Differential regulation of telomerase activity by six telomerase
subunits
Joseph Tung-Chieh Chang
1
, Yin-Ling Chen
2
, Huei-Ting Yang
2
, Chi-Yuan Chen
2
and Ann-Joy Cheng
2
1
Department of Radiation Oncology, Chang Gung Memorial Hospital, Taoyuan, Taiwan;
2
School of Medical Technology and
Graduate School of Basic Medical Science, Chang Gung University, Taoyuan, Taiwan
Telomerase is a specialized reverse transcriptase responsible
for synthesizing telomeric DNA at the ends of chromo-
somes. Six subunits composing the telomerase complex have
been cloned: hTR (human telomerase RNA), TEP1
(telomerase-associated protein 1), hTERT (human telom-
erase reverse transcriptase), hsp90 (heat shock protein 90),
p23, and dyskerin. In this study, we investigated the role of
each the telomerase subunit on the activity of telomerase.
Through down- or upregulation of telomerase, we found
that only hTERT expression changed proportionally with
the level of telomerase activity. The other components,
TEP1, hTR, hsp90, p23, and dyskerin remained at high and
unchanged levels throughout modulation. In vivo and in vitro
experiments with antisense oligonucleotides against each

most immortalized and human cancer cells exhibit stabil-
ized telomere lengths, and are positive for telomerase
activity [5–7]. The above evidence suggests that mainten-
ance of telomeric length is required for cells to escape from
replicative senescence and to acquire the ability to
proliferate indefinitely. Telomerase reactivation thus
appears to play an important role in cellular immortality
and oncogenesis.
The subunits comprising the human telomerase complex
have been identified: human telomerase RNA (hTR),
telomerase-associated protein 1 (TEP1), and human
telomerase reverse transcriptase (hTERT). hTR functions
as a template for telomere elongation by telomerase [8].
TEP1, which is homologous to the gene of Tetrahymena
telomerase component p80, contains WD40 repeats [9,10].
As p80 interacts with telomerase RNA, the function of
TEP1 is suspected to be associated with RNA and protein
binding. hTERT contains reverse transcriptase motifs and
functions as the catalytic subunit of telomerase [11,12].
Recently, other proteins associated with the telomerase
holoenzyme have been reported. Heat shock protein 90
(hsp90) and molecular chaperon p23 have been demon-
strated to bind to hTERT and contribute to telomerase
activity [13]. Another nucleolar protein, dyskerin, which is
the pseudouridine synthase component of the box
H + ACA snoRNAs, also interacts with hTR [14,15]. It
is conceivable that dyskerin mediates interaction of the
telomerase ribonuclear protein with the nucleolus to
facilitate hTR processing or assembly of the telomerase
complex [14–16]. Greater expression of hTERT, but less of

unclear whether other telomerase subunits are essential for
the holoenzyme function. Down-regulation of telomerase
activity by induction of differentiation of HL-60 cells has
been reported [24,25]. To understand the role of each
telomerase subunit in enzyme activity, we used down-
regulation of telomerase by inducing differentiation of
HL-60 cells, and up-regulation of telomerase by stimulating
proliferation of peripheral blood mononuclear cells
(PBMC) to evaluate changes in the telomerase components.
We then investigated alterations in telomerase activity after
blocking each telomerase component by antisense oligonu-
cleotides using in vitro and cellular models. We also
examined normal and malignant human tissue samples for
the expression of each telomerase subunit and correlated it
with telomerase activity.
MATERIALS AND METHODS
Chemicals
Dimethylsulfoxide, phorbol-12-myristate-13 acetate (PTA),
phenylmethanesulfonyl fluoride, and Wright and Trypan
blue dyes were from Sigma. Giemsa stain was from Aldrich,
and phytohaemagglutinin (PHA) and lipofectin reagent
were from Gibco BRL.
Oligonucleotides
The oligonucleotides used for PCR amplification are listed
in Table 1. The antisense oligonucleotide against the hTR
gene (anti-hTR) was designed to be complementary to the
template region sequences. Other antisense oligonucleo-
tides against TEP1 (anti-TEP1), hsp90 (anti-hsp90), p23
(anti-p23), dyskerin (anti-dkc) were designed to be comple-
mentary to the region )2 to +20 of the coding sequences.

as the surviving fraction. In all experiments, the cell viability
rates were >75%.
Induction and assessment of differentiated HL-60 cells
Induction of differentiation in HL-60 cells was performed
either by treatment with 1.4% dimethylsulfoxide or with
100 ngÆmL
)1
TPA for up to 4 days. The differentiated
HL-60 cells were assessed by morphological change.
Induction with dimethylsulfoxide led to granulocytic differ-
entiation, which was assessed using Wright–l Giemsa stain.
Cells (5 · 10
4
) were prepared on slides by Cytospin
(Shandon Southern) and stained, then examined under a
light microscope (·1000). Granulocytic differentiation was
determined according to the presence of an eccentric or
segmented nucleus and the increase in the nucleus/
cytoplasm ratio. Induction with TPA led to monocytic
differentiation and attachment to the bottom of the culture
flask. For morphological assessment, the supernatant was
aspirated and the TPA-treated cells were examined with a
phase contrast microscope (·400). Monocytic cells were
identified by the presence of dendriform cytoplasm.
Isolation, culture, and activation of PBMC
Heparinized peripheral blood was drawn from normal
volunteer donors, and the PBMC were separated and
isolated from the interface of Ficoll-Hypaque (Pharmacia
Biotech). The isolated PBMC were washed three times with
Table 1. Names and the sequences of oligodeoxyribonucleotides used for

) were cultured in the presence or absence
of PHA (20 lLÆmL
)1
PBMC), and incubated at 37 °Cina
humidified atmosphere containing 5% CO
2
.
Transfection with antisense and nonspecific
oligonucleotides
For the transfection of HL-60 cells, cells were seeded at a
density of 2 · 10
6
per well in a six-well culture plate in
0.8 mL serum-free medium (SFM). Antisense or nonspecific
oligonucleotides in a final concentration of up to 0.7 l
M
and
20 lL of lipofection reagent in a total of 0.2 mL SFM were
mixed gently at room temperature for 45 min. The DNA
mixture was added to the HL-60 cells and incubated for
20 h at 37 °CinaCO
2
incubator. The culture medium was
then replaced with fresh complete RPMI and further
incubated for 3 days. For the transfection of PBMC, cells
were seeded at a density of 2 · 10
6
cellsÆmL
)1
in SFM,

14 000 g for 30 min at 4 °C, the supernatants were trans-
ferred to fresh tubes for the telomerase activity assay.
Protein concentrations were determined using the Coomas-
sie Protein Assay Reagent (Pierce).
Assay of telomerase activity was performed by the
telomeric repeat amplification protocol-enzyme immunoas-
say (TRAP/EIA) as described previously [27]. Telomerase
activity was determined by the ability to produce telomere
repeats by a PCR-based TRAP assay and measuring the
PCR products using a EIA-based assay. Briefly, 0.3 lg
proteinextractwasaddedto30lL of the TRAP reaction
buffer and incubated at 25 °C for 15 min, followed by
amplification by 25 cycles of PCR at 94 °C for 30 s, 55 °C
for 30 s, and 72 °C for 1 min in a DNA Thermal Cycler.
After the PCR reactions, 5 lL of the PCR products were
dispensed into streptavidin-coated wells and incubated with
100 lL of antidigoxigenin antibody conjugated with horse-
radish peroxidase (10 mUÆmL
)1
) at room temperature for
60 min in an EIA reaction buffer. After washing, enzyme
reactions were initiated by the addition of 100 lLof
tetramethylbenzidine substrate solution to each well. Ten
min later, the reactions were stopped by the addition of
100 lL2
M
HCl to each well. Colorimetric signals were
determined by measuring the absorbance at 450 nm using
an automatic microwell reader.
RNA extraction and analysis of telomerase subunit genes

)1
TPA, > 90% of the
HL-60 cells became attached to the flask and developed
dendriform cytoplasm, indicating successful induction
(Fig. 1C and D). Longer treatment led to cell death, with
an increased fraction of floating rather than attached cells.
Therefore, we harvested cells treated only for up to 4 days
for evaluation of telomerase activity and the subunit
expression studies.
hTERT expression after modulation of telomerase
activity
DMSO treatment led to a decrease in telomerase activity to
 70% of baseline after 1 day, 40% after 2 days, and
> 10% after 4 days (Fig. 2A). The expression of hTERT
was dramatically decreased after 1 day of treatment,
indicating that the hTERT subunit was significantly corre-
lated with the decrease in telomerase activity, and was
an earlier event than the change in holoenzyme acti-
vity.However, other telomerase components remained
unchanged during the entire course of treatment (Fig. 2B).
Similar results were found in the TPA-treated HL-60 cells.
Telomerase activity was gradually decreased over 4 days of
treatment, accompanied by diminished hTERT expression
but little change in other telomerase components (data not
shown). Both of these results indicate that hTERT is
the component primarily responsible for regulation of
telomerase activity.
As shown in Fig. 3, telomerase activity was up-regulated
after 8 h of PHA treatment of PBMC, reaching the highest
level at 2–4 days, and gradually decreasing after 4 days.

nonspecific oligonucleotides did not inhibit telomerase
activity, except for slight inhibition with non-TEP1 (to
80% of control values). A low dose (5 n
M
)ofantisense
oligonucleotides, resulting in lower levels of subunit inhibi-
tion, led to variable but significant effects on telomerase
activity for hTR, TEP1 and p23, whereas inhibition of
dyskerin had the least effect. From these antisense studies, it
appears that all of the telomerase subunits contribute to the
full activity of the holoenzyme, although dyskerin plays a
lesser role.
Transfection of HL-60 cells with anti-TEP1 led to a
specific inhibition of TEP1 (Fig. 5A). There was no obvious
effect on the expression of hTR after transfection of HL-60
cells with anti-hTR, as this antisense oligonucleotide was
designed to be complementary to the template region
sequence (Fig. 5A). Telomerase activity was gradually
decreased in cells transfected with specific antisense oligo-
nucleotides, to  60% after 2 days and to almost undetect-
able levels after 3 days (Fig. 5B). However, transfection
with nonspecific oligonucleotides had no effect on telom-
erase activity (Fig. 5B). For the effects of hTR and TEP1 on
the activation of telomerase, the model of stimulating
PBMC was applied. As shown in the Fig. 5C, the addition
of anti-hTR or anit-TEP1 to PHA-stimulated PBMC
resulted in significantly reduced activation of telomerase
after 48 h.
For the other cell lines studied (OECM1, KB, OC2, and
HeLa), inhibition of the various telomerase subunits (hTR,

Telomerase activity and the expression of each subunit
in normal and malignant tissues
In four pairs of normal and malignant tissue from oral
cancer patients, telomerase activity, as expected, was found
in all the malignant tissue samples but was absent in the
normal counterparts. Results of analysis of the expression of
each telomerase subunit are shown in Fig. 7. hTERT
expression correlated with telomerase activity, that is, it was
expressed in all telomerase-positive malignant tissue but was
undetectable in all telomerase-negative normal tissue. Other
telomerase subunits, however, were found to be more
constantly expressed in both normal and malignant tissue.
DISCUSSION
Telomerase activation is stringently repressed in normal
human somatic tissues but reactivated in immortal cells,
suggesting that up-regulation of telomerase participates
in cellular aging and oncogenesis. Therefore, understand-
ing telomerase regulatory mechanisms is valuable in
understanding tumour biology as well as in defining
molecular targets for clinical application. Thus far, six
major components of telomerase have been identified;
Fig. 2. Changes in telomerase subunits and telomerase activity in
response to induction of differentiation in HL-60 cells with dimethyl-
sulfoxide. HL-60 cells were treated with 1.4% dimethylsulfoxide for
4 days. Cells were harvested, and RNA and protein fractions were
extracted for subunit expressions and telomerase activity analysis.
(A) Relative telomerase activity on each day. (B) RNA expression of
telomerase subunits analysed by RT-PCR and resolved in 1.5%
agarose gel. Genes are listed on the left. Actin expression was analysed
as a control. See Materials and methods for experimental details.

are built [29]. Recently, a novel protein containing WD40
repeats was cloned and found to be overexpressed in
breast cancer [30]. Moreover, a cytoplasmic ribonucleo-
protein complex Vaults also shares a common subunit of
TEP1 [31,32]. Therefore, TEP1 protein in telomerase may
play a role in ribonucleoprotein structure, assembly, or
may also be involved in cancer progression.
The essential roles of hTR and TEP1 in telomere length
maintenance and telomerase activity have been investigated
in vivo, using mouse embryonic stem cells lacking mouse
telomerase RNA or the mouse TEP1 (mTEP1) gene.
Functional analysis of mouse embryonic stem cells with-
out mouse telomerase RNA shows a lack of detectable
Fig. 6. Telomerase activity after introduction of antisense oligonucleo-
tides into various cells. Resultsarepresentedasthemeansoftwo
independent experiments. Antisense oligonucleotides at 0.2 l
M
(anti-
TEP1 or anti-hTR) or 0.5 l
M
(anti-hsp90, anti-p23 or anti-dyskerin)
were transfected into various cells and telomerase activity was meas-
ured after 2 days by TRAP/EIA. (A) OECM1 cells were transfected
with each antisense oligonucleotide and the expression of each
telomerase subunit gene was measured. Actin expression for each
treatment was determined as an mRNA control. C, Control sample,
with lipofectin transfection only; A, antisense transfected sample.
(B) Cells included OECM1, HeLa, KB, and HL-60, and OC2 as
indicated each at the top of the figure. Relative telomerase activity
was obtained by comparison with the untreated control sample.

only a fraction of the total telomerase activity, or other
telomerase-associated proteins may share a redundant role
with mTEP1, so that its disruption might have no overt
phenotypic consequence [34]. In our in vitro experiment,
because of the shortage of cellular salvage pathways and the
complete inhibition of TEP1 function by high concentra-
tions of antisense oligonucleotides, telomerase activity was
dramatically diminished. Alternatively, hTR and hTERT
may play a minimal catalytic activity in telomerase, while
the assembly of other telomerase subunits may amplify the
enzyme function. In this scenario, deletion of mTEP1 in
embryonic stem cells would have no effect on telomerase
activity, and the level may be sufficient for mouse develop-
ment. In our experiments, the relatively high levels of
telomerase present in cancer cell lines were significantly
decreased upon inhibition of TEP1 by antisense oligonu-
cleotides. A similar example can be found in transcription
factor TFIID. TFIID contains a core TFIID-binding
protein (TBP) plus several TBP-associated factors (TAFs).
TBP alone stimulates minimal transcriptional activity in the
TATA box region of the promoter, but when it is associated
with complete TAFs, it strongly facilitates transcriptional
activity [35]. Recently, an in vitro reconstitution study has
been reported to support this hypothesis. A reconstituted
complex of hTERT and hTR was detected by EMSA, and
its activity was stimulated more than 30-fold by the addition
of cell extract, indicating the presence of a cellular factor
contributing to the stimulatory effect of telomerase activity
[36].
Hsp90 and molecular chaperon p23 have been demon-

of telomerase subunits hTERT, hTR and TEP1 has been
reported. Expression of hTERT, and less so of hTR or
TEP1, has been found to correlate with telomerase activity
in many cancer cells [17,18]. We further studied telomerase
activity in relation to the expression of hsp90, p23 and
dyskerin in human tissue samples and found no correlation.
These results indicate that hTERT is strongly associated
with telomerase activity while other components are more
constantly expressed in cells.
As described above, the experiments with ectopic expres-
sion of hTERT suggests that the level of hTERT in cells is a
rate-limiting component for the regulation of enzyme
activity. Nevertheless, these results still cannot rule out the
Fig. 7. Expressions of telomerase activity and the subunits in human
tissues. Four pairs of normal (N) and tumour (T) tissues from oral
cancer patients were examined. (A) Relative telomerase activity of each
sample compared to the OC2 cancer cell line. Telomerase activities are
determined by PCR/EIA. Each sample indicated as Ôpatient : tissueÕ
below each bar represents the specific patient and tissue. (B) The
expression of six telomerase subunits in the samples determined by
RT-PCR. Each sample is indicated at the top of the figure. Lane C
indicates the control experiment which contained all of the RT-PCR
reagents except tissue RNA. Six telomerase subunits were examined
by RT-PCR and are indicated at the left of the figure. See Materials
and methods for experimental details.
3448 J. Tung-Chieh Chang et al. (Eur. J. Biochem. 269) Ó FEBS 2002
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the enzyme. In the present study, we demonstrated that
hTERT is a regulatable component and responsible for the
activation of telomerase, as it responded to environmental

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