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 s
Trang 1Differential regulation of telomerase activity by six telomerase
subunits
Joseph Tung-Chieh Chang1, Yin-Ling Chen2, Huei-Ting Yang2, Chi-Yuan Chen2and Ann-Joy Cheng2
1
Department of Radiation Oncology, Chang Gung Memorial Hospital, Taoyuan, Taiwan;2School 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
telomerase component were also performed Telomerase
activity was decreased or abolished by antisense treatment
To correlate clinical sample status, four pairs of normal and malignant tissues from patients with oral cancer were examined Except for the hTERT subunit, which showed differential expression in normal and cancer tissues, all other components were expressed in both normal and malignant tissues We conclude that hTERT is a regulatable subunit, whereas the other components are expressed more constantly in cells Although hTERT has a rate-limiting effect on enzyme activity, the other telomerase subunits (hTR, TEP1, hsp90, p23, dyskerin) participated in full enzyme activi‘ty We hypothesize that once hTERT is expressed, all other telomerase subunits can be assembled to form a highly active holoenzyme
Keywords:hTERT; hTR; telomerase activity; telomerase subunit; TEP1
Normal human somatic cells have a limited proliferative
capacity Malignant cells, in contrast, have acquired the
ability to override senescence Telomere length and
telom-erase activity have recently been implicated in the control
of the proliferative capacity of normal and malignant cells
[1,2] Telomeres consist of hundreds to thousands of
tandem repeats of the sequence TTAGGG, which are
specifically extended by telomerase [1,2] In most human
somatic cells, except for regenerating tissues and activated
lymphocytes, telomerase activity is undetectable and
telo-mere length is progressively shortened during cell
replica-tion [3,4] Cell senescence is though to occur when the
telomere length is critically shortened On the other hand,
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 hTR or TEP1, has been reported to correlate with telomerase activity in cancer cells [17,18] An association
of telomerase activity with the expression of hsp90, p23 or dyskerin has not been reported
Correspondence to A.-J Cheng, School of Medical Technology,
Chang Gung University, 259 Wen-Hwa 1st Road,
Taoyuan 333, Taiwan.
Fax:+ 886 3328 0174,
E-mail:ajchen@mail.cgu.edu.tw
Abbreviations:hTR, human telomerase RNA; TEP1, telomerase
associated protein 1; hTERT, human telomerase reverse
transcriptase; hsp90, head shock protein 90; PTA,
phorbol-12-myristate-13 acetate; PHA, phytohaemagglutinin; PBMC,
peripheral blood mononuclear cells; SFM, serum-free medium;
TRAP/EIA, telomeric repeat amplification protocol-enzyme
immunoassay; TBP, TFIID-binding protein; TAF, TBP-associated
factors; mTEP1, mouse TEP1.
(Received 13 December 2001, revised 25 April 2002,
accepted 28 May 2002)
Trang 2The mechanism by which telomerase is activated in
human cancers is unclear In vitro reconstitution
experi-ments have shown that hTR and hTERT are sufficient for
telomerase activity, suggesting minimal catalytic function of
the enzyme [19] Ectopic expression of hTERT in normal
human cells restored telomerase activity, extended the
replicative life span of the cells [20,21], maintained telomere
length, and eliminated tumorigenicity [22,23] The level of
hTERT in cells seems to be a sole component necessary for
regulation of the enzyme’s activity Nevertheless, it remains
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
M A T E R I A L S A N D M E T H O D S
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 All gene sequences were found in the GenBank database Non-specific oligonucleotides, designated as hTR, non-TEP1, non-hsp90, non-p23, and non-dkc, having the same base composition as the antisense oligonucleotides but with different sequences, were used as controls Oligonucleotides used for transfection experiments were modified by phos-phorothiolation All oligonucleotides were from Genasia Scientific Inc., Taipei, Taiwan
Tissue samples, cell lines, and cell culture Human tissues used for this study were from oral cancer patients, admitted to Chang Gung Memorial Hospital of Taiwan Written consent was obtained before use of the tissues in our experiments For each patient, one sample each of malignant tissue and normal mucosa were surgically dissected and frozen immediately in liquid nitrogen until used for molecular assay
Cells used included oral cancer cell lines OECM1, OC2 [26], KB, the cervical cancer cell line HeLa, and the leukaemia cell line HL-60 HL-60 cells were grown in RPMI-1640 (Gibco BRL), while others were maintained in Dulbecco’s modified Eagle’s medium (Gibco BRL) Both media were supplemented with 10% fetal bovine serum and antibiotics, and the cells were cultured in a humidified atmosphere containing 5% CO2 Cultures from each cell were harvested every 24 h and monitored for cell number by counting cell suspensions with a haemocytometer Cell viability was determined by staining cells with 0.25% Trypan blue, with the fraction of stain-negative cells taken
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· 104) 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
RT-PCR analysis.
Gene Name Sequences (5¢ fi 3¢)
Annealing temperature hTR F2b tccctttataagccgactcg 58 C
R3c gtttgctctagaatgaacggtggaag
TEP1 TEP1.1 tcaagccaaacctgaatctgag 58 C
TEP1.2 ccccgagtgaatctttctacgc
hTERT LT5 cggaagagtgtctggagcaa 56 C
LT6 ggatgaagcggagtctgga
hsp90 hsp90-f tccttcgggagttgatctctaatgc 60 C
hsp90-r gaattttgagctctttaccactgtccaa
p23 p23-f accagttcgcccgtccc 60 C
p23-r ccttcgatcgtaccactttgcaga
Dyskerin dkc-f cctcggctgtggaccgg 60 C
dkc-r aaataattacttccgcatccgcca
Actin actin-s gtggggcgccccaggcacc 58 C
actin-a ctccttaatgtcacgcacgatttc
Trang 3HBSS solution (Gibco BRL) and resuspended at a
density of 2· 106cellsÆmL)1in RPMI-1640 supplemented
with 20% fetal bovine serum and antibiotics PBMC
(1· 106cellsÆmL)1) were cultured in the presence or absence
of PHA (20 lLÆmL)1PBMC), and incubated at 37C in a
humidified atmosphere containing 5% CO2
Transfection with antisense and nonspecific
oligonucleotides
For the transfection of HL-60 cells, cells were seeded at a
density of 2· 106 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 lMand
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 37C in a CO2incubator 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· 106 cellsÆmL)1 in SFM,
followed by transfection with the DNA mixture as described
above After DNA transfection, the culture medium was
replaced with fresh complete RPMI containing 20 lLÆmL)1
PHA, and incubated for a further 3 days Protocols for
transfection of other cells were similar as described above,
except that final concentrations of up to 0.7 lM
oligonuc-leotides and 15 lL lipofection reagent were used to
maintain cell viability
Cellular protein extraction and analysis of telomerase
activity
Treated cells were suspended in a lysis buffer (10 mMTris/
HCl, pH 7.5, 1 mM MgCl2, 1 mM EGTA, 0.5% Chaps,
10% glycerol, 0.1 mMphenylmethanesulfonyl fluoride and
5 mM 2-mercaptoethanol), and incubated for 30 min at
4C while being gently mixed After centrifuging at
14 000 g for 30 min at 4C, 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
protein extract was added to 30 lL of the TRAP reaction
buffer and incubated at 25C for 15 min, followed by
amplification by 25 cycles of PCR at 94C for 30 s, 55 C
for 30 s, and 72C 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 lL of
tetramethylbenzidine substrate solution to each well Ten
min later, the reactions were stopped by the addition of
100 lL 2M 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 The expression of each of the telomerase subunit genes (hTR, TEP1, hTERT, hsp90, p23 and dyskerin) were analysed by using RT/PCR Total RNA from cells or tissues was isolated by using the TRIzol reagent (Gibco BRL) following the manufacturer’s instructions The con-centration, purity, and amount of total RNA were deter-mined by ultraviolet spectrophotometry
The reverse transcription reaction was performed in a total volume of 30 lL containing 30 ng RNA, 100 pmol poly T oligonucleotide, 4 U avian myeloblastosis virus reverse transcriptase (HT Biotech Ltd, UK), 10 U RNase inhibitor (CalBiochem), and 25 mMdNTP at 42C for 1 h The number of PCR cycles was titrated to avoid reaching the amplification plateau PCR was performed with 30 cycles of denaturation at 94C for 40 s, annealing at 56–
60C for 40 s and extension at 72 C for 1 min PCR products were analysed by either 8% polyacrylamide or 1.5% agarose gel electrophoresis, stained with SyBr Green I (Molecular Probes), then visualized and photographed by illuminating with 254 nm UV
R E S U L T S
Cellular changes in HL-60 cells after induction
of differentiation Over 80% of the HL-60 cells remained viable over the entire course of treatment with dimethylsulfoxide The ratio of differentiated cells (Fig 1A and B) was estimated to be 25% after 2 days of treatment and 70% after 4 days After
4 days of treatment with 100 ngÆmL)1TPA, > 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
Trang 4hTERT expression increased with the increase in telomerase
activity, while expression of the other telomerase subunits
remained unchanged This result further demonstrates that
hTERT is the major component responsible for the
regulation of telomerase activity
Telomerase activity after blockade of telomerase
subunits
Antisense inhibition of each telomerase subunit was carried
out in vitro In this experiment, 5–200 nMof each antisense
or nonspecific oligonucleotide was added to the TRAP
reaction buffer containing the protein extract from HL-60
cells After brief incubation (5 min) on ice, the reaction
mixtures were subjected to telomerase activity assay as
described in Materials and methods Telomerase activity
was inhibited by antisense oligonucleotides in a
dose-dependent manner (Fig 4) For each gene treated with
antisense oligonucleotides at 200 nM, telomerase activity
was completely inhibited (< 5% of untreated control)
Treatment with 50 nM antisense oligonucleotides led to a
dramatic reduction of telomerase activity, to < 20% of the
untreated control, except for anti-dyskerin, which reduced
telomerase activity to only 60% Treatment with 50 nM
nonspecific oligonucleotides did not inhibit telomerase
activity, except for slight inhibition with non-TEP1 (to
80% of control values) A low dose (5 nM) of antisense
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, TEP1, hsp90, p23 and dyskerin) with antisense oligonucleo-tides resulted in a reduction of specific mRNA expression (Fig 6A) and inhibition of telomerase activity in a dose-and time-dependent manner (partial results shown in Fig 6B) The exact effect of each antisense oligonucleotide
on telomerase activity varied according to the cell line This may have resulted from differing endogenous cellular regulatory responses or differing transfection efficiency in the various cell types For example, OECM1 cells generally showed more significant inhibition than other cells (Fig 6B) Nevertheless, inhibition of each telomerase sub-unit caused a reduction of telomerase activity, suggesting
Fig 1 Changes in cell morphology after
induction of differentiation of HL-60 cells by
dimethylsulfoxide and TPA (A) HL-60 cells
cultured in RPMI for 3 days (control for B),
followed by cytospin analysis and staining
(·1000) (B) HL-60 cells treated with 1.4%
dimethylsulfoxide, followed by cytospin
analysis and staining (·1000) (C) HL-60 cells
in suspension after culture in RPMI for 3 days
(control for B) (·400) (D) HL-60 cells after
treatment with 100 ngÆmL)1TPA for 3 days,
attached to flask (·400) suspension and
photographed the attached cells.
Trang 5that each component plays a distinct role in the full enzyme
function
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
D I S C U S S I O N
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.
Fig 3 Activation of telomerase activity by stimulating PBMC with PHA (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 N, sample was not determined See Materials and methods for experimental details.
Fig 4 In vitro analysis of changes in telomerase activity after intro-duction of antisense or nonspecific oligonucleotides Results are presented
as the means of duplicate experiments Antisense oligonucleotides were used at either 200, 50 or 5 n M , while nonspecific oligonucleotides were used at 50 n M as indicated at the top of the figure Relative telomerase activity was obtained after comparing the control sample without treatment with oligonucleotides Telomerase activity was measured by TRAP/EIA.
Trang 6except for hTR and hTERT, however, the roles of the
other subunits in enzyme function are still unclear TEP1
protein is thought to be associated with hTR, as the
N-terminal region of TEP1 is homologous to the gene of
Tetrahymena telomerase component p80, which interacts
with telomerase RNA [9,10] The WD40 repeats are found
in proteins involved in a wide variety of cellular processes
ranging from signal transduction to RNA processing [28]
Proteins containing WD repeats are often physically associated with other proteins and are believed in many cases to act as scaffolds upon which multimeric complexes 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 Results are presented as the means of two 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.
Fig 5 Telomerase activity after introduction of antisense
oligonucleo-tides (A) Antisense oligonucleotides against either TEP1 (anti-TP1) or
hTR (anti-hTR) were transfected into HL-60 cells and TEP1 and hTR
expression was measured after 3 days by TRAP/EIA Actin expression
for each treatment was determined as mRNA control TEP1
expres-sion was inhibited significantly by anti-TP1 treatment The expresexpres-sion
of hTR has not much affected by anti-hTR, because this antisense
oligonucleotide was designed to be complementary to the template
region sequence (B) Antisense oligonucleotides against either TEP1
(anti-TP1) or hTR (anti-hTR), or nonspecific oligonucleotides
(non-TP1 and non-hTR) were transfected into HL-60 cells and telomerase
activity was measured by TRAP/EIA cells 3 days later (C) Telomerase
activity after introduction of antisense oligonucleotides into
PHA-stimulated lymphocytes Ficoll-Hypaque isolated lymphocytes were
treated with PHA with or without anti-TP1 or anti-hRT and cultured
for up to 48 h.
Trang 7telomerase activity but maintenance of telomere length [33].
These results demonstrate the necessity for hTR in
telom-erase and suggest a telomtelom-erase-independent pathway in
maintaining telomere length Our results of antisense
manipulation of the hTR subunit are in agreement with
this finding Embryonic stem cells without mTEP1 reveal no
alteration in telomerase activity compared to wide-type
cells, suggesting a redundant role for mTEP1 [34] When we
inhibited TEP1, however, there was complete inhibition of
telomerase activity in vitro and significant inhibition in cells,
indicating that this protein is required for full activity of the
telomerase Several possibilities may explain this finding In
the in vivo mouse model, mTEP1 may be associated with
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-strated to bind to hTERT and are considered to be telomerase subunits [13] P23 was first identified as a component of progesterone and glucocorticoid receptor complexes [37] Subsequently, it was found that p23 is associated with hsp90 in these complexes and that the presence of both molecules is required to maintain these receptors in a ligand binding state [37] These observations led to the concept of a molecular chaperon machine or foldosome that mediates assembly of a biologically active protein complex Similarly, hsp90 and p23 in the telom-erase complex may also serve this foldosome function to assemble the active holoenzyme Geldanamycin, an hsp90 inhibitor, has been found to reduce the activity of reconstituted telomerase in cell extracts, demonstrating the role of hsp90 in the holoenzyme complex [36] In our
in vitro and cellular study of antisense oligonucleotide inhibition, blockage of hsp90 or p23 significantly decreased telomerase activity, further supporting the inference that these molecules play a role in the assembly of active telomerase Whether these molecules, like TEP1, have an additional stimulatory effect on telomerase requires further investigation Antisense inhibition of dyskerin, although showing comparable inhibition of telomerase function in cells, exhibited a weaker inhibition of telomerase activity
in the in vitro assay As dyskerin is believed to be involved
in hTR processing or assembly into the telomerase complex [14], the weaker inhibition leads us to suspect that this processing occurs at an earlier step of telomerase holoen-zyme assembly
The correlation of telomerase activity with the expression
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 patient :tissue
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.
Trang 8necessity of other telomerase components for full activity of
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
stimulation in both up- and down-regulation models The
other telomerase components retained expression at
relat-ively constant levels in both human tissue samples and cell
lines, and showed a lesser response to environmental
changes In addition, our antisense experiments showed
that inhibition of any component could result in the
reduction of telomerase activity, suggesting that each
telomerase subunit is necessary for full enzyme activity
We conclude that hTERT is a regulatable subunit, while the
other components are more constantly expressed We
hypothesize that once hTERT is expressed, all of the other
telomerase subunits can be assembled to form a highly
active holoenzyme
A C K N O W L E D G E M E N T S
This work was supported by National Science Council Research Grant
NSC89-2314-B-182-068 of Taiwan and Chang Gung Medical Research
Grant CMRP869 We thank M J Buttrey for critical reading and
correction of the manuscript.
R E F E R E N C E S
1 Blackburn, E.H (1991) Structure and function of telomeres.
Nature 350, 569–573.
2 Blackburn, E.H (1992) Telomerase Annu Rev Biochem 61,
113–129.
3 Cheng, A.J., Tang, R., Wang, J.I., See, L.C & Wang, T.C.V.
(1998) Possible role of telomerase activation in the cancer
predis-position of patients with hereditary nonpolyposis colorectal
can-cers J Natl Cancer Inst 90, 316–321.
4 Bacchetti, S (1996) Telomere dynamics and telomerase activity in
cell senescence and cancer Semin Cell Dev Biol 7, 31–39.
5 Cheng, A.J., Lin, J.D., Chang, T & Wang, T.C.V (1998)
Telom-erase activity in benign and malignant human thyroid tissues Br.
J Cancer 77, 2177–2180.
6 Tang, R., Cheng, A.J., Wang, J.Y & Wang, T.C.V (1998) Close
correlation between telomerase expression and adenomatous
polyp progression in multistep colorectal carcinogenesis Cancer
Res 58, 4052–4054.
7 Shay, J.W & Bacchetti, S (1997) A survey of telomerase activity
in human cancer Eur J Cancer 33, 787–791.
8 Feng, J., Funk, W.D., Wang, S.S., Weinrich, S.L., Avilion, A.A.,
Chiu, C.P., Adams, R.R., Chang, E., Allsopp, R.C., Yu, J et al.
(1995) The RNA component of human telomerase Science 269,
1236–1241.
9 Collins, K., Kobayashi, R & Greider, C.W (1995) Purification of
Tetrahymena telomerase and cloning of genes encoding the two
protein components of the enzyme Cell 81, 677–686.
10 Nakayama, J., Saito, M., Nakamura, H., Matsuura, A &
Ishikawa, F (1997) TEP1:a gene encoding a protein component
of mammalian telomerase is a novel member of WD repeats
family Cell 88, 875–884.
11 Meyerson, M., Counter, C.M., Eaton, E.N., Ellisen, L.W., Steiner,
P., Caddle, S.D., Ziaugra, L., Beijersbergen, R.L., Davidoff,
M.J., Liu, Q., Bacchetti, S., Haber, D.A & Weinberg, R.A (1997)
hEST2, the putative human telomerase catalytic subunit gene, is
up-regulated in tumor cells and during immortalization Cell 90,
785–795.
12 Linger, J., Hughes, T.R., Shevchenko, A., Mann, M., Lundblad,
V & Cheh, T.R (1997) Reverse transcriptase motifs in the
cata-lytic subunit of telomerase Science 276, 561–567.
13 Holt, S.E., Aisner, D.L., Baur, J., Tesmer, V.M., Dy, M., Ouellette, M., Trager, J.B., Morin, G.B., Toft, D.O., Shay, J.W., Wright, W.E & White, M.A (1999) Functional requirement of p23 and hsp90 in telomerase complexes Genes Dev 13, 817–826.
14 Mitchell, J.R., Cheng, J & Collins, K (1999) A box H/ACA small nuclear RNA-like domain at the human telomerase RNA 3¢ end Mol Cell Biol 19, 567–576.
15 Mitchell, J.R., Wood, E & Collins, K (1999) A telomerase component is defective in the human disease dyskeratosis con-genita Nature 402, 551–555.
16 Shay, J.W & Wright, W.E (1999) Mutant dyskerin ends relationship with telomerase Science 286, 2284–2285.
17 Nakayama, J., Tahara, H., Tahara, E., Saito, M., Ito, K., Nakamura, H., Nakanishi, T., Tahara, E., Ide, T & Ishikawa, F (1998) Telomerase activation by hTRT in human normal fibro-blasts and hepatocellular carcinomas Nature Genet 18, 65–68.
18 Dome, J.S., Chung, S., Bergemann, T., Umbricht, C.B., Saji, M., Carey, L.A., Grundy, P.E., Perlman, E.J., Breslow, N.E & Sukumar, S (1999) High telomerase reverse transcriptase (hTERT) messenger RNA level correlates with tumor recurrence
in patients with favorable histology Wilm’s tumor Cancer Res 59, 4301–4307.
19 Beattie, T., Shou, W., Robinson, M.O & Harrington, L (1998) Reconstitution of human telomerase activity in vitro Curr Biol 8, 177–180.
20 Bodnar, A.G., Ouellette, M., Frolkis, M., Holt, S.E., Chiu, C.P., Morin, G.B., Harley, C.B., Shay, J.W., Lichtsteiner, S & Wright, W.E (1998) Extension of life-span by introduction of telomerase into normal human cells Science 279, 349–352.
21 Counter, C.M., Meyerson, M., Eaton, E.N., Ellisen, L.W., Caddle, S.D., Haber, D.A & Weinberg, R.A (1998) Telomerase activity is restore in human cells by ectopic expression of hTERE (hEST2), the catalytic subunit of telomerase Oncogene 16, 1217– 1222.
22 Hahn, W.C., Stewart, S.A., Brooks, M.W., York, S.G., Eaton, E., Kurachi, A., Beijersbergen, R.L., Knoll, J.H.M., Meyerson, M & Weinberg, R.A (1999) Inhibition of telomerase limits the growth
of human cancer cells Nature Med 5, 1164–1170.
23 Zhang, X., Mar., V., Zhou, W., Harrington, L & Robinson, M.O (1999) Telomere shortening and apoptosis in telomerase-inhibited human tumor cells Genes Dev 13, 2388–2399.
24 Cheng, A.J., Liao, S.K., Chow, S.E., Chen, J.K & Wang, T.C.V (1997) Differential inhibition of telomerase activity during induc-tion of differentiainduc-tion in hematopoietic, melanoma, and glioma cells in culture Biochem Biophys Res Commun 237, 438–444.
25 Sharma, H.W., Sokoloski, J.A., Perez, J.R., Maltese, J.Y., Sartorelli, A.C., Stein, C.A., Nichols, G., Khaled, Z., Telang, N.T.
& Narayanan, R (1995) Differentiation of immortal cells inhibits telomerase activity Proc Natl Acad Sci U.S.A 92, 12343–12346.
26 Wong, D.Y.K., Chang, K.W., Chen, C.F & Chang, R.C.S (1990) Characterization of two new cell lines derived from oral cavity human squamous cell carcinomas-OC1 and OC2 J Oral Max-illofac Surg 48, 385–390.
27 Cheng, A.J., Tang, R., Wang, J.Y., Chang, J.T & Wang, T.C (1999) Polymerase chain reaction-based enzyme immunoassay for quantitation of telomerase activity:application to colorectal cancers Jpn J Cancer Res 90, 280–285.
28 Vander, V.L & Ploegh, H.L (1992) The WD-40 repeat FEBS Lett 307, 131–134.
29 Neer, E.J., Schmidt, C.J., Nambudripad, R & Smith, T.F (1994) The ancient regulatory protein family of WD-repeat proteins Nature 371, 297–300.
30 Matsuda, S., Katsumata, R., Okuda, T., Yamamoto, T., Miyazaki, K., Senga, T et al (2000) Molecular cloning and characterization of human MAWD, a novel protein containing WE-40 repeats frequently overexpressed in breast cancer Cancer Res 60, 13–17.
Trang 931 Kickhoefer, V.A., Stephen, A.G., Harrington, L., Robinson,
M.O & Rome, L.H (1999) Vaults and telomerase share a
com-mon subunit, TEP1 J Biol Chem 274, 32712–32717.
32 Kickhoefer, V.A., Liu, Y., Kong, L.B., Snow, B.E., Stewart, P.L.,
Harrignton, L & Rome, L.H (2001) The
telomerase/vault-asso-ciated protein TEP1 is required for vault RNA stability and its
association with the vault particle J Cell Biol 152, 157–164.
33 Niida, H., Shinkai, Y., Hande, M.P., Matsumoto, T., Takehara,
S., Tachibana, M., Oshimura, M., Lansdorp, P.M & Furuichi, Y.
(2000) Telomere maintenance in telomerase-deficient mouse
embryonic stem cells:characterizaiton of an amplified telomeric
DNA Mol Cell Biol 20, 4115–4127.
34 Liu, Y., Snow, B., Hande, M.P., Baerlocher, G., Kicihoefer, V.A.,
Yeung, D., Wakeham, A., Itie, A., Siderovski, D.P., Lansdorp,
P.M., Robinson, M.O & Harrington, L (2000)
Telomerase-associated protein TEP1 is not essential for telomerase activity or
telomere length maintenance in vivo Mol Cell Biol 20, 8178– 8184.
35 Burey, S.K & Roeder, R.G (1996) Biochemistry and structural biology of transcription factor IID (TFIID) Ann Rev Biochem.
65, 769–799.
36 Masutomi, K., Kaneko, S., Hayashi, N., Yamashita, T., Shirota, Y., Kobayashi, K & Murakami, S (2000) Telomerase activity reconstituted in vitro with purified human telomerase reverse transcriptase and human telomerase RNA component J Biol Chem 275, 22568–22573.
37 Hutchison, K.A., Stancato, L.F., Owens-Grillo, J.K., Johnson, J.L., Krishna, P., Toft, D.O & Pratt, W.B (1995) The 23-kDa acidic protein in reticulocyte lysate is the weakly bound compo-nent of the hsp foldosome that is required for assembly of the glucocorticoid receptor into a functional heterocomplex with hsp90 J Biol Chem 270, 18841–18847.