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From the Department of Obstetrics and Gynecology,* Kanazawa University, School of Medicine, Kanazawa, Japan; and the Virology Division, National Cancer Center Research Institute, Tokyo, Japan
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Abstract |
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The lack of a stable in vitro culture system of endometrial cells also renders studying the molecular carcinogenesis of the endometrium difficult. Most endometrial cancers arise from endometrial glandular cells via the multistep accumulation of abnormalities in oncogenes and tumor suppressor genes, including PTEN, Ras, and p53.4 These factors are frequently mutated or deregulated in endometrial cancers or even in its precursors. However, in vitro experiments to investigate the role of these factors in endometrial carcinogenesis have been impossible because of the extremely short life span of primary cultured endometrial epithelial cells in vitro.
One major mechanism that accounts for the limited life span of primary cultured cells is telomere-based replicative senescence. Telomeres are the specialized nucleoprotein structures at the ends of chromosome that play pivotal roles in chromosome stability.5,6 Without new synthesis, telomeres undergo progressive shortening with each cell division, according to Watson’s7 model. Critically short telomeres arising after a considerable number of cell divisions induce specific signals for cell-cycle arrest via DNA damage check point, resulting in replicative senescence of cells.8,9 Telomerase is a ribonucleoprotein complex that extends and maintains the telomeres. Activation of this enzyme is therefore required for cells to overcome replicative senescence and obtain the ability to divide without limit.10,11 Studies of the telomerase enzyme complex have revealed the presence of two major subunits contributing to enzymatic activity—a structural RNA component (hTR) that contains a template region binding to TTAGGG repeats in telomeres12 and a catalytic subunit with reverse transcriptase activity (hTERT).13,14 Although hTR is constitutively present in normal and cancer cells, expression of hTERT is almost exclusively limited to cancer cells.15-18 Introduction of the hTERT gene into telomerase-negative normal cells is sufficient to induce telomerase activity and to immortalize some types of normal cells.19-21 These findings indicate that hTERT expression is the rate-limiting step in telomerase activity and cellular immortalization.
Although telomere-based replicative senescence is a critical barrier to cell immortalization, some epithelial cells appear to senesce at an earlier stage, at 
20 population doublings (PDs) in which telomeres do not reach their critical short length.22,23
Introduction of hTERT into these cells did not result in extension of their life span.22
This telomere-independent senescence is thought to be controlled by the Rb/p16 pathway, because it can either be prevented by introduction of viral oncoproteins that bind Rb and inhibit its function, such as human papillomavirus (HPV) E7, or a few cells spontaneously escape from senescence with reduced p16 expression because of promoter methylation.24-26
Based on these findings, we previously reported that both Rb/p16 inactivation and telomerase activity are required to immortalize some types of human epithelial cells.22
A more recent report27
demonstrated a two-stage, p16- and p53-dependent senescence mechanism of keratinocytes that limits replicative potential independent of telomere status, suggesting a role of the p53 pathway in early-stage senescence of epithelial cells.
Establishment of immortalized human endometrial epithelial cells will greatly benefit the study of endometrial biology and carcinogenesis. Thus, we attempted to immortalize human endometrial epithelial cells. Because high-risk type HPV E6 and E7 seem to be useful to target both Rb and p53 pathways, we introduced HPV16 E6/E7 and/or hTERT into endometrial glandular cells. We successfully isolated immortalized endometrial epithelial cells expressing HPV 16 E6/E7 and hTERT. Of particular interest is that these cells do not have transformed phenotypes and retain the natural characteristics of endometrial glands without severe chromosomal abnormalities, and thus are potentially useful as an experimental model with which to research hormone functions, implantation, and endometrial carcinogenesis.
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Materials and Methods |
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Human endometrial tissue samples were obtained from two patients (42 and 52 years of age) undergoing hysterectomy as a treatment for uterine myoma. They had regular menstrual cycles and were in late proliferative phase at the time of operation. Minced endometrial tissue was placed in Dulbecco’s modified Eagle’s medium (DMEM) containing 350 U/ml of deoxyribonuclease I (Takara, Ohstu, Japan) and 180 U/ml of collagenase type 3 (Washington Biochemical Corp., Lakewood, NJ) in plastic dishes and gently shaken for 40 minutes at 37°C.28,29 Individual glands on the bottom of the dishes were directly picked up one by one under a microscope, collected into Eppendorf tubes, and seeded onto 24-well dishes for subsequent gene transfection by viral vectors.
Vector Construction and Retroviral Transfection of HPV16 E6, E7, and hTERT
pCMSCVpuro comprises the CMV/LTR fusion promoter, packaging signal Psi+, and the multicloning sequence from pCLXSN (Imgenex Corp., San Diego, CA) followed by the PGK-puro cassette and 3' long terminal repeat of murine embryonic stem cell virus from pMSCVpuro (Clontech, Palo Alto, CA). The Gateway system (Invitrogen, Carlsbad, CA) was used for subcloning genes into the retroviral vectors. The destination vectors pCLXSN-DEST and pCMSCVpuro-DEST were constructed by inserting a modified cassette containing attR sites and ccdB (Invitrogen) between the EcoRI and BglII sites of pCLXSN and pCMSCVpuro, respectively. Cloning of the full-length hTERT cDNA has been described previously.22 After cloning segments of HPV16 E7 (16E7) and a deletion mutant of HPV16 E6 (16E6SD-D151)30 into pDONR201 (Invitrogen), these segments were recombined into the retroviral vectors via the LR reaction (Invitrogen) to generate pCMSCVpuro-hTERT, pCMSCVpuro-16E6SDD151, pCLXSN-16E6SDD151, and pCLXSN-16E7. pCLXSN-16E6E7 was constructed by inserting the EcoRI-BamHI segment containing HPV16 E6 and E7 between the EcoRI and BglII sites of pCLXSN. Production of recombinant retroviruses has been described.31 Briefly, retroviral vector and packaging construct, pCL-10A1, were co-transfected into 293T cells using TransIT-293 (Mirus Co., Madison, WI) according to the manufacturer’s instructions, and the culture fluid was harvested at 48 to 72 hours after transfection. The titer of the recombinant viruses was greater than 5 x 105 drug-resistant colony forming units/ml on HeLa cells. One ml of the culture fluid was added to primary endometrial glandular cells seeded on 24-well dishes in the presence of polybrene (4 µg/ml). After inoculation with viruses, cells were grown without drug selection as mock-infected cells and stopped growing within 2 weeks. For combinations of retroviral infections, cells were first transduced with E6 and/or E7, and then transduced with hTERT.
Cell Culture
Stably transduced endometrial epithelial cells were maintained in DMEM/F12 (1:1) supplemented with 10% fetal bovine serum and ITS (BD Biosciences, Bedford, MA) in an atmosphere of 5% CO2 at 37°C. Three-dimensional culture was performed on Millicell CM filter inserts (Millipore, Bedford, MA) containing Matrigel (Becton Dickinson Labware, Bedford, MA) as described previously.1 Ishikawa cells and HEC1 cells were provided by Dr. Masato Nishida (Kasumigaura National Hospital, Tsuchiura City, Japan) and Dr. Hiroyuki Kuramoto (Kitasato University, Tokyo, Japan), respectively. C33A cells were obtained from the American Type Culture Collection (Rockville, MD). These cells were grown in DMEM with 10% fetal bovine serum. PDs were calculated as follows: PD = log(number of cells obtained/initial number of cells)/log2.
Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
The expression of HPV16 E6, E7, and hTERT mRNAs as well as estrogen receptor (ER)-
, ER-ß, and progesterone receptor (PR) mRNAs was analyzed by RT-PCR amplification. The primer pairs used were 5' to 3' and 5'-GCAACAGTTACTGCGACGTG-3' (forward), 5'-GGACACAGTGGCTTTTGACA-3' (reverse) for E6, 5'-TTCCCGGGATCCTTATGGTTTCTGAGAACAGAT-3' (forward) and 5'-TTCCCGGGATCCATGCATGGAGATACACCTACAT-3' (reverse) for E7, 5'-CGGAAGAGTGTCTGGAGCAA-3' (forward) and 5'-GGATGAAGCGGAGTCTGGA-3'(reverse) for hTERT, 5'-AGAGATGCTCCATGCCTTTG-3' (forward), 5'-GCAGACAGGGAGCTGGTTCA-3' (reverse) for ER, and 5'-TCACATCTGTATGCGGAACC-3' (forward), 5'-CGTAACACTTCCGAAGTCGG-3' (reverse) for ERß, and 5'-AACACGTCAGTGGGCAGATG-3' (forward), 5'-GCAGCAATAACTTCAGACATC-3' (reverse) for PR. Total RNA was isolated from the cells using Isogen (Nippon Gene, Tokyo, Japan) according to the manufacturer’s protocol, and cDNA was synthesized from 1 µg of RNA using the RNA PCR kit version 2 (TakaRa, Ohtsu, Japan) with random primers. Typically, 2-µl aliquots of the reverse-transcribed cDNA were amplified by 28 cycles of PCR in 50 µl of 1x buffer [10 mmol/L Tris-HCl (pH 8.3), 2.5 mmol/L MgCl2, and 50 mmol/L KCl] containing 1 mmol/L each of dATP, dCTP, dGTP, and dTTP, 2.5 U of Gene Taq (Nippon Gene), and 0.2 µmol/L primers. Each cycle consisted of denaturation at 94°C for 30 seconds, annealing at 55°C (for HPV16 E6 and E7), 62°C (for PR and ER), and 60°C (for hTERT) for 30 seconds and extension at 72°C for 45 seconds. The PCR products were resolved by electrophoresis in 7% polyacrylamide gels and stained with SYBR green I (FMC BioProducts, Rockland, ME). The efficiency of cDNA synthesis from each sample was estimated by PCR using GAPDH-specific primers as described previously.21
Western Blot Analysis
Cytoplasmic extracts from cells with extended life span (EM cells) were prepared using the method of Schreiber and colleagues.32 Briefly, 1 x 106 cells were collected, washed with PBS, and resuspended in 400 µl of buffer containing 10 mmol/L HEPES (pH 7.9), 10 mmol/L KCl, 0.1 mmol/L ethylenediaminetetraacetic acid, 0.1 mmol/L EGTA, 1 mmol/L dithiothreitol, and 0.5 mmol/L phenylmethyl sulfonyl fluoride, and allowed to swell for 15 minutes, after which 25 µl of a 10% solution of Nonidet P-40 (Sigma-Aldrich, St. Louis, MO) is added and the tube is vigorously vortex for 10 seconds. The homogenate is centrifuged at 12,000 rpm for 30 seconds. The supernatant is recovered as cytoplasmic extracts. Then, 50 µg of cytoplasmic extracts were electrophoresed on a sodium dodecyl sulfate-polyacrylamide gel and transferred to polyvinylidene difluoride membranes. Membranes were blocked in TBST (150 mmol/L NaCl, 20 mmol/L Tris-Cl, pH 7.5, 0.1% Tween) containing 5% nonfat dried milk, and then incubated with specific antibody against PTEN (clone 6H2.1; Cascade Bio Science, Winchester, MA), followed by reaction with horseradish peroxidase-linked anti-mouse IgG. Immunoreactive bands were visualized using the ECL detection system (Amersham), as suggested by the manufacturer.
Telomeric Repeat Amplification Protocol (TRAP) Assay
TRAP assays were performed using the TRAPEZE telomerase detection kit (Intergen, Purchase, NY) according to the manufacturer’s protocol.
Immunocytochemistry and Immunohistochemistry
EM cells were cultured on LAB TEK chamber slides (Nalge Nunc International, Naperville, IL) for 24 hours, fixed with methanol, and subjected to immunocytochemistry. Gland-like structures of EM cells grown in Matrigels were picked up by microscopic manipulation, formalin-fixed, and paraffin-embedded, and thin sections were prepared for immunohistochemistry. Antigen retrieval was then performed for 10 minutes in 1x antigen retrieval solution (Biogenex, San Ramon, CA). These samples were washed with PBS and incubated with mouse monoclonal antibody against pan-cytokeratin (4/5/8/10/13/18) (NCL-C11; Novocastra Laboratories, Newcastle, UK) at a 1:10 dilution, vimentin (NCL-VIM, Novocastra Laboratories) at a 1:100 dilution, CD10 (sc-7632; Santa Cruz Biotechnology, Inc, Santa Cruz, CA), and CD34 (NCL-VIM, Novocastra Laboratories) at a 1:50 dilution and control serum for 1 hour at room temperature. After incubation with an anti-mouse secondary antibody, the immune complex was visualized using the ABC-elite kit (Vector Laboratories Inc., Burlingame, CA).
Telomere Length Analysis by Southern Blotting
Telomere length was analyzed by the terminal restriction fragment (TRF) length assay using a TeloTTAGGG Telomere Length Assay kit (Roche). Briefly, genomic DNA was isolated from cells, and then analyzed by Southern blots probed with a telomere-specific probe to visualize the TRF.
ß-Gal Assay
The ß-gal assay was performed as previously described.33 Briefly, cells were fixed for 5 minutes at room temperature in 3% formaldehyde followed by incubation at 37°C with senescence-associated ß-gal stain solution: 1 mg of 5-bromo-4 chloro-3-indolyl ß-D-galactoside (X-Gal) per/ml/40 mmol/L citric acid/sodium phosphate, pH 6.0/5 mmol/L potassium ferrocyanide/5 mmol/L potassium ferricyanide/150 mmol/L NaCl/2 mmol/L MgCl2. After a 6- to 12-hour incubation, positive staining was confirmed by microscopy.
WST-1 Assay
The proliferative activity of cells treated with sex-steroid hormones was examined using the WST-1 assay according to the manufacturer’s protocol (Boehringer Mannheim, Indianapolis, IN). Briefly, the cells were seeded at 2.5 x 103 cells/well in 96-well flat-bottomed plates and incubated with 100 µl of the growth media overnight at 37°C. After starvation with phenol red-free media containing charcoal-treated fetal bovine serum for 24 hours, the cells were treated with 17ß-estradiol (E2) or 6-methyl-17-hydroxy-progesterone acetate (MPA) at various concentrations for different time periods. A 10-µl aliquot of WST-1 reagent was then added to the media and incubated for 1 to 2 hours at 37°C. The absorbance at wavelengths between 420 to 480 nm was measured using a microplate reader. The relative WST-1 activity was calculated and shown as WST-1 index in which the activity of untreated control samples was normalized to 1.
Anchorage Independence of Growth
A total of 1 x 104 endometrial cancer HEC1 and Ishikawa cells as well as EM-E6E7TERT-1, -2, and -3 cells were seeded onto 6-cm dishes with 0.33% noble agar in DMEM supplemented with 10% fetal calf serum on top of 0.5% base agar in DMEM supplemented with 10% fetal calf serum. Colonies larger than 0.125 mm were counted after incubation for 2 weeks.
Nude Mice Xenograft Experiments
Endometrial cancer HEC1 and Ishikawa ells and as well as EM-E6E7TERT-1, -2, and -3 cells were resuspended in growth media (107/ml) and subcutaneously injected (0.1 ml) at the base of the left flank of female BALB/c nu/nu mice (7 to 9 weeks of age) (SLC, Hamamatsu, Japan). Tumor growth was monitored weekly, and tumor formation was confirmed 60 days after inoculation.
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Results |
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We hypothesized that the early senescence of endometrial epithelial cells that occurs within a couple of passages in primary culture is because of cell-cycle arrest via the Rb pathway. One way to effectively block the Rb pathway is the use of high-risk type HPV E7, which specifically binds Rb and abrogates its functions. High-risk type HPV E6 that binds p53 and interferes with its normal functions is known to support the various activities of E7. Thus, we sought to introduce HPV 16 E6 and/or E7 genes into primary endometrial epithelial cells, with or without introduction of the hTERT gene. Human endometrial samples in late proliferative phase were collected from the 42- and 52-year-old women with regular menstrual cycle, who underwent hysterectomy as a treatment of uterine myoma. These samples were minced and digested in a collagenase solution, and endometrial glands were then isolated from the stromal cells. The isolated glands were directly collected one by one by microscopic manipulation (Figure 1A)
. Approximately 10 glands were seeded on one well of plastic dishes and infected with retroviral vectors for expression of HPV16 E6, E7 and hTERT in various combinations (Figure 1B)
. To dissect the role of E6, mutant E6 (E6
151) in which leucine at amino acid 151 was deleted, was also introduced, instead of wild-type E6. This mutant lacks the transforming ability of E6 but retains the ability to bind and abrogate p53 function.31
Infection was performed once per each sample isolated from the two patients, and stable infectants were then screened by drug-selection. Introduction of hTERT gene alone failed to generate clones with extended life span. Similarly, transfection of E7 or E6 gene alone was not able to produce such clones. Combinatorial transfection of these genes successfully isolated a total of five independent clones (Figure 1D)
, three of which were transfectants harboring the E6, E7, and hTERT genes (EM-E6/E7/TERT-1, -2, and -3), one harbored the E6151, E7, and hTERT genes (EM-E6151/E7/TERT) and the other contained only the E6 and E7 genes (EM-E6E7). Morphologically, these cells exhibited the round shape typical of epithelial cells (Figure 1, B and C)
. Gene expression in primary glands and isolated clone was analyzed by RT-PCR (Figure 2)
. As expected, EM-E6/E7/TERT clones and EM-E6151/E7/TERT cells expressed E6, E7, and hTERT mRNAs, whereas EM-E6E7 cells expressed only E6 and E7 mRNAs (Figure 2A)
. Primary endometrial glands cultured for retroviral infection were also examined, and no expression of E6/E7 mRNA or hTERT mRNA was detected. Our previous study demonstrated that endometrial glands in proliferative phase exhibit telomerase activity.28
However, once they are cultured on dishes, telomerase activity rapidly decreases, suggesting that telomerase activity in endometrial glands cannot be maintained in in vitro culture. Steroid hormone receptor mRNA expression was next examined by RT-PCR (Figure 2B)
. All isolated clones expressed estrogen receptor- (ER-) and progesterone receptor (PR) mRNA, whereas expression of ER-ß mRNA was basically negative but faint expression was observed only in EM-E6/E7/hTERT-3 cells. PTEN protein expression was also examined by Western blot analysis (Figure 2C)
. All clones were confirmed to express significant levels of PTEN protein. The TRAP assay revealed that EM-E6/E7/TERT and EM-E6151E7/TERT cells exhibited telomerase activity while EM-E6/E7 cells did not (Figure 2D)
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, these cells were positive for pan-cytokeratin and vimentin, but not for stromal marker, CD10, or vascular endothelial marker, CD34. Thus, these clones were most likely derived from endometrial epithelial cells, but not from other sources such as fibroblasts or endothelial cells.
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and revealed that EM-E6/E7/TERT-1 and -3 cells had completely normal karyotypes whereas EM-E6/E7/TERT-2 and EM-E6151/E7/TERT had basically diploid normal chromosome number but also contained several nonclonal aberrations. In contrast, EM-E6E7 cells exhibited abnormal chromosome numbers, varying between 43 to 47 at PD 31 when telomere length was 5 to 6 kb.
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All EM cell lineages grew more than PD 20 without any morphological changes. Even EM-E6/E7, which lacked hTERT expression, was able to divide throughout this period, suggesting that expression of E6/E7 allows endometrial epithelial cells to pass through early senescence, without the absolute requirement for hTERT expression. The growth of these cells was further monitored and thereafter we found changes in the growth pattern between cells with and without hTERT expression. The growth pattern of EM-E6/E7/TERT-1, -2, and EM-E6/E7 derived from the same patient is shown in Figure 4
. EM-E6/E7/TERT-1 and -2 passed through PD 40 and finally grew more than PD 100. In contrast, EM-E6E7 cells stopped dividing at PD 40 with morphological changes producing enlarged and flat cells (Figure 5)
. The growth of EM-E6/E7/TERT-3 and EM-E6151/E7/TERT cells derived from another patient was basically similar to that of EM-E6/E7/TERT-1, and -2 cells.
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The telomere length was then examined by Southern blot analysis using telomere probes (Figure 6)
. The telomere length of the EM-E6/E7/TERT-1, -2, and -3 cells was 11 kb, 11 kb, and 8 kb, respectively at PD 20, whereas that of EM-E6/E7 cells was 6 to 7 kb. With the progression of PD, the telomere length of the EM-E6/E/7TERT cells never shortened, and particularly interesting is that EM-E6151/E7/TERT cells had elongated telomeres. In contrast, the telomere length of the EM-E6E7 cells progressively shortened and reached 5.5 kb at PD 40. These findings suggest that telomere-based replicative senescence occurred at PD 40 in EM-E6E7 cells, and that EM cells with hTERT expression overcame this senescence, probably through the maintenance of telomere length.
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EM cells cultured on plastic dishes maintained the round shape typical of epithelial cells. However, on plastic dishes, they failed to compose gland-like structures, the natural characteristics of glandular cells. We therefore examined the ability of EM cells to reproduce the morphological characteristics of glands using the support of an extracellular matrix. EM cells were grown on Matrigel-coated Millicell CM filters. When grown on top of the Matrigel, the cells rapidly expanded on the surface of the Matrigel, exhibiting tight lateral junctions, resulting in cluster formation with a mesh-like network (Fig. 7A, 1)
. When cells were grown inside the Matrigel, they formed colonies with spherical structures (Fig. 7A; 2 and 3)
. To further examine the morphological characteristics of these colonies, each spherical colony was picked up by microscopic manipulation, fixed, and histologically examined. Hematoxylin and eosin staining of sections revealed gland-like structures, in which Matrigel clumps were coated with a cell monolayer (Figure 7B)
. Some cells were found in the center of this structure, reminiscence of stromal or mesenchymal-like cells. However, immunohistochemical analysis revealed that CD-10 was negative in these cells while pan-cytokeratin and vimentin were positive (Figure 7C)
. Although primary endometrial epithelial cells cultured in the same conditions are propagated for only a finite period (usually 2 to 3 weeks), the EM cells were able to survive up to confluence in Matrigel culture, and this was repeated by at least 10 passages as we confirmed.
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We next examined the responsiveness of EM cells to sex-steroid hormones. EM-E6/E7/TERT-2 cells were incubated with phenol red-free media with charcoal-treated fetal calf serum for 24 hours and then treated with 17ß-estradiol (E2) at 1 to 100 nmol/L for different time periods. Cell growth was examined by WST-1 assays. ER- and PR-positive Ishikawa cells and C33 A cells lacking them were used as positive and negative controls (Figure 8)
. Treatment with E2 at 10 to 100 nmol/L promoted cell growth by approximately twofold of WST-1 activity in Ishikawa and EM-E6/E7/TERT-2 cells. Time-course experiments using EM-E6/E7/TERT-2 cells revealed that the effect of E2 appeared at 24 hours and peaked at 48 hours with a gradual decrease to 72 hours in this assay system. The cells were then treated with progesterone (medroxyprogesterone acetate: MPA) in a similar manner. MPA at 100 nmol/L significantly inhibited growth of Ishikawa and EM-E6/E7/TERT-2 cells. Of particular interest is the time-dependent effect of MPA in EM-E6/E7/TERT-2 cells; it transiently activated cell growth in a short period within 24 hours, thereafter inhibiting growth with maximal inhibition at 72 hours after the treatment. Thus, the effect of MPA was biphasic. Similar findings were also observed in EM-E6/E7TERT-1 or -3 cells (data not shown). No regulation was observed in C33A cells in response to E2 or MPA stimulation. These findings suggest that the immortalized EM cells preserved their responsiveness to sex-steroid hormones.
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Although EM-cells exhibited the characteristics of glands in Matrigel culture and sustained the responsiveness to sex-steroid hormones, these cells obtained unlimited replicative capacity more than PD 100 and it was unclear whether these cells had transformed phenotypes. We thus sought to examine the growth properties of EM cells on soft agar or in nude mice. A total of 104 EM-E6/E7/TERT-1, -2, and -3 cells were seeded in soft agar on 6-cm dishes and colonies with diameters greater than 0.125 mm counted 14 days after seeding. Endometrial cancer HEC-1 and Ishikawa cells were similarly examined as positive controls. Although HEC-1 and Ishikawa cells formed distinct colonies (mean colony number, 116 ± 13.9 and 287 ± 23.9, respectively), EM-E6E7TERT cells did not (Figure 9)
. Next, we evaluated tumorigenicity using nude mice. EM-E6/E7/TERT-1, -2, and -3 cells (106) as well as Ishikawa cells were subcutaneously injected into five nude mice and tumor formation then monitored 60 days after injection. Tumors formed in all of the mice injected with Ishikawa cells, whereas no tumor formed in mice injected with EM cells (Figure 9)
. These findings suggest that EM cells did not obtain transformed phenotypes.
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Discussion |
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What are the molecular mechanisms by which HPVE6 and E7 enable endometrial epithelial cells to overcome this early senescence? Rb-mediated cell-cycle control mechanisms have been demonstrated as a major event toward immortalization of epithelial cells,22,34
and blockage of the Rb pathway by HPV E7 is likely to play a critical role in extended life span. However, E7 alone seems to be insufficient, because introduction of E7 alone failed to isolate any clones with extended life span. What is the role of E6 in overcoming early senescence? It is possible that abrogation of p53 function by E6 may cooperatively act to block the Rb pathway. A recent study demonstrated that activated E2F released by E7-induced Rb inactivation up-regulates p14ARF expression, which relocalizes MDM2 to the nucleolus and inhibits the ability of MDM2 to degrade p53.35-37
Eventually, HPV E7 enhances p53 function via induction of p14 ARF, which may induce apoptosis in some cell types. Abrogation of p53 function by E6 may block these adverse effects of E7. As shown in the present study, an E6 mutant (E6151) lacking the transforming ability of E6 but retaining the ability to target p53 for degradation had a similar effect to wild-type E6, suggesting that p53 inactivation is the major function of E6 to support E7. Based on these findings, we propose that two critical pathways, the Rb and p53 pathways, are involved in early senescence of endometrial glandular cells. This is consistent with the recent observation that a two-stage, p16- and p53-dependent senescence limits the replicative potential of keratinocytes.27
Thus, the mechanisms of telomere-independent senescence may be basically common among epithelial cells. The requirement for both Rb and p53 inactivation to bypassing senescence is reminiscent of the roles of SV40 large T (LT). Generally, normal cells can bypass senescence through inactivation of Rb and p53 by SV40 LT,38
and HPV E6/E7 may have a similar function. However, SV40 LT-introduced cells frequently have severe genetic abnormalities including aneuploidy and lose the normal structural and functional characteristics of the original cells.39
In contrast, our E6/E7 system appears to minimize the genetic abnormalities, successfully isolating EM-clones with normal karyotypes (EM-E6/E7/TERT-1 and -3) and therefore has the great advantage of establishing immortalized cell lines that sustain the natural features of the primary cells.
The failure of E6/E7-expressing clones (EM-E6/E7) to overcome the second senescence is obviously because of the lack of hTERT expression. The telomere length of the EM-E6/E7 cells finally reached 5 to 6 kb at the time of senescence, whereas the other clones expressing hTERT maintained telomere length and overcame the second senescence. We thus conclude that endometrial glandular cells have multistep senescence programs that can be overcome by both Rb/p53-inactivaiton and telomerase activation. Telomerase activation seems to be necessary not only to overcome the second senescence, but also to maintain chromosome integrity because EM-E6/E7 cells lacking telomerase activity had severe chromosome abnormalities.
Several previous studies have demonstrated that HPV E6 can activate telomerase.40 The molecular mechanisms of this activation are complex and our previous study showed that E6 activates the hTERT promoter in a c-Myc-independent manner.41 It is known that HPV E6 and E7 can immortalize certain types of normal cells, in which E6 substitutes for telomerase to overcome telomere-dependent senescence22 cells. However, we confirmed that HPV E6 failed to activate telomerase in endometrial epithelial cells as shown in EM-E6/E7 cells that lack detectable telomerase activity. Thus, the immortalization by HPV E6 and E7 appears to be a cell type-specific phenomenon, and only special cell types that allow telomerase activation by E6 may conform to the above model.
Our immortal endometrial epithelial cells may prove useful for various experimental models. EM cells maintain responsiveness to sex steroids, as estrogen activated growth of EM cells, while progesterone inhibited it, which mimics the physiological roles of estrogen and progesterone in endometrium. The growth inhibition by progesterone is of particular interest, considering the role of progesterone in endometrial biology. Progesterone inhibits growth or promotes differentiation of endometrial epithelial cells in the secretory phase of the menstrual cycle. It has widely been used as an anti-cancer drug for endometrial cancers.42
Various signaling pathways such as the MAP kinase pathway, are activated on short-term exposure to progesterone, resulting in transient proliferation of breast cancer cells,43
while progesterone induces p21 expression in the long term, associated with cell growth inhibition.44,45
EM cells were able to reproduce this biphasic pattern of progesterone action, as described in Figure 8
, and will therefore be a desirable model with which to research signaling pathways or the functions of progesterone.
EM cells will be available for organotypic culture or mice transplantation systems, which might be useful as implantation model. Alternatively, these cells may be used to establish a carcinogenesis model. As shown in the present study, these cells do not have transformed phenotypes. What factors are required for transformation of these cells? One of the major factors involved in endometrial carcinogenesis is PTEN, which is frequently mutated in endometrial cancers.4,46 We have confirmed no PTEN mutation in a series of EM cells (data not shown). Therefore, we are currently targeting PTEN in EM cells by the introduction of dominant-negative PTEN or siRNA techniques to define the genetic elements required for transformation.
In summary, we successfully immortalized human endometrial glandular cells by the introduction of HPV E6/E7 and hTERT. Our data show that endometrial glandular cells require Rb/p53 inactivation and telomerase activation for immortalization. The lack of transformed phenotypes of these cells supports the notion that additional factors are required for complete endometrial carcinogenesis. Our system to introduce HPV E6/E7 and hTERT provides the potential to generate relatively normal immortalized cells for research endeavors that have been limited so far by a limited life span of primary cells. We anticipate that our established lines will be useful in various experimental models with which to investigate endometrial biology and carcinogenesis.
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Acknowledgements |
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Footnotes |
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Supported in part by a grant-in-aid for the Second Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health and Welfare, Japan.
Accepted for publication August 5, 2003.
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 T. Uekita, L. Jia, M. Narisawa-Saito, J. Yokota, T. Kiyono, and R. Sakai CUB Domain-Containing Protein 1 Is a Novel Regulator of Anoikis Resistance in Lung Adenocarcinoma Mol. Cell. Biol., November 1, 2007; 27(21): 7649 - 7660. [Abstract] [Full Text] [PDF]
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 M. Kutsukake, R. Ishihara, M. Yoshie, H. Kogo, and K. Tamura Involvement of insulin-like growth factor-binding protein-related protein 1 in decidualization of human endometrial stromal cells Mol. Hum. Reprod., October 1, 2007; 13(10): 737 - 743. [Abstract] [Full Text] [PDF]
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T. Yugawa, K. Handa, M. Narisawa-Saito, S.-i. Ohno, M. Fujita, and T. Kiyono Regulation of Notch1 Gene Expression by p53 in Epithelial Cells Mol. Cell. Biol., May 15, 2007; 27(10): 3732 - 3742. [Abstract] [Full Text] [PDF]
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K. Kato, M. Yoshimoto, K. Kato, S. Adachi, A. Yamayoshi, T. Arima, K. Asanoma, S. Kyo, T. Nakahata, and N. Wake Characterization of side-population cells in human normal endometrium Hum. Reprod., May 1, 2007; 22(5): 1214 - 1223. [Abstract] [Full Text] [PDF]
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 C.-H. Cui, T. Uyama, K. Miyado, M. Terai, S. Kyo, T. Kiyono, and A. Umezawa Menstrual Blood-derived Cells Confer Human Dystrophin Expression in the Murine Model of Duchenne Muscular Dystrophy via Cell Fusion and Myogenic Transdifferentiation Mol. Biol. Cell, May 1, 2007; 18(5): 1586 - 1594. [Abstract] [Full Text] [PDF]
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 K. Handa, T. Yugawa, M. Narisawa-Saito, S.-i. Ohno, M. Fujita, and T. Kiyono E6AP-Dependent Degradation of DLG4/PSD95 by High-Risk Human Papillomavirus Type 18 E6 Protein J. Virol., February 1, 2007; 81(3): 1379 - 1389. [Abstract] [Full Text] [PDF]
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Y.-l. Wang, W. Qiu, H.-c. Feng, Y.-x. Li, L.-z. Zhuang, Z. Wang, Y. Liu, J.-q. Zhou, D.-h. Zhang, and G. S.W. Tsao Immortalization of normal human cytotrophoblast cells by reconstitution of telomeric reverse transcriptase activity Mol. Hum. Reprod., July 1, 2006; 12(7): 451 - 460. [Abstract] [Full Text] [PDF]
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P. Chapdelaine, J. Kang, S. Boucher-Kovalik, N. Caron, J. P. Tremblay, and M. A. Fortier Decidualization and maintenance of a functional prostaglandin system in human endometrial cell lines following transformation with SV40 large T antigen Mol. Hum. Reprod., May 1, 2006; 12(5): 309 - 319. [Abstract] [Full Text] [PDF]
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 S Hombach-Klonisch, A Kehlen, P A Fowler, B Huppertz, J F Jugert, G Bischoff, E Schluter, J Buchmann, and T Klonisch Regulation of functional steroid receptors and ligand-induced responses in telomerase-immortalized human endometrial epithelial cells J. Mol. Endocrinol., April 1, 2005; 34(2): 517 - 534. [Abstract] [Full Text] [PDF]
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M. Terai, T. Uyama, T. Sugiki, X.-K. Li, A. Umezawa, and T. Kiyono Immortalization of Human Fetal Cells: The Life Span of Umbilical Cord Blood-derived Cells Can Be Prolonged without Manipulating p16INK4a/RB Braking Pathway Mol. Biol. Cell, March 1, 2005; 16(3): 1491 - 1499. [Abstract] [Full Text] [PDF]
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