Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Culture medium – per se
Reexamination Certificate
1999-06-25
2002-03-26
Clark, Deborah J. R. (Department: 1633)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Culture medium, per se
C435S069100, C435S325000, C435S070100, C435S405000, C530S350000, C536S023500
Reexamination Certificate
active
06361998
ABSTRACT:
BACKGROUND OF THE INVENTION
Erythropoiesis
Erythropoiesis is the production of red blood cells. Under normal physiological conditions, erythropoiesis is principally regulated by erythropoietin (Epo), a hormone produced by the kidney in response to hypoxia. Erythropoietin, produced by the renal peritubular endothelium, circulates to the bone marrow where it stimulates committed stem cell progeny called erythroid progenitors to produce red blood cells.
Two distinct types of erythroid progenitors have been identified based on their abilities to form morphologically recognizable colonies when grown in semi-solid media such as methylcellulose. The burst forming unit-erythroid (BFU-E) represents the earliest identifiable progenitor fully committed to erythropoiesis. The BFU-E forms large multi-lobular hemoglobinized colonies, possesses a capacity for self-renewal and most (80-90%) are quiescent. BFU-E differentiate to give rise to the colony forming unit-erythroid (CFU-E). The CFU-E is a more differentiated erythroid progenitor which forms smaller hemoglobinized colonies and lacks the capacity of self-renewal. The majority of CFU-E are actively dividing. As BFU-E differentiate into CFU-E there is a loss in the expression of the primitive stem cell surface glycoprotein CD34, and an increase in the expression of the erythropoietin receptor (EpoR) and the transferrin receptor (CD71). Although BFU-E express low numbers of receptors for erythropoietin, they are stimulated by Epo to proliferate and differentiate into CFU-E which, in turn, express higher levels of the Epo receptors.
Erythroid Cell Proliferation and Differentiation
Erythroid proliferation and the differentiation beyond the CFU-E stage is dependent upon erythropoietin and is characterized by the expression of the red blood cell membrane protein glycophorin A, the accumulation of additional erythroid-specific membrane proteins, and the induction of hemoglobin synthesis. The later stages of erythroid differentiation are best characterized by the accumulation of hemoglobin, which accounts for approximately 95% of the protein present in the mature red call. Erythropoietin-stimulated hemoglobin synthesis is normally coordinated within differentiating red cell precursors so that the synthesis of the constituent alpha and beta globin chains is concurrent with the synthesis of heme.
Globin genes, as well as other genes encoding multiple enzymes along the heme synthesis pathway are transactivated by the major erythroid transcription factor, GATA-1, which is expressed following the activation of the Epo receptor by the binding of Epo. Whether Epo will support primarily erythroid differentiation or proliferation appears to depend on the concentration of Epo and the status of the cell cycle. Low concentrations of Epo support &bgr;-globin production and prolong the G1 phase of the cell cycle, whereas higher Epo concentrations promote cell proliferation and shorten the G1 phase.
Erythropoietin Receptor
Erythropoietin stimulates erythroid proliferation and differentiation by interacting with a specific receptor expressed almost exclusively on erythroid progenitors. The murine and human EpoR genes and cDNAs have been cloned (D'Andrea et al. (1989) Cell 57:277, Winkelmann et al (1990) Blood 76:24, Jones et al (1990) Blood 76:31). Sequence analysis of the isolates cDNAs revealed that the murine and human EpoRs are 507 and 508 amino acids long respectively, sharing an overall 82% amino acid identity. The topology of the EpoR is such that there is an amino terminal extracellular domain consisting of 226 amino acid (after cleavage of the 24 amino acid signal peptide), a 22 amino acid transmembrane domain and a 236 amino acid intracellular domain. The EpoR is a member of the cytokine receptor superfamily and possesses the characteristic pentapeptide WSXWS motif along with four conserved cysteine residues within the extracellular domain.
The binding of erythropoietin to the EpoR results in the phosphorylation of the intracellular tyrosine kinase, JAK2, which, in turn, phosphorylates several intracellular proteins including STAT5, PI3 kinase and vav. Evidence suggests that activation of second messengers by phosphorylation contributes to the Epo-induced proliferative response; however, the molecular basis which determines whether an erythroid cell will either proliferate or differentiate in response to Epo is unknown.
Characterization of EpoR Mutations
Various mutations have been described which render the murine EpoR either hypersensitive to Epo or constitutively active. Most studies into the functionality of the mutated EpoRs have been conducted using the BaF3 cell line. BaF3 cells are a murine IL-3-dependent pre-B cell line. These cells can be rendered IL-3-independent by over-expressing the EpoR and supplanting murine IL-3(“mIL-3”) with human Epo. Using this model, a frame-shift mutation resulting in the replacement of the C-terminal 42 amino acids of EpoR with Ala-Leu was shown to render the murine EpoR hypersensitive (Yoshimura et al (1990) Nature 348:647). This truncated EpoR, when expressed in BaF3 cells, is 3-5 times more responsive to Epo than the wild-type EpoR. It has been demonstrated that this C-terminal truncation removes a negative regulatory domain from the intracellular domain of the EpoR (Klingmuller et al (1995) Cell 80:729-738, D'Andrea (1991) Mol Cel Biol 11:1980). Normally, the hematopoietic protein tyrosine phosphatase SH-PTP1 docks to the C-terminal, dephosphorylating and inactivating JAK2 and thereby decreasing the signalling of the activated EpoR. Removal of this C-terminal negative regulatory domain prevents SH-PTP1 from binding to the EpoR thus resulting in prolonged signalling due to the delayed inactivation of JAK2. Transgenic mice have been generated which express a C-terminal truncated hypersensitive EpoR under the control of the &bgr;-actin promoter (Kirby et al (1996) Proc Natl Acad Sci 93:9402). Phenotypically the trangenic mice were normal; however, upon treatment with exogenous Epo there was a marked increase in pluripotent, clonogenic hematopoietic cells (CFU-S) in the transgenic mice as compared to the normal controls. CFU-S are pluripotent hematopoietic progenitors which give rise to granulocytes, erythroid cells, macrophage and megakaryoctes. The number of committed erythroid progenitors (BFU-E and CFU-E) were not significantly different between the transgenic and control mice.
A constitutively active form of the murine EpoR (but not the human EpoR) has also been previously identified. A point mutation whereby Arg129 (position is relative to the putative amino terminus at residue 25), which resides within the extracellular domain of the murine EpoR, is replaced with a Cys moiety (EpoR(R129C)) rendering this receptor constitutively active. Over-expression of EpoR(R129C) permits cytokine-independent growth of BaF3 cells and renders these cells tumourigenic in nude mice (Yoshimura et al (1990) Nature 348:647). Mechanistically it is thought that the R129C mutation within the murine EpoR renders it constitutively active by allowing the receptors to dimerize. Similarly, mutation of either Glu132 or Glu133 (position is relative to the putative amino terminus at residue 25) to a Cys residue within the extracellular domain of the murine EpoR also results in a constitutively active EpoR (Watowich et al (1994) Mol Cell Biol 14:3535). A truncated murine EpoR containing an R129C mutation has also been identified and is constitutively active (Yoshimura et al (1990) Nature 348:647).
In vivo studies whereby the env gene of the spleen focus-forming virus is replaced by EpoR(R129C) have demonstrated that the modified virus induces transient thrombocytosis and erythrocytosis in infected mice and that the EpoR(R129C) stimulates the proliferation of committed megakaryocytic and erythroid progenitors as well as nonerythroid multipotent progenitors (Longmore et al (1994) Mol Cell Biol 14:2266-2277). Eight different multiphenotypic immortal cell lines, including primitive erythroid, lymphoid and monocytic cells, were is
Bell David N.
Matthews Kathryn Emma
Mueller Susan G.
Bereskin & Parr
Chen Shin-Lin
Clark Deborah J. R.
Gravelle Micheline
Hemosol Inc.
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