Investigation of glucocorticoid and dissociated glucocorticoid activity in hepatoma cell lines with specific reference to regulation of the corticosteroid binding globulin (CBG) proximal promoter'

Allie-Reid, Fatima (2003-12)

Thesis (PhD)--Stellenbosch University, 2003.

Thesis

ENGLISH ABSTRACT: This study investigated the effect of several hormones on the rat corticosteroid binding globulin proximal promotor and for the first time showed that modulation occurs at the promotor level and can be correlated with changes in corticosteroid binding globulin mRNA and protein levels. The effect of various physical and psychological stressors on rat liver corticosteroid binding globulin mRNA levels was also tested and it was shown that voluntary running had no effect on rat corticosteroid binding globulin levels but that involuntary swimming and immobilization decreased rat corticosteroid binding globulin mRNA levels. Glucocorticoid responsiveness of the corticosteroid binding globulin promoter was investigated further by using truncated contructs of the corticosteroid binding globulin proximal promoter. Glucocorticoid responsiveness was delineated to between -296 and -145bp from the transcription start site an area that contains putative binding sites for D-site binding protein, hepatic nuclear factor-3 and CAAT/enhancer binding protein suggesting that these transcription factors may be involved in glucocorticoid responsiveness of the corticosteroid binding globulin proximal promoter. The dissociative glucocorticoid activity of medroxyprogesterone acetate and Compound A, both putative dissociated glucocorticoids, was compared to standard glucocorticoids by examining transactivation of glucocorticoid response element-containing reporter constructs and transrepression of corticosteroid binding globulin gene expression in hepatic cell lines. Results showed that medroxyprogesterone acetate, but not Compound A, trans activates only in the presence, but not in the absence, of co-transfected glucocorticoid receptor. Medroxyprogesterone acetate down modulated dexamethasone transactivation while the modulatory effect of Compound A depends on the order of addition of Compound A. If added together Compound A has no effect on dexamethasone transactivation, however, if Compound A was added before dexamethasone, Compound A significantly decreased dexamethasone transactivation. Both medroxyprogesterone acetate and Compound A, like glucocorticoids, transrepressed the rat corticosteroid binding globulin proximal promoter. The potency of repression was similar but Compound A repressed with a higher efficacy than medroxyprogesterone acetate. We conclude that Compound A is a completely dissociated glucocorticoid in contrast to medroxyprogesterone acetate that displays only partial dissociation, which is dependent on glucocorticoid receptor levels.

AFRIKAANSE OPSOMMING: Tydens hierdie ondersoek is die effek van verskeie hormone op die rot kortikosteroied bindings globulien proksimale promoter ondersoek en vir die eerste keer is getoon dat modulering plaasvind op promoter-vlak en dat repressie korrileer met die verandering in kortikosteroied bindings globulien mRNA-en proteinvlakke. Die effek van verskeie fisiese en fisiologiese stressors op rotlewer kortikosteroied bindings globulien-mRNAvlakke is ook getoets en daar is getoon dat willekeurige hardloop geen effek op rot kortikosteroied bindings globulien-mRNA-vlakke het nie maar dat gedwonge swem en immobilisering rot kortikosteroied bindings globulien-mRNA-vlakke verlaag. Glukokortikoied responsiewiteit van die kortikosteroied bindings globulien proksimale promoter is verder ondersoek deur verkorte konstrukte van die kortikosteroied bindings globulien te toets. Glukokotikoied responsiewiteit is afgebaken tot tussen -296 en - 145bp vanaf die transkripsie beginplek 'n area wat beweerde bindings setels vir D-setel bindings protein, hepatosiet faktoor-3 en CCAAT-bindings protein-2 bevat en dus suggereer dat hierdie transkripsie faktore betrokke mag wees met glukokortikoied effekte op die kortikosteroied bindings globulien-proksimale promoter. Die dissosiatiewe glukokortikoied aktiwiteit van medroksiprogesteroon asetaat en Verbinding A, beide beweerde dissosiatiewe glukokortikoiede, relatief tot standaard glukokortikoiede is vergelyk deur transaktivering van glukokortikoied reseptor e1elment-bevattende konstrukte en onderdrukking van kortikosteroied bindings globulien geen ekspressie in lewersellyne te bestudeer. Medroksiprogesteroon asetaat, maar nie Verbinding A nie, transaktiveer slegs in die teenwoordigheid, maar nie in die afwesigheid, van ko-getransfekteerde glukokortikoied reseptore. Medroksiprogesteroon asetaat moduleer deksametasoon transaktivering afwaarts terwyl die modulerende effek van Verbinding A afhanklik van die orde van Verbinding A byvoeging is. Indien saam bygevoeg het Verbinding A geen effek op deksametasoon transaktivering nie, maar indien Verbinding A voor deksametasoon bygevoeg word verlaag Verbinding A deksametasoon transaktivering. Beide medroksiprogesteroon asetaat and Verbinding A, soos glukokortikoiede, onderdruk die rot kortikosteroied bindings globulien-proksimale promoter. Die sterkte van onderdrukking is dieselfde maar Verbinding A onderdruk met 'n hoër effektiwiteit as medroksiprogesteroon asetaat. Ons toon dat Verbinding A 'n totale dissosiatiewe glukokortikoied is in teenstelling met medroksiprogesteroon asetaat, wat slegs gedeeltelik dissosiatief is afhangende van glukokortikoied reseptor-vlakke.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/53475
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