Full Text Available
Note: Clicking the button above will open the full text document at the original institutional repository in a new window.
Comparative study of the molecular mechanism of action of the synthetic progestins, Medroxyprogesterone acetate and Norethisterone acetate
Thesis (PhD (Biochemistry))--University of Stellenbosch, 2010.
Saved in:
| Main Author: | |
|---|---|
| Other Authors: | |
| Format: | Thesis |
| Language: | en_ZA |
| Published: |
Stellenbosch : University of Stellenbosch
2010
|
| Subjects: | |
| Tags: |
No Tags, Be the first to tag this record!
|
| _version_ | 1867613874222530561 |
|---|---|
| access_status_str | Open Access |
| author | Africander, Donita Jean |
| author2 | Hapgood, Janet P. |
| author_browse | Africander, Donita Jean Hapgood, Janet P. |
| author_facet | Hapgood, Janet P. Africander, Donita Jean |
| author_sort | Africander, Donita Jean |
| collection | Thesis |
| dc_rights_str_mv | University of Stellenbosch |
| description | Thesis (PhD (Biochemistry))--University of Stellenbosch, 2010. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/4585 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA |
| last_indexed | 2026-06-10T12:43:03.527Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2010 |
| publishDateRange | 2010 |
| publishDateSort | 2010 |
| publisher | Stellenbosch : University of Stellenbosch |
| publisherStr | Stellenbosch : University of Stellenbosch |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/4585 Comparative study of the molecular mechanism of action of the synthetic progestins, Medroxyprogesterone acetate and Norethisterone acetate Africander, Donita Jean Hapgood, Janet P. Louw, Ann University of Stellenbosch. Faculty of Science. Dept. of Biochemistry. Synthetic progestins Hormone replacement therapy Contraceptives Medroxyprogesterone Norethindrone Dissertations -- Biochemistry Theses -- Biochemistry Biochemistry 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. Doctoral 2010-02-23T15:11:46Z 2010-09-03T07:15:56Z 2010-02-23T15:11:46Z 2010-03 Thesis http://hdl.handle.net/10019.1/4585 en_ZA University of Stellenbosch application/pdf Stellenbosch : University of Stellenbosch |
| spellingShingle | Synthetic progestins Hormone replacement therapy Contraceptives Medroxyprogesterone Norethindrone Dissertations -- Biochemistry Theses -- Biochemistry Biochemistry Africander, Donita Jean Comparative study of the molecular mechanism of action of the synthetic progestins, Medroxyprogesterone acetate and Norethisterone acetate |
| title | Comparative study of the molecular mechanism of action of the synthetic progestins, Medroxyprogesterone acetate and Norethisterone acetate |
| title_full | Comparative study of the molecular mechanism of action of the synthetic progestins, Medroxyprogesterone acetate and Norethisterone acetate |
| title_fullStr | Comparative study of the molecular mechanism of action of the synthetic progestins, Medroxyprogesterone acetate and Norethisterone acetate |
| title_full_unstemmed | Comparative study of the molecular mechanism of action of the synthetic progestins, Medroxyprogesterone acetate and Norethisterone acetate |
| title_short | Comparative study of the molecular mechanism of action of the synthetic progestins, Medroxyprogesterone acetate and Norethisterone acetate |
| title_sort | comparative study of the molecular mechanism of action of the synthetic progestins medroxyprogesterone acetate and norethisterone acetate |
| topic | Synthetic progestins Hormone replacement therapy Contraceptives Medroxyprogesterone Norethindrone Dissertations -- Biochemistry Theses -- Biochemistry Biochemistry |
| url | http://hdl.handle.net/10019.1/4585 |
| work_keys_str_mv | AT africanderdonitajean comparativestudyofthemolecularmechanismofactionofthesyntheticprogestinsmedroxyprogesteroneacetateandnorethisteroneacetate |