Characterisation of Phosphatidate Phosphohydrolase Activity in Human Colorectal Cancer

Scott, Pamela Helen (1994) Characterisation of Phosphatidate Phosphohydrolase Activity in Human Colorectal Cancer. PhD thesis, University of Glasgow.

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Established chemotherapeutic agents often produce dose limiting toxicities in normal tissues due to lack of selectivity for tumour cells. Identification of abnormalities in the control of signal transduction in tumour cells may suggest more specific targets. Phosphatidate phosphohydrolase (PAP) is involved in the generation of diradylglycerol (DRG) from phosphatidylcholine (PC) in response to a mitogenic signal. The aims of the present studies were firstly to determine whether the activity of PAP is altered in transformed cell lines and in human tumours and secondly to determine whether PAP is a target for anticancer drug development. An assay was established and characterised to measure PAP activity. Two activities are present in rat liver. PAP1 is mainly cytosolic and is involved in glycerolipid synthesis. It requires Mg2+ and is inhibited by the thiol-blocking reagent n-ethylmaleimide (NEM). PAP2 is associated with the plasma membrane and is thought to be involved in mitogenic signal transduction. PAP activity can be measured in tissue homogenates and the two activities distinguished by their differential sensitivity to NEM. PAP activity was shown to be present in a range of mouse tissues The highest activities were present in the brain and the lowest in muscle tissue. Transformation of mouse fibroblast cell lines by a mutant ras oncogene results in an increase in DRG mass. This DRG could be derived from the breakdown of either phosphatidylinositol (PI) by a phospholipase C activity or PC by the sequential activity of phospholipase D and PAP. PAP2 activity was shown to be decreased in the transformed cells and clearly could not account for the increased DRG mass. However, the level of phosphatidate was also increased and this was consistent with the decreased activity of PAP2. The association of a mutant ras oncogene and decreased activity of PAP2 was also observed in human colon tumour cell lines. Cell lines DLD-1 and LoVo contain a mutant Ki-ras oncogene and were shown to have lower PAP2 activities than HT29 and Colo320DM which express wild type Ki-ras. The phosphatidate and DRG mass were also higher in the cell lines with a mutant ras oncogene. Thus, in both cell models a decrease in PAP2 activity is associated with increased levels of phosphatidate, a second messenger with potent mitogenic activity. PAP activity was measured in human colon tumours and in adjacent pathologically normal colonic mucosa. Two activities, with properties representative of PAP1 and PAP2, were identified. A wide range of activities were observed in the normal colon tissue. Both activities were significantly increased in the majority of tumours. Furthermore, the phosphatidate and DRG mass were decreased in the tumour tissue. An increase in PAP activity is consistent with the decreased phosphatidate mass but does not account for the decreased DRG mass. These changes are in contrast to those observed in the ras transformed cell lines and it is probable that they are unrelated to ras since only 50% of the tumours contained a mutation in the Ki-ras oncogene. However, the tumours with a ras mutation tended to have the highest activities of PAP2. In an attempt to account for the decreased levels of DRG in the colon tumours two enzymes that are responsible for the metabolism of DRG were measured. A wide range of activities of both DRG kinase and DRG lipase were observed in the normal colonic mucosa. A DRG kinase activity was increased in the tumour tissue but the substrate specificity of this activity suggests that it metabolises DRG derived from PI rather than PC. In contrast, a DRG lipase activity was also increased in the tumour tissue and the substrate specificity of this activity indicated a preference for PC derived DRG. Thus there is evidence for a general increase in DRG turnover in colon tumours. Studies of PAP activity in intact cell would be aided by a specific inhibitor. A number of cationic amphiphilic agents, including propranalol and sphingosine, were shown to inhibit PAP activity in rat liver homogenates and to inhibit proliferation of normal and ras transformed mouse fibroblast cell lines. These agents are known to inhibit other enzymes such as protein kinase C and thus lack specificity for PAP. Sphingosine was identified as the lead compound for future development of a more specific inhibitor of PAP. The role of PAP in the control of proliferation in transformed cells remains unclear. However, the consistent increase in activity observed in colon tumours suggests that this may be a target for anticancer drug development.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Jane Plumb
Keywords: Medicine, Oncology
Date of Award: 1994
Depositing User: Enlighten Team
Unique ID: glathesis:1994-75532
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Dec 2019 09:15
Last Modified: 19 Dec 2019 09:15

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