DIFFERENTIATION SIGNAL

Abstract: The present invention relates to the field of haematopoietic disorders, in particular to the regulation or proliferation and/or differentiation of specific cell-lineages of cells of haematopoietic origin. The invention relates specifically to the proliferation and/or differentiation of erythroid cells. 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.

Description: Title: Differentiation signal The present invention relates to the field of haematopoietic disorders, in particular to the regulation or proliferation and/or differentiation of specific cell- lineages of cells of haematopoietic origin. The invention relates specifically to the proliferation and/or differentiation of erythroid cells. 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 α-thalassemia, β-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. Several factors that regulate processes of proliferation followed by switches that initiate differentiation are known. It is for example known that proteins belonging to the GATA family are involved in regulating tissue-specific proliferation and absence of GATA factors may induce a default apoptotic pathway instead of differentiation. The 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. 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. Furthermore, it is generally understood that erythroid precursor cells have an intrinsic requirement for functional retinoblastoma protein (pRb) . 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. It has previously been postulated that 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 . Paradoxically, however, pRb is in general not considered to play an intrinsic role in the differentiation of eryrthroid cells, since generation of chimeric mice has demonstrated that pRb null cells (although being devoid of pRb) can contribute to the definitive erythroid lineage and differentiate normally, suggesting that loss of pRb affects an unknown stromal cell type present in the foetal liver which is required to support the differentiation of erythroid cells. Furthermore, 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. In general terms, 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. Herein we demonstrate that 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 . We show that GATA-1 overexpression accelerates Gl to S phase transition, activates hyperphosphorylation of pRb and blocks induced erythroid differentiation in vi tro . Using X- inactivation of a GATA-1 transgene and chimaeric animals, we show that GATA-1 overexpressing cells differentiate normally in the presence of wild-type erythroid cells in vivo . Thus, erythroid GATA-1 overexpression recapitulates the non- autonomous defect in pRb null cells. Herewith, 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. O 00/34329 As further explained in detail in the experimental part of the description, the invention provides a substance (herein also called REDS) , derived from cells capable of erythroid differentiation, regulating non-autonomous erythroid differentiation. Although the not-autonomous effect of inactivity of pRb has been assumed to be due to an effect on stromal cells, said signal overrides or bypasses the lack of functional retinoblastoma protein (pRb) , at least in those cases wherein said impaired or hampered cells are impaired or hampered because of the lack of functional retinoblastoma protein, for example because of overphosphorylation of retinoblastoma protein. In a preferred embodiment, 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. Overexpression of 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. When brought in contact with capable cells (or at least one substance derived thereof) , such as wild-type erythroid cells, for example in vivo in chimeric mice or in vi tro, via co-cultivation with capable cells, such ES-cells differentiate into definitive erythrocytes, i.e. the differentiation capacity is restored. Furthermore, the invention provides a method for inhibiting proliferation of erythroid cells, comprising contacting said cells with erythroid cells capable of erythroid differentiation or at least one substance secreted by said cells capable of erythroid differentiation. For example, proliferating erythroid precursors can be stopped in their proliferating activity by a method according to the invention. Said cells can especially be stopped by method according to the invention when said erythroid cells to be inhibited are incapable of differentiation and/or show (putative) malignant proliferation. 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. For example, it is now possible to expand erythroid cells in in vi tro culture systems to be used for autologous and heterologous transplants and blood transfusions by neutralizing REDS activity and to induce terminal differentiation of such cells with REDS. Said substance normally autoregulates the balance of proliferation and differentiation of erythroid cells. The activity of a substance such as REDS becomes for example apparent in experiments as herein further described in the experimental part of this description studying differentiation of cells with sustained expression of GATA-1, bypassing the downstream effect of GATA-1 on pRb activity. Furthermore, the invention provides a substance further characterised in that it is capable of triggering of enucleation (which is an essential last step in erythroid differentiation) . Enucleation occurs very inefficiently in Rb-null and GATA-1 overexpressing cells, and said substance as provided by the invention is capable of triggering enucleation, for example in these cells. Furthermore, REDS for example triggers enucleation of erythroid cells, enucleation of haematopoietic cells or enucleation of common cells . In a preferred embodiment, the invention provides a substance capable of inducing erythroid differentiation in erythroid cells impaired or hampered in said differentiation, wherein said substance is obtainable from wild-type erythroid cells. Said substance, herein also called REDS, can for example be isolated from tissue such as foetal calf liver, from adult mouse spleen, adult mouse bone marrow, human bone marrow, from tissue-culture supernatants derived from primary cultures of erythroid cells isolated from wild-type mouse and/or human foetal liver, spleen or bone marrow. The molecular composition of purified REDS is determined by gel electrophoresis, protein sequencing and mass spectrometry . 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. Furthermore, the invention provides a substance capable of inhibiting proliferation of erythroid cells, said substance being obtainable from wild-type erythroid cells. For example, proliferating erythroid precursors can be stopped in their proliferating activity by a substance, such as REDS, according to the invention. Also, 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. REDS is for example isolated from E12.5 wild-type foetal mouse liver extracts. It is purified and characterised biochemically from tissue, cells and/or supernatant using affinity chromatography (i.e. on heparin agarose columns), ion exchange chromatography (i.e. on mono-S and mono-Q columns) and/or size fractionation (i.e. on sephadex columns) . In particular, the invention provides a substance, such as REDS, capable of inhibiting proliferation of erythroid cells, said substance being characterised in that it is secreted by erythroid cells capable of inducing differentiation in erythroid cells impaired or hampered therein. In a preferred embodiment, 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. Alternatively, 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-α, 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. In a preferred embodiment, 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 also provides the use of the REDS gene or the REDS coding sequence in gene therapy of genetic and acquired diseases. Herewith the invention for example provides the use of genomic sequences, or functional fragments thereof, of the REDS locus to create an autoregulatory loop in erythroid cells or in haematopoietic cells. Furthermore, the invention provides the use of genomic sequences, or functional fragments thereof of the REDS locus to express transgenes in erythroid cells or in haematopoietic cells . Furthermore, 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. For example, 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. Such 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. Death receptor ligands, such as FasL, TRAIL and TNF-α, 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. Thus, in a preferred embodiment, the invention provides use of REDS substances such as death receptor ligands, such as FasL, TRAIL and TNF-α, 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-α, can be used alone, or in combination, for said treatment. 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. The invention is further explained in the experimental part of this description without limiting the invention. Experimental part. The mechanism by which the cell cycle is regulated during the proliferation and differentiation of specific cell lineages is poorly understood. By examining the role of the GATA-1 protein during erythroid differentiation, this description discusses the function of a lineage-restricted factor in regulating cell cycle progression. 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) . Although this demonstrates that 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). In mammals, 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) . The integrity of (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) . However, transcription of putative 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) . Also, expression of 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) . Certainly, 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). 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. However, 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. Gain-of-function experiments have demonstrated that 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). The level of cyclin proteins regulates Gl to S phase transition (reviewed in Sherr, 1996) . 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. Thus, 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. Different cyclins are active at different stages of the cell cycle. A, D and E-type cyclins are important in Gl to S phase progression. These cyclins regulate specific cdks. D-type cyclins bind and activate cdk4 and cdk6 , while A and E-type cyclins activate cdk2. High levels of cyclin-dependent kinase inhibitors (ckis) , which include pl6, pl8, pl9, p21 and p27, downregulate the kinase activity of cyclin/cdk complexes (reviewed in Sherr and Roberts, 1995) . 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) . We have previously shown that MEL cells proliferating in response to GATA-1 contain elevated levels of hyperphosphorylated pRb (Whyatt et al . , 1997) . However, this effect may merely reflect the fact that GATA-1 overexpressing cells fail to stop proliferating in response to DMSO. To determine if GATA-1 actually activates Gl to S phase progression and regulates the activity of cell cycle proteins such as pRb, the effect of GATA-1 on cell cycle progression in MEL cells was examined in detail. O 00/34329 14 We were unable to produce a line of transgenic mice overexpressing GATA-1 in the erythroid lineage (D.J.Whyatt, unpublished data) . If GATA-1 overexpression results in lethality by directly activating pRb phosphorylation (ie. inactivating pRb function) , one would expect such a transgenic line to resemble the pRb knockout. Loss of pRb results in the reduction in circulating definitive erythroid cells and death in utero (Clarke et al . , 1992; Jacks et al . , 1992; Lee et al . , 1992) . Interestingly this effect is non- autonomous, since pRb null cells contribute efficiently to the definitive erythroid lineage in a wild-type environment (Maandag et al . , 1994; Williams et al . , 1994). From this, we reasoned that a transgenic mouse overexpressing GATA-1 in a subset of the erythroid lineage could be viable. If the subset of cells overexpressing GATA-1 failed to terminally differentiate, an animal might survive on a partially effective erythroid lineage. Alternatively, if GATA-1 overexpression results primarily in the inactivation of pRb, it is possible that GATA-1 overexpression could also result in a non-autonomous effect on erythroid differentiation. To investigate these possibilities, we exploited the normal process of X-inactivation (reviewed in Kuroda and Meller, 1997) to generate a transgenic line of mice that overexpressed GATA-1 both in a pancellular and a heterocellular manner. An X-linked GATA-1 transgene should be transcriptionally silenced in approximately 50% of differentiating erythroid precursors in XX cells, leading to a subset of the erythroid cells overexpressing GATA-1. However, males should overexpress GATA-1 pancellularly. The effect of GATA-1 overexpression on erythroid differentiation in these two situations was then examined. Results Overexpression of GATA-1 in MEL cells. 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. The rate of growth of control MEL cells, after induction with DMSO, compared to GATA-1 overexpressing cells and the cell cycle distribution of these cultures is shown in Figures 1A and IB. After one day in the presence of DMSO, both control and GATA-1 overexpressing cells are more than 90% in the Gl phase of the cell cycle. At day 2 of induction, a proportion of both control and GATA-1 overexpressing cells have undergone Gl to S phase progression. However, alongside no detectable increase in the number of cells at day 2, a significantly higher number of GATA-1 overexpressing cells are in S phase at day 2 compared to controls (35% versus 18%) . Subsequently, 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. However, the cell cycle distribution of GATA-1 overexpressing cells never returned to a profile identical to that of uninduced cells. While control MEL cells induce erythroid markers such as β ma:ι 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. To investigate the activation of Gl to S phase transition in GATA-1 overexpressing MEL cells, the levels of GATA-1 and the cell cycle regulating proteins during DMSO induction were determined by Western blot analysis (Figure 1C) . 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. At day 1 of DMSO induction, pRb is virtually completely hypophosphorylated, consistent with the observed Gl arrest. A minor proportion of pRb then becomes phosphorylated from day 2 in control cells. However, a marked increase in hyperphosphorylated pRb (and a decrease in hypophosphorylated pRb) is detectable at day 2 in GATA-1 overexpressing MEL cells. From day 2, the difference in pRb phosphorylation between controls and GATA-1 overexpressors persists. The pRb related protein pl07 is also hypophosphorylated at day 1. At day 2 in control cells, pl07 returns to a hyperphosphorylated state, then progressively becomes hypophosphorylated from day 3 to day 4 of induction. In GATA-1 overexpressing cells, 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. To determine if activation of Gl to S phase progression and pRb phosphorylation could be due to activation of a specific cyclin, the levels of the cyclin proteins was then examined (Figure 1C) . MEL cells do not express cyclin Dl (Kiyokawa et al . , 1994), but do express D2 and D3. Cyclin D2 levels are gradually downregulated during the induction of control cells. GATA-1 overexpressing cells show a similar pattern of cyclin D2 expression up until day 2. From day 3 onwards, cyclin D2 levels increase in GATA-1 overexpressing cells. Cyclin D3 levels are downregulated in both control and GATA-1 overexpressing cells at day 1, followed by an increase at day 2 in both populations. This level of cyclin D3 then persists in control cells at day 3 and day 4. However, cyclin D3 levels decrease at day 3 and day 4 in GATA-1 overexpressing cells. Cyclin E levels do not markedly change in control cells during DMSO induction. However, in GATA-1 overexpressing cells, cyclin E levels increase from day 3 of induction. Unlike the single cyclin A gene found in humans, mice have two cyclin A genes. Cyclin Al is germ cell specific, while cyclin A2 is expressed in somatic tissue and has the highest sequence identity with human cyclin A (Sweeney et al . , 1996) . Cyclin A2 is downregulated at day 1 of induction in both control and GATA-1 overexpressing cells. However, GATA-1 overexpressing cells consistently express higher levels of cyclin A2 at day 1 of induction, prior to any detectable increase in the number of cells in S phase. In control cells, cyclin A2 levels increase weakly at day 2, but are still low compared to pre-induced levels. In contrast, GATA-1 overexpressing cells induce cyclin A2 levels at day 2 to pre-induced levels. This level of cyclin A2 then persists. Taken together, up until day 2 of DMSO induction, the levels of all cyclin proteins examined are identical in control and GATA-1 overexpressing MEL cells with the exception of cyclin A2. No significant effect on the level of the cyclin- dependent kinase inhibitors pl6 (Figure 1C) , pl8 or pl9 levels (data not shown) is detected either during DMSO induction or in response to GATA-1 overexpression. The level of p27 is markedly increased at day 1 of induction in both control and GATA-1 overexpressing cells, before reduction in both cases from day 2 (Figure 1C) . Thus, the high number of cells in Gl at day 1 correlates with high p27 levels. In summary, activation of 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. Generation of GATA-1 overexpressing transgenic mice To test if GATA-1 overexpressing cells could contribute to the mature erythroid compartment in chimeric mice, male ES cell clones containing a modified version of PEV3 -GATA-1 (where the neomycin-resistance cassette was replaced with the puromycin-resistance cassette) 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) . 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. In clone G4 , 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) . Eight chimeric 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. Analysis by DNA-fluorescence in-situ hybridization (FISH) and karyotyping demonstrated that this animal contained two X chromosomes (one with the transgene integration) and one Y chromosome (data not shown) , so it presumably arose from a karyotypically abnormal XY sperm in the chimeric male. FI females were then mated to FVB males and sacrificed at various time points during pregnancy or allowed to go to term. At 11.5 days post coitum (dpc), 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. However, beginning at 12.5 dpc, male transgenics display an anaemic phenotype . No other abnormalities are detectable. Subsequently, 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) . Expression of the transgene-derived GATA-1 in mice Figure 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. Since the protein expression data suggested that X- inactivation of transgene expression was indeed occurring in the transgenic females, we wished to confirm this by examining RNA expression. We performed RNA-FISH on 12.5 dpc fetal liver samples using GATA-1 and Xist probes (Figure 3B) . In 12.5 dpc fetal livers of both wild-type and GATA-1 overexpressing mice, approximately 80% of the cells are erythroid (Figure 4D) . GATA-1 transcription, appearing as a bright nuclear dot, is detectable in 66% (+/-3%, n=3) of cells from male transgenic fetal livers. This suggests that the majority of erythroid precursors in the fetal liver of transgenic males are expressing the GATA-1 transgene. The number of GATA-1 positive cells decreases to 32% (+/-2%, n=3) in female transgenic fetal liver cells. No GATA-1 RNA is detectable in wild-type littermates, suggesting that endogenous transcript levels are lower than the sensitivity of this assay. The decrease in GATA-1 positive cells in the female transgenic fetal liver cells is consistent with protein expression data and suggests that females are expressing the transgene in a heterocellular manner. Co-staining for Xist RNA shows a single punctate Xist signal in male fetal liver cells, corresponding to transcription from the Xist allele on the active X chromosome. Furthermore, female fetal liver cells also contain a large area of nuclear staining, corresponding to accumulated Xist RNA present on the inactive X chromosome (or Barr body) (Sheardown et al . , 1997) . It has been demonstrated that nascent transcripts from X-inactivation sensitive genes apparently co- localize with an Xist positive Barr body in less than 4% of differentiated ES cells (Clerc and Avner, 1998) . An Xist positive Barr body is detectable in approximately 50% of GATA-1 positive cells in female transgenics (Figure 3B) . Apparent co-localisation of these two signals occurs in less than 5% (n=3, >150 cells counted per sample) of such cells. If the GATA-1 transgene were escaping X-inactivation in females, then the GATA-1 positive signal would be expected to co-localise with the Barr body in close to 50% of such double positive cells. Since this is not observed, the data is consistent with GATA-1 transgene expression being restricted to the active X chromosome. We conclude that expression of the X-linked PEV3 -GATA-1 transgene is subject to X-inactivation. Pancellular overexpression of GATA-1 in transgenic males apparently results in the downregulation of endogenous GATA-1. Endogenous GATA-1 protein levels are also detectably downregulated in transgenic females, even though transgene- derived GATA-1 is expressed heterocellularly . This suggests that GATA-1 is negative regulator of its own expression in vivo . Phenotype of GATA-1 overexpressing mice The fetal livers of transgenic mice appeared normal at 12.5 dpc and in embryos that had not died at 13.5 dpc (Figure 4A) . To examine the apparent defect in erythropoiesis in the male transgenics, cytospins of blood and disaggregated fetal livers were prepared and stained with neutral benzidine and a modified Giemsa-like stain (Figure 4B) . At 12.5 dpc, the majority of the circulating erythrocytes are primitive nucleated cells. Compared to wild-type mice, the primitive erythrocytes of the transgenic males have a more open chromatin-staining pattern. This mild phenotype is presumably not affecting the function of these cells in maintaining the development of the embryo up to 12.5 dpc. Female transgenic animals display an intermediate phenotype, in that a proportion of the cells has an open chromatin-staining pattern. In wild-type animals, definitive enucleated erythrocytes have significantly replaced primitive cells at 13.5 dpc and 68% (+/-5%, n=4 , >250 cells counted per sample) of the erythrocytes are enucleated. However, in transgenic males definitive enucleated cells are reduced to 36% (+/-5%, n=3) of the circulating erythroid cells. Female transgenics have normal numbers of enucleated cells at 13.5 dpc, ie. 68% (+/-2%, n=3) . Furthermore, nucleated cells morphologically similar to definitive erythroid precursors, which are virtually absent in wild- type animals, can be found in the circulation of male transgenic (approximately 3% of all cells) and rarely in female transgenic mice (<1% of all cells) . 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) . However, the proportions of these precursors are altered in transgenic male animals compared to wild-type animals. Using the number of proerythroblasts as an internal standard, 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 . To confirm that there was an alteration in the differentiation profile of erythroid cells in transgenic males, 12.5 dpc fetal livers were then disaggregated and analysed by fluoresence-activated cell sorting (FACS) . 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) . However, the size distribution of TER119 + /PI " cells, as measured by forward scatter profile, is significantly different in transgenic males compared to wild-type animals. The proportion of such viable erythroid cells with higher than the median wild-type forward scatter value in the transgenic males is 60% (+/-3%, n=7) (Figure 4D and 4E) . This is consistent with an increase in the number of early (larger) precursor cells relative to late (smaller) precursors. Female transgenics have a distribution of cell size that is not significantly different from wild-type samples (Figure 4D) . The anaemia in male transgenics appears to be due a failure of the definitive erythroid precursors to differentiate efficiently beyond the basophilic erythroblast stage. However, 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. However, there is variability in the distribution of erythroid precursors in female transgenics, suggesting that heterocellular overexpression of 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. Colony forming uni t -erythroid assay To investigate the differentiation profile of GATA-1 overexpressing erythroid precursors in vi tro, fetal livers were dissected from 12.5 dpc embryos and then disaggregated and plated in methylcellulose in the presence of Epo (Wong et al . , 1986) . Colonies were grown for three days and then counted. No significant change in the number of colony- forming unit-erythroid (CFU-E) colonies is observed in the transgenic samples compared to controls (data not shown) . This suggests that differentiation to the CFU-E precursor stage (thought to be roughly analogous to the proerythroblast, reviewed in Quesenberry, 1983) is unaffected. Colonies grown for three days were harvested, stained with TER119 and PI then FACS analysed as above. 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) . Although the entire range of cell size is represented in both, the peak of small cells found in wild-type samples is dramatically reduced. Furthermore, 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. Instead, 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) . This profile is identical to that found when wild-type and transgenic male fetal liver cells are mixed 1:1 and co- cultured in a CFU-E assay (data not shown) . Apparently, there is a disruption in the differentiation of 50% of the CFU-Es derived from female fetal livers. Compared to male transgenics, proportionally half of erythroid precursors in female transgenic 12.5 dpc fetal livers overexpress GATA-1 (see above) . Therefore, this indicates that GATA-1 overexpressing erythroid precursors fail to undergo effective differentiation when disaggregated and plated as individual cells in methylcelllulose . Fa te of GATA-1 overexpressing cells in adul t females To determine the contribution of GATA-1 overexpressing cells to the erythroid compartment in adult females, GATA-1 transgenic females were bred with male Btk lacZ mice. 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). Male 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%. Females transgenic for both the GATA-1 transgene and the Btk lacZ insertion were compared with females containing the Btk lacZ insertion alone. As shown in Figure 6A and 6B, 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. This suggests that when there is a distortion in the X-inactivation pattern in the early pluripotent precursors, this is reflected in the number of GATA-1 overexpressing cells. Thus, there appears to be a normal representation of cells with an active lacZ gene in the early erythroid compartment of GATA-1 overexpressing females and we conclude there is no selection for or against GATA-1 overexpressing cells at this stage in erythroid differentiation. No change in the size distribution of the erythroid cells in the bone marrow is detectable (data not shown) . Thus, unlike in the male GATA-1 transgenics, 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. Function and regulation of GATA-1 during erythropoiesis Our data indicates that endogenous GATA-1 protein levels are downregulated in response to high levels of exogenous GATA-1 in both transformed proerythroblasts and transgenic mice. However, it has previously been shown that GATA-1 can activate its own transcription in transient transfection assays (Hannon et al . , 1991; Nicolis et al . , 1991; Schwartzbauer et al . , 1992). This suggests that although GATA-1 may be involved in activating its own transcription, at peak levels GATA-1 negatively regulates its own expression. Although we have previously shown that GATA-1 can downregulate endogenous GATA-1 mRNA levels (Whyatt et al . , 1997), this 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 activation of Gl to S phase transi tion 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) . It is not known how DMSO induces a transient Gl arrest, yet we suggest that the induction of p27, accumulation of both hypophosphorylated pRb and hypophosphorylated pl07 and the reduction in cyclin A2 levels that we have observed all play a role . Activation of Gl to S phase transition in response to GATA-1 is associated with the hyperphosphorylation of pRb. pRb regulates Gl to S phase transition (Goodrich et al . , 1991; Qin et al . , 1992). Phosphorylation of pRb is thought to activate Gl to S phase transition by disrupting pRb/E2F complexes and is considered synonymous with inactivation of pRb function (reviewed in Sherr, 1996) . Therefore, we wished to establish a link between GATA-1 and a regulator of the cell cycle apparatus known to activate pRb phosphorylation. 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) . Cyclin A2 accumulates as S phase progresses and cyclin A2/kinase complexes are thought to maintain the phosphorylation status of pRb during S phase (reviewed in Sherr, 1996) and play a role in G2 to M phase transition (Pagano et al . , 1992) . Therefore, the high level of cyclin A2 at day 2 of induction in GATA-1 overexpressing cells may be due to the higher number of cells in S phase relative to controls. However, cyclin A2 is already elevated in GATA-1 overexpressing cells at day 1 of induction, when both control and GATA-1 overexpressing cells are more than 90% in Gl . It is possible that the increase in cyclin A2 levels at this time reflects an undetectable difference in the percentage of cells already in S phase. However, there is biochemical evidence that increasing cyclin A2 levels activate pRb phosphorylation in late Gl, arguing that the increase in cyclin A2 is not merely the consequence of the increase in S phase cells. Both cyclin A2/cdk2 and cyclin Dl/cdk4 kinase activities are negatively regulated by the cki p27 (Toyoshima and Hunter, 1994) , which we show to be markedly elevated at day 1 of induction. 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. Suggesting a direct effect of cyclin A2/cdk2 on pRb/E2F complexes, it has been shown that 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 . Since cyclin A2 regulates both pRb phosphorylation and Gl to S phase transition, we propose a role for cyclin A2 in the pathway linking GATA-1 to the activation of pRb phosphorylation and Gl to S phase transition. 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). 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. Recapi tulation of pRb knockout phenotype by GATA-1 overexpress! on The effect of loss of pRb on erythropoiesis is remarkably similar to the effect we observe in the overexpression of GATA-1. Maximal expression of pRb in the developing fetal liver occurs between 12.5 and 15.5 dpc (Jiang et al . , 1997) and is upregulated during commitment of haematopoietic progenitor cells to the erythroid lineage (Condorelli et al . , 1995). Along with neurogenic defects, 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) . As in GATA-1 transgenic males, 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). Analysis of the CFU-E colonies derived from the fetal livers of pRb null mice revealed an increase in the number of immature precursors, at around the erythroblast stage (Jacks et al . , 1992) , similar to the stage we have identified as expanded in male GATA-1 transgenics. Such pRb null CFU-E colonies eventually lyse and disappear after five days in culture, presumably undergoing apoptosis (Jacks et al . , 1992) . Similarly, GATA-1 overexpressing CFU-E colonies are undergoing apoptosis after three days in culture. However, pRb is not considered to play an intrinsic role in the differentiation of erythroid cells, since generation of chimeric mice has demonstrated the pRb null cells can contribute to the definitive erythroid lineage and differentiate normally. From this, it has been suggested that loss of pRb affects a cell type present in the stroma of the fetal liver which is required to support the differentiation of erythroid cells (Maandag et al . , 1994; Williams et al . , 1994). 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. Utilization of an X-linked transgene to investigate non -autonomous cellular defects Generation of an 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). Clearly, 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 . Despite prolonged attempts, we were unable to generate a stable line of GATA-1 overexpressing mice via conventional oocyte microinjection (D.J.Whyatt, unpublished results) . However, one other transgenic line expressing a GATA-1 cDNA under the control of LCR/globin promoter elements has been described (Li et al . , 1997). This line appeared difficult to generate, suggesting that this construct could also induce lethality. No significant effect on erythroid differentiation was- observed, possibly due to either low level or heterocellular expression of the transgene. Although heterocellular overexpression of GATA-1 in the erythroid lineage may have no effect, pancellular overexpression is obviously lethal . O 00/34329 32 Male 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. Furthermore, in females, 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 It is unlikely that the effect of GATA-1 overexpression on erythropoiesis 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, arguing 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 33 overexpressing cells to terminally differentiate in vivo (Figure 7, REDS) . These observations have profound implications in interpreting the phenotype of the pRb null chimeric mice. It has been previously postulated that erythroid precursors have an intrinsic requirement for pRb activity in order to undergo normal differentiation (Condorelli et al . , 1995; Zhuo et al., 1995). We have demonstrated that GATA-1 activates the phosphorylation of pRb in erythroid cells. GATA-1 overexpression in the erythroid lineage recapitulates many aspects of the pRb knockout, most notably in being a non-autonomous effect. Although the non-autonomous effect of the pRb knockout on erythroid differentiation has been assumed to be due to an effect on stromal cells, our results now provide an alternative explanation. 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. Based on the data presented here, a model of the role of GATA-1 in regulating erythroid differentiation is shown (Figure 7) . During normal erythropoiesis, GATA-1 maintains the level of cyclin A2 and pRb phosphorylation during the proliferative period of normal differentiation. As GATA-1 activity drops, cyclin A2 levels decrease and pRb dephosphorylation occurs. As a result, 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. However, differentiation capacity is restored in the presence of normally differentiating erythroid cells. We show the existence of a signal emanating from these cells, here tentatively designated REDS (red cell differentiation signal) , which normally autoregulates the balance of proliferation and differentiation. In our experiments, REDS activity becomes apparent as it drives the differentiation of cells with sustained expression of GATA-1, bypassing the downstream effect of GATA-1 on pRb activity. 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. Treatment with varying doses of FasL has no significant effect on the number or viability of the erythroid cells in these cultures. Furthermore, FasL does not affect the differentiation of wild-type (31%, 31%, 34%, 34% of small TER119 positive cells after two days of culture with 0, 10, 40 and 100 ng/ml of FasL respectively, i.e. an increase of 10% at the highest dose tested (n=3)) or transgenic male cultures (an increase of 10% at the highest dose tested (n=3)) . However, a significant dose-dependent increase, instead of the expected decrease, in the number of differentiated erythroid cells was found in female transgenic cultures (18%, 21%, 26%, 26% of small TER119 positive cells after two days of culture with 0, 10, 40 and 100 ng of FasL respectively, i.e. an increase of >40% (n=2)), demonstrating that FasL could reverse the defect induced by GATA-1 overexpression in these cells. That 13.5 dpc male transgenic samples do not respond to FasL suggests the accumulated effect of GATA-1 overexpression in the absence of FasL signalling has irreversibly inhibited erythroid differentiation. Thus, death receptor ligands are components of REDS. To obtain further evidence for the differentiating role of death receptor ligands in erythroid differentiation, we added 200 and 1000 ng/ml of FasL to GATA-1 overexpressing erythroid cells in liquid culture and assessed the viability of the cells after two days. No statistically significant changes in viability were observed. We then added a combination of 1000 ng/ml of FasL, 200 ng/ml of TRAIL and 200 ng/ml of TNF-α and we observed a decrease of 15% (±3, n=4) in the number of viable erythroid cells in wildtype cultures, an 8% decrease in the number of viable erythroid cells in cultures of male transgenic cells (7% and 9%, n=2), but an increase of 15% (±9, n=3) in the number of viable erythroid cells in cultures of transgenic female cells. Thus, the loss of viable erythroid cells found in cultures of transgenic female cells is reversed by death receptor activation, confirming that 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. Male transgenic cells did not significantly respond to death receptor ligands. This again shows that overexpression of GATA-1 in the absence of REDS induces a loss in death receptor responsiveness. Immature erythroid cells are sensitive to the anti-differentiative and apoptosis-inducing effects of death receptor activation and GATA-1 degradation, particularly in the absence of Epo (erythropoietine , De Maria et al . , 1999a, De Maria et al . , 1999b). However, more mature polychromatic and orthochromatic erythroblasts express both death receptors and their ligands (De Maria et al . , 1999a, De Maria et al . , 1999b) . A high endogenous level of receptor activation in these cells may explain why differentiating wild-type erythroid cells do not readily respond to exogenous FasL (De Maria et al . , 1999a) . 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. According to the invention, REDS signalling by death receptor activation regulates both proliferative arrest and/or pyknosis during late erythroid differentiation. Materials and methods Plasmids , probes and primers 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. DNA-FISH and RNA-FISH was performed using a 4.3 kb fragment spanning exon 2 to exon 6 of the genomic GATA-1 gene, a 21 kb human beta-globin LCR probe and a 6.5 kb fragment corresponding to the Xist RNA (gift of N.Brockdorff , London) . Embryos were sexed using PCR primers specific for the Zfy gene (Nagamine et al . , 1989) . MEL cell s 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 μg/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 The puromycin resistant version of PEV3 -GATA-1 was linearized with Pvul and electroporated into 129 0A -derived ES cells as previously described (Pandolfi et al . , 1995) and individual clones were selected in 1 μg/ml puromycin. Mice 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) . Haemoglobin analysis Globin chain representation in chimeric animals was analyzed by Triton-acid-urea electrophoresis as previously described (Maandag et al . , 1994) . DNA and RNA FISH 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 . , 1995) . CFU-E assay 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 μM 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 Flow cytometry analysis of MEL cell DNA content was performed as previously described (Whyatt et al . , 1997) . Single cell suspensions of bone marrow, fetal livers and cultured cells were prepared, stained with FDG and/or antibodies and FACS analyzed as described previously (Hendriks et al . , 1996) . Antibodies used: R-PE-conjugated TER119 (Pharmingen, cat # 09085B) , biotin-conjugated ER-MP20 (de Bruijn et al . , 1994) and TriColor-streptavidin (Calatag Laboratories, CA) . l-5xl0 s cells were scored per sample. Figure legends Figure 1 Cell cycle progression during DMSO-induced differentiation of MEL cell populations stably transfected with control plasmid PEV3 or GATA-1 expression construct PEV3 -GATA-1. (a) Growth rates of MEL cells. The y-axis represents the number of cells per ml medium divided by the number of cells the previous day. Cultures were split to lxlO 6 cells/ml each day. Cells were cultured with 2% DMSO (v/v) from day 0. Each curve represents the average of three independent experiments and the error bars indicate the standard deviation. (b) Bar graph of MEL cells in the Gl , S and G2/M phases of the cell cycle, as determined by flow-cytometry analysis of DNA distribution, at each day of DMSO induction. Each bar represents the average of three independent experiments and error bars indicate the standard deviation. (c) Western blot analysis of GATA-1 and cell cycle regulating proteins. Nuclear extract (lμg) was prepared from MEL cell populations on each day of DMSO induction, subjected.to SDS-PAGE alongside appropriate molecular weight markers, blotted and probed for GATA-1, pRb, pl07, cyclin A2 , cyclin D2 , cyclin D3 , cyclin E, pl6 or p27. Indicated are the positions of myc-tagged transgene-derived GATA-1 ( trans. GATA-1) , endogenous GATA-1 (endo . GATA- 1 ) , hyperphosphorylated pRb (hyper. pRb), hypophosphorylated pRb (hypo.pRb), hyperphosphorylated pl07 (hyper. pl07) and hypophosphorylated pl07 (hypo.pl07) . Each blot is a representative example from three independent experiments . Figure 2 GATA-1 overexpressing ES clones can contribute to the definitive erythroid lineage in chimeric mice. However, X- linked GATA-1 overexpression induces embryonic lethality in male transgenics . (a) Blood from C57bl6 (host-blastocyst , Hb single) , 129 OA (ES cell strain, Hb diffuse) or clone G4 derived chimera number 8 (G4ch#8) was lysed and subject to triton- acid-urea PAGE. The gel was stained with Coomassie blue. Indicated are the positions of the alpha-type globin chains (α) and beta-type globin chains (β) . (b) Cytospins of C57bl6, 129 OLA and G4ch#8 blood, stained with a modified Giemsa-like stain and neutral benzidine . (c) 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. (d) Summary of the observed genotype and phenotype of the embryos and adults . Figure 3 Expression of GATA-1 transgene expression in the fetal liver is subject to X-inactivation. (a) Western blot analysis of nuclear extracts (5μg) from wild-type (wt) and transgenic (tg) 13.5 dpc fetal livers from either sex. As controls, 2.5μg of nuclear extract from day 4 DMSO induced control (PEV3) and GATA-1 overexpressing (PEV3 -GATA-1) MEL cells was included. The position of myc-tagged transgene-derived GATA-1 (trans. GAT - 1) and endogenous GATA-1 (endo . GATA-1) is shown. (b) RNA-FISH on 12.5 dpc disaggregated fetal liver cells. Cells were fixed and stained with FITC-labeled GATA-1 (green) and Texas Red-labeled Xist (red) probes. A representative cell from fetal livers of wild-type (wt) and transgenic (tg) embryos of either sex is shown. Beneath is indicated the percentage of 12.5 dpc fetal liver cells positive for transgene-derived GATA-1 RNA (>300 cells counted per sample) . The number of embryos examined is shown and standard deviations are indicated. Figure 4 Effect of GATA-1 overexpression on erythropoiesis. (a) Morphology of fetal livers of 12.5 dpc and 13.5 dpc embryos, either transgenic (tg) of either sex or male wild- type (wt) . (b) Cytospins of blood and disaggregated fetal livers of 12.5 dpc and 13.5 dpc embryos, either transgenic (tg) of either sex or male wild-type (wt) . Cells were stained with a modified Giemsa-like stain and neutral benzidine. Indicated by the key is the morphology of the various circulating erythroid cells and fetal liver erythroid precursors. 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 . (c) Summary of the number of fetal liver erythroid precursors found in wild-type and transgenic 12.5 dpc embryos. The number of basophilic erythroblasts (b.e.), polychromatic and orthochromatic erythroblasts (pc.e.+oc.e.) and enucleated cells (en.c.) have been normalized to the number of proerythroblasts in each sample (>250 cells counted per sample) . Each data point is the average of three independent embryos and the standard deviation is indicated. (d) Summary of FACS analysis of disaggregated fetal livers from 12.5 dpc wild-type and transgenic embryos of either sex. Cells were stained with R-PE-conjugated TER119 antibody and PI. The percentage of erythroid precursors (TER119) was determined by TER119 staining. The size distribution (size) is represented by the percentage of TER119 + /PI ' cells having a forward scatter (FSC) value higher than the average median forward scatter value of similarly staining wild-type cells. The number of individual embryos examined is shown and standard deviations are indicated. (e) Representative 5% probability contour plots of TER119 staining (log scale) versus FSC (linear scale) of cells in the fetal livers of 12.5 dpc wild-type male and transgenic male embryos, after PI + cells have been gated out. TER119 * erythroid cells were gated and histograms of the forward scatter profile of these cells are shown and the percentage of cells with higher than the average wild-type median FSC is indicated. Figure 5 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. (a) Summary of FACS analysis. TER119 " cells were gated out and then divided into four quadrants based on size (large is FSC>312, arbitrary units) and PI staining. The percentage of cells found in each quadrant in wild- type cultures and GATA-1 transgenic cultures of either sex is shown . (b) Shown on the left are representative 10% contour plots of PI staining (log scale) versus FSC (linear scale) for wild-type and GATA-1 transgenic samples of either sex, after TER119 " cells are gated out . The quadrants used to generate data in Figure 5(a) are shown. The percentage of cells in the PI + /large quadrant for each representative sample is shown. On the right are histograms of counts versus FSC of all cells shown in the contour plots on the left. The percentage of all cells with FSC>312 is indicated for each sample. Figure 6 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. (a) Summary of lacZ staining in large erythroid cells (TER119 + /FSC hιgh ) , medium-size erythroid cells (TER119 + /FSC raedlum ) , the B-cell lineage (ER-MP20 ~ /TER ~ ) , the granulocytic lineage (ER-MP20 + /TER119 " ) and the monocytic lineage (ER-MP20 hιgh /TER119 " ) of wild-type, male Btk lacZ , female Btk lacZ/+ and GATA-1 transgenic female Btk lacZA mice, (b) Representative 5% probability contour plots of adult mouse bone marrow cells. 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 hιgh 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 44 Figure 7 Model of the function of GATA-1, cyclin A2 and pRb during definitive erythropoiesis. Development from a proerythroblast to a differentiating erythroblast is presented as a predominantly proliferative stage where GATA- 1, cyclin A2 and hyperphosphorylated (inactive) pRb levels are high (top) . Terminal differentiation is associated with the downregulation of GATA-1 and cyclin A2 , followed by an increase in hypophosphorylated (active) pRb and Gl arrest. 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Claims: CLAIMS 1. 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 . 2. A method according to claim 1, wherein said impaired or hampered cells are impaired or hampered because of the lack of functional retinoblastoma protein. 3. A method according to claim 1 or 2 , wherein said impaired or hampered cells are impaired or hampered because of overphosphorylation of retinoblastoma protein. 4. A method according to any one of the aforegoing claims wherein said impaired or hampered cells are impaired or hampered because of overexpression of GATA-1 or a functional equivalent or derivative thereof. 5. A method according to anyone of claims 1-4, wherein said capable cells are wild-type erythroid cells. 6. A method for inhibiting proliferation of erythroid cells, comprising contacting said cells with erythroid cells capable of erythroid differentiation or at least one substance secreted by said cells capable of erythroid differentiation. 7. A method according to claim 6, wherein said erythroid cells to be inhibited are incapable of differentiation. 8. A substance capable of inducing erythroid differentiation in erythroid cells impaired or hampered in said differentiation, said substance being characterized in that it is secreted by erythroid cells capable of inducing differentiation in erythroid cells impaired or hampered therein. 9. A substance capable of inducing erythroid differentiation in erythroid cells impaired or hampered in said differentiation, said substance being obtainable from wild-type erythroid cells. 10. A substance capable of inhibiting proliferation of erythroid cells, said substance being obtainable from wild- type erythroid cells. 11. A substance capable of inhibiting proliferation of erythroid cells, said substance being characterised in that it is secreted by erythroid cells capable of inducing differentiation in erythroid cells impaired or hampered therein. 12. A substance according to any one of claims 8-11, which is proteinaceous . 13. Use of at least one substance according to any one of claims 8-12 for interfering with erythroid cell proliferation or differentiation.

Inventors: GROSVELD FRANKLIN GERARDUS, LINDEBOOM FOKKE ALBERT, PHILIPSEN JACOBUS NICOLAAS JOZ, WHYATT DAVID JOHN, GROSVELD, FRANKLIN, GERARDUS, LINDEBOOM, FOKKE, ALBERT, PHILIPSEN, JACOBUS, NICOLAAS, JOZES, WHYATT, DAVID, JOHN
Applicants: GROSVELD FRANKLIN GERARDUS, LINDEBOOM FOKKE ALBERT, PHILIPSEN JACOBUS NICOLAAS JOZ, UNIV ERASMUS, WHYATT DAVID JOHN, ERASMUS UNIVERSITEIT ROTTERDAM, GROSVELD, FRANKLIN, GERARDUS, LINDEBOOM, FOKKE, ALBERT, PHILIPSEN, JACOBUS, NICOLAAS, JOZES, WHYATT, DAVID, JOHN
Application year: 1999
Main classification: C07K14
IPC codes: C12N5