[CANCER RESEARCH 56. 3796-3802.
August 15, 1996]
Selective Inhibition of Telomerase Activity during Terminal Differentiation of
Immortal Cell Lines1
Leslie J. Bestilny, Christopher
B. Brown, Yutaka Miara, Laurie D. Robertson, and Karl T. Riabowol2
Departments of Medical Biochemistry and Oncology and Southern Alberta Cancer Research Center. University of Calgary, 3330 Hospital Drive N.W.. Calgary, Alberta. T2N 4NI
Canada
ABSTRACT
Telomerase, the enzyme that maintains the ends of linear eukaryotic
chromosomes, is active in human germ cells and in a majority of tumor
tissues and immortalized cell lines. In contrast, most mature somatic cells
and tissues contain low or undetectable telomerase activity, implying a
stringent negative regulatory control mechanism. We report here that
telomerase activity is dramatically inhibited during the terminal differen
tiation of HL-60 human promyelocytic leukemia cells to monocytic and
granulocytic lineages. A loss of telomerase activity was seen in response to
three different inducers of differentiation, was independent of differenti
ation-induced apoptosis, and occurred in the presence of unaltered ex
pression of the RNA component of telomerase. Reduction in telomerase
activity was also observed during the differentiation of murine F9 teratocarcinoma and C2C12 myoblast cells. In contrast, induced differentiation
of murine pl9 embryonal carcinoma and Neuro 2a neuroblastoma cells
did not result in a loss of telomerase activity. These results are therefore
consistent with the absence of telomerase activity in human somatic cells
and the presence of telomerase activity in many somatic murine cells and
tissues.
INTRODUCTION
Telomeres, the specialized structures present at the ends of eukary
otic chromosomes, consist of tandem arrays of highly conserved
hexameric (TTAGGG) repeats in vertebrates, and telomeres in hu
mans are an average of 10-15 kbp in length (1, 2). Telomeres have
been implicated in stabilizing chromosomes from exonucleolytic deg
radation and chromosome-to-chromosome
fusions, preventing other
forms of aberrant recombination, the attachment of chromosomes to
the nuclear matrix (reviewed in Ref. 3), and as a "mitotic clock" in
determining the maximum replicative capacity of human somatic cells
(4-6).
Normal diploid somatic cells lose approximately 50-200 bp of
telomeric DNA/mean population doubling due to the inability of DNA
polymerase to completely replicate the ends of linear chromosomes
(5, 7). In contrast, germ line and most immortal cell lines maintain
their telomeres at a constant length irrespective of the number of
divisions they undergo due to the enzyme activity of telomerase, a
ribonucleoprotein that is able to synthesize and add telomeric repeats
onto chromosomal termini. Because telomerase activity is absent in
most normal human somatic cells and tissues that have limited repli
cative life spans (8) but is activated during cellular immortalization
(9), the telomere hypothesis of cell aging and immortalization has
been proposed, in which the attrition of telomeric sequences ulti
mately interferes with the expression of genes required for continued
cell growth (4-6, 10). Strong correlative support for this hypothesis
was provided by the observation of telomerase activity in 98% of
Received 1/31/96: accepted 6/11/96.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
' Supported by grants to K. T. R. from the Medical Research Council of Canada and
the National Cancer Institute of Canada. L. J. B. received support from the Leukemia
Research Fund of Canada and is the recipient of a studentship from the Alberta Heritage
Foundation for Medical Research.
2 To whom requests for reprints should be addressed. Phone: (403) 220-8695: Fax:
(403) 283-8727; e-mail: [email protected].
established (immortal) cell lines and 90% of tumors tested and the
absence of telomerase activity in over 50 normal human somatic
tissues (8).
In contrast to human somatic cells, many rodent cell types display
telomerase activity (11, 12), and the length of telomeres in mouse
cells derived from different normal somatic tissues has been reported
to be hypervariable (13, 14). These results, combined with the obser
vation that rodent cells undergo spontaneous immortalization at a
frequency that is approximately 1,000,000-fold higher than that in
human cells (15), have led to the suggestion that the expression of
telomerase activity is a requisite for immortal human cancer cell
formation (8). Therefore, the absence of telomerase activity in normal
human somatic cells seems to reflect stringent controls that act developmentally to repress enzyme activity, perhaps as a mechanism for
limiting cell growth. Because reacquisition of telomerase activity
seems to be related to the emergence of immortal human cancer cells,
this enzyme may offer a unique target for cancer therapies. To better
understand the conditions leading to the suppression of telomerase
activity, we used five independent model systems of differentiation.
The HL-60 human promyelocytic leukemia cell line can be chemi
cally induced to differentiate into a variety of cell types that function
ally and morphologically resemble normal monocytes/macrophages
and granulocytes (reviewed in Ref. 16). We found that differentiation
of human HL-60 cells by several different agents is associated with
the rapid inhibition of telomerase activity. The effect is also seen in
the murine F9 (teratocarcinoma) and C2C12 (myoblast) cell lines;
however, two of four murine cell lines, P19 (embryonal carcinoma)
and Neuro 2a (neuroblastoma), did not display decreased telomerase
activity when induced to differentiate.
MATERIALS
AND METHODS
Cells and Cell Culture. HL-60 cells were maintained as a suspension in
RPM1 1640 supplemented with 10% FBS1 (Life Technologies, Inc.) at 37°C,
5% CO2/95% air. Cells for differentiation studies were seeded at 2 x IO5
cells/ml with or without 1 JIM RA and 5 x IO5 cells/ml with or without 160
nM TPA (a gift from D. Edwards, University of Calgary. Calgary. Alberta.
Canada) or 1.25% v/v DMSO on 15-cm tissue culture plates (Coming Glass).
Cells were monitored for viability by trypan blue exclusion and were counted
with a hematocytometer. Morphological assessment of differentiated HL-60
cells was made from Cytospin slide preparations stained with a LeukoStat stain
kit (Fisher Scientific). Murine F9 teratocarcinoma, P19 embryonal carcinoma,
and Neuro 2a neuroblastoma cells were maintained and differentiated as
described (17-19). C2C12 myoblast cells were maintained in DMEM supple
mented with 10% FBS and were differentiated as described (20). Differentiated
C2C12 cells were processed for telomerase activity assays after 10-14 days in
differentiation medium. Differentiated PI9 cells were incubated in the absence
of FBS for up to 60 days, with media changes every 3-4 days to ensure
complete differentiation as well as the death of undifferentiated PI9 cells
before analysis of telomerase activity.
NBT Reduction and CHI Mi Determination. Biochemical maturation of
RA- and DMSO-treated HL-60 cells was assessed by NBT reduction as
1The abbreviations used are: FBS. fetal bovine serum; RA. retinoic acid; TPA.
l2-O-tetradecanoy!phorbol-l3-acetate;
NBT. nitro blue telra/olium; TRAP, telomere re
peat amplification protocol; GAPDH. glyceraldehyde-3-phosphatc
dehydrogenase; RTPCR. reverse transcription-PCR.
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DIFFERENTIAL
Fig. 1. Morphological maturation of terminally
differentiating HL-60 cells. A. HL-60 cells were
treated with the phorhol ester TPA ( 160 iw) for 4
days to induce features of differentiated monocytes/macrophages or with RA ( 1 fXM)for 7 days or
DMSO (1.25% v/v) for 8 days to induce granulocytic characteristics as described. Panels I and 3.
untreated control cells. Arnm: a prominent nucleolus; Panel 2, TPA-treated for 48 h; Panel 4.
RA-treated for 6 days. The efficiency of differen
tiation with TPA. RA. and DMSO was determined
hy CDllb expression |TPA (D)-treated cells] or
NET staining [RA ( 0 )- and DMSO (O)-treated
cells]. B. the percentage of total GDI In-positive
and NBT-positive cells at different times after ad
dition of the inducing agent.
LOSS OF TELOMERASE
ACTIVITY
WITH DIFFERENTIATION
100
B
?
50-
described previously (2l ). Briefly. 2 X IO6 cells/ml were incubated for 25 min
at 37°Cwith an equal volume of 0.2% NBT dissolved in PBS containing TPA
protein (Boehringer Mannheim). O.I mg/ml BSA, 2 units of Taq DNA polymerase (Pharmacia Biotech), and 0.4 p.\ [a-"P]dCTP
(IO /J.CÌ//J.1.
3000 Ci/
(200 ng/ml). Cytospin preparations were made and were stained with Leukostat. and the percentage of cells that contained reduced blue-black formazan
mmol) in a total volume of 50 /j.l. Reactions were incubated for 20 min at
23°C.Control reactions were performed with cell extract incubated with I
deposits was determined. Expression ot the CD I Ib surface marker was meas
ured using a commercially available antibody (Beclon Dickinson, San Jose,
CA) to stain cells followed by fluorescence-activated
cell sorting analysis
mg/ml RNase A or with TPA, RA. or DMSO at concentrations used to induce
differentiation for 20 min at room temperature. Reactions were placed in a
thermal cycler for 27 cycles at 94°Cfor 30 s, 50°Cfor 30 s, and 72°Cfor 90 s.
using standard parameters.
Telomerase Assays. Cells from each time point after induction of differ
entiation with TPA, RA. or DMSO were collected and counted, and viability
was assessed as described above. In the case of TPA-treated HL-60 cells at
Reverse primer (CX. complementary to telomeric repeats) was added manually
to each reaction tube when the temperature reached 92°Cduring the first cycle.
later time points, only the most adherent cells were assessed. Pellets of
3-5 X IO6 cells were collected, and SlOO extracts were prepared (8). In every
After amplification, 10 /il of stop buffer ( 100 mM EDTA, 50% glycerol. 0.02%
xylene cyanol. 0.02% bromphenol blue, and 0.02% SDS) was added to each
tube, and 30 fi\ of the contents were size-fractionated on a 15% nondenaturing
case, unless otherwise indicated, cell extracts from the different cell types
represented l x IO6 cells/20 jxl of lysis buffer. To assess telomerase activity,
polyacrylamide gel. All TRAP assays on differentiating cell systems were
performed at least twice, with comparable results between trials.
Quantitation of TRAP Assays. TRAP assays were performed on HL-60
the TRAP assay was used (8) with minor modifications. Aliquots containing 2
(¿Iof SlOO extract were added to tubes containing 20 mM Tris-HCl (pH 8.3).
63 mM KCl, 0.005% Tween-20. I mM EGTA (pH 8.0). 50 /J.Mdeoxynucleoside
cell extracts representing various numbers of viable cells to determine the
optimal cell number for linear PCR amplification, and these studies indicated
that I x IO6 cells/20 fj.1 of lysis buffer gave accurate, reproducible results.
triphosphates.
Individual bands of TRAP assays were quantitated by computerized
0.1 fig of telomerase substrate oligonucleotide.
I /xg of T4g32
3797
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scanning
DIFFERENTIAL LOSS OF TELOMERASE ACTIVITY WITH DIFFERENTIATION
Cont.+
TPA
B
24487296
i
i
i
.M
Fig. 2. Loss of telomeruse activity in differen
tiating HL-60 cells. A, HL-60 cells were induced
to differentiate along the monocytic pathway with
TPA, and (elomerase activity was assessed at var
ious time points with the TRAP assay, as described
in "Materials and Methods." Lune I, HS68 normal
diploid fibroblast cell extract; Lanes 2-5. uninduced HL-f>0 cell extracts incubated with increas
ing concentrations of TPA; Ltine 6. uninduced cell
extract; Lane 7, uninduced extract treated with 1
mg/ml RNase; Lanes 8-11, extracts from TPAtreated cells. HL-60 cells were also induced lo
differentiate toward the granulocytic lineage with
RA (B) or DMSO (Ci. and telomera.se activity was
assessed as described. In each case: Lane I, unin
duced cell extract incubated with RA or DMSO;
Lane 2, uninduced (control) extraci: Lune 3. unin
duced extract incubated with RNase. Days of drug
treatment are indicated. Drug-free cells were al
lowed to grow in culture for 5 days without subcultivation to ensure loss of activity was not re
lated to excessive cell density or poor culture
conditions (C, Lane 12). Relative telomerase ac
tivity during differentiation was determined and
was expressed as a fraction of the undifferentiated
HL-60 cell signal. A),signal intensities of untreated
(A). TPA (O)-. RA O-. and DMSO ( 0 »-treated
HL-60 cells were compared to the standard and
were expressed as a fraction of the maximum
value of 100.
s
£ j
hours
jg
RA (days)
00tt1234567
i •¿ <
:
123456789
C
|ç|
12345
78
9 10
O
(A
g
O o i
123456
1011
DMSO (days)
1234
^
5678U
6
7 8 9 1011 12
densitometry using a Hewlett Packard Scanjet IIC and were analyzed using
NIH Image software. Results were corrected for background, and a standard
value of 1(X) was given to the undifferentiated HL-60 cell signal. Signal
intensities of TPA-, RA-, and DMSO-treated HL-60 cells were compared to
the standard and were expressed as a fraction of the maximum value of 100.
PCR Analysis of RNA. Total RNA was obtained by the acid guanidinium
method (22). and PCR was performed as described (23). Aliquots of each
reaction were equalized for the internal control GAPDH and were run on 2%
agarose gels containing ethidium bromide (0.2 mg/ml). Primers for hTR were
synthesized using the TRC 3 sequence (24). Amplification was carried out for
28-32 cycles for hTR and 19-22 cycles for GAPDH and c-myc. Gels were
scanned by computerized densitometry, and relative concentrations of hTR and
c-myc were determined by comparing the ratio of hTR or c-myc to GAPDH in
each lane. An arbitrary value of I(X) was given to the untreated HL-60 hTR and
c-myc levels. Experiments were repeated in two to five independent trials, and
values were either averaged or the SE was determined.
Quantitäten of Apoptosis by Flow Cytometry. Cells were assessed for
apoptosis by the ethanol-fixation method, as described (25). Briefly. 3 X IO6
HL-60 cells were collected at various stages of TPA-. RA-. or DMSO-induced
differentiation and were centrifuged at 200 X g. and the cell pellet was fixed
in 2 ml of ice-cold 70% ethanol for I h. After centrifugation, cells were washed
in 1 ml of PBS and were resuspended in 0.5 ml of PBS. A volume of 0.5 ml
of RNase A (Sigma Chemical Co.; I mg/ml in PBS) was added, and the cell
suspension was mixed gently, followed by the addition of 1 ml of propidium
iodide (Sigma; I mg/ml in PBS). Cells were incubated in the dark for 15 min
at room temperature and were stored in the dark for 24 h at 4°Cbefore flow
cytometry was performed.
Photomicroscopy. All cells were photographed using phase contrast or
differential interference optics and Kodak Technical Pan film set at 160 DIN.
RESULTS AND DISCUSSION
HL-60 cells differentiating into mature monocytes/macrophages
upon treatment with TPA increased in size, became adherent, and
displayed prominent pseudopodia within 24 h. whereas cells stimu
lated with RA or DMSO decreased in size and exhibited less prom
inent nucleoli along with segmented or lobulated nuclei (Fig. IA,
Panels 1-4). Cells treated with TPA exhibited maximal expression of
CD1 Ib (a surface marker of differentiation) 36 h after stimulation, at
which point 91% of the cells scored as positive (Fig. Iß).The
majority of cells treated with RA remained functionally undifferenti
ated until 4 days after the addition of RA, when their ability to
produce Superoxide aniónand subsequently reduce the water-soluble
dye NBT increased from 30% to 70%. Maximal NBT reduction was
seen after 7 days of RA treatment, when the majority (—85%)of cells
stained, indicating that the cells had acquired biochemical character
istics of granulocytic maturation. Cells treated with DMSO displayed
morphological and functional characteristics similar to those of RA-
3798
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DIFFERENTIAL
LOSS OF TELOMERASE
ACTIVITY
WITH DIFFERENTIATION
200
TPA
0
6
12
24
48
72
TPA
96 hours
100 -
c-myc
GAPDH
*
50
hTR
B
Fig. 3. hTR and c-mvc expression in differ
entiating HL-60 cells by RT-PCR. Tolal RNA
was prepared, and "primer-dropping" RT-PCR
was performed as described (23). Aliquols of
each reaction were equalized for the internal
control GAPDH and were run on 2% agarose
gels containing ethidium bromide (0.2 mg/mll.
A-C, times of treatment with different drugs are
as indicated. Graphs show the relative concen
trations of hTR (»-hilebars) and c-mvc (solid
bars) mRNA as determined by computeri/ed
densitometry by comparing the ratio of hTR or
c-myc to GAPDH in each lane. An arbitrary
value of lOO was given to untreated HL-60 hTR
and c-myc levels, and changes in response to
differentiating agents were plotted as a function
of time.
RA
01234567
days
-GAPDH
- c-myc
-GAPDH
-hTR
DMSO
150
01234567
DMSO
days
-GAPDH
•¿
c-myc
|
GAPDH
4
10,
50-
-hTR
456
Days
treated cells and showed comparable levels of NBT-positive cells over
the course of differentiation. Thus, all three agents (TPA, RA, and
DMSO) efficiently induced morphological and biochemical matura
tion of HL-60 cells.
To determine the effect of differentiation upon telomerase activity,
HL-60 cells were induced to differentiate with TPA, and telomerase
activity was assessed using the TRAP assay (8). Telomerase activity
declined rapidly during the course of differentiation, with changes
becoming evident at the first time point examined, 24 h after drug
treatment (Fig. 2A, Lane X). Telomerase activity in extract from
uninduced HL-60 cells was not inhibited by adding TPA directly to
the extract itself (Limes 2-5), indicating that the loss of activity was
differentiation specific and not due to chemical or direct biochemical
effects. Furthermore, treatment with trypsin/EDTA, which was re
quired to remove adherent TPA-induced HL-60 cells, was similarly
unrelated to the loss of telomerase activity seen in differentiating cells
because in parallel experiments, untreated cells incubated in trypsin/
EDTA for extended periods of time showed no decline in telomerase
activity (data not shown). Telomerase activity was not detected in the
normal HS68 human fibroblast cell strain (Lane I), as expected.
To determine whether the loss of telomerase activity was specific to
a particular pathway of differentiation, HL-60 cells were induced to
differentiate along the granulocytic lineage with RA (Fig. IB) or with
DMSO (Fig. 2C), followed by assessment of telomerase activity. In
both cases, telomerase activity was seen to decline rapidly over the
course of treatment, similar to results seen with TPA treatment (Fig.
2D), indicating that the mechanism(s) responsible for suppressing
telomerase activity is activated by differentiation along both granulo
cytic and monocytic pathways. The loss of telomerase activity in
DMSO-treated cells occurred more rapidly and more completely than
in RA-treated cells. Cells treated with DMSO exhibited a total loss of
detectable telomerase activity after 3 days, whereas after 7 days,
RA-treated cells still expressed very low levels of residual telomerase
activity (as indicated by the faint low molecular weight bands) that
were approximately equal to 3.5% that of uninduced HL-60 but still
higher than that of normal diploid cells, in which activity was undetectable. This residual activity may be due to a small proportion of the
HL-60 population that failed to respond to RA. Telomerase activity in
the uninduced HL-60 cell extract was not inhibited by adding RA and
DMSO at concentrations that induced cell differentiation, again indi-
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DIFFERENTIAL
LOSS OF TELOMERASE
ACTIVITY
WITH DIFFERENTIATION
HL-60 CONTROL
TPA1D
TPA2D
Ml=Apoptotic cells
t
i^U
Fig. 4. Levels of apoptosis in differentiating
HL-60 cells. DNA contenÃ-of HL-60 cells induced
to differentiate with TPA, RA. and DMSO was
assessed by flow cytometry before addition of
TPA (Control), or 1 (TPA ID), 2 (TPA 2D), 3
(TPA JD). or 4 (TPA 4D) days after TPA addition.
Ml. the DNA fluorescence of cells with a subdiploid DNA content that constitute the apoptotic cell
population (33). Values derived from fluores
cence-activated cell sorting analysis for TPA.
DMSO. and RA were plotted in Table 1.
»l"*"*"^^^^^11 ' 111u
e
E« 4ee
6W
eèe tee
DNA Content
K.
TPA 3D
eating that the loss of activity was differentiation specific and not due
to direct chemical or biochemical effects (Figs. 2A and 25, Lane /).
Telomerase is a complex enzyme containing both protein and an
RNA component that acts as a template for telomere elongation.
Although the cloning of genes encoding the protein components of
telomerase has not yet been reported for any vertebrate (perhaps due
to the rarity of the enzyme), the RNA component has been isolated
and cloned from Tetrahymena. Oxyiricha, Euplotes, mouse, and hu
man sources (24, 26-30). To assess the potential role of the RNA
component of human telomerase (hTR) in the loss of telomerase
activity, the levels of hTR RNA were determined in differentiating
cells by RT-PCR. as shown in Fig. 3. Cells differentiating toward
monocytes/macrophages
in response to TPA (Fig. 3/4) and toward
granulocytes in response to RA (Fig. 35) or DMSO (Fig. 3O showed
no significant differences in the amount of hTR expressed when
compared with the internal control "housekeeping" gene GAPDH. In
contrast to the unaltered levels of hTR and in agreement with other
reports (16), the levels of the c-myc gene transcript decreased very
rapidly in response to all three chemical inducers (Fig. 3/4-O, cor
roborating the previous observations of morphology, surface antigen
expression, and dye reduction that differentiation was being induced
efficiently.
Terminal differentiation of HL-60 cells is associated with a loss of
proliferative capacity and increased programmed cell death or apop
tosis (31, 32). To investigate whether the decreased telomerase activ
ity observed during treatment with TPA, RA. or DMSO might result
from increased apoptosis, HL-60 cells induced to differentiate with
TPA4D
TPA, RA, and DMSO were analyzed for DNA content by How
cytometry. Apoptotic cells show decreased DNA staining with a
variety of fluorochromes as a result of the activation of an endogenous
endonuclease and the subsequent diffusion of low molecular weight
DNA from the cells. As a result, cells undergoing apoptosis frequently
display a subdiploid DNA content on a DNA-content frequency
histogram (33). Fig. 4 shows the DNA content of HL-60 cells under
going TPA-induced differentiation. Inhibition of cell growth is re
flected by the accumulation of the vast majority of cells in the G,/G]
phase of the cell cycle and the loss of cells in the S phase and G,-M
phase after 2 days of treatment. Also evident is a small apoptotic cell
population that displays a sub-G,/G, DNA content that increases with
time of exposure to TPA. The percentages of apoptotic cells accumu
lating in response to TPA or RA, although increasing over the course
of differentiation, did not display levels that could adequately account
for the loss of telomerase activity seen upon differentiation (Table 1).
In addition, DMSO-treated cells, which do not exhibit morphological
characteristics of apoptosis such as membrane blebbing. cell shrink
age, or nuclear fragmentation (32), also did not display a subdiploid
DNA content that was significantly different from that of uninduced
cells, demonstrating that the loss of telomerase activity was not due to
apoptosis in this model system.
These data demonstrate that the terminal differentiation of HL-60
human promyelocytic leukemia cells to monocytic and granulocytic
lineages is accompanied by a loss of telomerase activity. This is most
likely the result of regulatory changes incurred as a result of differ
entiation rather then drug-induced apoptosis or changes in the levels
Table 1 Percentage of operatic cells in terminall\ differentiating HL-60 cells
Data generated by flow cytometry analysis as outlined in Fig. 4 is plotted for TPA-RA- and DMSO-treated HL-60 cells as a function of time after induction of differentiation.
apoptoticAgentTPARADMSO00.60.60.711.81.00.722.81.50.435.40.90.3Time
Percentage
(days)49.73.50.5cells5_1.60.66_6.24.47_12.32.08__4.2
3800
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DIFFERENTIAL
LOSS OF TELOMERASE
PRE
ACTIVITY
WITH DIH-KRIiNTlATION
POST
Fig. 5. Differential loss of telomerase activity
during differentiation of murine cell lines. F9 leratocarcinoma. C2CI2 myohlasl, PI9 embryonal
carcinoma, and Neuro 2a neuroblastoma cells were
induced to differentiate, and telomerase activity
was assessed with the TRAP assay. A-D contain
photomicrographs of undifferentiated (PRE\ and
differentiated (POST) cells as well as the TRAP
assay results of undifferentiated ( —¿}
and differen
tiated ( + ) cell extracts and RNase controls (R). A.
photomicrographs of uninduced and RA ( I ¿J.M
)treated F9 cells at 4 days. Lane I of the TRAP
assay, uninduced cell extract control incubated
with 1 /¿MRA. Lane 2, RNase control. Lane 3,
uninduced F9 cells. Lanes 4-6. F9 cells treated
with RA for 4, 8. and 12 days, respectively. All
extracts represent I ^g of protein. B. photomicro
graphs of uninduced C2C12 and differentiated
myoblasts after 1 week in differentiation medium.
Lane I of the TRAP assay. RNase control. Lanes
2-4. extracts representing I x IO4. 5 x IO4, and
1OSuntreated cells. Lanes 5-7, extracts represent
ing 1 X IO4. 5 X IO4, and IO5 cells grown for 10
R
-
H-
H-
H.
1234567
days in differentiation medium. C. photomicro
graphs of undifferentiated PI9 and cells treated
with RA for 4 days and allowed to form neurite
outgrowths. Lane I. RNase control. Lanes 2 and 3.
untreated P19 cells at 1 X 106and5 x 10" cells/20
jil of lysis buffer. Lane 4. differentiated P19 cells
at I X IO6 cells/20 /¿Iof lysis buffer. Cells were
maintained under serum-free conditions for 60
days before assessment of lelomerase activity to
ensure complete differentiation. D. photomicro
graphs of uninduced and 3-day serum-starved
Neuro 2a cells. Lane I of the TRAP assay, RNase
control. Lam1 2, uninduced cells. Lanes 3 and 4.
cells treated with 20 /i M of RA for 3 and 4 days.
Lane 5, serum-starved cells at 4 days.
,—
1234
R
-
+
12345
the changes in phenotype associated with differentiation of the murine
cell lines and the TRAP assays performed on cell extracts both pre- and
postdifferentiation. Treatment of F9 teratocarcinoma cells with RA and
growth of C2C12 myoblast cells in low-serum medium induces their
differentiation to cells with properties of parietal endoderm (17) and
skeletal muscle (20), respectively. Both differentiated cell types exhibited
a dramatic down-regulation of telomerase activity (Figs. 5A and fl). In
contrast, telomerase activity was retained in RA-treated P19 cells that
were induced to differentiate to neurons and glial cells by RA treatment
3801
of the RNA component of telomerase, although alterations in the RNA
secondary structure cannot be ruled out as a factor in blocking
telomerase activity.
In addition to HL-60, a variety of human and mouse cell lines can be
induced to differentiate, including the mouse F9 teratocarcinoma, C2C12
myoblast, PI9 embryonal carcinoma, and Neuro 2a neuroblastoma cell
lines. To determine if the loss of telomerase activity observed in HL-60
cells was specific to this cell type, we determined the effect of differen
tiation on telomerase activity in these murine cell lines. Fig. 5A-D shows
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DIFFERENTIA!.
LOSS Oh TE-ILOMERASK ACTIVITY
(18) and in Neuro 2a cells induced to differentiate into cells with a
neuronal phenotype by RA or by serum starvation (Ref. 19; Figs. 5C and
D). The possibility of residual, contaminating, undifferentiated. telomerase-positive cells interspersed among the differentiated cells was ad
dressed using the P19 system. Differentiated PI9 cultures were incubated
in serum-free conditions for up to 60 days before telomerase activity was
assessed. Cells were not subcultured during this time, demonstrating that
their morphological differentiation was also associated with loss of
growth. In other experiments, a large proportion of untreated PI9 cells
maintained under serum-free conditions died within 3 days, however, a
small population of cells remained for weeks after serum withdrawal.
This extensive incubation in serum-tree conditions ensured that residual
P19 cells that did not respond to RA died and that only a fully pure
population of differentiated cells was assessed with the TRAP assay.
These results clearly show that loss of telomerase activity is not a
requisite feature of the differentiation of all cells in vitro.
It is unclear at present why some cell types suppress telomerase
activity during differentiation and others retain it. It is possible that the
different results obtained with these cell lines may reflect the obser
vation that many mature mouse tissues, in contrast to human tissues,
express telomerase activity and may lack mechanisms to efficiently
repress enzyme activity during development. This has been proposed
as one possible explanation for the considerably greater rate
(1 X 10 versus 1 X 10"l2 for human cells) at which rodent cells
spontaneously immortalize in culture (15). Although this is an attrac
tive possibility, a recent report in which telomerase activity was
assayed in additional systems indicates that HL-60 cells and at least
two mouse cell lines, F9 murine teratocarcinoma cells and CCE-24
murine embryonal stem cells, also lose telomerase activity upon the
induction of differentiation (34). Our results confirm the observations
made in differentiating HL-60 cells and in differentiated F9 cells
using the protein concentrations described (34), however, we noted a
marked increase in telomerase activity in differentiated F9 cells when
higher protein concentrations were analyzed, indicating that repres
sion of activity may not be complete in this system. It is also unclear
whether some neuronal lineages in mice lack telomerase repression
mechanisms because those cell lines that retained activity formed
neuronal cell types upon differentiation. Testing of additional human
and murine cell lines that are capable of differentiating will be
required to better understand the factors responsible for the loss of
telomerase activity in some, but not all, differentiated cell types and
whether there are species differences or developmental differences in
the propensity to lose telomerase activity upon differentiation. It also
remains to be seen whether the specific pathways or the components
involved in TPA-, RA-, and DMSO-induced differentiation of HL-60
cells and other cell types overlap those responsible for the repression
of telomerase activity in normal human somatic cells. If so, such cell
lines will provide powerful in vitro model systems for studying the
regulation of human telomerase.
ACKNOWLEDGMENTS
We wish (o (hank Dr. R. Woodman and F. Johnston for helpful advice on
NBT staining. Drs. T. Tamaoki and G. Schultz for the gifts of P19, C2C12, and
F9 cells, and Shari Berridge for typing the manuscript.
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Selective Inhibition of Telomerase Activity during Terminal
Differentiation of Immortal Cell Lines
Leslie J. Bestilny, Christopher B. Brown, Yutaka Miura, et al.
Cancer Res 1996;56:3796-3802.
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