HAEMATOPOIESIS GROUP

Haematopoiesis Group - Principal Investigator - Professor T R J Lappin

Haematopoiesis is the process by which primitive stem cells proliferate and differentiate to produce mature blood cells. It is driven by highly co-ordinated patterns of gene expression under the influence of growth factors and hormones.  Dysregulated gene expression is the causative factor in many haematological disorders and detailed knowledge of the regulation of gene expression is central to the understanding of the mechanisms that cause anaemia, leukaemia and other blood disorders.

Most patients who suffer from cancer are also anaemic. Erythroppoietin  (EPO), a glycoprotien hormone produced in the kidney is the primary inducer of red cell production.  Although EPO circulates only at picomolar levels in the plasma it effectively regulates red cell production by the bone marrow.  In cancer and certain chronic diseases EPO production is blunted but inflammatory cytokines are increased. These two factors retard red blood cell production.  Fortunately recombinant human EPO has been developed for treating aneamia in end-stage renal desease and is now used increasingly to treat patients with cancer. The response to EPO varies from patient to patient and further work is required to elucidate the specific pathogenic mechanisms underlying the onset and progression of the anaemia.

We are investigating EP0-induced gene expression during terminal differentiation of human and murine erythroid cells. Using the technique of differential display PCR (DD-PCR) we have identified a group of fourteen genes regulated by EPO which match uncharacterised ESTs. Current efforts are focused on the characterisatian of one of these gene designated ERIC- I , for erythropoietin-induced cDNA. ERIC- I is expressed in the spleen and bone marrow and the highest levels are found in the testes.

The EPO receptor spans the membrane of developing erythroid cells. The extracellular domain responds to circulating EPO and the intracellular domain contains a C-terminal regulatory region that acts as a brake on red cell production. A mutation in the receptor DNA that causes truncation of the inhibitory region is enough to produce an obvious increase in the number of red cells. This type of mutation was first found in Finland, in the family of an Olympic cross-country skiing gold medalist who has a "natural" advantage: a gene that allows his blood to carry more oxygen. All affected members of the family are descended from one couple born in the 1850's. Our group has identified a similar mutation in an unrelated English boy, and is currently investigating the molecular basis of other myoloproliferative disorders.

Homeobox (HOX) genes play a major regulatory role in many different developmental processes. Human ceIIs contain 39 major HOX genes, grouped in clusters (HOX A to HOX-D) on four separate chromosomes at 7p15, 17q21, 12q13, and 2q3I These genes share extensive homology with the Hom-C genes of Drosophila. At least 22 of the 39 HOX genes are expressed by different subpopulations of blood progenitor cells. In the most primitive haematopoictic stem cells, high levels of expression of the 3' genes in the A and B clusters are subsequently down regulated. and the more 5' genes are expressed as the progenitor cells become committed to the myeloid or erythroid lineage. Maintenance of I HOX gene expression requires several types of transcription factor including a positive regulator, MLL and a negative regulator, Bmi- I. Chimeric oncoproteins associated with chromosomal translocations are involved in leukaemogenesis and probably act by disrupting the activity of their normal counterparts in HOX gene regulation. For example MLL is expressed in normal haematopoietic cells and may be required for normal differentiation. MLL is frequently rearranged in acute myeloid leukaemia (AML) and acute lymphoid leukaemia (ALL) in association with translocations involving chromosome IIq23. In collaboration with Professor Finbarr Cotter, at St Bartholomew's School of Medicine, our group is investigating the role of MLL on the expression of individual HOX genes and the downstream targets of these genes.

Cytogenetic studies have shown that certain non-random balanced chromosomal translocations represent independent prognostic factors for patients with acute leukaemia. The associated translocation-generated oncogenes can be detected by reverse transcriptase polymerase chain reaction (RT-PCR) techniques. Molecular approaches have certain advantages. Firstly, the analyses can be carried out on a few cells without the need for culture, and secondly between 10 and 20% of cryptic translocations (patients with a normal karyotype but bearing the molecular abnormality) have been identified for each translocation. PCR analyses also allow detection of residual leukaemia cells bearing these fusion genes during the course of therapy i.e. minimal residual disease (MRD) The clinical value of qualitative RT-PCR analyses in MRD has been established for chronic myeloid Icukaemia (CML), but their clinical relevance in other diseases remains unclear. Recently a new technique, real time quantitative PCR (RQ-PCR) has emerged that allows precise measurement of the target to be amplified and may revolutionise the field of MRD studies. We have set up a DNA bank for samples from patients with leukaemia and are currently examining the clinical correlates of MRD, as measured by RQ-PCR, in a number of different forms of leukaemia.