Date of Award

Spring 2009

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program/Concentration

Biomedical Sciences

Committee Director

Frank J. Castora

Committee Member

Darlene Dixon

Committee Member

Robert J. Swanson

Committee Member

Andrei O. Zalensky

Abstract

It is thought that the growth of uterine leiomyomas may be mediated by the interaction of estrogen receptor alpha (ERα) and growth factor pathways and that phosphorylation of ERα at serine 118 (ERα-phospho-Ser118) is important in this interaction. In tissue, immunoblotting and immunohistochemistry were used to investigate the expression of ERα-phospho-Ser118, phosphorylated p44/42 mitogen-activated protein kinase (phospho-p44/42 MAPK), and proliferating cell nuclear antigen (PCNA) in human leiomyoma and myometrial tissues during the proliferative and secretory phases of the menstrual cycle. In vitro studies to assess proliferation of uterine leiomyoma (UtLM) and uterine smooth muscle (UtSMC) cells and expression of ERα-phospho-Ser118 and phospho-p44/42 MAPK were done using western blotting and a cell proliferation assay, respectively, alter treatment with estrogen (E2) and progesterone (P4), or with the clinical compounds, raloxifene and asoprosnil in the presence and absence of the MAPK inhibitor, PD98059. The tissue studies showed that tumors taken from the proliferative phase expressed significantly higher levels of ERα-phospho-Ser118, phospho-p44/42 MAPK, and PCNA compared to patient-matched myometrial samples and had significantly higher ERα-phospho-Ser118 and PCNA expression compared to secretory phase tumors. Also, enhanced colocalization and association of phospho-p44/42 MAPK and ERα-phospho-Ser118 were observed in proliferative phase tumors by confocal microscopy and immunoprecipitation, respectively. The in vitro studies showed that ERα-phospho-Ser118 and phospho-p44/42 MAPK protein expression levels are increased in UtLM cells in a prolifeative phase versus secretory phase hormonal milieu and are reduced in UtLM cells after treatment with PD98059 in the proliferative phase. Also, the in vitro studies showed more interaction and co-expression of ERα-phospho-Ser118 and phospho-p44/42 MAPK proteins in leiomyoma cells treated with proliferative versus secretory phase hormones. These in vitro studies demonstrated that in uterine leiomyoma cells phospho-p44/42 MAPK phosphorylates ERα at serine 118 and this phosphorylation is increased when leiomyoma cells are treated with the proliferative phase (E2 predominant) hormones. Increased proliferation and enhanced ERα-phospho-Ser118 expression was observed in UtLM cells following treatment with E2. Also, the clinical compound raloxifene, but not asoprisnil was found to inhibit E2-induced leiomyoma cell growth in part by decreasing phosphorylation of ERα at serine 118.

Comments

Dissertation submitted to the Faculty of Eastern Virginia Medical School and Old Dominion University in Partial Fulfillment of the Requirement for the Degree of Doctor of Philosophy in Biomedical Sciences.

DOI

10.25777/2rg1-vh65

ISBN

9781109217551

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