Molecular and cellular studies examining the biological significance of different isoforms of the receptor tyrosine kinase, c-Kit / Antony Charles Cambareri.
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Date
2004
Authors
Cambareri, Antony Charles
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Abstract
c-Kit is a member of the Receptor Tyrosine Kinase Type III family and has four
naturally occurring isoforms. The work presented in chapter 3 utilised full-length
human or murine c-Kit cDNA expressed in murine cells. Over-expression of normal
c-Kit was capable of contributing to oncogenic transformation. The analysis of
human c-Kit isoforms demonstrated dissociation of various indicators of
transformation (anchorage independence, loss of contact inhibition, tumourigenicity)
in the NIH3T3 cell model.
Biochemical analysis of the c-Kit signalling revealed qualitative and quantitative
differences between the GNNK+ and GNNK- c-Kit isoforms. The GNNK- isoform
was hyperphosphorylated more extensively and rapidly, and was also more efficiently
ubiquitinated and degraded than the GNNK+ counterpart. PI3-K was recruited and
activated equally by both isoforms. Phosphorylation of MAPK paralleled that of the
c-Kit isoform's phosphorylation.
In Chapter 4, a new model was developed using a chimaeric human extracellular
c-Kit/murine transmembrane+ intracellular c-Kit. This new molecule in conjunction
with a murine Myb Immortalised Haemopoietic Cell (MIHC) line was used to
investigate a number of biological outcomes stimulated by scF simultaneously. A
MIHC line lacking Lyn was also analysed.
Chimaeric c-Kit displayed the same signalling characteristics exhibited by its' full-length
human counterpart. The model showed that the GNNK- isoform was superior in its survival stimulus to GNNK+, but both were equivalent in promoting
proliferation. The absence of Lyn reduced the ability of both isoforms to promote
survival.
The aim of work in Chapter 5 was to elucidate the expression patterns of the c-Kit
isoforms in subsets of normal human haemopoietic cells. Methodology was
developed to detect GNNK+/- c-Kit mRNA from rare subsets of cells from bone
marrow. As c-Kit is known to be down-modulated in mobilised peripheral blood stem
cells, mobilised CD34+ cells were also investigated. In all haemopoietic cells
analysed, there was no significant difference in expression patterns of the c-Kit
isoforms, with all samples expressing approximately 90% of total c-Kit transcripts as
the GNNK- isoform. c-Kit downmodulation observed in mobilisation of CD34+ cells
was not influenced at the level of transcription, but at the protein level.
School/Discipline
Dept. of Medicine
Dissertation Note
Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine, 2005
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Description
"October 2004"
Includes bibliographical references (leaves 201-256)
xiv, 256 leaves, [9] p. : ill., plates (col.) ; 30 cm.
Includes bibliographical references (leaves 201-256)
xiv, 256 leaves, [9] p. : ill., plates (col.) ; 30 cm.