Freeburn, Robin William; (1997) A study of the granulocyte-macrophage colony stimulating factor receptor (GM-CSFR) in leukaemia. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Mutation of signaling molecules such as Ras have been shown to confer a growth advantage in blast cells from patients with acute myeloid leukaemia (AML) and may contribute to the pathogenesis of the disease. Alterations in other signaling molecules could also play a role in leukaemogenesis. GM-CSF is a key growth factor in haemopoiesis which exerts its influence on target cells via membrane bound receptors. The GM-CSF receptor (GM-CSFR) is composed of oligomers of ligand-specific a chains and p chains which are common to the GM-CSF/IL-3 and IL-5 receptors (βC). The intracytoplasmic tail of the βC chain is essential for the activation of several downstream signaling pathways and alterations in this region could deregulate normal signaling processes. RT-PCR-SSCP analysis was used to look for mutations in the βC chain tail (nts 1281-2816) in RNA from 35 patients with acute myeloid leukaemia (AML) and 10 haematologically normal controls. Six nucleotide substitutions were detected, three of which were silent (Ser426, Pro648 and Pro800) and three which altered the amino acid residue at that position in the receptor sequence (Gly647>Val, Val652>Met and Pro603Thr). However all substitutions were detected in normal controls and were thought to be polymorphisms, with allele frequencies of 0.23 and 0.13 found for two of the most common silent substitutions. RNA from patients with juvenile chronic myeloid leukaemia (JCML) was also screened for mutations in the entire GM-CSFR α and β chain coding sequences using SSCP analysis as studies have shown progenitors from JCML patients have a hypersensitive growth response to GM-CSF in culture. Two nucleotide substitutions accounted for all a chain abnormalities (Ala17Gly and silent Val333) with both previously detected in normal controls. Four base substitutions were detected in the β chain. Three were polymorphisms previously described in AML patients and normal controls. A further nucleotide mutation which resulted in a Glu249Gln substitution was also found but was not thought to be of pathological significance as a Gin residue is present at this amino acid in both mouse β chains. During the course of these studies a novel isoform of the βC chain with a truncated intracytoplasmic tail (βIT) was isolated by our group. Transcripts of this alternatively spliced isoform were shown to be present in relatively high levels in blast cells from AML patients. Polyclonal antibodies were raised against a novel 23 amino acid sequence in the tail of the βIT chain and screened in COS-7 cells transfected with plasmids coding for βC or βIT chain before being used to demonstrate expression of the truncated receptor in a range of haemopoietic cells. The βIT-specific antibodies were then used to investigate GM-CSFR α and β chain stoichiometry and demonstrated that α chain homodimerization occurred without ligand stimulation in primary haemopoietic cells or GM-CSFR α chain in transfected COS-7 cells.

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