In vivo and in vitro regulation of plasminogen activator inhibitor type-1
type: CreativeWork
description: The human leukaemic cell line, K562 can be induced to differentiate along the megakaryoblastic pathway by the addition of phorbol myristate acetate and, during this process, the cells switch from producing a mixture of active tissue plasminogen activator (t-PA) and urokinase (u-PA) to producing active u-PA alone. I performed a series of experiments designed to examine the mechanism of down regulation of t-PA activity that occurred with differentiation. The results of these experiments showed that, in the K562 system at least, differentiation-linked changes in t-PA activity are incidental to plasminogen activator inhibitor type-1 (PAl-1) synthesis and release and that the inhibitor is a critical regulatory protein in this model system. I therefore chose to study the regulation of PAl-1 both in vitro and in vivo. To perform the in vitro regulation studies, DNA constructs derived from the PAl-1 promoter and 5' flanking sequence were cloned upstream of a luciferase reporter gene. These constructs were transfected into human hepatoma cells, the cells treated with TGFB for 24 hours after which luciferase activity was measured as a function of promoter activity. The active regions were also analyzed by gel retardation to investigate the transcription factors that bound to these regions. The functional data taken together with the DNA binding data supported the hypothesis that the response of the PAl-1 promoter to TGFP was mediated by two conserved regions of the promoter and 5' flanking sequence, both of which contain DNA sequences with homology to the activator protein-1 (AP-1) consensus sequence. I then focused on a single region from the PAl-1 5' flanking region in an attempt to define the role of the AP-1-like sites in the TGFB response. The direct response of this region to AP-1 was assessed by co-transfection of plasmids containing the genes for c-fos and c-jun together with the PAl-1 /reporter constructs. My data indicates that the full TGFB response of this region …
abstract: The human leukaemic cell line, K562 can be induced to differentiate along the megakaryoblastic pathway by the addition of phorbol myristate acetate and, during this process, the cells switch from producing a mixture of active tissue plasminogen activator (t-PA) and urokinase (u-PA) to producing active u-PA alone. I performed a series of experiments designed to examine the mechanism of down regulation of t-PA activity that occurred with differentiation. The results of these experiments showed that, in the K562 system at least, differentiation-linked changes in t-PA activity are incidental to plasminogen activator inhibitor type-1 (PAl-1) synthesis and release and that the inhibitor is a critical regulatory protein in this model system. I therefore chose to study the regulation of PAl-1 both in vitro and in vivo. To perform the in vitro regulation studies, DNA constructs derived from the PAl-1 promoter and 5' flanking sequence were cloned upstream of a luciferase reporter gene. These constructs were transfected into human hepatoma cells, the cells treated with TGFB for 24 hours after which luciferase activity was measured as a function of promoter activity. The active regions were also analyzed by gel retardation to investigate the transcription factors that bound to these regions. The functional data taken together with the DNA binding data supported the hypothesis that the response of the PAl-1 promoter to TGFP was mediated by two conserved regions of the promoter and 5' flanking sequence, both of which contain DNA sequences with homology to the activator protein-1 (AP-1) consensus sequence. I then focused on a single region from the PAl-1 5' flanking region in an attempt to define the role of the AP-1-like sites in the TGFB response. The direct response of this region to AP-1 was assessed by co-transfection of plasmids containing the genes for c-fos and c-jun together with the PAl-1 /reporter constructs. My data indicates that the full TGFB response of this region …
author: Keeton, Mark R
keywords: Clinical Laboratory Sciences
date published: 1992
date modified: 2026-07-22T22:23:06Z
provider: University of Cape Town
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: Department of Clinical Laboratory Sciences

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property ID: Handle
value: http://hdl.handle.net/11427/40589