Cellular and Molecular Characterisation of a Novel Murine Erythroleukaemia System

Qiu, Jin (1997) Cellular and Molecular Characterisation of a Novel Murine Erythroleukaemia System. PhD thesis, University of Glasgow.

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The ELM erythroleukaemia system is unusual in that the erythroleukaemia cells growing in vivo in the spleen cannot be maintained in vitro except in contact with bone marrow-derived stromal cells. They are also unusual in that they retain the ability to undergo erythroid differentiation in response to physiological growth factors, such as Epo and IL-3. Clonal stroma-dependent ELM cells (ELM-D) have been isolated in the presence of stroma. By selecting for growth of ELM-D cells without stroma, a series of stroma-independent clones (ELM-Is) have also been obtained. They are more tumorigenic when injected into mice compared with their stroma-dependent parent. All of the stroma-independent clones, apart from one (i.e. ELM-I/1), are still able to differentiate into mature erythroid cells in response to Epo or IL-3. This particular cell line, that is blocked in differentiation, also displays the highest tumorigenicity in vivo. The ELM system therefore provides a unique opportunity to analyse the molecular nature of stromal control of erythropoiesis, and identify genetic events involved in confering stroma-independent growth, differentiation arrest and increased tumorigenicity. The work presented in this thesis has demonstrated that, upon stroma-withdrawal, the majority of the ELM-D population dies out with characteristics of apoptosis. However, the fact, that a-globin expression in ELM-D cells is largely increased when the stroma is removed, indicates that ELM-D cells undergo erythroid differentiation in the absence of the stroma. Therefore, at least one mechanism whereby the stromal cells maintain the long-term growth of ELM-D cells is to prevent them from further maturation. Gene expression and functional studies of the ELM system have revealed the following: (1) The stroma-independent cells display an elevated expression of an ets oncogene, erg, at the mRNA level in comparison to ELM-D cells. However, only ELM-I/1 cells seem to express detectable level of the Erg protein. (2) The expression of another ets oncogene, fli-1, in the ELM-I/1 cells is significantly activated at both mRNA and protein levels, but is absent in all the other ELM cells; (3) The bcl-2 expression at the mRNA level in the stroma-independent cells is higher than that in ELM-D cells, especially in ELM-I/1 which is the only cell line wherein the Bcl-2 protein is expressed at a detectable level. Functional analysis indicate that over-expression of bcl-2 is not sufficient to induce differentiation arrest or increased tumorigenicity in ELM-I cells. Finally, transient transfection assays have demonstrated that the Fli-1 protein can transactivate a partial bcl-2 promoter in a quail fibroblast cell line. The bcl-2 promoter activity is higher in ELM-I/1 cells, which express high levels of Fli-1 protein, than that in ELM-I/2 cells, which do not express fli-1 at all. This implies that Fli-1 may be responsible for the up-regulation of bcl-2 expression in ELM-I/1 cells. DNase I footprinting analysis has identified six binding sites for the ELM nuclear proteins within the minimum bcl-2 promoter region. However, gel retardation assays and "supershift" analysis using a Fli-1-specific antibody do not reveal the binding of Fli-1 to any of the six nuclear protein binding sites. The tentative conclusion is, therefore, that Fli-1 may transactivate the bcl-2 promoter by an indirect mechanism.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Paul Harrison
Keywords: Cellular biology
Date of Award: 1997
Depositing User: Enlighten Team
Unique ID: glathesis:1997-74874
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 27 Sep 2019 15:44
Last Modified: 27 Sep 2019 15:44
URI: https://theses.gla.ac.uk/id/eprint/74874

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