Molecular Human Reproduction, Vol. 8, No. 3, 246-254,
March 2002
© 2002 European Society of Human Reproduction and Embryology
Uterine physiology |
Identification of genes with higher expression in human uterine leiomyomas than in the corresponding myometrium
1 Division for Reproductive Endocrinology, Department of Woman and Child Health, Karolinska Hospital L5:01, S-171 76 Stockholm, 2 Department of Clinical Sciences, Division for Obstetrics and Gynecology, Huddinge Hospital, S-141 86 Huddinge and 3 Department of Molecular Medicine, Karolinska Hospital L8:01, S 171 76 Stockholm, Sweden
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Abstract |
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We used a PCR-based subtraction method, representational difference analysis of cDNA (cDNA–RDA), to identify genes with a higher expression in leiomyomas in comparison with the corresponding myometrium during the proliferative phase of the menstrual cycle. Increased expression of the genes for pregnancy-associated plasma protein A (PAPPA), tomoregulin, cellular retinoid acid binding protein 1 (CRABP1), zinc finger protein 185 (ZFP 185) and latent transforming growth factor ß binding protein 2 (LTBP2) was demonstrated in individual leiomyoma samples compared with corresponding myometrium. Additionally, a specific positive immunostaining of LTBP2 was found in the smooth muscle cells of both leiomyomas and myometrium. These genes may be part of previously unidentified molecular mechanisms responsible for the selective growth advantage of leiomyomas compared with myometrium. This work expands our knowledge about the molecular nature of leiomyomas and provides novel candidate genes to further explore in relation to their function during leiomyoma growth.
differential gene expression/leiomyomas/uterus
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Uterine leiomyomas are the most common tumours in the reproductive tract, affecting 30% of women during the reproductive period (Carlson et al., 1993
). Although benign, these tumours often cause heavy menstrual bleeding, pelvic pressure symptoms and reproductive failure, causing them to be the leading indication for pelvic surgery.
Leiomyomas are believed to be monoclonal. The factors involved in their initiation and growth remain poorly understood. Large chromosomal abnormalities are absent in a large portion of leiomyomas. Nevertheless, the clonal origin of these tumours suggests that somatic mutations not yet identified may constitute a potential initial event in tumorigenesis by offering a selective growth advantage to the mutated myocyte. Heterogeneous cytogenetic abnormalities and specific translocations have been observed in 36% of uterine leiomyomas (Pandis et al., 1991) and are believed to be secondary to the clonal expansion. The cytogenetic aberrations include a specific reciprocal translocation involving chromosomes 12 and 14, another rearrangement involving the long arm of chromosome 12, deletion of a portion of the long arm of chromosome 7, trisomy 12, and rearrangements involving the short arm of chromosome 6 (Nilbert and Heim, 1990). Recently, Ligon and Marton demonstrated that expression of HMGI-C or HMGI-(Y) genes is a common feature in leiomyomas associated with 12q15 and 6p21 chromosomal alterations (Ligon and Marton, 2001).
Traditionally, estrogen has been considered to be the major promoter of leiomyoma growth but a growing amount of evidence support a role for progesterone in this process (Rein, 2000). Sex steroid regulation of leiomyoma growth is mediated by the binding of hormones to their respective receptors with subsequent activation of oncogenes, mitogenic factors, growth factors and their specific receptors.
The identification of genes differentially expressed in leiomyomas when compared with normal myometrium will help us to better understand the pathophysiology of these tumours. We have used an unbiased approach to identify genes with a higher expression in leiomyomas than in myometrium during the proliferative phase of the menstrual cycle. The use of the PCR-based subtraction method, representational difference analysis of cDNA (cDNA–RDA), has allowed us to identify a number of genes with higher expression in leiomyomas than in the corresponding myometrium. We found five candidate genes: pregnancy associated plasma protein A (PAPPA), tomoregulin, cellular retinoid acid binding protein 1 (CRABP1), zinc finger protein 185 (ZFP 185) and latent transforming growth factor (TGF) ß binding protein 2 (LTBP2), which may represent candidates for regulators of leiomyoma growth.
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Materials and methods |
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Patients
The study was approved by the ethics committee of Huddinge University Hospital and Karolinska Institute, and informed consent was obtained from all participating women. Leiomyomas and corresponding myometrium were collected following hysterectomy from a total of 15 premenopausal patients. Serum samples were collected on the morning of surgery. Indications for surgery in patients suspected to have uterine leiomyomas included menorrhagia, pelvic discomfort, rapid tumour growth or uncertainty about the diagnosis, or a combination of these indications. None of the patients received any medication influencing the levels of circulating sex steroid hormones, GnRH analogues or antagonists or corticosteroids. The phase of the menstrual cycle was determined by the serum levels of 17ß estradiol (E2) and progesterone, menstrual cycle pattern data, the date of the last menstrual period and endometrial histology. Leiomyomas and myometrial tissue were collected immediately at hysterectomy or at enucleation of leiomyomas. Tissue for mRNA extraction was immediately frozen in liquid nitrogen and stored at –70°C. Samples were also collected for immunohistochemistry and fixed in buffered formaldehyde solution.
For the basal cDNA–RDA analysis, a single leiomyoma and corresponding myometrium were collected from a 50-year-old nulliparous woman in the proliferative phase of the menstrual cycle, named as the first patient. The leiomyoma was submucous with a volume of ~60 cm3. It has been shown that significantly higher numbers of estrogen receptors (ER) and progesterone receptors (PR) are present in leiomyomas than in the surrounding myometrium (Englund et al., 1998). One of the reasons for selecting this specific patient was that the leiomyoma had a 15-fold higher content of ER and a 2.4-fold higher content of PR per mg protein, measured by enzyme immunoassay, as compared with that in the corresponding myometrium (data not shown).
For screening of false positive clones, myometrial and leiomyoma tissues were collected from 11 women in the proliferative phase of the menstrual cycle and from four patients in the secretory phase. Material for the subsequent analysis of expression of the remaining subset of genes with individual samples was available for seven out of the 11 patients in the proliferative phase of the menstrual cycle. One or two leiomyomas and the corresponding myometrium were analysed for each of these patients. Patient characteristics are shown in Table I.
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cDNA synthesis
Total RNA was isolated from leiomyomas or corresponding myometrium of the first patient, using TRIZOL reagent (Life Technologies Inc., Grand Island, NY, USA), according to the protocol supplied by the manufacturer. mRNA was purified from 1 mg total RNA using oligo-(deoxythymidine) paramagnetic beads (Dynal AS, Oslo, Norway). cDNA was synthesized from 2 µg mRNA using a cDNA synthesis kit purchased from Promega (Madison, WI, USA).
RDA
RDA was performed as previously described (Odeberg et al., 2000). cDNA was obtained from a single pair of myometrium and leiomyoma samples (Figure 1A) and used as the driver and tester for several rounds of PCR amplification and subtraction. This allowed the identification of gene transcripts with expected over-expression in leiomyomas. Putative differentially expressed gene fragments were generated by two rounds of subtraction and amplification, using hybridization tester–driver ratios of 1:100 (DP2) and 1:800 (DP3) (Figure 1B). Difference products were run in a 1.8% agarose gel to have a qualitative estimate of the sample heterogeneity. Since DP2 and DP3 showed a similar pattern, the individual bands were excised, eluted from the gel and the products were cloned into the BamHI site of the pBluescript II SK+ vector (Stratagene, La Jolla, CA, USA). One hundred isolated colonies were picked for each excised gel slice and were checked for inserts by PCR using vector-specific primers. Plasmid minipreparations were made using the Wizard system (Promega).
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Sequencing and sequence comparison
Sequence analysis of differentially expressed cDNA products was performed using cycle sequencing with dye-labelled nucleotides (Big-Dye; Perkin-Elmer, Norwalk, CT, USA), and loaded on a PE Applied Biosystems 377 DNA sequencer (Perkin-Elmer). The sequences were analysed for homologies with published sequences in the non-redundant and expressed sequence tags (EST) division of Gene Bank using the BlastN software (www.ncbi.nlm.nih.gov/BLAST).
Hormone determinations
Serum levels of E2 and progesterone were determined by enhanced luminescence competitive immunoassay using commercial kits (Amerlite; Amersham International, Little Chalfont, Bucks, UK). Detection limits and inter- and intra-assay coefficients were for E2, 50 pmol/l, 13.3 and 14.8% and for progesterone, 0.35 nmol/l, 11.1 and 16.9% respectively. In the statistical calculations, values below the detection limits were set to 25 pmol/l for E2 and to 0.20 nmol/l for progesterone. The serum levels are shown in Table I
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Solution hybridization analysis
The expression of genes identified through the RDA procedure were measured using a solution hybridization/RNase protection assay. Patient samples were processed to obtain either total RNA (Table III and Figure 2) or total nucleic acids (Table IV). Total RNA was isolated using Trizol reagent (Life Technologies) according to the manufacturer's instructions. The concentration of the total RNA samples was adjusted to 0.1 µg/µl prior its use for expression measurements. Total nucleic acids (TNA) were prepared as previously described (Tollet-Egnell et al., 2000). Briefly, tissue specimens were homogenized using a Polytron PT-2000 (Kinematica AG, Littau, Switzerland), extracted with chloroform and phenol and precipitated with ethanol. Nucleic acid concentration was measured spectrometrically. For expression measurements, equal amounts of TNA samples were pooled together according to the menstrual cycle. Transcript-specific 35S-labelled cRNA probes were transcribed in vitro from the respective cDNA vector construct, using the Riboprobe System (Promega).
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: myometrium; 
: leiomyomas).
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Hybridization of aliquots of total RNA or TNA samples was performed in 40 µl containing 0.6 mol/l NaCl, 20 nmol/l Tris–HCl (pH 7.5), 4 mmol/l EDTA, 0.1% (wt/vol) sodium dodecyl sulphate, 10 mmol/l dithiothreitol, 25% formamide and 5000–15 000 counts per minute (cpm) probe/incubation. After overnight incubation at 70°C, the samples were exposed to ribonucleases (RNase A and RNase T1), and the hybrids were precipitated by the addition of 100 µl of 6 mol/l trichloroacetic acid, collected on a glass-fibre filter (GF/C; Whatman Ltd, Madison, UK), and counted in a liquid scintillation counter.
Samples were analysed in triplicate, and the results are expressed as cpm of specific mRNA/µg total RNA. mRNA levels of ß-actin were measured using a specific cRNA probe. When comparing the expression in RNA samples from individual patients, data are presented as the ratio between the expression of each gene (cpm/µg total RNA) and ß-actin expression.
Immunohistochemistry
The biopsy samples were embedded in paraffin and cut into 5 µm sections. The sections were dewaxed in Bioclear (Bio-Optica, Milan, Italy) and rehydrated in decreasing concentrations of ethanol. Sections were pre-treated in 0.01 mmol/l citrate buffer in a microwave oven. Normal horse serum was used as blocking serum. The sections were then incubated with the LTBP2 primary antibody, diluted 1:1000 in 2% bovine serum albumin, at 4°C overnight. The rabbit polyclonal antibody (Ab-178) was a kind gift from Professor C.-H.Heldin (BMC, Uppsala University, Uppsala, Sweden). The antibody was raised against a peptide corresponding to amino acids 818–834 of the LTBP2 sequence (Moren et al., 1994), and does not cross-react with LTBP1 (Michel et al., 1998). Horse serum served as a negative control. As the secondary antibody, a biotinylated horse anti-rabbit antibody was used (Vector Laboratories, Burlingame, CA, USA), and the slides were thereafter incubated with horseradish peroxidase–avidin–biotin complex (Vectastain ABC Elite; Vector Laboratories). The site of bound enzyme was visualized by 3,3-diaminobenzidine (DAB-kit; Vector Laboratories). The sections were counterstained with haematoxylin and dehydrated before mounting with Pertex (Histolab, Gothenburg, Sweden).
Statistical analysis
The results are reported as mean ± SD. When more than one leiomyoma was analysed from one patient, the average expression was calculated before further statistical evaluation. The Wilcoxon's signed rank test was used for paired comparisons between leiomyomas and myometrium. P < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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cDNA–RDA
RDA was performed on cDNA obtained from a single woman with a leiomyoma in order to identify genes over-expressed in leiomyoma tissue when compared with the surrounding myometrium during the proliferative stage of the menstrual cycle, which is characterized by increasing estrogen levels and low progesterone levels. Following extensive sequencing of the RDA clone products, 34 different cDNA clones were identified; 29 corresponded to genes of assigned function, while the remainder showed high homology to EST found in public databases (Table II
). The majority of these sequences were homologous to EST sequences characterized from rat, mouse or human tissues. It should be noted that additional products might still be identified from this experiment, as gene products such as, for example, ER and PR (Englund et al., 1998), which are expressed at higher levels in leiomyomas than in myometrium, are missing from the list of putative up-regulated clones. The fact that just a subset of the differentially expressed genes is expected to be identified reflects the limitation of all differential cloning methods. The reasons behind this have been analysed elsewhere (Carulli et al., 1998; review) and will not be further discussed here.
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Screening for false positive clones
The fact that the RDA analysis was carried out using only one sample of leiomyoma and myometrial tissues could be criticized. We reasoned that due to the level of genetic variability within leiomyomas, performing RDA using pooled samples would make the analysis of the results difficult. Therefore, we chose one leiomyoma patient who had a much higher expression of ER and PR in a leiomyoma compared with myometrium, since steroid regulation of growth is a common feature shared by most leiomyomas. To rule out artefacts during the RDA procedure, we analysed the expression levels of a subset of genes in the total RNA extracted from the same samples used to perform the RDA procedure. 35S-labelled cRNA probes were prepared from 16 cDNA clones identified by the RDA product. The probes were hybridized to total RNA samples and the relative expression of these genes was determined using an RNase protection/solution hybridization assay. Higher expression in the leiomyoma (>130%) when compared to myometrium was observed in almost 50% of the selected genes (Table III
). Based on previous experience using the solution hybridization method, this is the minimum basic difference where data can be expected to be reproducible in a larger material. We were forced to select a subset of the cloned genes for the initial analysis (Table III) since our attempts to perform a parallel measurement of expression levels of all cloned genes using 32P-labelled cDNA hybridized on microarrays were not as sensitive as the RPA, where the hybridization occurs in solution with an excess of a gene-specific labelled probe (Welford et al., 1998; Tollet-Egnell et al., 2000).
Our intention was to identify genes with a general importance for tumour growth. Accordingly, the expression of a subset of candidate genes (defined in the previous experiment) was measured in TNA samples pooled from several samples of leiomyomas and myometrium collected in either the proliferative or the secretory phase of the menstrual cycle (Table IV). In these pooled samples, the expression of the seven genes was higher in leiomyomas than in myometrium when analysed in either phase of the menstrual cycle. The list of genes that were differentially expressed both in the basic material and in the pooled samples included PAPPA, tomoregulin, CRABP1, ZFP 185, regulator of G protein signalling 12, placental bikunin and LTBP2 (Table IV), and these were selected for further analysis using individual leiomyoma and myometrial samples.
Differential gene expression in leiomyomas
Taking into account their putative cellular function as well as the previous expression data, a more careful study of five selected genes was performed with individual samples. The mRNA expression of specific genes in total RNA from leiomyomas and matched myometrium was determined by a solution hybridization assay (Figure 2). The tissues studied were collected in the proliferative phase of the menstrual cycle. The expression transcripts coding for LTBP2, ZFP 185, tomoregulin, CRABP1 and PAPPA were significantly higher in leiomyomas compared with those in corresponding myometrium when these data were calculated according to specific mRNA levels normalized to ß-actin (P < 0.05).
Immunohistochemistry for LTBP2
Five genes were identified as displaying increased expression in several different leiomyomas. Of these, we were able to obtain an antibody only for LTBP2. In an immunohistochemical analysis, LTBP2 was primarily detected in the cytoplasm of cells. Mild specific LTBP2 immunostaining was observed in the myometrium (Figure 3a), leiomyoma (Figure 3b) and vascular smooth muscle cells (Figure 3c, arrow). Strong immunostaining of LTBP2 was found in vascular endothelium (Figure 3d, arrowhead). LTBP2 was not localized to the bands of connective tissue intervening between the smooth muscle cell bundles in myometrium and leiomyomas. The negative controls for myometrium and leiomyoma provided negative results (Figure 3e). From the immunohistochemical results, no observable difference in LTBP2 protein expression was noted. However, due to the limited sample size (three pairs of leiomyoma and myometrial samples) used in this study and the non-quantitative method used, the abundance and function of this protein in uterus and leiomyomas needs to be further investigated.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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We have performed RDA of cDNA on a sample pair of leiomyoma and surrounding myometrium collected in the proliferative phase of the menstrual cycle. We also describe the identification of genes with putative over-expression in leiomyomas. The identity could be described for 85% (29/34) of these RDA clones. Among the identified gene products, five (17%) have previously been characterized as differentially expressed in leiomyomas, including: collagen (Stewart et al., 1994
); adrenergic receptor (Adolfsson et al., 1998, 2000); tropomyosin (Giometti and Anderson, 1984; Rush et al., 2001); glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Verneaux et al., 1992) and ribosomal RNA (Pollard et al., 1987). Moreover, our results support previous findings regarding the expression in uterus and myometrium of six (21%) of the identified genes, these being: integrin 
(Terpe et al., 1994); interferon /ß receptor (Han et al., 1997); serotonin receptor 2 (Jeffrey et al., 1991); neuropsin (Chen et al., 1998); neurosin enzyme (Yousef et al., 1999) and placental bikunin (Butzow et al., 1986). The RDA products with unknown functions might also include new differentially expressed genes in leiomyomas, but the identities of those must await further analysis. Subsequently we have shown that PAPPA, tomoregulin, CRABP1, ZFP 185 and LTBP2 have significantly higher expression in leiomyomas than in myometrium when analysed by solution hybridization in the first patient, in the pooled samples of multiple patients during the menstrual cycle and in the individual paired samples of leiomyoma and myometrium.
One theory concerning the development of leiomyomas proposes that ovarian hormones, particularly estrogen and progesterone, mediate leiomyoma development, and that dysregulation of growth factors and their receptors is important in the growth of leiomyomas. PAPPA is a glycoprotein present in the serum of pregnant women in increasing concentrations throughout pregnancy (Lin et al., 1974). PAPPA mRNA can be synthesized in reproductive as well as non-reproductive tissues (Overgaard et al., 1999), indicating an additional function of PAPPA outside pregnancy. PAPPA can specifically cleave insulin-like growth factor (IGF) binding protein (BP)-4, which results in release of IGF from IGFBP-4. It has been shown that in leiomyomas, IGFBP-4 is the most abundant of the IGFBPs (Giudice et al., 1993). Intact IGFBP-4 is a potent inhibitor of IGF action in vitro, and PAPPA cleavage of IGFBP-4 may lead to increased bioavailability of IGF in leiomyomas (Conover et al., 1999; Lawrence et al., 1999; Mazerbourg et al., 2000). The IGF family has previously been proposed as being involved in the growth of leiomyomas, since these tumours have higher levels of IGF-I mRNA and protein than in myometrium (Englund et al., 2000), and exogenous IGF-I stimulates proliferation of leiomyoma-derived cell lines (Howe et al., 1996).
Retinoic acid (RA) is the biologically active form of vitamin A (Jetten et al., 1979). The gene regulatory actions of RA are mediated by its binding to the RA receptor (RAR) and retinoid X receptor (RXR), members of the nuclear receptor family. In the uterus, retinoic acid can inhibit estrogen-induced uterine smooth muscle cell proliferation (Boettger-Tong and Stancel, 1995; Boettger-Tong et al., 1997; Benson et al., 2000). Therefore, RA and selective ligands have been used in the treatment of hyperproliferative disorders (Fenaux et al., 2000; Recchia et al., 2000). The idea of a pharmaceutical therapy, using LGD 1069, a RXR selective ligand, to treat leiomyomas has been tested in both in-vitro and in-vivo models (Boehm et al., 1994; Tsibris et al., 1999; Gamage et al., 2000). Antagonistic effects on RA activity appear to be mediated by two distinct classes of proteins: a family of nuclear receptors that regulates gene transcription in a ligand-dependent fashion and a group of cellular RA-binding proteins (CRABPs). CRABP can bind intracellular RA (Napoli, 1993, 1996; Ong, 1994) in competition with the RA-binding nuclear receptors. The higher expression of CRABP detected in leiomyomas could limit the amount of RA available for the actions of RAR and/or RXR (Boylan and Gudas, 1991; Bucco et al., 1996; Wardlaw et al., 1997; Wei et al., 1997) resulting in the promotion of cell proliferation. Several prior studies support the importance of CRABP in protecting smooth muscle cells and inducing cell proliferation in the uterus (Bucco et al., 1996; Wardlaw et al., 1997).
TGF-ß has been suggested to be important for leiomyoma growth since it cannot only stimulate the expression of extracellular matrix (ECM) formation, but also inhibits the proteolytic degradation of matrix components of the ECM leading to fibrosis (Lyons and Moses, 1990). In animal models, sex steroids, especially estrogen, have been shown to modulate the expression of TGF-ß (Takahashi et al., 1994). The expression of TGF-ß is higher in leiomyomas than in myometrium (Dou et al., 1996). LTBP can positively regulate the bioactivity of TGF-ß (Mecham and Davis, 1994) by controlling its deposition and by targeting the latent and active form of TGF-ß into ECM and connective tissues (Saharinen et al., 1999). LTBP2 is characterized by the possession of 16–18 EGF-like motifs and eight cysteine residues, a common feature of the LTBP family. In reproductive systems, LTBP2 could have a yet undiscovered function in implantation and early development (Shipley et al., 2000). To the best of our knowledge, this is the first time any member of the LTBP family has been shown to be expressed in myometrium and to be up-regulated in leiomyomas. Although we were unable to observe any tissue difference in expression of the LTBP2 protein, it is still possible that LTBP2 has an important function in myometrium and leiomyomas. LTBP2 interaction with TGF-ß may play a role in both the fibrogenic process and the induction of cell proliferation in these tumours (Arici and Sozen, 2000; Lee and Nowak, 2001).
Tomoregulin is a transmembrane protein containing two follistatin modules and a single epidermal growth factor (EGF)-like domain. Tomoregulin can potentially bind to TGF-ß-related growth factors through the follistatin modules. The EGF-like domain is closely related to that found in the EGF/neuregulin family of growth factors. Tomoregulin is able to promote tyrosine phosphorylation of erbB-4, a receptor tyrosine kinase from the EGF receptor family (Uchida et al., 1999). In agreement with the character of the EGF/neuregulin family, tomoregulin may constitute a potential modulator of extracellular signalling, with putative functions in the regulation of cell growth, differentiation or apoptosis in myometrium. Indeed, EGF and its receptor are believed to play a crucial role in regulating leiomyoma growth, influenced by estrogen and progesterone (Shimomura et al., 1998).
The significance of the over-expression of the LIM domain ZFP 185 in leiomyomas can only be speculated upon due to the scarce functional information regarding this gene. Construction of a transcript map in the DXS52 region in Xq28 has led to the isolation of ZFP 185 cDNA with a LIM zinc finger domain in the carboxyl terminus (Heiss et al., 1997). The family of ZFP with LIM domains has previously been identified as having an important role in transcriptional regulation and cellular differentiation (Schmeichel and Beckerle, 1997). They have been involved in a wide range of cellular functions including transcriptional activation (Karlsson et al., 1990), somatic patterning (Freyd et al., 1990), focal cell adhesion (Sadler et al., 1992) and immediate–early response to serum stimulation (Feuerstein et al., 1994).
The changes in gene expression between myometrium and leiomyoma as reported above are of a small magnitude, but within the range of those reported for other genes, such as IGF-I (Englund et al., 2000), that are believed to be important for leiomyoma growth. Nevertheless, the biological significance of these changes requires further investigation using experimental strategies specific for individual genes. A useful first step would be to determine whether the changes in gene expression could be translated into changes in protein levels and whether these genes are under the influence of sex steroids.
In summary, we have used RDA to identify five genes which could possibly mediate growth-promoting effects in leiomyomas. The experimental strategy used has also highlighted the variability in gene expression among different leiomyoma samples. This should be taken into account in future attempts to obtain global gene expression profiles from this type of tumour.
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Acknowledgements |
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This work was supported by grants from the Swedish Medical Research Council (03972) and the Swedish Cancer Society. We thank Professor C.-H.Heldin for the generous gift of the LTBP2 antibody. We thank Petra Tollet-Egnell for reading the manuscript and for many helpful discussions.
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4 To whom correspondence should be addressed. E-mail: Amilcar.Flores{at}molmed.ki.se
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References |
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Adolfsson, P.I., Dahle, L.O., Berg, G. and Svensson, S.P. (1998) Characterization of alpha2-adrenoceptor subtypes in pregnant human myometrium. Gynecol. Obstet. Invest., 45, 145–150.[ISI][Medline]
Adolfsson, P.I., Haug, I., Berg, G. and Svensson, S.P. (2000) Changes in beta(2)-adrenoceptor expression and in adenylyl cyclase and phosphodiesterase activity in human uterine leiomyomas. Mol. Hum. Reprod., 6, 835–842.
Arici, A. and Sozen, I. (2000) Transforming growth factor-beta 3 is expressed at high levels in leiomyomas where is stimulates fibronectin expression and cell proliferation. Fertil. Steril., 73, 1006–1011.[ISI][Medline]
Benson, S., Padmanabhan, S., Kurtz, T.W. and Pershadsingh, H.A. (2000) Ligands for the peroxisome proliferator-activated receptor-gamma and the retinoid X receptor-alpha exert synergistic antiproliferative effects on human coronary artery smooth muscle cells. Mol. Cell. Biol. Res. Commun., 3, 159–164.[Medline]
Boehm, M.F., Zhang, L., Badea, B.A., White, S.K., Mais, D.E., Berger, E., Suto, C.M., Goldman, M.E. and Heyman, R.A. (1994) Synthesis and structure-activity relationships of novel retinoid X receptor-selective retinoids. J. Med. Chem., 37, 2930–2941.[ISI][Medline]
Boettger-Tong, H.L. and Stancel, G.M. (1995) Retinoic acid inhibits estrogen-induced uterine stromal and myometrial cell proliferation. Endocrinology, 136, 2975–2983.[Abstract]
Boettger-Tong, H., Shipley, G., Hsu, C.J. and Stancel, G.M. (1997) Cultured human uterine smooth muscle cells are retinoid responsive. Proc. Soc. Exp. Biol. Med., 215, 59–65.[Abstract]
Boylan, J. and Gudas, L.J. (1991) Overexpression of the cellular retinoic acid binding protein-1 (CRABP-1) results in a reduction in differentiation-specific gene expression in F9 teratocarcinoma cells. J. Cell. Biol., 112, 965–979.
Boylan, J. and Gudas, L.J. (1992) The level of CRABP I expression influences the amounts and types of all-trans-retinoic acid metabolites in F9 teratocarcinoma stem cells. J. Biol. Chem., 267, 21486–21491.
Bucco, R.A., Zheng, W.L., Wardlaw, S.A., Davis, J.T., Sierra-Rivera, E., Osteen, K.G., Melner, M.H., Kakkad, B.P. and Ong, D.E. (1996) Regulation and localization of cellular retinal-binding protein, retinal-binding protein, cellular retinoic acid-binding protein (CRABP), and CRABP II in the uterus of the pseudopregnant rat. Endocrinology, 137, 3111–3122.[Abstract]
Butzow, R., Alfthan, H., Julkunen, M., Rutanen, E.M., Bohn, H. and Seppala, M. (1986) Human endometrium and menstrual fluid contain placental protein 5 (PP5). Hum. Reprod., 1, 287– 289.
Carlson, K.J., Nichols, D.H. and Schiff, I. (1993) Indications for hysterectomy. N. Engl. J. Med., 328, 856–860.
Carulli, J., Artinger, M., Swain, P.M., Root, C.D., Chee, L., Tulig, C., Guerin, J., Osborne, M., Stein, G., Lian, J. et al. (1998) High throughput analysis of differential gene expression. J. Cell. Biochem., 30–31 (Suppl.), 286–296.
Chen, Z.L., Momota, Y., Kato, K., Taniguchi, M., Inoue, N., Shiosaka, S. and Yoshida, S. (1998) Expression of neuropsin mRNA in the mouse embryo and the pregnant uterus. J. Histochem. Cytochem., 46, 313–320.
Conover, C.A., Oxvig, C., Overgaard, M.T., Christiansen, M. and Giudice, L.C. (1999) Evidence that the insulin-like growth factor binding protein-4 protease in human ovarian follicular fluid is pregnancy associated plasma protein-A. J. Clin. Endocrinol. Metab., 84, 4742–4745.
Dou, Q., Zhao, Y., Tarnuzzer, R.W., Rong, H., Williams, R.S., Schultz, G.S. and Chegini, N. (1996) Suppression of transforming growth factor-beta (TGF beta) and TGF beta receptor messenger ribonucleic acid and protein expression in leiomyomata in women receiving gonadotropin-releasing hormone agonist therapy. J. Clin. Endocrinol. Metab., 81, 3222–3230.[Abstract]
Englund, K., Blanck, A., Gustavsson, I., Lundkvist, U., Sjoblom, P., Norgren, A. and Lindblom, B. (1998) Sex steroid receptors in human myometrium and fibroids: changes during the menstrual cycle and gonadotropin-releasing hormone treatment. J. Clin. Endocrinol. Metab., 83, 4092–4096.
Englund, K., Lindblom, B., Carlström, K., Gustavsson, I., Sjöblom, P. and Blanck, A. (2000) Gene expression and tissue concentrations of IGF-I in human myometrium and fibroids under different hormonal conditions. Mol. Hum. Reprod., 6, 915–920.
Fenaux, P., Chevret, S., Guerci, A., Fegueux, N., Dombret, H., Thomas, X., Sanz, M., Link, H., Maloisel, F., Gardin, C. et al. (2000) Long-term follow-up confirms the benefit of all-trans-retinoic acid in acute promyelocytic leukaemia. European APL group. Leukemia, 14, 1371–1377.[ISI][Medline]
Feuerstein, R., Wang, X., Song, D., Cooke, N.E. and Liebhaber, S.A. (1994) The LIM/double zinc-finger motif functions as a protein dimerization domain. Proc. Natl Acad. USA., 91, 10655–10659.
Freyd, G., Kim, S.K. and Horvitz, H.R. (1990) Novel cysteine-rich motif and homeodomain in the product of the Caenorhabditis elegans cell lineage gene line-11. Nature, 344, 876–879.[Medline]
Gamage, S.D., Bischoff, E.D., Burroughs, K.D., Lamph, W.W., Gottardis, M.M., Walker, C.L. and Fuchs-Young, R. (2000) Efficacy of LGD1069 (Targretin), a retinoid X receptor-selective ligand, for treatment of uterine leiomyoma. J. Pharmacol. Exp. Ther., 295, 677–681.
Giometti, C.S. and Anderson, N.L. (1984) Tropomyosin heterogeneity in human cells. J. Biol. Chem., 259, 14113–14120.
Giudice, L.C., Irwin, J.C., Dsupin, B.A., Pannier, E.M., Jin, I.H., Vu, T.H. and Hoffman, A.R. (1993) Insulin-like growth factor (IGF), IGF binding protein (IGFBP), and IGF receptor gene expression and IGFBP synthesis in human uterine leiomyomas. Hum. Reprod., 8, 1796–1806.
Han, C.S., Mathialagan, N., Klemann, S.W. and Roberts, R.M. (1997) Molecular cloning of ovine and bovine type I interferon receptor subunits from uteri, and endometrial expression of messenger ribonucleic acid for ovine receptors during the estrous cycle and pregnancy. Endocrinology, 138, 4757–4767.
Heiss, N.S., Gloeckner, G., Bachner, D., Kioschis, P., Klauck, S.M., Hinzmann, B., Rosenthal, A., Herman, G.E. and Poustka, A. (1997) Genomic structure of a novel LIM domain gene (ZNF185) in Xq28 and comparisons with the orthologous murine transcript. Genomics, 43, 329–338.[ISI][Medline]
Howe, S.R., Pass, H.I., Ethier, S.P., Matthews, W.J. and Walker, C. (1996) Presence of an insulin-like growth factor I autocrine loop predicts uterine fibroid responsiveness to tamoxifen. Cancer. Res., 56, 4049–4055.
Jeffrey, J.J., Ehlich, L.S. and Roswit, W.T. (1991) Serotonin: an inducer of collagenase in myometrial smooth cells. J. Cell. Physiol., 146, 399–406.[ISI][Medline]
Jetten, A.M., Jetten, M.E.R., Shapiro, S.S. and Poon, J.P. (1979) Characterization of the action of retinoids on mouse fibroblast cell lines. Exp. Cell Res., 119, 289–299.[ISI][Medline]
Karlsson, O., Thor, S., Norberg, T., Ohlsson, H. and Edlund, T. (1990) Insulin gene enhancer binding protein Isl-1 is a member of a novel class of proteins containing both a homeo- and a Cys-His domain. Nature, 344, 879–882.[Medline]
Lawrence, J.B., Oxvig, C., Overgaard, M.T., Sottrup-Jensen, L., Gleich, G.J., Hays, L.G., Yates, J.R. 3rd and Conover, C.A. (1999) The insulin-like growth factor (IGF)-dependent IGF binding protein-4 protease secreted by human fibroblasts is pregnancy-associated plasma protein-A. Proc. Natl Acad. Sci. USA., 96, 3149–3153.
Lee, B.S. and Nowak, R.A. (2001) Human leiomyoma smooth muscle cells show increased expression of transforming growth factor-beta 3 (TGF beta 3) and altered response to the antiproliferative effcts of TGF beta. J. Clin. Endocrinol. Metab., 86, 913–920.
Ligon, A.H. and Morton, C.C. (2001) Leiomyomata: heritability and cytogenetic studies. Hum. Reprod. Update., 7, 8–14.
Lin, T.M., Galbert, S.P., Kiefer, D., Spellacy, W.N. and Gall, S. (1974) Characterization of four human pregnancy-associated plasma proteins. Am. J. Obstet. Gynecol., 118, 223–236.[ISI][Medline]
Lyons, R.M. and Moses, H.L. (1990) Transforming growth factors and the regulation of cell proliferation. Eur. J. Biochem., 187, 467–473.[ISI][Medline]
Mazerbourg, S., Zapf, J., Bar, R.S., Brigstock, D.R. and Monget, P. (2000) Insulin-like growth factor (IGF)-binding protein-4 proteolytic degradation in bovine, equine, and porcine preovulatory follicles: regulation by IGFs and heparin-binding domain-containing peptides. Biol. Reprod., 63, 390–400.
Mecham, R.P. and Davis, E.C. (1994) Extracellular Matrix Assembly and Structure. Academic Press Inc., San Diego, pp. 281–314.
Michel, K., Roth, S., Trautwein, C., Gong, W., Flemming, P. and Gressner, A.M. (1998) Analysis of the expression pattern of the latent transforming growth factor beta binding protein isoforms in normal and diseased human liver reveals a new splice variant missing the proteinase-sensitive hinge region. Hepatology, 27, 1592–1599.[ISI][Medline]
Moren, A., Olofsson, A., Stenman, G., Sahlin, P., Kanzaki, T., Claesson-Welsh, L., Dijke, P., Miyazono, K. and Heldin, C.H. (1994) Identification and characterization of LTBP-2, a novel latent transforming growth factor-beta-binding protein. J. Biol. Chem., 269, 32469–32478.
Napoli, J.L. (1993) Biosynthesis and metabolism of retinoic acid: roles of CRBP and CRABP in retinoic acid homeostasis. J. Nutr., 123, 362–366.
Napoli, J.L. (1996) Biochemical pathways of retinoid transport, metabolism, and signal transduction. Clin. Immunol. Immunopathol., 80, S52–S62.[ISI][Medline]
Nilbert, M. and Heim, S. (1990) Utereine leiomyomas cytogenetics. Genet. Chromosome Cancer, 2, 3–13.
Odeberg, J., Wood, T., Blucher, A., Rafter, J., Norstedt, G. and Lundeberg, J. (2000) A cDNA RDA protocol based on solid-phase technology suited for analysis of gene expression in small tissue samples. Biomol. Eng., 17, 1–9.[ISI][Medline]
Ong, D.E. (1994) Cellular transport and metabolism of vitamin A: roles of the cellular retinoid-binding proteins. Nutr. Rev., 52, S24–S31.[ISI][Medline]
Overgaard, M.T., Oxvig, C., Christiansen, M., Lawrence, J.B., Conover, C.A., Gleich, G.J., Sottrup-Jensen, L. and Haaning, J. (1999) Messenger ribonucleic acid levels of pregnancy-associated plasma protein-A and the proform of eosinophil major basic protein: expression in human reproductive and nonreproductive tissues. Biol. Reprod., 61, 1083–1089.
Pandis, N., Heim, S., Bardi, G., Floderus, U.M., Willen, H., Mandahl, N. and Mitelman, F. (1991) Chromosome analysis of 96 uterine leiomyomas. Cancer Genet. Cytogenet., 55, 11–18.[ISI][Medline]
Pollard, J.W., Pacey, J., Cheng, S.V. and Jordan, E.G. (1987) Estrogens and cell death in murine uterine luminal epithelium. Cell. Tissue. Res., 249, 533–540.[ISI][Medline]
Recchia, F., Sica, G., De, Filippis, S., Rosselli, M., Saggio, G., Guerriero, G.Pompili, P. and Rea, S. (2000) Cisplatin, vindesine, mitomycin-C and 13-cis retinoic acid in the treatment of advanced non small cell lung cancer. A phase II pilot study. Anticancer Res., 20, 1985–1990.[ISI][Medline]
Rein, M.S. (2000) Advances in uterine leiomyoma research: the progesterone hypothesis. Environ. Health. Perspect., 108 (Suppl. 5), 791–793.
Rush D.S., Tan J., Baergen R.N. and Soslow R.A. (2001) h-Caldesmon, a novel smooth muscle-specific antibody, distinguishes between cellular leiomyoma and endometrial stromal sarcoma. Am. J. Surg. Pathol., 25, 253–258.[ISI][Medline]
Sadler, I., Crawford, A.W., Michelsen, J.W. and Beckerle, M.C. (1992) Zyxin and cCRP: two interactive LIM domain proteins associated with the cytoskeleton. J. Cell. Biol., 119, 1573–1587.
Saharinen, J., Hyytiainen, M., Taipale, J. and Keski-Oja, J. (1999) Latent transforming growth factor-beta binding proteins (LTBPs)—structural extracellular matrix proteins for targeting TGF-beta action. Cytokine Growth Factor Rev., 10, 99–107.[ISI][Medline]
Schmeichel, K.L. and Beckerle, M.C. (1997) Molecular dissection of a LIM domain. Mol. Biol. Cell, 8, 219–230.[Abstract]
Shimomura, Y., Matsuo, H., Samoto, T. and Maruo, T. (1998) Up-regulation by progesterone of proliferating cell nuclear antigen and epidermal growth factor expression in human uterine leiomyoma. J. Clin. Endocrinol. Metab., 83, 2192–2198.
Shipley, J.M., Mecham, R.P., Maus, E., Bonadio, J., Rosenbloom, J., McCarthy, R.T., Baumann, M.L., Frankfater, C., Segade, F. and Shapiro, S.D. (2000) Developmental expression of latent transforming growth factor beta binding protein 2 and its requirement early in mouse development. Mol. Cell. Biol., 20, 4879–4887.
Stewart E.A., Friedman A.J., Peck K. and Nowak R.A. (1994) Relative overexpression of collagen type I and collagen type III messenger ribonucleic acids by uterine leiomyomas during the proliferative phase of the menstrual cycle. J. Clin. Endocrinol. Metab., 79, 900–906.[Abstract]
Takahashi, T., Eitzman, B., Bossert, N.L., Walmer, D., Sparrow, K., Flanders, K.C., McLachlan, J. and Nelson, K.G. (1994) Transforming growth factors ß1, ß2, and ß3 messenger RNA and protein expression in mouse uterus and vagina during estrogen-induced growth: a comparison to other estrogen-regulated genes. Cell. Growth Differ., 5, 919–935.[Abstract]
Terpe, H.J., Stark, H., Ruiz, P. and, Imhof, B.A. (1994) Alpha 6 integrin distribution in human embryonic and adult tissues. Histochemistry, 101, 41–49.[ISI][Medline]
Tollet-Egnell, P., Flores-Morales, A., Odeberg, J., Lundeberg, J. and Norstedt, G. (2000) Differential cloning of growth hormone-regulated hepatic transcripts in the aged rat. Endocrinology, 141, 910–921.
Tsibris, J.C., Porter, K.B., Jazayeri, A., Tzimas, G., Nau, H., Huang, H., Kuparadze, K. Porter, G.W., O'Brien, W.F. and Spellacy, W.N. (1999) Human uterine leiomyomata express higher levels of peroxisome proliferator-activated receptor gamma, retinoid X receptor alpha, and all-trans retinoic acid than myometrium. Cancer Res., 59, 5737–5744.
Uchida, T., Wada, K., Akamatsu, T., Yonezawa, M., Noguchi, H., Mizoguchi, A., Kasuga, M. and Sakamoto, C. (1999) A novel epidermal growth factor-like molecule containing two follistatin modules stimulates tyrosine phosphorylation of erbB-4 in MKN28 gastric cancer cells. Biochem. Biophys. Res. Commun., 266, 593–602.[ISI][Medline]
Verneaux, V., Jouvenot, M., Compagnone, P., Propper, A.Y. and Adessi, G.L. (1992) Expression of c-fos and c-myc genes in uterus and nonreproductive organs during the implantation period in the rat. Biochem. Cell Biol., 70, 142–148.[ISI][Medline]
Wardlaw, S.A., Bucco, R.A., Zheng, W.L. and Ong, D.E. (1997) Variable expression of cellular retinal- and cellular retinoic acid-binding proteins in the rat uterus and ovary during the estrous cycle. Biol. Reprod., 56, 125–132.[Abstract]
Wei L.N., Lee C.H., Filipcik P. and Chang L. (1997) Regulation of the mouse cellular retinoic acid-binding protein-I gene by thyroid hormone and retinoids in transgenic mouse embryos and P19 cells. J. Endocrinol., 155, 35–46.[Abstract]
Welford, S.M., Gregg, J., Chen, E., Garrison, D., Sorensen, P.H., Denny, C.T. and Nelson, S.F. (1998) Detection of differentially expressed genes in primary tumor tissues using representational differences analysis coupled to microarray hybridization. Nucleic Acids Res., 26, 3059–3065.
Yousef, G.M., Obiezu, C.V., Luo, L.Y., Black, M.H. and Diamandis, E.P. (1999) Prostase/KLK-L1 is a new member of the human kallikrein gene family, is expressed in prostate and breast tissues, and is hormonally regulated. Cancer Res., 59, 4252–4256.
Submitted on June 19, 2001; resubmitted on September 21, 2001; accepted on November 30, 2001.
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