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The regulation of inositol lipid breakdown during the differentiation of HL60 myeloid leukaemia cells

Porfiri, Emilio; (1993) The regulation of inositol lipid breakdown during the differentiation of HL60 myeloid leukaemia cells. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

The binding of certain growth factors and mitogens to their cell-surface receptors triggers the phospholipase C (PLC)-catalysed hydrolysis of a rare inositol lipid, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). PLC-mediated PtdIns(4,5)P2 breakdown yields two classes of second messengers: inositol polyphosphates which trigger intracellular Ca2+ mobilization and Ca2+ entry, and diacylglycerol (DAG) which activates protein kinase C. Growth factor or mitogen-stimulated inositol lipid breakdown represents a signal critical for the initiation of cascades of biochemical events which culminate in mitosis. Autonomous, i.e. growth factor-independent, hydrolysis of inositol lipids and generation of inositol phosphates and DAG has been detected in several cell types following the transformation with oncogenes whose products were localized to the cell membrane. These observations have suggested the participation of inositol lipid turnover in triggering or maintaining the neoplastic process. This thesis describes investigations on the autonomous generation of inositol lipid- derived second messengers in the HL60 human promyelocytic leukaemia cell line. The possible roles of these species in regulating proliferation and during the induction of differentiation of these cells was also addressed. In proliferating HL60 cells, brief inhibition of inositol phosphate phosphatases with LiCl, led to the accumulation of inositol phosphates, suggesting the occurrence of autonomous inositol lipid hydrolysis. Induction of granulocytic differentiation by the treatment with retinoic acid (RA) or dimethyl sulphoxide (DMSO), or of monocytic differentiation with phorbol myristate acetate (PMA), abolished LiCl-induced inositol phosphate accumulation suggesting that down-regulation of inositol lipid hydrolysis had occurred. Consistent with these observations, the steady state levels of inositol polyphosphates declined within the same time frame. Importantly, down-regulation of inositol lipid breakdown and the decline of inositol polyphosphate levels always preceded the cessation of HL60 cell proliferation or the decline of DNA synthesis which accompanied the induction of terminal differentiation. In addition, down-regulation of inositol lipid hydrolysis was probably due to subtle regulatory changes triggered by the compounds used to induce differentiation, since no alterations of the absolute levels and enzymological characteristics of key enzymes of inositol lipid turnover (Ptdlns(4,5)P2-PLC, inositol phosphate phosphatases) were detected. These experiments also showed that PtdIns(4,5)P2 represents the substrate preferentially hydrolysed by PI-PLC in HL60 cells. The ability of the fluoroaluminate ion, a direct activator of G proteins, to reactivate inositol lipid breakdown in HL60 cells treated with RA or PMA suggested that these changes may involve a G protein-linked mechanism. Nevertheless, the participation of PKC in the process of PMA-induced differentiation could not be excluded. These observations suggested that autonomous, PLC-mediated, hydrolysis of inositol lipids occurred in HL60 cells and that this process involved the preferential hydrolysis of PtdIns(4,5)P2- therefore possible that autonomous generation of PtdIns(4,5)P2-derived second messengers may play a regulatory role in signalling HL60 cell proliferation and that the early shutdown of this turnover following the induction of differentiation, constitutes a regulatory signal important in securing the cessation of proliferation associated with differentiation.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: The regulation of inositol lipid breakdown during the differentiation of HL60 myeloid leukaemia cells
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Health and environmental sciences
URI: https://discovery.ucl.ac.uk/id/eprint/10102340
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