(The FASEB Journal. 1999;13:2091-2104.)
© 1999 FASEB
Studies of the molecular mechanisms in the regulation of telomerase activity
JUN-PING LIU1
Molecular Signaling Laboratory, Baker Medical Research Institute, Prahran, Victoria, Australia
1Correspondence: Molecular Signaling Laboratory, Baker Medical Research Institute, P.O. Box 6492, St. Kilda Road Central, Melbourne 8008, Australia. E-mail: jun-ping.liu{at}baker.edu.au
 |
ABSTRACT
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Telomerase, a specialized RNA-directed DNA polymerase that extends
telomeres of eukaryotic chromosomes, is repressed in normal human
somatic cells but is activated during development and upon neoplasia.
Whereas activation is involved in immortalization of neoplastic cells,
repression of telomerase permits consecutive shortening of telomeres in
a chromosome replication-dependent fashion. This cell cycle-dependent,
unidirectional catabolism of telomeres constitutes a mechanism for
cells to record the extent of DNA loss and cell division number; when
telomeres become critically short, the cells terminate chromosome
replication and enter cellular senescence. Although neither the
telomere signaling mechanisms nor the mechanisms whereby telomerase is
repressed in normal cells and activated in neoplastic cells have been
established, inhibition of telomerase has been shown to compromise the
growth of cancer cells in culture; conversely, forced expression of the
enzyme in senescent human cells extends their life span to one typical
of young cells. Thus, to switch telomerase on and off has potentially
important implications in anti-aging and anti-cancer therapy. There is
abundant evidence that the regulation of telomerase is multifactorial
in mammalian cells, involving telomerase gene expression,
post-translational protein–protein interactions, and protein
phosphorylation. Several proto-oncogenes and tumor suppressor genes
have been implicated in the regulation of telomerase activity, both
directly and indirectly; these include c-Myc, Bcl-2,
p21WAF1, Rb, p53, PKC, Akt/PKB, and protein phosphatase 2A.
These findings are evidence for the complexity of telomerase control
mechanisms and constitute a point of departure for piecing together an
integrated picture of telomerase structure, function, and regulation in
aging and tumor development—Liu, J.-P. Studies of the molecular
mechanisms in the regulation of telomerase activity.
Key Words: telomerase • telomere • structure • function • gene expression • protein phosphorylation • tumorigenesis • cancer • aging
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INTRODUCTION
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FROM STUDIES OVER the last two decades, it has become
clear that telomeres (1)
, the physical ends of eukaryotic
chromosomes, play a key role in maintaining the integrity of
chromosomal structures, in mediating the processes of chromosomal
replication, and in regulating the timing of cellular aging process and
thereby the life span of eukaryotes. Given these functions, telomeres
have attracted enormous attention, as detailed in various reviews
(2
3
4
5
6
7
8
9
10
11
12
13
14
15)
. Telomeres are primarily controlled by a highly
specialized DNA polymerase, termed telomerase, which is activated in
most human cancers and immortal cell lines (16
, 17)
; the
mechanisms of activation and regulation of telomerase, however, have
yet to be established. The purpose of this review is to briefly detail
our current understanding on the molecular mechanisms of telomerase
activation and of its inter- and intramolecular regulation.
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TELOMERES IN SIGNALING
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Telomeres are extended arrays of tandem repeat sequences of
G- and C-rich complementary hexanucleotide strands and
their binding proteins. Telomeric DNA possesses a general structure of
(T or A)m(G)n, with the vertebrate sequence being (TTAGGG)n. The
telomere binding proteins that have been identified to date include the
double-strand telomere binding proteins Rap1 (18)
and
Taz1p (19)
and single-strand telomere binding proteins
Rlf6p (20)
, Cdc13 (21)
, Est1p
(22)
, and Est2p (23
, 24)
in yeast, and in
humans the duplex telomere TTAGGG repeat binding factors TRF1 and TRF2
(25)
, the single-strand telomere binding proteins
telomerase reverse transcriptase (TERT) (26
, 27)
,
telomerase-associated protein 1 (TEP1) (28
, 29)
, and hnRNP
A1 (30)
. It is possible that most (if not all) of these
telomere proteins are involved in the regulation of telomere structure
and function. Recent studies with electron microscopy have revealed
that TRF2 remodels linear telomeric DNA into large duplex loops (t
loops) in vitro (31)
. These telomere t loops
formed through insertion of the 3' telomeric overhang into the duplex
telomeric repeat array may reflect a mechanism for the replication of
telomeric DNA and protection of telomeres against degradation and
damages (31)
.
Telomeres, intact or unwounded, function to protect chromosome ends
from recombination, fusion, and degradation by exonucleases and
ligases; to regulate mitotic chromosome recognition and separation; and
to position and anchor chromosomes with the nuclear machinery to
facilitate DNA replication at various stages of the meiotic and mitotic
cell cycle (10)
. Telomeres are metabolized by progressive
shortening as a function of chromosomal DNA replication in normal human
cell cultures (32
33
34)
and with age in vivo
(35
, 36)
. In each round of chromosome replication, for
instance, telomeres typically lose ~150 base pairs of nucleotide
sequence at the 5' end of a DNA molecule, reflecting the inability of
conventional DNA polymerases to replicate the extreme ends of telomeres
(37
, 38)
and the effects of a putative 5'-3' exonuclease
(39
, 40)
. Thus, an increasing cell division number is
usually accompanied by declining telomere length; in a given
population, the more that cells have undergone cell division, the
shorter their telomeres will be. This DNA replication-dependent loss of
telomere length has accordingly been proposed to operate as a mitotic
clock in order to count the number of cell divisions and to signal
cellular senescence. When the number of cell division is high, the
telomeres become so short that the cells are then permanently directed
to exit the cell division cycle, characteristic of replicative
senescence (32
, 41
, 42)
. Thus, degradation of telomeres
appears to constitute a signal with which a cell is no longer able to
undergo cell division.
Although the signaling pathways whereby telomere shortening induces
cellular senescence remain ill defined, it has been proposed that short
telomeres may lead to activation of multiple signaling mechanisms
(43)
. One possible mechanism may be that with shortening
telomere, transcription of genes nearby is affected. Repression of
euchromatic gene transcription by repetitive sequences of condensed
heterochromatin is known as position effect variegation
(44)
. In organisms such as Saccharomyces
cerevisiae (45)
, transcription of genes near
telomeres is reversibly repressed, a phenomenon called telomere
position effect (TPE). Extensive cutting or elimination of telomeres
may, on the other hand, activate gene transcription (Fig. 1
). Little is known about what genes are regulated by TPE in human
chromosomes and whether or not these genes are involved in signaling
cellular senescence in response to telomere length reduction.
Identification of the telomere shortening-sensitive genes (TSSG) is
therefore important for our understanding the cellular effects of
telomere metabolism. Recent studies in mice have shown that significant
shortening of telomeres results in activation of the tumor suppressor
p53 expression in association with cell growth arrest and apoptosis
(46)
. In contrast, elimination of p53 allows direct
oncogene-induced cell immortalization (47)
. Thus, p53 may
be one of TSSG involved in regulating cell growth arrest and apoptosis.
In addition, it is conceivable that telomere shortening may be
associated with production and accumulation of molecules regulating
permanent exit from the cell cycle into senescence (Fig. 1)
.
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Figure 1. A model of telomere shortening and associated signaling events. In
normal human somatic cells, where telomerase is inactive, telomeres
composed of TTAGGG repeats and their binding proteins undergo
progressive shortening with DNA replication. The shortening of
telomeres may shed telomere binding proteins (TBPs) and provide less
frequent binding sites in new generations of cells. Consecutive
shortening of telomeres is thus likely to be accompanied by the
production and accumulation of ‘free’ TBPs, which potentially
interact with other machinery serving as signaling molecules.
Comprehensive shortening of telomeres may also affect the transcription
of adjacent genes, an action termed telomere position effect or TPE.
Changes in expression of telomere shortening-sensitive genes (TSSG) may
thus be involved in telomere shortening-related signaling events that
ultimately lead to permanent arrest of cell proliferation prior to
chromosome damage.
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To accommodate telomere shortening, organisms have evolved
complex mechanisms to compensate for the loss of telomeres during cell
replication under particular circumstances. Three different
telomere-lengthening mechanisms have been described. In human cells,
de novo synthesis of telomeres by telomerase is the
predominant mechanism in telomere lengthening. In Drosophila
melanogaster, telomeric DNA can be elongated by transposition of
specific retrotransposons (HeT-A and TART) to chromosome ends
(48)
. In yeast, telomere extension can occur by homologous
recombination of nonreciprocal telomeric DNA between telomeres of
homologous or heterologous chromosomes (8
, 49
50
51)
. Thus,
telomere length in cells is regulated not only by the gradual loss of
telomeric DNA with each round of cell replication, but also by
differential gains of telomeric repeats through various mechanisms
across different animal species (51)
. At a given time in
the development of a eukaryotic organism, the length of telomeres is
thus a dynamic equilibrium between the rate of the DNA
replication-dependent shortening and the degree of a compensatory (or
anomalous) lengthening of telomeres through processes such as
telomerase activation. Erroneous activation of telomerase has been
widely believed to be involved in telomere stabilization or elongation
in humans, triggering the continuous division and proliferation
characteristic of immortal cells (16
, 52
53
54
55)
.
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STRUCTURE AND FUNCTION OF TELOMERASE
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Telomerase is a large ribonucleoprotein complex (56
, 57)
containing an RNA subunit (58
59
60
61
62
63)
and several
protein components. The RNA moiety is essential for the enzymatic
function of telomerase. Human telomerase RNA (hTR) is 445 nucleotides
long with an 11 nucleotide putative template sequence
(5'-CUAACCCUAAC-3') coding for the telomere repeats of (TTAGGG)n
(57)
. In vitro mutagenesis studies suggest that
the region required for minimal function is located between residues 44
and 203, with mutations between residues 170–179, 180–189, or
190–199 inhibiting both templating function and telomerase activity
(64)
. Besides serving as a template for reverse
transcription in telomere DNA synthesis, the RNA subunit is also
involved in the enzyme active site, probably with specific nucleotides
interacting with structural components of the DNA primer substrate and
protein subunits (65)
. Interspecies substitution of
telomerase RNA with an identical template base sequence from the
ciliate Glaucoma chattoni into Tetrahymena
thermophila cells produces a functional but aberrant telomerase,
suggesting that non-template RNA domains play roles in regulating
enzyme structure and function via intermolecular interactions within
the telomerase ribonucleoprotein complex (66)
. Removal or
down-regulation of the RNA subunit leads to inhibition of telomerase,
erosion of telomeres, compromise of growth capacity of highly
proliferative embryonic stem cells (67)
, testicular cells,
and hematopoietic cells (68)
in the mouse, and death of
both cultured HeLa cells (60)
and malignant human glioma
cells (69)
.
Although only a single gene for the telomerase RNA subunit has
been identified, there appear to be at least three genes that have been
cloned coding telomerase protein components in various organisms. In
the unicellular ciliate Tetrahymena thermophila, the
proteins p95 and p80 have been copurified with telomerase activity
(70)
. Biochemical studies suggest that p80 binds to the
telomerase RNA component, whereas p95, which has some sequence
similarity to two viral reverse transcriptases, interacts with the DNA
primer of the telomere substrate (70
, 71)
. In the ciliate
Euplotes aediculatus, two proteins of 120 kDa and 43 kDa
have been copurified with telomerase activity (72)
.
Although the sequence identity of the 43 kDa protein remains unknown,
the higher molecular mass protein has been cloned and found to be the
same as a yeast protein, Est2p (23)
. The EST2
gene had previously been identified by genetic analysis in that
mutation of this molecule induces a massive loss of telomere DNA and
senescence in yeast (73)
. Furthermore, both
Euplotes p123 and yeast Est2p contain sequences with strong
homology and six highly conserved regions commonly found in reverse
transcriptases; mutations of the three invariant Asp residues in the
conserved regions of both proteins result in loss of telomerase
activity, telomere shortening, and cellular senescence in yeast. These
studies have been interpreted as evidence that p123 and Est2p are the
catalytic subunit of telomerase in Euplotes and yeast,
respectively (23
, 24)
.
Although neither yeast homologues of Tetrahymena p80 or p95
nor a mammalian homologue of Tetrahymena p95 have been
reported, mammalian homologues of Tetrahymena p80 and
Euplotes p123/Est2p have recently been described based on
amino acid similarity. First, the p80 homologue telomerase associated
proteins 1 (TEP1) in human, mouse (28)
, and rat
(29)
have high homology and identical overall structures.
TEP1 is considerably larger than p80, with 2627 predicted amino acid
residues in humans. In addition, the p80 homology region and other
structures characteristic of regions putatively involved in molecular
interactions have allowed several domains of TEP1 to be described. The
modular structure of TEP1 thus includes a p80 homology domain, preceded
by a series of four tandem repeats of a 30 amino acid residue sequence
at the NH2 terminus, a centrally located
nucleotide binding motif, and a carboxyl-terminal region comprising a
large number of WD-40 repeats (Fig. 2
). Resembling Tetrahymena p80, TEP1 binds to the telomerase
RNA subunit (28)
, suggesting that the p80 homology domain
is involved in this interaction. Furthermore, human TEP1 (hTEP1)
coimmunoprecipitates with telomerase activity (28)
and
with the human homologue of p123/Est2p (human telomerase reverse
transcriptase or hTERT) (74
, 75)
. Given its interactions
with telomerase RNA and catalytic subunits and with the mosaic of WD-40
repeats implicated in mediating protein–protein interactions, TEP1 may
thus play a role in coordinating telomerase holoenzyme tertiary or/and
quaternary structures and/or serve as a docking/scaffold protein in
recruiting telomerase regulatory factors. Identification of the
molecules with which hTEP1 interacts would thus shed light on the
structures of telomerase holoenzyme and the mechanisms regulating
telomerase function.
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Figure 2. Domain structures of telomerase proteins. Two telomerase-related
proteins have been identified. The human catalytic subunit of
telomerase, hTERT, contains a unique motif (T motif) found in all TERT
molecules across different animal species and six conserved reverse
transcriptase motifs found in different reverse transcriptases. The
telomerase-associated protein 1, on the other hand, possesses a modular
structure including, from the NH2 terminus, the 1–4 random
repeat domain, the Tetrahymena homology domain, a
central ATP/GTP binding motif, and the C-terminal large WD-40 repeat
domain, implicated as playing roles in complex molecular
interactions.
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Given its homology with p123/Est2p, hTERT was independently identified
by several laboratories on the basis of sequence homology searching
(26
, 27
, 74
, 76
, 77)
. The hTERT is a polypeptide of 1132
amino acid residues with a molecular mass of ~120 kDa on sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (Fig. 2)
. Sequence
analysis and comparison with p123, Est2p, and Trt1p (a telomerase
catalyst in the fission yeast Schizosaccharomyces pombe)
indicate that hTERT shares extensive amino acid sequence identity
(46–49%) with the telomerase catalytic subunits of lower eukaryotes
throughout their various conserved regions. All these telomerase
catalytic subunits possess a unique conserved region called the T motif
that is amino-terminal to the six conserved reverse transcriptase
motifs (Fig. 2)
. As in the yeast telomerase reverse transcriptase Est2p
(23)
, mutations of the invariant Asp residues (D712, D868,
and D869) in the conserved reverse transcriptase regions of hTERT
abolish telomerase activity (77
, 78)
. In vitro
reconstitution experiments also show that hTERT and hTR constitute the
minimum core structure of telomerase (78
79
80)
. These
studies suggest that the intrinsic structures and properties of TERT
may be sufficient to complex with the telomerase RNA subunit and to
reverse transcribe telomere DNA from the RNA subunit to extend
telomeres; adding to the complexity are the findings of several
alternative splicing variants of hTERT mRNA (76
, 81
, 82)
.
These hTERT variants include hTERT
with an in-frame deletion of 12
amino acids from the reverse transcriptase domain A, hTERTß with a
182-nucleotide deletion resulting in a premature stop codon, and
another variant encoding an alternative carboxyl terminus (76
, 81)
. It is possible that the alternative splicing variants may
substitute for full-length hTERT to alter telomerase activity during
tissue and organ development of humans (83)
. A variety of
telomerase holoenzymes may therefore exist with varied hTERT proteins
to subserve diverse developmental and physiological functions.
To examine telomerase holoenzyme components by classical peptide
affinity chromatography, human breast cancer cell nuclear telomerase
was selectively enriched into particular fractions (75)
.
Protein analysis of these fractions showed several novel proteins
containing amino acid sequences homologous to reverse transcriptases of
lower order eukaryotes (unpublished observations), consistent with the
possibility that additional telomerase catalytic subunits or ancillary
factors may also exist in human cancer cells. In addition, sequencing
of a ~100 kDa protein from the telomerase fractions of human breast
cancer cells revealed the presence of the molecular chaperone gp96,
implicating its potential involvement in telomerase holoenzyme
structure and function (unpublished data). Recently it has been
reported that the molecular chaperones p23 and Hsp90 bind to the
catalytic subunit of telomerase; interference of this interaction with
geldanamycin inhibits telomerase activity (84)
. Given that
the estimated molecular mass of mammalian telomerase holoenzyme is
greater than 1000 kDa (29
, 63
, 85)
and that in
Euplotes crassus, the size can vary from 280 kDa to 5000 kDa
depending on the initiation of macronuclear development
(86)
, these data are consistent with the possibility that
the telomerase may contain proteins in addition to hTERT and hTEP1
under physiological and/or pathophysiological conditions in the
regulation of telomerase structure and function. Further studies are
required to elucidate these molecules and the roles of hTEP1 and
different hTERT proteins in telomerase holoenzymes.
The mode of telomerase action in synthesizing and elongating telomeres
is incompletely understood. It is believed that the telomerase
holoenzyme interacts with the single strand of 3' GT-rich telomeric
primer and polymerizes deoxynucleoside triphosphates in the 5' to 3'
direction. This reaction appears not to require ATP in yeast, but
utilizes the telomerase RNA as a guiding template complementary to the
telomeric DNA repeats (58
, 59)
. Once telomerase binds to
telomere primer and positions itself in such a way that the RNA
template sequence aligns with telomeric DNA primer, the enzyme
transcribes one DNA nucleotide onto the telomere at a time according to
the complementary coding sequence of the RNA template. When a full
telomeric repeat TTAGGG is formed, the telomerase may then translocate
to the next site with the process cycling to allow telomerase to add
iterative telomere DNA repeats. Both the catalytic activity and
processitivity require optimal conditions in temperature, ionic
strength, and enzyme–substrate interactions. Whereas the catalytic
activity is enhanced, the processitivity is restrained by increasing
temperature (up to 37°C) and the concentrations of monovalent cation
and substrate telomere DNA (87)
. Use of synthetic TTAGGG
oligodeoxynucleotide or reverse transcriptase inhibitors (such as
azidothymidine and carbovir) inhibits telomerase activity and induces
cell programmed death (57
, 88
89
90
91)
. Currently, neither the
tertiary/quaternary structures of operating telomerase nor the kinetics
of the interactions between telomerase and telomeres are known,
although it seems likely that the process involves dynamic changes in
telomerase configurations and its interactions with both single
substrate telomeric DNA and other factors. Other issues that remain to
be resolved include how the telomerase holoenzyme is assembled in the
nucleus; whether it undergoes a dynamic assembly and disassembly
process, with or without telomere substrate, during the DNA replication
cycle; and whether force generation is involved in telomerase
operation, especially given the presence of an ATP/GTP binding motif in
TEP1.
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MOLECULAR REGULATION OF TELOMERASE ACTIVITY
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Many lines of evidence indicate that telomerase is reversibly
regulated. For example, telomerase activity is undetectable in most
normal human somatic cells, but in ~85% of human cancers and
immortalized cell lines, telomerase becomes highly activated (16
, 17)
. Resting lymphocytes express little telomerase activity, but
stimulation of specific antigen receptors on the cell plasma membrane
markedly increases telomerase activity (92
93
94
95)
.
High-level sun exposure of normal human skins results in an increased
incidence of telomerase activation (96)
. Bombarding
cultured Chinese hamster cells with UV (97)
, human
hematopoietic cells with 
-rays (98)
, or human carcinoma
cell lines with X-rays (99)
induces the activation of
telomerase. Activated telomerase in cancer cells is repressed when the
cells exit the cell cycle and become quiescent (100
101
102
103
104)
.
In an animal model, treatment with an antagonist of growth
hormone-releasing hormone dramatically decreased telomerase activity in
xenografted U-87MG human glioblastoma cells (105)
. The
ensuing paragraphs examine the current evidence for factors involved in
regulating telomerase gene expression, structure, and function at the
molecular level. Although the mechanisms of telomerase regulation, such
as those underlying its suppression in normal human somatic tissues and
activation in neoplastic cells, are far from established, a better
understanding of the regulation of telomerase activity will provide a
basis for further investigation and manipulation of telomerase activity
as a potential therapeutic modality.
TELOMERASE GENE EXPRESSION
Whereas hTR and hTEP1 are widely expression in normal human
tissues (28
, 29
, 60
, 62
, 106
107
108)
, the expression of TERT
is repressed in most normal human somatic tissues after birth
(26
, 27
, 83
, 109)
but becomes activated in most (if not
all) telomerase-positive primary tumors and immortal cell lines so far
tested (26
, 27
, 107
, 108
, 110)
. Moreover, ectopic
expression of hTERT in telomerase-negative cells is sufficient to
reconstitute telomerase activity (77
, 78
, 111
112
113
114)
, to
elongate telomeres (111
, 112)
, and to extend cellular life
span (111
112
113)
. Correlating with telomerase activity and
cellular function, de novo activation of hTERT gene
transcription in neoplastic and immortalized cells may thus be the
dominant, rate-limiting step in telomerase activation, a step that is
potentially regulated by changes in the levels and functions of
multiple oncogene and tumor suppressor gene products. Thus, the widely
present hTEP1 and hTR may form an inactive telomerase complex
(70
, 71
, 74)
that becomes activated on the incorporation
of hTERT during the process of telomerase activation (Fig. 3
).
The mechanism of regulation of TERT gene expression is currently under
intensive investigation. Recent studies have shown that the hTERT
promoter is inactive in normal human somatic cells, but becomes
activated during cell immortalization (115)
. Sequence
analysis has revealed that the hTERT promoter contains binding sites
for several transcription factors, suggesting that hTERT expression may
be subject to multiple levels of control and regulated by different
factors in different cellular contexts (82
, 115)
. Several
groups have shown that c-Myc (a proto-oncogene product with
transcriptional activity) activates telomerase (110
, 116
117
118)
. Expression of hTERT in both normal human mammary
epithelial cells and normal human diploid fibroblasts is stimulated by
c-Myc (116)
, an effect attributed to direct interaction of
c-Myc with the hTERT promoter (117
, 118)
. Treatment of
human leukemic cells with antisense pentadecadeoxynucleotides targeted
against c-Myc mRNA leads to inhibition of telomerase activity
(119)
. These studies provide evidence for additional
layers of control in terms of telomerase expression and activation.
Equally interesting, on the other hand, is the recent finding of a
potential key element in telomerase suppression in normal somatic
tissues. The hTERT repression activity has been localized to chromosome
3 (but not chromosomes 7, 8, 11, 12, or 20) (120
121
122
123
124)
.
Introduction of chromosome 3 into telomerase positive cell lines, human
renal carcinoma (121
, 122)
, or breast cancer cells
(123)
induces repression of hTERT expression,
down-regulation of telomerase activity, up-regulation of telomere
shortening, and cessation of cell growth. The putative telomerase
repressor gene has been further mapped to chromosome region
3p14.2-p21.3 (122
, 125)
. Thus, expression of TERT and the
consequent activation of telomerase are regulated tightly at the
promoter machinery of TERT gene transcription by multiple elements
including oncogenes and an as yet unidentified suppressor activity.
In addition, up-regulation of telomerase is associated with
introduction of the human papillomavirus (HPV) type-16 E6 protein in
early-passage human keratinocytes and mammary epithelial cells
(126
, 127)
, with expression of the oncogenes SV40 or
v-Ki-ras in human prostate epithelial, prostate
endothelial, or umbilical vein fibroblast cells (128
, 129)
and with overexpression of Bcl-2 in human cancer cells
(130)
and rat pheochromocytoma cells (91)
. In
contrast, reestablishment of expression of the retinoblastoma protein
(Rb) in Rb-/p53- tumor
cells is associated with telomerase inhibition and cellular senescence
(131)
; overexpression of full-length Rb in human squamous
carcinoma cell lines also results in down-regulation of telomerase
activity (132)
. Moreover, overexpression of the universal
cyclin-dependent kinase inhibitor p21WAF1 induces
suppression of telomerase activity in human immortalized keratinocytes
(133)
and human glioma cell lines (134)
.
Although the mechanisms of telomerase up-regulation by HPV-16 E6
protein, SV40, v-Ki-ras or Bcl-2, and
down-regulation by Rb are still elusive, the novel function of
p21WAF1 in reducing telomerase activity appears
to be mediated by down-regulation of hTR gene expression
(133)
. Taken together, these data suggest that activation
of telomerase and the maintenance of its activity in cancer cells be
elicited by many factors at multiple levels on various targets.
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INTRA- AND INTERMOLECULAR INTERACTIONS
|
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Considerable evidence suggests that telomerase activity is
also controlled by post-transcriptional mechanisms (75
, 84
, 135
136
137
138
139)
. After translation, the assembly, maintenance, and
disassembly of functionally active telomerase holoenzyme presumably
require interaction with other proteins. Since telomerase is slowly
metabolized, with a half-life of more than 24 h
(101)
, telomerase activity may also be subject to
modulation by the holoenzyme changing in its conformation by direct
protein–protein interactions. Although little is known about how
proteins interact within the telomerase holoenzyme, it is noteworthy
that whereas hTEP1 binds to hTERT and the complex exhibits telomerase
activity (74
, 75)
, a peptide from the amino-terminal
region of hTEP1 specifically inhibits telomerase activity in
vitro (139)
. This hTEP1 peptide (aa 385–399), termed
telomerase inhibitory polypeptide 1 or TEIPP1, binds intact full-length
hTEP1 on affinity columns, suggesting the possibility of potential
hTEP1 oligomer formation in vivo (139)
. The
possibility of hTEP1 oligomerization is also supported by
immunoprecipitation experiments in which hTEP1 sometimes shows a
molecular mass of more than 1000 kDa (unpublished data). These
observations are consistent with the view that hTEP1 is a regulatory
subunit of telomerase, potentially serving as a scaffold for recruiting
and organizing hTR, hTERT, telomere substrates, and other potential
regulatory factors.
When potential telomere and telomerase regulatory factors are isolated
by molecular genetic approaches, various studies have shown that human
TRF1 (25
, 135)
, yeast Rap1p (140)
, and Taz1p
(19)
play crucial roles in telomerase activity. Long-term
overexpression of TRF1 in telomerase-positive tumor cells results in
gradual and consecutive telomere shortening, probably through a
cis-mediated inhibition of telomerase activity
(135)
. The binding of Rap1p to telomeres in yeast is also
associated with telomere shortening in proportion to the numbers of
bound Rap1p molecules (140)
, and disruption or deletion of
the Taz1 gene causes an increase in telomere length (19)
;
both thus appear to be negative regulators of telomere lengthening,
perhaps through a mechanism similar to that underlying the action of
TRF1. In addition, a TRF1 interacting protein (tankyrase) has also
recently been identified as potentially affecting telomerase via TRF1
(141
, 142)
. Tankyrase is a poly(ADP-ribose) polymerase
that catalyzes ADP-ribosylation of TRF1 at telomeres, leading to the
dissociation of both tankyrase and TRF1 from telomeres. This mechanism
promoting TRF1 dissociation may thereby allow telomerase to act on
telomere extension (141)
; nevertheless, whether or not
such telomere binding proteins directly interact with telomerase awaits
formal demonstration.
Given that hTEP1 may potentially serve as a scaffold for telomerase
regulatory factors, we have attempted to identify telomerase
interactive proteins by affinity chromatography with synthetic peptides
derived from the sequences of hTEP1 and have screened nuclear proteins
from human breast cancer cells for binding to the column. Several
proteins have been observed, among them a 53 kDa species on
immunological criteria the tumor suppressor protein p53
(139)
. Additional studies show that both endogenous
nuclear and recombinant p53 proteins coimmunoprecipitate with hTEP1 and
that recombinant p53 not only binds to hTEP1 peptide affinity column
specifically but also inhibits telomerase activity in vitro,
with an intact carboxyl terminus of p53 being essential for inhibition.
Moreover, the inhibitory effect of p53 on telomerase is abrogated by
TEIPP1, a peptide capable of inhibiting telomerase activity, further
suggesting a direct interaction between p53 and hTEP1. These data
suggest that telomerase may be a downstream element of p53, with its
activity being regulated by the tumor suppressor, a conclusion
consistent with the findings that p53 specific mutations are involved
in telomerase activation in sun-exposed human skins (96)
and that expression of a p53 mutant in human mammary epithelial cells
is occasionally associated with telomerase activation on additional
genetic events (143)
. Although further direct in
vivo demonstration is required, it is tempting to speculate that
p53 plays a guardian role in cellular senescence (47
, 144
145
146)
, at least in part through controlling telomerase
activity, and down-regulation of p53 or interference of its interaction
with telomerase favors telomerase activation and cell immortalization
(Fig. 4
).
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Figure 4. Potential interactions between p53 and telomerase in cellular
senescence and immortalization. A) The tumor repressor
p53 may interact directly with telomerase holoenzyme, resulting in
inhibition of telomerase activity in favor of cellular senescence.
B) Down-regulation of p53 or inhibition of its
interaction with telomerase may play a permissive role for telomerase
activation in cellular immortalization.
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ROLES OF PROTEIN PHOSPHORYLATION AND DEPHOSPHORYLATION
|
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Protein phosphorylation is an important post-translational
mechanism commonly used in controlling protein structure and function
(147)
. To determine whether protein phosphorylation plays
a role in telomerase activity, we analyzed telomerase activity of
cultured human breast cancer cells and partially purified telomerase by
affinity chromatography. In the presence of protein phosphatase 2A
(PP2A), but not of protein phosphatase 1 or protein phosphatase 2B,
telomerase activity is markedly inhibited in vitro
(136)
. This inhibition, also observed in human melanoma
cell lysates (137)
, is mimicked by nonspecific protein
phosphatase alkaline phosphatase and prevented by the PP2A inhibitor
okadaic acid, suggesting that dephosphorylation locks telomerase into
an inactive conformation, with protein phosphorylation able to restore
telomerase activity (136)
. These results suggest a model
in which telomerase exists in two different configurations that can be
switched on and off by reversible phosphorylation and dephosphorylation
(Fig. 3)
. Whereas protein phosphorylation is essential for telomerase
activity, PP2A appears to be the protein phosphatase involved in the
negative regulation of telomerase activity. It is so far unclear
whether the phosphorylation is required for assembly of the holoenzyme
or post assembly for the correct configuration for activity.
On affinity chromatography prepared with peptide sequences
derived from hTERT and hTEP1, no binding of cdc2 kinase, casein kinase
II, and ERK was obtained. In contrast, significant binding of
protein kinase C (PKC), but not PKCß1, PKC
, or PKC
, was
detected, suggesting the possible involvement of PKC in regulating
telomerase structure and function (75)
.
Immunoprecipitation studies show that both hTERT and hTEP1 are
phosphoprotein, dephosphorylated by PP2A, and rephosphorylated by
PKC (75)
. Furthermore, analysis of telomerase activity
shows that purified recombinant PKC markedly stimulates basal and
PP2A-inhibited telomerase activity in an ATP-dependent manner,
suggesting that PKC-induced phosphorylation is involved in
telomerase activation during the assembly of telomerase holoenzyme into
a functionally active configuration. In addition, although cdc2 kinase
and DNA-dependent protein kinase have no effect, PKC
, PKC and
PKC are all capable of reactivating telomerase post PP2A
dephosphorylation. What remains to be explored includes whether
different protein kinase C isoforms might play particular roles in the
regulation of telomerase activity in different types of cell
(75)
.
In cultured peripheral blood mononuclear cells, the PKC activator
phorbol myristate acetate stimulates telomerase activity, whereas
increased telomerase activity during T cell activation is inhibited by
the PKC inhibitor bisindolylmaleimide (148)
. Similarly,
the PKC inhibitor bisindolylmaleimide I also inhibits telomerase
activity in cultured nasopharyngeal cancer cells (149)
.
Given these reports and the finding that okadaic acid stimulates
(136)
and bisindolylmaleimide I inhibits (unpublished
observation) telomerase activity in cultured human breast cancer cells,
it is possible that PKC and PP2A are generally involved in reciprocally
controlling telomerase activity through protein phosphorylation and
dephosphorylation in intact neoplastic cells. Such a reciprocal effect
of PKC and PP2A on telomerase activity in human breast cancer cells
is highly consistent with the findings that PKC activity is markedly
elevated (150
151
152
153)
and PP2A inhibited
(153
154
155)
in the nucleus of human breast cancer cells in
response to various oncogene products and tumor promoting compounds
(Fig. 5
), which in turn is consistent with the notion that a balance between
PKC activation (156
, 157)
and PP2A inhibition
(153
154
155)
plays an important part in tumorigenesis.
In addition, studies have shown that besides PKC, protein kinase B (PKB
or Akt) is also involved in up-regulating telomerase activity
(137)
. In vitro, PKB phosphorylates the hTERT
peptide 817AVRIRGKSYV (826) and
stimulates telomerase activity. Treatment of human melanoma cells with
the protein phosphatase inhibitor okadaic acid stimulates both hTERT
peptide phosphorylation and telomerase activity, whereas treatment of
the cells with PI3 kinase inhibitor Wortmannin inhibits the
phosphorylation and telomerase activity. These observations suggest
that the serine residue at 824 of hTERT may be phosphorylated by PKB in
human cancer cells and that the phosphorylation is involved in
mediating cellular signaling produced by growth factor activation of
the PI3 kinase pathway (137)
. PKB may thus act on multiple
molecular targets including telomerase in the processes of apoptosis,
cell survival, and proliferation during aging and tumorigenesis.
|
IMPLICATIONS FOR AGING AND TUMORIGENESIS
|
|---|
Aging is a process associated with progressive changes, ultimately
leading to death, and the mechanisms involved in aging are still far
from well understood. Why some animal species live longer than others
has been the subject of speculation for centuries. What is currently
becoming clear is that aging is predominantly determined by genetic
factors, with aging and longevity genes activated and deactivated as
aging progresses. Activation of aging genes and repression or
deactivation of longevity genes is presumably controlled over time by
the gradual loss of positive/negative regulation and/or of genetic
material. Different animal species may have different levels of the
genetic material involved, different regulation of the aging and
longevity genes, and/or different defense mechanisms against
potentially lethal factors. The search for aging and longevity genes
has long been a focus in biomedical research. Since telomeres shorten
as a function of age in vivo and telomerase antagonizes the
process of telomere shortening, whether or not telomere shortening
serves as a timer with different settings in different species to
control the onset of cell senescence, and thus life span, has been the
subject of intense debate (14
, 142
, 158
159
160
161
162)
.
Telomeres are shorter in certain human tissues in older people than in
younger people (32
, 33
, 35
, 41)
, and aging in human
diploid fibroblasts and hematopoietic cells is accompanied by the
progressive loss of telomeres (32
, 34
, 36
, 41)
. Short
telomeres may activate multiple signaling pathways to induce cell
senescence (43)
, including activation of the p53 and p21
cell cycle checkpoint pathway (163
, 164)
. In two diseases
of very marked premature aging, Hutchinson-Gilford progeria
(32)
and Down syndrome (33)
, short telomeres
have been detected. It is thus possible that an onset of the major
aging-related diseases (such as atherosclerosis, hypertension, and
Alzheimer’s disease) and debilitating diseases (such as degenerative
joint disease and sensory impairment) may result from early cellular
senescence in the relevant tissues. Whether or not patients with these
diseases are born with short telomeres in relevant tissues ab
initio, due to growth retardation in uterus or due to altered
timing of telomerase activation/suppression during development in
uterus, requires further investigation. Recent studies in mice bearing
a germ line knockout of the mTR gene and thus null telomerase activity
show that short telomeres trigger multiple aging-related processes
including cell growth arrest, apoptosis, and/or decreased capacity in
response to stresses in highly proliferative organs, demonstrating a
critical role for telomere length in genomic stability, cell
replicative life span, and aging (68
, 165)
.
Consistent with the involvement of telomeres in cellular aging,
telomerase activity is absent during aging, and introducing the gene
for the telomerase catalytic subunit hTERT into senescent human cells
extends both their life span and their telomeres to lengths typical of
young cells (111
112
113)
. Such cells then display all the
identifiable characteristics of healthy young cells without genetic
instability (111
112
113)
or the hallmarks of malignant
transformation (128
, 166
167
168
169)
. These findings reinforce
the hypothesis that telomeres constitute the central timing mechanism
for cellular aging, and for the first time demonstrate that such a
mechanism can be reset to extend cell replicative life span in
vitro. Evidence against telomere length being directly correlated
with aging is that mice have long telomeres (50–150 kb) but a short
life span (~2 years) in comparison with the human’s relatively short
telomeres (10–15 kb) but long life span (~80 years)
(6)
. What is unclear, though, is whether telomere
signaling pathways are differentially regulated in different species
and whether cellular growth arrest in mouse is more sensitive to the
signals produced by telomere shortening than human or induced by
differentially integrated signals. Even in human cells, evidence has
also been presented showing a lack of relationship between adult human
age and telomere length (42
, 170
, 171)
. These findings
are, nonetheless, consistent with the notion that aging is a complex
process, development of which is determined by multiple mechanisms,
probably with telomere shortening being one of the programs that
becomes to be a predominant driving force under certain circumstances
such as in rapidly renewal and highly proliferative cells and in
premature aging of Hutchinson-Gilford progeria, Down syndrome, and
ataxia telangiectasia (32
, 33
, 35
, 36
, 41)
. With the
identification of the structures and regulatory mechanisms of telomeres
and telomerase, it has become possible to explore the fundamental
mechanisms underlying human aging, targeting telomerase components to
clarify the roles played by the machinery of telomeres and telomerase
in cell replicative senescence in aging.
In contrast with aging, cellular immortalization in the germ line and
the early steps of tumorigenesis are commonly associated with the
activation of telomerase and the ensuing stabilization of telomeres
(16
, 17
, 52
53
54
55)
. Expression of telomerase activity and
the relative stabilization of telomeres are thereby likely to confer
immortality on germ line and cancer cells when further oncogenic
signal(s) are present; inhibition of telomerase allows telomere
shortening and growth inhibition in cultured neoplastic cells
(60
, 69)
. In vivo, deletion of telomerase RNA
subunit results in short dysfunctional telomeres and impaired
tumorigenesis in the INK4a
2/3 cancer-prone
mouse (172)
. Injection of nude mice bearing xenografts of
U-87MG human glioblastoma with the growth hormone-releasing hormone
antagonist MZ-5–156 induces inhibition of telomerase activity with
hTERT down-regulation and of tumor growth (105)
. These
findings suggest that activation of telomerase is a pivotal element in
tumor development in response to the continuous presence of various
tumorigenic factors. Determination of intracellular factors that
control telomerase activity therefore appears to be an important issue
for both cancer research and therapy.
It is also worth noting, however, that deletion of the telomerase
RNA subunit in the mouse does not prevent telomerase-deficient cells
from being immortalized in culture, transformed by viral oncogenes, and
generating tumors in nude mice despite the very long telomeres in mouse
cells being consistently shortened (173)
. Considerable
evidence also shows that in most human cancer cells, chromosome
terminal restriction fragments, a measure of telomere length, are
dramatically reduced to 2–4 kb (52
, 174
175
176
177
178
179
180)
; in marked
contrast, those in the germ line and fetal cells are over 20 kb
(33
, 42
, 181)
. Although the mouse knockout model may
reflect different mechanisms in the maintenance of the long telomeres
and in signaling cellular senescence in this species, the shorter
telomere sequences in human cancer cells may incur genomic instability
by mechanisms including TPE (Fig. 1)
as a factor in the activation of
telomerase and in the cause of cancer, a condition that increases in
incidence as a function of age. In addition, it is also possible that
the shorter telomeres in cancer cells may result from a higher turnover
rate, with the telomere mitotic clock reset by mutation or telomerase
activation. Thus, the telomere clock may tick at different rates, with
different alarm settings determined by telomerase under different
cellular conditions in different species. However, human epithelial
cells do not become immortalized unless telomerase is activated and
Rb/p16 is inactivated (127)
. Furthermore, cells having
activated telomerase and overcome cellular senescence do not form
tumors in nude mice either until they are transformed with a second
agent with either v-Ki-ras or X-ray
(128)
. Clearly, tumorigenesis is a process of multiple
steps and more studies are needed to show whether the synthesis of
telomeres by telomerase in human cancer cells may at least be partly
involved in overcoming the cellular mechanisms controlling senescence;
experiments similar to those in which telomerase activity was inhibited
to suppress cancer growth are required.
|
CONCLUDING REMARKS
|
|---|
Telomerase constitutes an important mechanism in stabilizing the
structure of telomeres. Comprising the RNA subunit, TERT catalytic
subunit, and TEP1 regulatory protein, activation of telomerase requires
de novo transcription of TERT gene and correct incorporation
of full-length TERT protein into the holoenzyme. The expression of
telomerase activity is likely to be regulated by various oncogenes and
tumor suppressor genes, both directly and indirectly. The
proto-oncogene c-Myc is a transcription factor for the TERT gene.
Post-translation, telomerase proteins are phosphorylated for activity
by PKB and PKC; the phosphorylation is reversed by PP2A-mediated
dephosphorylation. Inhibition of telomerase activity is accompanied by
shortening of telomeres, compromised growth in highly proliferative
normal and neoplastic cells in culture, and reduced tumorigenesis in a
mouse model. Ectopic expression of telomerase in normal diploid cells
elicits extension of the cellular life span toward immortalization,
without the hallmarks of malignant transformation in vitro.
If telomeres could be selectively stabilized, chromosomes might
replicate forever and individuals be forever young; and if telomerase
could be selectively blocked in cancer cells, their telomeres might
shorten to the point of being no longer able to divide. Much work
remains to be done, and time may provide the answers, in the technical
if not the ethical domain, to such questions.
|
ACKNOWLEDGMENTS
|
|---|
The author wishes to thank Drs. John Funder, He Li, and Roger
Reddel for discussion. This work was supported by grants from the
Australia Research Council, National Health and Medical Research
Council of Australia, and the Anti-Cancer Council of Victoria.
|
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TOP
ABSTRACT
INTRODUCTION
