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Author

Josie Ursini

Date of Award

4-1999

Degree Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Medical Sciences

Supervisor

Professor P. Whyte

Abstract

The SH2 containing inositol polyphosphate 5-phosphatase (SHIP) has been implicated in the negative regulation of growth and differentiation of multiple hematopoietic cell types. In this thesis, I describe the cloning and biochemical characterization of the human SHIP cDNA. SHIP expression was found to be restricted to cell lines of hematopoietic origin at both the protein and RNA level. The K562 erythroleukemia cell line was identified which lacked detectable SHIP protein and message. The absence of endogenous SHIP in K562 cells provides a useful in vitro system to study the contribution of SHIP to the process of growth and differentiation in this cell line. Hemin stimulation of the K562 cell line results in the induction of an erythroid differentiation program. When stably overexpressed in K562 cells, SHIP was found to be constitutively tyrosine phosphorylated and associated with endogenous Shc, Grb-2 and Bcr/Abl. Overexpression of SHIP did not affect the overall growth rate of the cells but resulted in decreased synthesis of hemoglobin protein and [varepsilon]-globin mRNA in response to hemin stimulation. This effect was not due to increased cell death or cell cycle arrest in the SHIP-expressing lines following hemin stimulation, but was likely the result of an impaired differentiation program in these cells. Mutational studies indicated that SHIP must retain both an intact catalytic domain and Shc binding site to efficiently inhibit K562 erythroid differentiation.

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