Human Reproduction, Vol.30, No.12 pp. 2816–2828, 2015
Advanced Access publication on October 25, 2015 doi:10.1093/humrep/dev267
ORIGINAL ARTICLE Gynaecology
Human endometrial epithelial
telomerase is important for epithelial
proliferation and glandular formation
with potential implications in
endometriosis
A.J. Valentijn 1,†, G. Saretzki 2,†, N. Tempest1,3, H.O.D. Critchley4,
and D.K. Hapangama 1,3,*
1
Department of Women’s and Children’s Health, Institute of Translational Medicine, University of Liverpool, Liverpool L8 7SS, UK
Institute for Cell and Molecular Biosciences and Institute for Ageing, Campus for Ageing and Vitality, Newcastle University, Newcastle upon
Tyne NE4 5PL, UK 3Liverpool Women’s Hospital NHS Foundation Trust, Liverpool, UK 4MRC Centre for Reproductive Health,
University of Edinburgh, Edinburgh EH16 4TJ, UK
2
*Correspondence address. Department of Women’s and Children’s Health, Institute of Translational Medicine, University of Liverpool,
Liverpool L8 7SS, UK. E-mail: [email protected]
Submitted on March 1, 2015; resubmitted on August 27, 2015; accepted on September 11, 2015
study question: How does regulation of telomerase activity (TA) in human endometrial epithelial cells (EEC) by ovarian hormones impact
on telomere lengths (TL) and cell proliferation?
summary answer: Healthy endometrial epithelial cell proliferation is characterized by high TA and endometrial TL changes according to
the ovarian hormone cycle, with shortest TL observed in the progesterone dominant mid-secretory phase, when TA is lowest, implicating progesterone in the negative regulation of TA and TL.
what is known already: Critical shortening of telomeres may result in permanent cell cycle arrest while the enzyme telomerase maintains telomere length (TL) and replicative capacity of cells. Telomerase expression and activity change in the human endometrium with the ovarian
hormone cycle, however the effect of this on endometrial TL and cell growth is not known.
study design, size, duration: A prospective observational study, which included endometrial and blood samples collected from
196 women.
participants/materials, setting, methods: We studied endometrial samples from five different groups of women. Endometrial and matched blood TL and circulating steroid hormones were studied in samples collected from 85 women (Group 1). Fresh epithelial and
stromal cell isolation and culture in vitro for TL and TA was done on endometrial biopsies collected from a further 74 healthy women not on hormonal therapy (Group 2) and from 5 women on medroxyprogesterone acetate (MPA) for contraception (Group 3). The epithelial TL and telomerase protein expression was examined in active, peritoneal, ectopic endometriotic and matched uterine (eutopic) endometrial samples
collected from 10 women with endometriosis (Group 4); the in vivo effect of mifepristone on telomerase protein expression by immunohistochemistry (IHC) was examined in endometrium from 22 healthy women in mid-secretory phase before (n ¼ 8), and after administering
200 mg mifepristone (n ¼ 14) (Group 5). TA was measured by telomere repeat amplification protocol (TRAP) assay; TL by qPCR, and
Q-FISH; cell proliferation was assessed by immunoblotting of histone H3 and 3D-culture to assess the ability of EECs to form spheroids; telomerase reverse transcriptase protein levels and Ki-67 (proliferative index) were assessed with IHC.
main results and the role of chance: Endometrial TLs correlated negatively with serum progesterone levels (n ¼ 58,
r ¼ 20.54) and were significantly longer than corresponding blood TLs (4893 + 929 bp versus 3955 + 557 bp, P ¼ 0.002) suggesting a
tissue-specific regulation. High TA and short TLs were observed in proliferating EECs in vivo and in vitro. During the progesterone dominant
mid-secretory phase endometrial TL were significantly shorter compared with the proliferative phase (P ¼ 0.0002). Progestagen treatment
†
The first two authors contributed equally.
& The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
For Permissions, please email: [email protected]
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Endometrial telomerase regulation
suppressed EEC TA in vivo and reduced endometrial TA in explant (P ¼ 0.01) and in vitro cultures (P ¼ 0.02) compared with untreated cells. Mifepristone (progesterone receptor antagonist) increased telomerase protein levels in vivo (P , 0.05). In 2D culture, Imetelstat inhibited EEC TA
(P ¼ 0.03), proliferation (P ¼ 0.009) and in 3D culture disrupted endometrial glandular architecture (P ¼ 0.03).
limitations, reasons for caution: The in vitro telomerase inhibition data were tested in a mono-cellular system for a short-term.
Further confirmation of the results in an in vivo model is necessary. The women in group 2 included a high proportion of women although with a
regular menstrual cycle, with an increased BMI (.25) therefore this may affect extrapolation of data to other groups.
wider implications of the findings: The observed effects of telomerase inhibition in vitro on epithelial cell proliferation, suggest
that telomerase might be an attractive target in developing new therapies for proliferative disorders of the endometrium, such as endometriosis.
study funding/competing interest(s): The work was supported by Wellbeing of Women project grant RG1073 (D.K.H./
G.S.), RG1487 (D.K.H./G.S.), MRC/DFID funded Special Project Grant No: G9523250 (H.O.D.C.) and Geron PLC (A.J.V./D.K.H.). Geron
PLC, USA covered expenses for the Imetelstat experiments (A.J.V.) described in this manuscript. The terms of this arrangement have been
reviewed and approved by the University of Liverpool, UK in accordance with its policy on objectivity in research. The authors have no other
conflicts of interest to declare.
trial registration number: N/A.
Key words: endometrium / telomere length / telomerase / progesterone / Imetelstat / endometriosis / endometrial epithelium / proliferation / cell culture / 3D culture
Introduction
The human endometrium is a highly dynamic tissue undergoing repetitive
monthly cycles of growth, differentiation, shedding, and regeneration
throughout a woman’s reproductive life. This cycle of endometrial cell
proliferation and growth is regulated by ovarian steroid hormones (Hapangama et al., 2015). Telomerase remains inactive in most somatic cells
apart from those with self-renewing abilities such as the endometrium
(Williams et al., 2001; Hapangama et al., 2008). Telomerase is a ribonucleoprotein complex consisting of two core components, the RNA-part
containing the template region for telomere synthesis, hTERC and the
catalytic telomerase reverse transcriptase (hTERT). The main function
of telomerase is the maintenance of the telomeres that cap the ends of
chromosomes thereby regulating the proliferative lifespan of a cell;
when telomeres shorten to a critical length the cell undergoes senescence or apoptosis. In addition to maintaining telomere length, telomerase may also have a role in proliferation and modulation of the expression
of growth-promoting genes (Belair et al., 1997; Greider, 1998; Smith
et al., 2003). In healthy endometrium telomerase activity (TA) correlates
with proliferation; is typically associated with the glandular epithelial cells
and is regulated in a menstrual-phase dependent manner, suggesting a
regulatory role for sex steroids (Tanaka et al., 1998; Williams et al.,
2001). hTERT is a critical determinant of TA (Kyo et al., 1999) and is a
target of estrogen and progesterone (Kyo et al., 1999; Wang et al.,
2000). This has implications for endometrial proliferative diseases,
such as endometriosis, defined as the presence of endometrial cells
outside the uterine cavity and endometrial cancer; both pathologies
associated with estrogen action and high TA. Progesterone antagonizes
estrogen-induced proliferation in the endometrium and lack of progesterone activity on estrogen primed endometrium increases the risk of
endometrial cancer whilst endometriosis is commonly associated with
progesterone resistance (Bulun, 2010). Telomere length (TL) in peripheral blood mononuclear cells has been proposed as a potential biomarker of ageing and disease risk. Shortened TL has been linked to
endometrial cancer (Smith and Yeh, 1992; Akbay et al., 2008) while elongated TL has been observed in the endometrium of women with
endometriosis (Hapangama et al., 2008, 2009). Although progesterone
is therapeutically given to inhibit the growth of estrogen-dependent
cancers and in the management of endometriosis, little is known about
the impact of progesterone on normal endometrial TA and TL. We
report herein that like TA, TL changes with cyclical (hormone dependent) endometrial epithelial cell (EEC) proliferation in vivo and in ectopic
lesions in endometriosis. Furthermore, inhibition of TA compromises
endometrial epithelial cell proliferation and affects the ability of such
cells to form glandular-like structures in a 3D culture.
Materials and Methods
Patient groups
Human endometrial samples for the measurement of TL were obtained from
85 fertile women who have each had at least one live birth, not on any hormonal therapy and a matched venous blood samples were obtained simultaneously from 70 of these women (Group 1). Peripheral blood mononuclear
cell (PBMC) TL was available for 53 women and circulating estradiol (E2)
and progesterone were also measured on 70 samples by radioimmunoassay,
as previously described (Hapangama et al., 2008).
Endometrial biopsies were collected from a further 74 healthy women,
who have had at least one full term pregnancy, without a history of recurrent
miscarriage or infertility, not on hormonal therapy (Group 2) and from
5 women on medroxyprogesterone acetate (MPA) for contraception
(Group 3) for fresh cell isolation and culture in vitro. All above mentioned
women (Groups 1 – 3) underwent surgery for benign, non-endometrial
gynaecological conditions (e.g. female sterilization) with no endometriosis
(surgically excluded).
Additionally, ectopic endometriotic and matched uterine (eutopic) endometrial samples were collected from 10 women with symptomatic peritoneal
endometriosis, not on hormonal treatment, undergoing surgical excision of
endometriosis (Group 4).
Endometrial biopsies were also collected from healthy fertile women
in mid-secretory phase before (n ¼ 8), and after 200 mg mifepristone
(n ¼ 14) as previously published (Hapangama et al., 2002) (Group 5) and
examined with immunohistochemistry (IHC).
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Valentijn et al.
Endometrial biopsies were dated using histological criteria (Noyes et al.,
1975), the date of last menstrual period and hormone profile. All women
were of reproductive age (18– 46 years) with further demographic data presented in Table I. Ethical approval for the study was obtained from Liverpool
Adult Research Ethics committee (LREC09/H1005/55 and 11/H1005/4)
and Lothian Research Ethics Committee (Institutional Review Board) (Hapangama et al., 2008).
Immunohistochemistry and
immunofluorescence
Three-micrometre paraffin sections underwent antigen retrieval at pH6 in
citrate buffer (Hapangama et al., 2012). Primary antibodies used: Ki67
(clone MM1, Leica Biosystems Ltd, Newcastle Upon Tyne UK) and telomerase (ab27573, Abcam, Cambridge UK). Detection was with ImmPRESS
anti-mouse/rabbit polymer and visualization with ImmPACT DAB (Vector
Laboratories, Peterborough, UK). Non-immune IgG was used as negative
control. Ki67 was evaluated as percentage of immunostaining positive cells
and an arbitrary four-point semi-quantitative scoring scale (0, negative/no
staining; 1, weak; 2, strong; 3, very strong) was used to analyse telomerase
staining as previously described (Hapangama et al., 2008). For immunofluorescence (IF), EECs in monolayer were fixed in 10% (w/v) NBF
(neutral-buffered formalin, Sigma-Aldrich, Dorset, UK) and immunostained
for pan-cytokeratin (C9231, Sigma-Aldrich, Dorset, UK) and F-actin (FITCPhalloidin, P5282, Sigma-Aldrich, Dorset, UK). Images were collected and
processed as described previously (Valentijn et al., 2013).
TA and TL assays
TA was measured using the TeloTTAGGG TRAP assay (Telomere Repeat
Amplification Protocol assay; Roche Diagnostics, Ltd, Burgess Hill, UK)
using 1 mg of lysate and mean TL was measured with qPCR in the blood,
tissue and cells as previously described (Hapangama et al., 2008, 2009; Valentijn et al., 2013). Telomerase activity was measured as absorbance at 450 nm
in a Fluostar Omega Plate reader (BMG Labtech) and presented as arbitrary
units (AU).
were used at 1 mM for 72 h to inhibit TA in monolayer cultures of epithelial
cells. Dosing regime was based on previous publications (Goldblatt et al.,
2009; Marian et al., 2010; Mender et al., 2013) and a trial on epithelial cells
derived from a patient (in-house) with endometrial adenocarcinoma maintained in monolayer culture.
Telomere Q-FISH
Quantitative fluorescence in situ hybridization (Q-FISH) was performed on
3 mm-thick sections using a Cy-3 labelled PNA (peptide nucleic acid) telomere oligonucleotides (Panagene, Daejeon, Korea) (Hewitt et al., 2012).
Nuclei were stained with DAPI (4′ ,6-Diamidino-2-Phenylindole, Dihydrochloride, Vector Laboratories, Peterborough, UK). Digital images were
captured using a Nikon IF Microscope Lens-Nikon Plan Fluor 100x/1.30
oil OFN25 DIC H/N2 MRH01902 using the programme NCIS and analysed
using Image J. The mean fluorescence intensity of telomere spots in each of
the nuclei was quantified relating TL to pixel brightness. Freehand drawing
around the nuclei on the DAPI image was transposed to the PNA red filtered
image and measurement of the mean relative TL within each nuclei was
gained. All epithelial cell nuclei (confirmed by the expression of cytokeratin
in sequential sections) in the high magnification and at least 10 fields of
vision per section were analysed. A control sample of mouse gut was included
with each run to ensure uniformity and reproducibility.
Explant culture
Endometrial biopsies were washed several times in Dulbecco’s modified
Eagle’s medium (DMEM)/F12 (Phenol-red free, Life Technologies, Paisley,
UK) to remove blood. The tissue was cut into 1 – 2 mm3 pieces and placed
into 24-well plates with medium [DMEM/F12, 5% charcoal-stripped FBS
(fetal bovine serum, Sigma-Aldrich, Dorset, UK), Primocin (Source Bioscience, Nottingham, UK)], containing 1028 M b-estradiol (E2) or 1026 M
medroxyprogesterone acetate (MPA). All hormones were from
Sigma-Aldrich, Dorset, UK and prepared as 1 mg/ml stocks in ethanol.
Treatment was for 24 h, after which harvested tissue was stored at 2808C
for TRAP assay. Control treatments received ethanol only.
Isolation of human endometrial epithelial cells
Imetelstat treatment
Imetelstat (GRN163L) is a 5′ palmitoylated 13-mer thiophosphoramidate
oligonucleotide, composed of the sequence 5′ -TAGGGTTAGACAA-3′ ,
complementary to the template region of human telomerase RNA component (hTR) (Herbert et al., 2005). It is a highly specific and potent competitive
inhibitor of telomeric repeat addition. Imetelstat and a mismatch control
(GRN832) were provided by Geron Corporation, California, USA. Both
Epithelial and stromal fractions were separated from freshly harvested endometrial tissue as previously described (Valentijn et al., 2013). Freshly isolated
epithelial cells were positively selected using EpCAM (epithelial cell adhesion
molecule, CD326) microbeads (#130-061-101; Miltenyi Biotec Ltd, Surrey,
UK) according to the manufacturer’s instructions and were pooled and
stored at 2808C. EpCAM-depleted stromal cells were plated in DMEM/
F12 medium (Sigma-Aldrich, Dorset, UK), 10% FBS (Sigma-Aldrich,
Table I Patient demographics, median value and the range are shown.
Age (years)
Height (m)
Weight (kg)
BMI (kg/m2)
Parity 11 (%)
.............................................................................................................................................................................................
Group 1
(n ¼ 85)
36
(18–46)
1.58
(1.5–1.78)
73
(50–119)
28
(18– 45)
100
Group 2
(n ¼ 74)
38
(23–45)
1.60
(1.48–1.75)
74
(49–112)
30.7
(19.9– 42)
100
Group 3
(n ¼ 5)
26
(22–30)
1.62
(1.5–1.72)
57
(49–72)
21.7
(19.6– 29.7)
80
Group 4
(n ¼ 10)
38
(24–47)
1.64
(1.53–1.7)
60
(51–76)
24.5
(21.5– 27.9)
70
Group 5
(n ¼ 22)
34
(26–45)
1.64
(1.55–1.77)
70
(51–90)
27.6
(21– 40)
100
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Endometrial telomerase regulation
Dorset, UK), Primocin (Source Bioscience, Nottingham, UK) for 24 h to minimize the amount of contaminating blood cells. Stromal cell were collected by
trypsinisation and stored at 2808C. Representative aliquots of the epithelial
and stromal fractions (sorted by EpCAM microbeads) were analysed by
sodium dodecyl sulfate-Poly acrylamide gel electrophoresis (SDS-PAGE)
and immunoblotting for expression of cytokeratin and vimentin. Cells maintained in culture were assessed morphologically and by flow cytometry for
the expression of CD9, a glandular epithelial marker and CD13, a stromal
marker (Valentijn et al., 2013).
Primary culture (2D and 3D) of human
endometrial epithelial and stromal cells
Primary human epithelial and stromal cells were maintained in monolayer
culture in DMEM/F12, 10% FBS, and Primocin. For epithelial cells, medium
was supplemented with 50 ng/ml EGF (epidermal growth factor,
Sigma-Aldrich, Dorset, UK). For 3D culture, short-term (16– 36 h postplating) monolayer epithelial cultures were embedded in Matrigel (BD Biosciences, Oxford, UK) and maintained in DMEM/F12 medium supplemented
with insulin-transferrin-selenite (ITS (insulin-transferrin-seleniumG); Life
Technologies, Paisley, UK) and 50 ng/ml EGF with medium changes every
3 days (Valentijn et al., 2013). For inhibition of TA, spheroids were allowed
to form for 3 – 5 days. Imetelstat and a control mismatch oligonucleotide,
both at 5 mM were added on Day 5 of culture and the spheroids monitored
daily for a further 5 days. Spheroids that had formed on Day 5 of culture with a
diameter of at least 50 mm were assessed by microscopy and scored. On Day
5 of treatment, the spheroids were assessed morphologically by microscopy
and the number of intact spheroids with obvious lumen was counted. The
number of intact spheroids remaining on Day 5 of treatment was expressed
as a percentage of spheroids that had formed on Day 5 of the initial culture.
Monolayer endometrial cultures comprising stromal and epithelial cells
were grown in DMEM/F12 with 2% charcoal-stripped FBS (Sigma-Aldrich,
Dorset, UK) for 24 h prior to treatment with E2, P4 (progesterone;
Merck, Nottingham, UK). After a further 24 h, cells were treated with estrogen (E2; 1028 M) alone or in combination with progesterone (P4; 1026 M)
for 5 days. The medium containing the hormones was changed every 1 – 2
days. Cells were subsequently analysed for TA by TRAP.
MTT and senescence assays
Cell viability was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma-Aldrich, Dorset, UK) assay. Briefly primary
epithelial cells were seeded into 96-well plates (10 000 cells/well) and
allowed to attached overnight prior to treating with Imetelstat or the mismatch control for 72 h. MTT solution (5 mg/ml) was added to each well
(10 ml/100 ml medium) for 4 h. The medium was removed and then
100 ml acidified isopropanol was added to dissolve the formazan precipitate
before absorbance was measured at 570 nm using a microplate reader
(Multiskan Ascent, Thermo Scientific, UK). Cell viability was expressed as a
percentage of the untreated control.
Senescent human endometrial cells in culture were identified using a
pH-dependent b-galactosidase assay (Dimri et al., 1995).
SDS-PAGE and immunoblotting
Cells were extracted in RIPA (radioimmunoprecipitation assay) buffer supplemented with protease (P8340, Sigma-Aldrich, Dorset, UK) and phosphatase (PhosSTOP, Roche Diagnostics Ltd, Burgess Hill, UK) inhibitors. Lysates
were analysed by SDS-PAGE under reducing conditions on precast 4 – 15%
gradient gels (Mini-PROTEAN TGX, Bio-Rad, Hertfordshire, UK) and transferred to polyvinylidene difluoride membrane (Bio-Rad, Hertfordshire, UK).
Primary antibodies used included: cytokeratin 18 (M7010, Dako, Ely, UK),
vimentin (ab45939, Abcam, Cambridge, UK), GAPDH (Glyceraldehyde
3-phosphate dehydrogenase, G9545, Sigma-Aldrich, Dorset, UK) and phosphohistone H3 [(Ser10), ab5176, Abcam, Cambridge, UK]. HRP (horse
radish peroxidase)-linked secondary antibodies were from ThermoScientific,
Loughborough, UK. Signal detection was performed using SuperSignalTM
West Dura chemiluminescent substrate (Thermo Scientific, Loughborough,
UK) and CL-Xposure film (Thermo Scientific, Loughborough, UK).
Statistical analysis
Statistical analysis was performed using GraphPad prism. Student’s t-test and
non-parametric equivalent tests were used as appropriate to determine significance between groups, paired samples and treatments. Pearson’s correlation coefficient was used to test the association between TL with
demographic and endocrine data. Criterion for significance was P , 0.05.
All data are presented as mean + standard error of the mean (SEM).
Results
Endometrial mean tissue TLs are longer
than donor-matched peripheral blood
mononuclear cells TLs and correlate
with serum progesterone levels suggesting
a tissue-specific regulation
To test the tissue-specific regulation of the endometrial TL, the mean TL
(TL) was measured in endometrium collected from 85 women (Group 1,
proliferative phase, n ¼ 18; mid-secretory phase, n ¼ 38, and latesecretory phase n ¼ 17) and in matched PBMC collected simultaneously
in 53 of these women. The PBMC TL was significantly shorter than in
the matched endometrial tissue TL (3955 + 557 bp (base pairs) versus
4893 + 929 bp, n ¼ 53 pairs; paired t-test ***P , 0.0001, Fig. 1A) from
the same woman when all phases of the menstrual cycle were analysed,
and there was no correlation between the PBMC and endometrial tissue
TL (r ¼ 20.05, Fig. 1B) of individuals suggesting a tissue-specific regulation
of TL in endometrial cells. There was no correlation between endometrial
TL and the demographic features such as age (Fig. 1C) and BMI (Fig. 1D).
To evaluate the effect of ovarian hormones on endometrial TL,
the data were analysed according to the menstrual cycle phase. The
endometrial TL changed according to the cycle and were shortest in
the progesterone dominant mid-secretory phase (Kruskal – Wallis test,
4 groups, P ¼ 0.0003, Fig. 1E) where progesterone levels were either
absent or low. While there was no correlation with serum estrogen
(data not shown), endometrial TL negatively correlated with circulating
progesterone levels (n ¼ 70 pairs, r ¼ 20.54, Fig. 1F).
High TA and short TLs are features of
proliferating EECs in vivo and in vitro
To confirm that high TA is associated with EEC proliferation, freshly isolated endometrial epithelial and stromal cells (n ¼ 5) in the proliferative
phase (the epithelial proliferation in these samples was confirmed by
Ki67 expression by IHC; Supplementary data Fig. S1A) were examined.
Routinely cultured epithelial cells were .80% positive for CD9; stromal
cells were .95% positive for CD13 (Supplementary data, Fig. S1B and C).
The EpCAM+ sorted EECs showed significantly higher TA (1.57 + 0.16
versus 0.93 + 0.36, n ¼ 5 pairs, paired t-test, P ¼ 0.01; Fig. 2A,
AU for arbitrary units of absorbance at 450 nm) with a shorter TL
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Valentijn et al.
Figure 1 Endometrial telomere length (TL) varies according to the menstrual cycle and does not correlate with peripheral blood mononuclear cells
(PBMC) TL, age or BMI. The TL of endometrial tissue and PBMC TL in normal fertile healthy women were measured by qPCR. (A) In paired samples endometrial TL was significantly longer than PBMC TL (n ¼ 53, ***P ¼ 0.0001). Endometrial TL did not correlate with PBMC (B; n ¼ 53 pairs), age (C; n ¼ 69
pairs) or BMI (D; n ¼ 67 pairs). (E) In endometrial tissue TLs were shortest in the mid-secretory phase (Kruskal –Wallis test, ***P ¼ 0.0005) of the
menstrual cycle when progesterone levels are maximal. Proliferative phase (n ¼ 13), early-secretory phase (n ¼ 12), mid-secretory phase (n ¼ 33),
late-secretory phase (n ¼ 17). Mean + SEM are presented. (F) Negative correlation of endometrial TL with serum progesterone levels in secretory
phase samples (n ¼ 70 pairs, r ¼ 20.54, *P , 0.0001). Serum progesterone was measured by radioimmunoassay (RIA).
Endometrial telomerase regulation
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Figure 2 Epithelial cells of the endometrium are characterized by high telomerase activity (TA) and short telomeres. (A) TA (arbitrary units of absorbance at 450 nm [AU]) in freshly isolated epithelial and stromal cells from proliferative phase endometrium (n ¼ 5 pairs, Mann –Whitney test, *P ¼ 0.01).
(B) Telomere length (TL) in base-pairs (bp) in epithelial and stromal cells (n ¼ 5 pairs, Mann– Whitney test, *P ¼ 0.01). (C) Correlation of TA with TL in
epithelial cells in the proliferative phase (n ¼ 5, r ¼ 20.98, ***P ¼ 0.0005). (D) TA in freshly isolated epithelial and stromal cells from secretory phase
endometrium (n ¼ 4). (E) TL (bp) in epithelial and stromal cells from secretory phase (n ¼ 5 pairs, Mann – Whitney test, *P ¼ 0.03). (F) Lack of correlation
of TA with TL in epithelial cells in the secretory phase (n ¼ 5 pairs, r ¼ 0.54, P ¼ 0.46).
(5167 + 1332 bp versus 8598 + 1422 bp, n ¼ 5 pairs, paired t-test,
P ¼ 0.0002, Fig. 2B) compared with the corresponding EpCAMdepleted stromal population from the same sample. TA and TL in the
proliferating EEC were negatively correlated (n ¼ 5 pairs, r ¼ 20.994,
P ¼ 0.0005; Fig. 2C), suggesting that the proliferating epithelial cells
with short TL were associated with high TA. EpCAM+ EEC TLs in the
secretory phase showed no correlation with TA (n ¼ 5 pairs, r ¼ 0.24,
Fig. 2F) and had lower TA levels [these epithelial cells were not proliferating shown by absent or low Ki67 (Supplementary data, Fig. S1)].
Stromal cell TL remained significantly longer than epithelial cell TL in
both proliferative and secretory phases (Fig. 2B and E).
Effect of Imetelstat on epithelial
and stromal cells
Since TA is associated with cell proliferation we tested the effect of telomerase inhibition on proliferation of epithelial cells in culture. In cultures
of epithelial and stromal cells, TA was higher in epithelial cells where it
increased initially and persisted for longer (Fig. 3A and B). Long-term
culture of EEC was associated with a senescent phenotype (Fig. 3C;
enlarged cell morphology and b-galactosidase staining). Imetelstat produced a dose-dependent inhibition of telomerase (IC50 1.5 mM) in a
primary endometrial epithelial cancer cell line with high telomerase activity (Fig. 3D). The IC50s for telomerase inhibition ranges from 0.1 to 1 mM
in most tumour cell lines tested (Joseph et al., 2010). 1.0 mM Imetelstat
inhibited TA on average by .60% in monolayer cultures of normal
healthy primary EECs treated for 72 h (n ¼ 4 pairs, Paired t test,
*P ¼ 0.02; Fig. 3E) and this was associated with a decrease in the expression of the mitotic marker, phosphohistone H3 (Ser10) (n ¼ 5 pairs,
t-test, **P ¼ 0.009, Fig. 3G). Note that while Imetelstat inhibited TA in
the primary endometrial epithelial cancer cell line (Fig. 3C), it did not
affect the phosphorylation status of phospho-histone H3 (Fig. 3G). Imetelstat only affected epithelial cell viability at 100 mm as assessed by MTT
assay (**P ¼ 0.002, Fig. 3F). Inhibition of TA in stromal cells by 1.0 mM
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Valentijn et al.
Figure 3 Imetelstat affects telomerase activity (TA) and proliferation, but not viability of endometrial epithelial cells. (A) Epithelial cells and (B) stromal
cells were maintained in monolayer culture for the indicated times prior to harvesting for telomere repeat amplification protocol (TRAP) assay. For each
time point, n ≥ 4; Patient group 2. (C) Epithelial cells maintained in long-term culture had a phenotype consistent with senescence. Note the enlarged cells
and positive blue stain for b-galactosidase in the micrographs (representative of n ¼ 5). (D) Epithelial cells were isolated from an adenocarcinoma of the
human endometrium and maintained in culture as a cell line. The cells were treated with the concentrations of Imetelstat indicated for 72 h prior to TRAP.
TRAP activity is expressed as a percentage relative to the activity of the mismatch control (mean + SEM for n ¼ 3 separate experiments). (E) Epithelial cells
were maintained in culture for up to 3 days and then treated with 1 mM Imetelstat or mismatch control oligonucleotide for a further 72 h prior to TRAP
assay. TRAP activity is expressed as a percentage of the mismatch control. (n ¼ 4). T-test, *P ¼ 0.02. (F) Human endometrial epithelial cells (HuEEC)
(n ¼ 5) were directly seeded into 96-well dishes, allowed to attach and treated the next day with Imetelstat or the mismatch control at the concentrations
indicated for 72 h. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Note significant loss in cell
viability at 100 mM (Mann – Whitney test, P ¼ 0.002). (G) Cultures of normal epithelial cells and an adenocarcinoma of the endometrium treated with
Imetelstat or mismatch control as before, and immunoblotted for phospho-H3 [phosphohistone H3 (Ser10)]. Histone H3 is only phosphorylated on
Ser 10 during mitosis. Shown is a representative blot (top) of normal epithelial cells (n ¼ 5) and the adenocarcinoma (representative of two separate experiments) and densitometric analysis (bottom). T-test, **P ¼ 0.009. (H) Stromal cells were grown for 24 h and then treated with 1 mM Imetelstat or mismatch
control oligonucleotide for 72 h prior to TRAP assay. Telomerase activity is expressed as a percentage of the mismatch control. T-test, ***P ¼ 0.0004.
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Endometrial telomerase regulation
Imetelstat in monolayer culture for 72 h was on average .90% (n ¼ 4
pairs, paired t-test, ***P ¼ 0.0004; Fig. 3H) when measurable TA was
present.
To test if telomerase inhibition affects formation of glands, primary
EECs forming gland-like spheroids in 3D after 3–5 days in culture
(Fig. 4A) were treated with 5 mM Imetelstat or the mismatch control
for 5 days. Imetelstat significantly affected the architecture of the
glands reducing the number of intact spheroids (Fig. 4B) compared
with control (n ¼ 3 pairs, paired t-test; *P ¼ 0.03). Imetelstat treated
spheroids failed to proliferate and increase in size beyond Day 5 of treatment. Given the effect of Imetelstat on the architecture of the spheroids
we speculated that Imetelstat might affect the cytoskeleton. We treated
EECs in monolayer with 5 mM Imetelstat (same concentration as that
used for spheroids) or the mismatch control for 5 days and immunostained for F-actin (Phalloidin) and cytokeratin. After 5 days of treatment
with Imetelstat, the cells adopted a rounded morphology and actin filaments were concentrated along the cell membrane (Fig. 4C). The distribution of cytokeratin was similarly affected (Fig. 4C).
In vivo and in vitro treatment with
progesterone inhibits endometrial
epithelial TA
The effects of endogenous and exogenous progesterone in vivo on EEC
TA were determined on freshly isolated EECs from women in the proliferative phase (n ¼ 8), secretory phase (n ¼ 8) and MPA-treated (n ¼ 5).
TA was significantly higher in the proliferative phase samples compared
with the secretory phase and MPA-treated samples (Kruskal –Wallis
test, ***P ¼ 0.0005; Fig. 5A). In explant cultures of endometria collected
during the proliferative phase, MPA treatment (24 h) in vitro produced a
significant reduction in TA (n ¼ 4 pairs, paired t-test, *P ¼ 0.01; Fig. 5B).
Rather than look at separate cultures of epithelial and stromal cells, we
reasoned that a combined culture of the two cell types physically interacting would provide more useful information on the effect of progesterone on estrogen induced TA than separate cultures. Endometrial
cultures comprised of both epithelial and stromal cells showed .50%
reduction in TA (Fig. 5C) after 5 days of treatment with progesterone
Figure 4 Imetelstat affects the morphology of endometrial epithelial cells (EECs) in monolayer and 3D culture. (A) Micrographs showing the formation of
an endometrial epithelial spheroid embedded in Matrigel on the days indicated. Scale bar, approx. 100 mm. (B) On Day 5 of culture the number of spheroids
was counted and 5 mM imetelstat or mismatch control added and cultured for a further 5 – 10 days. The number of intact spheroids remaining after 5 days of
treatment was scored and expressed as a percentage of those formed on Day 5 before treatment started. N ¼ 3 for each treatment group (T-test,
*P ¼ 0.03). Micrographs show positive nuclei stained with 4′ ,6-Diamidino-2-Phenylindole, dihydrochloride (DAPI) of spheroids in 3D culture on Day 5
of treatment. Scale bar 100 mm. (C) Effects of 5 mM Imetelstat or mismatch control on F-actin and cytokeratin in EECs in monolayer treated for 5 days.
EECs were seeded in chamber slides (Nunc, Sigma-Aldrich, Dorset, UK) and allowed to attach overnight prior to treatment and analysis of the cytoskeleton
by immunofluorescence. Representative of three separate experiments.
2824
Valentijn et al.
Figure 5 Progesterone is associated with changes in endometrial telomerase. (A) Telomerase activity (TA; arbitrary units of absorbance at 450 nm [AU])
in freshly isolated endometrial epithelial cells (EECs) is repressed by endogenous progesterone in secretory phase normal cycling endometrium (Mann –
Whitney test, **P ¼ 0.005, n ¼ 8/phase) and in endometrium treated exogenously with synthetic progestagen (medroxyprogesterone acetate [MPA],
Group 3, n ¼ 5, Mann– Whitney test *P ¼ 0.001). (B) In explant cultures of human endometrium, MPA (1026 M) but not estradiol (E2) (1028 M) had
a significant impact on TA after 24 h. n ¼ 4, paired t-test, *P ¼ 0.01 (vehicle versus MPA). (C) In monolayer cultures comprised of both epithelial (E)
and stromal (S) cells (micrograph), E2 and P4 significantly reduced TA compared with E2 alone (n ¼ 3, paired t-test, *P ¼ 0.02). (D) Progesterone antagonism with mifepristone increases telomerase immunostaining intensity in endometrial glands in vivo. For each time point, n ≥ 3, Group 5, **P ¼ 0.003.
Micrographs of telomerase immunostaining in mid-secretory phase (0 h after) and 36 h after 200 mg mifepristone administered in mid-secretory phase.
Also shown are a positive control for telomerase expression (endometrial cancer) and a negative control (post-menopausal endometrium). The telomerase immunoreactivity in the stromal cells did not change significantly after mifepristone treatment.
2825
Endometrial telomerase regulation
Figure 6 Peritoneal ectopic lesions have longer telomeres and higher telomerase expression. (A) Telomerase length (TL) in matched secretory phase
uterine (uterine) and ectopic endometrium was assessed by fluorescence in situ hybridization (FISH) using peptide nucleic acid (PNA) telomere probe. The
relative fluorescence intensity was stronger in the glandular structures of the ectopic lesion compared with uterine (eutopic) endometrium (paired t-test,
P ¼ 0.01, n ¼ 5, Group 4). Data from at least 3000 epithelial nuclei per field/section and around 6000 – 10 000 per matched sample were analysed.
(B) Secretory phase endometrium and peritoneal ectopic lesions were stained by immunohistochemistry for telomerase. Representative micrographs
for eutopic (left) and ectopic (right), n ¼ 10. Note the strong staining in the gland-like structures in the ectopic lesion. Scale bar 100 mM.
and E2 compared with E2 alone (n ¼ 3 pairs, paired t-test, *P ¼ 0.02).
Administration of 200 mg of the progesterone antagonist mifepristone
in the mid-secretory phase in healthy women increased immunoreactivity for the telomerase protein hTERT in endometrial glands after 24 h,
with significant changes in semi-quantitative scoring at 24 and 36 h
(n ¼ 22, Kruskal –Wallis test, **P ¼ 0.003; Fig. 5C). Telomerase immunoreactivity in stromal cells did not change significantly after mifepristone
treatment (Fig 5D).
Longer relative mean TL in ectopic
glandular-like epithelium
Progesterone resistance is a hallmark of ectopic endometriotic lesions
and we examined glandular epithelial cells for differences in TL with
qFISH. Ectopic endometriotic lesions (Group 4) were associated
with significantly higher epithelial telomeric signal than in matched corresponding uterine (eutopic) endometrium from the same women
(n ¼ 5 pairs; paired t-test, P ¼ 0.01) suggesting longer relative mean
TL in ectopic endometriotic glands (Fig. 6A). Agreeing with this observation, the ectopic peritoneal endometriotic lesions showed increased
immunoreactivity for telomerase protein hTERT compared with the
matched functional uterine (eutopic) endometrium (n ¼ 10 pairs,
Fig. 6B).
Discussion
The dynamic changes in TA according to the ovarian cycle have been well
documented in the human endometrium (Kyo et al., 1997; Tanaka et al.,
1998; Williams et al., 2001) but little is known about its downstream
effects particularly on endometrial TL. In humans, mean PBMC TL
decreases with age (Frenck et al., 1998) and in addition, there seems
to be a correlation between TL in different tissues of the same individual
(Friedrich et al., 2000; von Zglinicki et al., 2000; Takubo et al., 2002) suggesting that PBMC TL could serve as a surrogate for relative TL in other
tissues. Although there is a positive correlation between reproductive life
span and PBMC TL (Aydos et al., 2005; Lin et al., 2011), we demonstrate
herein that endometrial TL does not correlate with matched PBMC TL in
a large, well characterized group of fertile women not on any hormonal
therapy and without any endometrial pathology. This suggests a tissue
specific, possibly hormonal regulation of the endometrial TL. Since a
fully functional endometrium can be regenerated from the thin post-
2826
menopausal endometrium with the provision of exogenous ovarian
steroid hormones (Paulson et al., 2002), it is the only reproductive
organ not reported to show irreversible age-related changes. This endometrial age-defiance might include the apparent tissue specific TL preservation, presumably due to the ability of the hormonal induction of TA.
The human endometrium is a complex, multi-cellular, hormoneresponsive tissue. The observation that isolated epithelial and stromal
cells from dissociated endometrium showed differential TLs and TA
levels is a novel finding adding a further level of complexity to endometrial
telomere biology.
We now demonstrate, we believe, for the first time that endometrial
TL changes according to the ovarian hormone cycle using a reproducible
and validated qPCR method (Martin-Ruiz et al., 2014), with the shortest
TL observed in the progesterone dominant mid-secretory phase, when
TA is known to be at its lowest level (Tanaka et al., 1998; Kyo et al., 1999;
Williams et al., 2001). Fractionated endometrium showed that EEC TL is
shorter than that of the corresponding stroma throughout the cycle,
whilst TA was higher in the EEC than in the corresponding stroma
during the proliferative phase. In normal human somatic cells, telomere
attrition triggers replicative senescence (Bodnar et al., 1998). Shorter TL
would impose a limit on the proliferative life span in EECs and thus higher
TA in these cells would allow them to maintain TL at a certain length
thereby preventing mitotic arrest. Studies have shown that high TA, telomerase mRNA and hTERT protein are features of the endometrial proliferative disease, endometriosis (Kim et al., 2007; Hapangama et al.,
2008; Mafra et al., 2014) and we now further demonstrate that ectopic
lesions with a postulated hallmark of progesterone resistance phenotype
(Bulun, 2010) have relatively longer TLs than the eutopic functionalis
cells. Therefore, direct inhibition of TA with Imetelstat in EEC may
bypass the pathological progesterone resistance and may prevent EEC
proliferation and propagation of endometriotic lesions.
Endometrial TA as assessed by TRAP was reported to be localized to
the glandular epithelial cells (Tanaka et al., 1998; Yokoyama et al., 1998);
however, we now demonstrate measureable TA in the stromal compartment of the endometrium using the more sensitive TRAP-ELISA (Fajkus,
2006). TA is a marker of cell proliferation (Belair et al., 1997) and persisted in our short-term cultures of EECs while the cells were proliferating
but was reduced upon senescence, which is consistent with previous
work (Tanaka et al., 1998; Yokoyama et al., 1998). Moreover, inhibition
of TA in short-term proliferating EEC cultures with 1 mM Imetelstat for
72 h was associated with a reduction in the expression of the mitosis
marker, phosphorylated histone H3. This was in contrast to the adenocarcinoma where TA was affected by Imetelstat, but the phosphorylation
status of histone H3 was not, suggesting a disconnection between TA and
proliferation in endometrial cancer. Although our work focused mainly
on the endometrial epithelial compartment, the direct inhibition of TA
in stromal cells in culture appears to be potentially relevant and warrants
further investigation. The stromal cells of the endometrium are persistently proliferative throughout the menstrual cycle, while the epithelial
cells are highly proliferative specifically during the proliferative phase (Jurgensen et al., 1996).
It is now well established that a 3D cell culture system more closely
mimics natural tissues and organs than cells grown in 2D (Yamada and
Cukierman, 2007). For this reason EECs were grown in 3D culture in
order to recapitulate the architecture of the glands in vivo, to study the
effects of telomerase inhibition. In 3D culture, under defined serum-free
conditions with proliferation being driven by EGF, epithelial cells formed
Valentijn et al.
spheroid structures. There was a more profound effect of TA inhibition in
3D culture compared with 2D culture. Inhibition of TA in 3D was associated with an inability of spheroids to proliferate and expand. Additionally, the architecture of the spheroids was adversely affected such that
after 5 days of treatment there was a loss of spherical form, suggesting
an effect on the cytoskeleton which was confirmed by treating 2D cultures of EECs with Imetelstat. Imetelstat altered the cell morphology
and growth in breast (Goldblatt et al., 2009) and lung (Mender et al.,
2013) cancer cell lines. The profound effects of telomerase inhibition
in 3D culture suggest an important role for telomerase in normal glandular epithelial function of the endometrium.
Observational studies suggest that TA in the endometrium is activated
by estrogen and repressed by progesterone. TA is at its highest during the
proliferative phase and progressively decreases during the secretory
phase, becoming undetectable during the mid-secretory when progesterone levels peak (Kyo et al., 1997; Saito et al., 1997; Tanaka et al.,
1998; Williams et al., 2001). In further support of these observations,
we have demonstrated that women treated with MPA (as DepoProvera) for 6 months, had a marked reduction in endometrial epithelial
TA. Depo-Provera is known to induce pronounced reduction in endometrial epithelial proliferation (Lu et al., 2013). Also, in monolayer endometrial cultures consisting of both stromal and epithelial cells that allow
these cell types to physically interact, we demonstrated that estrogen and
progesterone significantly reduced TA compared with estrogen alone.
Although there was a modest effect of MPA on TA in our explant cultures, the culture time was short and in addition to epithelial and
stromal cells have endothelial cells and lymphocytes, both of which are
reported to possess TA (Norrback et al., 1996; Hsiao et al., 1997).
The anti-proliferative effect of progesterone in the endometrium is
exerted via the stroma, and both progesterone receptor (PR) and glucocorticoid receptor (GlucR) are postulated to be involved in progesterone
and MPA action in the endometrium (Henderson et al., 2003).
The mechanisms by which hTERT expression is regulated by progesterone appear to be complex because the hTERT promoter lacks a canonical progesterone-responsive element (Wang et al., 2000). In a
breast cancer cells line, MPA inhibited hTERT mRNA transcription
even in the presence of estrogen (Wang et al., 2000; Lebeau et al.,
2002) and arrested cells in the late G1-phase (Lange et al., 1999) with
the induction of p21 (Wang et al., 2000). It is known that cell cycle
arrest is associated with down-regulation of telomerase (Belair et al.,
1997). Interestingly p21 is induced in the secretory phase of the menstrual cycle when progesterone levels are high (Toki et al., 1998).
There is evidence for cell cycle-dependent regulation of telomere synthesis in human cells (Tomlinson et al., 2006). The telomerase
complex consisting of, hTR and hTERT, is targeted to telomeres specifically in the late S phase of the cell cycle when telomeres are lengthened
(Hug and Lingner, 2006). We suggest that the different telomere lengths
in proliferative and mid-secretory phase endometrium might reflect the
negative effect of progesterone on cell cycle during the mid-secretory
phase with cells accumulating in the late G1 phase. The mid-secretory
phase is characterized as being metabolically active with differentiation
of the stromal cells (Yang et al., 2011). Therefore, at this juncture the
negative effects of progesterone on estrogen induced hTERT in the
endometrial epithelium might also be indirect. In contrast, treatment
with mifepristone (a PR antagonist) during the progesterone dominant
mid-secretory phase when TA is low/undetectable (Williams et al.,
2001), was associated with a significant increase in endometrial glandular
2827
Endometrial telomerase regulation
telomerase immunoreactivity after 24–36 h. This coincides with
reduced serum progesterone levels (Hapangama et al., 2002) and
increased endometrial cell proliferation (Cameron et al., 1997). All
women receiving mifepristone at mid-luteal phase developed vaginal
bleeding and endometrial shedding by 48 h, therefore these 48 h
samples may contain endometrial cells that were undergoing apoptosis
and necrosis (associated with shedding). We speculate that this is the
reason for the lack of a persisting increase in telomerase expression by
48 h. As mifepristone is capable of inhibiting glucocorticoid and progesterone receptors, its effect on telomerase expression is unclear.
However, in healthy women progesterone had been previously shown
to reverse the mid-cycle effects of mifepristone (Batista et al., 1992).
Consistent with the antagonistic effects of progesterone on estrogeninduced proliferation in the endometrium (Hapangama et al., 2015), our
study further implicates progesterone in the negative regulation of both
TA and TL. The mechanisms by which progesterone regulates telomerase activity in the endometrium requires further investigation which
might reveal novel pathways that can be targeted in treating endometrial
proliferative diseases. Of particular interest was the effect of telomerase
inhibition in vitro on epithelial cell proliferation in 2D and 3D cultures, suggesting that telomerase might be an attractive target in developing new
therapies for proliferative disorders of the endometrium.
Supplementary data
Supplementary data are available at http://humrep.oxfordjournals.org/.
Acknowledgements
Imetelstat was a generous gift from Geron Corporation. Authors are
grateful to Professor DT Baird, University of Edinburgh, for supporting
access to archival endometrial biopsies from women administered
mifepristone in the luteal phase of their menstrual cycles; Professor T
von Zglinicki, Drs C Martin-Ruiz and D Jurk from Newcastle University
for assistance with TL and qFISH measurements; Mrs J Drury,
Ms J Harrold, Ms R Campbell, Ms L Heathcote and Dr A Kamal of
University of Liverpool for their technical assistance.
Authors’ roles
D.K.H. obtained the Ethical approval, and conceived the study design.
D.K.H., A.J.V., G.S. formulated experiments, analysed and interpreted
data, produced figures and produced the first draft. The samples were
collected by D.K.H., H.O.D.C. and N.T. Experiments were carried
out and data collected by A.J.V., D.K.H., N.T., and G.S. and D.K.H.
and G.S. were involved in obtaining funding. H.O.D.C. was involved in
data interpretation, and manuscript preparation and revision. All
authors approved the submitted final version of the manuscript.
Funding
The work was supported by Wellbeing of Women project grant RG1073
(D.K.H./G.S.), RG1487 (D.K.H./G.S.), MRC/DFID funded Special
Project Grant No: G9523250 (H.O.D.C.) and Geron PLC (A.J.V./
D.K.H.). Geron PLC, USA covered expenses for the Imetelstat experiments (A.J.V.) described in this manuscript. The terms of this
arrangement have been reviewed and approved by the University of
Liverpool, UK in accordance with its policy on objectivity in research.
Conflict of interest
None declared.
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