The Oncologist, Vol. 1, No. 1_2, 116–118,
February 1996
© 1996 AlphaMed Press
Special Education
Hiroyuki Kozutsumi, Ph.D.
Pharmaceutical Division Kirin Brewery Co., Ltd. Fax: 81-2-5485-6318
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HEMOPOIETIC FACTORS AND BLOOD CELL PROLIFERATION AND DIFFERENTIATION
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Blood cells are generally classified into three cell lineages: erythrocytes, granulocytes and megakaryocytes. In the bone marrow, pluripotent stem cells differentiate into either the lymphoid stem cell line, where they are further induced to differentiate into B- or T-derived lymphocytes, or the myeloid stem cell (CFU-GEMM) line, where they are further induced to become erythrocytes, granulocytes (neutrophils, eosinophils or basophils), macrophages or megakaryocytes (platelets).
Proliferation and differentiation of blood cells in the bone marrow are regulated by hemopoietic factors. Hemopoietic factors include those that are continuously produced, such as EPO, G-CSF and thrombopoietin (TPO), and those that are produced on demand in response to inflammation and infection, such as IL-3, IL-11 and GM-CSF. In recent years the genes for hemopoietic factors which regulate erythrocytes and granulocytes have been cloned using the techniques of genetic engineering. In 1994 the gene for TPO was cloned. TPO acts specifically on megakaryocytes.
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PROLIFERATION AND DIFFERENTIATION OF ERYTHROCYTIC CELLS
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The earliest cells destined to become erythrocytes which differentiate from the myeloid stem cells (CFU-GEMM) are early phase erythroblast progenitor cells called BFU-E cells. After the BFU-E cells have undergone several divisions, they differentiate into late phase erythroblast progenitor cells called CFU-E cells. After passing through the proerythroblast stage, the CFU-E cells become erythroblasts. Erythroblasts can be confirmed by light microscope as belonging to the erythroid cell line. Erythroblasts mature and become enucleated reticulocytes, which are then released from the bone marrow into the blood, thus becoming mature erythrocytes.
Proliferation and differentiation of the erythroid progenitor cells are regulated by erythropoietin (EPO), which is primarily produced by the kidneys. In 1985 genomic DNA and cDNA for human EPO were cloned, and it was learned that the mature protein is a glycoprotein consisting of 165 amino acids and having a molecular weight of about 30,000.
There is powerful evidence to suggest that EPO is produced by peritubular cells of the renal cortex. When the hematocrit drops for some reason and hypoxia occurs, the number of EPO-producing cells increases and EPO production rises in the kidneys.
CFU-E cells are the main target cells for EPO. EPO receptors are expressed along the lineage from BFU-E cells to proerythroblasts, with peak expression found in CFU-E cells. The EPO receptor, which was cloned in 1989, belongs to the cytokine receptor family, transduces the EPO signal to the interior of the cell, and brings about the proliferation and differentiation of CFU-E cells.
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PROLIFERATION AND DIFFERENTIATION OF GRANULOCYTIC CELLS
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The earliest cells destined to become neutrophils and macrophages which differentiate from the pluripotent stem cells are called granulocyte-macrophage progenitor (CFU-GM) cells. The CFU-GM cells are affected by colony-stimulating factors and become either CFU-G or CFU-M cells. Ultimately, they differentiate into mature neutrophils or macrophages. The main factor stimulating the proliferation and differentiation of neutrophils is the granulocyte colony-stimulating factor (G-CSF). CFU-GM cells are stimulated by G-CSF in the bone marrow, pass through the CFU-G stage, and become myeloblasts, which are the most primitive neutrophils that can be morphologically distinguished. Myeloblasts continue to divide and differentiate, and they mature into neutrophils, which then lose their ability to divide.
Mature neutrophils are not immediately released into the blood, but rather are stored within the bone marrow. Neutrophils that have been released into the blood reside in the marginal granulocyte pool or the circulating granulocyte pool, and they later egress into tissues.
G-CSF is produced by cells such as monocytes, macrophages and bone marrow stromal cells, and its action is almost entirely selective for the proliferation of neutrophils. The cDNA for G-CSF was cloned in 1986, and it was learned that the mature protein is a glycoprotein consisting of 174 amino acids and having a molecular weight of about 20,000. When G-CSF is administered to a patient it causes the release of mature neutrophils from the marrow into the peripheral blood. G-CSF also enhances neutrophil function in the presence of bacterial products, and it acts on mature neutrophils to enhance cellular motility, the production of bioactive oxygen, and microbicidal activity.
The cDNA for the G-CSF receptor was cloned in 1990, and its receptor belongs to the cytokine receptor family. The human G-CSF receptor consists of 813 amino acids and has an approximate molecular weight of 100,000 to 130,000. The G-CSF receptor signal is mediated by the JAK-1 and JAK-2 tyrosine kinases. 
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PROLIFERATION AND DIFFERENTIATION OF MEGAKARYOCYTIC CELLS
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The bone marrow megakaryocytic progenitor (CFU- Meg) cells differentiate from pluripotent stem cells and mature to become megakaryocytes in the bone marrow. At some stage the CFU-Meg cells cease to divide and begin to undergo endomitosis. The ploidy of the nuclei increases from 2N to 4N, 8N, 16N and 32N as the megakaryocytes differentiate and mature. Approximately 2,000 platelets are produced by a mature megakaryocyte after it reaches a ploidy of 8N or more.
The existence of a proliferation and differentiation factor for the megakaryocyte-platelet cells implied from the plasma of animals with thrombocytopenia and this factor was given several different names, such as megakaryocyte colony-stimulating factor (Meg-CSF), megakaryocyte potentiator (Meg-POT), and TPO, based upon its activity. It was already known that cytokines such as IL-6, IL-11 and LIF could regulate megakaryocyte development, but it was not believed that any of these cytokines acted specifically on the megakaryocytic cells.
In 1993 strong evidence suggested that c-MPL, which belongs to the cytokine receptor family, had a role in megakaryopoiesis. Research began, therefore, to find the ligand for c-MPL. In 1994 the cloning of the TPO/c-MPL ligand was announced simultaneously by four different groups. Miyazaki and co-workers did not approach the problem by searching for the c-MPL ligand, but they purified TPO from rat plasma as an in vitro index for megakaryocyte proliferation and differentiation activity, and they successfully cloned the cDNA for rat and human TPO.
The amino acid sequence for the proposed human TPO/c-MPL ligand consists of 353 amino acids, including a signal sequence of 21 amino acids. The C-terminus has six potential sites for N-linked glycosilation, and the N-terminus demonstrates approximately 24% homology with EPO. Recombinant human TPO protein (rhTPO) expressed in mammalian cells exhibits megakaryocyte colony-stimulating activity in vitro, but does not induce colony stimulation in other blood cell lines. Moreover, when rhTPO is administered to normal mice, the platelet count more than quadruples, but other blood cell lineages remain unaffected. These results strongly suggest that TPO is a lineage-specific regulatory factor for the megakaryocytopoiesis and platelet production. (For further discussion of thrombopoietin and megakaryocytopoiesis please see review by D. Kuter, The Oncologist 1996;1:98-106).
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