Biological and Genetic Characterisation of a Novel Murine Erythroleukaemia System

The ELM erythroleukaemia is novel in that long-term survival of leukaemic cells in culture (ELM-D cells) is dependent on contact with a bone marrow-derived stromal feeder cell layer. A collaborative study has shown that the transmembrane form of stem cell factor is a crucial component of the stromal cell-derived survival signal. A number of stroma-independent (ELM-I) mutants have been derived that vary in their tumorigenicity and their ability to differentiate in vitro in response to erythropoietin and IL-3. The work in this thesis has attempted to define the genetic changes responsible for these different phenotypes. At the p53 locus in the primary leukaemic cells, one copy of the gene has been lost whilst the other contains an 18bp deletion, implicating its mutation as an early step in the development of the leukaemia. Changes in ets gene expression have also been found. The Fli-l locus is rearranged in the primary tumour due to the insertion of a retrovirus upstream of one Fli-l allele. However, this does not result in Fli-l gene activation in any of the ELM- D or ELM-I cell lines, except one, which significantly is the only cell line to have lost the ability to differentiate in response to erythropoietin. In addition, up-regulation of another ets family member, erg, is associated with stromal cell-independent growth since all ELM-I mutants have moderate levels of erg mRNA, whereas only low or undetectable levels are found in primary leukaemic cells in vivo or in ELM-D cells in vitro. This up-regulation of erg mRNA seems to be important for stromal cell- independent growth since ELM-D cells, which exhibit short-term viability after separation from stromal cells, show elevated expression of the erg gene. This seems to be made permanent in ELM-I mutants since, unlike ELM-D cells, they do not down-regulate erg mRNA when grown in contact with stromal cells. These experiments suggest that alterations in ets gene expression may affect both the survival and the differentiation of erythroid cells. Future work designed to assess the validity of this hypothesis is discussed