title: The Evi1 proto-oncoprotein blocks endomitosis in megakaryocytes by inhibiting sustained cyclin-dependent kinase 2 catalytic activity.
abstract: The 3q21q26 syndrome leukaemias are characterised by dystrophic megakaryocytes, elevated platelet counts, ectopic EVI1 protein production and poor prognosis. To investigate the molecular basis of this disease, we developed a model system to examine the biological activity of EVI1 in a megakaryocyte progenitor cell line. For this purpose, Evi1 was conditionally expressed in human erythroleukaemia cells (HEL) that progress along the megakaryocyte lineage in the presence of 12-O-tetradecanoylphorbol 13-acetate (TPA). TPA-stimulated HEL cells normally undergo: (1) growth arrest; (2) altered morphology; (3) endomitosis and (4) characteristic changes in gene expression, including reduction of the erythroid-specific glycophoryn A and elevation of the specific glycoproteins GPIIIa and GPVI. Enforced Evi1 expression alone had no effect upon HEL cell proliferation or differentiation but a phenotype was manifest upon stimulation to differentiate. Evi1-expressing, TPA-treated HEL cells still showed growth arrest, had reduced and enhanced glycophoryn A and GPIIIa mRNA's, respectively, but failed to significantly elevate GPVI mRNA. This was accompanied by inhibition of endomitosis and altered cell morphology. Sustained CDK2 catalytic activity, typically associated with megakaryocyte endomitosis, was dramatically decreased in TPA-stimulated Evi1-expressing HEL cells because of significantly reduced levels of cyclin A. Therefore, enforced Evi1 expression could inhibit megakaryocyte differentiation although retention of some characteristic molecular changes, in combination with a block in endomitosis and altered morphology, suggest a defect in lineage progression. These results suggest that ectopic Evi1 expression contributes to a defective megakaryocyte differentiation programme and is likely to contribute to the phenotype observed in 3q21q26 syndrome leukaemias.
authors.affiliation: Department of Biological and Biomedical Sciences, Glasgow Caledonian University, Glasgow, UK.
authors.forename: Anna
authors.initials: A
authors.lastname: Kilbey
authors.forename: Hadi
authors.initials: H
authors.lastname: Alzuherri
authors.forename: Joan
authors.initials: J
authors.lastname: McColl
authors.forename: Carmela
authors.initials: C
authors.lastname: Calés
authors.forename: Jonathan
authors.initials: J
authors.lastname: Frampton
authors.forename: Christopher
authors.initials: C
authors.lastname: Bartholomew
mesh_terms: D042846:CDC2-CDC28 Kinases / Q000037:antagonists & inhibitors* / Q000502:physiology; D002453:Cell Cycle; D002454:Cell Differentiation; D049109:Cell Proliferation; D051357:Cyclin-Dependent Kinase 2; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D006410:Hematopoiesis; D006801:Humans; D004915:Leukemia, Erythroblastic, Acute / Q000201:enzymology / Q000378:metabolism / Q000473:pathology*; D000074008:MDS1 and EVI1 Complex Locus Protein; D008533:Megakaryocytes / Q000166:cytology* / Q000201:enzymology; D008938:Mitosis; D009363:Neoplasm Proteins / Q000378:metabolism; D011519:Proto-Oncogenes / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014157:Transcription Factors / Q000378:metabolism / Q000502:physiology*; D014407:Tumor Cells, Cultured
chemical_list: D004268:DNA-Binding Proteins; D000074008:MDS1 and EVI1 Complex Locus Protein; C063575:MECOM protein, human; D009363:Neoplasm Proteins; D014157:Transcription Factors; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2