a. Transcription Factors
Transcription factors contribute to the proliferation and differentiation signals required for haematopoiesis by regulating the expression of genes. The expression of each transcription factor involved in regulating haematopoiesis is not necessarily restricted to single cell lineage. For example, the development of erythrocytes and megakaryocytes are both under regulation of the zinc-finger transcription factor GATA-1. The expression of GATA-1 mRNA coding for GATA-1 is detected in multipotent progenitors and the erythroid, megakaryocyte, eosinophil and mast cell lineages (Tsai et al, 1989;Romeo et al, 1990;Zon et al, 1993). Disruption of the GATA-1 gene in mouse embryonic stem cells results in a block in the production of mature erythroid cells and whilst GATA-1' embryonic stem cells can generate erythroid colonies containing proerythroblasts, they are unable to mature properly and apoptose (Pevny et al, 1991;Weiss & Orkin, 1995). Shivdasani et al (1997) also demonstrated a development of megakaryocytes with reduced ploidy and a failure to generate platelets in mice lacking expression of GATA-1 (Shivdasani et al, 1997). Pu.1 (Purine rich box-1) belongs to the Ets (erythroblastosis)-family of transcription factors. Evidence of Pu.1 as a key regulator of haematopoiesis has been provided from mice knock-out studies. Pu.1-1- mice lacked cells from
the myeloid and lymphoid lineages resulting in prenatal lethality (Scott et a/, 1994). Transcription factors also contribute towards the self renewal capacities of HSCs. For example, Homeobox (HOX) genes encode DNA-binding transcription factors. They are organised into four genomic clusters (A-D) and were first identified as regulators of positional identity along the anterior-posterior body axis in embryos (as reviewed by (Krumlauf, 1994). The majority of the A, B and C clusters are expressed in haematopoietic cells, and most of these HOX genes are preferentially expressed in HSCs and primitive progenitor cells, decreasing in expression as the cells differentiate and mature (Moretti et al, 1994;Sauvageau et al, 1994;Pineault et al, 2002). Southern blot analysis of cDNA identified HOX genes located at the 3' regions of the A and B clusters as enhanced in expression in primitive CD34* cells whereas those located at the 5* end were expressed at relatively equal levels in all the primitive CD34* sub-populations (Sauvageau et al, 1994). Mice knock-out studies have demonstrated the various effects of HOX genes on proliferation, differentiation and HSC-renewal. Knock-out studies revealed subtle reductions in the HSC and progenitor cell numbers without disrupting haematopoiesis in /-/OXB4-deficient mice and a more pronounced decrease in the proliferation capacity of these cells in HOXB3- and HOXB4-deficient mice (Bjomsson et al, 2003;Brun et al, 2004). HOXB3 and HOXB4 are both highly expressed in primitive CD34+ cells, but a decrease in their expression is observed as the cells differentiate and mature (Sauvageau et al, 1994). Despite their similar expression patterns and a decrease in HSC proliferation resulting from
their underexpression, the overexpression of these two genes generates very different results. An overexpression of HOXB3 blocks the development of T- and B-cells whilst an overexpression of HOXB4 results in the selective expansion of primitive haematopoietic cells (Sauvageau etal, 1995;Sauvageau et al, 1997).
b. Cytokines
Many of the regulatory pathways controlling the haematopoietic system are controlled by cytokines, a large family of extracellular ligands, binding to and activating a family of cytokine receptors. Cytokines are secreted from or presented on the surface of mesenchymal cells within the bone marrow, known as stromal cells, to promote the survival, proliferation and differentiation of haematopoietic stem cells and progenitors. Colony-stimulating factors (CSF) are synthesised locally by stromal cells and include macrophage-CSF (M-CSF), granulocyte-CSF (G-CSF) and granulocyte-macrophage-CSF (GM-CSF). M-CSF and G- CSF are relatively lineage-specific contributing to the differentiation and proliferation of macrophages and granulocytes. GM-CSF is produced by osteoblasts at relatively low levels and functions at earlier stages of lineage commitment, promoting the proliferation and maturation of granulocytes and macrophages and working with other cytokines as a growth factor for erythroid and megakaryocyte progenitors (Barreda et al, 2004). The multi-CSF is more commonly known as interleukin-3 (IL- 3) contributing to the production of macrophages, neutrophils, eosinophils, basophils, mast cells, megakaryocytes, and erythrocytes
(as reviewed by (Barreda et al, 2004). Several cytokines work in synergy with other cytokines to promote haematopoiesis. Stem cell factor (SCF) functions with G-CSF, GM-CSF, IL-3 and EPO (erythropoietin) to support the growth of colony-forming units (CFU) in semisolid media (Martin etal, 1990).
c. Cytokine Receptors
Cytokines interact with specific membrane receptors that transmit a series of intracellular signals to the target cell following the cytokine- receptor interaction. The receptors for M-CSF and SCF, M-CSFR (also known as c-fms) and c-kit respectively, belong to the class III receptor tyrosine kinase (RTK) family (Yarden et al, 1987). Class III receptor RTKs are characterised by an extracellular ligand-binding domain containing five immunoglobulin-like domains, a single transmembrane domain, a juxtamembrane domain, two intracellular domains and a C- terminal domain (Ullrich & Schlessinger, 1990). Binding of a ligand to a class III RTK results in the activation of intrinsic tyrosine kinase activity (Ullrich & Schlessinger, 1990;Weiss & Schlessinger, 1998). The receptors mediating the effects of G-CSF, GM-CF and IL-3 all belong to the type 1 cytokine receptor superfamily. Unlike M-CSF, G-CSFR, the receptor for G-CSF, has no intrinsic kinase activity. The GM-SCF receptor, GM-CSFR, and the IL-3 receptor are members of the gp140 family of type 1 cytokine receptors composed of two distinct chains; a and p. The a-chain is the primary binding chain of the ligands whilst the P-chain is necessary for signal transduction (Chiba et al, 1990;Barreda
et al, 2004). Binding of the ligands to their receptors results in the activation of kinases from the Janus kinase family (JAK) resulting in a cascade of phosphorylation events through the initiation of several pathways, for example the p21Ras (rat sarcoma)/MAP (mitogen- activated protein) kinase and PI-3 (phosphoinositol-3) kinase/PKB (protein kinase B) pathways, to promote cell survival, proliferation and differentiation (Ihle & Kerr, 1995).
d. Cell Cycle Regulation
An important characteristic of haematopoietic stem cells is their relative proliferative quiescent state. The clonal-succession model proposed by Kay et al (1965) hypothesised that only one or a few HSC clones from the large pool of HSCs gives rise to mature blood cells at any time whilst the remainder of HSC clones remain quiescent until they are needed due to the exhaustion of the proliferative capacity of the long term HSC (LT-HSC) clone (Kay, 1965). Studies by Abkowitz et al (1990) and Guttorp et al (1990) supported the clonal-succession model. Safari cats heterozygous for the X chromosome linked enzyme glucose- 6-phosphate dehydrogenase (G6PD) received autologous marrow transplantations with limited numbers of cells following lethal doses of radiation. The data suggests that haematopoiesis resumed to the state observed before irradiation but fewer stem-cell clones were contributing (Abkowitz et al, 1990;Guttorp et al, 1990). A later in vivo model studying the bromodeoxyuridine (BrdU) incorporation by replicating cells did not support the model. Cheshier et al (1999) proposed that long term-HSCs
enter the cell cycle in an asynchronous manner and that a few clones did not dominate proliferation. From their results they calculated that 99% of long term-HSCs divided, on average, every 57 days (Cheshier et al, 1999). The self-renewal capacity of HSCs requires maintaining the coordination of HSC progression through the cell cycle and HSC-fate choices. A direct control of the HSC cycle is by the activation of a group of enzymes known as cyclin-dependent kinases (CDK). CDKs are positive regulators inducing cell cycle progression; their activity partly controlled by their association with cyclins to form a cyclin-dependent kinase complex. The activities of the complexes are further regulated by cyclin-dependent kinase inhibitors (CDKIs) acting as negative regulators inhibiting the progression of the cell cycle. CDKIs belong to one of two families depending on their targets; the Cip/Kip family, including p21 and p27, inhibit a number of cyclin-dependent kinases whilst the Ink4 (inhibitors of CDK4) family, including p15 and p16, inhibits CDK4 and CDK6 (Harper et al, 1995;Sherr & Roberts, 1999;Attar & Scadden, 2004). The Cip/Kip family members inhibit the kinase activity of the cyclin/CDK2 complexes and over-expression of these inhibitors induces a cell cycle arrest in the Gi phase (Harper et al, 1995;Lee & Yang, 2001). The p21 family member is under transcriptional control of p53 and appears to play a role in the inhibition of stem cell proliferation acting as a switch. Early studies with irradiated mice demonstrated the targeted disruption of the gene encoding p21 impaired the ability of cells to achieve cell cycle arrest (Deng et al, 1995). Study of mice deficient in p21 demonstrated the number of
primitive haematopoietic cells in the G0 phase was reduced yet the number of primitive cells that gave rise to long-term multipotent colonies in culture was increased indicating the role p21 in controlling the quiescence of HSCs (Cheng et al, 2000).
1.2 Disorders of Haematopoiesis
A characteristic hallmark of leukaemia is the inhibition of cell differentiation. Unable to terminally differentiate, these cells instead retain their proliferative capacity (Andreeff, 1986). Some cells may only display the phenotype of hyperproliferation (myeloproliferative disorders) or defective differentiation (myelodysplasia) and therefore are not true leukaemias but these disruptions in the haematopoietic process can result in the progression to several forms of leukaemia (Sawyers et al, 1991). Depending on the symptoms patients present with, and the cells involved, leukaemias can be sub-grouped into acute and chronic and further classified depending on the cell of origin into lymphoid and myeloid leukaemia (Sawyers et al, 1991).