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Biology of Reproduction 67, 506-514 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Telomerase Immortalization of Human Myometrial Cells1

Jennifer Condona, Su Yina, Bobbie Mayhewa, R. Ann Worda, W.E. Wrightb, J.W. Shayb, and William E. Rainey2,,a,b

a Departments of Obstetrics and Gynecology b Cell Biology, University of Texas, Southwestern Medical Center at Dallas, Dallas, Texas 75390-9032


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Several strategies have been described for the primary culture of human myometrial cells. However, primary cultures of myometrial cells have a limited life span, making continual tissue acquisition and cell isolation necessary. Recent studies have demonstrated that cell culture life span is related to chromosomal telomere length, and cellular senescence results from progressive telomere shortening and the lack of telomerase expression. Transfection of cells with expression vectors containing the human telomerase reverse transcriptase (hTERT) maintains telomere length and effectively gives normal cells an unlimited life span in culture. In addition, hTERT extends the life span of cultured cells far beyond normal senescence without causing neoplastic transformation. In the present study, we developed a cell line from hTERT-infected myometrial cells (hTERT-HM). Cells were isolated from myometrial tissue obtained from women undergoing hysterectomy, and retroviral infection was used to express the catalytic subunit of telomerase in myometrial cells. Cells expressing hTERT have been in continuous culture for >10 mo, whereas the control culture senesced after approximately 2 mo. Telomerase activity was monitored in cells with a polymerase chain reaction-based telomerase activity assay. Telomerase-expressing cells contained mRNA for {alpha} smooth muscle actin, smoothelin, oxytocin receptor, and estrogen receptor {alpha}, but the estrogen receptor ß receptor was lost. Immunoblotting analysis identified the expression of calponin, caldesmon, {alpha} smooth muscle actin, and oxytocin receptor. Although estrogen receptor expression was below the level of detection with immunoblotting, transfection experiments performed with reporter constructs driven by estrogen response elements demonstrated estrogen responsiveness in the hTERT-HM. In addition, treatment of hTERT-HM with oxytocin caused a concentration-dependent increase in intracellular calcium levels, confirming the presence of functional oxytocin receptors. Myometrial cells immortalized with hTERT retained markers of differentiation that are observed in primary cultures of smooth muscle cells. The expression of various smooth muscle/myometrium cell markers suggests that these cells may be an appropriate model system to study certain aspects of human myometrial function.

female reproductive tract, oxytocin, parturition, pregnancy, uterus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The myometrium constitutes a major portion of the uterus and is composed of smooth muscle cell bundles, connective tissue, and blood vessels. The alterations that occur in the myometrium as it progresses from a nonpregnant to a pregnant phenotype and at parturition relate to a variety of factors, including the differential expression of numerous genes. Although the expression of these genes can be examined using in vivo models, the determination of molecular mechanisms that control expression would be greatly facilitated through the development of in vitro cell culture model systems. Human myometrial cells have been isolated and placed in primary culture [1]. However, the use of normal human cells in primary culture is restricted in part by the limited proliferative potential exhibited by diploid cells [2]. In addition, human myometrial tissue is not readily available to all laboratories, making the development of a readily available human myometrial cell culture model an important step toward facilitating research in this area.

Several strategies have been used to develop myometrium-like cell lines that have extended proliferative potential. Cell lines have been developed from human and mouse uterine leiomyomas [3, 4], and viral oncogenes have been used to produce immortalized human, mouse, and porcine myometrium cell lines [5–7]. These cells exhibit the extended cell culture life span expected of immortalization and have retained some of the differentiated aspects of normal myometrium. However, the insertion of viral oncogenes can result in the development of characteristics associated with cancer cells, including altered cellular signaling pathways and a loss of normal cell-cycle checkpoints [8].

Recently, telomerase expression vectors have been used to immortalize normal human fibroblast and epithelial cells [9–13]. Expression of telomerase maintains telomere length, which normally decreases each time a cell divides. Without complete replication of telomeric DNA, the telomeres slowly shorten until cell division is arrested by what has been termed the Hayflick limit [14]. Telomerase is expressed in high levels in fetal tissues but is normally lost in normal non-stem cells during fetal development [15]. The results of these studies suggest that the loss of telomere length results in cellular senescence observed in cells that have been maintained for extended periods in monolayer culture. The ability of human telomerase reverse transcriptase (hTERT) to stabilize telomere length and extend cellular proliferative capacity has been demonstrated [9–13]. Many human cell types that express hTERT are currently being produced. These cells have thus far been karyotypically stable, with no indication of cancer-associated changes and with normal cellular responses to DNA damage signals [10, 13, 15]. We undertook the present investigation to develop an in vitro model system for the purpose of investigating the complex molecular, hormonal, and cellular processes associated with the myometrium. Myometrial cells immortalized with hTERT retained markers of differentiation that are observed in myometrial tissue. The expression of various smooth muscle and myometrial cell markers suggests that these cells may be an appropriate model system for investigating the molecular mechanisms that regulate uterine smooth muscle cell gene expression.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Construction of Retroviral Vectors and Production of Retroviruses

The hTERT cDNA [9, 10] was modified to remove the 5' untranslated region (UTR) and the 3' UTR and to provide the cDNA with an optimized initiation codon identical to the Kosak consensus. This modified molecule was flanked by EcoRI sites to facilitate subsequent cloning. This fragment was then inserted into the EcoRI site of the retroviral vector pBABEpuro [16]. The final construct was electroporated in the mouse ecotropic packaging cell line PE501. The ecotropic viruses produced by the PE501 cells were supplemented with polybrene (4 µg/ml 1,5-dimethyl-1,5-diazaundecamethylene polymethobromide; Sigma, St. Louis, MO) and utilized to infect the mouse amphotropic packaging cell line PA317. Following infection, the PA317 cells were placed under puromycin selection (4 µg/ml; Sigma), and after 1 wk of selection supernatants containing amphotropic viruses were collected and then used to infect primary cultures of human myometrium cells.

Myometrial Cell Isolation and Culture

All tissues were obtained in accordance with the Donors Anatomical Gift Act of the State of Texas with informed written consent from women undergoing hysterectomy. In each case, myometrial tissue was obtained from uteri of reproductive age (i.e., premenopausal) women. Indications for hysterectomy were conditions other than uterine disease. Myometrial tissue was obtained from the anterior wall of the uterine fundus and placed in Hanks balanced salt solution (buffered with 20 mM Hepes and sodium bicarbonate to pH 7.4). Tissue was minced and digested enzymatically with type 1 collagenase (150 mg/ml; Worthington Biochemical, Freehold, NJ) and 15 mg/ml DNase (Sigma) in Dulbecco modified Eagle/F12 low glucose medium (DME/F12; Gibco BRL, Grand Island, NY) at 37°C with agitation for 4 h. The nondispersed tissue fragments were separated by filtration of the mixture through two sterile gauze layers. The cells were placed in multiwell dishes in DME/F12 medium with 10% fetal bovine serum (FBS; Gibco BRL) and antibiotic/antimycotic (10 000 units/ml; Gibco BRL) at 37°C in 95% air with 5% CO2. Cells were subcultivated with trypsin/EDTA at a 1:3 split after reaching confluence, approximately once every 10 days.

Retroviral Vectors and Infection

On Day 10 of culture, cells were subcultured onto six-well dishes and allowed to recover for 4 days. Cells were then infected for 16 h with the amphotrophic hTERT retroviral vectors in the presence of polybrene (4 µg/ml) and selected in puromycin (600 ng/ml) for 6 wk. The development of the defective retroviral vector has been previously described in detail [16] and is shown in Figure 1. Following infection with virus, puromycin was used to kill all noninfected cells to allow colony formation of the hTERT-infected cells. Cell lines were generated by clonal selection and were maintained in DME/F12 medium with 10% FBS and antibiotic/antimycotic (10 000 units/ml). Six of these infected human myometrium cell lines (hTERT-HM) were isolated, but only four maintained sufficient growth for further study. A nonclonal population of hTERT-infected myometrial cells (hTERT-HM mixed) and primary noninfected (HM) cells were also propagated under the same conditions described above.



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  		FIG. 1. Construction of the hTERT retroviral vectors, production of retroviruses, and infection of myometrial cells. Recombinant viruses containing the hTERT cDNA were packaged in an amphotropic cell line. The packaged retroviral recombinant vector was used to infect passage 2 myometrium cells, forcing hTERT expression, telomere elongation, and cell immortalization

Indirect Immunoflourescence

The hTERT-HM cells were cultured for 3 days on Labtek multichambered slides (Nalgene/Nunc International Corp., Naperville, IL) at 37°C. Cells were fixed and permeabilized by immersion in -20°C methanol for 5 min and air dried. The cells were incubated with {alpha} smooth muscle actin antibody (1:500 dilution) for 1 h and rinsed three times with PBS for 10 min each. The cells were then incubated for 1 h with a 1:200 dilution of fluorescein isothiocyanate-conjugated donkey anti-mouse IgG secondary antibody (Jackson Laboratories, Bar Harbor, ME). All antibody dilutions were made in PBS. The slides were mounted in FA Mounting Buffer (Bio-Rad, Hercules, CA) and coverslipped. Photographs were taken with a Zeiss microscope (Carl Zeiss MicroImaging Inc., Thornwood, NY) equipped for epifluorescence. Cells incubated in the absence of actin antibody served as negative controls.

Measurement of Telomerase Activity

Cells (1000 cells/µl) were resuspended in lysis buffer (10 mM Tris-HCl pH 8.0, 1 mM MgCl2, 1 mM EGTA, 1% NP-40, 0.25 mM sodium deoxycholate, 10% glycerol, 150 mM NaCl, 5 mM ß-mercaptoethanol, and 0.1 mM PMSF), incubated on ice for 30 min, and then centrifuged at 14 000 x g for 20 min as previously described [15]. Supernatants were used for the detection of telomerase (0.5–2 µl/assay) or flash frozen and stored at -80°C. Telomerase activity was determined with the TRAP-eze telomerase detection kit (Intergen, Norcross, GA) with the telomerase-specific (TS) primer (5'-AATCCGTCGAGCAGAGTT-3') as the substrate. Following the extension of the substrate by telomerase (for 30 min at room temperature), the products were amplified by polymerase chain reaction (PCR) in the presence of an end-labeled [32P]TS primer, resolved on a 10% polyacrylamide gel, and revealed by exposure to a PhosphorImaging cassette (Molecular Dynamics, Piscataway, NJ). Telomerase activity was calculated as the ratio of the intensity of the telomerase ladder to the intensity of the 36-base pair (bp) internal standard.

Measurement of Telomere Length

Total genomic DNA was isolated from frozen pellets of cells. The DNA was digested (2–4 µg) with a mixture of restriction enzymes that do not cut the telomeric DNA: AluI, CfoI, HaeIII, HinfI, MspI, and RsaI [17]. Digested samples were resolved on a 0.7% agarose gel. The gel was dried and hybridized to a telomeric probe (an end-labeled [32P]-(TTAGGG)4 oligonucleotide), washed in 0.1x saline sodium citrate (twice for 15 min at room temperature), and then exposed to a phosphoimaging cassette.

Reverse Transcription PCR

Total RNA was isolated from the hTERT-HM cell lines and myometrial tissue in a one-step 4 M guanidinium thiocyanate extraction and 5.7 M cesium chloride ultracentrifugation at 42 000 rpm (Beckman SW60 rotor) for 16–24 h. The first-strand cDNA synthesis reaction is catalyzed by SUPERSCRIPT II RNase H- reverse transcriptase (RT) (Gibco BRL). The first strand cDNA synthesis reaction was primed by random hexamers. Amplification of the target cDNAs from first-strand synthesis reaction was performed with PCR. Estrogen receptor {alpha} (ER{alpha}), estrogen receptor ß (ERß), oxytocin receptor (OXR), calponin, smoothelin (short form), progesterone receptor (PR), and glyceraldehyde 3-phosphate dehydrogenase (G3PDH) cDNAs were amplified using specific primers for each transcript (Table 1) [18–23].


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  		TABLE 1. Primers used for RT-PCR analysis of myometrial markers

Semiquantitative RT-PCR

A semiquantitative measurement of the relative abundance of ER{alpha} and PR in the hTERT-HM cell lines and HM tissue was performed. PCR primers were used in this semiquantitative assessment. Master PCR mix (for 30 PCRs) contained 75 µl 10x PCR buffer, 45 µl 25 mM MgCl2, 15 µl 10 mM dNTPs, 15 µl (10 pmole) sense gene-specific primer, 15 µl (10 pmole) antisense gene-specific primer, 7.5 µl Taq DNA polymerase, 510 µl water, and 37.5 µl dimethyl sulfoxide. Master mix (24 µl) was aliquoted in each tube with 1 µl from the original first-strand cDNA synthesis reaction. PCR amplification was programmed as follows: denaturing at 94°C for 1 min, annealing at primer-specific temperatures (ER{alpha}, 51°C; OXR, 49°C; PR, 61°C; G3PDH, 64°C) for 1 min, and extension at 72°C for 1 min. The constitutively expressed G3PDH transcript was used as a reference gene to normalize mRNA levels and to evaluate data from the exponential phase of the PCR amplification. PCRs were terminated at various cycle points to decipher the exponential phase of the PCR amplification. ER{alpha} PCRs were terminated at 17, 20, 22, 25, 27, and 30 cycles, PR PCRs were terminated at 15, 20, 25, and 30 cycles, and G3PDH PCRs were terminated at 10, 15, 17, 20, 22, and 25 cycles.

Protein Immunoblotting Analysis

PAGE was carried out on cell and tissue lysates with a precast Novex gel electrophoresis system with 4–12% bis-tris NuPage gels (Invitrogen, San Diego, CA). Proteins were electrophoretically transferred onto polyvinylidene fluoride membranes by wet transfer for 1 h at 25 V. Following transfer, membranes were incubated for 1 h at room temperature with antibodies directed against calponin, {alpha} smooth muscle actin, h-caldesmon, OXR, progesterone A/B receptors, or ER{alpha}. Membranes were incubated with horseradish peroxidase-conjugated secondary antibodies, and immunoreactive bands were visualized. Calponin antibody (1:200) was obtained from Innogenex (San Ramon, CA), {alpha} smooth muscle actin antibody (1:40 000) and the caldesmon antibody that recognizes the high-molecular-weight isoform (h-caldesmon, 1:5000) were obtained from Sigma, and OXR antibody (1:2000) was kindly provided by Dr. K. Whittington (University of Bristol, Bristol, U.K.) and has been previously characterized [24]. The PR antibody that recognizes both A and B isoforms (1:400) and ER{alpha} antibody (1:600) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Myometrial Cell Transient Transfection

Human myometrial cells (hTERT-HM, hTERT-HM mixed, and HM) were cultured in DME/F12 medium (buffered with 20 mM Hepes and sodium bicarbonate to pH 7.4) supplemented with 10% FBS and antibiotic/antimycotic (10 000 units/ml; Gibco BRL) at 37°C in 95% air and 5% CO2. Cell monolayers were subcultured onto 12-well culture dishes in DME/F12 phenol red-free, low-glucose medium (Gibco BRL) and transfected 24 h later. Transfection was carried out with 2 µl of Fugene 6 (Boehringer Mannheim, Indianapolis, IN) per 1 µg of the pGL3-EREc38-luciferase reporter vector [25] in 1 ml DME/F12 phenol red-free, low-glucose medium for 6 h at 37°C. The total amount of plasmid DNA transfected was kept constant by addition of empty pGL3-pro vector. Following 6 h of transfection, cells were incubated with 1.0 ml low-serum medium (DME/F12 phenol red-free, low-glucose medium containing 0.1% FBS) overnight at 37°C. Following overnight recovery, cells were treated with agonists (1 nM 17ß-estradiol) for 6 h. Cells were then lysed and assayed for reporter activity with a luciferase assay system (Promega, Madison, WI). Results are expressed as a percentage of basal luciferase activity and represent the mean ± SEM of determinations from six independent experiments, each performed in triplicate.

Determination of [Ca2+]i Responses to Oxytocin

Confluent hTERT-HM (clone 4) cells were removed from the culture flask by incubation at 37°C for 3 min in Hanks balanced salt solution containing trypsin (0.05%) plus EDTA (0.02%). Cells were pelleted and resuspended in fluorescent buffer (4.8 mM KCl, 130 mM NaCl, 1.0 mM MgCl2, 1.5 mM CaCl2, 1.0 mM Na2HPO4, 15 mM glucose, 10 mM Hepes (pH 7.4) supplemented with 0.1% BSA). Cells were loaded with fura-2 by incubation with fura-2/AM (1 µM) for 15 min at 25°C. Cells (106 cells/ml) were washed twice in fluorescent buffer without albumin and used for assay within 1 h of fura-2 incubation. Fura-2-containing myometrial cells were suspended at 25°C in a cuvette equipped with an electronically controlled, minimotorized Teflon rotor (Instech Laboratories, Horsham, PA). Fluorescence was recorded with a Perkin-Elmer 650-10S fluorescence spectrophotometer (Perkin-Elmer, Norwalk, CT) at an excitation wavelength of 340 nm, and emission was monitored at 510 nm. Measurements were corrected for autofluorescence and extracellular fluorescence as previously described [26]. Intracellular free Ca2+ concentration was calculated according to the following formula: [Ca2+]i = Kd(F - Fmin)/(Fmax - F), where F is the experimentally determined fluorescence, Fmax is the maximum fluorescence in the presence of 50 µM ionomycin, and Fmin is the minimum fluorescence in the presence of 12 mM EGTA plus 20 mM Tris base [27]. The Ca2+ dissociation constant (Kd) for fura-2 is 224 nM [28].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Morphological Characteristics of Myometrial Cell Lines

After infection of cultured myometrial cells with the hTERT retrovirus, six puromycin-resistant colonies were isolated. Of these, four maintained growth after subcloning, and these four clones (designated hTERT-HM1 to hTERT-HM4) were further characterized. In addition, a nonclonal group of hTERT-infected myometrial cells was maintained in puromycin-containing medium (designated hTERT-HM mixed). The hTERT-HM mixed population as well as the clonal population of cells retained morphological characteristics of proliferating smooth muscle cells in culture, including an elongated cell shape with a central nucleus, a sheetlike growth pattern at confluence (Fig. 2A), and expression of smooth muscle-specific actin (Fig. 2C). This morphology and expression of {alpha} smooth muscle-specific actin was not observed in fibroblast cell lines developed with expression of hTERT (Fig. 2, B and D). Staining was not observed in the absence of primary antibody directed against {alpha} smooth muscle actin (data not shown). The cell clones have been maintained in culture for up to 10 mo (approximately 80 population doublings) without loss of cell division. In contrast, in nonimmortalized human myometrium from the same patient cultured under the same conditions as the hTERT-HM cell growth decreased over 8 wk in culture after approximately 15 population doublings.



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  		FIG. 2. Telomerase-immortalized HM and HF cells in culture. A) Phase contrast image of immortalized hTERT-HM cells in monolayer culture. x200. B) Phase contrast image of immortalized hTERT-HF cells in monolayer culture. x200. C) Immunoflourescence for {alpha} smooth muscle actin in hTERT-HM cells (x200) demonstrating positive staining in myofilaments and stress fibers. D) Absence of {alpha} smooth muscle actin immunofluorescence in hTERT-HF cells. x200

Telomerase Activity Measurements

The telomeric repeat amplification protocol (TRAP) assay was used to examine telomerase activity in primary cultures of myometrial cells and the hTERT retrovirus-infected cells (Fig. 3). Non-hTERT-infected cultures of human myometrial cells did not have telomerase activity. However, human myometrial cells infected with the hTERT retrovirus (hTERT-HM1 through hTERT-HM4) had reconstituted telomerase activity, as did the nonclonal population of hTERT-HM cells. The hTERT-HM cells continuously expressed telomerase activity over time at various population doublings. Telomerase-infected human fibroblast cell lines (hTERT-HF) were used as a positive control for this assay. The hTERT-HF cells have been maintained in culture for >400 population doublings and continue to express telomerase RT. However, the hTERT-HF were transfected with a plasmid-based system, which over time in culture has become progressively silenced, leading to telomere shortening because of reduced telomerase activity [29].



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  		FIG. 3. Telomerase activity in myometrial cells and the hTERT immortalized HM and HF cell lines. Telomerase activity was determined with a TRAP assay. Activity in myometrial cells and the four hTERT-HM cell lines (HM1–HM4) was compared with that of hTERT-HF cells after 202 and 483 population doublings. The noninfected myometrial cells served as a telomerase negative control. This representative TRAP assay reveals the characteristic 6-bp ladder indicative of enzymatic activity. The 36-bp internal standard serves to quantitate relative telomerase activity levels

Telomere Length Analysis

Telomere length was measured with the terminal restriction fragment length (TRL) assay (Fig. 4). Genomic DNA was extracted from cultures of myometrial cells that had not been virally infected, the non-clonal population of hTERT-HM cells, four independent clonal populations of hTERT-HM [1–4], and two clonal populations of hTERT-HF at early and late (202 and 483, respectively) population doublings. The myometrial cells that were cultured for 8 wk had shorter telomeric DNA (10.5–4.8 kilobases [kb]) as indicated by a telomeric smear. In contrast, the telomerase-containing myometrial cell lines, hTERT-HM clones 1–4, maintained a longer and more uniform telomere length (24.0, 16.6, 28.5, and 19.8 kb, respectively, when examined at population doublings 50–80. The telomerase-containing hTERT-HF cell lines at 202 population doublings continued to have a relatively uniform telomere length (11–14 kb), whereas the hTERT-HF cell line at 483 population doublings demonstrated considerable loss of telomere length (3.7 kb) and telomerase activity (Fig. 3) but continued to divide. These results demonstrate that forced expression of hTERT in normal human myometrial cells results in telomerase activity, elongation of telomeres, and an extended life span.



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  		FIG. 4. Analysis of telomere length using the TRL assay in myometrial cells in culture and in telomerase-immortalized human myometrial cell lines (hTERT-HM). The four hTERT-HM cell lines maintained telomere length compared with noninfected HM cells. Lower passage hTERT-HF cells (202 population doublings) were included as a positive control. Extended culture (483 population doublings) of the hTERT-HF cells decreases telomere length. Molecular weight markers are shown to allow the estimation of telomere length

Examination of mRNA for Myometrial and Smooth Muscle Markers

Initially we characterized the expression of transcripts for several myometrial cell markers with total RNA isolated from myometrial tissue, cultures of myometrial cells, and the nonclonal hTERT-HM cell line with RT-PCR (Fig. 5). We analyzed the expression of calponin, smoothelin, OXR, ER{alpha}, and ERß. Myometrial tissue RNA was used as a positive control for each transcript. The hTERT-HM cells expressed all the transcripts in abundance except for ERß, which was present at very low levels relative to myometrial tissue (Fig. 5).



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  		FIG. 5. Analysis for mRNA for myometrial cell markers. Total RNA was isolated from the nonclonal population of hTERT-HM, the cultures of HM cells, and myometrial tissue. Transcripts for ER{alpha}, ERß, OXR, calponin (Calp), and smoothelin (SME) were examined with RT-PCR. The expected 481-bp fragment for ERß was present in myometrial tissue, but this product was reduced in myometrial cells in culture

To compare relative expression levels of ER{alpha} and PR, we analyzed the hTERT-HM cell lines by semiquantitative RT-PCR (Fig. 6). G3PDH was used as a reference gene to normalize mRNA levels. ER{alpha} transcript levels differed between hTERT-HM cell lines but were expressed at levels comparable to that expressed in myometrial tissue. PR mRNA expression was analyzed in the same manner and was expressed at levels similar to that of myometrial tissue.



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  		FIG. 6. Analysis of ER{alpha} and PR transcripts in human myometrial tissue and four hTERT-HM clonal cell lines (hTERT-HM1 to hTERT-HM4). Semiquantitative RT-PCR analysis was performed for ER{alpha} and PR mRNA, with G3PDH as a reference. Myometrial tissue served as a positive control for relative myometrial mRNA expression levels. Results are presented as a function of the number of PCR cycles and are representative of a minimum of three independent experiments

Examination of Protein Expression for Myometrial and Smooth Muscle Markers

The expression of smooth muscle-specific proteins ({alpha} actin, calponin, and h-caldesmon) and OXR was evaluated in hTERT-HM cells relative to hTERT-HF cells, epithelial cells (T47D), and human myometrial tissue (Fig. 7). Immunoblotting was also used to examine PR A, PR B, and ER{alpha} protein expression in nuclear extracts of primary cultures of myometrial cells and hTERT-HM cell lines. The {alpha} smooth muscle actin (40 kDa) was found in abundance in myometrial tissue and in all four hTERT-HM clonal cell lines, with lower levels of expression present in the primary cultures (HM) and hTERT-HM mixed cells; {alpha} smooth muscle actin was absent in the telomerase-containing hTERT-HF cells and epithelial cells. Expression of calponin was decreased in the nonclonal hTERT-HM and normal HM cells compared with myometrial tissue. However, this smooth muscle-specific marker protein was enriched in hTERT-HM clones 1 and 3 relative to normal HM cells in culture. All myometrium-derived cultures expressed higher levels of calponin than were observed in fibroblasts or epithelial (T47D) cells (Fig. 7). Likewise, the expression of h-caldesmon was significantly increased in hTERT-HM cell lines relative to the low levels expressed in HM cells in primary culture or to the nondetectable levels in fibroblasts and the T47D epithelial cells. In fact, hTERT-HM clone 3 maintained h-caldesmon expression levels similar to those in myometrial tissue. These results indicate that the expression of smooth muscle-specific genes is maintained in hTERT-HM cells, and the levels of smooth muscle-specific proteins in some clonal isolates are greater than those in cells in mixed primary culture.



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  		FIG. 7. Immunoblot analysis for myometrial and smooth muscle cell markers. Levels of expression for {alpha} smooth muscle actin, calponin, h-caldesmon, and OXR were examined with Western analysis. Expression was examined in four hTERT-HM cell lines, the nonclonal population of hTERT-HM (mixed), cultures of HM cells, myometrial tissue, and hTERT-HF cells and an epithelial cell line (T47D). Eight micrograms of protein was added to each lane for immunoblot analysis

Western analysis was used to determine whether the myometrial cell lines maintained the expression of three receptors that are observed in myometrium, OXR, PR, and ER. OXR expression was also retained in the myometrial cell lines. However, OXR gene expression was observed in the hTERT-HF cell lines (hTERT-HF 202 and 483-PD). The expression of OXR in the fibroblast cell lines was not surprising because of previous reports of OXR expression in rat atrial fibroblasts [30].

Examination of Estrogen Responsiveness

In contrast to the mRNA data (Figs. 5 and 6), PR and ER protein levels were present at very low levels compared with those observed for lysates of myometrial tissue (data not shown). To determine whether functional ER activity was present, a transient transfection assay was carried out with a luciferase reporter vector and driven by a series of estrogen response elements (EREs). The hTERT-HM cell clone 4 was transiently transfected with either the promoterless pGL3-basic reporter vector (empty vector) or the same reporter construct containing three EREs (Fig. 8).



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  		FIG. 8. Examination of estrogen responsiveness with reporter constructs driven by EREs. The hTERT-HM cells (clone 4) were transiently transfected with a luciferase reporter vector driven by three EREs. Following recovery, the cells were incubated with 1 nM 17ß-estradiol for 6 h. Cells were lysed and assayed for luciferase activity. Results are expressed as percentage luciferase activity and represent the mean ± SEM determined from three independent experiments, each performed in triplicate. *Significant differences from all other treatments (P < 0.05, ANOVA)

Transfection of the myometrial cell line (hTERT-HM4) with the ERE reporter construct did not result in a significant increase in reporter activity relative to the promoterless vector alone (Fig. 8). However, treatment of transfected cells with 1 nM estradiol resulted in 4- to 5-fold increases in luciferase activity, which was significantly higher than that observed with the promoterless reporter vector or the ERE reporter construct alone. These results indicate that hTERT-HM cells are responsive to estrogen through estradiol-dependent activation of DNA response elements.

Examination of Oxytocin Responsiveness

To demonstrate the presence of functional OXRs, fura-2-loaded hTERT-HM cells were treated with increasing concentrations of oxytocin (1–100 nM). [Ca2+]i was significantly elevated with all concentrations of oxytocin (Fig. 9). A maximal increase in [Ca2+]i (2.5-fold greater than basal) was observed using 10 nM oxytocin. These results indicate that hTERT-HM4 cells are responsive to oxytocin and therefore may provide a model system for studying this signaling pathway.



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  		FIG. 9. Effect of oxytocin on increases in [Ca2+]i in hTERT-HM cells. Cell suspensions were treated with oxytocin, and fluorescence was monitored continuously for 1 min. Maximal increase in [Ca2+]i in response to oxytocin was quantified with fura-2 fluorescence. Data represent mean ± SEM of three or four determinations. *Significant differences from baseline (P < 0.05, ANOVA). Inset: Representative tracing of [Ca2+]i transient in hTERT-HM cells treated with oxytocin (10-7 M, arrow)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Through the introduction of an hTERT cDNA, we produced myometrial cell lines with greatly extended life spans and that retain the expression of a number of smooth muscle cell markers. Insertion of hTERT resulted in reconstitution of telomerase activity, elongation of telomere length, and extended life span. Whereas control cells stopped dividing after about 15–20 doublings, the hTERT-expressing myometrial cells have exceeded 80 population doublings and are continuing to divide at normal rates. Recent studies have shown that expression of hTERT proteins produced immortalized human fibroblasts and keratinocytes without transformation to a malignant phenotype [9–13]. Consistent with these observations, the hTERT-HM cell lines demonstrated growth characteristics of normal cells but, unlike the parent cells, did not cease to grow after extended culture time.

Smooth muscle cells exhibit a variety of phenotypes ranging from fully differentiated quiescent contractile cells to synthetic proliferating cells expressing low levels of contractile proteins [31]. In adult tissues, smooth muscle cells are normally quiescent, exhibit very low rates of division, and express high levels of contractile proteins and myofilaments. These differentiated contractile smooth muscle cells are characterized by unique isoforms of contractile proteins (e.g., {alpha} and {gamma} smooth muscle actin, h-caldesmon, calponin, and telokin) [22, 32–34]. Once in culture, smooth muscle cells undergo phenotypic modulation, during which they change from a contractile quiescent state characteristic of smooth muscle cells in vivo to a synthetic phenotype characteristic of smooth muscle cells under various pathologic conditions [22]. Thus, the maintenance of contractile protein expression in primary cultures of smooth muscle cells and cell lines has been challenging [35, 36].

Several strategies have been utilized to develop immortalized differentiated uterine smooth muscle cell lines. Lines have been derived from leiomyosarcoma, Syrian hamster myometrium [4], and myometrial cells transformed with SV-40 large T antigen [6, 37]. Retroviral infection of primary human myometrial cell cultures with constructs containing E6/E7 open reading frames of the human papillomavirus type 16 has also been used to derive immortalized myometrial cell systems [5, 7]. Although these myometrial cell lines maintain some smooth muscle characteristics, sensitive markers of smooth muscle differentiation have not been examined. In the present study, immortalization of uterine smooth muscle cells derived from nonpregnant myometrium with hTERT resulted in maintenance of several sensitive markers of smooth muscle differentiation (calponin, h-caldesmon, and smoothelin). Moreover, the cells maintained an elongated cell shape characteristic of smooth muscle cells in vivo and distinct from the phenotype of fibroblasts in culture. The relationship between telomerase and expression of smooth muscle-specific genes is difficult to extrapolate from the current studies. In most in vivo models of vascular, airway, and intestinal injury, smooth muscle cells are subject to mechanical and/or cytological trauma resulting in exposure to growth factors and reentry into the cell cycle. Together these agents increase smooth muscle cell proliferation and cause phenotypic modulation altering the contractile properties of the muscle. However, contractile smooth muscle cells can proliferate in vivo without downregulating contractile protein expression [38]. Perhaps inhibition of cellular senescence through telomerase expression results in adaptations of cellular function, including maintenance of a differentiated phenotype. Further studies are necessary to clarify the relationships among cell senescence, proliferation, telomerase, and cell type-specific differentiation.

Myometrium is a heterogeneous tissue, and the isolation technique used to remove the myometrium from its surrounding cell layers may have yielded multiple cell types. This concern would certainly apply to the nonclonal populations of hTERT-expressing cells that resulted from the initial cell cultures prepared from the myometrial tissues. Because of this concern, we produced the clonal lines. In addition, to ensure that the hTERT-expressing cells were of smooth muscle origin, we used Western analysis to examine the expression of several smooth muscle cell markers. One of the more highly expressed markers for smooth muscle cells is {alpha} smooth muscle-specific actin. The actin isoforms are divided into two classes, muscle and nonmuscle actins. The {alpha} smooth muscle actin represents a major portion of actin isoforms expressed in vascular smooth muscle cells. However, {alpha} smooth muscle actin is also expressed in differentiating heart, early skeletal muscle, and a variety of non-smooth muscle and myofibroblast-like cell types [39–41]; thus, this isoform of actin does not always act as a specific marker for smooth muscle. Using immunoblot analysis, high levels of {alpha} smooth muscle actin expression were demonstrated in the hTERT-HM cell lines and myometrial tissue. Whereas lower levels were expressed in the hTERT-HM mixed or HM cell lines, smooth muscle actin expression was not detectable in hTERT-HF cells or T47D human breast cancer cells.

Calponin expression is a sensitive indicator for smooth muscle contractile phenotype and is possibly related to smooth muscle cell differentiation. Calponin binding to actin, myosin, Ca2+-binding proteins, and tropomyosin inhibits the actinomyosin ATPase and the movement of actin filaments over myosin in vitro [34]. Immunoblotting analysis demonstrated that human myometrial tissues expressed high levels of calponin. Examination of the hTERT-HM lines demonstrated high calponin protein expression with lower levels of expression found in cultured normal myometrial cells. Two caldesmon isoforms have been identified; h-caldesmon (the high relative molecular mass form) is dominantly expressed in differentiated smooth muscle cells, whereas l-caldesmon (low relative molecular mass form) is widely distributed in nonmuscle tissue and cells [33]. In particular, the isoform conversion of caldesmon is strongly associated with phenotypic modulation of smooth muscle cells, in which the caldesmon isoform converts from the l form to the h form during differentiation. Caldesmon is therefore considered a useful marker for studying phenotypic modulation of smooth muscle cells. In previous studies, human myometrium expressed high levels of h-caldesmon [42]. Immunoblot analysis demonstrated expression of the h-caldesmon isoform in our hTERT-HM cell lines (clones 2, 3, and 4), suggesting that these cells have maintained a smooth muscle-like phenotype.

Calponin, h-caldesmon, and {alpha} smooth muscle actin are markers that are retained in smooth muscle-like cell lines; however, they occasionally occur in other cell types. The characterization of the expression patterns of the cytoskeletal protein smoothelin suggests that this protein may indeed be specific to smooth muscle tissue (including myometrium) [43]. Smoothelin expression drops in both primary and long-term smooth muscle cell cultures because of a decrease in mRNA expression [43]. RT-PCR analysis of the hTERT-HM cell lines indicated that smoothelin mRNA was still being transcribed in the cell line, suggesting that at least some of the cells present in the hTERT-HM cell lines are in a differentiated phenotype. Studying the regulation of smoothelin expression within the hTERT-HM may help define the growth factors and hormones that regulate myometrium differentiation. The expression of smoothelin has not been examined in any of the currently available myometrial cell lines.

In addition to general markers of smooth muscle cells, we also examined the expression of several markers that appear to be preferentially expressed in myometrial smooth muscle cells. OXR expression has been demonstrated in the myometrium of pregnant and nonpregnant human uteri [44, 45]. The presence of OXRs in the late pregnant uterus may account for increased myometrial cell sensitivity to oxytocin and consequently uterine contractions. The hTERT myometrial cell line responded to oxytocin with an increase in intracellular calcium levels. These findings suggest that the hTERT myometrial cell lines have functional OXRs that may allow these cells to act as a model for studying OXR expression and function.

ER{alpha} and ERß isoforms have been detected in the myometrium in nonpregnant, pregnant, and term human uteri [46]. Estrogens have many different functions in many different cell types. Both ER{alpha} and ERß bind estradiol and then bind to classical EREs. Estradiol and the ERs have been investigated for the physiological role they play in the expression of connexin-43 in cultured myometrium [47] and c-jun and c-fos [48, 49]. Rapid loss of ERs has been demonstrated in myometrial explant cultures [50]. Our ER{alpha} and ERß data demonstrated decrease of ERß mRNA in hTERT-HM cell lines compared with myometrial tissue as detected by RT-PCR. ER{alpha} mRNA expression was maintained, as demonstrated by our semiquantitative RT-PCR. However, immunoblot analysis suggested that the cell lines have much less ER{alpha} protein expression compared with myometrial tissue. Nevertheless, transient transfection analysis of the hTERT-HM cell line with a reporter construct controlled by EREs demonstrated estradiol responsiveness. This finding suggests that the hTERT-HM cell lines have the ability to promote an estrogen response under the right conditions.

PRs exist as two protein isoforms, A and B. Both isoforms are expressed from a single gene in humans as a result of transcription from two alternative promoters [51]. The selective physiological roles of the two isoforms of PR are unknown. PR A and PR B are both expressed in the human myometrium, and RNase protection assays indicated no difference in the concentrations of mRNA encoding PR A and PR B, though Western blotting demonstrated a consistent dominance of PR A over PR B [52]. PRs are expressed in the epithelial, stromal and myometrial compartments of the uterus, and their expression is controlled by estrogen [51]. Our semiquantitative RT-PCR analysis demonstrated mRNA expression of the PR, although our immunoblot analysis was unable to detect PR protein.

The hTERT-HM cell lines exhibit a number of markers of smooth muscle cells, including {alpha} smooth muscle actin, smoothelin, h-caldesmon, and calponin expression. Myometrial properties of the hTERT-HM cell lines include the expression of these smooth muscle markers and the expression of OXR. Our data also demonstrate a functional estrogen response in these cells, further supporting their potential to act as an appropriate in vitro model for studies related to myometrial function. The hTERT-HM cell lines also illustrate the usefulness of telomerase for immortalizing normal cells. Cells generated with hTERT methodology could provide a readily available human model for investigators in the field of reproductive biology.


   ACKNOWLEDGMENTS
 
We thank Charles Kresge for consulting on the microscopy and Louella Hupp for editorial assistance with the article.


   FOOTNOTES
 
First decision: 2 August 2001.

1 This work was supported by awards from the National Institutes of Health (HD11149). Back

2 Correspondence: William E. Rainey, Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9032. FAX: 214 648 8066; william.rainey{at}utsouthwestern.edu Back

Accepted: February 14, 2002.

Received: July 5, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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