Carcinogenesis vol.17 no.12 pp.2631-2634, 1996
Telomerase activation during the linear evolution of human
fibroblasts to tumorigenicity in nude mice
Michael C.Montalto and F.Andrew Ray1
Department of Microbiology, Immunology and Molecular Genetics, Albany
Medical College, Albany, NY 12208, USA
'To whom correspondence should be addressed
The ribonucleoprotein enzyme telomerase is active in most
immortal cell lines, most tumors and all tumor-derived cell
lines. The enzyme is important because it prevents continual
shortening of telomeres and therefore plays a significant
role in chromosome maintenance. In man, telomerase is
not active in most somal cells with finite lifespans. Using
the SV40 T antigen we immortalized and transformed to
fully tumorigenic a human fibroblast cell strain. We wished
to determine when telomerase was activated during this
progression to tumorigenicity. Using the PCR-based TRAP
assay we found that eight of eight immortal cell lines that
were either not tumorigenic or rarely formed tumors were
telomerase positive at the time of inoculation. Additionally,
10 of 11 newly immortal cell lines contained telomerase
activity within the first 25-33 population doublings after
crisis. None of the precrisis cells from which these immortal
cells were derived were positive for telomerase activity.
Thus we found that telomerase activation is not the final
in vivo step in the transformation of these cells and the
window of activation is usually near the escape from
crisis or M2. These results strengthen the hypothesis that
telomerase activation may allow the rare cell to escape
from crisis in those immortal cell populations dependent
on telomerase for telomere maintenance.
Introduction
The ends of human chromosomes, called telomeres, are composed of a six nucleotide repeating unit (TTAGGG)n with
associated proteins and probably serve a functional role in
maintaining the structural integrity of chromosomes (\—4). It
has been hypothesized that the loss of telomeric repeats during
successive cell divisions triggers cellular senescence (5-10).
Thus telomere shortening may act as a molecular counter that
determines the lifespan of a mortal cell.
All immortal cells examined thus far can maintain telomeric
repeats above a minimal level, indicating that telomere maintenance is necessary for immortality (10,11). In most immortal
cells telomere length can be maintained by the enzyme
telomerase, which adds telomeric repeats to the ends of human
chromosomes (11,12). This enzyme is active in all tumorderived cell lines and most tumor tissues, yet most mortal
somatic tissues do not have detectable enzyme activity (11).
Because a tumor is the result of multiple mutations widiin a
lineage of cells, it is difficult to determine retrospectively
when telomerase became active in that lineage. Avilion et al.
•Abbreviations: HEK, human embryonic kidney; CPD, cumulative population
doublings; Ml, mortality stage 1; M2, mortality stage 2; TRAP, telomeric
repeat amplification protocol; PMSF, phenylmethylsulfonyl fluoride.
© Oxford University Press
have reported the existence of a single human embryonic
kidney (HEK*) cell line that contains telomerase activity
during crisis, indicating that telomerase activation may coincide
with immortalization in cells that maintain telomeres by
telomerase (13). To determine the role telomerase plays in
tumorigenesis it is important to identify when it is activated.
Normal human fibroblasts have a defined proliferative capacity of ~75 cumulative population doublings (CPD) (14,15).
Senescence or mortality stage 1 (Ml) has recently been shown
to be associated with increased expression of the DNA damageresponsive p53 protein (16). It has been postulated that critically
short telomeres may be analogous to DNA damage and through
unknown mechanism(s) trigger p53 and subsequent cell cycle
arrest (8). The viral oncoproteins that bind and inactivate p53
result in Ml bypass and an extended lifespan (17,18). After
an extended lifespan the cells enter a second proliferative
blockade called mortality stage 2 (M2) or crisis, which is
characterized by excessive cell death (17,19). Escape from
crisis occurs at very low frequency (~ 1 cell in 3 000 000) in
fibroblasts expressing the SV40 T antigen (20,21). The molecular basis that allows this rare resumption of growth is not
understood. It has been hypothesized that the activation of
telomerase may be responsible for the infrequent escape from
crisis (9).
We wished to determine when during tumorigenesis telomerase was activated in SV40 T antigen-positive immortal human
fibroblasts. We assayed telomerase activity using the telomeric
repeat amplification protocol (TRAP) assay (22). Precrisis
(mortal) cells, newly immortal cells, high passage (immortal)
cells that were used for tumorigenicity assays and resulting
tumor-derived cells were tested. We found that in all (eight of
eight) cell lines tested for tumorigenicity, telomerase was
activated prior to injection into nude mice. Therefore, telomerase activation was not the transforming step that occurred
in vivo. Ten of 11 newly immortal cell lines had telomerase
activity and none of the precrisis cell strains had detectable
activity. Therefore, the activation of telomerase occurred
frequently during or immediately after crisis.
Materials and methods
Cells and culture conditions
Cell lineages, plasmids and transfections were as previously described (18,21).
Briefly, the HSF 43 human diploid fibroblast parental strain was electroporated
with various SV40 T antigen constructs and colonies were selected with
medium containing G418 (Gibco BRL). CT lineages express SV40 T antigen
only, E lineages express SV40 large T and small t antigens and SV lineages
express SV40 T and t antigens and have the SV40 origin of replication
(18,21). Eight T antigen-positive precrisis cell strains, 11 newly immortal cell
lines, eight high passage immortal cell lines that were injected into nude mice
and representative tumors derived from them were thawed and cultured
(Figure 1; 21). Cells were cultured in a MEM (Gibco BRL) supplemented
with 109b fetal bovine serum, 50 U/ml penicillin (Gibco BRL) and 50 Hg/ml
streptomycin (Gibco BRL) in a 5% CO2 incubator Tumor-derived cells from
nude mice were passaged in 0.4 mg/ml G418 (Gibco BRL) for al least two
passages prior to lysis in order to ensure no contaminating mouse cells were
present. Adenovirus-immortalized 293 cells (ATCC CRL 1573) known to
have active telomerase were cultured as above (10,22).
2631
M.CMontalto and F.A.Ray
Ml
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T2
T3
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Electroporation I
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Fig. 1. The acquisition of telomerase activity during the linear evolution of
human fibroblasts. Telomerase activity was measured in parental HSF 43,
precrisis (PC), newly immortal (NI), high passage (HP, at the time of
inoculation) and tumor-derived cells (T). The time that non-inoculated high
passage lineages were assayed for telomerase activity are indicated by filled
circles. A subset of E2 cells were cultured after the time of inoculation until
>200 CPD (21). Ml and M2 represent the Hayflick limit of replication
(senescence) and cellular crisis respectively (14,17). Positions of Ml and
M2 relative to each cell line are approximations based on the time of
senescence of the parental HSF 43 strain and time of crisis of the SV40 T
antigen-positive cells (21).
Cell extract preparations
Cells were either plated onto two 150 mm diameter tissue culture dishes or
two T75 cm2 tissue culture flasks and grown to near confluence. Cells were
harvested at 4°C by scraping or trypsinization (Gibco, BRL) and pelleted by
centrifugation at 1800 g. No variation in telomerase activity was observed
between cells which were scraped versus cells which were treated with trypsin
(data not shown). Cell pellets were washed in cold phosphate-buffered saline
(2.7 mM KC1, 1.5 mM KH2PO4, 137 mM NaCl, 8 mM Na2HPO4), pelleted
and resuspended in ice-cold wash buffer [10 mM HEPES-KOH, pH 7.5,
1 mM MgCl2, 10 mM KCI, 1 mM dithiothreitol, 1 mM EGTA, 0.1 mM
phenylmethylsulfonyl fluoride (PMSF)] and pelleted at 4°C The cells were
then resuspended in 20 |il lysis buffer (10 mM Tns-HCI, pH 7.5, 1 mM
MgCl z 1 mM EGTA, 0.1 mM PMSF, 5 mM P-mercaptoethanol, 0.59b CHAPS,
10% glycerol) per 10* cells. This suspension was incubated for 30 min on ice
and then centnfuged at 100 000 g for 35 min at 4°C. The supernatant was
aliquoted, quick frozen and stored at -70°C. The protein concentrations were
determined by the Bradford protein assay (Coomassie Protein Assay; Pierce).
TRAP assay
The TRAP assay was performed as previously described (22) with some
minor modifications. Briefly, 50 |il reactions contained 1 X TRAP reaction
buffer (20 mM Tris-HCl, pH 8, 1.5 mM MgCl2, 63 mM KCI, 0.005% Tween
20, 1 mM EGTA), 50 HM each dNTP, 0.1 ug TS primer, 0 1 mg/ml BSA,
2 u.Ci [oe-32P]dGTP (10 uCi/nl) and 6 ng CHAPS cell extract (1-3 |il). After
incubation at room temperature for 30 min, 2 U Primezyme (Biometra) were
added. Reactions were then heated to 90°C for 1 min and 0.1 ng CX primer
was added per reaction. Telomerase extension products were amplified by
PCR in a thermocycler at 94°C for 30 s, 50°C for 30 s and 72°C for 45 s,
for a total of 28 cycles. PCR products were resolved on a 10% non-denaturing
polyacrylamide gel for 3.5 h at 250 V. TRAP products were visualized by
autoradiography with exposure times of between 8 and 16 h. Positive control
293 cells were assayed in parallel with every experiment. For RNase
pretreatments of cell extracts 1 |il 10 mg/ml RNase (Boehringer Mannheim)
was added to 5 ul of each extract and incubated at room temperature for
20 min. For heat pretreatments 5 (il each extract were incubated at 75°C for
20 min. Cells were scored telomerase-positive if a continuous 6 bp ladder
was observed from 40 bp upward and the ladder was eliminated by RNase
and heat pretreatments (22).
Results
Parental fibroblasts, transfected SV40 T antigen-positive cell
strains, newly immortal cell lines, high passage cells inoculated
2632
Fig. 2. Representative TRAP assay for telomerase activity at various times
during transformation. Parental fibroblasts (HSF 43), prior to
electroporation, T antigen-positive precnsis cells (PC), high passage
immortal cells (HP) and the resulting tumor-derived cells (T) for each
lineage were thawed and assayed using the TRAP assay. HP and control
(293) cells were pretreated with RNase (+) or heat (A).
into nude mice and their tumorigenic descendants (Figure 1)
were tested for telomerase activity using the TRAP assay
(Figures 2 and 3). The parental HSF 43 and eight precrisis
mortal T antigen-positive cell strains were telomerase negative,
as expected. Newly immortal cell lines ranged from 25 to 33
population doublings (PD) beyond crisis. The population
doubling levels of the high passage cells used for inoculations
ranged from 126 to 300 CPD (21). [The number of population
doublings before the cells were considered high passage was
variable and depended on an observed increase in growth rate
(21).] All (eight of eight) high passage immortal lines that
were thawed at the population doubling levels of inoculation
were positive for telomerase activity. No tumors were derived
from three of these immortal lineages. All (11 of 11) tumors
tested were also telomerase positive, as expected. Ten of 11
newly immortal cell lines were telomerase positive. Interestingly, one newly immortal cell line (E2) did not have telomerase activity (Figure 3), but gained activity by the time of
inoculation (155 CPD) (data not shown). All other newly
immortal cell lines had strong telomerase activity, which could
be retained when diluted to 0.6 |J.g protein, indicating that
activation was an early event in immortalization (data not
shown). Thus, we conclude that telomerase activation is
frequently an early event in the acquisition of the immortal
phenotype of human fibroblasts and is not the in vivo tumorigenicity step.
Telomerase activation in fibroblasts
Fig. 3. Representative TRAP assay for telomerase activity of newly
immortal cells. Newly immortal cells of each lineage were thawed and
assayed for telomerase activity within 25-33 PD after crisis.
Discussion
Telomerase is active in 100% of all tumor-derived cell lines
(11,22), 85-90% of primary rumor tissue (9) and 72% of
immortal cell lines (22). This activation is likely involved
in tumorigenesis. Therefore, we wished to determine when
telomerase activation occurred. For example, our earlier studies
showed that there is an in vivo transformation event that allows
tumors to arise in nude mice (21). In those studies, SV40 T
antigen-immortalized cells that had been cultured in vitro for
many CPD were injected into pre-implanted collagen sponges.
Tumors arose at low frequencies with long latencies, if at all.
When inoculated into nude mice, the cells from these tumors
caused tumors to form rapidly, at high incidence. We postulated
that an additional mutation(s) occurred in vivo that represented
a final step(s) in the tumorigenic process. Here we show that
telomerase activation occurred prior to inoculation and was
not the final in vivo transforming event.
Our results support the possibility that telomerase activation
and escape from the M2 block are the same event, as suggested
by Wright et al. (9). Counter et al. showed that telomere length
is maintained immediately after crisis of HEK cells and more
recently Avilion et al. cultured the same parental cell strain
through crisis and found that the cells become telomerase
positive during crisis (10,13). There are several other reports
in which the authors did not measure telomerase activity
immediately after crisis, but show that telomere shortening
was arrested soon after immortalization (23-25). Here we
confirm that telomerase is active by at least 25-33 CPD after
crisis in 10 different cell lines. The intensity of telomerase
activity from diluted lysates of all 10 newly immortal clones
indicates that telomerase was likely to be active well before
the cells were frozen, strongly supporting the possibility that
activation occurred during crisis. The absence of varying
intensity levels among clones soon after crisis favors the
possibility that activation occurred at the same time after
immortalization (and possibly at the time of immortalization)
for each lineage. If escape from M2 and telomerase activation
are concomitant, this will provide important clues about
telomerase regulation. For example, it was recently shown that
fusions between telomerase-positive immortal cells and normal
cells that lack telomerase activity result in hybrids that have
repressed telomerase activity, indicating the existence of a
trans-dommaxti telomerase inhibitor (9). The rarity of escape
from M2 might suggest that such an inhibitor is autosomal
and that loss of both copies of the putative inhibitor gene are
required for immortalization. Mixing experiments between
telomerase-positive and -negative lysates indicate that if such
an inhibitor exists, it does not completely'inhibit telomerase
activity under in vitro conditions (10,22,26,27).
Relatively rare telomerase-negative immortal cell lines exist,
yet the mechanism of telomere maintenance in these cell lines
is not known (22,26). In this report we also show the existence
of a telomerase-negative cell line (E2). Interestingly, this cell
line expressed detectable telomerase activity later, at the time
of inoculation into nude mice. Since most immortal cells
described here and by others have telomerase activity, the
possibility remains that these cell lines arise by telomerase
activation and the rest by developing an alternative mechanism^) of telomere extension. No immortal cells have yet been
described that do not have a mechanism to maintain telomere
length, indicating that telomere maintenance is an absolute
requirement of the immortal phenotype.
A recent report by Klingelhutz et al. suggests that expression
of the human papillomavirus type 16 E6 protein can activate
telomerase in precrisis (prior to M2) keratinocytes and epithelial cells (28). In a previous study the authors were not able
to immortalize cervical epithelial cells with E6 alone and
therefore suggest that E6-mediated activation of telomerase is
insufficient for immortalization. E6 alone can immortalize
mammary epithelial cells, yet at a lower frequency than E6/
E7 together, suggesting that both the p53 and Rb pathways
may be involved in escape from M2 (29). It has been shown
that E6 expression alone can extend the lifespan of keratinocyes
(28). It is possible that mutations which mimic the Rb binding
functions of E7 may arise during this extended lifespan. In
such rare instances the expression of telomerase would impart
additional growth advantages leading to immortalization.
Klingelhutz et al. subcloned three cell strains and found that
they were telomerase negative, suggesting that only a fraction
of cells in the population were expressing telomerase. Since
no telomerase-positive clones in this study were isolated and
tested for immortalization, it is possible some cells in the
population were immortal. Examination of telomerase activation in individual clones as they escape M2 (crisis) will resolve
this issue.
Kim et al. and Bryan et al. have reported that some SV40
T antigen-immortalized fibroblasts (two of three and nine of
16 respectively) do not have telomerase activity (18,22). We
found that all of our high passage SV40-immortalized cell
lines are telomerase positive. The conditions used for immortalization are generally the same and probably do not account
2633
M.C.Montalto and F.A.Ray
for such discrepancies (18,22,26,30). We cannot eliminate the
possibility that differences between specific parental cell strains
influence the reactivation of telomerase in SV40 T antigenimmortalized fibroblasts.
It has been suggested that telomerase activation is a late
event in tumorigenesis, because telomeres are often shorter in
tumors than surrounding normal tissue and telomerase activity
is not usually observed until the late stage of progression of
the disease (11,31). Because tumor formation requires multiple
mutations, it is likely that telomerase imparts a selective
advantage to cells by allowing replication of telomeres. Therefore, it is possible that telomerase activation coincides with
the immortalization step required for development of most
malignant tumors. Although this step can be late in the
replicative history of the tumor cell lineage, it may be early
in the acquisition of the malignant phenotype.
In summary, we conclude that telomerase activation in S V40
T antigen-immortalized human fibroblasts is an event which
occurs during or immediately after crisis and is not responsible
for the in vivo transformation to tumorigenicity step observed
in nude mice.
Acknowledgements
We thank Drs Robert Laffin and Tom Friedrich for critical reading of the
manuscript and Dr John Lehman and Ms Judy Laffin for helpful discussions
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Received on June 27, 1996; revised on July 29, 1996; accepted on August
14, 1996