Mol. Hum. Reprod. Advance Access originally published online on July 8, 2004
Molecular Human Reproduction 2004 10(9):685-695; doi:10.1093/molehr/gah086
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Immortalization and characterization of human myometrial cells from term-pregnant patients using a telomerase expression vector
1Department of Obstetrics and Gynecology, 2Sealy Center for Molecular Science and 3Department of Pediatrics, University of Texas Medical Branch, Galveston, TX 77555-1062, USA
4 To whom correspondence should be addressed. Email: msoloff{at}utmb.edu
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Abstract |
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An examination of cellular processes involved in myometrial function has been greatly assisted by the use of human myometrial cells in primary culture. However, these cells can be used only for several passages before they senesce, and responses to various agents change with time in culture. The use of transformed cells is limited, as they can be polynucleated and can lose or gain chromosomes. We have developed three telomerase-immortalized cell lines from term-pregnant human myometrium to eliminate variability between passage numbers and allow genetic manipulations of myometrial cells to fully characterize signal pathways. These cells have a normal karyotype and were verified to be uterine smooth muscle by immunocytochemical staining for smooth muscle cell-specific
-actin and high affinity oxytocin antagonist binding sites. The three cell lines and the cells in primary culture from which they were derived were examined by cDNA microarray analysis. Of >10 000 expressed genes, there were consistent changes in the expression of 
1% in the three immortalized cell lines. We were unable to detect any significant differences between primary and immortalized cells in signal pathways such as epidermal growth factor-stimulated epidermal growth factor receptor phosphorylation, insulin-stimulated Akt phosphorylation, oxytocin and lysophosphatidic acid-stimulated extracellular signal-regulated kinase 1 and 2 phosphorylation, myosin light chain phosphorylation, and interleukin-1 induction of I
B degradation. The immortalized cells should be useful for a range of studies, including high throughput analyses of the effects of environmental agents on the human myometrium. Key words: human myometrial cells/immortalization/oxytocin/signal pathways/telomerase
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Introduction |
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The myometrium is a dynamic structure: uterine smooth muscle cells undergo proliferation and hypertrophy during pregnancy, resulting in an
20-fold increase in weight (Nilsson et al., 1998
). As part of this process, the myometrium must stretch to accommodate the growing fetal–placental unit. During most of pregnancy, the myometrium is maintained in a relatively quiescent state, but becomes highly contractile at the end of gestation resulting in the initiation of labour. Concomitantly, there is an accelerated disruption in collagen fibrils in the extracellular matrix of the uterine cervix that contributes to a marked increase in cervical distensibility. Smooth muscle cells of the cervix and lower segment of the myometrium are thought to be involved in producing proinflammatory cytokines, chemokines, and matrix metalloproteinases that participate in the collagenolytic process (Watari et al., 2000). Following parturition, the uterus undergoes extensive remodelling and reduction in size to that of pre-pregnancy levels. In the non-pregnant uterus, leiomyomas develop clonally as masses of smooth muscle-like tissue embedded in the myometrium. These benign growths are probably the most common tumours among humans, occurring in as many as 30% of women aged >30 years and are the most frequent indication for hysterectomy.
Relatively little is known regarding factors that regulate these various myometrial processes in humans because of the lack of ready availability of biological samples at different gestational stages for study. Over the past 20 years, a number of studies have been performed on human myometrial cells, mainly in primary culture, to characterize myometrial functions (Table I). Although these cells are highly differentiated, they have the ability to proliferate under standard cell culture conditions. Unfortunately, myometrial cells in primary culture are useful for fewer than ten passages before they senesce. Several laboratories have immortalized human myometrial cell lines by viral-based transformation (Matsuo et al., 1989; Sourla et al., 1994; Monga et al., 1996). However, a detailed analysis of phenotype of these cells, as compared to cells in primary culture, has not been carried out. While transformed cells, in general, have been useful for some purposes, many lines are modified by alterations, such as anchorage independence, aneuploidy, a high level expression of certain proteins, and the loss of the p53-mediated cell-cycle DNA repair checkpoint. Because of these cell modifications, experimental findings using these cells might not accurately reflect normal cell characteristics.
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More recently, several laboratories have shown that several primary cell types, immortalized by stable transfection with an expression vector for the catalytic subunit of human telomerase (hTERT), maintain normal karyotype and phenotype (Greider, 1996
; Bodnar et al., 1998; Vaziri and Benchimol, 1998; Wang et al., 1998). Telomeres are specialized DNA repeats at the ends of chromosomes that gradually become shorter with succeeding cell division cycles (Blackburn, 1991). At some critical reduced telomere length, the cells stop dividing (Harley et al., 1990). Unlike mature normal cells, embryonic stem cells, germline cells and cancer cells express telomerase, a reverse transcriptase that restores telomere length after each cell division. Several telomerase-immortalized cell lines offered commercially (Infinity; BD Biosciences Clontech, USA) have been reported by the vendor to maintain a normal phenotype and are genetically stable. In the present studies, we have telomerase-immortalized three myometrial cell lines derived from myometrial cells in primary culture prepared from pieces of lower uterine segment from three separate patients undergoing Caesarean section near the end of gestation. We have compared the phenotypes of these cells with those of the cells in primary culture from which they were derived, using cDNA microarray analysis, and examination of several distinct signal pathways to show that the cell lines maintain essentially all of the characteristics of the cells in primary culture. Selected signal pathways chosen for examination include those involved in stimulation of growth [epidermal growth factor (EGF), insulin, lysophosphatidic acid], contractile activity (oxytocin, lysophosphatidic acid), and proinflamatory responses (interleukin-1). The availability of immortalized myometrial cells that share the properties of cells in primary culture will allow an examination of cellular process under conditions where the properties of the cells do not change with successive passages, and the framework to produce stable transfectants to elucidate physiological and pathophysiological process in the myometrium in future studies.
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Materials and methods |
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Reagents
Antibodies used for immunoblotting and their sources are as follows: anti-Akt, anti-phospho-Akt (Ser 473), and anti-phospho-p44/42 MAP kinase (Thr 202/Tyr 204) (Cell Signaling Technology, USA), anti-phosphotyrosine and anti-EGF receptor (Upstate, USA), anti-p42 (Santa Cruz Biotechnology, USA), and monoclonal antibody to myosin light chain 20 (Sigma, USA). Rabbit antibody to phosphorylated MLC20 was obtained from Dr James M.Staddon, Eisai London Research Laboratories, University College, London, UK. Oxytocin and oxytocin antagonist [d(CH2)5, Tyr(Me)2, Thr4, Tyr-NH29]ornithine vasotocin were obtained from Peninsula Laboratories, Inc. (USA). Fetal bovine serum (FBS) was procured from Atlanta Biological (USA); MEM and cell culture reagents came from Life Technologies, Inc. (USA). Forskolin and other chemicals were obtained from Sigma (USA).
hTERT retrovirus
hTERT retrovirus vector pLPC-hTERT was obtained from Geron Corp. (USA). This Moloney murine leukaemia virus-based retroviral vector expresses the catalytic subunit of human telomerase from the CMV promoter and contains the puromycin selection marker. The plasmid DNA was amplified in bacteria, and purified DNA was transfected into AmphoPack-293 cells (Clontech), which provide the gag, pol and env genes necessary for particle formation and replication. Retroviral particles cannot replicate within target cells because they lack these viral genes. Several dilutions of medium containing retroviral particles were used to infect myometrial cells. Myometrial cells in primary culture were prepared as described earlier (Park et al., 2002), using myometrial samples taken from women by elective Caesarean section (not in labour) for non-medical reasons near term, as approved by The University of Texas Medical Branch Committee on Research Involving Human Subjects. Early passage cells (second or third) were seeded at 50–70% confluence in 6-well culture dishes and infected with serially diluted virus to cover a range of multiplicities of infection. After several days in culture, the cells were treated with puromycin (200 ng/ml). Cell lines were generated by clonal selection.
Validation of hTERT expression
Expression of telomerase activity in the immortalized cells was verified using the TRAPeze Telomerase Detection kit provided by Serologicals Corp. (USA). This is a highly sensitive in vitro assay in which active telomerase present in a cell lysate adds an increasing number of telomeric repeats (5'-TTAGGG-3') onto the 3' end of a substrate oligonucleotide. The extension products were then amplified by PCR and visualized using 5% polyacrylamide electrophoresis and ethidium bromide staining. Human myometrial cells in primary culture, lacking telomerase activity, were used as negative controls, as were heat-denatured extracts. A tumour cell extract supplied in the kit was used as a positive control.
Cytogenetic analysis
Cytogenetic studies were carried out using standard procedures (Priest, 1997). Once a sufficient number of mitotic cells were observed, the cell lines were treated with colcemid (10 µg/ml) (Roche, USA) to arrest the dividing cells at metaphase. The cells were lifted using trypsin–EDTA (0.25% solution) (Irvine Scientific, USA) and the cell suspension was transferred to a centrifuge tube. After hypotonic treatment with 0.075 mol/l KCl solution (Sigma) and fixation in 3:1 methanol:glacial acetic acid (Sigma), the harvested cell suspension was applied by drop onto pre-cleaned, chilled glass slides. The slides were aged by baking at 80°C for 30 min. The chromosomes were G-banded using pancreatin (0.05%) (Sigma) for 3 min, followed by Giemsa staining (Biomedical Specialty Laboratories, USA) for 1.5 min (Pearson, 1972). For each karyotypic analysis, a minimum of eight metaphases were analysed.
cDNA array analysis
The Molecular Genomics Core at UTMB carried out gene chip analysis. Cells were seeded at 5 x 106 per 10 cm dish, and RNA was extracted using the Ambion (USA) RNAqueous kit. RNA (25 µg) from each of three cell lines and the corresponding primary cells from which they were derived were reverse-transcribed using an oligo dT primer encoding a bacteriophage T7 RNA polymerase promoter. Following second strand synthesis, the double-stranded DNA was used as a template for in vitro transcription using T7 RNA polymerase and biotin-tagged nucleoside triphosphates. The resulting biotin-labelled cRNA was used for hybridization to gene probes using Affymetrix HG-U133 A gene chips. The A chip contains >22 000 of the best-characterized human genes. Before hybridization, the biotin-labelled RNA were fragmented to a mean size of 200 bases to facilitate interaction with the probe sequences. Control RNA from four prokaryotic genes were added to the hybridization mix as internal controls. These controls were used to normalize expression levels between experiments and to estimate relative abundance of RNA transcripts in the sample. After hybridization, arrays were rinsed using successive non-stringent (1 mol/l NaCl, 24°C) and stringent (1 mol/l NaCl, 50°C) conditions prior to analysis. Detection of expressed genes was performed using a phycoerythrin streptavidin stain and analysed using the Affymetrix Gene Chip Analysis software (Suite 5.0).
Immunocytochemistry
Cells were grown to confluence in chamber slides (Fisher Scientific, USA), and fixed in absolute methanol. Endogenous peroxidase activity was inactivated with 0.1% H2O2. Non-specific staining was blocked with normal goat serum (Sigma) diluted 1:60 in 0.01 mol/l phosphate-buffered saline (PBS). To detect smooth muscle -actin, cells were incubated for 1 h with a monoclonal mouse antibody (AM128M-5, 1:10 dilution; BioGenex, USA) at room temperature. The complex was detected using supersensitive multilink-HRP/DAB kit from BioGenex (QD000-5L) following the vendor's procedure. Cells were counterstained for 1 min using diluted Mayer's haematoxylin solution (Sigma).
Iodinated oxytocin antagonist binding
Whole cell assays for oxytocin receptor binding were performed on confluent serum-starved (24 h) cells subsequently untreated (basal), or treated with either 5% FBS or 5% FBS containing 20 µmol/l forskolin for 16 h. Briefly, cells were rinsed twice in PBS and then incubated in 0.5 ml PBS containing a saturating (or near-saturating) concentration of [125I]oxytocin antagonist (OTA) (Hinko and Soloff, 1992) at room temperature for 1 h. Non-specific binding was determined by adding unlabelled oxytoxin (OT) (1 µmol/l) in combination with [125I]OTA. Cells were then rinsed three times with PBS and solubilized with 0.5 ml 1 N NaOH. Radioactivity in the extracts was determined using a gamma counter. The amount of radioactive OTA bound specifically (difference in cpm between total and non-specific [1 µmol/l oxytocin] binding) per well was determined, and the results are expressed as specific binding relative to basal levels (serum-free).
Immunoblotting
Immunoblotting procedures were carried out using methods previously described (Strakova et al., 1998), except that incubations with primary antibodies were carried out for 2 h. Cells were plated at a density of 1 x 106 per 60 mm dish. After 1 day in culture, the cells were serum-starved overnight and treated with various agents. For analysis of myosin light chain phosphorylation, the cells were lysed in situ by the addition of boiling Laemmli (1970) sample preparation solution. Immunoblots were stripped in Tris–HCl buffer (62.5 mmol/l, pH 8.0) containing 2% sodium dodecyl sulphate and 100 mmol/l 2-mercaptoethanol for 10 min at 65°C, rinsed, and then reprobed with antibodies to non-phosphorylated proteins.
DNA determination
DNA was quantified using the intercalating dye Hoecht 33258 and a fluorometric assay (DYNAquant 200; Hoffer Pharmacia Biotech Inc., USA).
Statistical methods
Assays were carried out in triplicate, and the results are expressed as mean±SEM. Matched sets of primary and immortalized cells from at least two different patients, usually three, were used. A t-test was used to compare treated groups with control groups. All tests were made at the P<0.05 level of statistical significance. In instances where immunoblots are shown, each is representative of the results from at least two primary/immortalized cell sets from different patients.
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Results |
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Eleven cell lines from eight patients having the overall morphology of smooth muscle cells and showing bundles of smooth muscle actin, as determined immunocytochemically, were resistant by puromycin selection. The first three cell lines characterized, arising from three different patients, were selected for further characterization. These cells were passaged over >100 doublings to ensure that they were immortalized. The three immortalized cell lines (passages 40 to 50) were compared with the primary cells (passages 3 to 5) from which they were derived, and which had been stored under liquid nitrogen until used.
Expression of telomerase in hTERT adenovirus-infected, antibiotic-selected human myometrial cells
The telomeric repeat amplification protocol (TRAP) assay was used to examine telomerase activity in primary cultures of myometrial cells and hTERT retrovirus-infected human myometrial cells. The non-infected cells did not express telomerase activity, while the infected cell lines did (Figure 1). A cancer cell extract provided in the kit was used as an assay positive control (Figure 1). Heating the C and I lysates to 65°C for 10 min resulted in a loss of telomerase activity.
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Doubling time and karyotype of TERT-immortalized myometrial cells
Cells were seeded (10 000 per well) and harvested every 24 h for DNA quantification.
There was no significant difference in doubling time between two immortalized cell lines and the cells from which they were derived. The immortalized and primary cell doubling times for patients 3 and 6 were 2.01±0.6 (mean±SE, n=3) and 2.33±0.12, 2.36±0.2 and 2.42±0.06 days respectively.
Karyotypic analysis was carried out on cells in primary culture, and on two immortalized cells lines, which were examined both after several doublings and after >100 doublings. Analysis of myometrial cells in primary culture from patient 3 showed a normal 46,XX karyotype in all 10 cells examined. Analysis of immortalized cells from lines 6 (passage 6) and 3 (passage 20) also showed normal 46,XX karyotypes in all of the eight metaphase chromosomes examined from each cell line. A later karyotypic analysis of lines 3 (passage 29) and 6 (passage 32) demonstrated normal 46,XX karyotypes in at least eight of the 10 cells analysed from each cell line. In line 3, two of the 10 cells analysed had a tetraploid karyotype.
Verification of the smooth muscle character of immortalized myometrial cells
The myometrium is made up of several cell types, and initial cultures might be contaminated with decidual tissue (endometrium). However, the identity of smooth muscle cells in culture was apparent from their typical fusiform shape. To ensure that the cells were of smooth muscle origin, we examined the expression of smooth muscle specific -actin by immunocytochemistry. Both primary and immortalized cells were stained for -actin (Figure 2). While it is true that other smooth muscle cell types, including vascular smooth muscle, also express -actin, uterine cells also expressed relatively high levels of oxytocin receptors, which was apparent from the specific binding of iodinated oxytocin antagonist (Figure 3). Oxytocin antagonist binding was determined by incubating cells with a near-saturating concentration of tracer in the presence and absence of 1 µmol/l oxytocin to measure specific binding. All the primary and immortalized cells showed the presence of high affinity, low capacity specific binding sites for the ligand in the presence of 5% FBS, confirming their identity as myometrial cells (Figure 3). The number of binding sites was markedly reduced after incubation of the cells overnight in medium lacking FBS, or upon addition of forskolin, 20 µmol/l, to cells containing FBS (Figure 3). The relative effects of FBS and forskolin were essentially the same on primary and the corresponding immortalized cell counterpart (Figure 3).
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Comparison of primary and immortalized human myometrial cells by cDNA array analysis
We compared the gene expression of three sets of immortalized human myometrial cells with the matching cells in primary culture from which the immortalized cells were derived. Each set of cells was established from a different patient's myometrium in late gestation. Of a total of 22 263 probe sets examined, an average of 10 273 (range 9948–10 768 for the six samples) were expressed. Of these, 32 were expressed at a higher level (
2-fold), and 71 were expressed at a lower level (at least one-half less) in the immortalized cells. The five mRNA with the highest level of relative expression in all three immortalized cell lines were forehead box E1 (thyroid transcription factor), early growth response 4 (EGR4), solute carrier family 16 (monocarboxylic acid transporter), oxytocin receptor, and cyclin D2. Five mRNA with the greatest reduction in expression by the three immortalized cell lines were hypothetical protein FLJ21212, pgH3 proteoglycan, potassium voltage-gated channel G1, alpha 1 collagen type XV, and prostacyclin synthase. Estrogen receptor mRNA was detectable in primary cells from all three patients, but was absent in immortalized cells. Several other genes involved in glycoprotein expression and prostaglandin production were also down-regulated. Of the 32 genes that were up-regulated, only six were undetectable in the primary cells, but increased to detectable levels in the immortalized cells. Of the 71 genes that were down-regulated in the three immortalized lines, 37 were down-regulated to non-detectable levels. The changes in the expression of the 103 genes out of a possible 10 273 probe sets in the three immortalized cell lines represent 1% of the expressed genes. If one considers that the Affymetrix chip variance is 0.5%, the changes in gene expression in the immortalized cells is relatively small. In support of this finding, we were unable to detect any differences in signalling related to EGF, insulin, oxytocin, lysophosphatidic acid, or interleukin-1 (see ensuing paragraphs). These findings indicate that there are no substantive changes in gene expression in myometrial cells upon telomerase immortalization.
Effect of EGF on phosphorylation of the EGF receptor
Occupancy of epidermal growth factor receptor (EGFR) with EGF or other ligands results in its dimerization and autophosphorylation of specific tyrosine residues in the receptor intracellular domain. Treatment of human myometrial cell in primary culture or telomerase-immortalized with 50 nmol/l EGF for 5 min resulted in the phosphorylation of the EGFR, as shown by immunoblotting using an antibody specific for phosphotyrosines (Figure 4). The identity of the stained band was indicated by reprobing the polyvinyl membrane with antibody to EGFR (Figure 4).
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Insulin stimulation of Akt (protein kinase B) phosphorylation
Protein kinase B (PKB), the cellular homologue of v-Akt, has a catalytic domain that is similar to that of cAMP-dependent protein kinase (PKA) and protein kinase C (PKC). Isoforms of Akt are phosphorylated by serine/threonine kinases in response to insulin or growth factors by a phosphatidylinositol 3-kinase-dependent process (Alessi and Cohen, 1998
). Treatment of both primary and immortalized myometrial cells with insulin, 50 nmol/l, resulted in the rapid (5 min) phosphorylation of Akt (Figure 5). Akt was also phosphorylated by addition of 1% fetal bovine serum (Figure 5). As a negative control, there was no change in Akt phosphorylation after oxytocin (100 nmol/l) treatment for 5 min. Pretreatment of the cells with the phosphatidylinositol 3-kinase inhibitor wortmannin, 10 µmol/l for 30 min, completely blocked Akt phosphorylation, as has been shown in many insulin target cells. The control group received vehicle (dimethylsulphoxide) alone.
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Oxytocin and lysophosphatidic acid stimulation of extracellular signal-regulated kinase (ERK) phosphorylation
Treatment of cells with either oxytocin (100 nmol/l) or LPA (10 µmol/l) resulted in the phosphorylation of ERK1 and 2 in both cell types (Figure 6). The temporal pattern of phosphorylation was different between the two treatments. Oxytocin caused a transient increase in ERK1/2 phosphorylation, peaking at
5 min (Figure 6). In contrast, ERK1/2 phosphorylation stimulated by LPA was continuous, reaching a plateau at 5 min and beyond (Figure 6). Virtually identical results were seen with both cell types.
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Stimulation of myosin light chain phosphorylation
Lysophosphatidic acid stimulates actomyosin contraction by increasing phosphorylation of myosin light chains (MLC) by a Rho/Rho-associated kinase signalling in a variety of cell types (Chrzanowska-Wodnicka and Burridge, 1996
; Manning et al., 1998; Retzer and Essler, 2000; Yanase et al., 2000). These actions of lysophospholipids on MLC phosphorylation appear to be the result of inhibition on myosin light chain phosphatase (Essler et al., 2002; Schmidt et al., 2002). Oxytocin also stimulates myosin light chain phosphorylation; but through the activation of calcium–calmodulin-mediated myosin light chain kinase activity (MacKenzie et al., 1990). Treatment of human myometrial cells, either in primary culture or immortalized, with either LPA (10 µmol/l) or oxytocin (100 nmol/l), resulted in increased myosin light chain phosphorylation (Figure 7). These results show that both the kinase and inhibitory phosphatase pathways are equally operational in primary and immortalized cells.
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Treatment of myometrial cells with interleukin-1 results in the reduction in I
B levelsTreatment of transformed human myometrial cells (Belt et al., 1999
) or myometrial cells in primary culture (Soloff et al., 2004) with interleukin-1 activates NF-B association with cognate sites on the prostaglandin endoperoxide synthase-2 (COX-2) gene. Interleukin-1 activates the IB kinase complex, which phosphorylates IB (inhibitor of B) resulting in the targeting of phospho-IB for ubiquitination and degradation through the 26S proteasomal pathway (Liou and Baltimore, 1993). In this manner, NF-B, a transcription factor, is released from being bound to IB and is translocated to the nucleus where it interacts with promoter elements. These events are apparent by the reduction in IB concentration in myometrial cell lysates at 15 and 30 min after IL-1 (500 ng/ml) treatment (Figure 8). Treatment with the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), also causes degradation of IB in some cell types (Molitor et al., 1990; Schmitz et al., 1995). However, unlike IL-1, PMA was ineffective in stimulating a reduction in IB concentration in uterine myocytes (Figure 8). Virtually identical results were obtained with cells in primary culture and those that were immortalized (Figure 8).
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Discussion |
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As shown by a number of criteria, the hTERT-immortalized cells exhibit a phenotype that is essentially indistinguishable from that of cells in primary culture. In addition to signal pathways found in many cell types, such as those associated with EGF and insulin action, these cells also exhibit properties characteristic of highly differentiated smooth muscle cells. Thus, they respond to oxytocin, which has a far more limited number of targets, including uterine smooth muscle. The karyotype analysis is a further assurance that these cells are truly representative of normal myometrial cells.
Myometrial cells in primary culture from a non-pregnant woman have also been immortalized by infection with hTERT (Carney et al., 2002; Condon et al., 2002). A comparison of the phenotype of the non-pregnant cells with the pregnant hTERT-immortalized cells in future studies might elucidate important phenotypic changes in myometrial cells that occur during pregnancy.
Pregnant women are a population at particular risk with respect to environmental impact. Exposure to certain agents can result in spontaneous abortion as well as birth defects, and perturbations in growth and development, including intellectual functioning. Several environmental agents with estrogenic or anti-estrogenic activity could cause toxicological effects on reproductive and developmental processes. Rather than testing the effects of potentially harmful agents in laboratory animals, whose response may not reflect what occurs in humans, the effects of these agents can be tested directly on human myometrial cells in culture. A limitation of this approach has been the absence of myometrial cell lines with the phenotype of non-transformed cells. Preparation of myometrial cells in primary culture requires a limited amount of clinical material, and is very labour intensive. The availability of the cell lines will allow high-throughput analysis of numerous environmental agents. Immortalized myocytes will also allow an examination of a number of signal pathways involved in reproductive processes.
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Acknowledgements |
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This research was supported by NIH grant RO3HD043031 (MSS) and funds from the Department of Obstetrics and Gynecology. We thank Geron Corp. for the hTERT retroviral expression system, and Ms Jennifer Desormeaux for expert secretarial assistance.
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Submitted on April 9, 2004; resubmitted on June 1, 2004; accepted on June 10, 2004.
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