Molecular Cancer Research Infection and Cancer: Biology, Therapeutics, and Prevention Bridging the Lab and the Clinic in Cancer Medicine
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Cell Growth & Differentiation
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Harada, H.
Right arrow Articles by Rustgi, A. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Harada, H.
Right arrow Articles by Rustgi, A. K.
Molecular Cancer Research 1:729-738 (2003)
© 2003 American Association for Cancer Research

Cell Cycle, Cell Death, and Senescence

Telomerase Induces Immortalization of Human Esophageal Keratinocytes Without p16INK4a Inactivation1

Hideki Harada1,4, Hiroshi Nakagawa1,4, Kenji Oyama1,4, Munenori Takaoka1,4, Claudia D. Andl1,4, Birgit Jacobmeier6, Alexander von Werder6, Gregory H. Enders1,2,3,5, Oliver G. Opitz6 and Anil K. Rustgi1,2,3,4

1 Gastroenterology Division, 2 Genetics Department, 3 Abramson Cancer Center, 4 Abramson Family Cancer Research Institute, and 5 Program in Cell and Molecular Biology, University of Pennsylvania, Philadelphia, PA and
6 Department of Medicine, University of Freiburg, Freiburg, Germany

Requests for reprints: Anil K. Rustgi, 6 CRB, University of Pennsylvania, 415 Curie Boulevard, Philadelphia, PA 19104. Phone: (215) 898-0154; Fax: (215) 573-5412. E-mail: anil2{at}mail.med.upenn.edu or Oliver G. Opitz, Medizinische Klinik, Abteilung Innere Medizin II, Labor B5, University of Freiburg, 79106 Freiburg, Germany. Phone: 49-761-270-3540; Fax: 49-761-270-3610. E-mail: opitz{at}med1.ukl.uni-freiburg.de


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Normal human somatic cells have a finite life span and undergo replicative senescence after a limited number of cell divisions. Erosion of telomeric DNA has emerged as a key factor in senescence, which is antagonized during cell immortalization and transformation. To clarify the involvement of telomerase in the immortalization of keratinocytes, catalytic subunit of telomerase (hTERT) expression was restored in normal human esophageal epithelial cells (EPC2). EPC2-hTERT cells overcame senescence and were immortalized without p16INK4a genetic or epigenetic alterations. p16INK4a was expressed at moderate levels and remained functional as evidenced by induction with UV treatment and binding to cyclin-dependent kinase 4 and 6. There were no mutations in the p53 gene, and p53 was functionally intact. Importantly, senescence could be activated in the immortalized EPC2-hTERT cells by overexpression of oncogenic H-ras or p16INK4a. Furthermore, the EPC2-hTERT cells yielded basal cell hyperplasia in an innovative organotypic culture system in contrast to a normal epithelium from parental cells. These comprehensive results indicate that the expression of telomerase induces immortalization of normal human esophageal keratinocytes without inactivation of p16INK4a/pRb pathway or abrogation of the p53 pathway.

Key Words: telomerase • p16INK4a • keratinocyte • immortalization • senescence


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Immortalization is a necessary step toward malignant transformation of normal human somatic cells, which have intrinsic mechanisms that monitor cell divisions and limit their life span. Progressive shortening of telomeres with each cell division has been found to limit replicative life span of human cells in culture (1–4).

Telomeres are structures at the ends of mammalian chromosomes that provide a protective function, preventing end-to-end chromosomal fusions (5–8). Because conventional DNA polymerase replicates DNA only in the 5'-3' direction, the so-called DNA end replication problem would occur on lagging strands at the ends of chromosomes. Because no DNA is synthesized beyond the last RNA primer hybridized to the DNA template, removal of the RNA primer generates a persisting gap. Moreover, 100–200 nucleotides of the telomeric DNA are removed from both ends of chromosomes with each round of replication, perhaps due to the action of a 5'-3' exonuclease (9). When the telomere length reaches a critical threshold, short telomeres are thought to be recognized as damaged DNA and evoke a permanent cell cycle arrest that is termed replicative senescence (10). Cells lacking mechanisms that maintain telomeres ultimately enter a cell death phase termed crisis.

Independent of replicative age, factors such as oncogenic activation, DNA damage, chromatin remodeling, cellular stress, overexpression of cyclin-dependent kinase (Cdk) inhibitors, and deprotection of chromosome ends can also induce a phenotype resembling senescence (11–16). Senescence also has the potential to function in tumor suppression. Consistent with this notion, senescence is alleviated by ablation of either the p16INK4a/pRb or the p14ARF/p53 tumor suppression pathways (17).

Human somatic cells are believed to be immortalized as long as their telomeres are maintained. In fact, most human cancer cells maintain their telomeres through activation of telomerase (18, 19), while some immortalized and cancer cells employ alternative mechanisms to maintain telomeres (20–22). Enforced expression of the catalytic subunit of telomerase (hTERT) can immortalize a variety of normal human somatic cells such as fibroblasts (2), endothelial cells (23), mesothelial and retinal pigment epithelial cells (2), and mammary epithelial cells (24). However, it is controversial whether telomerase alone is sufficient to induce immortalization of human keratinocytes. The factors limiting proliferation of such cells are of particular interest, because squamous cell cancers derived from such cells are among the most common fatal malignancies worldwide and frequently demonstrate p16INK4a inactivation (25–28). Some reports have concluded that inactivation of the p16INK4a/pRb pathway is required for immortalization of human keratinocytes in addition to the expression of telomerase (29, 30). By contrast, it has been reported that tissue culture conditions using a fibroblast feeder layer system do not require inactivation of the p16INK4a/pRb pathway in the induction of immortalization of human keratinocytes by ectopic expression of hTERT (31). When hTERT immortalized human oral keratinocytes were examined under the same conditions, it was observed that the ability of hTERT-expressing cells to proliferate was limited by expression of p16INK4a (32).

To address the requirement for immortalization of normal keratinocytes without inactivation of p16INK4a, we retrovirally transduced primary human esophageal cells or keratinocytes (esophageal epithelial cells [EPC2]) with hTERT, immediately and most importantly preceding senescence, and examined the functional roles of the p16INK4a/pRb and p53 pathways in a comprehensive manner not done previously. We observed that EPC2 cells had a limited life span, whereas retroviral introduction of hTERT immortalized EPC2 (EPC2-hTERT) in standard keratinocyte growth conditions (Keratinocyte-sfm; Invitrogen, Carlsbad, CA) in the absence of a fibroblast feeder layer system. EPC2-hTERT cells overcame replicative senescence and have continuously proliferated more than 200 population doublings (PD), 5-fold longer than the life span of parental cells and without evidence of crisis. In addition, p16INK4a is not mutated in coding regions, methylated in the promoter region, or lost due to allelic deletion. In fact, p16INK4a is functionally intact in immortalized EPC2-hTERT cells. Moreover, there was no evidence of alteration in the p53 tumor suppressor gene. However, the senescence program can be activated in these immortalized cells through overexpression of oncogenic H-ras or p16INK4a. This is unexpectedly accompanied by down-regulation of p14ARF. Our results suggest that expression of hTERT is sufficient to immortalize human keratinocytes and does not require inactivation of p16INK4a. Additionally, replicative senescence is suppressed through hTERT expression but can be activated by overexpression of oncogenic H-ras or p16INK4a.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
EPC2 Cells Show Progressive Shortening of Telomere Length and Have a Limited Replicative Life Span
EPC2 cells have normal diploid status (33). In these cells, hTERT gene expression was detected in an extremely low level by reverse transcription-PCR (data not shown), and no telomerase activity was found by the telomeric repeat amplification protocol (TRAP) assay (Fig. 1A). Consistent with these observations, the length of telomeres in parental EPC2 cells shortened with passage (Fig. 1B). Three independent experiments demonstrated that EPC2 cells underwent replicative senescence by 40–44 PD, confirmed by senescence-associated ß-galactosidase (SABG) staining (Fig. 2, A and B).



View larger version (78K):
[in this window]
[in a new window]
  		FIGURE 1. EPC2-hTERT cells have increased telomerase activity and telomere length compared with parental EPC2 cells. A. Cell extracts from EPC2-hTERT and EPC2-hyg cells were assayed for telomerase activity by the TRAP assay. Samples of equal mass were heat inactivated as a negative control. Strong telomerase activity was demonstrated by a 6-bp ladder in EPC2-hTERT extracts but was undetectable in extracts of EPC2-hyg cells infected with a control virus. IC indicates amplification of a 36-bp internal control. B. Telomere length was measured by Southern blot using telomeric repeats as a probe. Telomere shortenings occurred after serial passage of parental EPC2 cells. Ectopic expression of hTERT increased telomere length.

 



View larger version (17K):
[in this window]
[in a new window]
  		FIGURE 2. Ectopic expression of hTERT induces immortalization of EPC2 cells. A. The downward arrow indicates the time of transduction. While EPC2-hyg cells underwent senescence at a similar PD to parental cells, EPC2-hTERT cells overcame senescence and continuously grew, without a slow growth phase. {lozenge}, EPC2; •, EPC2-hTERT; {blacktriangleup}, EPC2-hyg. B. Senescence is evaluated by SABG staining in EPC2 cells (9 and 44 PD) and EPC2-hTERT cells (55 PD) and depicted in a quantitative fashion. Bars, SD.

 
Ectopic hTERT Expression Induces Immortalization of EPC2 Cells
We transduced hTERT in parallel with an empty vector in EPC2 cells at 42 PD, just before senescence, to determine if hTERT expression alone is sufficient to induce immortalization. Telomerase activity in hTERT-transduced cells was confirmed by the TRAP assay (Fig. 1A). As expected, telomeres were elongated in hTERT-expressing cells (Fig. 1B). Whereas cells transduced with the empty vector underwent senescence at 44 PD, similar to parental cells, hTERT-expressing cells did not undergo senescence and have continuously grown more than 200 PD (Fig. 2, A and B). These cells have never demonstrated a slow growth phase (30). These results were confirmed in five additional independent transduction experiments at different PD (data not shown). We next checked the karyotype of EPC2-hTERT cells and parental EPC2 cells. EPC2-hTERT cells, which overcame senescence, exhibited normal karyotype, consistent with that found in parental cells (data not shown).

Senescent and Immortalized EPC2 Cells Express Normal p16INK4a
To explore the molecular basis underlying senescence of EPC2 cells, we examined the state of the p16INK4a/pRb and p14ARF/p53 pathways. p16INK4a was strongly up-regulated in senescent EPC2 cells (Fig. 3A). Interestingly, cyclin D1 and cyclin D2 were also up-regulated in senescent cells. However, pRb expression was reduced and the protein was hypophosphorylated (Fig. 3A). Whereas p53 and p21WAF1/CIP1 expression was unaffected, p14ARF expression decreased with serial passages and was strongly down-regulated during senescence (Fig. 3B). These findings are consistent with a p16INK4a-mediated senescence.



View larger version (25K):
[in this window]
[in a new window]
  		FIGURE 3. Expression and functional analysis of the p16INK4a/pRb pathway and the p14ARF/p53/p21WAF1/CIP1 pathway in EPC2, EPC2-hyg, and EPC2-hTERT cells. Western blot for (A) p16INK4a, pRb phosphorylation status, cyclin D1, and cyclin D2 and for (B) p14ARF, p53, and p21WAF1/CIP1 in serial passages of parental EPC2, EPC2-hyg, and EPC2-hTERT cells. Hyperphosphorylation (pp) and hypophosphorylation (p) forms of pRb are indicated. Tubulin served as a protein loading control. The esophageal cancer cell line TE11, which has a mutated p53 gene, was used as a positive control for the detection of p53 protein (data not shown).

 
Because inactivation of p16INK4a has been reported to play a pivotal role in immortalization of human keratinocytes, we determined if p16INK4a inactivation occurred in the immortalized hTERT-expressing cells. The expression of p16INK4a protein remained at moderately high levels in early and intermediate PD of immortalized hTERT-expressing EPC2 cells (Fig. 3A). In late PD of immortalized cells, p16INK4a expression decreased but remained higher than in early passage parental cells (Fig. 3A). We asked if this down-regulation of p16INK4a found in late PD was caused by a genetic or epigenetic alteration such as deletion, mutation, or promoter hypermethylation that is frequently observed in human cancers, especially esophageal squamous cell carcinoma. DNA sequencing revealed no mutations in the coding sequence, and a PCR-based assay showed no loss of heterozygosity (LOH) adjacent to or within the p16INK4a gene by a PCR-LOH assay (Table 1). We determined the methylation status of the p16INK4a promoter in early and late PD immortalized cells by methylation-specific PCR (MSP). Only unmethylated DNA was found at both time points (Fig. 4A).


View this table:
[in this window]
[in a new window]
  		Table 1. Genetic and Epigenetic Analysis

 


View larger version (36K):
[in this window]
[in a new window]
  		FIGURE 4. p16INK4a promoter and functional assays in immortalized EPC2-hTERT cells. A. MSP (unmethylated [U] or methylated [M]) of the p16INK4a gene was evaluated on 2% agarose gel stained with ethidium bromide. The p16INK4a promoter was not methylated in EPC2-hTERT cells. CaCO2 colon cancer cells served as a positive control for methylation. B. Immunoprecipitations (IP) of EPC2-hTERT at 68 PD with Cdk2, Cdk4, or Cdk6 antibody were immunoblotted (IB) and probed with p16INK4a, p21WAF1/CIP1, or p27Kip1 antibody. p16INK4a expressed in immortalized EPC2-hTERT cells maintained the ability to bind Cdk4 or Cdk6. C. Western blot for Cdk4 and Cdk6 in serial passages of parental EPC2, EPC2-hyg, and EPC2-hTERT cells. Cdk4 and Cdk6 remained expressed in serial passages. Tubulin served as a protein loading control. D. EPC2-hTERT cells (176 PD) were treated with 60 J/m2 of UV. Expression of p16INK4a was up-regulated after UV treatment in immortalized EPC2-hTERT cells. ß-actin served as a protein loading control.

 
p16INK4a is a specific inhibitor of the cyclin D1/Cdk4 or cyclin D1/Cdk6 complexes, and tumor-associated mutations regularly disrupt p16INK4a binding to Cdks (34). To determine whether p16INK4a expressed in immortalized cells was functional, we asked whether the protein was bound to Cdk4 and Cdk6. Cell lysates from immortalized cells were immunoprecipitated with antibodies against either Cdk4 or Cdk6 followed by immunoblotting for p16INK4a. p16INK4a was readily detected in such immunoprecipitates as were other Cdk inhibitors, such as p21WAF1/CIP1 and p27Kip1 (Fig. 4B). The binding of p16INK4a to Cdk4 or Cdk6 was observed even in late PD of immortalized cells (data not shown). Of note, Cdk4 and Cdk6 levels remained constant in parental or immortalized cells (Fig. 4C). We then asked whether the p16INK4a promoter in immortalized cells remained responsive to stimuli known to induce p16INK4a expression as in skin keratinocytes (35, 36). UV irradiation induced expression of p16INK4a in EPC2-hTERT cells, demonstrating that the p16INK4a promoter remains responsive in these cells (Fig. 4D). Taken together, these results indicate that the p16INK4a gene and protein remained intact during immortalization of EPC2 cells induced by hTERT.

Enforced Expression of p16INK4a Induces Senescence in Immortalized EPC2 Cells
An adenovirus vector expressing p16INK4a was infected in late PD of EPC2-hTERT cells (Fig. 5A). Consistent with a previous report (13), the p16INK4a-expressing vector shifted pRb to a hypophosphorylated status, inhibited DNA synthesis (data not shown), and induced morphological changes of flattening and spreading in 100% of cells (data not shown), whereas a control lacZ-expressing vector did not affect morphology and cell growth. These senescent changes were reinforced by the presence of SABG staining (Fig. 5B). Therefore, the p16INK4a/pRb pathway is functional in immortalized EPC2-hTERT cells.



View larger version (15K):
[in this window]
[in a new window]
  		FIGURE 5. Adenoviral overexpression of p16INK4a in immortalized EPC2-hTERT cells. A. Cell lysates were prepared from EPC2-hTERT cells at 215 PD infected with 100 multiplicities of infections of adenovirus vector expressing either p16INK4a (adp16) or lacZ 72 h after infection. Enforced expression of p16INK4a induced hypophosphorylation of pRb in EPC2-hTERT cells. Hyperphosphorylation (pp) and hypophosphorylation (p) forms of pRb are indicated. Tubulin was served as a protein loading control. B. Enforced expression of p16INK4a induced senescence in immortalized EPC2-hTERT cells. Senescence cells were evaluated 2 weeks after adenovirus infection by positive SABG staining. Bars, SD.

 
Immortalized EPC2-hTERT Cells Have No Changes in the p14ARF/p53 Pathway
Although it has been reported that the p53 pathway is less involved in senescence of human keratinocytes than in fibroblasts and not necessarily inactivated during immortalization of human keratinocytes (29, 30), expression of dominant negative p53 extends the life span of human keratinocytes (21). Therefore, we asked whether abrogation of this pathway is involved in immortalization of hTERT-expressing EPC2 cells. Western blot analysis showed equivalent p53 levels in parental EPC2 and immortalized EPC2-hTERT cells (Fig. 3B). There were no appreciable changes in the expression of p14ARF and p21WAF1/CIP1 proteins during immortalization (Fig. 3B). In addition, there were no mutations found in the p53 gene by direct DNA sequencing (Table 1). We also checked the function of p53 in immortalized EPC2-hTERT cells (176 PD). Hydroxyurea and UV irradiation are known to induce DNA damage and activate p53. As shown in Fig. 6, p53 induction was observed after either UV irradiation or hydroxyurea treatment, resulting in up-regulation of p21WAF1/CIP1 (Fig. 6). Thus, immortalized hTERT-expressing EPC2 cells maintain an intact p53 pathway.



View larger version (42K):
[in this window]
[in a new window]
  		FIGURE 6. DNA damage activates the p53 pathway. UV and hydroxyurea (HU) treatment induced stabilization of p53 in EPC2-hTERT cells (176 PD), resulting in induction of p21WAF1/CIP1. ß-actin served as a protein loading control.

 
Additional Genetic Alterations Are Required for Transformation
Because immortalization is a critical step toward malignant transformation, we asked whether immortalized hTERT-expressing cells were prone to transformation or acquired other features of transformed cells. First, we examined the phenotype of immortalized EPC2-hTERT cells in organotypic culture. As shown in Fig. 7, EPC2-hTERT cells exhibit complete stratified epithelia with evidence of basal cell hyperplasia. However, none of the cells are dysplastic or invade through the basement membrane, characteristics of malignant transformation.



View larger version (23K):
[in this window]
[in a new window]
  		FIGURE 7. H&E staining of organotypic culture. Organotypic culture demonstrates basal cell hyperplasia (arrows) in EPC2-hTERT cells (A) versus a normal phenotype in parental EPC2 cells (B). Squamous epithelium (E) and fibroblasts (F) in collagen matrix are indicated. (Scale bar corresponds to 50 µm.)

 
Because immortalized EPC2-hTERT cells demonstrated basal cell hyperplasia but not malignant transformation in organotypic culture, we transduced mutant H-ras into immortalized EPC2-hTERT cells to determine if transformation could be induced. Expression of oncogenic H-ras induced up-regulation of p16INK4a, hypophosphorylation of pRb (Fig. 8A), and inhibition of DNA synthesis (Fig. 8B), and 100% of cells had a large and flat morphology consistent with senescence (data not shown), the latter of which was confirmed by SABG staining (Fig. 8C). Consistent with findings in senescent parental EPC2 cells, p14ARF expression was down-regulated in H-ras-induced senescence in immortalized EPC2-hTERT cells and accompanied by increased cyclin D1 expression (Fig. 8A). However, p21WAF1/CIP1 was mildly increased (Fig. 8A). Expression of oncogenic H-ras can transform a variety of immortalized cells but activates senescence in cells expressing functional p16INK4a and p53 (12). Our immortalized EPC2-hTERT cells were resistant to transformation and remained susceptible to senescence by expression of oncogenic H-ras, presumably due to the expression of functional p16INK4a. Supporting the observed resistance of immortalized EPC2-hTERT cells to transformation by oncogenic H-ras, we did not find up-regulation of c-myc by Western blot analysis (data not shown). c-myc can cooperate with oncogenic ras to induce transformation and has been reported to be up-regulated in cells immortalized by hTERT (37). These results verify that the p16INK4a/pRb response pathway is intact in immortalized cells.



View larger version (21K):
[in this window]
[in a new window]
  		FIGURE 8. Retroviral overexpression of H-ras activates a senescence phenotype in immortalized EPC2-hTERT cells. A. Expression of the ras, p16INK4a, pRb, cyclin D1, p14ARF, p53, and p21WAF1/CIP1. Cell lysates were prepared from EPC2-hTERT at 215 PD infected with a retrovirus vector expressing oncogenic H-ras or empty vector on day 13 after infection. Hyperphosphorylation (pp) and hypophosphorylation (p) forms of pRb are indicated. Tubulin served as a protein loading control. B. Thirteen days after infection, cells were incubated with the growth media containing [3H]thymidine for 24 h. [3H]Thymidine incorporation assay indicated inhibition of DNA synthesis in EPC2-hTERT cells by expression of H-ras. Bars, SD. C. Transduction of H-ras induced senescence in immortalized EPC2-hTERT cells. Senescent cells were confirmed by positive SABG staining on day 13 after adenoviral infection. Bars, SD.

 

   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
Because somatic cells do not express telomerase, telomeres progressively shorten with each cell division. Telomeric attrition causes disruption of the telomere structure, which normally protects the ends of chromosomes. Critically short telomeres are then thought to trigger the process of replicative senescence (1–3). Consistent with this notion, telomeres have been found to be short in a variety of senescent human cells (3, 38, 39). Parental EPC2 cells approaching senescence have short telomeres and no detectable telomerase activity. Although the mechanisms through which short telomeres are recognized remain to be fully elucidated, cell cycle checkpoint proteins such as p16INK4a, pRb, and p53 have been reported to be involved in cell cycle arrest during senescence (17, 40, 41). Up-regulation of p16INK4a and hypophosphorylation of pRb were observed in EPC2 cells, without accumulation of p53. These results suggest that the p16INK4a/pRb pathway fosters senescence in human keratinocytes. Furthermore, we found up-regulation of cyclin D1 and cyclin D2 in senescent cells. It is conceivable that cyclin D1 and cyclin D2 in this context bind to and inactivate Cdk2 (42–44). Thus, there may be a potential contribution of high cyclin D1 and cyclin D2 levels in promoting senescence in this pathway for esophageal keratinocytes, although, generally, cyclin D1 and cyclin D2 will promote proliferation or extend the replicative life span of cells in other contexts.

In addition, a senescence phenotype can also be induced by aberrant expression of oncogenes, damaged DNA, chromatin remodeling, cellular stress, overexpression of Cdk inhibitors, or deprotection of chromosome ends (11–16, 45). In this study, we observed a senescence phenotype in immortalized EPC2-hTERT cells with enforced expression of either p16INK4a or oncogenic H-ras. Interestingly, senescence induced by oncogenic H-ras also revealed up-regulation of p16INK4a and hypophosphorylation of pRb, without accumulation of p53. This result supports a previous report showing that p16INK4a-deficient human fibroblasts are resistant to oncogenic ras-induced senescence (46). p21WAF1/CIP1 was mildly up-regulated during activation of senescence by oncogenic H-ras. Interestingly, p21WAF1/CIP1-associated senescence results in a different cellular morphology than morphological changes associated with p16INK4a/pRb-induced senescence (47). By contrast, we observed morphological differences between cells associated with replicative senescence compared with oncogenic H-ras-activated senescence (data not shown). Our present results suggest that both telomere-dependent and telomere-independent senescence is preferentially regulated through the p16INK4a/pRb pathway in human keratinocytes.

We also observed down-regulation of p14ARF in telomere-dependent replicative senescence in parental EPC2 cells and telomere-independent oncogenic H-ras-activated senescence in immortalized EPC2-hTERT cells. Accordingly, it has been previously reported that senescent epidermal keratinocytes express little p14ARF (48). It is conceivable that hypophosphorylated pRb results in decreased E2F activity and therefore decreased p14ARF transcription (49). One apparent consequence of down-regulation of p14ARF during senescence is that p53 is not induced, although p53 could still be induced and targeted for subsequent degradation by MDM2.

Several lines of evidence reveal that the reconstitution of telomerase into certain human somatic cells prevents telomeric attrition and senescence, resulting in immortalization (2, 19, 23). By contrast, ectopic expression of hTERT in human keratinocytes was insufficient in some studies to induce immortalization, suggesting that they have telomere-independent mechanisms that limit their life span (29, 30, 32). In this study, we have demonstrated that immortalization of human esophageal keratinocytes can be mediated by ectopic expression of hTERT immediately preceding senescence. The immortalized cells have intact p16INK4a/pRb and p53 pathways, suggesting that ectopic expression of telomerase is sufficient to induce their immortalization and that inactivation of the p16INK4a/pRb pathway is not necessary. Thus, our data indicate that keratinocytes do not possess a telomere-independent clock that activates p16INK4a-mediated replicative senescence. Differences in biological properties between esophageal and other keratinocytes, such as those originating in the epidermis, oral cavity, cornea, and conjunctiva (29, 30, 32), may include donor age and latent diseases. These and other factors may account in part for the observed discrepancies in keratinocyte response and tissue culture conditions, which reportedly abolished the requirement of the p16INK4a inactivation for immortalization of epidermal keratinocytes and mammary epithelial cells (24, 31). Another possibility may relate to the timing of hTERT overexpression in human oral keratinocytes that transduced hTERT in early PD as represented in previous studies, whereas we transduced hTERT just before replicative senescence. Our study is the first to provide a thorough evaluation of the integrity of the p16INK4a pathway during hTERT-induced immortalization, which had been lacking in previous studies with oral or epidermal keratinocytes.

Because hTERT expression has been detected even in squamous cell carcinoma in situ (50), telomerase reactivation might take place as an early step in the development of esophageal squamous cell cancer. This is supported by findings that 80–100% of esophageal cancers express telomerase (51, 52). Extended cell replication facilitated by telomerase might increase the risk of other genetic or epigenetic alterations, leading to malignant transformation. In fact, EPC2-hTERT cells (late PD) demonstrated basal cell hyperplasia in organotypic culture but were not yet transformed. Thus, hTERT expression appears to be important for immortalization but insufficient for malignant transformation (53, 54), consistent with the failure of oncogenic H-ras to transform immortalized EPC2-hTERT cells. Whether hTERT expression acts exclusively through telomere elongation in EPC2 cells or mediates other functions remains to be determined (55, 56).

In summary, replicative senescence in human esophageal keratinocytes depends on telomere length and is induced by the p16INK4a/pRb pathway. Ectopic hTERT expression is sufficient to induce immortalization of human esophageal keratinocytes without inactivation of p16INK4a or abrogation of p53, which is underscored by the robustness of our data and not done in a comprehensive fashion previously. Importantly, the senescence program can be imposed in immortalized human esophageal keratinocytes by overexpression of p16INK4a or oncogenic H-ras, indicating that the senescence program remains intact.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
Cell Culture, Retroviral Vectors, and Retroviral Transduction
Normal human esophageal keratinocytes (EPC2) were isolated and cultured in Keratinocyte-sfm (Invitrogen) (33). Organotypic culture was performed as previously described (33). The pBabe hygromycin-resistant retrovirus vector expressing hTERT was obtained from R. Weinberg (Whitehead Institute, Cambridge, MA). The pBabe puromycin-resistant retrovirus vector expressing H-ras V12 was obtained from S. Lowe (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). Each vector and empty vectors were transfected into phoenix retrovirus producer cells (33). Retroviral supernatants were collected 48 and 72 h posttransfection. Parental EPC2 cells were prepared for retroviral transduction at 42 PD, just before senescence. After transduction with either pBabe-hygro-hTERT (EPC2-hTERT) or pBabe-hygro (EPC2-hyg), cells were incubated with 10 µg/ml of hygromycin B (Roche, Basel, Switzerland). Clones were pooled and expanded. For H-ras infection, immortalized EPC2-hTERT cells at 215 PD were used and incubated with 0.5 µg/ml of puromycin.

Adenoviral Vectors and Infection
Recombinant adenoviral vectors expressing p16INK4a and lacZ were generated as previously described (57). We prepared EPC2-hTERT cells at 215 PD. Cells (3 x 105) were plated on 6-cm dishes 24 h before infection. Infection was performed by adding 100 multiplicities of infections of adenovirus.

Determination of Replicative Life Span
Parental EPC2, EPC2-hTERT, and EPC2-hyg cells were routinely maintained in subconfluent conditions. The number of PD was determined using the following formula: PD = log(N/N0) / log2, where N is the number of collected cells and N0 is the number of seeded cells.

[3H]Thymidine Incorporation Assay
We prepared 5 x 104 H-ras-transduced cells and empty vector-transduced cells per well into 12-well plates 24 h before the assay. Growth media containing 1 µCi/ml of methyl-[3H]thymidine were added to the cells and incubated for 24 h. The cells were lysed with 0.5-N NaOH, and high molecular weight nucleic acid was precipitated with cold 10% TCA. Precipitated cells were collected by filtration through Whatman GF/C filters (Clifton, NJ). Dried filters were placed into Ready Value Liquid Scintillation Cocktail, and the radioactivity was counted with LS6500 Multi-Purpose Scintillation Counter. The assay was done in a triplicate fashion.

Senescence-Associated ß-Galactosidase Activity
Senescent cells were identified by SABG staining using the Senescence ß-Galactosidase Staining Kit (Cell Signaling, Beverly, MA) according to the manufacturer's protocol. Stained cells were scored by counting five high-power fields under light microscopy.

Telomerase Activity and Telomere Length Assays
Telomerase activity was determined by the TRAP assay using TRAPEZE Telomerase Detection Kit (Intergen Co., Purchase, NY). Telomere length was measured by Southern blot of HinfI- and RsaI-digested genomic DNA from parental and derivative cells using the Telomere Length Assay Kit (PharMingen, San Diego, CA).

DNA Damage Treatment
UV irradiation was performed using a Stratalinker 1800 UV Crosslinker (Stratagene, La Jolla, CA) with 60 J/m2, and protein was extracted after 24 h. Hydroxyurea treatment was done at 20 mM for 48 h.

Western Blot Analysis
Ten micrograms of protein from whole-cell extracts were separated by SDS-PAGE and transferred to Immobilon-P membranes (Millipore, Billerica, MA). The membranes were incubated with antihuman p16INK4a (PharMingen), G3-245 against pRb (PharMingen), DCS-6 against cyclin D1 (PharMingen), G132-43 against cyclin D2 (PharMingen), H-22 against Cdk4 (Santa Cruz, Santa Cruz, CA), C-21 against Cdk6 (Santa Cruz), 14P02 against p14ARF (Oncogene Science, Boston, MA), DO-1 against p53 (Oncogene Science, Boston, MA), EA10 against p21WAF1/CIP1 (Oncogene Science), Kip1/p27 (Transduction Laboratories, Lexington, KY), AC-74 against ß-actin (Sigma Chemical Co., St. Louis, MO), and DMA1A + DM1B against tubulin (Neo Markers, Union City, CA). Staining was detected by using ECL Plus (Amersham, Piscataway, NJ).

Immunoprecipitation
Cell lysates were prepared by lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, protease inhibitors cocktail [complete, Mini, Roche]) and incubated with anti-Cdk2 (M2; Santa Cruz), Cdk4 (H-22, Santa Cruz), or Cdk6 (C-21, Santa Cruz) antibody. Bound proteins were collected on Protein G Agarose (Invitrogen). Pellets were washed three times with wash buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl) and resuspended in electrophoresis sample buffer.

LOH Analysis
Genomic DNA was extracted from EPC2 and EPC2-hTERT cells using the PUREGENE DNA Isolation Kit (Gentra Systems, Minneapolis, MN). Three microsatellite markers (D9S126, D9S1748, and D9S162) were used to detect LOH in the CdkN2/INK4a locus. PCR mixtures contained 25-ng genomic DNA, 10 pmol each of forward primer labeled with [{gamma}32]ATP and reverse primer, 1.5-mM MgCl2, 200-µM deoxynucleotide triphosphates, and 0.5-U Taq DNA polymerase. After denaturation at 94°C for 5 min, PCR was performed with 35 cycles at 94°C for 15 s, 65°C for 15 s, and 72°C for 30 s, with a final extension at 72°C for 7 min. After heat denaturation at 95°C for 5 min, PCR products were loaded on 6% denaturing polyacrylamide gels. Gels were dried and exposed to X-ray film for evaluation of LOH.

Methylation-Specific PCR
The methylation status of the p16INK4a gene promoter was examined by MSP. Bisulfite modification of genomic DNA and MSP were performed as published previously (58). Primer sets used for MSP were 5'-CAGAGGGTGGGGCGGACCGC-3' and 5'-CGGGCCGCGGCCGTGG-3' for unmodified DNA, 5'-TTATTAGAGGGTGGGGCGGATCGC-3' and 5'-GACCCCGAACCGCGACCGTAA-3' for methylated DNA, and 5'-TTATTAGAGGGTGGGGTGGATTGT-3' and 5'-CAACCCCAAACCACAACCATAA-3' for unmethylated DNA (58). DNA extract from CaCO2 was served as a positive control of methylated promoter (59).

DNA Mutational Analysis
We amplified exons 3–9 of the p53 gene and exons 1–3 of the p16INK4a gene from genomic DNA and sequenced the PCR products for detection of any mutations in coding sequences.


   Acknowledgements
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
We thank the morphology, molecular biology, and cell culture core facilities of the NIH/NIDDK Center for Molecular Studies in Digestive and Liver Diseases. We thank E.Z. Tomczak, A. Ganguly, G. Goessel, J. Mitra, and G. Swain for excellent technical assistance and advice and T. Deramaudt, C.N. Johnstone, B. Rhodes, H.E. Blum, and M. Imamura for helpful discussions and support.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
1 NIH grants R01-DK5337 (A.K.R.), P01-DE12467 (A.K.R.), P01-CA098101 (A.K.R.), Deutsche Krebshilfe 10-1656-Op 1 and D/96/17197 (O.G.O.), NIH R21 DK64249-01 (H.N.), AGA/FDHN Fiterman Award and American Cancer Society (G.H.E.), and NIH/NIDDK Center for Molecular Studies in Digestive and Liver Diseases (P30 DK50306). Back

Received March 24, 2003; revised May 16, 2003; accepted June 10, 2003.


   References
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 

  1. Allsopp, R. C., Vaziri, H., Patterson, C., Goldstein, S., Younglai, E. V., Futcher, A. B., Greider, C. W., and Harley, C. B. Telomere length predicts replicative capacity of human fibroblasts. Proc. Natl. Acad. Sci. USA, 89: 10114–10118, 1992.[Abstract/Free Full Text]
  2. Bodnar, A. G., Ouellette, M., Frolkis, M., Holt, S. E., Chiu, C. P., Morin, G. B., Harley, C. B., Shay, J. W., Lichtsteiner, S., and Wright, W. E. Extension of life span by introduction of telomerase into normal human cells. Science, 279: 349–352, 1998.[Abstract/Free Full Text]
  3. Harley, C. B., Futcher, A. B., and Greider, C. W. Telomeres shorten during ageing of human fibroblasts. Nature, 345: 458–460, 1990.[Medline]
  4. Vaziri, H., Dragowska, W., Allsopp, R. C., Thomas, T. E., Harley, C. B., and Lansdorp, P. M. Evidence for a mitotic clock in human hematopoietic stem cells: loss of telomeric DNA with age. Proc. Natl. Acad. Sci. USA, 91: 9857–9860, 1994.[Abstract/Free Full Text]
  5. Counter, C. M., Avilion, A. A., LeFeuvre, C. E., Stewart, N. G., Greider, C. W., Harley, C. B., and Bacchetti, S. Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity. EMBO J., 11: 1921–1929, 1992.[Medline]
  6. Sandell, L. L. and Zakian, V. A. Loss of a yeast telomere: arrest, recovery, and chromosome loss. Cell, 75: 729–739, 1993.[Medline]
  7. Blasco, M. A., Lee, H. W., Hande, M. P., Samper, E., Lansdorp, P. M., DePinho, R. A., and Greider, C. W. Telomere shortening and tumor formation by mouse cells lacking telomerase RNA. Cell, 91: 25–34, 1997.[Medline]
  8. van Steensel, B., Smogorzewska, A., and de Lange, T. TRF2 protects human telomeres from end-to-end fusions. Cell, 92: 401–413, 1998.[Medline]
  9. Makarov, V. L., Hirose, Y., and Langmore, J. P. Long G tails at both ends of human chromosomes suggest a C strand degradation mechanism for telomere shortening. Cell, 88: 657–666, 1997.[Medline]
  10. Hemann, M. T., Strong, M. A., Hao, L. Y., and Greider, C. W. The shortest telomere, not average telomere length, is critical for cell viability and chromosome stability. Cell, 107: 67–77, 2001.[Medline]
  11. von Zglinicki, T., Saretzki, G., Docke, W., and Lotze, C. Mild hyperoxia shortens telomeres and inhibits proliferation of fibroblasts: a model for senescence? Exp. Cell Res., 220: 186–193, 1995.[Medline]
  12. Serrano, M., Lin, A. W., McCurrach, M. E., Beach, D., and Lowe, S. W. Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a. Cell, 88: 593–602, 1997.[Medline]
  13. Dai, C. Y. and Enders, G. H. p16 INK4a can initiate an autonomous senescence program. Oncogene, 19: 1613–1622, 2000.[Medline]
  14. Yeo, E. J., Hwang, Y. C., Kang, C. M., Kim, I. H., Kim, D. I., Parka, J. S., Choy, H. E., Park, W. Y., and Park, S. C. Senescence-like changes induced by hydroxyurea in human diploid fibroblasts. Exp. Gerontol., 35: 553–571, 2000.[Medline]
  15. Dumont, P., Royer, V., Pascal, T., Dierick, J. F., Chainiaux, F., Frippiat, C., de Magalhaes, J. P., Eliaers, F., Remacle, J., and Toussaint, O. Growth kinetics rather than stress accelerate telomere shortening in cultures of human diploid fibroblasts in oxidative stress-induced premature senescence. FEBS Lett., 502: 109–112, 2001.[Medline]
  16. Smogorzewska, A. and de Lange, T. Different telomere damage signaling pathways in human and mouse cells. EMBO J., 21: 4338–4348, 2002.[Medline]
  17. Shay, J. W., Pereira-Smith, O. M., and Wright, W. E. A role for both RB and p53 in the regulation of human cellular senescence. Exp. Cell Res., 196: 33–39, 1991.[Medline]
  18. Kim, N. W., Piatyszek, M. A., Prowse, K. R., Harley, C. B., West, M. D., Ho, P. L., Coviello, G. M., Wright, W. E., Weinrich, S. L., and Shay, J. W. Specific association of human telomerase activity with immortal cells and cancer. Science, 266: 2011–2015, 1994.[Abstract/Free Full Text]
  19. Meyerson, M., Counter, C. M., Eaton, E. N., Ellisen, L. W., Steiner, P., Caddle, S. D., Ziaugra, L., Beijersbergen, R. L., Davidoff, M. J., Liu, Q., Bacchetti, S., Haber, D. A., and Weinberg, R. A. hEST2, the putative human telomerase catalytic subunit gene, is up-regulated in tumor cells and during immortalization. Cell, 90: 785–795, 1997.[Medline]
  20. Bryan, T. M., Englezou, A., Gupta, J., Bacchetti, S., and Reddel, R. R. Telomere elongation in immortal human cells without detectable telomerase activity. EMBO J., 14: 4240–4248, 1995.[Medline]
  21. Opitz, O. G., Suliman, Y., Hahn, W. C., Harada, H., Blum, H. E., and Rustgi, A. K. Cyclin D1 overexpression and p53 inactivation immortalize primary oral keratinocytes by a telomerase-independent mechanism. J. Clin. Invest., 108: 725–732, 2001.[Medline]
  22. Henson, J. D., Neumann, A. A., Yeager, T. R., and Reddel, R. R. Alternative lengthening of telomeres in mammalian cells. Oncogene, 21: 598–610, 2002.[Medline]
  23. Yang, J., Chang, E., Cherry, A. M., Bangs, C. D., Oei, Y., Bodnar, A., Bronstein, A., Chiu, C. P., and Herron, G. S. Human endothelial cell life extension by telomerase expression. J. Biol. Chem., 274: 26141–26148, 1999.[Abstract/Free Full Text]
  24. Herbert, B. S., Wright, W. E., and Shay, J. W. p16(INK4a) inactivation is not required to immortalize human mammary epithelial cells. Oncogene, 21: 7897–7900, 2002.[Medline]
  25. Okamoto, A., Demetrick, D. J., Spillare, E. A., Hagiwara, K., Hussain, S. P., Bennett, W. P., Forrester, K., Gerwin, B., Serrano, M., Beach, D. H., and Harris, C. C. Mutations and altered expression of p16INK4 in human cancer. Proc. Natl. Acad. Sci. USA, 91: 11045–11049, 1994.[Abstract/Free Full Text]
  26. Otterson, G. A., Khleif, S. N., Chen, W., Coxon, A. B., and Kaye, F. J. CDKN2 gene silencing in lung cancer by DNA hypermethylation and kinetics of p16INK4 protein induction by 5-aza 2'deoxycytidine. Oncogene, 11: 1211–1216, 1995.[Medline]
  27. Enders, G. H., Koh, J., Missero, C., Rustgi, A. K., and Harlow, E. p16 inhibition of transformed and primary squamous epithelial cells. Oncogene, 12: 1239–1245, 1996.[Medline]
  28. Linardopoulos, S., Street, A. J., Quelle, D. E., Parry, D., Peters, G., Sherr, C. J., and Balmain, A. Deletion and altered regulation of p16INK4a and p15INK4b in undifferentiated mouse skin tumors. Cancer Res., 55: 5168–5172, 1995.[Abstract/Free Full Text]
  29. Kiyono, T., Foster, S. A., Koop, J. I., McDougall, J. K., Galloway, D. A., and Klingelhutz, A. J. Both Rb/p16INK4a inactivation and telomerase activity are required to immortalize human epithelial cells. Nature, 396: 84–88, 1998.[Medline]
  30. Dickson, M. A., Hahn, W. C., Ino, Y., Ronfard, V., Wu, J. Y., Weinberg, R. A., Louis, D. N., Li, F. P., and Rheinwald, J. G. Human keratinocytes that express hTERT and also bypass a p16(INK4a)-enforced mechanism that limits life span become immortal yet retain normal growth and differentiation characteristics. Mol. Cell. Biol., 20: 1436–1447, 2000.[Abstract/Free Full Text]
  31. Ramirez, R. D., Morales, C. P., Herbert, B. S., Rohde, J. M., Passons, C., Shay, J. W., and Wright, W. E. Putative telomere-independent mechanisms of replicative aging reflect inadequate growth conditions. Genes Dev., 15: 398–403, 2001.[Abstract/Free Full Text]
  32. Rheinwald, J. G., Hahn, W. C., Ramsey, M. R., Wu, J. Y., Guo, Z., Tsao, H., De Luca, M., Catricala, C., and O'Toole, K. M. A two-stage, p16(INK4A)- and p53-dependent keratinocyte senescence mechanism that limits replicative potential independent of telomere status. Mol. Cell. Biol., 22: 5157–5172, 2002.[Abstract/Free Full Text]
  33. Andl, C. D., Mizushima, T., Nakagawa, H., Oyama, K., Harada, H., Chruma, K., Herlyn, M., and Rustgi, A. K. Epidermal growth factor receptor mediates increased cell proliferation, migration, and aggregation in esophageal keratinocytes in vitro and in vivo. J. Biol. Chem., 278: 1824–1830, 2003.[Abstract/Free Full Text]
  34. Koh, J., Enders, G. H., Dynlacht, B. D., and Harlow, E. Tumor-derived p16 alleles encoding proteins defective in cell-cycle inhibition. Nature, 375: 506–510, 1995.[Medline]
  35. Wang, X. Q., Gabrielli, B. G., Milligan, A., Dickinson, J. L., Antalis, T. M., and Ellem, K. A. Accumulation of p16CDKN2A in response to ultraviolet irradiation correlates with late S-G(2)-phase cell cycle delay. Cancer Res., 56: 2510–2514, 1996.[Abstract/Free Full Text]
  36. Pavey, S., Conroy, S., Russell, T., and Gabrielli, B. Ultraviolet radiation induces p16CDKN2A expression in human skin. Cancer Res., 59: 4185–4189, 1999.[Abstract/Free Full Text]
  37. Wang, J., Hannon, G. J., and Beach, D. H. Risky immortalization by telomerase. Nature, 405: 755–756, 2000.[Medline]
  38. Hastie, N. D., Dempster, M., Dunlop, M. G., Thompson, A. M., Green, D. K., and Allshire, R. C. Telomere reduction in human colorectal carcinoma and with ageing. Nature, 346: 866–868, 1990.[Medline]
  39. Lindsey, J., McGill, N. I., Lindsey, L. A., Green, D. K., and Cooke, H. J. In vivo loss of telomeric repeats with age in humans. Mutat. Res., 256: 45–48, 1991.[Medline]
  40. Alcorta, D. A., Xiong, Y., Phelps, D., Hannon, G., Beach, D., and Barrett, J. C. Involvement of the cyclin-dependent kinase inhibitor p16 (INK4a) in replicative senescence of normal human fibroblasts. Proc. Natl. Acad. Sci. USA, 93: 13742–13747, 1996.[Abstract/Free Full Text]
  41. Hara, E., Smith, R., Parry, D., Tahara, H., Stone, S., and Peters, G. Regulation of p16CDKN2 expression and its implications for cell immortalization and senescence. Mol. Cell. Biol., 16: 859–867, 1996.[Abstract]
  42. Dulic, V., Drullinger, L. F., Lees, E., Reed, S. I., and Stein, G. H. Altered regulation of G1 cyclins in senescent human diploid fibroblasts: accumulation of inactive cyclin E-Cdk2 and cyclin D1-Cdk2 complexes. Proc. Natl. Acad. Sci. USA, 90: 11034–11038, 1993.[Abstract/Free Full Text]
  43. Lucibello, F. C., Sewing, A., Brusselbach, S., Burger, C., and Muller, R. Deregulation of cyclins D1 and E and suppression of cdk2 and cdk4 in senescent human fibroblasts. J. Cell Sci., 105 (Pt 1): 123–133, 1993.[Abstract]
  44. Meyyappan, M., Wong, H., Hull, C., and Riabowol, K. T. Increased expression of cyclin D2 during multiple states of growth arrest in primary and established cells. Mol. Cell. Biol., 18: 3163–3172, 1998.[Abstract/Free Full Text]
  45. Serrano, M. and Blasco, M. A. Putting the stress on senescence. Curr. Opin. Cell Biol., 13: 748–753, 2001.[Medline]
  46. Brookes, S., Rowe, J., Ruas, M., Llanos, S., Clark, P. A., Lomax, M., James, M. C., Vatcheva, R., Bates, S., Vousden, K. H., Parry, D., Gruis, N., Smit, N., Bergman, W., and Peters, G. INK4a-deficient human diploid fibroblasts are resistant to RAS-induced senescence. EMBO J., 21: 2936–2945, 2002.[Medline]
  47. Alexander, K. and Hinds, P. W. Requirement for p27(KIP1) in retinoblastoma protein-mediated senescence. Mol. Cell. Biol., 21: 3616–3631, 2001.[Abstract/Free Full Text]
  48. Munro, J., Stott, F. J., Vousden, K. H., Peters, G., and Parkinson, E. K. Role of the alternative INK4A proteins in human keratinocyte senescence: evidence for the specific inactivation of p16INK4A upon immortalization. Cancer Res., 59: 2516–2521, 1999.[Abstract/Free Full Text]
  49. Bates, S., Phillips, A. C., Clark, P. A., Stott, F., Peters, G., Ludwig, R. L., and Vousden, K. H. p14ARF links the tumor suppressors RB and p53. Nature, 395: 124–125, 1998.[Medline]
  50. Koyanagi, K., Ozawa, S., Ando, N., Mukai, M., Kitagawa, Y., Ueda, M., and Kitajima, M. Telomerase activity as an indicator of malignant potential in iodine-nonreactive lesions of the esophagus. Cancer, 88: 1524–1529, 2000.[Medline]
  51. Takubo, K., Nakamura, K., Izumiyama, N., Mafune, K., Tanaka, Y., Miyashita, M., Sasajima, K., Kato, M., and Oshimura, M. Telomerase activity in esophageal carcinoma. J. Surg. Oncol., 66: 88–92, 1997.[Medline]
  52. Koyanagi, K., Ozawa, S., Ando, N., Takeuchi, H., Ueda, M., and Kitajima, M. Clinical significance of telomerase activity in the non-cancerous epithelial region of esophageal squamous cell carcinoma. Br. J. Surg., 86: 674–679, 1999.[Medline]
  53. Jiang, X. R., Jimenez, G., Chang, E., Frolkis, M., Kusler, B., Sage, M., Beeche, M., Bodnar, A. G., Wahl, G. M., Tlsty, T. D., and Chiu, C. P. Telomerase expression in human somatic cells does not induce changes associated with a transformed phenotype. Nat. Genet., 21: 111–114, 1999.[Medline]
  54. Morales, C. P., Holt, S. E., Ouellette, M., Kaur, K. J., Yan, Y., Wilson, K. S., White, M. A., Wright, W. E., and Shay, J. W. Absence of cancer-associated changes in human fibroblasts immortalized with telomerase. Nat. Genet., 21: 115–118, 1999.[Medline]
  55. Blasco, M. A. Telomerase beyond telomeres. Nat. Rev. Cancer, 2: 627–633, 2002.[Medline]
  56. Stewart, S. A., Hahn, W. C., O'Connor, B. F., Banner, E. N., Lundberg, A. S., Modha, P., Mizuno, H., Brooks, M. W., Fleming, M., Zimonjic, D. B., Popescu, N. C., and Weinberg, R. A. Telomerase contributes to tumorigenesis by a telomere length-independent mechanism. Proc. Natl. Acad. Sci. USA, 99: 12606–12611, 2002.[Abstract/Free Full Text]
  57. Mitra, J., Dai, C. Y., Somasundaram, K., El-Deiry, W. S., Satyamoorthy, K., Herlyn, M., and Enders, G. H. Induction of p21(WAF1/CIP1) and inhibition of Cdk2 mediated by the tumor suppressor p16(INK4a). Mol. Cell. Biol., 19: 3916–3928, 1999.[Abstract/Free Full Text]
  58. Herman, J. G., Graff, J. R., Myohanen, S., Nelkin, B. D., and Baylin, S. B. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc. Natl. Acad. Sci. USA, 93: 9821–9826, 1996.[Abstract/Free Full Text]
  59. Hentosh, P., Yuh, S. H., Elson, C. E., and Peffley, D. M. Sterol-independent regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in tumor cells. Mol. Carcinog., 32: 154–166, 2001.[Medline]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
L. A. Feagins, H. Y. Zhang, X. Zhang, K. Hormi-Carver, T. Thomas, L. S. Terada, S. J. Spechler, and R. F. Souza
Mechanisms of oxidant production in esophageal squamous cell and Barrett's cell lines
Am J Physiol Gastrointest Liver Physiol, February 1, 2008; 294(2): G411 - G417.
[Abstract] [Full Text] [PDF]

Home page
 Genes Dev.Home page
T. Okawa, C. Z. Michaylira, J. Kalabis, D. B. Stairs, H. Nakagawa, C. Andl, C. N. Johnstone, A. J. Klein-Szanto, W. S. El-Deiry, E. Cukierman, et al.
The functional interplay between EGFR overexpression, hTERT activation, and p53 mutation in esophageal epithelial cells with activation of stromal fibroblasts induces tumor development, invasion, and differentiation
Genes & Dev., November 1, 2007; 21(21): 2788 - 2803.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
B. G. Goldstein, H.-H. Chao, Y. Yang, Y. A. Yermolina, J. W. Tobias, and J. P. Katz
Overexpression of Kruppel-like factor 5 in esophageal epithelia in vivo leads to increased proliferation in basal but not suprabasal cells
Am J Physiol Gastrointest Liver Physiol, June 1, 2007; 292(6): G1784 - G1792.
[Abstract] [Full Text] [PDF]

Home page
 FASEB J.Home page
Y. Yang, B. G. Goldstein, H. Nakagawa, and J. P. Katz
Kruppel-like factor 5 activates MEK/ERK signaling via EGFR in primary squamous epithelial cells
FASEB J, February 1, 2007; 21(2): 543 - 550.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Pathol.Home page
M. Lioni, P. Brafford, C. Andl, A. Rustgi, W. El-Deiry, M. Herlyn, and K. S.M. Smalley
Dysregulation of Claudin-7 Leads to Loss of E-Cadherin Expression and the Increased Invasion of Esophageal Squamous Cell Carcinoma Cells
Am. J. Pathol., February 1, 2007; 170(2): 709 - 721.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
D. Kranz and M. Dobbelstein
Nongenotoxic p53 Activation Protects Cells against S-Phase-Specific Chemotherapy
Cancer Res., November 1, 2006; 66(21): 10274 - 10280.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
S.-H. Kim, H. Nakagawa, A. Navaraj, Y. Naomoto, A. J.P. Klein-Szanto, A. K. Rustgi, and W. S. El-Deiry
Tumorigenic Conversion of Primary Human Esophageal Epithelial Cells Using Oncogene Combinations in the Absence of Exogenous Ras
Cancer Res., November 1, 2006; 66(21): 10415 - 10424.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
C. D. Andl, B. B. Fargnoli, T. Okawa, M. Bowser, M. Takaoka, H. Nakagawa, A. Klein-Szanto, X. Hua, M. Herlyn, and A. K. Rustgi
Coordinated Functions of E-Cadherin and Transforming Growth Factor {beta} Receptor II In vitro and In vivo.
Cancer Res., October 15, 2006; 66(20): 9878 - 9885.
[Abstract] [Full Text] [PDF]

Home page
 FASEB J.Home page
R. Maurelli, G. Zambruno, L. Guerra, C. Abbruzzese, G. Dimri, M. Gellini, S. Bondanza, and E. Dellambra
Inactivation of p16INK4a (inhibitor of cyclin-dependent kinase 4A) Immortalizes Primary Human Keratinocytes By Maintaining Cells in the Stem Cell Compartment
FASEB J, July 1, 2006; 20(9): 1516 - 1518.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
M. Takaoka, C. E. Smith, M. K. Mashiba, T. Okawa, C. D. Andl, W. S. El-Deiry, and H. Nakagawa
EGF-mediated regulation of IGFBP-3 determines esophageal epithelial cellular response to IGF-I
Am J Physiol Gastrointest Liver Physiol, February