39 research outputs found
A morphological study of human endometrial stroma in vivo and in vitro.
Despite its crucial role in fertility, relatively little has been published on the human
endometrial stroma. The first experiment reported in this thesis was designed to provide
quantitative baseline data on the human stroma during the mid to late luteal phase, when it plays
a major part in normal pregnancy. Subsequent chapters investigate the effects of an antioestrogen
(clomiphene citrate-CC); growth hormone (GH) supplementation of patients on
hormone replacement therapy (HRT) due to lack of endogenous ovarian steroids; and patients
with unexplained recurrent miscarriage. Finally a novel 3-dimensional in vitro model of human
stroma is described along with the effects of steroid supplementation on stromal cells grown in
the model.
In all cases of in vivo studies, conventional stereological methods were used to obtain
quantitative morphological data from at least 6 subjects per group, from both control (fertile)
and experimental (infertile) subjects using light and electron microscopy. In addition, several
staining techniques were also used to demonstrate qualitative changes that occur in human
endometrial stroma.
During the mid to late luteal phase, endometrial stroma and blood vessels underwent
substantial changes, and thus quantitative and qualitative baseline data have been established to
fill a gap in this important area of reproductive biology. CC caused no substantial changes in
stromal structure in women of proven fertility and therefore its advantageous effects on
ovulation are not negated at the level of the endometrium. GH supplementation had no effects
on infertile human endometrial stroma from subjects on HRT, (in either premature ovarian
failure or Turner's syndrome groups). However, both infertile groups had endometrial stroma
which significantly differed from matched fertile endometrium, suggesting an impaired
endometrial development in infertile subjects which was not reversed by HRT, either with or
without Gil treatment. Stromal morphology did not differ between the recurrent miscarriage
groups, however data suggested the existence of several subgroups which made firm
conclusions difficult. The tissue culture model provided preliminary data suggesting it to be a
potentially very useful technique for the study of stromal cell biology
Isolation and characterization of androgen receptors from male target cells
Androgens exert their action after binding to cytoplasmic
receptors resulting in the formation of androgenreceptor
complexes. This initial event is followed by activation,
translocation to the nucleus and interaction with
chromatin acceptor sites of the androgen-receptor comulexes.
Bound to chromatin, the androgen-receptor complex stimulates
many biochemical events resulting in gene expression and
starting with RNA-synthesis. Further detailed understanding
of the complex processes requires a purified receptor preparation.
Due to the low amount of receptors present ln androgen
target organs, large scale isolation of these receptors
requires a suitable source. Thusfar it seems that androgen
receptors from different sources have similar characteristics.
In this respect seminal vesicles of the ram contain
an androgen receptor comparable to the receptor present in
rat prostate (chapter 4.3 and appendix paper II). Studies
were performed to purify the receptor present ln ram seminal
vesicles, and an almost two thousand fold purified receptor
preparation has been obtained (chapter 4.4 and appendix
paper III). Nuclear localization and acceptor sites of
androgen-receptor complexes on the chromatin in target cells
have hardly been studied. To gain more insight in the mechanism
of interaction of androgen-receptor complexes with
chromatin acceptor sites, the usefulness of purified androgen
receptors was investigated. In preliminary studies high
affinity interaction of androgen-receptor complexes with
isolated chromatin was observed (chapter 5) and the possibilities
for further investigation are discussed (chapters 6.3
and 6.4)
Molecular basis of anti-hormonal treatment and resistance in breast cancer
Le cancer du sein est le plus fréquent cancer chez les femmes dans le monde. Environ 70% des tumeurs mammaires expriment le récepteur des oestrogènes alpha (REa) et sont considérées comme hormono-sensibles. La thérapie hormonale a longtemps été le traitement de choix. Toutefois, les effets agonistes sur le REa et le développement de la résistance des thérapies actuels, tels que le tamoxifène nécessitent le développement de nouveaux traitements qui agissent par des mécanismes distincts. L'objectif de notre étude était de concevoir des outils qui peuvent aider à comprendre les mécanismes moléculaires impliqués dans la modulation ligand-dépendante ou dans la dégradation du REa. Nous avons choisi quelques anti-oestrogènes avec différentes structures et nous avons comparé leurs effets sur: 1. la dégradation du REa.2. La localisation intra-cellulaire du REa. 3. La régulation de la transcription des gènes cibles du REa 4. La régulation de la transcription dans les mutants de REa. Grace à notre appproche mécanistique, nous avons pu classer ces anti-estrogènes en trois groupes sur la base de leur fonction : SERM, SERD et un nouveau groupe EM. Les SERM (selective estrogen receptor modulator) stabilisent le REa, induisent une re-localisation du REa dans le noyau, augmentent l'activité transcriptionnelle de mutants qui affectent le recrutement de cofacteurs et qui altèrent la liaison avec la chaîne latérale de l'anti-œstrogène et, enfin, manquent de pouvoir d'inhibition de l'expression basale de gènes endogènes. Les SERD (selective estrogen receptor disruptor) induisent une dégradation protéasome dépendante du REa dans le noyau en formation des foyers nucléaires qui colocalisent avec le proteasome, et inhibent l'expression basale du gène endogène du récepteur de la progestérone (PGR). Enfin, EM652 est un composé qui affecte le devenir du RE comme les SERM mais qui a un effet inhibiteur sur l'expression basale du gene endogene PGR. Cette approche peut être utilisée pour cribler de nouveaux anti-oestrogènesBreast cancer is the most common type of malignancy among women in the world. Approximately 70% of breast tumours express the estrogen receptor alpha (ERa) and are considered hormone-responsive. Endocrine therapies have long been the treatment of choice. However, the estrogen- like agonist effect and development of resistance of the available selective estrogen receptor modulator such as tamoxifen require developing new treatments that act through different mechanisms. The objective of our study is to design tools that can help to understand the molecular mechanisms involved in ligand-dependent modulation or degradation of ERa. We selected a set of anti-estrogens with different structures and compared their effect on:
1. ERa degradation. 2. Intra-cellular localisation of ERa. 3. Regulation of transcription of ERa- endogenous target genes. 4. Regulation of transcription in the mutants of ERa.
Using this mechanistic study we could classify the tested anti-estrogens into three groups based on their function: SERM, SERD and a new group for EM652. SERM (selective estrogen receptor modulator) include compounds such as OH-tamoxifen and RU39411, that stabilise ERa, that re-localize ERa into the nucleus upon binding, that increase transcriptional activity in mutants affecting the recruitment of cofactors or the binding of their side chain and that lack inhibitory capacities of the basal expression of endogenous genes. SERD (selective estrogen receptor modulator) include compounds such as ICI182580 or RU58668, that induce nuclear proteasome-dependent degradation ERalpha which occur in large nuclear foci that colocalize with the proteasome and that inhibit basal gene expression of the endogenous progesterone receptor gene (PGR). Finally, EM652 was found to affect ER degradation and localisation similarly to SERM but inhibited basal gene expression of the endogenous PG
Oestrogen receptor dynamics and cell signalling
Oestrogen receptors (ER) have classically been described as ligand-inducible nuclear
transcription factors. The pleiotrophic effects of ER function have a predominant role
in the direct regulation of the growth, differentiation and development of tissues of
the human reproductive system. There are two ER subtypes, ER and ER which
differ in their specificity for ligand and the consequent actions they orchestrate.
Moreover, the latter exists in multiple splice variants of which ER is the only fully
functional homologue. Research into the underlying differences in subtype responses
to ligand has involved examination of the intranuclear dynamics of individual
receptor subtypes. Studies into the mobility of ER in response to ligand have
exclusively focused on studies of full length ER and ER independently in
transfected cell lines. The studies described in this thesis have investigated the
kinetics of ER using Fluorescence Recovery After Photobleaching (FRAP) in
infected cell lines which lends itself to more precise expression of the subtype of
interest. The morphological impact of natural oestrogenic and synthetic ligands on
ERs was examined and the influence on the intranuclear dynamics assessed. Further
to this, the effect of co-expression of different ER subtype combinations was
examined.
Studies on the intranuclear mobility of ER have confirmed and extended the findings
of others. Previous work on the development of ER agonists and antagonists has
been to target specific overexpressing ER subtypes in a physiological setting. In this
study, we demonstrated for the first time an overwhelming ER -selective effect in
slowing the rate of mobility within the nucleus, suggesting the study of intranuclear
dynamics is an important parameter for the examination of efficacy of a compound.
Differential responses to ligand based on co-infected partnerships indicate that
heterodimerisation has a profound effect in augmenting ligand-dependent regulation
and activity
Modification of oestrogen signalling during the acquisition of hormone resistance in breast cancer cells
The oestrogen receptor (ER) mediates both normal and malignant breast
development. The ER can exert its ligand-dependent gene regulatory functions
directly by association with target genes and subsequent dynamic formation of a
transcription complex through its oestrogen response element. The complex receptorligand signalling network has received much interest as a potential therapeutic target.
Endocrine agents, such as tamoxifen, have been developed to reverse oestrogen (E2)
stimulated gene transcription and tumour growth, however progression to endocrine
resistance provides a major obstacle in breast cancer treatment. This study
investigates changes in endocrine response and ER transcription activation during the
acquisition of endocrine resistance.Breast cancer cell lines were selected that encompassed the range of oestrogen
and anti-oestrogen sensitivities from the E^-dependent and tamoxifen-sensitive MCF7 cell line through the less sensitive LCC-1 and LCC-2 lines to the insensitive LCC-9
and independently derived LY2 line. All lines had been derived from MCF-7 cells.
These models represent different endocrine phenotypes and were designed to reflect
sequential changes in the clinical progression from hormone sensitive to hormone
insensitive and antioestrogen resistant. MDA-MB-231 breast cancer cells were used
as ERα negative controls. Growth assays confirmed these phenotypes and
proliferative behaviour in response to E₂ and tamoxifen.To help evaluate the role of the ERα in the development of endocrine resistance,
quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and Western
blot analysis was carried out to assess transcriptional and translational receptor status
for each phenotype. All resistant lines possessed greater levels of ERα mRNA than
wild-type MCF-7 cells. E₂ downregulated ERα mRNA and protein. Results suggest
ERa functionality in these cell lines. Analysis of mRNA levels of several ER target
genes (pS2, progesterone receptor (PR), cathepsin D and MYC) indicated
informative differences between lines. pS2 was highly expressed in resistant lines as
was PR in most lines compared to MCF-7 cells. E2 and tamoxifen produced
attenuated or no effects in pS2 expression compared to the marked induction
produced by E2 in MCF-7 cells. Minor changes were also observed in the expression
levels of various coactivators (SRC-1, SRC-2, SRC-3) and corepressors (NCoR,
SMRT and RIP140) investigated by qRT-PCR and Western blot.A subset of three cell lines (MCF-7, LCC-1 and LCC-9) was used to examine
transcription complex assembly at the pS2 promoter in response to E2 utilizing
chromatin immunoprecipitation. This identified a dynamic cycle of increasing H4
acetylation (indicative of active transcription) and ERα as well as cofactor
recruitment upon E₂ addition in MCF-7 cells. A distinctive H4 acetylation pattern
was revealed for LCC-1 and LCC-9 cells. While ERα recruitment was similar to
MCF-7 cells, particularly strong SRC-1 and SRC-3 recruitment was detected in
LCC-1 but most markedly in LCC-9 cells, implying altered pS2 transcription
complex assembly.The data suggest that a functional oestrogen receptor may remain in these
endocrine resistant models. Cell proliferation and E₂ target gene expression in LCC-1
cells proved to be oestrogen and antioestrogen independent but responsive in the
presence of the ligand whereas LCC-9 cells manifest complete endocrine resistance.
In addition, changes have been detected in the assembly of a gene transcription
complex at the pS2 promoter. Taken together, this is evidence for modified ER
mediated transcription activity. These results may help to identify potential
mechanisms of endocrine resistance
Role of mast cells in women's health and disorders of the endometrium
During the normal menstrual cycle, the human endometrium undergoes
extensive tissue remodelling under the influence of ovarian-derived hormones. The
endometrium has well defined stromal and epithelial compartments with the former
containing both a well-developed vasculature as well as a diverse population of
immune cells. Mast cells (MCs) are long-lived tissue resident immune cells
characterised by the presence of granules containing proteases. Mast cells have been
detected in the human uterus but little is known about their regulation or the impact of
steroids on their differentiation status. Recently MCs have been implicated as key
players in physiological and pathological pain pathways but little is known about their
role in endometrial pathologies. Endometriosis is a chronic incurable condition
characterized by the presence of endometrial tissue outside the uterine cavity: women
with endometriosis can suffer from a debilitating range of symptoms including chronic
pain. Whilst the aetiology of endometriosis is uncertain, close proximity between MCs
and nerves has implicated them in aberrant activation of pain pathways.
The aims of the current project were: 1. To determine the spatial and temporal
location of uterine MCs and to explore their phenotype including expression of steroid
receptors. 2. To explore the activation status of MCs in women with endometriosis
and/or pain, 3. To explore the use of cells and mice as models to investigate the
phenotype of mast cells and their regulation by steroids.
Mast cell proteases tryptase and chymase were detected by RTPCR and
immunohistochemistry in “full thickness” (uterine lumen to endometrial-myometrial
junction) biopsies from women undergoing hysterectomy. In agreement with previous
findings MCs were most abundant in the myometrium. Uterine MCs were
predominantly of the classical MC subtypes: tryptasepos/chymaseneg and
tryptasepos/chymasepos but a rare third subtype was also identified as
tryptaseneg/chymasepos. Mast cell activation/degranulation was cycle stage dependent
and for the first time their steroid receptor phenotype was identified as
ERαneg/ERβpos/GRpos, suggesting potential regulation by the uterine steroid
microenvironment. Studies on tissue samples from women with endometriosis revealed MCs with
an altered activation status in the pelvic peritoneal wall, compared to controls, which
showed an intense diffuse immunoexpression of chymase suggestive of MC activation
and release of this protease during normal physiology of the peritoneum. Surprisingly,
analysis of peritoneal fluids from controls, women with pain but no endometriosis, and
pain with endometriosis did not detect differences in numbers of MCs or
concentrations of tryptase or chymase. Analysis of peritoneal biopsies also provided
the first evidence for a striking increase in immunoexpression of PAR-2, a protease-activated
receptor, in women suffering from chronic pelvic pain and/or endometriosis
which may provide a mechanism by which mast cell derived factors may alter pain
pathways.
Studies in a mouse model of endometriosis identified MCs within endometria-llike
lesions and offer a platform for future studies. In vitro explorations using MCs
derived from peripheral blood precursors and HMC-1, a cell line derived from a patient
with MC leukaemia confirmed expression of ERβ but did not support previous studies
claiming cells were ERαpos.
In summary, this study has provided novel insights into the phenotype of
endometrial mast cells in the normal cycling endometrium and contrasted them with
those in women with endometriosis and pelvic pain. This is the first study to identify
MCs as ERβpos. Further studies are required to determine whether inhibition of PAR-
2 might offer a therapeutic target in women with chronic pelvic pain