Title: Genetic and Epigenetic Changes in Human Epithelial Cells Immortalized by Telomerase
Abstract: Exogenous expression of hTERT, the catalytic component of telomerase, is sufficient for the immortalization of human fibroblasts but insufficient for the immortalization of human foreskin keratinocytes (HFKs) and human mammary epithelial cells (HMECs). These latter cell types can overcome senescence by coexpression of hTERT and human papillomavirus (HPV) E7 or by expression of hTERT and loss of p16INK4a expression, indicating that the retinoblastoma (Rb. pathway, along with a telomere maintenance pathway, plays a role in determining the life span of epithelial cells. In this study, we further characterize hTERT-immortalized HFKs and human adenoid epithelial cells (HAKs) for genotypic and phenotypic alterations that are associated with immortalization. Of five hTERT-immortalized HFK and HAK cell lines examined, four exhibited repression of p16INK4a expression by promoter methylation or specific large-scale deletion of chromosome 9p, the location of p16INK4a. Interestingly, one cell line exhibited complete down-regulation of expression of p14ARF, with only slight down-regulation of expression of p16INK4a. Yet, all of the immortal cells lines exhibited hyperphosphorylated Rb. Cytogenetic analysis revealed clonal chromosome aberrations in three of the five cell lines. All of the cell lines retained a growth block response with the expression of mutant ras. When grown on organotypic raft cultures, however, the hTERT-immortalized cells exhibited a maturation delay on terminal differentiation. Our results indicate that immortalization of epithelial cells may require both activation of telomerase and other genetic and/or epigenetic alterations that abrogate normal differentiation. Exogenous expression of hTERT, the catalytic component of telomerase, is sufficient for the immortalization of human fibroblasts but insufficient for the immortalization of human foreskin keratinocytes (HFKs) and human mammary epithelial cells (HMECs). These latter cell types can overcome senescence by coexpression of hTERT and human papillomavirus (HPV) E7 or by expression of hTERT and loss of p16INK4a expression, indicating that the retinoblastoma (Rb. pathway, along with a telomere maintenance pathway, plays a role in determining the life span of epithelial cells. In this study, we further characterize hTERT-immortalized HFKs and human adenoid epithelial cells (HAKs) for genotypic and phenotypic alterations that are associated with immortalization. Of five hTERT-immortalized HFK and HAK cell lines examined, four exhibited repression of p16INK4a expression by promoter methylation or specific large-scale deletion of chromosome 9p, the location of p16INK4a. Interestingly, one cell line exhibited complete down-regulation of expression of p14ARF, with only slight down-regulation of expression of p16INK4a. Yet, all of the immortal cells lines exhibited hyperphosphorylated Rb. Cytogenetic analysis revealed clonal chromosome aberrations in three of the five cell lines. All of the cell lines retained a growth block response with the expression of mutant ras. When grown on organotypic raft cultures, however, the hTERT-immortalized cells exhibited a maturation delay on terminal differentiation. Our results indicate that immortalization of epithelial cells may require both activation of telomerase and other genetic and/or epigenetic alterations that abrogate normal differentiation. Telomerase is a ribonucleoprotein that maintains telomere length during cell division.1Greider CW Telomeres.Curr Opin Cell Biol. 1991; 3: 444-451Crossref PubMed Scopus (111) Google Scholar, 2Blackburn EH Telomerases.Annu Rev Biochem. 1992; 61: 113-129Crossref PubMed Scopus (610) Google Scholar Increased telomerase activity is thought to be a key event in the immortalization of cells and has been detected in many transformed human cell lines, precancerous lesions, and carcinomas.3Shay JW Bacchetti S A survey of telomerase activity in human cancer.Eur J Cancer. 1997; 33: 787-791Abstract Full Text PDF PubMed Scopus (2446) Google Scholar, 4Kim NW Piatyszek MA Prowse KR Harley CB West MD Ho PLC Coviello GM Wright WE Weinrich SL Shay JW Specific association of human telomerase activity with immortal cells and cancer.Science. 1994; 266: 2011-2015Crossref PubMed Scopus (6663) Google Scholar Introduction of the reverse transcriptase component of human telomerase (hTERT) and subsequent telomerase activation has been shown to extend the life span of human fibroblasts and retinal pigment epithelial (RPE. cells.5Bodnar AG Ouellette M Frolkis M Holt SE Chiu CP Morin GB Harley CB Shay JW Lichtsteiner S Wright WE Extension of life-span by introduction of telomerase into normal human cells.Science. 1998; 279: 349-352Crossref PubMed Scopus (4203) Google Scholar It has been proposed that telomerase activation is the only step required for the immortalization of cells.6Morales CP Holt SE Ouellette M Kaur KJ Yan Y Wilson KS White MA Wright WE Shay JW Absence of cancer-associated changes in human fibroblasts immortalized with telomerase.Nature Genet. 1999; 21: 115-118Crossref PubMed Scopus (689) Google Scholar, 7Jiang XR Jimenez G Chang E Frolkis M Kusler B Sage M Beeche M Bodnar AG Wahl GM Tlsty TD Chiu CP Telomerase expression in human somatic cells does not induce changes associated with a transformed phenotype.Nature Genet. 1999; 21: 111-114Crossref PubMed Scopus (581) Google Scholar Attempts to prolong the life span of epithelial cells such as human foreskin and human mammary epithelium, however, have not been successful, except in conjunction with alterations in the retinoblastoma (Rb) pathway.8Kiyono T Foster SA Koop JI McDougall JK Galloway DA Klingelhutz AJ Both Rb/p16INK4a inactivation and telomerase activity are required to immortalize human epithelial cells.Nature. 1998; 396: 84-88Crossref PubMed Scopus (1095) Google Scholar Specifically, expression of the cyclin D/cdk inhibitor p16INK4a was down-regulated in the cells when they became immortal. Loss of p16INK4a expression is commonly observed in epithelium-derived cancers,9Kamb A Gruis NA Weaver-Feldhaus J Liu Q Harshman K Tavtigian SV Stockert E Day III, RS Johnson BE Skolnick MH A cell cycle regulator potentially involved in genesis of many tumor types.Science. 1994; 264: 436-440Crossref PubMed Scopus (2866) Google Scholar but its precise role in immortalization and malignant progression is unclear. Expression of p16INK4a steadily increases as cells are passaged in culture, and up-regulation of p16INK4a has been associated with cellular senescence in both fibroblasts and epithelial cells.10Hara E Smith R Parry D Tahara H Stone S Peters G Regulation of p16CDKN2 expression and its implications for cell immortalization and senescence.Mol Cell Biol. 1996; 16: 859-867Crossref PubMed Scopus (664) Google Scholar, 11Reznikoff CA Yeager TR Belair CD Savelieva E Puthenveettil JA Stadler WM Elevated p16 at senescence and loss of p16 at immortalization in human papillomavirus 16 E6, but not E7, transformed human uroepithelial cells.Cancer Res. 1996; 56: 2886-2890PubMed Google Scholar, 12Loughran O Malliri A Owens D Gallimore PH Stanley MA Ozanne B Frame MC Parkinson EK Association of CDKN2A/p16INK4A with human head and neck keratinocyte replicative senescence: relationship of dysfunction to immortality and neoplasia.Oncogene. 1996; 13: 561-568PubMed Google Scholar, 13Noble JR Rogan EM Neumann AA Maclean K Bryan TM Reddel RR Association of extended in vitro proliferative potential with loss of p16INK4 expression.Oncogene. 1996; 13: 1259-1268PubMed Google Scholar, 14Alcorta DA Xiong Y Phelps D Hannon G Beach D Barrett JC Involvement of the cyclin-dependent kinase inhibitor p16 (INK4a) in replicative senescence of normal human fibroblasts.Proc Natl Acad Sci USA. 1996; 93: 13742-13747Crossref PubMed Scopus (817) Google Scholar, 15Yeager TR DeVries S Jarrard DF Kao C Nakada SY Moon TD Bruskewitz R Stadler WM Meisner LF Gilchrist KW Newton MA Waldman FM Reznikoff CA Overcoming cellular senescence in human cancer pathogenesis.Genes Dev. 1998; 12: 163-174Crossref PubMed Scopus (125) Google Scholar Yet, fibroblasts can be immortalized by telomerase activation alone without p16INK4a loss, suggesting cell type-specific roles for p16INK4a in senescence. It is possible that p16INK4a is involved in a terminal differentiation program that needs to be overcome for the immortalization of epithelial cells. It has been demonstrated, for example, that restoration of p16INK4a induces myogenic differentiation in rhabdomyosarcoma cells.16Urashima M Teoh G Akiyama M Yuza Y Anderson KC Maekawa K Restoration of p16(INK4A) protein induces myogenic differentiation in RD rhabdomyosarcoma cells.Br J Cancer. 1999; 79: 1032-1036Crossref PubMed Scopus (17) Google Scholar It has also been shown that p16INK4a is directly involved in regulating αVβ3, an integrin that plays a role in extracellular matrix interactions and differentiation.17Fahraeus R Lane DP The p16(INK4a) tumour suppressor protein inhibits alpha(v)beta(3) integrin-mediated cell spreading on vitronectin by blocking PKC-dependent localization of alpha(v)beta(3) to focal contacts.EMBO J. 1999; 18: 2106-2118Crossref PubMed Google Scholar Fibroblasts are considered to be relatively undifferentiated and may not require abrogation of a differentiation associated proliferation block to become immortal, although it has been suggested that p16INK4a is involved in fibroblast-specific differentiation.18Stein GH Drullinger LF Soulard A Dulic V Differential roles for cyclin-dependent kinase inhibitors p21 and p16 in the mechanisms of senescence and differentiation in human fibroblasts.Mol Cell Biol. 1999; 19: 2109-2117Crossref PubMed Scopus (617) Google Scholar Another possibility is that p16INK4a is involved in a stress response to accelerated proliferation. It has been demonstrated, for example, that overexpression of mutant ras in fibroblasts causes premature senescence and that this is associated with up-regulation of p16INK4a and p14ARF,19Serrano M Lin AW McCurrach ME Beach D Lowe SW Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a.Cell. 1997; 88: 593-602Abstract Full Text Full Text PDF PubMed Scopus (4062) Google Scholar, 20Serrano M Gomez-Lahoz E DePinho RA Beach D Bar S Inhibition of ras-induced proliferation and cellular transformation by p16INK4.Science. 1995; 267: 249-252Crossref PubMed Scopus (408) Google Scholar, 21Lin AW Barradas M Stone JC van Aelst L Serrano M Lowe SW Premature senescence involving p53 and p16 is activated in response to constitutive MEK/MAPK mitogenic signaling.Genes Dev. 1998; 12: 3008-3019Crossref PubMed Scopus (778) Google Scholar a gene that shares exons with p16 but utilizes a different promoter and is read in an alternative reading frame.22Quelle DE Zindy F Ashmun RA Sherr CJ Alternative reading frames of the INK4a tumor suppressor gene encode two unrelated proteins capable of inducing cell cycle arrest.Cell. 1995; 83: 993-1000Abstract Full Text PDF PubMed Scopus (1334) Google Scholar It is unknown, however, if natural senescence in culture, particularly in human epithelial cells, is in any way related to the mutant ras-mediated stress response observed in fibroblasts. DNA damage has also been hypothesized to play a role in p16INK4a up-regulation, but the mechanism has not been well characterized.23Robles SJ Adami GR Agents that cause DNA double strand breaks lead to p16INK4a enrichment and the premature senescence of normal fibroblasts.Oncogene. 1998; 16: 1113-1123Crossref PubMed Scopus (391) Google Scholar, 24Shapiro GI Edwards CD Ewen ME Rollins BJ p16INK4A participates in a G1 arrest checkpoint in response to DNA damage.Mol Cell Biol. 1998; 18: 378-387Crossref PubMed Scopus (157) Google Scholar Whether telomerase activation and subsequent immortalization can predispose cells to malignant transformation is an issue that remains controversial. Earlier studies have concluded that immortalization of human fibroblasts and RPE cells by hTERT was not associated with cancer-specific alterations.6Morales CP Holt SE Ouellette M Kaur KJ Yan Y Wilson KS White MA Wright WE Shay JW Absence of cancer-associated changes in human fibroblasts immortalized with telomerase.Nature Genet. 1999; 21: 115-118Crossref PubMed Scopus (689) Google Scholar, 7Jiang XR Jimenez G Chang E Frolkis M Kusler B Sage M Beeche M Bodnar AG Wahl GM Tlsty TD Chiu CP Telomerase expression in human somatic cells does not induce changes associated with a transformed phenotype.Nature Genet. 1999; 21: 111-114Crossref PubMed Scopus (581) Google Scholar For example, the hTERT-immortalized cells still maintained the ability to hypophosphorylate Rb under conditions that cause cell cycle blocks, such as DNA damage or confluency. Telomerase activation was also suggested to confer karyotypic stability. If this is true, an argument could be made that constitutive telomerase activation is protective against the development of cancer because it prevents the genetic instability associated with telomere loss. Telomerase activation and subsequent immortalization of cells, however, clearly would provide a selective growth advantage for the development of malignancy. Telomerase activation occurs in up to 97% of in vivo malignancies and the vast majority of immortal cell lines,3Shay JW Bacchetti S A survey of telomerase activity in human cancer.Eur J Cancer. 1997; 33: 787-791Abstract Full Text PDF PubMed Scopus (2446) Google Scholar, 4Kim NW Piatyszek MA Prowse KR Harley CB West MD Ho PLC Coviello GM Wright WE Weinrich SL Shay JW Specific association of human telomerase activity with immortal cells and cancer.Science. 1994; 266: 2011-2015Crossref PubMed Scopus (6663) Google Scholar and it has been demonstrated that expression of hTERT can predispose human embryonic kidney cells to tumorigenic transformation by coexpression of SV40 large T antigen and mutant H-ras.25Hahn WC Counter CM Lundberg AS Beijersbergen RL Brooks MW Weinberg RA Creation of human tumour cells with defined genetic elements.Nature. 1999; 400: 464-468Crossref PubMed Scopus (2001) Google Scholar Nevertheless, the ability to immortalize cells by hTERT could provide the potential to generate relatively normal immortal cells for research endeavors that have been limited by a finite supply of primary cells and tissue. Our observation, however, that immortalization of HFKs and HMECs by hTERT requires abrogation of the Rb pathway raises the question of whether the immortal cells are actually “normal.” In this study, we have analyzed hTERT-immortalized HFKs and human adenoid epithelial cells (HAKs) to characterize events associated with immortalization and to determine whether the cells exhibit phenotypic alterations that make them abnormal. Our results indicate that both epigenetic and genetic changes occur on immortalization by hTERT and that immortalization is associated with an aberrant delay in the epithelial program of terminal differentiation. Human adenoid epithelial cells (HAKs) and human foreskin keratinocytes (HFKs) were isolated as previously described.26Blanton RA Perez-Reyes N Merrick DT McDougall JK Epithelial cells immortalized by human papillomaviruses have premalignant characteristics in organotypic culture.Am J Pathol. 1991; 138: 673-685PubMed Google Scholar Cells were grown in keratinocyte serum-free media (K-SFM; Gibco-BRL, Gaithersburg, MD) in 50 μg/ml G418 after retroviral infection. Clones were picked by cylinder isolation of colonies as previously described and split into 1:4 or 1:8, using trypsin/EDTA. Cells in crisis or senescence were fed three to four times weekly, split if necessary, and maintained until there was no visible sign of proliferation (usually 2 months). All experiments described were performed within 100 population doublings (pd) of crisis. The protocol for organotypic rafts has been described.26Blanton RA Perez-Reyes N Merrick DT McDougall JK Epithelial cells immortalized by human papillomaviruses have premalignant characteristics in organotypic culture.Am J Pathol. 1991; 138: 673-685PubMed Google Scholar, 27Asselineau D Bernard BA Bailly C Darmon M Prunieras M Human epidermis reconstructed by culture: is it “normal”?.J Invest Dermatol. 1986; 86: 181-186Abstract Full Text PDF PubMed Scopus (152) Google Scholar, 28Kopan R Traska G Fuchs E Retinoids as important regulators of terminal differentiation: examining keratin expression in individual epidermal cells at various stages of keratinization.J Cell Biol. 1987; 105: 427-440Crossref PubMed Scopus (303) Google Scholar Briefly, dermal equivalents are generated with collagen and normal human fibroblasts. Epithelial cells are seeded on top of the dermal equivalents and grown submerged for 7 days in Rheinwald Green media. The “rafts” are then raised to the air-liquid interface, using a stainless steel grid on organ culture plates, and fed from beneath daily. After 12 days, the rafts were frozen in OCT or fixed in methacarn for immunohistochemical analyses. Total RNA was isolated from subconfluent cells with a kit (Qiagen). Twenty micrograms was run on a 1.2% gel and transferred to Hybond N membrane. For detection of p14ARF expression, exon 1 of the INK4 locus was amplified and labeled by random priming with [32P]CTP. Blots were hybridized and washed as described.29Church GM Gilbert W Genomic sequencing.Proc Natl Acad Sci USA. 1984; 81: 1991-1995Crossref PubMed Scopus (7898) Google Scholar The 36B4 probe was generated by polymerase chain reaction (PCR), labeled by random priming as described,8Kiyono T Foster SA Koop JI McDougall JK Galloway DA Klingelhutz AJ Both Rb/p16INK4a inactivation and telomerase activity are required to immortalize human epithelial cells.Nature. 1998; 396: 84-88Crossref PubMed Scopus (1095) Google Scholar and hybridized to the same stripped blot as above. The pCTV3-H-Ras61L construct was made by cloning the H-Ras61L cDNA from the pAX142 vector into pCTV3, and viral supernatants were generated in Phoenix amphotropic packaging lines as described.30Der CJ Pan BT Cooper GM rasH mutants deficient in GTP binding.Mol Cell Biol. 1986; 6: 3291-3294Crossref PubMed Scopus (89) Google Scholar, 31Whitehead I Kirk H Kay R Expression cloning of oncogenes by retroviral transfer of cDNA libraries.Mol Cell Biol. 1995; 15: 704-710Crossref PubMed Google Scholar, 32Zohn IE Symons M Chrzanowska-Wodnicka M Westwick JK Der CJ Mas oncogene signaling and transformation require the small GTP-binding protein Rac.Mol Cell Biol. 1998; 18: 1225-1235Crossref PubMed Scopus (69) Google Scholar Exponentially growing cells were infected overnight in 4 μg/ml polybrene. Infected cells were washed in regular media and refed. Cells were passed on the following day, and selective medium containing 8 μg/ml hygromycin (Boehringer-Mannheim) was added on the following day. After 5–10 days, cells were fixed and stained for β-galactosidase expression and collected for protein and RNA. β-Galactosidase staining of cell cultures was performed as described.33Dimri GP Lee X Basile G Acosta M Scott G Roskelley ABC Medrano EE Linskens M Rubelj I Pereira-Smith O A biomarker that identifies senescent human cells in culture and in aging skin in vivo.Proc Natl Acad Sci USA. 1995; 92: 9363-9367Crossref PubMed Scopus (5926) Google Scholar To test TGF-β sensitivity, subconfluent newly passaged cells were treated with various concentrations of porcine TGF-β (0, 0.1, 1.0, 10.0, and 100.0 μmol/L) (R&D Systems, Minneapolis, MN) for 6 days, after which cells were collected for protein. Cells were treated with 5-azadeoxycytine (Sigma) at 1.5 × 10−6 mol/L at subconfluency for 7 days before collection for Western or Northern analyses. Untreated subconfluent cells were collected at the same passage. Western analyses were performed as described, using monoclonal antibodies for p16INK4 (Pharmingen, San Diego, CA), Rb (Pharmingen), p27KIP (Transduction Labs), cyclin D (Pharmingen), cdk4 (Transduction Labs), or p53 (Oncogene Research). Forty micrograms of protein lysate for Rb analysis and 20 μg of lysate for analysis of other proteins were run on polyacrylamide gels of the appropriate concentration and blotted onto polyvinyl- idine fluoride (PVDF) membranes (Millipore, Bedford, MA). Detection was performed using a Renaissance chemiluminescence kit (NEN, Boston, MA). Standard immunocytohistochemistry and hematoxylin and eosin (H&E. staining techniques were used on methacarn-fixed or OCT frozen and acetone-fixed sections as described.34Coltrera MD Wang J Porter PL Gown AM Expression of platelet-derived growth factor B-chain and the platelet-derived growth factor receptor beta subunit in human breast tissue and breast carcinoma.Cancer Res. 1995; 55: 2703-2708PubMed Google Scholar Antibodies were AE2 (Biodesign) for K1 analysis and clone A9 (a gift from Tom Carey, University of Michigan) for α6β4 analysis. Cells that were 30–50 pd postcrisis were injected into 4- to 6-week-old athymic nude female mice (Simonson) at 3 × 106 cells/site, one site per animal, two animals per cell line, subcutaneously into the dorsal anterior quadrant. Mice were observed for more than 3 months for tumorigenic growth at the site of injection. Comparative genome hybridization (CGH) was performed as described, with modifications.35Kallioniemi A Kallioniemi OP Sudar D Rutovitz D Gray JW Waldman F Pinkel D Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors.Science. 1992; 258: 818-821Crossref PubMed Scopus (2837) Google Scholar, 36Pinkel D Straume T Gray JW Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization.Proc Natl Acad Sci USA. 1986; 83: 2934-2938Crossref PubMed Scopus (3087) Google Scholar, 37Weber RG Sabel M Reifenberger J Sommer C Oberstrass J Reifenberger G Kiessling M Cremer T Characterization of genomic alterations associated with glioma progression by comparative genomic hybridization.Oncogene. 1996; 13: 983-994PubMed Google Scholar Reference DNA was isolated from precrisis hTERT HFK cells, and test DNA was isolated from hTERT postcrisis cells. Test DNA was labeled with biotin-14-dATP (Gibco BRL. and reference DNA with digoxigenin-11-dUTP (Boehringer-Mannheim), using the standard protocol provided with the Boehringer-Mannheim nick translation kit. Normal male metaphase spreads were pretreated and hybridized for 3–5 days at 37°C as described.35Kallioniemi A Kallioniemi OP Sudar D Rutovitz D Gray JW Waldman F Pinkel D Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors.Science. 1992; 258: 818-821Crossref PubMed Scopus (2837) Google Scholar, 36Pinkel D Straume T Gray JW Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization.Proc Natl Acad Sci USA. 1986; 83: 2934-2938Crossref PubMed Scopus (3087) Google Scholar, 37Weber RG Sabel M Reifenberger J Sommer C Oberstrass J Reifenberger G Kiessling M Cremer T Characterization of genomic alterations associated with glioma progression by comparative genomic hybridization.Oncogene. 1996; 13: 983-994PubMed Google Scholar Biotinylated DNA sequences were detected using avidin-fluorescein isothiocyanate (FITC) (Vector Labs). Signals were amplified once. Digoxigenin-labeled DNA sequences were visualized using rhodamine-conjugated sheep anti-digoxigenin Fab fragments (Boehringer-Mannheim). The preparations were then counterstained with 4,6-diamidino-2-phenylindole-dihydrochloride (DAPI) (50 ng/ml) for 10 minutes at room temperature and mounted in Vectashield (Vector Labs). Image acquisition and processing were performed using an epifluorescence microscope (Nikon Microphot-SA) equipped with a 100-W mercury lamp and a standard CCD camera interfaced to a DELL PC. Gray-level images were recorded separately for each fluorochrome, using specifically aligned filter sets for DAPI, FITC, and rhodamine. Digital images were processed with Cytovision software developed by Applied Imaging. After correction of centromere placement and determination of the chromosome axis, individual FITC/rhodamine profiles were calculated for each chromosome. Mean ratio profiles were then determined from 9–25 metaphase spreads. Chromosomes were identified by inspection of inverted digital DAPI images. After digestion with EcoRI,1.5 μg of genomic DNA was bisulfite modified as previously described, with minor modifications.38Herman JG Graff JR Myohanen S Nelkin BD Baylin SB Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands.Proc Natl Acad Sci USA. 1996; 93: 9821-9826Crossref PubMed Scopus (5272) Google Scholar PCR amplification of the p16INK4a promoter was performed with a sense primer (5′ GTA GGT GGG GAG GAG TTT AGT T) binding −355 to −334 relative to the translation start site {43}, and one of two antisense primers (5′ TCT AAT AAC CAA CCA ACC CCT CC or 5′ CTA CCT AAT TCC AAT TCC CCT ACA) binding at positions −95 to −73 or +209 to +233.39Gonzalgo ML Hayashida T Bender CM Pao MM Tsai YC Gonzales FA Nguyen HD Nguyen TT Jones PA The role of DNA methylation in expression of the p19/p16 locus in human bladder cancer cell lines.Cancer Res. 1998; 58: 1245-1252PubMed Google Scholar Reaction conditions were 34 cycles at 95°C for 30 s, 60°C for 15 s, 72°C for 75 s, and 34 cycles at 95°C for 30 s, 64°C for 15 s, and 72°C for 75 s. PCR products were cloned, and at least 10 individual epigenotypes per sample were determined by automated sequencing. Analysis of non-CpG cytosines indicated the efficiency of bisulfite conversion at 99%. Metaphase spreads of HAK and HFK cell lines between 10 and 50 pd postcrisis were prepared on glass coverslips by standard cytogenetic methods, and 10–40 G-banded cells were analyzed for karyotypic abnormalities. The sequences of the coding regions and splice junctions of exons 1 and 2 of p16INK4a were determined by automated sequencing after amplification as described, with minor modifications.40Kamb A Gruis NA Weaver-Feldhaus J Liu Q Harshman K Tavtigian SV Stockert E Day RS Johnson BE Skolnick MH A cell cycle regulator potentially involved in genesis of many tumor types.Science. 1994; 264: 436-440Crossref PubMed Google Scholar Primer sequences were as follows: exon 1. sense 5′ GAA GAA AGA GGA GGG GCT G, antisense 5′ GCG CTA CCT GAT TCC AAT TC; exon 2: sense 5′ GGA AAT TGG AAA CTG GAA GC, antisense TCT GAG CTT TGGAAG CTC T. Earlier studies indicated that loss of p16INK4a expression or abrogation of the Rb pathway by HPV E7 was necessary for immortalization of HFKs by hTERT.8Kiyono T Foster SA Koop JI McDougall JK Galloway DA Klingelhutz AJ Both Rb/p16INK4a inactivation and telomerase activity are required to immortalize human epithelial cells.Nature. 1998; 396: 84-88Crossref PubMed Scopus (1095) Google Scholar In this study we extend our observations on the mechanisms of epithelial cell immortalization by hTERT to human adenoid epithelium (HAK). As was the case with HFKs, retroviral mediated expression of hTERT in HAK cells was insufficient for immortalization. Telomerase-positive hTERT-expressing clones senesced or went through a severe crisis before becoming immortal. All of the hTERT HFK and HAK immortal cell lines were shown to have high telomerase activity and to maintain telomere lengths with passaging in culture (data not shown). As has been described previously,8Kiyono T Foster SA Koop JI McDougall JK Galloway DA Klingelhutz AJ Both Rb/p16INK4a inactivation and telomerase activity are required to immortalize human epithelial cells.Nature. 1998; 396: 84-88Crossref PubMed Scopus (1095) Google Scholar hTERT HFK clones (cl 22 and 398) that went through crisis and became immortal exhibited loss of p16INK4a expression as compared to normal HFKs (Figure 1a, lanes 1–3). Of three hTERT HAK clones that emerged from crisis, two (cl 7 and cl 12) exhibited loss of p16INK4a expression (Figure 1a, lanes 5 and 6). One clone (cl 41), however, did not show complete loss of p16INK4a, but instead exhibited loss of expression of p14ARF as assayed by Northern analysis (Figure 1b, lane 8). Western analysis failed to detect ARF in any of the lines (data not shown), presumably because of low ARF protein levels in keratinocytes in general.41Munro J Stott FJ Vousden KH Peters G Parkinson EK Role of the alternative INK4A proteins in human keratinocyte senescence: evidence for the specific inactivation of p16(INK4A) upon immortalization.Cancer Res. 1999; 59: 2516-2521PubMed Google Scholar Interestingly, the immortalized cell lines did not exhibit any consistent alterations in p21, p53, p27KIP, or cdk4 expression (Figure 1a). However, there was slightly lower expression of cyclin D as compared to normal HFK or HAK, particularly in those cell lines that had complete loss of p16INK4a expression, and a slightly higher expression of Mdm2 in all of the postcrisis cells. All of the immortal cells, including the ARF-negative cell line cl 41, also exhibited more hyperphosphorylated Rb as compared to normal precrisis cells when grown in subconfluent conditions, indicating a probable defect in the Rb pathway (Figure 1a). Examination of the hTERT-immortalized cells by cytogenetic analyses within 50 population doublings (pd) of crisis revealed clonal chromosome aberrations in some of the cell lines but not in others (Table 1). Two cell lines, TERT HFK cl 398 and TERT HAK cl 41, showed no evidence of cytogenetic abnormalities and had normal diploid chromosome numbers. One cell line, hTERT HAK cl 12, contained an extra chromosome 5, whereas another, hTERT HAK cl 7, contained translocations of chromosomes 2 and 7 and several nonclonal aberrations. Interestingly, the cell line hTERT HFK cl 22 exhibited an isochromosome 9q as the only cytogenetic abnormality visible by G-banding. Loss of heterozygosity (LOH) analyses using PCR primers that amplified polymorphic sequences on 9p, the location of p16INK4a, revealed that this cell line had sustained a deletion of one 9p