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Molecular and Cellular Biology, February 2000, p. 1436-1447, Vol. 20, No. 4
Division of Dermatology, Department of
Medicine and Harvard Skin Disease Research Center, Brigham and Women's
Hospital,1 Department of Adult Oncology,
Dana-Farber Cancer Institute, and Department of Medicine, Brigham and
Women's Hospital,3 Department of
Pathology and Neurosurgical Service, Massachusetts General
Hospital,4 and Department of Adult
Oncology, Dana-Farber Cancer Institute,6 Harvard
Medical School, Boston, Whitehead Institute for Biomedical
Research and Department of Biology, Massachusetts Institute of
Technology, Cambridge,2 and Laboratory
of Cell and Tissue Development, Organogenesis, Inc.,
Canton,5 Massachusetts
Received 11 August 1999/Returned for modification 11 October
1999/Accepted 18 November 1999
Normal human cells exhibit a limited replicative life span in
culture, eventually arresting growth by a process termed senescence. Progressive telomere shortening appears to trigger senescence in normal
human fibroblasts and retinal pigment epithelial cells, as ectopic
expression of the telomerase catalytic subunit, hTERT, immortalizes
these cell types directly. Telomerase expression alone is insufficient
to enable certain other cell types to evade senescence, however. Such
cells, including keratinocytes and mammary epithelial cells, appear to
require loss of the pRB/p16INK4a cell cycle control
mechanism in addition to hTERT expression to achieve immortality. To
investigate the relationships among telomerase activity, cell cycle
control, senescence, and differentiation, we expressed hTERT in two
epithelial cell types, keratinocytes and mesothelial cells, and
determined the effect on proliferation potential and on the function of
cell-type-specific growth control and differentiation systems. Ectopic
hTERT expression immortalized normal mesothelial cells and a
premalignant, p16INK4a-negative keratinocyte line. In
contrast, when four keratinocyte strains cultured from normal tissue
were transduced to express hTERT, they were incompletely rescued from
senescence. After reaching the population doubling limit of their
parent cell strains, hTERT+ keratinocytes entered a slow
growth phase of indefinite length, from which rare, rapidly dividing
immortal cells emerged. These immortal cell lines frequently had
sustained deletions of the CDK2NA/INK4A locus or otherwise
were deficient in p16INK4a expression. They nevertheless
typically retained other keratinocyte growth controls and
differentiated normally in culture and in xenografts. Thus,
keratinocyte replicative potential is limited by a
p16INK4a-dependent mechanism, the activation of which can
occur independent of telomere length. Abrogation of this mechanism
together with telomerase expression immortalizes keratinocytes without
affecting other major growth control or differentiation systems.
Normal human somatic cells have a
limited capacity to replicate in culture, even under conditions that
appear to satisfy their nutritional and mitogen requirements (53,
56). These cells proliferate initially but eventually enter a
state of permanent growth arrest termed senescence, clearly distinct
from differentiation, in which they can remain metabolically active
indefinitely. Progressive shortening of the telomeres, DNA-protein
structures located at the ends of linear eukaryotic chromosomes, occurs
during the 50- to 100-population-doubling (PD) life span of human
fibroblasts in culture (19). The erosion of telomeric DNA
with successive cell replications has led to the proposal that
telomeres not only function to protect the chromosomes from end-to-end
fusions but, when disrupted by shortening, also signal the onset of
senescence (2).
Unlike most normal human somatic cell types, most advanced-stage cancer
cells are replicatively immortal and express the enzyme telomerase.
Telomerase is a multimeric ribonucleoprotein containing an RNA
component that includes in its sequence the template for telomere
synthesis (14) and a catalytic protein subunit that is a
reverse transcriptase (34, 38). The expression of telomerase in immortal cancer cells apparently is responsible for their
maintenance of a stable telomere length through an indefinite number of
cell divisions (11). Although the telomerase RNA component
is expressed constitutively (19), the catalytic subunit,
hTERT, is expressed only in germ cells and in immortal cancer cells
(34, 38), suggesting that hTERT is the activity-limiting
component of the telomerase holoenzyme. Introduction of hTERT into
presenescent human fibroblasts and retinal pigment epithelial cells was
found to confer telomere maintenance and unlimited replicative
potential to these cell types (5), giving strong support to
the model that telomere shortening determines the onset of senescence.
This simple interpretation, however, may not apply to all cell types, as it was reported recently that ectopic expression of hTERT is not
sufficient to immortalize normal human keratinocytes and mammary epithelial cells (25).
We have sought to investigate the role of telomerase in cellular
senescence, to identify potential ancillary genetic alterations necessary for immortalization of epithelial cells, and to determine the
effects of immortalization on cell-type-specific growth control and
differentiation mechanisms. We have expressed hTERT in two different
types of epithelial cells, mesothelial cells and keratinocytes, both of
which exhibit a finite life span in vitro and have well-characterized growth control systems and differentiation programs (10, 16, 47,
52). Our experiments indicate that these two epithelial cell
types behave very differently in response to ectopically expressed
hTERT and that such expression is not sufficient to immortalize
keratinocytes. We have identified a complex pattern of
p16INK4a expression in keratinocytes associated with
senescence which functions independent of telomere shortening.
Keratinocytes that express hTERT and also acquire a defect in
triggering p16INK4a expression become immortalized but
otherwise display normal growth characteristics and differentiation
potential, indicating that the process of senescence in this cell type
is complex but separate from mechanisms that regulate growth and differentiation.
Cell lines and culture media.
Most of the cells studied
(Table 1) were cell strains (sometimes
termed primary cells) cultured from clinically and presumably genetically normal tissues (28, 45, 61). POE9n was cultured from a premalignant dysplastic oral epithelial lesion and was found to
have a homozygous deletion at the CDKN2A/INK4A locus, to
lack expression of p53, and to have an extended but finite life span
(J. Rheinwald, J. Benwood, A. Palanisamy, Y. Ino, D. Louis, R. Feldman, and E. Sauter, unpublished data). LiF-Ep was cultured from
phenotypically normal epidermis from an individual (National Cancer
Institute repository identifier no. 1010 from kindred no. 1 [27]) with pancreatic cancer and Li-Fraumeni syndrome (31). The inherited mutation in this individual is a
heterozygous, one-base-pair deletion at the exon 9-intron 9 junction of
the TP53 gene. Somatic cells sampled from this individual
previously were found by reverse transcription-PCR analysis not to
express detectable levels of p53 mRNA encoded by the mutant allele (S. Verselis, personal communication).
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Human Keratinocytes That Express hTERT and Also
Bypass a p16INK4a-Enforced Mechanism That Limits Life
Span Become Immortal yet Retain Normal Growth and
Differentiation Characteristics
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Human cell lines
10 M cholera
toxin, 2 × 1011 M triiodothyronine, and 1.8 × 104 M adenine [44, 46]). Keratinocytes
and derived transductants were subsequently cultured without feeder
cells in keratinocyte serum-free medium (K-sfm) (GIBCO/BRL) plus 30 µg of bovine pituitary extract per ml, 0.1 ng of EGF per ml and
additional CaCl2 to raise the [Ca2+] to 0.4 mM (52).
Dermal fibroblasts were cultured in medium 199 (M199) plus 15% CS plus
10 ng of EGF per ml. Mesothelial cells were cultured in M199 plus 15%
CS plus 10 ng of EGF per ml plus 0.4 µg of HC per ml (10).
3T3J2 cells and PT67 retroviral vector producer cells (17)
were cultured in pyruvate-free Dulbecco modified Eagle medium
(GIBCO/BRL)-10% CS. A multiple-drug-resistant fibroblast feeder cell
line, 3T3J2NHP, was generated by sequentially transducing 3T3J2 with
the retroviral vectors LXSN (35), BABE/hygro, and BABE/puro
(36) to confer resistance to G418, hygromycin, and puromycin, respectively. This line was used as feeder cells to support
transduced keratinocyte populations during selection with any of these drugs.
Retroviral vectors and transduction.
PT67 amphotropic
packaging cells producing BABE-hygro-hTERT and BABE-puro-hTERT
(17) or control BABE-hygro and BABE-puro (36)
retroviral vectors were used to generate retroviral supernatants in
K-sfm, which was passed through a 0.45-µm-pore-size filter and stored
at 
80°C until use. Human cells plated 1 day earlier at
~105 cells per 9-cm2 well were transduced by
refeeding them for 5 to 7 h with retroviral supernatant plus
Polybrene (4 µg/ml; Sigma). The treated cells were subcultured the
next day into 75-cm2 flasks. Drug selection (5 to 10 µg
of hygromycin per ml or 1 µg of puromycin per ml) was started 2 days
after transduction and continued for 7 to 14 days. Some hTERT
transductants were generated by coplating keratinocytes with
mitomycin-treated retroviral producer cells in FAD medium. Four days
later, the producer cells were selectively removed by brief incubation
with EDTA and vigorous pipetting, mitomycin-treated 3T3J2NHP cells were
added back to the cultures, and drug selection was started the next day.
Replicative life span determination. Cells were plated at 1 × 105 to 3 × 105 cells per T75 or T175 flask or p100 dish in their appropriate growth medium, refed every 2 to 3 days, and subcultured 5 to 8 days later, before high cell density slowed their growth. PD per passage was calculated as log2 (number of cells at time of subculture/number of cells plated). No correction was made for cells that failed to reinitiate growth at subculture. Cumulative PD was plotted against total time in culture to assess replicative life span, senescence, slow growth, or crisis, and immortalization, the latter judged to have occurred if cells grew for at least 50 PD beyond the life span of the parent cell line.
Telomerase assays and telomere length determination. Telomerase activity was detected using the PCR-based, telomerase repeat amplification protocol (TRAP) assay (24). Telomere length was measured by hybridizing a 32P-labeled telomeric (CCCTAA)3 probe to HinfI- and RsaI-digested genomic DNA separated on agarose gels (12).
CDKN2A/INK4A genomic analysis.
For genomic
analysis of keratinocyte cell lines (Table
2), DNA was extracted from cell pellets
Puregene DNA isolation kit (Gentra Systems). Allelic loss on the
chromosome 9p21 region was assessed by comparing parent cell
line-experimental cell line sets using microsatellite markers D9S126,
D9S741, and D9S1748 (3) as described previously
(30) (primer sequences are available from the genome
database [gdbwww.gdb.org]). Exons 1 through 3 of the
CDKN2A/INK4A gene were screened for mutations by
single-strand conformation polymorphism (SSCP) analysis, followed by
direct DNA sequencing of samples with shifted bands, as described
previously (58). CDKN2A/INK4A homozygous
deletions were detected by comparative multiplex PCR as described
previously (39). Hypermethylation of CpG islands of the
CDKN2A/INK4A promoter was assessed by methylation-specific PCR (20), with minor modifications as described previously
(8).
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Growth control assays. Cells were plated at 103 to 3 × 103 cells per 9-cm2 well in various medium formulations, refed every 3 to 4 days, and counted after 9 to 11 days of growth, when cells cultured in the control, permissive medium were still actively proliferating.
Keratinocytes.
To assess mitogen dependence, cells were
plated in K-sfm supplemented with the reduced bovine pituitary extract
concentration of 10 µg/ml with or without EGF (0.1 ng/ml). To assess
sensitivity to growth inhibitors, cells were cultured in K-sfm to which
109 M tetradecanoyl phorbol acetate (TPA; Sigma) or 0.3 ng of transforming growth factor 
1 (R&D Systems) was added. Cells
were cultured in control and experimental conditions for 7 to 9 days,
and their growth rates were calculated as [log2 (number of
cells at harvest/number of cells plated)]/number of days = PD/day.
Mesothelial cells. To assess mitogen dependence, cells were plated in M199 plus 15% CS with or without EGF and HC and in M199 medium plus EGF plus HC plus 1% CS (10, 57).
Immunochemical analysis of cell cycle regulatory protein expression. Cultured cells were trypsinized, rinsed in phosphate-buffered saline (PBS), pelleted, and lysed in 20 mM Tris buffer (pH 7.3)-2% sodium dodecyl sulfate-1 mM dithiothreitol. Twenty to 100 µg of protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using a polyacrylamide concentration of 14% for p16INK4a, 10 or 12% for cyclin D1, cdk4, and cdk6, or 7% for pRB. Gels were electrotransferred to nitrocellulose paper, and proteins were detected with antibodies specific for p16INK4a (JC1; a gift from E. Harlow, Massachusetts General Hospital, Boston), pRB (G3-245; PharMingen), p53 (DO-1), cdk4 (C-22), cdk6 (C21), and cyclin D1 (HD11) (the latter four antibodies all from Santa Cruz Biotechnologies), followed by peroxidase-labeled secondary antibody (Southern Biotechnologies) and enhanced chemiluminescence reagent (Amersham Corp.).
Cells growing on culture dishes, some which received 0.5 nM actinomycin D for the final 2 days to elicit a DNA damage-stimulated increase in p53 levels (23), were fixed in 4% paraformaldehyde in PBS for 30 min, permeabilized with 0.1% Triton X-100 in PBS for 5 min, and incubated for 30 min with antibodies specific for p16INK4a (JC8 or JC2 [8]; a gift from E. Harlow) or p53 (BP53.12; Zymed). Antibody binding was detected using ABC peroxidase (Vector Laboratories) with NovaRed colorimetric substrate.Cell differentiation and histogenesis assays. Keratinocyte differentiation-related proteins were detected immunocytochemically by ABC peroxidase staining (Vector Laboratories) as described elsewhere (52). Cultures were fixed in cold methanol and immunostained for involucrin (antibody SY5 [21]; from F. Watt, ICRF Laboratories, London, England), for keratin K10 (antibody AE20 [32]; from C. A. Loomis, New York University School of Medicine, New York, N.Y.), and for keratin K13 (antibody AE8 [13]; from T.-T. Sun, New York University School of Medicine).
The ability of hTERT-immortalized keratinocytes to form a differentiated, stratified squamous epithelium was assessed in organotypic culture (40, 52). Keratinocytes were seeded at 2 × 105 cells per ~1-cm2 surface area onto collagen gels containing human foreskin fibroblasts (strain B256), cultured submerged for 4 days, and then at the air-liquid interface for 10 days. Cell lines of oral epithelial origin (i.e., OKF6/TERT-1, OKF6/TERT-2, and POE9n/TERT-1) received 108 M retinoic
acid, which provides for more accurate recapitulation of in vivo
histology by oral mucosal keratinocytes (52). Cultures were
fixed in formalin and embedded in paraffin or were frozen in OCT
compound. Sections were stained with hematoxylin and eosin (H&E).
For grafting, athymic NIH Swiss (nu/nu) mice were
anesthetized with ketamine-xylazine, and a 1-cm2 area of
full-thickness skin was excised from the middle of the back. An
organotypic culture was transferred to the site and held in place with
Vaseline-impregnated gauze covered by a Band-Aid. Grafts were kept
covered for 1 week, after which they were exposed to the air. Mice were
sacrificed 24 or 48 days postgrafting; the grafts were excised, fixed
in formalin, paraffin embedded, sectioned, and stained with H&E or with
a human involucrin-specific antiserum (BTI-601 [48];
Biomedical Technologies, Inc.) to distinguish human from mouse epithelium.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Effect of hTERT expression on human mesothelial cells, a simple squamous epithelial cell type. To examine the effects of hTERT expression on the growth of epithelial cells, we used amphotropic retroviral vectors that transduced hTERT and a drug resistance selection marker, or a control vector expressing only the marker gene, into mid-life-span cultures of the normal mesothelial cell strain LP9. We also transduced these genes into the normal dermal fibroblast strain S1F to serve as a control for the function of our hTERT vector, since hTERT has previously been shown to immortalize normal human fibroblasts (5, 25). The resulting hTERT-expressing transductants exhibited readily detectable telomerase activity, whereas the control cells did not (data not shown). Later-passage LP9 and S1F control cells had short (~3-kb) telomeres (Fig. 1b), whereas the respective hTERT transductants acquired and maintained average telomere lengths of ~10 kb.
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), of control vector transductants (
), and of hTERT
transductants (
). The label in each panel is the name of the
immortal cell line that resulted from that hTERT transduction. In panel
c, +, +,
,
N/BABE-2G; 
, N/TERT-2F). In panel e, some
OKF6/TERT-1 cells cryopreserved at the onset of SGP (indicated by *)
were thawed and the serial passage was repeated (
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Effects of expressing hTERT in normal human keratinocytes. We then extended these experiments to the study of a different epithelial cell type, the keratinocyte. We used cells cultured from specimens of normal human epidermis (strain N) and from normal oral mucosal epithelium (strains OKF6 and OKF4). Mid-life-span cultures of N and OKF6 were infected with amphotropic retroviral vectors encoding hTERT or a drug resistance marker alone, as before. In each case, cells transduced with a retrovirus encoding hTERT demonstrated clearly detectable telomerase activity, while control cells were telomerase negative (Fig. 1a). Corresponding to this acquisition of telomerase activity, hTERT-expressing cells maintained their telomeres at lengths longer than those of control cells (Fig. 1b).
In each case, cells transduced with the control vector senesced between 35 and 55 PD, as evidenced by a lack of net population increase during passage (Fig. 2b to e) and complete cessation of division accompanied by enlargement of all cells in the culture. This senescence occurred after approximately the same number of cumulative PD as we had observed previously for the untransduced parent cell strains (data not shown). Senescent keratinocytes did not reattach efficiently when subcultured, resulting in progressive loss of cells at subsequent passages. A different outcome was observed for the hTERT-expressing keratinocytes. After the same cumulative PD at which control cells senesced, further expansion of the hTERT-transduced population was severely curtailed but not completely arrested. Instead, the cells entered a slow-growth phase (SGP) characterized by low colony-forming efficiencies (<1 to 5% of plated cells) and lower than normal colony growth rates, which resulted in minimal to modest net population increase during serial passage. The length of the SGP ranged from about 3 weeks to 3 months for the nine hTERT-transduced keratinocyte cultures that we examined, during which time the PD rate remained quite constant for each hTERT+ culture, ranging among cultures from 0.03 to 0.29 PD/day (Fig. 2; Table 3). Similar to normal keratinocyte cultures (4, 60), dividing cells in hTERT+ SGP cultures were relatively small, whereas nondividing cells became enlarged. hTERT-transduced populations examined before they had entered or during their SGP were found to have telomerase enzyme activity (Fig. 1, lanes 2, 3, 6, and 10) and to have an average telomere length substantially greater than that of control cells approaching senescence (Fig. 1b, lane 6; Fig. 1c, lanes 2, 7, and 9). We therefore concluded that the low PD rate exhibited by hTERT-transduced keratinocytes after they had reached their normal replicative life span limit was not the result of failure to reverse telomere erosion.RDI cells arise from hTERT+ keratinocyte populations. In eight of the nine hTERT-transduced keratinocyte populations that we examined, rapidly dividing cells eventually appeared in the SGP cultures and took over the population within several passages of their first visual detection. Their first appearance, typically seen as a single colony or small number of rapidly growing colonies within an otherwise slowly dividing population, suggested clonal origin as the progeny of a single cell that had undergone a permanent heritable alteration. The rapidly dividing variant cells were uniformly small and had a high colony-forming efficiency and population growth rate, typically similar to that of early-passage cells of the parent line. These rapidly dividing, immortalized (RDI) cells expressed telomerase activity (Fig. 1a) and maintained telomeres at lengths greater than or equal to that of control cells (Fig. 1c). They continued to divide for at least 50 PD beyond the normal life span of the parent line. We therefore concluded that they had undergone immortalization.
The emergence of RDI cells from hTERT+ SGP populations appeared to be stochastic. The duration of SGP varied greatly (Table 3) among three independent hTERT-transduced OKF6 cultures, with one SGP population ceasing growth before emergence of an RDI variant. Cryopreserved SGP cells of the same transduction from which the OKF6/TERT-1 RDI cell line had emerged were thawed and reanalyzed by serially passage again under the same conditions. We again observed eventual emergence of an RDI line after an SGP of long duration (Fig. 2e; Table 3). This second RDI line (OKF6/TERT-1R) had a lower proliferation rate than OKF6/TERT-1 (Table 3) and, as described below, carried different genomic alterations. We therefore concluded that the two RDI lines represented progeny of independent genetic events that occurred in this hTERT+ population during the SGP. We entertained the possibility that the two-stage immortalization process that we observed, and that was reported by others recently (25), might have been the result of the specific conditions that we were using to culture keratinocytes. Specifically, we wished to determine whether the absence of fibroblast feeder cells, previously found to be beneficial for long-term, albeit limited, serial propagation of normal human keratinocytes (2, 46, 47), might have caused the cells to have a limited expansion potential in spite of hTERT expression and telomere stabilization. In the experiment shown in Fig. 2d, N cells were transduced with hTERT or control vectors and the drug-selected populations then were divided, with half of the cells propagated subsequently in the feeder cell-FAD system and half placed in K-sfm medium without feeder cells. The PD levels at which control cells senesced and hTERT transductants entered SGP were very nearly the same in both culture systems, and in this experiment, RDI cells arose within the population passaged in K-sfm (designated N/TERT-2G) before they arose in the population passaged in the feeder cell-FAD system (designated N/TERT-2F) (Fig. 2d; Table 3). In another experiment, we transduced the normal oral keratinocyte strain OKF4 with the BABE-puro-hTERT vector and, after drug selection, serially passaged the hTERT transductants in the feeder cell-FAD system. The cells went through an SGP, and RDI cells (OKF4/TERT-1) emerged (Table 3), which we found were able to divide rapidly and indefinitely also in K-sfm medium (data not shown). We concluded that in both culture systems a second event, in addition to hTERT expression, is required for keratinocyte immortalization and that the same type(s) of second event can serve to complement hTERT in conferring an RDI phenotype to keratinocytes growing in either culture system.Loss of p16INK4a expression and deletions involving the CDKN2A/INK4A locus in hTERT-immortalized keratinocytes. We expressed hTERT in keratinocytes cultured from a premalignant oral lesion, strain POE9n, which was found to have a homozygous deletion of the CDKN2A/INK4A locus and to exhibit an abnormally extended but limited, replicative life span (J. Rheinwald, J. Benwood, A. Palanisamy, Y. Ino, D. Louis, R. Feldman, and E. Sauter, unpublished data). As shown in Fig. 2f, hTERT-transduced POE9n cells divided continuously for at least 50 doublings beyond the limit of control vector-expressing cells without exhibiting any period of slowed growth. We obtained similar results (i.e., direct immortalization without an SGP) when we expressed hTERT in strain N keratinocytes that had been previously transduced to express the human papillomavirus type 16 (HPV 16) E7 protein (N/E7 cells) (Tables 1 and 3), confirming a prior report (25). Since both POE9n cells and N/E7 cells lack a functional pRB cell cycle regulatory pathway (loss of p16INK4a and expression of the HPV16 E7 protein, which sequester and inactivate pRB, respectively), we hypothesized that defects in the pRB pathway complement the expression of hTERT in keratinocytes to bypass senescence and yield RDI cells.
To test this hypothesis, we examined expression of pRB pathway proteins in the RDI lines that ultimately had arisen following hTERT expression in the normal keratinocyte strains. Exponentially growing cultures of hTERT+ RDI lines of strains N, LiF-Ep, and OKF6 were compared with mid-life-span cultures of their parent cell lines for expression of p16INK4a and other cell cycle regulatory proteins that act in the pRB-mediated mechanism controlling the G1 restriction point. As shown in Fig. 3a, levels of cyclin D1, cdk4, cdk6, and pRB expressed by hTERT-immortalized keratinocytes were similar to those of the parent cell lines. Importantly, no evidence of cyclin D1 or cdk4/6 overexpression or loss of pRB expression was detected in the hTERT-expressing cells.
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Lack of a complementary role for p53 mutations in keratinocyte
immortalization by hTERT.
The TP53 gene product, p53,
has been implicated as playing an important role in initiating
senescence-associated growth arrest in fibroblasts and other cell types
(53). It has been reported that cells cultured from
individuals with Li-Fraumeni (inherited (p53+/) syndrome
may occasionally undergo spontaneous immortalization associated with
loss of the wild-type p53 allele (50, 55). We therefore
included in our transduction experiments LiF-Ep, a keratinocyte strain
that we had cultured from a specimen of normal skin from an adult with
Li-Fraumeni syndrome (see Materials and Methods). The pattern of
senescence of control LiF-Ep cultures and the frequency with which RDI
cells arose following transduction expression were indistinguishable
from those of the normal, p53+/+ strains (Fig. 2; Table 3).
/ cells first appeared in the
LiF-Ep/TERT-1 culture long after the event responsible for conversion
of SGP cells to RDI status. The LiF-Ep/TERT-1 population consisted of
at least 99% p53+ cells at the time rapidly dividing
variants first emerged from the SGP (at 39 PD). Fifty percent of the
cells in the RDI population were p53 negative three passages later (at
48 PD), and the culture consisted entirely of p53-negative cells by 66 PD (Fig. 4 and 2c).
A different hTERT transduction of LiF-Ep yielded an RDI line,
LiF-Ep/TERT-2 (Table 3), in which apparently all of the RDI cells
(i.e., 150 of 150 colonies examined immunocytochemically [data not
shown]) remained phenotypically normal for p53 expression and function
at least 20 PD after their emergence from SGP. We therefore concluded
that p53-deficient keratinocytes do not have a selective advantage in
hTERT+ populations during SGP and, therefore, that loss of
p53 does not complement hTERT for keratinocyte immortalization.
Growth regulation and differentiation characteristics of
hTERT-immortalized keratinocytes.
We wished to determine whether
any aspect of immortalization, especially p16INK4a
deficiency, which is a frequent characteristic of keratinocyte transformation in vivo (9) and of human squamous cell
carcinoma (SCC) cell lines that can grow in culture (29,
37), impaired specific keratinocyte growth control and
differentiation mechanisms. Four of the hTERT transductants were
compared with their respective parent cell lines in several in vitro
assays for function of several keratinocyte growth regulatory
mechanisms that are frequently lost in SCC cells (41, 45,
51). In control conditions, the hTERT lines, tested at 45 to 68 PD after emergence as RDI cells, all had higher PD rates than mid- to
late-life-span cells of their respective parent cell strains (Fig.
5). N/TERT-1, LiF-Ep/TERT-1, and
OKF6/TERT-1 cells still retained dependence upon EGF for growth and
sensitivity to growth inhibition by the phorbol ester TPA, exhibiting
only minimal growth (less than one (<1-PD increase over the plating
density during the course of the experiment) in the absence of EGF or
presence of TPA. The exception was OKF6/TERT-2, which remained TPA
sensitive but proliferated in the absence of EGF at 60% of the rate in
the presence of EGF, approaching the degree of EGF independence of
SCC-13. The four hTERT-transductants examined, including
OKF6/TERT-2, were growth inhibited by transforming growth factor (data not shown). We concluded that loss of normal keratinocyte growth
regulatory mechanisms is not a necessary consequence of
immortalization, even when the senescence-related
pRB/p16INK4a growth arrest mechanism has been abrogated, as
it is in N/TERT-1. However, our results could not eliminate the
possibility that homozygous deletion of the p16INK4a locus
was responsible for the EGF independence exhibited by OKF6/TERT-2 cells
(Fig. 5a) and by POE9n and POE9n/TERT cells (data not shown).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Several lines of evidence have implicated telomere erosion in limiting the proliferative potential of human cells, but the results presented here clearly indicate that a different mechanism is responsible for determining the replicative life span of human keratinocytes in culture. We have identified a precipitous increase in p16INK4a protein levels accompanying end-of-life-span growth arrest in normal keratinocytes, which appears to be triggered by a mechanism that is activated in cells with increasing probability as a cell population is serially propagated. Loss of this mechanism, whether by p16INK4a gene deletion, mutation, or altered regulation of expression, together with telomere stabilization effected by hTERT expression is necessary to enable a keratinocyte to become immortalized. Perhaps surprisingly, immortalization of keratinocytes by forced expression of telomerase and subsequent spontaneous events leading to loss of this p16INK4a-dependent mechanism generally does not disrupt other normal growth control mechanisms or affect the ability of the cells to form a differentiated epithelium. In contrast, senescence arrest is abrogated in cultured fibroblasts and retinal pigment epithelial cells by expression of hTERT (5), and our observations have added the mesothelial cell, a mesoderm-derived epithelial cell, to the group of cell types for which a telomere length-sensitive mechanism appears responsible for initiating senescence.
We have shown that expression of hTERT alone permits keratinocytes to escape complete growth arrest and to enter a phase of slow growth of variable length from which rapidly dividing immortal variants emerge. Such immortalized cells typically have identifiable defects in p16INK4a expression but retain functional p53. These results therefore confirm and extend those of Kiyono et al. (25), who demonstrated that expression of hTERT in combination with expression of the HPV16 E7 oncoprotein allowed human foreskin keratinocytes and mammary epithelial cells to bypass senescence. We have demonstrated that telomere length stabilization alone is unable to permit keratinocytes to bypass senescence, but that the subsequent slow, indefinite continued growth permitted by telomerase expression permits rare immortalized variants to arise.
Our observations clearly provide evidence supporting recent proposals (43, 62) that multiple "clocks" function to limit the proliferative capacity of human cells. The mechanism that triggers p16INK4a accumulation appears to sense the proliferative history of the keratinocyte, but preventing telomere erosion does not avoid its activation. In hTERT-expressing keratinocyte populations a small proportion of the cells indefinitely evade arrest, however. It is possible, therefore, that senescence-associated p16INK4a regulation is under two types of control, one that is tightly telomere length sensitive and another that is telomere length independent and stochastic. Whether the latter detects an aspect of cell aging related to number of cell divisions or to chronological time in culture remains to be determined because it is not possible to adjust culture conditions to slow or arrest keratinocyte growth without triggering irreversible commitment to terminal differentiation. The molecular mechanisms regulating p16INK4a gene expression are only beginning to be elucidated (18). It has been proposed that in vivo a subpopulation of keratinocytes serve as stem cells by possessing a very long or indefinite replicative potential (for example, see references 26 and 59). Whether stem cell status involves a special mechanism for avoiding senescence-related p16INK4a expression remains to be determined.
Mutations in the pRB/p16INK4a tumor suppressor pathway are found in the majority of human cancers (54). The observations presented here implicate this pathway as an essential control mechanism that must be subverted to create immortal cells. The role of p16INK4a in limiting epithelial cell proliferation is suggested by findings that variants of mammary epithelial cells that are unable to express p16 owing to promoter hypermethylation exhibit an extended replicative life span (6, 15); that p16 levels, but not p21 levels, progressively increase in keratinocyte cultures as they approach senescence (37); and that immortal SCC cell lines (29) and immortal variants of HPV16 E6-transfected prostate epithelial cell cultures (22) are found consistently to have lost p16 expression as a result of mutation or promoter hypermethylation.
It has been reported (25, 37) that p16INK4a levels, detected by Western blot analysis, increase in keratinocyte cultures as they are serially propagated. These data would support a model in which slowly increasing levels of p16INK4a in dividing keratinocytes eventually result in levels at which a subsequent G1-S transition can no longer occur. Our immunocytochemical analysis of normal and hTERT-transduced keratinocyte cultures revealed an unsuspected complexity in p16INK4a expression. We detected marked heterogeneity in p16INK4a levels within normal keratinocyte cultures at both early and late passage. p16INK4a protein was undetectable in small, rapidly dividing cells at any stage of the life span, at low to moderate levels in slightly larger cells in more slowly dividing colonies, and at very high levels in large, nondividing cells. This heterogeneous pattern of p16INK4a expression continued in hTERT+ keratinocyte populations during the SGP. These observations suggest that telomere-independent mechanisms signal senescence in keratinocytes by triggering a precipitous increase in p16INK4a levels that then causes cell cycle arrest. Moreover, they suggest that the incrementally increasing levels of p16INK4a observed in serially passaged keratinocyte cultures result from the increasing representation of cells that express high levels of p16INK4a amid a majority population of cells that remain p16INK4a negative. Since p21cip1 protein does not increase in senescent keratinocyte cultures (37), increased p16INK4a levels are likely to serve as the effector mechanism that enforces senescence-related growth arrest in keratinocytes.
In the majority of the rapidly dividing immortal variants that arose within the hTERT+ keratinocyte cultures examined here, we identified alterations compromising p16INK4a expression. Such lesions included homozygous deletion involving all p16INK4a exons, heterozygous deletion of the CDKN2A/INK4A locus with continued, heterogeneous expression of p16INK4a, and no detectable deletion, mutation, or promoter hypermethylation but complete loss of p16INK4a protein expression. The rapidly dividing immortal keratinocyte lines that continue to express p16INK4a may have mutations in other proteins involved in the pRB/p16INK4a pathway, or they may have acquired a lesion in a specific, senescence-related inducer of p16INK4a.
The deletions we detected at CDKN2A/INK4A also included loss of at least the second exon of p14ARF, another potential cell cycle inhibitor encoded in part by an alternate reading frame of the p16INK4a exon 2. p14ARF functions by preventing MDM2 from targeting p53 for degradation, thereby resulting in elevated levels of the latter and consequent cell cycle arrest (63). A recent study (37) has found that p14ARF levels do not increase during keratinocyte senescence and that all of 20 immortal human SCC lines examined had mutations compromising p16INK4a and none had mutations specifically affecting p14ARF. Our preliminary analyses also indicate that p14ARF is still expressed by hTERT-immortalized lines that still have at least one intact CDKN2A/INK4A allele (Z. Guo and J. Rheinwald, unpublished data), supporting the conclusion that it is loss of p16INK4a that is required to complement hTERT for keratinocyte immortalization.
We found no evidence that inactivation of any p53-dependent pathway is
necessary for immortalization of keratinocytes that express hTERT. In
neither of two hTERT transductions of the p53+/ LiF-Ep
strain did we find RDI conversion associated with loss of the wild-type
p53 allele. These observations are consistent with earlier studies
suggesting that p53 is an essential component of the telomere
length-sensitive growth arrest signal in fibroblasts (62),
in that hTERT-expressing keratinocytes would be expected to permanently
avoid activating such a signal.
Despite expression of hTERT and loss of p16INK4a function, the immortal keratinocytes described here are able to initiate their program of terminal differentiation, express suprabasal differentiation-specific proteins, and form differentiated epithelia in vitro and in vivo. In particular, the N/TERT-1 and LiF-Ep/TERT-1 lines continued to show a normal pattern of epidermal histogenesis in organotypic culture and in grafts to athymic mice. These results clearly indicate that p16 does not play an essential role in the irreversible growth arrest that precedes normal stratified squamous epithelial differentiation. Three of the four RDI TERT lines that we studied in detail also retained EGF dependence for growth at low density and sensitivity to irreversible growth arrest by TPA, which are normal keratinocyte growth control mechanisms consistently found to be lost in advanced SCCs (41, 45). Although OKF6/TERT-2, which had undergone homozygous deletion of the CDKN2A/INK4A locus, showed EGF-independent growth, our observations clearly demonstrate that the mechanisms involved in keratinocyte senescence and immortalization are distinct from those essential for other aspects of tissue growth control and differentiation.
Finally, the hTERT-immortalized keratinocyte lines that we have described here may have significant potential value for a wide variety of investigations into human epithelial biology, including characterization of the effects of expression or loss of specific gene products on acquisition of malignant phenotypes. Although we have demonstrated that hTERT-immortalized immortal cells can retain normal growth and differentiation control mechanisms, it is possible that the loss of the p16-mediated growth arrest mechanism and unlimited replicative potential predisposes such cells to further changes that may result in malignant transformation. Indeed, it has been shown recently that expression of hTERT cooperates with the simian virus 40 large T oncoprotein and oncogenic ras to transform human fibroblasts and kidney epithelial cells to tumorigenicity (17). For this reason, enthusiasm for potential clinical applications of hTERT-immortalized epithelial cell lines as therapeutic transplants should be tempered with caution.
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ACKNOWLEDGMENTS |
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We thank J. Benwood, D. Long-Woodward, and K. O'Toole for technical assistance and S. Verselis for information about the mutant p53 allele in our Li-Fraumeni cell line. We thank D. Galloway for the HPV16 E7 retroviral vector, J. Koh, S. C, Ngwu, and E. Harlow for p16INK4a antibodies, and T.-T. Sun, C. A. Loomis, and F. M. Watt for keratin and involucrin antibodies.
This research was supported by Oral Cancer Program Project grant PO1 DE12467 from the NIDCR, Skin Disease Research Center grant P30 AR42689 from the NIAMS, and a research grant from Organogenesis, Inc. (J.G.R.). In addition, portions of this work were supported by a Daniel K. Ludwig and American Cancer Society Professorship (R.A.W.), a Culpeper Biomedical Initiative Pilot Grant (W.C.H. and R.A.W.), a Damon-Runyon/Walter Winchell Cancer Research Fund award, a Howard Hughes Medical Institute postdoctoral fellowship, and a Herman and Margaret Sokol postdoctoral fellowship (W.C.H.), and a Starr Foundation and American Cancer Society Clinical Research Professorship (F.P.L.).
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FOOTNOTES |
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* Corresponding author. Mailing address: Harvard Institutes of Medicine, Room 664, 77 Ave. Louis Pasteur, Boston, MA 02115. Phone: (617) 525-5553. Fax: (617) 525-5571. E-mail: JRheinwald{at}rics.bwh.harvard.edu.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Dellambra, E., Golisano, O., Bondanza, S., Siviero, E., Lacal, P., Molinari, M., D'Atri, S., De Luca, M.
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