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Carcinogenesis Advance Access originally published online on July 8, 2006
Carcinogenesis 2007 28(1):174-182; doi:10.1093/carcin/bgl115
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Knockdown of p53 combined with expression of the catalytic subunit of telomerase is sufficient to immortalize primary human ovarian surface epithelial cells

Gong Yang1, Daniel G. Rosen1, Imelda Mercado-Uribe1, Justin A. Colacino1, Gordon B. Mills2, Robert C. Bast, Jr3, Chenyi Zhou1 and Jinsong Liu1,*

1 Department of Pathology, The University of Texas M. D. Anderson Cancer Center Houston, TX, USA
2 Department of Molecular Therapeutics, The University of Texas M. D. Anderson Cancer Center Houston, TX, USA
3 Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center Houston, TX, USA

*To whom correspondence and requests for reprints should be addressed at: Department of Pathology, Unit 85, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA. Tel: +1 713 745 1102; Fax: +1 713 792 5529; Email: jliu{at}mdanderson.org


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Ovarian cancer is developed from a single layer of thin epithelial cells covering the surface of ovary, named human ovarian surface epithelial cells. Like all primary human cells, human ovarian surface epithelial cells have a finite life span and will go into senescence and eventually die when cultured in vitro. Immortalized human ovarian surface epithelial cells will provide an important model system with which to study ovarian cancer initiation and progression. Here, we show that silencing p53 expression with retrovirus-mediated small interfering RNA can delay the senescence and extend cell passage number, but is not sufficient to immortalize normal ovarian surface epithelial cells. Introduction of the catalytic subunit of telomerase is similarly insufficient to achieve immortalization. However, concurrent disruption of p53 expression with small interfering RNA retroviral constructs and ectopic expression of the catalytic subunit of telomerase was sufficient to induce cellular immortalization in 3 of 3 human ovarian surface epithelial cell cultures tested. The immortalzation is associated with increased telomerase activity and telomere length, and attenuated response of cell-cycle regulatory proteins to irradiation. The resultant immortal cells continued to express the same specific cytokeratins 8 and 18 as parental cells did, indicating that the epithelial characters are still maintained in the immortal cells. In addition, the immortalized cells are non-tumorigenic and nearly diploid, which is in constrast with one immortalized by SV40 T/t antigens and hTERT. As both p53 pathway dysfunction and activation of telomerase are commonly present in human ovarian cancer, these immortal cells provide an authetic cell model system for the study of the human ovarian cancer initiation, progression, differentiation and chemoprevention.

Abbreviations: hTERT, human telomerase reverse transcriptase; RB, retinoblastoma; SV40 ER, simian virus 40 early region; TRAP, telomeric repeat amplification protocol


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
More than 22 000 women in the United States will develop ovarian cancer in 2006 and 60% of these women will eventually succumb to the disease (1). The dismal prognosis associated with ovarian cancer results from a lack of ability to detect disease at an early, treatable stage and a lack of effective therapies for advanced disease, which is in part due to our poor understanding of its etiology. Defining the molecular basis of ovarian cancer progression may provide a key to early detection and development of more effective treatments for this disease. It would be tremendously valuable to have model system that faithfully mimics the genetic change from ovarian cancer in patients to study the biology and etiology of this deadly disease. Recently, several mouse ovarian cancer models have been developed to address these questions. Orsulic et al. (2) developed a mouse model system in which an avian retroviral gene delivery technique is used to introduce several genes into mouse ovarian surface epithelial cells. In that system, introduction of any two of the oncogenes c-Myc, K-ras, or Akt onto a mutated p53 background led to the formation of ovarian tumors that were similar to human ovarian cancer. A more recent study showed that about half of female transgenic mice expressing the transforming region of SV40 under the control of the Mullerian inhibitory substance type II receptor gene promoter developed bilateral ovarian tumors (3). Mutations in K-ras and PTEN lead to the development of ovarian endometrioid carcinoma (4), whereas concurrent inactivation of p53 and RB leads to the development of serous carcinoma from mouse ovarian surface epithelial cells (5). Although these mouse models provide a powerful tool to study ovarian cancer initiation and progression, it remains to be determined how these murine systems faithfully recapitulate the development of ovarian cancer in the human patient, as human epithelial cells require the disruption of several additional pathways in order to achieve transformation and human cancers are different in multiple aspects from murine cancer, as these differences have been reviewed by Rangarajan and Weinberg (6).

Human ovarian surface epithelial cells are a single layer of thin epithelial cells covering the surface of the ovary and are the origin of most epithelial ovarian cancer. Several laboratories used cultured human ovarian surface epithelial cells as model system to study ovarian cancer progression. Unfortunately, like all human primary cells, human ovarian surface epithelial cells, can divide only a finite number of times in culture before going replicative senescence (7). Loss of spontaneous immortalization without viral oncogenes has never been reported for human ovarian surface epithelial cells. Therefore, it is urgently needed to develop immortalized human ovarian epithelial cell lines as an alternative model system to manipulate the genetic changes detected in human ovarian cancer. We report here that we have successfully achieved immortalization of human ovarian surface epithelial cells by applying retrovirus-mediated small interfering RNA-mediated silencing technology to knock down the expression of p53 in these cells and together with ectopic expression of hTERT. These immortal cells thus provided a first authentic viral protein-free model system to study the initiation and progression of human ovarian cancer.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Growth media
Normal human ovarian surface epithelial cells were grown in a 1:1 mixture of MCDB105 and M199 supplemented with 10–20% fetal bovine serum (FBS) (8,9). Previous formulations consisted of a 1:1 mixture of MCDB202 and M199 supplemented with a variety of agents [e.g. epidermal growth factor (10) at 20 ng/ml] to increase the cell proliferation (8,9). MCDB105 (a modification of medium F12) was found to enhance the growth potential of the cells while maintaining their epithelial phenotype.

Isolation of human primary ovarian surface epithelial cells
Surface epithelial cells from normal ovaries from surgical residual specimen were isolated using standard and Institutional Review Board (IRB)-approved protocols (8,9). Briefly, the surgical samples of ovarian tissue were immediately placed in plastic bags and kept on ice. Tissues were gently washed twice (5 min for each wash) with phosphate-buffered saline (PBS) containing 10% penicillin and streptomycin. Afterward, the outer surface of the ovarian tissues was scraped gently with a scalpel blade or more firmly with the blunt side of the blade. The scraped cells were gently transferred to a tissue culture dish containing the growth medium, and grown for 7–10 days at 37°C in 5% CO2 without changing the medium. Trypsinization was used to remove any stromal fibroblasts, after which the cells were usually split in a 1:3 ratio (33.33%, passage 1) when they reached 85% confluence in average. In this way, one passage is {cong}1.3 population doublings (PD), which was calculated by referring to the method reported in literature (11). Three primary cell cultures were maintained this way (OSE103, OSE137 and OSE151) and used for additional experimentations.

Senescence-associated ß-galactosidase staining
Cells cultured either in slide chambers or 24-well dishes were washed three times with PBS and fixed with buffer (PBS with 2% formaldehyde and 0.2% glutaraldehyde) for 4 min at room temperature. The cells were then washed three more times with PBS and incubated with citric acid/sodium phosphate buffer (pH 6.0) (composed of 0.15 M NaCl, 0.02 M MgCI2, 1 mg/ml X-gal, 0.005 M potassium ferricyanide and 0.0016 M citric acid) for 2–5 h at 37°C in the dark.

Viral vector construction
The first pair of DNA oligonucleotides used to generate siRNA-1 against p53mRNA was P1: 5'-GGCAGTCACAGCACATGACGttcaagagaCGTCATGTGCTGTGACTGCCCTTTTTg-3' and P2: 5'aattcAAAAAGGGCAGTCACAGCACATG ACGtctcttgaaCGTCATGTGCTGTGACTGCC-3' (lowercase letters indicate the loop nt, italic lowercase letters are EcoR1-compatible nt, and bold italic uppercase letters indicate the sequence of the U6 transcription terminator), which target the open reading frame of p53 mRNA at 492–510 nt. The second pair of DNA oligonucleotides used to generate siRNA-2 against p53 mRNA was P3: 5'- GGACTCCAGTGGTA ATCTACTtcaagagaAGTAGATTACCACTGGAGTCCCTTTTTg -3' and P4: 5'- aattcAAAAAGGGCAGTCACAGCACATGACGtctcttgaaCGTCATGTGCTGTGACTGCC-3', which target the open reading frame of p53 mRNA at 775–794 nt. Oligonucleotides were annealed in a buffer containing 100 mM Tris–HCl (pH 7.5) and 20 mM MgCl2 for 10 min at 95°C followed by 20 min at 65°C. The annealed DNA was ligated into pBabe-U6/puromycin vector that had been cut with ApaI, blunted with Klenow enzyme, and digested with EcoRI (12). pBabe-U6/GFP siRNA expression vector was described previously (13). pBabe-hTERT/hygromycin was a gift from Dr Robert Weinberg (MIT).

Packaging cell lines
Phoenix amphotropic packaging cells from the American Type Culture Collection (Manassas, VA) were maintained at 37°C in Dulbecco's modified Eagle's medium with 10% FBS, 1 mM sodium pyruvate, 1 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. The cell line stably expresses gag-pol and envelope proteins, which are necessary for the formation of active viral particles (14).

Generation of retrovirus and infection of primary cells
To create retroviruses expressing p53 siRNA, hTERT, GFPsiRNA and vector control, phoenix amphotropic cells (95% confluence) were subjected to calcium/chloroquine–mediated transfection with 20–25 µg of the retroviral constructs noted above, and the retroviruses were harvested as described previously (15). Breast cancer cells MDA-MB-435, known expressing wild-type p53 (16), were maintained at 37°C in Dulbecco's modified Eagle's medium containing 10% FBS, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 µg/ml streptomycin (all from Sigma Chemical, St Louis, MO), and used for virus infection at 40% of confluence (17). The primary ovarian epithelial cells OSE103, OSE137 and OSE151 were cultured in 60 mm dishes with complete medium containing 15% FBS and used for virus infection when they reached 50–85% confluence as described previously (15). The infected cells were then selected at 37°C in medium containing either puromycin (1–5 µg/ml) or hygromycin (20–100 µg/ml) twice for 48–72 h each time depending on cell lines and the efficiency of virus infection, after which the cells were grown in media without these drugs and used for the various analyses described below.

Telomerase activity assay
Telomerase activity was detected by using the TRAP Telomerase Detection Kit (S7700, Chemicon International, Temecula, CA) according to the kit manual. Briefly, 105–106cells were pelleted, washed twice with PBS, and resuspended in 200 µl of 1x CHAPS Lysis Buffer (included in the kit). The suspension was incubated on ice for 30 min and then spun in a microcentrifuge at 12 000x g for 20 min at 4°C. The supernatant (160 µl) was transferred into a fresh tube, and the protein concentration was determined by spectrometry (OD 595 nm) by using Bio-Rad protein assay dye reagent (Bio-Rad Laboratories, Hercules, CA). The protein extract (1 µg/sample) was used for PCR amplification, which consisted of 30 min at 30°C followed by two-step PCR (30 s at 94°C and 30 s at 59°C) for 30 cycles in a thermocycler. The resultant products were separated on a 12.5% non-denaturing SDS gel (no urea) in 0.5x Tris–borate–EDTA buffer by electrophoresis and visualized by staining the gel with ethidium bromide.

Telomere length assay
Genomic DNA was prepared from 107–108 cells incubated with 1 ml of digestion buffer containing 100 µg/ml proteinase K and 0.5% SDS (w/v) for 12–18 h at 50°C, followed by phenol–chloroform extraction and ethanol precipitation. Portions of genomic DNA (2 µg) were digested with HinfI/RsaI enzymes and separated on a 0.8% agarose gel, then transferred onto a positively charged nylon membrane in 20x sodium chloride–sodium citrate transfer buffer. After southern transfer, the transferred DNA on the wet blotting membrane was fixed by UV-crosslinking (120 mJ). Telomeric DNA was detected with a digoxigenin-labeled telomere probe (TAGGG) according to the instructions for the hybridization and chemiluminescence detection kit (TeloTAGGG Telomere Length Assay, Roche Applied Science, Indianapolis, IN). The positive control DNAs (low, 3.9 kb; and high, 10.2 kb) were purified genomic DNAs from immortalized cell lines supplied with the kit. The average lengths of terminal restriction fragments of telomeres were quantitated according to the instruction of the kit.

Immunohistochemical staining
Cells at passage 50 were cultured on chamber slides (LAB-TEK, Nalgene NUC International, Naperville, IL) for 24 h until they reached 60–70% confluence. The cells were fixed for 10 min with 10% formaldehyde and washed twice with PBS. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide for 10 min, followed by two washes with PBS. Next, the slides were incubated for 30 min with protein blocking solution (PBS with 5% normal goat serum and 0.5% bovine serum albumin) to avoid non-specific binding of the antibodies and to reduce background staining. The slides were then incubated for 1 h at room temperature with antibodies against vimentin (V9, 1:100; Biocare Medical, Concord, CA), pan-cytokeratin (LU-5, 1:100; Biocare Medical, Concord, CA), and p53 (D07, 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA), followed by two washes with PBS. Next, the slides were treated with the secondary antibody (universal link, Biocare Medical, walnut creek, CA) for 10 min and washed twice with PBS. Finally, a solution of streptavidin–horseradish peroxidase (Biocare Medical) was applied for 10 min, after which the cells were treated for 3–5 min with 0.05% 3',3-diaminobenzidine tetrahydrochloride (freshly prepared in 0.05 M Tris buffer at pH 7.6) and then counterstained with hematoxylin, dehydrated and mounted.

Western blot analysis
Precast 4–20% Tris–glycine gels (Cambrex, East Rutherford, NJ) were used to run protein samples made from cells at passage 50 under denaturing conditions. Proteins were transferred on to polyvinylidene fluoride (PVDF) membranes (Amersham Pharmacia Biotech Limited, UK), and the blot was blocked in 10% non-fat milk (Bio-Rad) overnight at 4°C. The antibodies used in this study to detect p15 (C-20/sc-612, 1:500), cyclin D1 (M-20/SC-718, 1:400), cdk2 (M-2/sc-163, 1:1000) and p53 (DO-1/sc126, 1:2000) were purchased from Santa Cruz Biotechnology. The antibodies against p27kip (G173–524, 1:1000) and pRb (G3–245, 1:1000) were from BD Pharmingen (San Diego, CA). The anti-p21waf1/cip1 (DCS60, 1:1000) was from Cell Signaling Technology (Danvers, MA). The ß-actin antibody (AC15/A5441, 1:50 000) was from Sigma. The anti-p16INK4A (Ab1, 1:5000) was from Lab Vision Corporation (Fremont, CA). Antibodies to vimentin [Ab-2 (V9); Fremont, CA] and cytokeratins 8 and 18 (C51; Biogenesis Ltd, Poole, UK) were used for western blot analysis. The secondary antibodies against mouse IgG (RPN4201) or rabbit IgG (RPN4301) conjugated with horseradish peroxidase were from Amersham Bioscience (Piscataway, NJ). Western blot reagents were from a western blot electrochemiluminescence kit (Amersham Pharmacia).

Soft agar assay
Immortalized cells (105) at passage 30 (PD 42) were suspended in 2 ml of ovarian epithelial cell medium with 0.35% agarose (Life Technologies, Rockville, MD), and the suspension was placed on top of 5 ml of solidified 0.7% agarose in 60mm dishes. Triplicate cultures for each cell type were maintained for 14 days at 37°C in an atmosphere of 5% CO2 and 95% air. Fresh medium was added after 1 week. Colonies larger than 50 µm in diameter were counted after 2 weeks. These experiments were repeated twice.

Tumorigensis assay in vivo
Equal numbers (5 x 106) of immortalized cells at passage 30 (PD 42) in which p53 had been silenced (T137 p53i, T103 p53i and T151 p53i) were harvested by trypsinization, washed twice with PBS, resuspended in 0.1 ml of saline, and injected either subcutaneously into 4–6-week-old BALB/c athymic nude mice (Jackson Laboratory, Bar Harbor, ME). T29H cells were used as a positive control. The mice were kept in a pathogen-free environment and checked every 2 days for 5 months. The date at which the first grossly visible tumor appeared was recorded, and tumor size was measured every 2 days thereafter. Mice were sacrificed when tumors reached 1.5 cm in diameter.

Cytogenetic karyotyping
Immmortal cells (T103p53i, T151p53i and T137p53i) at passage 45 (PD 58.5) were fed 24 h and harvested for chromosome preparation using the standard procedures: cells were exposed to Colcemid (0.04 µg/ml) for 1 h, subjected to hypotonic treatment (0.075 M KCl for 20–25 min at room temperature), and fixation in a mixture of methanol and acetic acid. Slides were stained in Giemsa and examined for structural and numerical abnormalities. A minimum of 30 metaphase spreads was analyzed for each cell line, and representative spreads were captured using a Genetiscan imaging system. Difference in proportions was calculated using chi-squared analysis of Fisher's exact test as appropriate.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Silencing of p53 expression by retrovirus-mediated siRNA against p53
Because the p53 pathway is commonly dysregulated in ovarian cancer (18), we used retrovirus-mediated expression of siRNA to stably silence p53 expression. We designed two pairs of oligonucleotides that encode siRNAs against p53 (Figure 1A). We first examined changes in p53 protein in MDA-MB-435 breast cancer cells, which normally express high levels of the wild-type p53 protein. After retroviral infection with the anti-p53 siRNA, the infected cells were selected with puromycin, and the resulting cells were analyzed by western blotting. Both anti-p53 siRNAs markedly decreased p53 protein expression level as compared with the parental or U6 vector controls (Figure 1B). In quantitation, the p53 protein was reduced by 92.5% in the siRNA-1-treated cells, and by 89% in the siRNA-2-treated cells. As a control for global effect of introduced small interfering RNA, we infected MDA-MB-435 cells with siRNA against green fluorescence protein (GFP) using GFPsiRNA viruses described from our previous study (13), and analyzed the cells after drug selection. The p53 expression level was not affected in these cells infected with retrovirus-expressing siRNA against GFP as detected by western blot (Figure 1B), suggesting that effect of siRNA against p53 is specific not due to global non-specific silencing from introduced siRNA. We chose to use the anti-p53 siRNA-1 for the primary cell infections in the subsequent study.


Figure 1
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  		Fig. 1 Retroviral constructs that express siRNA against p53 and their effect on MDA-MB-435 breast cancer cells. (A) Schematic overview of two different retroviral constructs against p53. The corresponding oligonucleotides were synthesized and ligated into a pBabe-U6 vector, which was made from pBluescript/U6 (13). (B) Western blot of p53 protein expression in parental, U6 vector, GFP siRNA (GFPi) and p53 siRNA-treated cells. Quantitation was performed by densitometry and analyzed by ImageQuant version 5.1 (Molecular Dynamics).

 
Extension of passage number in primary cells by using retrovirus-expressing siRNA against p53
To examine the effect of pBabe/U6/p53 siRNA on senescence, we cultured three human ovarian surface epithelial cell lines (OSE103, OSE137 and OSE151) for 4–8 passages (i.e. before senescence; Figure 2A, F and K) and then infected the cells with the virus expressing p53 siRNA-1, U6 vector and GFP siRNA. At 5–8 passages, almost all of the uninfected primary OSE cells displayed the senescence phenotype indicated by enlarged cell size (Figure 2B, G and L) and senescence-associated acidic ß-galactosidase (SA-ß-gal) staining (pH 6.0) (Figure 2C, H and M). Introducing the p53 siRNA led to another 3 or 4 passages compared with parental and U6 control cells (Figures 2D, I and N and 3A) before they went to senescence, suggesting that blocking p53 in this way is able to delay the senescence thus to extend the passage numbers although is not sufficient to bypass the senescence, as all cell growth did reach a plateau and became senescent after 8–13 passages. OSE cells infected with retrovirus expression of siRNA against GFP has no effect on p53 expression in the presence or absence of {gamma}-irradiation compared with U6 vector-treated cells (Figure 3C), demonstrating that observed effect is p53-siRNA-specific and not caused by global non-specific effect during infection of retrovirus or induced by non-specific siRNA expression.


Figure 2
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  		Fig. 2 Morphology of cultured human ovarian surface epithelial cells at various passages. Parental cells at P1 or P2 (A, F and K). Parental cells at P5 or P6 displaying typical enlarged, flattened senescent morphology (B, G and L) or acidic ß-galactosidase staining (C, H and M). Continued proliferation after exposure to retrovirus-mediated siRNA against p53 (D, I and N). Morphology of immortalized cells after retrovirus-mediated siRNA against p53 plus ectopic hTERT expression (E, J and O). 1 passage is {cong}1.3 cell PD.

 


Figure 3
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  		Fig. 3 Growth curve of various cells derived from OSE103, OSE137, and OSE151 (A). Senescence comparison between cells expressing GFPi and p53i at the same passage (P6) (B). p53 expression detected by western blot in U6 vector virus- (OSE137) or GFPi virus-infected cells (OSE137, OSE 103 and OSE151) at passage 4 in the presence or absence of irradiation, showing that GFP siRNA had no effect on expression of p53 (C).

 
Concurrent expression of hTERT and p53 siRNA results in immortalization
The experiment described above demonstrated that disruption of p53 by using retrovirus-mediated siRNA against p53 was not sufficient to immortalize human ovarian surface epithelial cells. Thus, we infected either primary cells or cells with siRNA against p53 with another retroviral expression vector to express hTERT. Introduction of hTERT into primary cells without siRNA against p53 failed to stimulate cell growth. In contrast, introduction of hTERT to cells in which p53 had been silenced led to continued robust growth even after 50 passages in all three cell lines tested (Figures 2E, J and O and 3A). To date, these cells have been in culture for >9 months without a decrease in division rate, suggesting that they have been immortalized. A telomere repeat amplification protocol (TRAP) assay showed marked increases in telomerase activity in cells with ectopic expression of hTERT and p53 siRNA (T103 p53i, T137 p53i and T151 p53i) at passages 45–49 (P45–49) as compared with the parental primary cells (OSE103, OSE137 and OSE151) at passages 2–3 (P2–3) (Figure 4A). As expected, telomeres in these immortalized cells were longer than those in the parental cells (Figure 4B). The average lengths of telomere were 6.8, 8.9 and 6.5 kb for OSE 137, OSE 151 and OSE 103 cells, respectively, while these for immortalized counterparts were at 10.4, 18.2 and 16.4 kb, respectively.


Figure 4
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  		Fig. 4 Telomerase activity as determined by TRAP assay (A) and telomere length (B) in primary (OSE103, OSE151 and OSE137) at passage 2 or 3 and immortalized ovarian surface epithelial cells expressing p53 siRNA (T103p53i, T151p53i and T137p53i) at passage 45 or 49. Low and high DNA controls used in the assay were purified genomic DNA from immortal cell lines supplied in the kit. The average length of TRFs was calculated according to the kit instructions.

 
Immunophenotyping of primary and immortalized ovarian surface epithelial cells
To characterize potential phenotypic changes during the immortalization process, we stained all three immortal cell lines T103p53i, T137p53i and T151p53i for pan-cytokeratin, vimentin, and p53 (Figure 5). All of three lines were positive for pan-cytokerain and vimentin, and negative for p53, similar to the parental primary culture (data not shown). We further confirmed these results using western blot with specific antibodies against cytokeratins 8 and 18, two commonly expressed cytokeratin for human ovarian surface epithelial cells, and vimentin. As shown in Figure 5 (WB), both parental culture and immortalized cells showed identical expression pattern for cytokeratins 8 and 18 and vimentin, while no p53 protein expression was detected. These data demonstrated that the epithelial properties were maintained in the immortal cell lines.


Figure 5
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  		Fig. 5 Immunophenotypes of immortalized ovarian surface epithelial cells. T103 p53i, T137 p53 or T151 p53i cells were expressing cytokerain and viementin at passage 50, but not p53. Vimentin and specific cytokeratins 8 and 18 were further detected from the protein samples made from the cells at the same PD levels, compared with those proteins in parental cells at passage 2.

 
Dysregulation of cell-cycle regulatory proteins by p53 siRNA expression alone or in combination with hTERT
To examine the mechanism in cell-cycle progression involved by silencing of p53, we examined the expression of the cell-cycle regulatory proteins p15, p16, p21, p27, cdk2 and cyclin D1 in OSE137 (parental), OSE137 expressing p53 siRNA and immortalized OSE137 cells expressing both p53 siRNA and hTERT. The primary OSE137 cells expressed very little p53 protein (Figure 6, parental). As expected, irradiation of these cells at a dose of 10 Gy resulted a marked increase in p53 protein. The levels of pRb, p15, p16, p21 and cdk2 were also increased following irradiation. However, cells in which p53 had been silenced showed only weak increase after irradiation (Figure 6, p53 siRNA IR). Interestingly, silencing of p53 led to accumulation of Rb relative to the parental (non-irradiated) control, suggesting that the Rb pathway was activated in the absence of a functional p53. The response of immortal cells to irradiation was attenuated for most of the cell-cycle regulatory protein including p53, p15, p16, p21 and cdk2. These results demonstrated that immortalization is associated with change of several cell-cycle regulatory proteins and attenuated response to irradiation.


Figure 6
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  		Fig. 6 Changes in cell-cycle proteins associated with introduction of retrovirus-mediated siRNA against p53 and ectopic expression of hTERT in OSE 137 cells at passage 50 compared with the parental cells at passage 2. Western blotting was used to assess the amounts of p53, pRb, p15, p16, p21, p27, cdk2 and cyclin D1. IR, irradiation.

 
Immortalized human ovarian surface epithelial cells are not tumorigenic
Three of the siRNA p53- and hTERT-immortalized ovarian surface epithelial cell lines (T103 p53i and T151 p53i) were tested for tumorigenicity on soft agar and in nude mice. As shown in Figure 7A, neither cell type formed colonies on soft agar after 21 days or formed tumors in nude mice after 5 months, while previously SV40 T/t, hTERT and HRAS transformed T29H cells formed numerous colonies on soft agar and tumors in all four of the nude mice tested (15).


Figure 7
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  		Fig. 7 (A) Colony formation assay and mice tumor burden of immortal cells compared with T29H cells previously transformed by a combination of SV40 T/t antigen hTERT and HRASV12 (15). (B) Representative cytogenetic karyotypes of T103 p53i, T151 p53i and T137 p53i cells. Arrows indicate rare aberrations. br, breakage; DM, double minutes; dic, dicentric.

 
Immortalized cells are genetically stable
As p53 has been implicated in the maintenance of genetic stability, we examined the level of genetic stability of the immortalized cell lines at passage 50. As shown in Table I, all three immortalized cells were nearly diploid: 88.2% for T103 p53i, 71.4% for T151 p53i and 95.2% for T137 p53i; T103 p53i and T151 p53i has 8% and 20% chromosomal aberrations, while no chromosomal aberrations were detected for T137 p53i in 31 cells analyzed. These results are in marked contrast with SV40 T/t and hTERT-immortalized cells, only 27.5% were normal diploid, and 30% of the analyzed cells displayed chromosomal aberrations, ~5% of these cells had chromosomal fusions. These results demonstrated that the cells immortalized with p53siRNA are genetically more stable than those (T29) immortalized with SV40 T/t antigen. Representative cytogenetic karyotypes of three newly immortalized ovarian surface epithelial cell lines are shown in Figure 7B. The stable karyotypes observed here are similar to those previously reported by other investigators (19), suggesting that silencing of p53 is not the key driver of genetic instability although it may facilitate its development, while the ectopic expression of hTERT protects human cells from chromosomal instability.


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  		Table I Karyotypic analysis of immortalized OSE103, OSE151 and OSE137 cells compared with T29 cells immortalized with SV40 T/t and hTERT

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Over the past 20 years, many investigators have used viral oncogenes to achieve immortalization of human ovarian surface epithelial cells as model system to study human ovarian cancer progression (9,20–22). Transfection of human ovarian epithelial surface cells with SV40 large T antigen or human papilloma virus 16 (HPV-16 E6/E7) can extend the cells' life span, but they eventually enter crisis and die. Occasionally, after several months, immortalized cells emerge, but are not transformed as they do not form colonies in soft agar or tumors in nude mice (21,23). Very rarely, these viral protein-expressing human ovarian surface epithelial cells can grow into colonies in soft agar or form tumors in nude mice (20,22) after many passages in tissue culture, presumably as a result of spontaneous mutation. Previously, we have successfully immortalized human ovarian surface epithelial cells using SV40 T/t antigen together with the catalytic subunit of telomerase (hTERT) (15). We showed that the SV40 T/t and hTERT-immortalized cells were sensitive to RAS-mediated transformation (15). The transformed human ovarian surface epithelial cells recapitulated many features of natural ovarian cancer including a subtype of ovarian cancer histology, formation of ascites, CA125 expression and NF-{kappa}B-mediated cytokine activation. These cells represent first generation of model system to study human ovarian surface epithelial cell transformation. However, one potential limitation in the previous model is involvement of SV40 T/t antigen that might complicate the effect of other genes involved in the immortalization and transformation, as SV40 are not involved in the development of ovarian cancer. In the present study, we overcome this limitation by using retrovirus-mediated siRNA against p53 in combination with hTERT to achieve the immortalization of human ovarian surface epithelial cells. As both p53 and hTERT pathway are frequently altered pathway in ovarian carcinoma (18,24–26), these cell lines immortalized with these two known genetic changes in ovarian cancer provide newer generation of cell model system with which to study human ovarian cancer initiation and progression.

Retrovirus-mediated expression of gene silencing is a powerful approach to study numerous signaling pathways in normal cell and cancer cell homeostasis. Here we show that siRNA against p53 can delay the senescence program and extend the passage number in epithelial cell cultures. Although it is not sufficient for immortalization, it does lead to immortalization when combined with hTERT. Other groups of investigators reported immortalization of human bronchial epithelial cells by either combined expression of cdk4 and hTERT or hTERT alone (27,28), and others have successfully immortalized primary prostate epithelial cells or human mammary epithelial cells with c-Myc or BMI through activation of hTERT expression (29). Our results are also consistent with previous study using hTERT-immortalized foreskin fibroblasts or keratinocytes which showed accumulation in both p53 pathways and p16/pRB pathway (30,31). We previously used siRNA successfully to knock down several key oncogenes involved in ovarian or breast cancer development (13,17). Here we show that this technology can be used to generate immortalized human ovarian surface epithelial cell and create a useful cellular model of cancer progression. This approach should be applicable to other tumor suppressor genes involved at the different stages of tumor progression.

The immortalized human ovarian surface epithelial cell lines described in this report represent an authentic cell culture model system with which to study human ovarian cancer initiation and progression, as p53 and hTERT pathways, two commonly altered pathways in ovarian cancer from patients, are faithfully modeled in these cells. It should now be possible to introduce additional oncogenes known or suspected of being involved in ovarian cancer in a stepwise manner to study their role in malignant transformation. In addition, as different types (serous, endometrioid, mucinous and clear cells) of human ovarian cancer are all derived from Mullerian origin, these cells should also be valuable in generating human ovarian cancer model of different histotypes upon introduction of right combination of genetic elements. Furthermore, these cell lines should also be useful as cell-based assays for high-throughput drug screens to identify the drugs when combined with isogenic transformed cells. Finally, as p53 dysregulation and activation of hTERT are common in several types of epithelial cancer, the approach described here to immortalize human ovarian epithelial cells should be applicable to other epithelial cell types, thus providing a general approach to generating the cellular agents needed to study human cancer initiation and progression and chemoprevention.


   Acknowledgments
 
We thank Drs Sandy Chang and Asha Multani from the Molecular Cytogenetics Core facility at M. D. Anderson for karyotyping the immortalized cells. J.L. is supported by a Research Scholar Grant (RSG-04-028-1-CCE) from the American Cancer Society and by an Ovarian SPORE grant (IP50CA83638). This work is also supported in part by Cancer Center Core grant (CA016672) from the National Cancer Institute.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received February 15, 2006; revised June 13, 2006; accepted June 20, 2006.


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