Molecular Human Reproduction, Vol. 5, No. 8, 748-756,
August 1999
© 1999 European Society of Human Reproduction and Embryology
Two-dimensional gel analysis of human endometrial proteins: characterization of proteins with increased expression in hyperplasia and adenocarcinoma
1 Center for Clinical and Basic Research, Ballerup Byvej 222, DK-2750 Ballerup, 2 The Centre for Proteome Analysis, International Science Park Odense, DK-5230 Odense M, and 3 Department of Gynaecology and Obstetrics, Hvidovre Hospital, DK-2650 Hvidovre, Denmark
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Abstract |
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In the search for new markers of human endometrial hyperplasia and adenocarcinoma the method of quantitative two-dimensional gel electrophoresis was applied to study the protein expression profiles of metabolically [35S]-methionine-labelled proteins of endometrial explants. Approximately 1700 protein spots were resolved by the two-dimensional gel electrophoresis, and the expression pattern of each of these proteins was assessed for increased expression during hyperplasia or adenocarcinoma. In total, six protein spots showed increased expression in hyperplasia, 19 in carcinoma, and eight in both hyperplasia and carcinoma. Twelve of these 33 differentially expressed proteins were identified by peptide mass mapping combined with sequence database searching. Among the identified proteins were proteins involved in cellular transport and chaperoning, i.e. heat shock protein 27 kDa protein, heat shock 70 kDa protein, heat shock cognate 71 kDa protein, and serotransferrin. Other identified proteins were: regulatory chain protein of cAMP-dependent protein kinase, prohibitin, and heterogeneous nuclear ribonucleoprotein A2/B1. Finally we identified proteins associated with the cytoskeleton, vimentin and tropomyosin isoform 3, and the glycolytic pathway,
enolase, and phosphoglycerate kinase. The remaining unidentified proteins were either not contained in the database and must be assumed to be novel proteins, or were present in too low amounts to allow characterization. 2-D gel electrophoresis/cancer/endometrium/hyperplasia/proteins
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Introduction |
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During the menstrual cycle, the endometrium is the organ in the body that shows the greatest changes under the influence of the sex hormones, oestradiol and progesterone. In the first 14 days of the cycle, the endometrium exhibits proliferation due to oestrogenic stimulation. Following ovulation, the granulosa cells of the corpus luteum begin to secrete progesterone which induces the transformation of the endometrium from proliferative phase to secretory phase. After full transformation, the endometrium breaks down and is shed during menstruation. Persistent unbalanced oestrogen stimulation is associated with increased risk of developing hyperplasia of the endometrial glands and subsequently endometrial adenocarcinoma (Hammond et al., 1979
; Gambrell, 1986; Persson et al., 1996). The increased risk during hormone replacement therapy can be eliminated by the inclusion of progestogen in the treatment regimen (Gambrell 1986; Persson et al., 1996). However, endometrial cancer remains the most frequent gynaecological disease in the Western World causing considerable morbidity and mortality with a 5 year survival rate of ~75% (Connelly et al., 1982; Abeler and Kjorsted, 1991). The prognosis is closely related to the stage of the disease with a 5 year survival rate ~82% if diagnosed in stage I, where the disease is most frequently diagnosed, decreasing to 63–73% in stage II, 44–52% in stage III and 17–27% in stage IV (Abeler and Kjorsted, 1991; Partridge et al., 1996).
Early diagnosis of endometrial cancer has not improved over the years due to the lack of suitable screening methodology (Partridge et al., 1996). At present endometrial hyperplasia and cancer are commonly diagnosed as a consequence of abnormal uterine bleeding, but there are a large number of factors which are currently under investigation or have been suggested to be of prognostic but not diagnostic value. It has been shown that serum concentrations of the tumour-associated antigen CA 125 alone or in combination with CA 15–3 or CA 19–9 may be used as predictors of myometrial invasion, extrauterine spread, for monitoring the chemotherapy response and as a prognostic risk factor in patients with endometrial cancer (Duk et al., 1986; Scambia et al., 1994; Takeshima et al., 1994). Also, another high molecular weight ovarian epithelial cancer cell antigen, the OVX1 protein, was found to be more frequently elevated in patients with deep myometrial invasion and poorly differentiated tumours (Xu et al., 1994) although in a later study there was no correlation between the serum concentration of OVX1 and the clinical stage of the disease (Beck et al., 1997). Of the markers based on analysis of tissue samples, several biochemical and immunohistochemical studies have shown that the concentrations of progesterone receptor (PR) and oestrogen receptor (ER) were inversely correlated with both the histological grade and the tumour stage as reviewed recently (Nyholm, 1996). In addition increased content of PR was a predictive factor of better disease-free survival (Creasman, 1993; Nyholm et al., 1995). Several markers investigated in other cancer diseases were also found to be related with the prognosis and clinical staging of endometrial carcinoma although some of the markers also varied with the menstrual cycle. Among these cancer markers are some of the growth factors, the epidermal growth factor receptor (EGFR) (Khalifa et al., 1994), the cell adhesional transmembrane glycoprotein CD44 (Saegusa et al., 1998; Tempfer et al., 1998), the extracellular matrix glycoprotein tenascin (Yamanaka et al., 1996), telomerase activity (Shay and Bacchetti, 1997; Shroyer et al., 1997; Yokoyama et al., 1998), and expression of HER-2/neu oncogene (Khalifa et al., 1994, Rasty et al., 1998). Also the use of p53 has been investigated in a number of papers, and it was recently described that nuclear overexpression of p53 was associated with poor patient survival in contrast to cytoplasmic overexpression of p53 which was associated with better patient survival (Soong et al., 1996).
Recently, in the search for new biochemical markers of the human endometrium, we studied the changes in the protein expression during the normal menstrual cycle using the method of quantitative two-dimensional gel electrophoresis of metabolically labelled proteins in endometrial tissue explants (Byrjalsen et al., 1995a). This two-dimensional gel electrophoresis technique encompassing the demand for both high sensitivity and high resolution, is excellent for studying the complex protein mixtures expressed in cells, and for identifying individual proteins altered in expression during, e.g. disease-processes. In the present study we applied this proteomic approach for the search of proteins having increased expression in endometrial hyperplasia or carcinoma with the aim of identifying new biochemical markers that in the future may facilitate the diagnosis, prognosis or choice of treatment of endometrial disorders.
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Materials and methods |
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Endometrial samples
Endometrial tissue samples were obtained from 16 pre- and post-menopausal women undergoing endometrial curettage (n = 9) or hysterectomy (n = 7) for medical reasons of menorrhagia, metrorrhagia, abnormality or malignancy of the endometrium. Based on the routine histological examination of the endometrial tissues the participants were assigned to the following three groups: Group IP (Irregular Proliferative/Proliferative; n = 6): five irregularly menstruating premenopausal (41–52 years old) and one postmenopausal woman (73 years old) having proliferative or irregular proliferative phase endometrium; Group H (Hyperplasia; n = 5): two irregularly menstruating pre-menopausal (44 and 47 years old) and three post-menopausal women (54–79 years old) of whom three had simple endometrial hyperplasia and two had complex endometrial hyperplasia; and Group C (adenocarcinoma; n = 5): one pre-menopausal (50 years old) and four post-menopausal women (70–77 years old) of whom three had endometrial adenocarcinoma with <50% myometrial invasion and two had endometrial adenocarcinoma with >50% myometrial invasion. Endometrial tissue samples from pre-menopausal, regular cycling women representing the various phases the endometrium undergoes during the normal menstrual cycle was part of the previously described study (Byrjalsen et al., 1995a
). These patients (Group M, n = 13) were healthy, normally menstruating women (35–50 years old) not receiving hormone contraception and representing the various phases that the endometrium undergoes during the normal menstrual cycle. Immediately after surgery a part of each endometrial sample was collected in cell culture medium (Dulbecco's modified Eagle's medium/F12) for metabolic radiolabelling of the proteins. The remaining portion was placed in neutral formalin for routine histological analysis. All participants gave their informed consent, and the study was performed in accordance with the Helsinki Declaration II and was approved by the local ethical committee.
Protein labelling and two-dimensional gel electrophoresis
Radiolabelling was initiated within few hours after obtaining the tissue and performed as previously described (Byrjalsen et al., 1995a). Briefly, the tissue sample was washed in phosphate-buffered saline (PBS) and three to four small pieces (
1 mm3) were incubated in the presence of 100 µCi/100 µl of [35S]-methionine for 20 hours at 37°C in 5% CO2. Following incubation, the radiolabelled pieces of tissue were washed in PBS and stored at –85°C until electrophoresis. Prior to electrophoresis the tissue was homogenized in 200 µl of ice-cold 20 mmol/l Tris–HCl buffer pH 7.5, containing 30 mmol/l sodium chloride, 5 mmol/l calcium chloride, 5 mmol/l magnesium chloride, 25 µg/ml RNase and 25 µg/ml DNase. After homogenization the samples were lyophilized and dissolved in lysis buffer [9.5 mol/l urea, 2% (w/v) Nonidet P-40, 5% (v/v) ß-mercaptoethanol, and 2% (v/v) ampholytes pH 7–9]. The incorporation of [35S]-methionine was determined by trichloroacetic acid precipitation. If possible, a sample containing 500 000 c.p.m. was used for electrophoresis. Otherwise the exposure time was adjusted to reach the same degree of exposure of the fluorograms. The two-dimensional gel electrophoresis was performed essentially as previously described (O'Farrell, 1975) with modifications (Fey et al., 1984) described in detail (Byrjalsen et al., 1995a). The first dimensional electrophoresis was carried out in glass tubes (internal diameter 1.5 mm, length 18 cm). First dimensional isoelectric focusing gels (IEF) were cast and run for resolving of proteins with isoelectric points (pI) ranging from 3.5 to 7, and non-equilibrium pH gradient gels (NEPHGE) gels were cast and run for resolving of proteins with pI ranging from 6.5 to 11. The second dimensional sodium dodecyl sulphate (SDS)–polyacrylamide gel electrophoresis (12.5% acrylamide) was done in slab gels (20 cm long, 18.5 cm wide, 1 mm thick) for resolution of proteins with relative molecular weights ranging from 10 to 300 kDa. After electrophoresis the gels were fixed, soaked in fluorographic reagent, dried and exposed to X-ray film.
Analysis of protein expression
The fluorograms of the two-dimensional gel electrophoresis were subjected to quantitative analysis using the Bio-Image system (Millipore, Bedford, MA, USA). The fluorograms were scanned using a Truvel scanner, and spots were detected and quantified on the resulting computer-images. Finally, all the gel-images were matched to the same master-image, whereby each protein was assigned a match number. All match vectors were checked and non-matched spots were matched manually. It was ensured that all the selected spots classified as having increased synthesis in endometrial hyperplasia or carcinoma were located on all gel-images even if the spot position had only a background value. Data of a given spot was determined as percentage integrated optical density (IOD%) which is the percentage IOD of the spot taken relative to the total IOD of the individual fluorogram. The expression pattern of each protein spot, i.e. the IOD% plotted against the menstrual cycle day and the three groups with endometrial disorders, was subjectively assessed independently by two persons to decide which proteins had increased synthesis in endometrial hyperplasia or adenocarcinoma. Subsequently histograms were drawn for the selected protein spots, of the geometric mean value of the IOD% grouped according to the assigned endometrial group. In case the selected spot varied also in a menstrual cycle-related manner, the menstrual cycle group was subdivided into a proliferative phase and a secretory phase group. The Kruskal–Wallis one-way analysis of variance was used to assess the statistical significance of difference among all the endometrial groups, and the Wilcoxon 2-sample test was used to compare the statistical significance (two-tailed) between hyperplasia or carcinoma with the combined group of the normal menstrual cycle and the proliferative/irregular proliferative group. In case the selected spot varied in a menstrual cycle-related manner, results of secretory phase endometrium were omitted from the combined group. In order to assess the strength of combining spots, firstly the individual spot ratio was calculated as the IOD% of the spot normalized by the geometric mean value of the spot in the menstrual cycle group. Secondly, for combination of two spots the individual ratios of the two spots were multiplied and the non-parametric statistical analysis were performed as described above. All statistical analyses were performed using the NPAR1WAY procedure of the SAS Institute programs (SAS, 1989).
Protein identification
Protein spots were localized and excised from dried two-dimensional gels (Byrjalsen et al., 1995b), and in-gel tryptic digestion was performed according to previously described methods (Rosenfeld et al., 1992; Shevchenko et al., 1996). The procedure consisted of washing the excised gel pieces, and an overnight incubation with trypsin (sequencing grade; Promega, Madison, WI, USA). A total of 2–5% of the peptide digest was then analysed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. If this direct analysis did not result in unambiguous identification, the peptide mixture was extracted from the gel pieces, and subsequently desalted and concentrated by micro-column chromatography prior to mass spectrometric analysis (Gobom et al., 1997). The 4-hydroxy--cyanocinnamic acid (HCCA) was used as the UV-absorbing MALDI matrix. Delayed-extraction MALDI peptide mass spectra were acquired using a Bruker REFLEX (Bruker–Franzen, Bremen, Germany) reflector time-of-flight mass spectrometer. Calibration was accomplished by using external mass standards or by using the trypsin autodigestion peptide signals and matrix-related ion signals as internal standards. The peptide mass error was <50 ppm when internal calibration was used.
Protein identification was performed by searching the peptide mass maps in a comprehensive non-redundant protein sequence database (nrdb, European Bioinformatics Institute, Hinxton, UK) using PeptideSearch software (Mann et al., 1993). The peptide mass maps and the protein identifications were evaluated as previously described (Jensen et al., 1998).
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Representative fluorograms of isoelectric focussing (IEF) and non-equilibrium pH gels (NEPHGE) are shown in Figure 1
. On the IEF gels a total of 1203 protein spots with pI of 3.5–7 and molecular weights of 10–300 kDa were matched. Correspondingly, a total of 555 protein spots (pI 6.5–11) were matched on the NEPHGE gels. The matched spots represented an average 96 and 92% of the total integrated optical density on the IEF-gels and NEPHGE-gels respectively. The non-matched spots were generally located in the buffer front or in the neutral edge on the NEPHGE-gels. For the IEF gels, a total of 217 spots representing 65% of the staining intensity were localized on all 29 fluorograms, and 618 spots corresponding to 90% of the staining intensity and 916 spots corresponding to 94% of the staining intensity were localized in 50 and 25% of the fluorograms respectively. A similar degree of detectability was found for the NEPHGE-gels.
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Out of the total number of ~1700 protein spots, 14 spots had statistically significant or showed a tendency toward increased synthesis in endometrial hyperplasia and 27 had, or showed a tendency toward, increased synthesis in carcinoma in comparison with the expression in endometrium during the normal menstrual cycle and irregular proliferative phase endometrium. Of these, eight spots were increased in both hyperplasia and carcinoma. The location of these 33 spots with increased synthesis in hyperplasia and/or carcinoma are indicated in Figure 1
. Figure 2 shows histograms of the geometric mean value (+SEM) of IOD% in the four endometrial groups of the six spots with increased expression exclusively in endometrial hyperplasia. One of the spots (no. 6) showed also menstrual cycle-related expression with maximum values during the proliferative phase. The results from the normal menstrual cycle endometrium of this spot were therefore subdivided into a proliferative phase and a secretory phase group. Table I lists the information obtained from the two-dimensional gel electrophoresis with respect to the pI and the molecular weight of the spots. The Table also gives the P values of the non-parametric statistical analysis of the one-way analysis of variance for comparison among all the endometrial groups and the P values of the comparison of hyperplasia with the combined group of normal menstrual cycle and the proliferative/irregular proliferative groups. Similarly, Figure 3 and Table II give the results of each of the 19 protein spots with increased synthesis exclusively in carcinoma. Spots nos. 7–11 showed the same tendency with lowest expression in normally menstruating women, a small increase in the proliferative/irregular proliferative group, more increase during hyperplasia, and highest increase in women with carcinoma. The five spots (nos. 21–25) shown in the lower row had a menstrual cycle-related expression with spots nos. 21–24 being maximally expressed in proliferative phase endometrium, and no. 25 being maximally expressed in secretory phase endometrium. Finally, Figure 4 and Table III give the results of the eight protein spots with increased synthesis in both hyperplasia and carcinoma.
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A combination of two spots, i.e. the relative expression of one spot increased in, e.g. cancer multiplied by the relative expression of one of the other spots increased in cancer generally allowed a higher degree of distinction between the endometrial groups (the spot expression was taken relative to the expression in the normal menstrual group and calculated for the individual patient). The 19 spots that were increased in carcinoma could in this way be combined in 171 different combinations, and the non-parametric statistical analysis (Wilcoxon 2-sample test) revealed that in 40% of the combinations P
0.01; in 50% of the combinations P 0.05–0.01; and 10% were not significant (i.e. P > 0.05). Figure 5 shows an example of combination of two of the spots increased in hyperplasia (panel A; spot nos. 1 and 3) and two of the spots increased in carcinoma (panel B; spot nos. 15 and 18). Thus the example of combining two of the spots increased in hyperplasia gave an increase by a factor of 10 in the group of hyperplasia as compared to the normal menstrual cycle group. Similarly, combining two of the spots increased in carcinoma gave an increase by a factor 19 in the carcinoma group as compared to the normal menstrual cycle group.
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Table IV
lists the proteins with increased expression in hyperplasia and carcinoma that were identified by peptide mapping using mass spectrometry. Twelve proteins out of the 33 differentially expressed proteins were unambiguously matched to an entry in the sequence database. We identified two out of the six proteins increased in hyperplasia, i.e. human vimentin (spot no. 3) and human enolase (spot no. 5). Altogether seven out of the 19 protein spots increased in endometrial carcinoma were identified. The identified proteins comprised proteins associated with the cytoskeleton, vimentin (spot no. 17) and tropomyosin isoform 3 (spot no. 9), and heat shock 70 kDa protein (spot no. 10) which is involved in chaperoning and protein folding. Spot no. 13 contained both heat shock 27 kDa protein, a protein that is involved in stress resistance, and prohibitin which inhibits DNA synthesis. Spot no. 21 also contained two proteins, i.e. a nuclear protein and in addition glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Finally, spot no. 11 was identified as serotransferrin precursor and spot no. 14 was the regulatory chain of cAMP-dependent protein kinase type I. Three out of the eight proteins increased in both hyperplasia and carcinoma was identified as heat shock 70 kDa protein (spot no. 26), human heat shock cognate 71 kDa protein (spot no. 33), and phosphoglycerate kinase (spot no. 32). Spot no. 26 contained in addition serum albumin precursor.
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Discussion |
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In the present study the protein expression patterns of endometrial hyperplasia and adenocarcinoma tissue samples were analysed with quantitative, high resolution two-dimensional gel electrophoresis. This proteomic technique is excellently suited for identification of proteins with altered expression during various conditions. It has wide applications, e.g. for comparison of protein expression patterns of different cell types, cell differentiation and transformation and alteration in response to various stimuli. We recently used the technique for identification of proteins exhibiting a menstrual cycle-related expression, and characterized several proteins not previously described to exhibit variation during the normal menstrual cycle in addition to proteins previously described to be cycle-related or influenced by the sex hormones (Byrjalsen et al., 1995b
). The two-dimensional gel electrophoretic technique combined with the high sensitivity of [35S]-methionine metaboliclabelling of proteins made it possible to compare the expression of ~1700 protein spots with pI ranging from 3.5 to 11 and relative molecular weights ranging from 10–300 kDa. In the present study we found that 33 of the protein spots corresponding to 1.9% of the total number of spots showed increased expression in hyperplasia or adenocarcinoma as compared to the expression in tissue from normally menstruating women or irregularly menstruating women with proliferative/irregular proliferative phase endometrium. Altogether six protein spots were found to have increased synthesis exclusively in endometrial hyperplasia, 19 had increased synthesis exclusively in carcinoma, and eight protein spots were increased in both hyperplasia and carcinoma. Although it would have been desirable to analyse more endometrial patient samples in each of the endometrial histology groups to reduce the individual variation, the non-parametric statistical analysis revealed statistically significant differences for several of these differentially expressed proteins. The risk of analysing a limited number of patient samples is, from a statistical point of view, that we have overlooked some proteins that in addition are being differentially expressed (Type II error), but not that the proteins we have classified as being differentially expressed proteins will be classified as non-differentially expressed when including more samples (Type I error). The idea behind combinations of spots, i.e. the relative expression of one of the spots increased in e.g. cancer multiplied by the relative expression of one of the other spots increased in cancer (calculated within the individual patient) was that this product could be a more reliable `marker' as it was based on information arising from two of the proteins instead of the information from a single protein spot only. The technique of combination generally gave more significant distinction of the diseased group, and it may be speculated that combination of a higher number of proteins would become even more significant. From a practical point of view it must however be aimed at keeping the number of proteins as low as possible.
By employing the technique of MALDI peptide mass mapping combined with sequence database searching, we unambiguously identified 12 out of the 33 differentially expressed proteins (corresponding to identification of 36%). The remaining un-identified proteins were either present in too low amounts or proteins that were not contained in the sequence database, i.e. novel proteins. Spot nos. 10 and 26 revealed identical identifications and contained both heat shock protein 70 kDa and serum albumin precursor. The latter is probably contamination due to the quantitatively larger amounts of serum albumin, which in the gel is localized immediately above the area of the gel pieces excised for the identification. The reason for the different localization of the same protein may be ascribed to post-translational modification influencing the iso-electric point, e.g. phosphorylation. Similarly, vimentin was identified in both spot no. 3 and spot no. 17 although the positions of these spots corresponded with a difference of ~10 kDa in the molecular weight, indicating that the protein of spot no. 3 could be a truncated form of vimentin. Two proteins were identified in spot no. 21, but probably the GAPDH was a contaminant as this protein is known to be localized at the position of the heavily stained protein spot just below this excised gel piece. The concurrent finding of both prohibitin and heat shock protein 27 in spot no. 13 does not allow us to determine whether both or only one of the proteins are differentially expressed. In a recent study where similar techniques were employed it was found that two or more proteins were identified in ~10% of the 335 excised gel pieces (Nawrocki et al., 1998). Several of the identified proteins have previously been described to be differentially expressed in the endometrium. Data have shown that oestrogen stimulation increased the level of heat shock protein 27 in the glandular epithelial cells of the endometrium suggesting that this protein could be a biochemical marker of the oestrogenic endometrial response of the epithelial cells (Ciocca et al., 1993; Padwick et al., 1994; Tabibzadeh et al., 1996). Recently it was also shown that the heat shock protein 27 was an independent prognostic indicator in patients with endometrial adenocarcinoma (Geisler et al., 1999). Some heat shock proteins have been reported to show menstrual-cycle dependent changes, suggesting a regulation by the sex steroid hormones, that is the heat shock protein 60 and the constitutive form of heat shock protein 70, but not the inducible form of heat shock 70 or heat shock protein 90 (Tabibzadeh et al., 1996). The intermediate filament protein, vimentin, especially found in mesenchymal cells and normally regarded as a stromal cell marker, have been found to be synthesized by the epithelial cells of the endometrium during all stages of the menstrual cycle. However, it was not expressed after the onset of pregnancy suggesting a role of vimentin in the proliferation and/or decidualization of the endometrium (Norwitz et al., 1991). The increase in the glycolytic enzymes, phosphoglycerate kinase and alpha enolase, may reflect an increased rate of glycolysis in the hyperplastic and neoplastic cells.
Further work will be carried out to characterize and identify the remaining proteins. The identification of the less abundant proteins may be carried out by isolation of the proteins using the immobilized pH gradient (IPG) based two-dimensional gel electrophoresis system which allows a higher sample load capacity as compared to the carrier ampholyte-based system (Görg et al., 1988). Additionally the IPG systems may provide greater separation distance of the proteins when running the narrow pH gradient gels, and hereby overcome the ambiguity with identification of two or more proteins within the same gel piece. Subsequent to identification and characterization, the clinical utility of the differentially expressed proteins must be evaluated to distinguish between the normal endometrial conditions and pathological conditions. Assessment of the clinical utility of marker proteins would require specific assays for the measurement of the constitutive concentrations of proteins and an extensive number of clinically relevant samples. Given that the endometrial synthesis of some of these proteins are reflected in the circulation it may be possible to have an easy serological screening tool for the detection of, e.g. endometrial hyperplasia before the progression of this condition into carcinoma, and an easy tool for the discrimination of hyperplasia and carcinoma. Furthermore, such markers might help to gain insight into the genesis and development of the disease.
In conclusion, by using two-dimensional gel electrophoresis we localized 33 proteins with increased synthesis in hyperplasia or adenocarcinoma and identified 12 of these proteins. Given the current improvements of characterization and identification by mass spectrometry and database searching of proteins available in minute amounts it seems feasible to expect that most of the remaining proteins with increased expression will be identified. Identification of the proteins will allow us to establish specific assays to be assessed for clinical usefulness hopefully leading to identification of new markers that may be used for the early diagnosis, prognosis or choice of treatment of endometrial disorders.
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Acknowledgments |
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Claus Christiansen is consultant for and stockholder in Osteometer Biotech A/S.
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4 To whom correspondence should be addressed at: Osteometer BioTech A/S, Herlev Hovedgade 207, DK-2730 Herlev, Denmark
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References |
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Submitted on March 10, 1999; accepted on June 8, 1999.
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