chain. A chimeric receptor containing the extracellular domain of the granulocyte colony-stimulating factor (G-CSF) receptor fused to the transmembrane and cytoplasmic domains of Mpl was able to induce G-CSF responsiveness when transfected into the interleukin 3 (IL-3)-dependent cell line BAF/BO3. This demonstrated that the cytoplasmic Mpl domain is most probably implicated in proliferative signal transduction.

Rearrangements of the Pim-1, c-myc, and p53 genes in Friend helper virus-induced mouse erythroleukemias.

The Friend helper leukemia virus (F-MuLV) induces in mice leukemias of the erythroid, lymphoid, and myeloblastic lineages. Erythroleukemic cell DNAs were examined for genetic alterations at loci described as common proviral integration regions in MuLV-induced myeloid or lymphoid leukemias or in Friend complex-induced erythroleukemias. No alteration of the Fim-1, Fim-2, Fim-3, pvt-1, and Spi-1 loci were detected in 17 erythroleukemias, p53 gene rearrangement was observed in 6 (30%) erythroleukemias and was always associated with a loss of the germ line allele. Interestingly, genetic alterations were also detected at two loci, c-myc and Pim-1, previously described as common provirus integration regions in T lymphoid leukemias. Rearrangements of these two genes were often associated with p53 gene alteration within the same tumor.

Early involvement of the fim-2 and fim-3 regions in mouse myeloblastic leukemogenesis.

Retroviruses lacking oncogenes induce tumors or leukemias after a long latency which generally exceeds several months. Cellular transformation most probably results from the activation of cellular oncogenes or putative proto-oncogenes due to proviral integration. Several genetic changes are likely to be necessary for the appearance of fully malignant cells. However, the sequence of genetic changes initiating and leading to malignant transformation is difficult to study since, in most experimental conditions, the only accessible cells are fully transformed cells. We have previously described an in vitro model of murine myeloblastic leukemogenesis during which several successive steps leading to fully malignant and transplantable cells have been identified. This in vitro transformation process develops over approximately a 1-year period. In this paper, we demonstrate that frequent cellular DNA rearrangements due to proviral integrations in specific regions occur early in the myeloblastic transformation process and remain stable throughout the in vitro leukemogenesis, and in tumors derived from in vitro fully transformed myeloblasts.

Evi-1 expression in leukemic patients with rearrangements of the 3q25-q28 chromosomal region.

The Evi-1 zinc-finger protein gene is normally not expressed in hematopoietic cells. However, high Evi-1 mRNA expression has been reported in mouse myeloblastic leukemias, due to transcriptional activation by proviral integration in either the Fim-3 or Evi-1 loci. The human Evi-1 gene is located on chromosome 3q24-q28. In this paper three examples are presented of human acute myelogenous leukemias presenting common characteristics: (i) high Evi-1 mRNA expression; (ii) chromosomal abnormalities t(3;3)(q21;q26) or inv(3;3)(q21-22;q26); and (iii) high platelet counts and dystrophic megakaryocytes. Thirty-four other patients with hematological malignancies, among which 11 had chromosomal rearrangements in the 3q24-q28 region did not exhibit such abnormalities. Of the 13 permanent hemopoietic cell lines tested, Evi-1 RNA expression was found in HEL and K-562 cell lines. Weak Evi-1 expression was also seen in fibroblasts and lung cells. This expression was affected neither in skin cells from a patient with a 3q27 constitutional translocation nor in a lung epithelioma cell line containing an excess of chromosome 3 long arm. Ectopic strong expression of Evi-1 in human leukemias could define an uncommon subclass of acute myelogenous leukemias with translocations involving the 3q25-28 chromosomal domain and abnormal megakaryopoiesis.

Intrinsic expression of 20 alpha-hydroxysteroid dehydrogenase in murine myelomonocytic cell lines.

The expression of a 20 alpha-hydroxysteroid dehydrogenase (20 alpha SDH) activity was investigated in various murine hematopoietic cells. Large differences appeared between cells belonging to the same hematopoietic lineage. Normal thymocytes were positive when splenocytes, B and T lymphoma cell lines were negative. In two stromal fibroblastic cell lines, the expression of 20 alpha SDH was very high, whereas it was negative in NIH/3T3 normal fibroblasts. Among myelomonocytic cell lines, six expressed high levels and two were negative. Contaminating lymphoid subpopulations were not detectable in these cell lines. The levels of detected enzymatic activity seemed unrelated with the virus infection. The presence of 20 alpha SDH activity in factor-independent myelomonocytic cell lines, and the lack of induction of the enzymatic activity by the IL-3 containing WEHI-3B conditioned medium in negative cell lines, led us to suggest that myelomonocytic cells intrinsically express the 20 alpha SDH enzymatic activity, as well as other hemopoietic and stromal cell lines.

Transforming growth factor inhibits erythropoiesis by blocking proliferation and accelerating differentiation of erythroid progenitors.

Erythropoiesis is positively regulated by stem cell factor, interleukin 3, and erythropoietin, which synergize to allow the production of hemoglobinized red blood cells from erythroid progenitors. In contrast, interferon gamma, tumor necrosis factor alpha, and transforming growth factor B(1), (TGF-beta(1)) are powerful inhibitors of erythropoiesis. Interferon gamma and alpha act principally by inducing apoptosis. The aim of this study was to elucidate the mechanisms by which TGF-beta(1) inhibits erythropoiesis. We used an in vitro serum-free system of human red blood cell production. From a virtually pure population of CD36(+) erythroid progenitors, stem cell factor, interleukin 3, and erythropoietin allowed massive proliferation (x300) and promoted terminal red blood cell differentiation. We show here that TGF-beta(1) (2 ng/mL) inhibited the growth of CD36(+) cells by 15-fold. TGF-beta(1) markedly accelerated and increased erythroid differentiation as assessed by hemoglobin and glycophorin expression. Furthermore, May-Grünwald-Giemsa staining and ultrastructural analysis revealed that TGF-beta(1) induced full differentiation toward normal enucleated red cells even in the absence of macrophages. This acceleration of erythroid differentiation did not modify the pattern of hemoglobin chains expression from adult or fetal erythroid progenitors. Analysis of apoptosis, cell cycle and Ki-67 expression showed that TGF-beta(1) inhibited cell proliferation by decreasing the cycle of immature erythroid cells and accelerating maturation toward orthochromatic normoblasts that are not in cycle. We showed that TGF-beta(1) is a paradoxical inhibitor of erythropoiesis that acts by blocking proliferation and accelerating differentiation of erythroid progenitors.

Analysis of megakaryocyte growth and development factor (thrombopoietin) effects on blast cell and megakaryocyte growth in myelodysplasia.

Thrombocytopenia is a frequent feature of myelodysplastic syndromes (MDS) that could be improved by the use of recombinant human megakaryocyte growth and development factor (rHuMGDF). Using short-term liquid cultures and progenitor assays, we have found that rHuMGDF stimulated DNA synthesis and potentiated leukemic cluster growth of bone marrow mononuclear cells in 10/38 MDS cases (26%). Cytogenetically malignant colonies were detectable in rHuMGDF-stimulated cultures (n=3) by fluorescence in situ hybridization. rHuMGDF was able to stimulate CFU-MK formation in 45% of the samples tested. Finally, rHuMGDF-induced blast cell proliferation correlated with elevated expression of c-MPL, previously identified as a bad prognosis factor in MDS.

Fluorescence in situ hybridization on methylcellulose cultured hematopoietic stem cells from myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are clonal malignancies characterized by peripheral blood pancytopenia and signs of maturation disturbances of one or several cell lineages in bone marrow. MDS present as chimeras associating normal polyclonal and malignant monoclonal progenitors cells in various proportions. Numerous cytogenetic abnormalities have been reported in MDS and can be detected by fluorescence in situ hybridization (FISH) on interphase cells. We have used this technique on methylcellulose cultured hematopoietic progenitors obtained from three patients suffering from MDS and exhibiting informative karyotypic features. Hematopoietic cells were cultured for 14 days, and individual clones (BFU-E, CFU-GM) were picked up and then cytocentrifuged for FISH analysis. We used centromeric probes realized and labeled in our laboratory by PCR to detect aneuploidies for chromosomes 7 and 11 in two patients. Furthermore, we could detect a 5q partial deletion on interphase cells from the third patient using a 5q31 specific probe visualized with the HNPP Fluorescent Detection Set from Boehringer Mannheim. In conclusion, FISH is a helpful method to detect malignant clones in hematopoietic progenitor cultures and hence to study the relative growth of normal vs. leukemic cells in MDS.

The in vitro autocrine secretion of CSFs alone does not account for the longterm growth of murine myeloid leukemic cells in suspension cultures.

To investigate the mechanisms supporting the in vitro longterm growth of murine leukemic myeloblastic cells, factor-dependent and autonomous myeloblastic cells have been examined for their CSF responsiveness, CSF secretion, and autostimulation of growth. Purified CSF-1, GM-CSF, and IL-3 stimulated cloning and proliferation of both autonomous (ACL) and factor-dependent cell lines (FDCL), and were unable to induce differentiation of these cells. Sensitivities to CSFs were similar for ACL, FDCL and normal bone marrow cells. Most of the cell lines secreted CSFs, stimulating colony formation from normal bone marrow cells in bilayer agar cultures. The number of induced granulo-macrophagic colonies was similar in the presence of either ACL or FDCL. The ability to stimulate their own proliferation was similar for both ACL and FDCL in a longterm suspension culture assay as well as in a shortterm cloning assay. These results strongly suggest that the autocrine secretion of CSF-related activities are not sufficient to fully account for the in vitro longterm proliferation of virus-transformed myelomonocytic cells.

[Radiocrystallographic study of 46 salivary calculi. Physiopathogenic hypotheses (author's transl)]. / Etude radiocristallographique de 46 calculs salivaires. Hypothéses physiopathogéniques.

The radiocrystallographic study of 46 salivary calculi using the Debye and Scherrer powder methods showed that such stones, whether submaxillary, parotid or "accessory" consist essentially of hydroxyapatite with the frequent presence of tricalcium and octocalcium phosphates, Whitlockite and rarely Brushite and Calcite. In order for a stone to form, the following conditions would seem to be necessary; transient supersaturation of the saliva in Ca++ and PO4--, a pH greater than normal, intracellular precepitation of amorphous tricalcium phosphate which is transformed into crystalline hydroxyapatite and, then, the fixation of crystals on a "matrix" such as desquamated cells, fibrils and collagens.