Thesis (PhD (Biochemistry))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Medroxyprogesterone acetate (MPA) and norethisterone (NET) and its
derivatives (norethisterone enanthate (NET-EN); norethisterone acetate (NETA)),
are used by millions of women as contraceptives and in hormone
replacement therapy (HRT). Although both progestins are widely used, very
little is known about their mechanism of action at the molecular level. In this
thesis, the differential regulation of gene expression and molecular
mechanism of action via different steroid receptors by these synthetic
progestons, as compared to progesterone (Prog) was investigated in human
cell lines. In the first part of the study, the effect of Prog, MPA and NET-A on
the expression of endogenous cytokine genes was investigated in two
epithelial cell lines of the human female genital tract, Ect1/E6E7 (an
ectocervical cell line) and Vk2/E6E7 (a vaginal cell line). Quantitative realtime
RT-PCR (QPCR) showed ligand-specific and cell-specific regulation of the
interleukin (IL)-6, IL-8 and RANTES (Regulated-upon-Activation, Normal T cell
Expressed and Secreted) genes with Prog, MPA and NET-A. Moreover, the
repression of the TNF -induced RANTES gene by MPA in the Ect1/E6E7 cell
line was found to be mediated by the androgen receptor (AR). The second
part of the study focused on elucidating the androgenic activities of these two
progestins, in comparison to Prog. Competitive binding in whole cells revealed
that Prog, MPA and NET-A have a similar binding affinity for the hAR as the
natural androgen dihydrotestosterone (DHT). Both transactivation and
transrepression transcriptional assays demonstrate that, unlike Prog, MPA
and NET-A are efficacious AR agonists, with activities comparable to DHT.
Using a mammalian two-hydrid assay, it was shown that MPA and NET-A
exert their androgenic actions by different mechanisms. NET-A, like DHT and
other well-characterised androgens, induces the ligand-dependent interaction
between the NH2- and COOH-terminal domains (N/C-interaction) of the AR
independent of promoter-context, while MPA does this in a promoterdependent
manner. In the third part of this study, competitive binding revealed
that MPA and NET-A have a similar binding affinity to each other, but about a
100-fold lower affinity than Prog for the human mineralocorticoid receptor
(hMR), while RU486 has an even lower affinity for the hMR. Promoter-reporter
assays showed that MPA, NET-A and RU486 are all antagonists of the hMR,
but unlike Prog, they have weak antagonistic activity. However, on the
endogenous MR-regulated Orm-1 (a-glycolytic protein or orosomucoid-1)
gene expressed in a rat cardiomyocyte cell line, NET-A and RU486, but not
MPA, has similar antagonistic activity as Prog. This study is the first to show
that, NET-A and RU486, but not MPA, can dissociate between
transrepression and transactivation via the hMR. Taken together, these
results show that natural Prog and the synthetic progestins, MPA and NET-A
display differential promoter-, cell- and receptor-specific effects on gene
expression. Furthermore they may have important implications for
cervicovaginal immune function, cardiovascular and other physiological
functions. / AFRIKAANSE OPSOMMING: Medroksieprogesteroon asetaat (MPA), noretisteroon (NET) en derivate
daarvan (noretisteroon enantaat (NET-EN); noretisteroon asetaat (NET-A),
word deur miljoene vroue gebruik as voorbehoedmiddels en vir hormoon
vervangingsterapie (HVT). Tenspyte daarvan dat beide hierdie progestiene
algemeen gebruik word, is min bekend oor hulle meganisme van werking op
molekulêre vlak. In hierdie proefskrif word die differensiële regulering van
geenuitdrukking asook die molekulêre meganisme van werking deur middel
van steroïedreseptore van beide hierdie sintetiese progestiene, ondersoek, en
vergelyk met progesteroon (Prog), in menslike sellyne. In die eerste deel van
die studie is die effek van Prog, MPA en NET-A op die uitdrukking van
endogene sitokinien gene ondersoek in twee epiteel sellyne van die menslike
vroulike geslagskanaal, Ect1/E6E7 (‘n ektoservikale sellyn) en Vk2/E6E7 (‘n
vaginale sellyn). Kwantitatiewe intydse RT-PKR het ligand-spesifieke en selspesifieke
regulering van interleukien (IL)-6, IL-8 en RANTES (Regulering-na-
Aktivering, Normale T-sel Uitgedrukte en Afgeskei) gene getoon met Prog,
MPA en NET-A. Verder is gevind dat die onderdrukking van die TNF- -
geïnduseerde RANTES geen deur MPA in die Ect1/E6E7 sellyn bemiddel
word deur die androgeen reseptor (AR). Die tweede deel van die studie het
gefokus op die toeligting van die androgeniese aktiwiteit van die twee
progestiene in vergelyking met Prog. Kompeterende binding in volselle het
getoon dat Prog, MPA en NET-A ‘n soortelyke bindings affiniteit vir die
menslike AR as die natuurlike androgeen dehidrotestosteroon (DHT) vir die
menslike AR het. Beide transaktiverings en transonderdrukkings
transkripsionele analieses toon dat, anders as Prog, MPA en NET-A
effektiewe AR agoniste is met aktiwiteite wat vergelykbaar is met die van
DHT. Deur die gebruik van ‘n soogdier twee-hibried toets, kon gewys word dat
MPA en NET-A hul androgeniese effekte uitoefen deur verskillende
meganismes. NET-A, soos DHT en ander goed gekarakteriseerde androgene,
induseer die ligand-afhanklike interaksie tussen die NH2- en COOH-terminale
domeine (N/C-interaksie) van die AR, onafhanklik van die promoter-konteks.
MPA, aan die ander kant, doen dit op ‘n promoter-afhanklike manier. In die
derde deel van die studie het kompeterende binding getoon dat MPA en NETA
soortelyke relatiewe bindings affiniteite vir die menslike mineralokortikoïed
reseptor (hMR) het, maar dat hierdie affiniteit ongeveer 100-voud laer is as
die van Prog en dat die affiniteit van RU486 vir hMR selfs nog laer is.
Promoter-rapporteerder toetse het getoon dat MPA, NET-A en RU486 almal
antagoniste van die hMR is, maar anders as Prog, is hierdie ‘n swak
antagonistiese aktiwiteit. Nietemin, op die endogene MR-gereguleerde Orm-1
( -glikolitiese proteïen of orosomukoïed-1) geen, uitgedruk in ‘n rot
kardiomiosiet sellyn, het NET-A en RU486, maar nie MPA nie, ‘n soortgelyke
antagonistiese aktiwiteit as Prog. Hierdie studie is die eerste om te wys dat
NET-A en RU486, maar nie MPA nie, kan onderskei tussen transrepressie en
transaktivering deur middel van die hMR. Samevattend toon die resultate dat
natuurlike Prog en die sintetiese progestiene, MPA en NET-A, ‘n differentiële
promoter-, sel- en reseptor-spesifieke effek op geenuitdrukking het. Verder
mag die resultate belangrike implikasies vir servikovaginale immuunfunksie,
asook kardiovaskulêre en ander fisiologiese funksies, inhou.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/4585 |
| Date | 03 1900 |
| Creators | Africander, Donita Jean |
| Contributors | Hapgood, Janet P., Louw, Ann, University of Stellenbosch. Faculty of Science. Dept. of Biochemistry. |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Rights | University of Stellenbosch |
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