EP1137756A2 - Signal de differentiation - Google Patents

Signal de differentiation

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Publication number
EP1137756A2
EP1137756A2 EP99960025A EP99960025A EP1137756A2 EP 1137756 A2 EP1137756 A2 EP 1137756A2 EP 99960025 A EP99960025 A EP 99960025A EP 99960025 A EP99960025 A EP 99960025A EP 1137756 A2 EP1137756 A2 EP 1137756A2
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EP
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Prior art keywords
cells
gata
erythroid
differentiation
prb
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EP99960025A
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German (de)
English (en)
Inventor
Franklin Gerardus Grosveld
David John Whyatt
Jacobus Nicolaas Jozes Philipsen
Fokke Albert Lindeboom
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Erasmus Universiteit Rotterdam
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Erasmus Universiteit Rotterdam
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Priority to EP99960025A priority Critical patent/EP1137756A2/fr
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Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0641—Erythrocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40—Regulators of development
    • C12N2501/48—Regulators of apoptosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors

Definitions

  • Erythropoiesis or the production of red blood cells, is one of the most important processes in higher eukaryotes, particularly in mammals. It is therefore highly important to have insight into the factors which play a role in said erythropoiesis. Particularly, since erythropoiesis is a process whereby proliferation and differentiation are intricately linked, it is crucial to know what factors regulates switches from proliferation to for instance apoptosis and/or what factors influence switches between proliferation and differentiation of erythroid cells.
  • Deficiencies or aberrance ' s in erythropoiesis leading to erythrocytes are associated with a number of disorders wherein said differentiation is impaired or hampered, such as ⁇ -thalassemia, ⁇ -thalassemia, sickle cell anaemia, aplastic anaemias such as Fanconi ' s anaemia, disorders affecting red blood cell stability such as G6PD-deficiency, erythroleukaemia, leukaemia (anaemia is very often a secondary effect of leukaemia) , any genetic or acquired disorder in which erythroid differentiation is impaired or hampered, and for example associated with medical treatment in which erythroid differentiation is impaired or hampered (such as chemotherapy) , neoplasia, aberrant cell growth, malignant: cell growth.
  • ⁇ -thalassemia ⁇ -thalassemia
  • sickle cell anaemia aplastic anaemias
  • Fanconi ' s anaemia disorders affecting red blood cell
  • GATA-1 protein which is the founding member of this family of proliferation and differentiation factors, for example is thought to be required for differentiation of the definitive erythroid lineage beyond the proerythroblast stage.
  • GATA-2 Another member of the family, GATA-2, is thought to be expressed in early pluripotent haematopoietic lineages and is required for the formation of all haematopoietic cell types. In a typical feedback fashion, GATA-2 expression is thought to be down- regulated by GATA-1 expression, thereby tipping the cellular balance in favour of terminal erythroid differentiation.
  • erythroid precursor cells have an intrinsic requirement for functional retinoblastoma protein (pRb) .
  • pRb retinoblastoma protein
  • Lack of functional pRb inhibits erythroid differentiation, leading to anaemia and embryonic lethality.
  • pRb, and related pl07 and pl30 proteins are thought to regulate the transcription of genes required for Gl to S phase progression in preparation of cell division. Hyperphosphorylation of pRb inactivates further differentiation whereas hypophosphorylation of pRb provides a switch to differentiate.
  • erythroid precursors have an intrinsic requirement for pRb activity to undergo normal differentiation. Loss of pRb results in the reduction of circulating definitive erythroid cells and death in utero .
  • GATA-1 may cause hyperphosphorylation of pRb, thereby promoting cell division instead of further differentiation, and thus counteracting the drive towards differentiation, as described above.
  • above conflicting observations can be understood within a framework of checks-and-balances, comprising factors that on the one hand comprise a drive towards proliferation and/or differentiation, and on the other hand comprise the necessary feedback factors to keep proliferation and/or differentiation at bay, however, these observations do not constitute enough understanding of erythropoiesis to arrive at sufficient remedies when erythroid differentiation is impaired or hampered.
  • the invention provides a method for stimulating erythroid differentiation of erythroid cells of which said differentiation is impaired or hampered, comprising contacting said cells with erythroid cells capable of erythroid differentiation or with at least one substance secreted by said cells capable of erythroid differentiation.
  • GATA-1 is a tissue-specific transcription factor required for the development of the definitive erythroid lineage.
  • GATA-1 overexpression inhibits erythroid differentiation, leading to anaemia and embryonic lethality, in a manner similar to the loss of functional retinoblastoma protein (pRb) .
  • the effect of pRb loss on erythropoiesis is non-autonomous and has therefore in general been attributed to a defect in stromal cells .
  • GATA-1 overexpression accelerates Gl to S phase transition, activates hyperphosphorylation of pRb and blocks induced erythroid differentiation in vi tro .
  • erythroid GATA-1 overexpression recapitulates the non- autonomous defect in pRb null cells.
  • the invention provides a signal substance derived from wild-type erythroid cells that rescues the differentiation of GATA-1 overexpressing and pRb null cells, bypassing loss of pRb function.
  • the invention provides a substance (herein also called REDS) , derived from cells capable of erythroid differentiation, regulating non-autonomous erythroid differentiation.
  • REDS a substance
  • pRb functional retinoblastoma protein
  • the invention provides a method stimulating erythroid differentiation of erythroid cells of which said differentiation is impaired or hampered, comprising contacting said cells with erythroid cells capable of erythroid differentiation or with at least one substance secreted by said cells capable of erythroid differentiation wherein said impaired or hampered cells are impaired or hampered because of overexpression of GATA-1 or a functional equivalent or derivative thereof.
  • GATA-1 for example, leads to loss of active pRb and results in impaired or hampered erythroid precursors that cannot efficiently switch from a proliferative to a differentiating cell-type.
  • Such impaired or hampered cells for example comprise ES cell-derived erythroid colonies overexpressing GATA-1.
  • capable cells such as wild-type erythroid cells, for example in vivo in chimeric mice or in vi tro
  • capable cells such as wild-type erythroid cells, for example in vivo in chimeric mice or in vi tro
  • capable cells such as wild-type erythroid cells, for example in vivo in chimeric mice or in vi tro
  • Said substance as provided by the invention is used to treat human genetic and acquired diseases in which erythroid differentiation is impaired or hampered, either as a direct consequence of the disease or as the consequence of treatment, i.e. because of chemotherapy. Also, it is used to treat disorders in which the cell cycle of erythroid cells is deregulated, such as erythroleukemias , by inducing terminal differentiation of such cells. Furthermore, it is used to treat disorders in which the cell cycle of haematopoietic cells is deregulated, such as leukemias, by inducing terminal differentiation and/or enucleation of such cells, and to treat disorders in which malignant growth of cells occurs, as in cancers, by inducing differentiation and/or enucleation of such cells. Furthermore, the invention provides a method to treat anaemia by stimulating the production of red blood cells by interfering with REDS activity for instance by manipulating REDS activity by functionally inactivating the REDS receptor.
  • the invention also provides a substance capable of inducing erythroid differentiation in erythroid cells impaired or hampered in said differentiation, said substance being characterised in that it is secreted by erythroid cells capable of inducing differentiation in erythroid cells impaired or hampered therein.
  • Said substance herein also called REDS, derived from cells capable of erythroid differentiation, is regulating non-autonomous erythroid differentiation.
  • REDS derived from cells capable of erythroid differentiation
  • REDS mRNA is obtained by subtractive hybridisation followed by cDNA cloning using wild-type, Rb null- and/or GATA-1 overexpressing erythroid cells as the starting material.
  • the invention provides a substance capable of inhibiting proliferation of erythroid cells, said substance being obtainable from wild-type erythroid cells.
  • proliferating erythroid precursors can be stopped in their proliferating activity by a substance, such as REDS, according to the invention.
  • REDS activity of the protein, its functional fragments and derivatives is monitored in methylcellulose colony assays wherein it rescues differentiation of erythroid cells that are impaired or hampered in differentiation, such as Rb null- and GATA-1 overexpressing cells.
  • the invention provides a nucleic acid encoding a substance as provided by the invention.
  • REDS is cloned using mammalian expression/cloning systems known in the art using for example the assay system as provided by the invention as described below.
  • the REDS gene and the REDS locus are identified and cloned from genomic DNA libraries such as cosmid, PAC and YAC libraries with the REDS cDNA by hybridisation.
  • Recombinant REDS protein and (other proteinaceous) functional fragments or derivatives thereof are provided by the invention from cloned DNA according to recombinant protein expression techniques known in the art.
  • REDS protein and functional fragments or derivatives thereof are derived via synthetic peptide synthesis.
  • REDS substances as provided by the invention are for example erythroid death receptors or death receptor ligands, such as FasL, TRAIL and TNF- ⁇ , being part of the REDS pathway, for interfering with erythroid cell differentiation or proliferation.
  • Derivatives include REDS agonists or antagonists, for example derived by creating and testing synthetic peptides, or peptides in a phage library, for their activity in the herein described REDS assay.
  • said REDS protein or functional fragments or derivatives are derived from cloned human DNA, thereby minimising the risk of adverse immune reactions when using said REDS protein and functional fragments or derivatives thereof in treatment of humans .
  • the invention provides the in vi tro use of a substance such as (recombinant) REDS, the REDS receptor and/or molecules closely related to these, for therapeutic and/or biotechnological purposes, for instance the use of REDS technology in the production of any substance from cultured cells and/or eukaryotic and prokaryotic organisms.
  • the invention provides the use of or inhibition of REDS activity, optionally in combination with other substances, such as erythopoietine, in in vi tro organogenesis systems to obtain erythroid cells or haematopoietic cells, or other in vi tro organogenesis systems.
  • haematopoietic cells can advantageously be used to produce mature erythrocyte populations, which can be made substantially free of nucleic acid, or at least of coding sequences, if so desired, by methods known in the art, for example by selecting those erythrocytes for lack of reaction or staining with a nucleic acid reactant or dye, such as thiazole orange.
  • Said mature erythrocyte populations, stemming from a selected individual haematopoietic stem cell population (s) or combinations thereof, optionally derived from an anaemic patient can for example be used as source of functional red blood cells in blood transfusions, optionally to treat said anaemic patient.
  • Other examples comprising use of at least one substance according to the invention for interfering with erythroid cell proliferation or differentiation comprise use of a substance such as (recombinant) REDS, a REDS receptor and/or molecules closely related to these, for therapeutic purposes, such as use of REDS substances such as erythroid death receptors or death receptor ligands, being part of the REDS pathway, for interfering with erythroid cell differentiation.
  • REDS substances such as erythroid death receptors or death receptor ligands, being part of the REDS pathway, for interfering with erythroid cell differentiation.
  • Death receptor ligands such as FasL, TRAIL and TNF- ⁇ , reactive with receptors expressed on erythroid cells are, for example by activating GATA-1 degradation, allowing interference of REDS substances with proliferation and/or differentiation of erythroid cells, especially in those cells of which said proliferation or differentiation is impaired or hampered.
  • the invention provides use of REDS substances such as death receptor ligands, such as FasL, TRAIL and TNF- ⁇ , for the preparation of a (if so required pharmaceutical) composition for in vivo or in vi tro treatment of (pre) erythroid cells, especially of those cells of which said proliferation or differentiation is impaired or hampered.
  • Death receptor ligands such as FasL, TRAIL and TNF- ⁇
  • Said death receptor ligands can of course also be used in in vi tro organogenesis systems to obtain erythroid cells or haematopoietic cells, or other in vi tro organogenesis systems, as described above.
  • GATA-1 protein In vertebrates, the GATA-1 protein is predominantly expressed in the erythroid lineage (Evans and Felsenfeld, 1989; Tsai et al . , 1989) . Knockout experiments have demonstrated that GATA-1 is required for differentiation of the definitive erythroid lineage beyond the proerythroblast (Pevny et al . , 1991; Weiss et al . , 1994). This committed erythroid precursor normally undergoes the final rounds of cell division before a proliferative arrest and formation of the mature erythrocyte (Bessis et al . , 1983). GATA-1 null proerythroblasts instead undergo apoptosis (Weiss and Orkin, 1995b) .
  • GATA-1 is required for proerythroblast survival, apoptosis does not normally occur at this stage of erythroid differentiation. Therefore, the role of GATA-1 in the formation of mature erythrocytes, beyond preventing the premature loss of erythroid precursors, remains obscure.
  • GATA-1 contains two zinc finger-like domains that direct its binding to DNA (Martin and Orkin, 1990) . A number of recognition sequences have been defined, the most important being the (A/T) GATA (A/G) motif (Ko and Engel, 1993; Merika and Orkin, 1993; Whyatt et al . , 1993).
  • GATA-1 is the founding member of the family of GATA proteins which share a high degree of homology in the zinc finger- like domains, all of which bind to (A/T) GATA (A/G) motifs (Ko and Engel, 1993; Merika and Orkin, 1993; Evans, 1997).
  • GATA-2 is expressed in early pluripotent haematopoietic lineages and is required for the formation of all haematopoietic cell types (Leonard et al . , 1993; Tsai et al. , 1994) .
  • A/T GATA (A/G) motifs is important for the function of transcriptional regulatory elements of a number of erythroid-specific genes, including the beta- globin locus control region (LCR) (Philipsen et al . , 1993) and the GATA-1 gene itself (Simon et al . , 1992) .
  • LCR beta- globin locus control region
  • GATA-1 target genes including that of the beta-globin locus, is not dramatically affected in GATA-1 null erythroid cells (Weiss et al . , 1994). It has been suggested that GATA-2 can compensate for the loss of GATA-1, since GATA-2 is upregulated in the GATA-1 knockout (Weiss et al . , 1994) .
  • heterologous GATA proteins can reverse aspects of the GATA-1 loss-of-function phenotype (Blobel et al . , 1995) .
  • the prevailing model for the role of GATA-1 and GATA-2 in the erythroid lineage suggests that during erythroid differentiation, rising levels of GATA-1 repress GATA-2 expression.
  • GATA-1 expression is then maintained by positive autoregulation.
  • the shift from GATA-2 to GATA-1 predominance tips the cellular balance in favour of terminal differentiation either through activation of GATA-1-specific target genes or downregulation of proliferative signals specifically mediated by GATA-2; cell division ceases and erythroid maturation ensues (Weiss and Orkin, 1995a) .
  • GATA-2 early haematopoietic precursors predominantly express GATA-2 and late erythroid precursors predominantly express GATA-1 (Leonard et al . , 1993).
  • Empirical evidence that GATA factors regulate proliferation and differentiation has mainly come from enforced expression of estrogen receptor (ER) fusion proteins in chicken erythroid precursors (Briegel et al . , 1993; Briegel et al . , 1996).
  • ER estrogen receptor
  • Enforced expression of a GATA-l/ER fusion protein in an immortalized murine GATA-1 null erythroid cell line will rescue aspects of terminal erythroid differentiation (Weiss et al . , 1997) .
  • Implicit in the above model is the suggestion that proerythroblasts, which are a proliferating cell type, are about to stop dividing in response to a GATA-1-dependent signal.
  • differentiation of the proerythroblast to the definitive erythrocyte is characterised by a number of rounds of cell division before cell cycle arrest (reviewed in Bessis et al . , 1983). Therefore, a role for GATA-1 in promoting cellular proliferation as part of the erythroid differentiation program has not been excluded.
  • GATA-1 can regulate the switch from proliferation to differentiation in the mammalian erythroid lineage.
  • Murine erythroleukemia (MEL) cells a virally transformed proerythroblast cell line, can be induced to undergo differentiation upon stimulation with chemical inducers such as dimethyl sulphoxide (DMSO) (Friend et al . , 1971).
  • MEL cells overexpressing GATA-1 under human beta-globin regulatory sequences fail to differentiate, and overcome differentiation-associated Gl arrest.
  • Overexpression of GATA-l in non-transformed erythroid precursors also blocks normal erythroid differentiation (Whyatt et al . , 1997).
  • the effect of GATA-1 overexpression on cell cycle progression is not a secondary phenomenon associated with loss of differentiation.
  • Fibroblasts constitutively expressing GATA-1 can display both a shortened Gl phase and an elongated S phase (Dubart et al . , 1996). Since GATA-1 influences cell cycle progression in a heterologous cell type, independent of differentiation, it has been suggested that it might directly regulate Gl to S phase progression in erythroid cells (Whyatt et al . , 1997).
  • Cyclins bind and activate cyclin dependent kinases (cdks) .
  • An important substrate for the cdks is the family of pocket proteins, which are the pRb and related pl07 and pl30 proteins.
  • Hypophosphorylated pRb binds to the E2F family of transcription factors, blocking their ability to activate the transcription of genes required for Gl to S phase progression.
  • Hyperphosphorylated pRb does not bind E2F.
  • activation of cdks by cyclins during the Gl phase of the cell cycle is thought to result in hyperphosphorylation of Rb, release of bound E2F and then Gl to S phase progression.
  • cyclin-dependent kinase inhibitors which include pl6, pl8, pl9, p21 and p27, downregulate the kinase activity of cyclin/cdk complexes (reviewed in Sherr and Roberts, 1995) .
  • ckis Activation of ckis is associated with the differentiation of a number of cell types, as is alterations in the levels of active cyclin/cdk complexes (reviewed in Gao and Zelenka, 1997; Zavitz and Zipursky, 1997) .
  • the construct (PEV3 -GATA-1) used to overexpress GATA-1 tagged with the myc-epitope has been previously described (Elefanty et al . , 1996).
  • the construct places GATA-1 transcription under the control of the erythroid-specific human beta-globin LCR and beta-globin promoter, which activate high level expression from the proerythroblast stage of erythroid differentiation.
  • GATA-1 overexpressing cells are proliferating faster than control cells on day 3 of induction and have returned to pre-induced proliferation rates by day 4, while the proliferation rate of control cells remains low.
  • the cell cycle distribution of GATA-1 overexpressing cells never returned to a profile identical to that of uninduced cells.
  • control MEL cells induce erythroid markers such as ⁇ ma: ⁇ globin mRNA forty-fold at day 4
  • GATA-1 overexpressing cells induce such expression only two- to three-fold (Whyatt et al . , 1997). Therefore, following a transient Gl arrest in response to DMSO, GATA-1 overexpression activates Gl to S phase transition, while preventing the activation of genes associated with terminal differentiation.
  • GATA-1 overexpressing cells have a low pre- induced level of transgene-derived GATA-1. Activation of the transgene is detectable at day 1 of induction, but does not reach high levels until day 2 of induction. Consistent with previous experiments (Whyatt et al . , 1997), the increase in GATA-1 transgenic protein levels is associated with downregulation of endogenous GATA-1 protein. Thus, increasing GATA-1 levels from day 1 to day 2 of induction correlates with the activation of Gl to S phase progression.
  • pl07 behaves identically up until day 1 of induction, then its protein levels are relatively increased from day 2. In this assay, we could not clearly show if the levels of hypophosphorylated pl07 change in GATA-1 overexpressing cells at day 2. However, the level of E2F/pl07 complex DNA binding activity is not significantly altered in GATA-1 overexpressing cells compared to controls,- suggesting that the level of hypophosphorylated pl07 does not change (data not shown) . We could not reproducibly detect pl30 in MEL nuclear extracts.
  • GATA-1 expression in MEL cells after DMSO-induced differentiation results in the acceleration of Gl to S phase progression alongside the loss of differentiation-associated gene activation.
  • GATA-1- induced activation of Gl to S phase progression is associated with increased in cyclin A2 levels and hyperphosphorylation of pRb.
  • ES cell clones containing a modified version of PEV3 -GATA-1 were generated. Independent single-copy ES cell clones were injected into blastocysts and the resulting chimeric animals were analyzed. Surprisingly, such cells were found to both efficiently contribute to the mature and fetal erythroid compartments and express transgene-derived GATA-1 in chimeric embryos (data not shown) .
  • FIG. 2A Such contribution (ES cell clone G4 , chimera number 8) is shown in Figure 2A, using triton-acid-urea gel electrophoresis to assay for ES cell-derived globin chains in adult blood. Up to 50% of the mature beta-type globin chains could be derived from clone G4. Contribution to the erythroid lineage by such ES cells was confirmed by glucose phosphate isomerase analysis (data not shown) . The morphology of the erythrocytes in such chimeric animals is normal ( Figure 2B) .
  • ES clones were screened for integration of the transgene into the X chromosome by DNA fluorescence-in-situ- hybridisation (DNA-FISH) , with probes to the human beta- globin LCR and the GATA-1 gene.
  • DNA-FISH DNA fluorescence-in-situ- hybridisation
  • signal indicating the integrated transgene apparently coincided with the endogenous GATA-1 locus (which is on the X chromosome (Chapman et al . , 1991)) in metaphase spreads, yet could be distinguished from the endogenous locus in interphase nuclei and on stretched DNA.
  • Southern blot analysis demonstrated no disruption to the endogenous GATA-1 locus (data not shown) .
  • mice derived from clone G4 were generated and mated to FVB females. Two of the eight chimeric males gave 100% germline transmission. Close to the expected male/female ratio in the progeny was observed (38 male, 34 female), all the females carrying the transgene and all the males (except one) being non-transgenic . One phenotypically male animal was found to be transgenic.
  • FISH DNA-fluorescence in-situ hybridization
  • transgenic embryos were then mated to FVB males and sacrificed at various time points during pregnancy or allowed to go to term.
  • dpc days post coitum
  • transgenic embryos are similar to wild-type littermates .
  • Transgenic females are indistinguishable from wild-type littermates at a gross morphological level at all time points examined.
  • male transgenics display an anaemic phenotype . No other abnormalities are detectable.
  • approximately 50% of male transgenic embryos are dead (scored by lack of heartbeat) at 13.5 dpc, and 100% at 14.5 dpc ( Figure 2C and 2D) .
  • FIG. 3A shows a Western blot of crude nuclear extract from 13.5 dpc fetal livers, probed with the anti-GATA-1 antibody N6. Only live male transgenics are included in this analysis. Male transgenic mice express a higher level of transgene-derived GATA-1 than females, as would be expected if X-inactivation results in heterocellular transgene expression in the female transgenic mice. Total GATA-1 levels in male transgenics is in the range of four to ten- fold increased over endogenous levels. Furthermore, female transgenics express lower than wild-type levels of endogenous GATA-1, and endogenous GATA-1 is almost undetectable in male transgenic fetal livers. It is difficult to determine if endogenous GATA-1 expression is completely ablated in male transgenic animals due to background bands in the male transgenic sample lanes that are apparently degradation products from the transgene- derived GATA-1.
  • the fetal liver is the major source of definitive erythrocytes during this period of development.
  • the recognisable erythroid precursors in order of differentiation and progressively reducing size are the proerythroblasts, the basophilic erythroblasts, the polychromatic erythroblasts, the orthochromatic erythroblasts and the enucleated reticulocytes.
  • Haemoglobin accumulation detectable by benzidine staining, begins at the polychromatic erythroblast stage (reviewed in Bessis et al . , 1983) .
  • Each of these precursors can be found in 12.5 and 13.5 dpc fetal livers in both transgenic and wild-type animals ( Figure 4B) .
  • transgenic male animals compared to wild-type animals.
  • proerythroblasts 12.5 dpc transgenic male fetal livers contain significantly more basophilic erythroblasts, fewer benzidine positive polychromatic and orthochromatic erythroblasts and fewer reticulocytes (Figure 4C) .
  • Female transgenics do not have significantly different proportions of erythroid precursors, yet the numbers are highly variable .
  • erythroid cells 12.5 dpc fetal livers were then disaggregated and analysed by fluoresence-activated cell sorting (FACS) .
  • FACS fluoresence-activated cell sorting
  • Cells were stained with R-phycoerythrin (R-PE) -conjugated TER119 antibody (a membrane-bound erythroid marker (Ikuta et al . , 1990)) and propidium iodide (PI, used to exclude dead cells from the analysis) . No significant change in the number of PI + cells is detectable in transgenic animals (data not shown) .
  • R-PE R-phycoerythrin
  • TER119 antibody a membrane-bound erythroid marker (Ikuta et al . , 1990)
  • PI propidium iodide
  • the anaemia in male transgenics appears to be due a failure of the definitive erythroid precursors to differentiate efficiently beyond the basophilic erythroblast stage.
  • definitive erythropoiesis is apparently not completely blocked, since some enucleated erythrocytes do enter the circulation.
  • Female transgenics appear to generate circulating definitive erythrocytes as efficiently as wild- type animals.
  • GATA-1 may have moderate effects on erythroid differentiation in 12.5 dpc fetal livers.
  • Such variability in females might arise due to varying X-inactivation balance in an earlier stem cell compartment.
  • TER119 cells were gated out and the remaining cells (>80%) were analyzed for size distribution and viability.
  • Cultured cells derived from transgenic male livers have a dramatically different forward scatter profile compared to those derived from wild-type livers ( Figure 5A and 5B) .
  • Figure 5A and 5B the entire range of cell size is represented in both, the peak of small cells found in wild-type samples is dramatically reduced.
  • the group of PI " small cells that have a forward scatter value similar to a group of intensely PI + bodies is significantly reduced in transgenic male samples. These are presumably recently extruded nuclei, which later become PI + , and enucleated reticulocytes.
  • transgenic male cultures contain an increased number of PI + cells that have a forward scatter value in the range of normally differentiating erythroid precursors. This suggests that GATA-1 overexpressing proerythroblasts undergo inefficient differentiation in culture and eventually apoptose .
  • TER119 + cells from transgenic female cultures show an intermediate forward scatter profile and an intermediate increase in large cells that are PI + ( Figure 5A and 5B) .
  • Btk lacZ mice contain an integration of the lacZ gene on the X chromosome in the Bruton's tyrosine kinase (Btk) locus (Hendriks et al . , 1996).
  • Btk lacZ mice express lacZ in approximately 50% of large erythroid progenitors in the bone marrow. Since this locus is X- inactivated, the number of such cells positive in heterozygous female mice is approximately 25%.
  • GATA-1 transgenic females transgenic for both the GATA-1 transgene and the Btk lacZ insertion were compared with females containing the Btk lacZ insertion alone.
  • the GATA-1 transgenic females express lacZ in a similar number of large erythroid precursors when compared to three normal heterozygous Btk l cZ/+ females.
  • the GATA-1 transgenic heterozygous female Btk lacZ/+ mouse (#1) expressing lacZ in a higher number of large erythroid precursors also expresses lacZ in a higher than expected number of cells in the B-cell and monocytic lineages.
  • GATA-1 overexpressing cells are differentiating normally in females expressing the transgene heterocellularly. Although erythroid precursors overexpressing GATA-1 are unable to differentiate in vi tro, they are rescued and normally represented in female transgenic mice.
  • GATA-1 may not be the only level at which GATA-1 is autoregulated. Since GATA-1 overexpression activates the proliferation of erythroid cells and inhibits differentiation, the downregulation of GATA-1 activity would appear to be crucial during terminal erythroid differentiation.
  • the data presented in this descriprion show that the prevailing model of GATA-1 function in erythroid differentiation (Weiss and Orkin, 1995a) must be modified, in that GATA-1 does not regulate itself only positively, nor does GATA-1 expression drive cells from a proliferating to a non-proliferating/differentiated cell type.
  • GATA-1 overexpression in differentiating MEL cells is associated with accelerated Gl to S phase transition following a transient Gl arrest.
  • the Gl arrest we observe at day 1 of induction is consistent with previous reports of a transient inhibition of DNA synthesis in MEL cells treated with DMSO.
  • Labeled-thymidine incorporation by MEL cells decreases rapidly 10 hours after DMSO addition, reaching a low at approximately 20 hours, before increasing as cells re-enter S phase (Marks et al . , 1978) .
  • GATA-1 overexpression specifically increases the level of cyclin A2 , while all other cyclins known to regulate pRb phosphorylation during Gl are unaffected up until one day after the GATA-1-induced Gl to S phase transition has occurred.
  • Cyclin A2 is an attractive candidate linking GATA-1 to activation of pRb phosphorylation and Gl to S phase transition. Cyclin A2 is expressed and translocates to the nucleus prior to detectable DNA synthesis (Girard et al . , 1991) . Cyclin A2 is required for Gl to S phase transition (Girard et al . , 1991; Pagano et al . , 1992), can overcome pRb-mediated Gl arrest (Hinds et al . , 1992) and is rate limiting for entrance into S phase in fibroblasts (Resnitzky et al . , 1995) .
  • Increasing cyclin A2 levels could indirectly result in pRb phosphorylation by titration of cki ' s away from cyclin D and E-dependent kinase complexes, as has been demonstrated in the case of cyclin Dl in inducing cyclin E-dependent kinase activity by sequestering p21 (Planas-Silva and Weinberg, 1997) .
  • SV40 large T antigen bound to the pocket domain of pRb has been used as a model for the Rb/E2F complex that is thought to be disrupted at the Gl to S phase transition.
  • cyclin A2/cdk2 can phosphorylate and disrupt a pRb/T antigen complex while cyclin Dl/cdk4 cannot (Zarkowska and Mittnacht, 1997) . Therefore, in addition to phosphorylation of pRb during Gl by cyclin D and E-dependent kinases, cyclin A2 may also be activated and disrupt pRb/E2F complexes in late Gl .
  • GATA-1 Loss of GATA-1 may result in the apoptosis of erythroid precursors (Weiss and Orkin, 1995b) due to a disruption in GATA-1-dependent cell cycle progression.
  • Other GATA family members have also been implicated in promoting proliferation and inhibiting apoptosis.
  • a GATA-2/ER fusion protein activates proliferation of chicken erythroblasts in a hormone-responsive manner (Briegel et al . , 1993).
  • GATA-4 protein Overexpression of the cardiac GATA-4 protein in P19 cells increases the number of cardiomyocytes following differentiation and loss of GATA-4 induces apoptosis (Grepin et al . , 1997). Elevated GATA-6 levels (also expressed in heart) in Xenopus delays cardiomyogenic differentiation and increases the number of cells in the myocardium (Gove et al . , 1997) . Therefore, GATA factors as a family may be responsible for regulating tissue-specific proliferation and the absence of GATA factors may induce a default apoptotic pathway.
  • mice deficient for pRb die due to anaemia between 12.5 and 15.5 dpc (Clarke et al . , 1992; Jacks et al . , 1992; Lee et al . , 1992) .
  • pRb null embryos have a reduction in the number of circulating enucleated erythrocytes, yet the appearance of some enucleated cells suggests that the block in differentiation is not absolute (Lee et al . , 1992) . Furthermore, the decondensed chromatin structure in nucleated embryonic erythrocytes in GATA-1 transgenic males is also observed in the embryonic erythrocytes of pRb null mice (Lee et al . , 1992).
  • ES cell-derived erythroid colonies overexpressing GATA- 1 have previously been shown to differentiate poorly into definitive erythrocytes (Whyatt et al . , 1997). We demonstrate here that such ES cells will contribute effectively to the circulating erythrocytes in chimeric mice. It is possible that those ES cell-derived circulating erythrocytes are descendants of precursors that for unknown reasons never expressed the GATA-1 transgene. However, the high level of contribution (around 50%) in a number of chimeras argues strongly against contribution from a small non-expressing subset of the ES cell-derived precursors.
  • X-linked transgenic mouse line allowed us to investigate the fate of GATA-1 overexpressing cells in situations where the expression is either heterocellular or pancellular, since the integrated PEV3 -GATA-1 transgene is subject to X- inactivation. It is significant that the element used to drive expression of GATA-1, the human beta- globin LCR, does not escape X-inactivation.
  • the LCR functions as a dominant regulator of chromatin remodelling and transcriptional activation and is resistant to inhibitory effects such as silencing upon intergration into heterochromatin (Milot et al . , 1996).
  • X- inactivation inhibits LCR function via a mechanism distinct from heterochromatin formation. Exploitation of the X-linked transgene approach described here may also be applicable to the study of other factors where the comparison of heterocellular and pancellular expression patterns is informative .
  • mice of our X-linked transgenic line overexpress GATA-1 pancellularly and fail to effectively differentiate definitive erythrocytes.
  • Female transgenic mice overexpress GATA-1 heterocellularly and are apparently unaffected. That females survive is not due to being female per se, since a transgenic XXY phenotypic male is also viable.
  • XXY cells are known to inactivate the extra X chromosome (reviewed in Kuroda and Meller, 1997) . Therefore, survival depends on the presence of a second non-transgenic X chromosome.
  • GATA-1 overexpressing cells differentiate normally, as do ES cell-derived GATA-1 overexpressing cells in chimeric animals. Together this suggests that the disruption of erythroid differentiation due to overexpression of GATA-1 is a non-autonomous event.
  • a putative signal regulating non -autonomous erythroid differentiation is a non-autonomous event.
  • GATA-1 overexpression is an effect on the stromal cells supporting erythropoiesis, for the following reasons.
  • the regulatory elements used to direct expression of the transgene are erythroid-specific (Grosveld et al . , 1987) .
  • GATA-1 overexpressing erythroid precursors from female fetal livers autonomously undergo inefficient differentiation and eventually apoptose in culture, while non-overexpressing cells from the same liver differentiate normally, imply that GATA-1 overexpression induces an intrinsic defect in erythroid cells. Therefore, there must be a signal rescuing the differentiation of the GATA-1 overexpressing cells in the female transgenics.
  • the source of this signal must be a cell type in the female transgenic mice that is absent in male transgenic mice.
  • the obvious difference between male and female GATA-1 transgenics is the presence of normally differentiating (and not overexpressing GATA-1) erythroid cells in the females. This shows that such erythroid cells produce a signal substance (REDS) that directs GATA-1 O 00/34329
  • pRb null proerythroblasts may be intrinsically unable to differentiate efficiently, yet may overcome this defect in a manner similar to GATA-1 overexpressing cells in response to a signal emanating from normally differentiating erythroid cells.
  • FIG. 7 a model of the role of GATA-1 in regulating erythroid differentiation is shown ( Figure 7) .
  • GATA-1 maintains the level of cyclin A2 and pRb phosphorylation during the proliferative period of normal differentiation.
  • cyclin A2 levels decrease and pRb dephosphorylation occurs.
  • pRb activates Gl arrest as the cells undergo terminal differentiation and enucleation.
  • Overexpression of GATA-1 leads to loss of active pRb and results in erythroid precursors that cannot efficiently switch from a proliferative to a late non- proliferating/fully differentiating state.
  • REDS red cell differentiation signal
  • Mature erythroid cells express so-called "death receptor” ligands FasL, TRAIL and TNF-alpha that are generally been thought to have an apoptotic effect on erythropoeisis (Rusten and Jacobsen, 1995, De Maria et al . , 1999a, De Maria et al . , 1999b). It was found that the important downstream effect of death receptor ligands on erythroid cells is to activate the caspase-mediated degradation of GATA-1. However, we tested the effects of these ligands in the context of the REDS signalling pathway on the differentiation of GATA-1 overexpressing erythroid cells in liquid culture (10 6 cells/ml) by addition of 10, 40 and 100 ng/ml FasL.
  • death receptor ligands are components of REDS, and, in the context of REDS signalling to proliferated cells, such as cells overexpressing GATA-1, and preferably being impaired in differentiation, also have differentiative activity, instead of apoptic activity only.
  • Fas is known to activate caspase-mediated nuclear condensation (Sahara et al . , 1999) and caspase inhibitors can block pyknosis (Zamzami and Kroemer, 1999) .
  • the lack of a severe erythroid phenotype in mice lacking individual death receptors and ligands may be due to the redundant expression of multiple death signalling molecules (Rusten and Jacobsen, 1995) .
  • Caspase-8 deficient mice display an accumulation of erythroid cells (Varfolomeev et al . , 1998), suggesting a role for caspases in suppressing erythroid expansion.
  • REDS signalling by death receptor activation regulates both proliferative arrest and/or pyknosis during late erythroid differentiation.
  • Plasmids PEV3 -GATA-1 and PEV3 have been previously described (Whyatt et al . , 1997) . Details of cloning steps to produce puromycin resistant variant of PEV3 -GATA-1 are available on request. Integration of PEV3 -GATA-1 in ES cells and mice was screened by Southern blot using a lkb EcoRl internal probe corresponding to the 5' end of the GATA-1 minigene.
  • MEL were grown and transfected with the neomycin- resistant version of PEV3 -GATA-1 and PEV3 as previously described (Whyatt et al . , 1997) .
  • Populations were selected in 400 ⁇ g/ml G418 and induced in 2% DMSO.
  • Wes tern blot ting Nuclear protein extracts were prepared as previously described (/Andrews and Faller, 1991) . Proteins were electrophoresed through appropriate percentage
  • SDS/polyacrylamide gels transferred onto nitrocellulose, then probed with appropriate primary and secondary antibodies before detection using chemiluminesence .
  • Antibodies used (purchased from Santa Cruz, CA) : GATA-1 N6 cat # sc-265, pRb C-15 cat # sc-050, pl07 C-18 cat # sc-318, cyclin A2 C-19 cat # sc-596, cyclin D2 C-17 cat # sc-181, cyclin D3 18B6-10 cat # sc-453, cyclin E M-20 cat # sc-481, pl6 M-156 cat # sc-1207, p27 F-8 cat # sc-164 ES cells
  • Chimeric mice were generated by injecting ES clones generated as above into C57bl6 blasocysts. Chimeras were then bred with wild-type FVB females and screened by coat colour for transmission of the ES clone and Southern blot for integration of the transgene. Transgenic females were then mated to wild-type FVB males and sacrificed during gestation or allowed to go to term. Blood and single cell suspensions of fetal livers were prepared on slides by cytocentrifugation and stained with neutral benzidine and a modified Giemsa-like stain as described previously (Beug et al. , 1982) .
  • DNA-FISH was performed as previously described (Mulder et al . , 19S5) .
  • RNA-FISH was performed on disaggregated 12.5 dpc fetal livers as previously described (Wijgerde et al . ,
  • CFU-E assays were performed as previously described (Wong et al . , 1986) . Fetal livers from 12.5 dpc embryos were disaggregated into single cells by passage through a 100 ⁇ M mesh and plated at a density of 3xl0 5 /ml in methylcellulose containing 1 U/ml Epo . Colonies were grown for times indicated and then collected and washed in PBS to remove residual methylcellulose before staining. FACS analysis
  • GATA-1 overexpressing ES clones can contribute to the definitive erythroid lineage in chimeric mice.
  • X- linked GATA-1 overexpression induces embryonic lethality in male transgenics .
  • FI transgenic females were mated to FVB males and sacrificed from 11.5 dpc to 14.5 dpc, or allowed to go to term. Embryos were dissected to leave yolk sacs intact. Representative wild-type males (wt) and transgenics (tg) of either sex are shown.
  • GATA-1 transgene expression in the fetal liver is subject to X-inactivation.
  • Fetal liver cells were scored as proerythroblasts (large, faintly basophilic) , basophilic erythroblasts (medium sized and basophilic) , polychromatic erythroblasts (medium sized, basophilic and benzidine positive) , orthochromatic erythroblasts (small and benzidine positive) or enucleated cells .
  • TER119 erythroid precursors
  • GATA-1 overexpressing CFU-Es fail to differentiate and undergo apoptosis in vi tro .
  • Single cell suspensions of fetal livers from 12.5 dpc embryos were grown for three days in methylcellulose cultures in the presence of Epo . Colonies were then harvested and stained with R-PE-conjugated TER119 antibody and PI.
  • GATA-1 overexpressing cells contribute to and differentiate normally in female transgenic mice.
  • Female GATA-1 transgenic mice were crossed with male Btk lacZ mice to produce double transgenic females. Bone marrow from adult animals was stained with fluorescein-di-b-D- galactopyranoside (FDG) for lacZ activity, biotin-conjugated ER-MP20 antibody (Tricolor-streptavidin secondary antibody) , R-PE-conjugated TER119 antibody and PI.
  • FDG fluorescein-di-b-D- galactopyranoside
  • the plot on the left shows the gate that selects the low side scatter and ER-MP20 " cells that are shown in the right-hand plot of TER119 staining versus forward scatter.
  • Representaive histograms of the lacZ staining of the FSC h ⁇ gh TER119 + cells (as defined by the gate shown in the right-hand plot) in wild-type, male Btk lacZ , female heterozygous Btk lacZ/+ and GATA-1 transgenic female heterozygous Btk lacZ/+ mice is shown. Percentage of lacZ positive cells in each sample is indicated. O 00/34329
  • GATA-2/estrogen receptor chimera arrests erythroid differentiation in a hormone-dependent manner. Genes & Dev. 7: 1097-109. Chapman, V.M. , D.A. Stephenson, L.J. Mullins, B.T. Keitz, C. Disteche and S.H. Orkin. 1991. Linkage of the erythroid transcription factor gene (Gf-1) to the proximal region of the X-chromosome of mice. Genomics 9 : 309-313.
  • Hemoglobin synthesis in murine virus-stimulated leukemic cells in vitro Stimulation of erythroid differentiation by dimethyl sulfoxide. Proc . Natl . Acad . Sci . USA 68: 378-382.
  • Tumor necrosis factor (TNF) -alpha directly inhibits human erythropoiesis in vitro: role of p55 and p75 TNF receptors. Blood, 85, 989-996.
  • GATA-1 Transcription factor GATA-1 permits survival and maturation of erythroid precursors by preventing apoptosis. Proc . Natl . Acad . Sci . USA 92: 9623-9627.

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Abstract

L'invention concerne le domaine des troubles hématopoïétiques, notamment la régulation ou la prolifération et/ou la différentiation de lignées cellulaires spécifiques des cellules d'origine hématopoïétique. L'invention se rapporte notamment à la prolifération et/ou à la différentiation des cellules érythroïdes. Elle concerne, entre autres, un procédé pour stimuler la différenciation d'érythroïdes des érythrocytes dont la différentiation d'érythroïdes a été perturbée ou freinée. Ce procédé consiste à mettre les cellules en contact avec des érythrocytes capables de la différentiation d'érythroïdes ou avec au moins une substance sécrétée par ces cellules et capable de différentiation d'érythroïdes.
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