Françoise Wendling

Peritubular cells are the site of erythropoietin synthesis in the murine hypoxic kidney.

Erythropoietin (Epo)-producing cells were identified in the murine hypoxic kidney by in situ hybridization. Profound anemia was induced in order to greatly increase Epo production. This resulted in high levels of Epo mRNA in the kidney. 35S-labeled DNA fragments of the murine Epo gene were used as probes for in situ hybridization. Control experiments conducted in parallel included kidneys of nonanemic mice, RNase-treated hypoxic kidney sections, and 35S-labeled non-Epo-related DNA. The Epo probe gave a specific hybridization signal in the hypoxic kidney in the cortex and to a lesser extent in the outer medulla. Glomerular and tubular cells were not labeled. All positive cells were identified as peritubular cells. Using immunofluorescence, we showed that cells with the same topography contained Factor VIII-related antigen. These data demonstrated that peritubular cells, most likely endothelial cells, constitute the major site of Epo production in the murine hypoxic kidney.

A putative truncated cytokine receptor gene transduced by the myeloproliferative leukemia virus immortalizes hematopoietic progenitors

The myeloproliferative leukemia virus (MPLV) is an acute leukemogenic murine replication-defective retrovirus. By sequencing the envelope gene of a biologically active MPLV clone, we found that this region comprises a novel oncogene named v-mpl in phase with two parts of the Friend murine leukemia virus envelope gene. The MPLV env region could encode an env-mpl fusion polypeptide that presents the characteristics of a transmembrane protein. We show that in vitro infection of bone marrow cells with helper-free MPLV readily yields immortalized factor-independent hematopoietic cell lines of different lineages. In mice, the c-mpl proto-oncogene is expressed in hematopoietic tissues as a 3 kb mRNA. Since v-mpl shares strong structural analogies with the hematopoietin receptor superfamily, it is likely that MPLV has transduced a truncated form of an as yet unidentified hematopoietic growth factor receptor.

Spi-1/PU.1 transgenic mice develop multistep erythroleukemias.

Insertional mutagenesis of the spi-1 gene is associated with the emergence of malignant proerythroblasts during Friend virus-induced acute erythroleukemia. To determine the role of spi-1/PU.1 in the genesis of leukemia, we generated spi-1 transgenic mice. In one founder line the transgene was overexpressed as an unexpected-size transcript in various mouse tissues. Homozygous transgenic animals gave rise to live-born offspring, but 50% of the animals developed a multistep erythroleukemia within 1.5 to 6 months of birth whereas the remainder survived without evidence of disease. At the onset of the disease, mice became severely anemic. Their hematopoietic tissues were massively invaded with nontumorigenic proerythroblasts that express a high level of Spi-1 protein. These transgenic proerythroblasts are partially blocked in differentiation and strictly dependent on erythropoietin for their proliferation both in vivo and in vitro. A complete but transient regression of the disease was observed after erythrocyte transfusion, suggesting that the constitutive expression of spi-1 is related to the block of the differentiation of erythroid precursors. At relapse, erythropoietin-independent malignant proerythroblasts arose. Growth factor autonomy could be partially explained by the autocrine secretion of erythropoietin; however, other genetic events appear to be necessary to confer the full malignant phenotype. These results reveal that overexpression of spi-1 is essential for malignant erythropoiesis and does not alter other hematopoietic lineages.

MPLV: a retrovirus complex inducing an acute myeloproliferative leukemic disorder in adult mice.

A novel murine retrovirus complex was derived from the in vivo passage of a molecularly cloned Friend ecotropic helper virus. The virus isolate, myeloproliferative leukemia virus (MPLV), causes an acute (2-3 weeks) and generalized myeloproliferative disorder in adult mice. All strains of mice examined, including the C57BL/6J strain, developed the acute syndrome. This syndrome is characterized by a rapid hepatosplenomegaly, no thymus or lymph node involvement, granulocytosis, thrombocytosis, and erythroblastosis leading to polycythemia. The most prominent feature at the terminal phase of the disease is a granulocytic hyperplasia. The MPLV isolate replicates in vitro on NIH 3T3 fibroblasts but does not induce foci of transformed cells. Thus, MPLV exhibits unique biological properties that distinguish it either from the Friend virus complexes or from acutely transforming sarcomatogenic murine retrovirus which also induced a rapid splenomegaly.

The human homolog of the myeloproliferative virus maps to chromosome band 1p34.

SummaryThe human homologue of the recently isolated myeloproliferative leukemia virus, a retrovirus that induces myeloproliferative disorder in mouse, has been mapped in man to chromosome band 1p34 by in situ hybridization.

Erythrocyte Production in Mice Infected by the Polycythaemia‐Inducing Friend Virus or by the Anaemia‐Inducing Friend Virus

Summary. The changes induced by infection with the anaemia‐inducing (FVA) or the polycythaemia‐inducing (FVP) strain of Friend virus on PCV, RBC, reticulocyte counts, red‐cell and plasma volume and reticulocyte maturation time were studied in Swiss mice for 7 weeks after viral infection. The data indicated that the increase in total blood volume is mainly due to the augmentation of the spleen and liver blood volume in FVA‐disease, whereas it results from the large numbers of red cells produced by the spleen in FVP‐disease.

Spi-1 transgenic mice develop a clonal erythroleukemia which does not depend on p53 mutation

Spi-1 transcriptional activation and wild-type p53 extinction are two oncogenic alterations involved in the malignant transformation of erythroblasts during the Friend acute erythroleukemia. To dissect the contribution of these alterations in the deregulation of the differentiation and proliferation of erythroblasts, we generated spi-1 transgenic mice. Analysis of these animals revealed that Spi-1 overexpression was directly involved in the block of proerythroblast differentiation. However, the erythroleukemia that develops in these animals evolved in two steps. During the early step (HS1 step), non tumorigenic proerythroblasts remained strictly dependent upon erythropoietin (Epo) for their survival and proliferation. Later on, Epo-independent and tumorigenic proerythroblasts emerged (HS2 step) suggesting that other oncogenes cooperate with Spi-1 to lead to a fully malignant phenotype. By provirus tagging, we demonstrate that the HS1 step was clonal indicating that a cell selection must occur in vivo. Analysis of the nature of p53 in both the in vivo HS1 and HS2 proerythroblasts and in cultured erythroblastic cell lines showed that–p53 was normal in the HS1 primary tissues but was mutated in the HS1 cultured cell lines–p53 was frequently altered in HS2 primary tissues but was found normal in some mice. These data indicate that (i) the blockage of the erythroblast differentiation by Spi-1 occurs independently of p53 alteration (ii) p53 alteration is not necessary to confer Epo independence and tumorigenicity to spi-1 transgenic proerythroblasts.

Viral glycoproteins synthesis in Friend cell lines producing polycythemic (FV-P) and anemic (FV-A) Friend viruses

Abstract Two tumor Friend cell lines producing anemia-inducing virus (TF-A line) or polycythemia-inducing virus (TF-P line) were compared for their viral-encoded glycoproteins. The envelope glycoproteins of the two viral populations differ by their electrophoretic mobilities. The gpr env precursors also differ by their relative mobilities. The TF-P cells contain the typical gp50–52 molecular species, which is coded for by Spleen Focus Forming sequences (SFFV) present in the genome of the polycythemia-inducing virus. The TF-A cells do not contain the gp50–52, but express in small amounts a species with a higher apparent molecular weight. This species which has been named FV-A gp55 could be equivalent to the gp50–52 coded for by the SFFV sequences. Very similar results were obtained with leukemic cells prepared from enlarged spleens of mice infected with the anemic or polycythemic Friend viruses.

Germ-line deletion of p53 reveals a multistage tumor progression in spi-1/PU.1 transgenic proerythroblasts

Activation of the spi-1/PU.1 proto-oncogene and loss of p53 function are genetic alterations associated with the emergence of Friend malignant erythroleukemic cells. To address the role of p53 during erythroleukemogenesis, spi-1 transgenic mice (spi-1-Tg) which develop erythroleukemia were bred with p53-deficient mice. Three classes of spi-1 transgenic mice differing in their p53 functional status (p53+/+, p53+/− and p53−/−) were generated. These mice developed a unique pattern of erythroleukemia. In wild-type p53 spi-1-Tg mice, none of the primary erythroleukemic spleen cells displayed autonomous growth in vitro and in vivo. In contrast, in p53+/− spi-1-Tg mice, erythroleukemic cells gave rise to growth factor-independent cell lines and generated tumors in vivo. Malignancy was associated with loss of the wild-type p53 allele. The p53−/− spi-1-Tg mice developed erythroleukemia with a total incidence and a reduced latency compared to the two other genotypes. Unexpectedly, 50% of p53−/− spi-1-Tg erythroleukemic spleens generated cell lines that were strictly dependent upon erythropoietin (Epo) for proliferation, whereas the remainder proliferated independently of cytokines. Moreover, only 70% of these spleen cells were tumorigenic. These findings indicate that p53 germ-line deletion did not confer malignancy to spi-1-transgenic proerythroblasts. Moreover Epo independence and tumorigenicity appear as separable phenotypic characteristics revealing that the spi-1-Tg proerythroblasts progress towards malignancy through multiple oncogenic events.

Preferential liver irradiation enhances hematopoiesis through a thrombopoietin-independent mechanism.

Liver synthesizes thrombopoietin, which is a major cytokine involved in the production of hematopoietic cells. The purpose of this study was to examine the effects of preferential liver irradiation on expression of thrombopoietin and production of hematopoietic cells. About 70% of the liver of C57BL6/J mice was irradiated with 20 Gy of gamma rays. Exposure to ionizing radiation enhanced hematopoietic progenitors and megakaryocyte frequency in bone marrow and induced a transient increase in platelet and neutrophil counts that peaked 14 days after irradiation. The concentration of thrombopoietin was increased in serum as early as 5 h after liver irradiation and was still elevated at day 14. By using Northern blot analysis and an RNase protection assay, we showed that thrombopoietin mRNA was increased in the irradiated liver. To determine whether thrombopoietin was involved in the stimulation of hematopoiesis, we irradiated mice in which thrombopoietin deficiency had been induced by homologous recombination. Platelet levels were increased in both heterozygous and homozygous thrombopoietin-deficient mice with a magnitude similar to that obtained in normal mice. In summary, our data demonstrate that local irradiation of the abdomen encompassing the liver leads to stimulation of hematopoiesis through a thrombopoietin-independent mechanism.