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Departments of Reproduction and Development,
1 Obstetrics and Gynecology,
2 Center for Biomics,
3 Bioinformatics, Erasmus University Medical Center, 3000 DR Rotterdam, The Netherlands,
4 OmniViz, Inc., Maynard, Massachusetts, USA and
5 Research and Development Laboratories, N V Organon, 5340 BH Oss, The Netherlands
(Requests for offprints should be addressed to P Hanifi-Moghaddam; Email: p.hanifi_moghaddam{at}erasmusmc.nl)
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In the premenopausal endometrium, estrogens induce proliferation which is counteracted by the differentiative activities of progestagens (Markiewicz & Gurpide 1990). For a hormone replacement therapy drug, it is important to display potent estrogenic activities for prevention of osteoporosis and climacteric complaints; however, for the endometrium, these estrogenic activities need to be balanced with appropriate progestagenic action. As indicated in literature (Kloosterboer 2001, de Gooyer et al. 2003), tibolone can be converted into estrogenic as well as progestagenic metabolites and for the endometrium, the progestagenic properties of tibolone seem to outweigh its estrogenic activities (de Gooyer et al. 2003). The reason for this can be twofold: first, tibolone may preferentially be converted into its progestagenic 
4-isomer (Tang et al. 1993) and the second, tibolone has been described to induce sulfotransferase activity (Tseng & Gurpide 1975) in the endometrium. Sulfotransferases are enzymes involved in the inactivation of endogenous estrogens and estrogenic compounds like tibolone’s 3
-and 3ß-reduced derivatives.
In order to investigate tibolone’s estrogenic and progestagenic properties, in earlier work, we have used two separate cell lines: a solely estrogen-sensitive endometrial cancer cell line (ECC1) and a solely progesterone-sensitive Ishikawa endometrial cell line. We have shown that the estrogenic activity of tibolone is potentially counterbalanced by the progestagenic metabolite of tibolone via differential regulation of similar cellular processes (Hanifi-Moghaddam et al. 2005). Because these experiments did not take into account the effects of estrogens or estrogen-like compounds on the progesterone receptor (PR) and effects of progestagens or progestagen-like compounds on the estrogen receptor (ER), we have set up a new study to evaluate the estrogenic and the progestagenic properties of tibolone in the same cell line expressing both PR and ER. Therefore, we have generated a progesterone- and estrogen-sensitive ECC1 cell line.
The central question in this study is whether the effect of tibolone on a human ECC1-PRAB72 is similar to, or comparable with, the effect of estrogen, progesterone, or a combination of both. In this paper, we analyze and compare the gene expression profiles reflecting the endometrial response to tibolone, E2, MPA, and a combination of both (E2 + MPA). The results indicate that the addition of tibolone to these cells results in a potent progestagenic response and, interestingly, also in a tibolone-specific response.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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, hERß , hPRA, and hPRB
Recombinant plasmids
The hPR-cDNA cloned into the pSG5 expression vector was a generous gift from Dr E Milgrom (Kremlin-Bicetre, Paris, France). This vector was used to create human progesterone receptor A (hPRA) and progesterone receptor B (hPRB) expression plasmids (Smid-Koopman et al. 2005).
Cell culture and stable transfection
ECC1 cells are derived from a well-differentiated human endometrial adenocarcinoma (Satyaswaroop et al. 1983) and were a generous gift from Dr B van den Burgh (Hubrecht laboratory, Utrecht, The Netherlands). The ECC1 cell line endogenously expresses high levels of hER (comparable to in vivo endometrial levels, not shown) and low levels of hERß (comparable to the situation in vivo) (Taylor & Al-Azzawi 2000). The cells were maintained, transfected, and selected for as described by Smid-Koopman et al.(2005). The initial selection resulted in three potentially useful clones. One clone, ECC1-PRAB72 was chosen for further characterization and used for the gene expression profiling experiments. All experiments were conducted with cells cultured for one whole passage in DMEM/F12 medium devoid of phenol red and supplemented with 5% dextran-coated charcoal-treated fetal calf serum (DCC-FCS) before the experiments were started in the same medium. The hormonal concentrations used are indicated in the figure legends.
Characterization of the ECC1-PRAB72 cells
Western immunoblotting
The cells were cultured in the presence or absence of tibolone, E2, MPA, or E2 + MPA. The hormonal concentrations are indicated in the figure legends. The antibodies used were: mouse monoclonal antibody recognizing PRB (hPRa2; Labvision Neomarkers, Fremond, CA, USA), rabbit polyclonal antibody recognizing PRA and PRB (hPgR Ab8; Neomarkers, Fremont, CA, USA), and mouse monoclonal antibody recognizing ER (sc-8002; Santa Cruz Biotechnology). All experimental conditions regarding western immunoblotting were described earlier by Smid-Koopman et al.(2005).
Cell proliferation experiments
The cells were cultured for 6 days in the presence or absence of increasing concentrations of tibolone, E2 or MPA, or in the presence of 1 nM E2 + increasing concentrations of MPA. The hormonal concentrations are indicated in Fig. 1
. The proliferation was measured using 3H-thymidine incorporation as described earlier by Gielen et al.(2005).
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After maintaining the cells for 1 week in phenol red-free DMEM/F12 medium (Gibco, Invitrogen Life Technologies), containing 5% DCC-FBS, they were passaged, allowed to attach, and subsequently cultured for 1, 6, 12, 24, 48, and 72 h in the absence (in total six control samples were generated) or presence of E2 (1 nM), MPA (1 nM), E2 + MPA (both 1 nM), or tibolone (100 nM). After removal of the medium, total RNA was isolated by lysing the cells with 3 M lithium chloride/6 M urea. Subsequently, the RNA was purified as described by Blok et al.(1995). After RNA cleanup (Qiagen), RNA levels, quality, and purity were assessed with the use of the RNA 6000 Nano assay on the Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA, USA). None of the samples showed RNA degradation or contamination by DNA.
Gene profiling and quality control
The samples were analyzed using Affymetrix U133plus2 GeneChips containing 54 614 probe sets, representing approximately 47 000 transcripts (30 000 genes). We used 1 µ g total RNA to prepare antisense biotinylated RNA according to Affymetrix protocol for gene chip experiments (Affymetrix). All GeneChips were visually inspected for irregularities. The global method of scaling or normalization was applied. All additional measures of quality indicated a high overall quality of the samples and assays.
Statistical analyses
Data normalization was done according to the quantile method (Bolstad et al. 2003). The level of expression of each probe set in every hormone-treated sample at each time-point was determined relative to its corresponding control sample and transformed logarithmically (on a base 2 scale). A threefold change deviation from the control reflects differential gene expression. Genes (probe sets) whose level of expression differed from the controls (reflecting up- or down regulation) in at least one sample were selected for further analysis. In total, 3067 out of 54 614 probe sets corresponding to 2063 known and 281 unknown genes were selected for further analysis. The Omniviz package (Omniviz) was used to perform and visualize the results of unsupervised cluster analysis. The list of differentially expressed genes is available at http://www2.eur.nl/fgg/rede/hanifi_moghaddam. Gene expression data were also analyzed using Time series regression analysis from http://linus.nci.nih.gov/brb-arraytools.html. The results from this analysis can be accessed from http://www2.eur.nl/fgg/rede/hanifi_moghaddam.
The classification of genes into biological processes was done using GoTree Machine (http://genereg.ornl.gov/gotm) and Celera database (https://panther.appliedbiosystems.com/). In order to identify biological processes with significantly enriched or depleted gene numbers, we need to compare, for each biological process, the distribution of genes in each hormone-regulated gene list with those in the reference gene list. It should be noted that an inappropriate reference list would lead to a wrong identification of regulated biological processes. Therefore, we used two reference lists: the Affymetrix 133Uplus2 gene list and the National Center for Biotechnology Information (NCBI) human gene list (n-23520). The enrichment or depletion of genes in a certain biological process indicates hormonal regulation. The significance of enrichment or depletion for a given process is determined by the P value (see references given on the websites). Those processes with P values < 10–4 were considered statistically significant and modulated.
Validation of array data by quantitative PCR
The validation of microarray expression data was accomplished by quantitative real-time reverse transcriptase (RT)-PCR on nine randomly selected genes MGP, FOS, MUC1, EGR1, CTSD, KRT18L1, IGFBP4, BCMP11, AREG and five tibolone-regulated genes PITX1, TFCP2L2, THADA, MCM2, and ID1. The experimental protocol and part of the results are available on our website (http://www2.eur.nl/fgg/rede/hanifi_moghaddam).
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The hormone responsiveness of the ECC1-PRAB72 cells was characterized by evaluating the effects of hormone treatments on the expression of PR and ER and the cell proliferation. Western blotting indicated that PRA, PRB, and ER expressions were reduced in the presence of tibolone, MPA, or E2 + MPA (Fig. 2). Furthermore, the ER expression was slightly increased upon culture in the presence of E2.
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); E2-stimulated proliferation of the cells, while tibolone and MPA resulted in a pronounced inhibition of proliferation. It should be noted, however, that tibolone is required at a concentration 100 times higher to show similar inhibitory effect as MPA (100 vs 1 nM). MPA was also able to counteract the E2-stimulated growth. Based on these results, we hypothesized that tibolone behaves much more similar to MPA and E2 + MPA treatment, than to E2 treatment. Profile similarities
In total, 2344 out of approximately 30 000 genes were differentially expressed (at least threefold deviation from the control levels in one or more time-points) and were selected for further analyses (Figs 3 and 4). The expression patterns of nine randomly selected genes were confirmed by real-time RT-PCR indicating that the microarray experiment was of high quality (http://www2.eur.nl/fgg/rede/hanifi_moghaddam; Fig. 5).
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). In addition, the magnitude of profile similarity between tibolone and the other hormones is indicated in terms of the number of regulated genes for each time-point in Fig. 4A–C. In addition, the magnitude of profile specificity for each hormone treatment in terms of the number of treatment-specific genes is indicated in Fig. 4D. Our definition of a treatment-specific gene is if a gene is only modulated by one hormonal treatment and not by any of the other hormonal treatments at any time points.
In the unsupervised cluster analysis (Fig. 3), three main clusters were defined by the software program and can be distinguished. The first main cluster contains all 1-h expression profiles of all hormonal treatments. Since the numbers of regulated genes are small, the only relevant conclusion drawn from this cluster is that at 1 h, gene regulation by the four hormonal treatments is clearly different from that measured after longer treatment times.
Interestingly, after 1 h, the E2 profiles are disassociated from the other profiles and form the second main cluster. On average, considering all time-points excepting 1 h, only 7% of the total number of genes regulated by tibolone is regulated by E2 also. This result was not unexpected because the cell proliferation data indicated a clear opposite response to tibolone when compared with E2 (Fig. 1A versus B).
The third main cluster is formed by the profiles generated after 6, 12, 24, 48, and 72 h of treatment of the ECC1-PRAB72 cells with tibolone, MPA, or E2 + MPA. Within this cluster, a number of sub-clusters can be defined. First, at 6 and 12 h, the profiles of tibolone, MPA, and E2 + MPA cluster together. Second, the 24, 48, and 72-h E2 + MPA treatment dissociates from the other treatments and forms its own sub-cluster. Third, at 24, 48, and 72 h, tibolone and MPA cluster closely together. These observations are strengthened by the percentages of the shared genes; tibolone and E2 + MPA share about 41% of regulated genes and this percentage increases to 55% for tibolone and MPA alone. Interestingly, despite these similarities, there is still a relatively large proportion of genes which is hormone treatment-specific (Fig. 4D). In total, 366 genes were exclusively regulated by tibolone, 67 genes were specifically regulated by E2 + MPA, 269 genes were MPA-specific, and 221 genes were E2-specific (Fig. 4D).
Physiological similarities
Cluster analysis groups genes based on their expression pattern and does not consider the functional relationships between genes. In theory, genes that are functionally unrelated could be co-regulated due to their physical vicinity on the genome and thus can be clustered together (Zhang et al. 2003). In order to understand the (dis)similarity of expression profiles in terms of biological processes, all differentially regulated genes were classified into biological processes and those processes with significantly enriched gene numbers were identified. ‘Cell growth’ and ‘morphogenesis’ were the most significantly regulated processes by E2 and E2 + MPA (P < 10– 4). The ‘cell cycle’ was found to be the most significantly regulated process by tibolone and MPA treatment (P < 10–15).
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Discussion |
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Upon administration of a hormone to a cellular system, usually some genes are transiently regulated, while others are more permanently (constitutively) regulated. It can be argued that the permanently regulated genes are generally those that are important for a sustained hormonal effect on a cellular model. Upon reviewing those genes that were regulated at least at the 24, 48, and 72-h time-points (permanently) in the present experiments, it was found that the percentages of overlapping genes between the hormonal treatments changed. It was calculated that 393 genes out of a total of 1384 tibolone-regulated genes were regulated permanently. The overlap with permanently E2-regulated genes was only 7%, with permanently E2 + MPA-regulated genes was 58%, and with permanently MPA-regulated genes was 77%. Interestingly, if these percentages are compared with the overall overlap-percentages, the overlap between tibolone and E2 remained unchanged, while that between tibolone and E2 + MPA, and tibolone and MPA markedly increased (E2 + MPA: 41% becomes 58%; MPA: 55% becomes 77%). Upon reviewing the permanently tibolone-regulated genes in more detail, only 21 genes were truly tibolone-specific (never regulated at any time-point by any of the other hormonal treatments). From six of these genes, no additional information was available in literature, which left 15 genes with a putative common element that links them to tibolone-signaling. A literature search indicated that most of the 15 genes could be linked to general processes like differentiation (TWSG1, TFCP2L2, SLC39A14, PITX1, THADA, ID1) and the cell cycle (PPP3CC, MCM2, ASF1B, ELL).
Upon reviewing the literature further, minichromosome maintenance deficient 2 (MCM2) turned out to be the most interesting because it is part of a complex, which is prerequisite for DNA replication (Bailis & Forsburg 2004). The minichromosome maintenance proteins (MCM2–MCM7) form a heterohexamer that is part of the prereplication complex. DNA replication begins with loading of the origin recognition complex, followed by recruitment of Cdc6/Cdc45, followed by loading of the MCM2–7 complex (Guida et al. 2005). Upon entering the S-phase, Cdc6 is released and kinase activity recruited (Cdc2-cyclinE and Dbf4-Cdc7) to initiate DNA synthesis. Because the prereplication complex dissociates from DNA, replication of each region on the genome occurs only once during a cell cycle (Guida et al. 2005). In general, replicating cells have higher levels of MCM proteins than quiescent cells. During the menstrual cycle, this finding was illustrated in a physiological setting; in the follicular (proliferative) phase, MCM2 and MCM3 levels were found to be significantly higher than during the luteal (differentiation) phase (Kato et al. 2003).
Upon analyzing our own data concerning prereplication complex-regulated genes (Table 1), it was observed that most of the above-mentioned genes were clearly downregulated by tibolone as well as MPA. Since a downregulation of components of the prereplication complex is highly correlated to downregulation of proliferation in the endometrium (Kato et al. 2003), these findings can at least partly explain the growth-inhibitory properties of tibolone and MPA.
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4-tibolone. This, however, is not the case. Earlier work showed that in ECC1 cells tibolone is converted at least for 30% into its estrogenic 3ß-hydroxy metabolite (Hanifi-Moghaddam et al. 2005). Earlier work also indicated that this amount of 3ß-hydroxy tibolone is enough to provide a clear estrogen-like response in ECC1 cells (Hanifi-Moghaddam et al. 2005). It is possible that over time 3ß-hydroxy tibolone is converted back into 4-tibolone (Schatz et al. 2005). However, our array data show that, like MPA, they are tibolone’s own progestagenic properties which are responsible for an increased expression of the aldoketo reductase family 1 member C1 (AKR1C1, an enzyme that catalyzes the formation of 3ß-hydroxy tibolone exclusively) (Steckelbroeck et al. 2004).
Another explanation for tibolone’s dominant progestagenic properties can be found in the first experiment that was performed to characterize the presently used cell line. In Fig. 2, it is shown that MPA, E2 + MPA, and tibolone treatments clearly result in a profound reduction in ER expression. It is very well possible that this reduction in ER expression is responsible for the diminished estrogenic response that is left after tibolone treatment of the cell line.
It is also possible that tibolone’s progestagenic dominance has to do with the fact that a cancer cell line was used. The presently used ECC1-PRAB72 endometrial carcinoma cell line has a relatively high proliferation rate. It is possible that due to this, estrogens are only capable of having a relatively minor growth stimulatory effect (twofold in Fig. 1), while tibolone and MPA are highly effective to induce inhibition of proliferation. The results presented in Fig. 1 and the high number of regulated genes by tibolone, MPA, and E2 + MPA treatments when compared with E2 treatment, as shown in Fig. 4, seem to support this hypothesis.
Whether, in vivo, tibolone’s progestagenic properties are dominant remains to be seen; Timmer & Houwing (2002) observed that in serum of tibolone-treated patients, the 3- and 3ß-hydroxy metabolites are the most predominant metabolites, while the 4-isomer can only be measured until 6 h after administration. Our own data (Klaassens et al. 2006) on the endometrial tissues of healthy postmenopausal women indicate that relatively short-term tibolone treatment (21 days) results in some estrogen-like stimulation of the human endometrium, while it is specifically after long-term treatment that tibolone’s progestagenic properties outweigh its estrogenic properties. To test this hypothesis, we are in the process of generating expression profiles of endometrial tissues from long-term tibolone users.
In summary, the answer to our main question regarding the similarity between E2, MPA, E2 + MPA, and tibolone is that in the estrogen- and the progesterone-responsive endometrial cancer cell line ECC1-PRAB72, tibolone acts very similar to MPA and is clearly different from E2. However, there are also hormone-specific differences between tibolone and MPA.
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Acknowledgements |
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Received 31 May 2006
Accepted 10 August 2006
Made available online as an Accepted Preprint 12 October 2006
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