An operational model for estrogenic action in the presence of sex hormone binding globulin (SHBG)

Vismer, Michael John (2007-03)

Thesis (MSc)--University of Stellenbosch, 2007.

Thesis

ENGLISH ABSTRACT: The aim of this study was to build a mathematical model that describes the binding of 17- -estradiol (E2) to estrogen receptor (ER- ) and the influence the sex hormone binding globulin (SHBG) has on this interaction. The influence of SHBG on the transactivation of an estrogen response element, via ligand bound ER- , was also studied. COS-1 cells, derived from the kidney of a green african monkey, were used to study the binding of E2 to ER- in the absence of SHBG. The influence of SHBG on the binding of E2 to ER- was studied using Hep89 cells, human hepatacoma carcinoma, which express SHBG endogenously and are stably transfected with the ER- gene. Human pregnancy plasma was used to study the interaction of E2 with SHBG in the absence of ER- . The results of this study have shown that the Kd (E2) for ER- was determined as between 3.4nM and 4.4nM in the absence of SHBG. With respect to the binding of E2 to ER- it was not possible to determine the Kd app and Bmax for ER- using the Hep89 experimental system. The Kd (E2) for SHBG was not determined using the human pregnancy plasma experimental system. With the aid of mathematical modelling, a model of the Hep89 and human pregnancy plasma experimental systems, was built. The results of the numerical modelling, using mathematical modelling, showed that the presence of albumin together with SHBG was the reason that the Kd app (E2) could not be determined in the Hep89 experimental system. With respect to the use of human pregnancy plasma to determine the Kd (E2) for SHBG it was shown that if the plasma was diluted 200 times it would have been possible to determine the Kd app (E2) for SHBG, in the presence of albumin. Ligand independent transactivation of an estrogen response element was shown to be a problem in the COS-1 cell system when promoter reporter gene assays were undertaken. As COS-1 cells were used as a control for the absence of SHBG no further promoter reporter gene assays were undertaken using the Hep89 experimental system.

AFRIKAANSE OPSOMMING: Die doel van hierdie studie was die bou van ‘n wiskundige model wat die verbinding van E2 met die estrogeenreseptor (ER- ) en die invloed wat die geslagshormoon-verbindingglobulien (SHBG) op hierdie interaksie het, beskryf. Die effek van SHBG op die transaktivering van ‘n estrogeen responselement, via die ligandverbonde ER- , is ook bestudeer. COS-1-selle uit die nier van ‘n groen afrika-aap is gebruik om die verbinding van E2 met ER- in die afwesigheid van SHBG te bestudeer. Die invloed van SHBG op die verbinding van E2 met ER- , is bestudeer deur gebruik te maak van Hep89-selle, die menslike lewergeswelkarsinoom, wat SHBG uitwendig afgee en wat stabiel getransfesteer kan word met die ER- geen. Menslike swangerskapplasma is gebruik om die interaksie van E2 met SHBG in die afwesigheid van ER- te bestudeer. Die uitslag van hierdie studie toon aan dat die Kd (E2) vir ER- vasgestel tussen 3.4nM en 4.4nM in die afwesigheid van SHBG. Met betrekking tot die verbinding van E2 met ER- , was dit nie moontlik om die Kd (E2) en Bmax app vir ER- met die gebruik van die Hep89 eksperimentele stelsel vas te stel nie. Die Kd (E2) vir SHBG is nie vasgestel deur die gebruik van die menslike swangerskapplasma eksperimentele stelsel nie. ‘n Model van die Hep89 en menslike swangerskapplasma eksperimentele stelsels is met behulp van wiskundige modellering gebou. Die uitslag van die numeriese modellering, met gebruik van wiskundige modellering, toon dat die teenwoordigheid van albumien, saam met SHBG, die rede was dat die Kd app (E2) nie in die Hep89 eksperimentele stelsel vasgestel kon word nie. Wat betref die gebruik van menslike swangerskapplasma om die Kd (E2) vir SHBG vas te stel, is daar aangetoon dat, indien die plasma 200 maal verdun was, dit moontlik sou gewees het om die Kd app (E2) vir SHBG in die teenwoordigheid van albumien vas te stel. Promotor verkilkkergeen toetse het ligandonafhanklike transaktiveering van ‘n estrogeen responselement aangetoon as ‘n probleem in die COS-1-selle stelsel. Omdat COS-1-selle gebruik is as ‘n kontrole vir die afwesigheid van SHBG, is geen verdere promotor verkilkkergeen toetse onderneem met die gebruik van die Hep89 eksperimentele stelsel nie.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/19887
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