Carcinogenesis vol.24 no.7 pp.1167±1176, 2003
DOI: 10.1093/carcin/bgg085
COMMENTARY
Transcriptional regulation of the telomerase hTERT gene as a target for
cellular and viral oncogenic mechanisms
Izumi Horikawa1 and J.Carl Barrett
Introduction
Laboratory of Biosystems and Cancer, Center for Cancer Research,
National Cancer Institute, National Institutes of Health, 9000 Rockville Pike,
Building 37, Room 5046, MSC-4264, Bethesda, MD 20892, USA
Normal human somatic cells, unlike cancer cells, undergo only
a limited number of cell divisions and eventually stop dividing
through the process termed cellular senescence or replicative
senescence (1,2). Cellular senescence can be a tumor suppressive mechanism in human cells, and immortalization is a
rate-limiting step for cancer cells to develop and grow (3,4).
Cellular senescence, aging at cellular levels in other words, is
also suggested to play a role in aging of multicellular organisms (5). Multiple mechanisms can trigger the cellular senescence program in human cells (2,6). A major mechanism
of cellular senescence involves shortening of telomeres, a
specialized structure at chromosome ends that consists of an
array of repetitive DNA sequences and the associated proteins
(5,7). Most normal human somatic cells do not have a mechanism to compensate for loss of telomeres associated with
each round of DNA replication (so-called `end replication
problem'), and critically short, dysfunctional telomeres eventually induce cells to senesce or die (5,8). In contrast, cancer
cells maintain their telomere length mainly through activation
of telomerase, an enzyme that synthesizes telomeric repeat
DNA (9). Telomere maintenance is essential for immortal
growth of cancer cells (1,4,7,8,10). These findings have established a crucial role of regulation of telomerase and telomeres
in human cell senescence, immortalization and carcinogenesis.
Among multiple protein components and an RNA component of human telomerase, the catalytic protein subunit, human
telomerase reverse transcriptase (hTERT), is the key determinant of the enzymatic activity of human telomerase (11±14).
Although various steps at post-transcriptional and posttranslational levels can modulate the hTERT function (15),
the transcriptional control of the gene is a major contributor
to the regulation of telomerase activity in many types of human
cells (11±13,16). Thus, investigation of activating and repressive mechanisms of the hTERT transcription in cancer and
normal cells, respectively, has become an area of intense
interest in cancer research. Several known transcription
factors, including some oncogene and tumor suppressor gene
products, are able to affect the hTERT transcription in an
experimental setting of over-expression or ectopic expression.
The existence of telomerase repressor proteins, which function
effectively at an endogenous expression level but are not yet
identified, has been proposed. At least one of those repressors
acts to repress the hTERT transcription through a specific DNA
element within the hTERT promoter. Recent data also suggest
that viral oncogenesis involves activation of the hTERT gene
transcription.
This review summarizes a growing number of data to show
that the hTERT gene is transcriptionally controlled by a variety
of signaling pathways that promote or suppress carcinogenic
processes in humans. It should also be noted that, however,
most of the candidate hTERT transcriptional regulators thus
far suggested still remain to be proven as `endogenous' or
1
To whom correspondence should be addressed
Email: [email protected]
Malignant transformation from mortal, normal cells to
immortal, cancer cells is generally associated with activation of telomerase and subsequent telomere maintenance.
A major mechanism to regulate telomerase activity in
human cells is transcriptional control of the telomerase
catalytic subunit gene, human telomerase reverse transcriptase (hTERT). Several transcription factors, including
oncogene products (e.g. c-Myc) and tumor suppressor gene
products (e.g. WT1 and p53), are able to control hTERT
transcription when over-expressed, although it remains to
be determined whether a cancer-associated alteration of
these factors is primarily responsible for the hTERT activation during carcinogenic processes. Microcell-mediated
chromosome transfer experiments have provided evidence
for endogenous factors that function to repress the telomerase activity in normal cells and are inactivated in cancer cells. At least one of those endogenous telomerase
repressors, which is encoded by a putative tumor suppressor gene on chromosome 3p, acts through transcriptional
repression of the hTERT gene. The hTERT gene is also a
target site for viruses frequently associated with human
cancers, such as human papillomavirus (HPV) and
hepatitis B virus (HBV). HPV E6 protein contributes to
keratinocyte immortalization and carcinogenesis through
trans-activation of the hTERT gene transcription. In at
least some hepatocellular carcinomas, the hTERT gene is
a non-random integration site of HBV genome, which activates in cis the hTERT transcription. Thus, a variety of
cellular and viral oncogenic mechanisms converge on transcriptional control of the hTERT gene. Regulation of chromatin structure through the modification of nucleosomal
histones may mediate the action of these cellular and viral
mechanisms. Further elucidation of the hTERT transcriptional regulation, including identification and characterization of the endogenous repressor proteins, should lead
to better understanding of the complex regulation of
human telomerase in normal and cancer cells and may
open up new strategies for anticancer therapy.
Abbreviations: 5-aza-C, 5-aza-20 -deoxycytidine; EMSA, electrophoretic
mobility shift assay; HAT, histone acetyltransferase; HBV, hepatitis B
virus; HDAC, histone deacetylase; HPV, human papillomavirus; hTERT,
human telomerase reverse transcriptase; MMCT, microcell-mediated
chromosome transfer.
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I.Horikawa and J.C.Barrett
`physiological' regulators, whose alteration during carcinogenesis plays a causative role in hTERT activation in human
cancer cells.
Known transcription factors as the candidate hTERT
transcriptional activators
The strong correlation between the expression of hTERT
mRNA and telomerase activity in human cancer and normal
cells has greatly stimulated investigation of regulatory
mechanisms of the hTERT gene expression. In 1999 several
groups reported the cloning of the 50 -regulatory region (promoter) of the gene (17±22). Reporter gene assays (e.g. luciferase assays) showed that the transcriptional activity of the
hTERT promoter fragment correlates well with the expression
level of hTERT mRNA in the cells used: the promoter activity
in hTERT-expressing cancer cells is much higher than that in
hTERT-negative normal cells, suggesting that the control at
transcriptional level represents a major mechanism to regulate
the amount of hTERT mRNA and protein, and the resulting
telomerase activity. Initial analyses of the hTERT promoter
depended largely on the prediction of transcription factor
binding sites through computer-assisted search. In particular,
two canonical E-box elements (CACGTG), located ~185 bp
upstream and ~25 bp downstream of the transcription initiation
site (17,22), looked attractive because they are potential binding sites of basic helix±loop±helix zipper (bHLHZ) transcription factors encoded by the Myc family of oncogenes (23),
which could give a direct scenario for a link between oncogene
activation and telomerase activation. Indeed, over-expression
of c-Myc protein resulted in a remarkable, E-box-dependent
increase in the hTERT promoter activity (19,20,24). The overexpressed c-Myc also induced an expression of endogenous
hTERT mRNA and telomerase activity in some normal human
cells (20,25). These findings suggest that the hTERT gene can
be a direct target of the c-Myc transcription factor when it is
over-expressed (Figure 1). A recombinant c-Myc protein was
shown to be able to bind the canonical E-box elements within
the hTERT promoter in the electrophoretic mobility shift assay
(EMSA) (19,21,24). However, whether an increased expression of c-Myc protein, which is observed in some types of
cancers, is required for activation and maintenance of the
hTERT expression still remains to be proven. Although in a
few published studies the binding of an endogenously
expressed, cellular c-Myc protein to the hTERT E-boxes was
detected by the EMSA (25,26), an increase in the binding
during transition from normal to cancer cells has not been
shown. In some experimental systems, including chromosome
transfer and E6 expression experiments described below, control of the hTERT transcription depends on the E-box element
but does not correlate with the amount of endogenous c-Myc
protein (27±29). On the other hand, Xiao et al. (30) recently
showed, by means of a PCR-based DNA±protein interaction
assay, the differential c-Myc binding to the hTERT promoter
between U937 leukemia cell subclones with high and low
hTERT/telomerase expression. Xu et al. (31) showed by chromatin immunoprecipitation assay that the c-Myc binding to the
hTERT promoter in vivo correlates with the induction of the
hTERT in proliferating HL60 leukemic cells (Mad1 binding in
differentiated, hTERT-repressed HL60 was also shown; see
below). To establish a causative relationship between c-Myc
function and hTERT transcription during malignant transformation of human cells, the in vivo binding of endogenous
c-Myc protein to the hTERT promoter should be examined in
normal versus cancer cells. A functional knock-out of c-Myc
aberrantly expressed in cancer cells by means of antisense
Fig. 1. Multiple mechanisms of the transcriptional regulation of the hTERT gene. Schematically shown are various mechanisms that can act on the hTERT promoter
to regulate the hTERT transcription. Sites of actions within the promoter are not in scale. See the text for the details. Some activators (e.g. Myc) and repressors (e.g.
Mad) may function through recruitment of HAT and HDAC, respectively. Note that, in addition to its activator function in cancer cells, Sp1 may recruit HDAC to
repress the hTERT transcription in normal cells (not shown in this figure). E, two canonical E-box (CACGTG) elements upstream and downstream of the
transcription initiation site (‡1; refs 17 and 27).
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Transcriptional regulation of the telomerase hTERT
RNA or small interfering RNA strategy (32) would also
address whether the endogenous c-Myc is responsible for
activation and maintenance of the hTERT expression.
The GC-rich hTERT promoter contains multiple binding
sites for a ubiquitously expressed, zinc finger transcription
factor Sp1 (17,18,20,22,24). Mutation of these Sp1 binding
sites dramatically reduced the hTERT promoter activity in
cancer cells (24), suggesting that they actually contribute to
the hTERT transcription (Figure 1). The EMSA analysis
revealed the binding of cellular Sp1 protein to the hTERT
promoter (17,18). An increase in the amount of Sp1 protein
was associated with telomerase activation during in vitro transformation of a fibroblast strain (24). However, in another
experimental system (27), hTERT-expressing and negative
cells showed similar levels of Sp1 binding to the hTERT
promoter. Because of its ubiquitous expression in a wide
range of normal cells the Sp1 protein by itself is unlikely to
be a determinant of hTERT activation during carcinogenic
processes. The Sp1 may interact with some other proteins
[e.g. p53 and histone deacetylase (HDAC); see below] to
negatively regulate the hTERT transcription in normal cells,
and inactivation of the interacting proteins and/or alteration in
other regulatory mechanisms may allow Sp1 to function as an
activator in cancer cells. Recent studies added the upstream
stimulatory factors (USF) (27,33), an abundantly expressed
family of E-box binding factors, and the Ets family of transcription factors (30,34), which is known to be upregulated in
breast and other cancers, to the list of candidates for hTERT
transcriptional activators (Figure 1) [for USF, an N-terminally
truncated form of USF2 is suggested to function as a dominant
negative repressor antagonizing the wild-type proteins (33)].
These factors also await further analyses to examine whether
they play a critical role in hTERT activation in human cancers.
Known transcription factors as the candidate hTERT
transcriptional repressors
A systematic search through an expression cloning strategy for
the candidate hTERT transcriptional repressors was carried out
by Oh et al. (35,36). Although this approach relies on activities
of over-expressed proteins rather than endogenous proteins, it
should allow a large-scale identification of the candidate
repressors. They first identified the WT1, a tumor suppressor
protein involved in a common pediatric cancer, Wilms' tumor
(Figure 1). The repressive effect on the hTERT promoter
activity by the over-expressed WT1 was observed in 293
embryonal kidney cells but not in Hela cervical cancer cells,
consistent with the restricted expression of WT1 in specific
tissues (kidneys, gonads and spleen) (37) and implying the
tissue-specificity of the transcriptional repression by WT1. A
recombinant WT1 protein was shown to bind the consensus
WT1 binding site within the hTERT promoter (35). However,
whether inactivation of the endogenous WT1 during development of Wilms' tumors contributes to activation of the hTERT
transcription remains to be examined to establish its role as
a physiological repressor in normal cells from which the
tumors arise.
Another candidate identified through the expression cloning
approach by Oh et al. (36) was an E-box binding factor Mad1
(Figure 1), which competes with c-Myc, the candidate hTERT
activator described above, for the common interacting partner
Max and for the binding DNA sequence E-box (23). The
role of endogenous Mad1, as well as c-Myc, in the hTERT
transcriptional regulation associated with cell differentiation is
well characterized. Upon the hTERT repression during differentiation of HL60 and U937 leukemic cells, the Mad1/Max
complex replaces the c-Myc/Max complex at the hTERT promoter E-boxes (26,31). Even though normal cells tend to
express higher levels of Mad1 protein than cancer cells
(26,36), evidence for the in vivo binding of endogenous
Mad1 to the hTERT promoter and its significance in the
hTERT repression in normal cells (versus cancer cells) is still
missing. Mxi1, another member of Mad family transcriptional
repressors, which may contribute more to cancer development
than Mad1 (38), showed a similar repressive effect on the
hTERT transcription in a reporter gene assay (our unpublished
data) and should also be examined as a possible endogenous
repressor of the hTERT transcription.
MZF-2 (39), a zinc finger transcription factor, and E2F-1
(40), an E2F family transcription factor that generally transactivates genes involved in cell-cycle progression, are also suggested to potentially bind the hTERT promoter and repress its
activity (Figure 1). EMSA experiments show binding of an
endogenously expressed MZF-2 or E2F-1 to their respective,
putative binding sites within the hTERT promoter; however,
only one cancer cell line was examined and no comparison
between cancer cells and their normal counterparts was made.
The repressive effect on hTERT transcription was evaluated
through overexpression of MZF-2 or E2F-1 in cancer cells
without any known aberrations (e.g. mutation or loss of
expression) of these proteins. Although a tumor suppressor
function of E2F-1 was suggested by tumor formation in
E2F-1 null mice (41), no mutations in the E2F-1 gene have
been found in human cancers (42,43). A possibility of the E2F1 regulation of both cell-cycle progression and telomerase
activity is interesting, but further analyses will be needed to
examine if the hTERT gene is a physiological target of the
repressor function of E2F-1 in normal cells. There is thus far
no evidence for a role of MZF-2 in human cancers. It is
important to find a condition, if any, in which the MZF-2's
repressive activity is of physiological significance.
Some tumor suppressor proteins or pathways may have an
ability to repress the hTERT transcription by acting through
potential activators such as c-Myc. An important effect by the
TGF-b/Smad signaling pathway is to down-regulate c-Myc
expression (44,45). The activation or suppression of this pathway in human cancer cells resulted in a significant reduction or
increase of hTERT expression and telomerase activity, respectively (46). Thus, activation of the TGF-b/Smad signaling
pathway is likely to negatively regulate the hTERT expression
at least in part through the downregulation of c-Myc (Figure 1).
Another possible function of this signaling pathway to control
telomerase activity is regulation of the alternative splicing of
hTERT mRNA (47). However, it is still important to examine
if inactivation of this pathway (e.g. by a mutation of genes
encoding TGF-b receptors or Smad proteins) is directly
involved in the hTERT/telomerase activation during transformation of normal (or pre-malignant) to cancer cells. BRCA1, a
tumor suppressor protein for hereditary breast and ovarian
cancers involved in DNA repair and transcriptional regulation
(48), is another candidate hTERT repressor acting through
regulation of c-Myc (Figure 1). Li et al. (49) found a complex
consisting of BRCA1, c-Myc and Nmi (originally identified
as N-Myc-interacting protein) that can inhibit the hTERT
promoter activity in breast cancer cells. Zhou and Liu (50)
also reported that BRCA1 abrogates the c-Myc-mediated
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enhancement of the hTERT promoter activity in ovarian cancer
cells. Although their studies relied on the activity of the transiently over-expressed protein and lacked data on endogenous
hTERT gene expression and telomerase activity, the hTERT
gene may be one of the target genes of the BRCA1 protein as a
transcriptional regulator. A tumor suppressor p53, which is
most frequently mutated in various types of human cancers,
is also suggested to have an ability to repress the hTERT
transcription. Restoration of the functional p53 in cervical
cancer, Burkitt lymphoma and breast cancer cells led to the
downregulation of telomerase activity through the repression
of the hTERT promoter activity (51,52). This repression is
likely to be mediated by an interaction of p53 and Sp1,
which inhibits the Sp1 binding to the hTERT promoter (51)
(Figure 1). Although these findings suggest that the repression
of hTERT (and therefore the repression of telomerase activity)
may be one of the mechanisms by which p53 contributes to
tumor suppression, as in the case of other candidate hTERT
repressors, it remains to be examined whether p53 inactivation
plays a causative role in the hTERT activation in carcinogenesis during conversion from telomerase-negative normal/
pre-malignant cells to telomerase-positive cancer cells.
Investigation of endogenous mechanisms for telomerase
repression: a telomerase repressor gene on chromosome 3
acts on the hTERT promoter
As described above, several transcription factors are suggested
to have the ability to activate or repress the hTERT transcription. However, there is little evidence that these factors are
active enough to control the hTERT transcription when endogenously expressed (versus over-expressed following transfection) and that a cancer-associated alteration of these factors
(e.g. an inactivating mutation of the repressors or an activating
mutation of the activators) plays a causative role in the hTERT
activation at a specific stage during multi-step carcinogenesis
(versus an additive effect in the cells already expressing an
enough level of telomerase for their immortal growth). To
investigate endogenous factors that are directly responsible
for the telomerase repression and activation in normal and
cancer cells, respectively, we and others have used an experimental system based on microcell-mediated chromosome
transfer (MMCT) (53,54). Because inactivation of tumor suppressor genes, including putative telomerase repressor genes,
generally involves loss or mutation of both alleles of the
genes (so-called `two hit'), introduction of a single copy of
the genes under the physiological expression control into
cancer cells via MMCT is expected to restore their suppressor
function. The cells generated by MMCT (microcell hybrids)
are a good material for functional analyses of the genes on the
transferred chromosomes even before they are cloned. Several
normal human chromosomes (i.e. chromosomes 3, 4, 6, 7, 10
and 17) have thus far been shown to repress the telomerase
activity in some, but not all, cancer cells (55±63), suggesting
that these normal chromosomes carry a gene whose expression
under a physiological setting is responsible for the telomerase
repression in normal cells and can compensate for a defect in
the telomerase repression that certain cancer cells sustained.
Such cancer-associated defects are likely to vary among individual tumors and/or in cell type-specific manners. Once a
specific chromosome is identified to cause the telomerase
repression in a given cancer cell line, comparative
analyses of the telomerase-positive cancer cells and the
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telomerase-negative microcell hybrids allow one to focus on
a single, specific mechanism of telomerase regulation that
involves the function of a telomerase repressor gene on the
transferred chromosome, instead of comparing normal cells
with cancer cells containing a number of genetic and epigenetic changes, which could have the indirect effects on the
telomerase expression [e.g. a change in telomerase activity
associated with cell proliferation rate (64)].
Repression of telomerase via MMCT is associated with the
repression of hTERT mRNA expression in all the cases so far
examined, including chromosome 3 in renal and breast carcinoma cells (55,57), chromosome 6 in immortalized keratinocytes and cervical carcinoma cells (60), chromosome 7 in
SV40-transformed mesothelial cells (61) and chromosome 10
in hepatocellular carcinoma cells (62). These findings suggest
that the endogenous, repressive mechanisms, which are
restored by transfer of these chromosomes, act through regulation of the hTERT gene expression. Indeed, as described
below, our recent work has shown that an endogenous mechanism restored by a telomerase repressor gene on chromosome 3
acts on a specific DNA element within the hTERT gene promoter to repress the hTERT transcription (27).
Our cell system to investigate an endogenous mechanism for
telomerase repression consists of a telomerase-positive, human
renal cell carcinoma cell line (RCC23) and its telomerasenegative counterpart (RCC23‡3, generated by transfer of a
normal human chromosome 3 into RCC23) that shows progressive telomere shortening associated with cell division and
eventually undergoes senescent growth arrest (55,65). The
lack of telomerase activity in RCC23‡3 is due to lack of
hTERT mRNA expression, which is explained by the transcriptional repression of the hTERT gene (27). By examining
the activity of various deleted or mutated hTERT promoter
fragments, the E-box element downstream of the transcription
initiation site was found to be critical to differential hTERT
transcription between RCC23 and RCC23‡3 (27). This E-box
element-mediated repression of hTERT transcription in
RCC23‡3 but not in RCC23, suggesting that it is a site
where the telomerase repressive mechanism restored by normal chromosome 3 targets (Figure 1). Although this E-box
element is a potential binding site of c-Myc and Mad1 as
described above, expression level or binding activity of the
endogenous c-Myc or Mad1 protein did not appear to be
responsible for the differential hTERT transcription between
RCC23 and RCC23‡3 (27). Importantly, enhancement of the
repression in an E-box copy number-dependent manner in
RCC23‡3 and the presence of a RCC23‡3-specific E-box
binding factor, which appears distinct from Myc/Mad and
USF families, in the EMSA experiment (27) support for the
presence of an E-box-binding, transcriptional repressor that
actively functions in the telomerase-negative RCC23‡3 and
is defective in the telomerase-positive RCC23. It should be
noted that the E-box-mediated repression also functions in
various types of normal human cells including fibroblast,
mammary epithelial cells and prostate epithelial cells, while
it is inactive in some, but not all, telomerase/hTERT-positive
cancer cells (27). These findings provide evidence for an
endogenous mechanism of the hTERT transcriptional repression, which becomes inactivated during carcinogenic processes (e.g. by an inactivation of the telomerase repressor
gene on chromosome 3). Cloning and characterization of the
E-box-binding repressor would greatly facilitate our understanding of the endogenous mechanism by which normal
Transcriptional regulation of the telomerase hTERT
human cells tightly repress the hTERT transcription and therefore the telomerase activity. Of another interest will be to
examine whether the putative telomerase repressor genes on
the other chromosomes also act on the hTERT promoter, and,
if so, whether the same E-box element is involved.
Viral activation of the hTERT gene in trans
It is well known that some virus-encoded proteins contribute to
human cell transformation and carcinogenesis by modulating
cellular signaling pathways that regulate cell-cycle progression, cell proliferation and differentiation, and cell death.
Recent evidence suggests that the activation of telomerase
through transcriptional activation of the hTERT gene is another
way by which human tumor-associated viruses can work.
Human papillomaviruses (HPV), especially type 16 and
type 18, play an important role in immortalization and transformation of human keratinocytes to cause uterine cervical
cancers. The oncogenic activity of HPV is mainly attributed
to its oncoproteins E6 and E7, which can abrogate the tumor
suppressive function of p53 and Rb signaling pathways. We
and others (28,29,66) have revealed that the expression of E6
protein of HPV type 16 upregulates the hTERT promoter
activity in normal human keratinocytes, explaining the previously reported ability of E6 to induce the telomerase activity
in these cells (67). The E-box elements within the hTERT
promoter, especially one downstream of the transcription
initiation site (the same E-box where the above-mentioned,
endogenous repressive mechanism acts), seem to be at least in
part responsible for the E6 activation of the hTERT transcription (28,29, and our unpublished data) (Figure 1). However, as
in the chromosome 3-mediated repression, no evidence was
thus far obtained for a role of either c-Myc or Mad1 protein in
the hTERT activation by E6 (28,29,66). Some of the cellular
proteins known to interact with E6 (e.g. E6TP1 and E6AP) are
suggested to be involved in the hTERT activation by E6
(29,68). Further analyses will be needed to clarify the molecular details and to identify the cellular protein(s) that mediates the E6 action. Interestingly, we have recently found that
another HPV early gene product E2 can directly repress the
hTERT transcription through the Sp1 binding sites within the
hTERT promoter (69) (Figure 1). The HPV E2 protein, which
is essential for episomal replication of the viral DNA in precancerous lesions and is usually lost upon the viral DNA
integration into the cellular genome during progression to
cervical cancers (70,71), may play a role in regulating the
virus's life cycle and transforming ability in the host cells by
directly repressing the hTERT transcription [in addition to the
indirect inhibition through E6 downregulation (72)], as well as
by its well-known functions in replication and transcription of
the viral genome. Although it is clear that the HPV proteins are
the important hTERT regulators for development of uterine
cervical cancers, alterations of cellular factors may be also
required for the full activation of hTERT and telomerase.
Supporting this notion is that human cervical keratinocytes
transduced with the HPV E6/E7 gene show progressive
increases in hTERT and telomerase expression during their
successive passages, despite that the cells keep expressing
the similar amounts of E6/E7 proteins (73). Among these
alterations is likely an inactivation of a telomerase repressor
gene, as suggested by the repression of the hTERT expression
and telomerase activity via MMCT of normal chromosome 6
in the E6/E7-expressing cervical cancer cells (60).
The other examples of viral proteins that trans-activate the
hTERT transcription include: KSHV LANA protein (latencyassociated nuclear antigen of Kaposi's sarcoma-associated
herpesvirus), which appears to target and affect the Sp1 protein
bound to the hTERT promoter (74); and an adenovirus type 12
E1A mutant, which seems to function through a modification
of histone acetylation (75).
Viral activation of the hTERT gene in cis
A classic way by which tumor-associated viruses contribute to
carcinogenesis is to transcriptionally activate cellular genes
with an oncogenic potential (e.g. proto-oncogenes) by the
activity of the regulatory sequences (i.e. promoter or enhancer)
of the viral genome integrated in the cellular genome (76).
This viral cis-activation mechanism, as well as the transactivation mechanism by the virus-encoded proteins as
described above, can target the hTERT gene transcription.
We found that the hepatitis B virus (HBV) genome is integrated in the hTERT promoter region in a hepatocellular carcinoma (HCC) cell line huH-4, in which the hTERT mRNA is
transcribed from both the endogenous promoter and the HBV
promoter (77). The HBV enhancer sequence, located ~1.6 kb
upstream of the hTERT transcription start site, was responsible
for the activation in cis of the hTERT transcription in the HCC
cells (Figure 1). This finding for the first time showed that a
structural alteration in the telomerase component genes
resulted in the activation of telomerase in human cancers,
adding the hTERT gene to the list of cellular targets that are
activated in cis by oncogenic viruses. Moreover, Gozuacik
et al. (78) reported one case (out of 18 HBV-positive HCC
patients) with the HBV genome integrated upstream of the
hTERT gene. Another group found that a primary HCC
tumor (out of 10 cases) and an HCC cell line SNU449 (out
of eight cell lines) had the HBV genome integrated in the
hTERT gene locus (Lewis Roberts et al., personal communication). It is most likely that the hTERT gene is a non-random
integration site of, and a target for cis-activation by, the viral
genome in a subset (at least 5±10%) of HBV-positive HCCs.
Other human tumor-associated viruses also deserve investigation for the integration into the hTERT gene.
These findings have implications in a recent debate on
whether the telomerase is an oncogene. The telomerase `cooperates' with the other factors (e.g. SV40 T antigen and
activated Ras) to transform normal human cells to a fully
malignant state (79,80), thus behaving like an oncogene at
least in a model of human cell transformation with
multiple factors. On the other hand, the telomerase has not
been regarded as an oncogene `in its classical sense' (81),
in that the ectopic expression of telomerase `by itself' in otherwise telomerase-negative, normal human cells can lead to their
unlimited but controlled growth and does not result in
any malignant phenotypes (e.g. lowered serum-requirement,
anchorage-independent growth, and tumorigenicity in
immune-deficient animals) or chromosomal abnormalities
(82,83). [It should be noted that, however, a mutated ras gene
would not be regarded as an oncogene in this sense, either,
because its ectopic expression `by itself' in normal human
cells induces premature senescence instead of any
malignancy-related phenotypes (84)]. One important issue to
be addressed in this debate is whether any of the telomerase
component genes undergoes primary genetic changes
resulting in its over-expression (e.g. rearrangement and
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gene amplification as observed in c-myc) or its functional
activation (e.g. point mutation as observed in ras) during
carcinogenic processes. It is clear that the hTERT gene is
subject to such a genetic alteration directly associated with
cancer-causing agents (in the cases described above, the
genomic rearrangements resulting from the HBV integration
and leading to activation of the hTERT gene expression). In
addition, increased copy number or gene amplification of the
hTERT gene is associated with the hTERT activation in a subset
of human cancers (85,86). These structural and numerical
changes of the hTERT gene observed in human cancers
provide the evidence for a causative, oncogenic role of the
activated telomerase in human carcinogenesis (at least in liver
carcinogenesis).
DNA methylation- and histone acetylation-mediated
regulation of the hTERT expression
Methylation of CpG sites at promoter regions is well known to
be associated with repression of gene expression. The promoter regions of some tumor suppressor genes (e.g. p16INK4a ,
hMLH1 and E-cadherin) become methylated during
tumor development and their repression contributes to tumorassociated phenotypes (e.g. aberrant cell-cycle control, genomic instability and cell invasion/metastasis) (87). The presence
of a cluster of CpG sites (CpG island) in the hTERT promoter
region (16) prompted us to examine a possible role of CpG
methylation in regulation of the hTERT transcription in normal
and cancer cells. By means of a bisulfite genomic sequencing
and a methylation-specific PCR-based assay, we and others
(88,89) found no general correlation between the hTERT
expression and the methylation status either overall or at a
specific site: most of hTERT-negative, normal human cells, as
well as some hTERT-expressing cancer cells, had the
unmethylated or hypomethylated hTERT promoter, while
other cancer cells had the highly methylated promoter. These
findings suggest that normal cells have a mechanism(s) to
tightly repress the hTERT transcription independently of promoter methylation, and that the hTERT gene can be transcribed
from the densely methylated promoter in some cancer cells.
However, in the immortal cell lines with the highly methylated
hTERT promoter and no expression of hTERT transcript and
telomerase activity [their telomeres are maintained through the
`alternative lengthening of telomeres', or ALT (90)], a DNA
demethylating agent 5-aza-20 -deoxycytidine (5-aza-C)
induced demethylation of the hTERT promoter and expression
of the hTERT transcript (88,89), which was enhanced by combination with a HDAC inhibitor trichostatin A (TSA) (88).
This may suggest that the promoter methylation can potentially function to repress the hTERT transcription (Figure 1),
even though such a methylation-mediated mechanism is not
critical to the hTERT repression in normal human cells.
To explain an apparent discrepancy between data from normal cells and the immortal ALT cells, one possible scenario is
that: (i) CpG sites within the hTERT promoter are methylated
during neoplastic development of some (but not all) human
tumors, just like the promoter of tumor suppressor genes (e.g.
p16INK4a , hMLH1 and E-cadherin); (ii) in contrast to the tumor
suppressor genes, the promoter methylation of the hTERT gene
does not contribute to tumor development but instead would
function as a fail-safe mechanism against carcinogenesis by
preventing telomerase-mediated cell immortalization; and (iii)
cancer cells could arise by a mechanism that allows expression
1172
of the hTERT gene from its highly methylated promoter.
Another apparent contrast to a repressive role of the promoter
methylation is that 5-aza-C inhibited (versus induced) the
hTERT expression and telomerase activity in some cancer
and immortal dysplastic cells (91,92). In these cases the
methylation status of the hTERT promoter was not examined,
and the 5-aza-C inhibition of the hTERT expression was likely
an indirect effect that originated from altered expression of
other factors affecting the hTERT transcription [e.g. inhibition
of c-Myc by up-regulation of p16INK4a (91)].
A growing body of evidence suggests that methylation of
DNA and modifications (such as acetylation and methylation)
of nucleosomal histones are physically and functionally linked
and cooperate to regulate chromatin structure and gene transcription (93,94). The enhancement of 5-aza-C-induced
hTERT activation by the HDAC inhibitor TSA, as mentioned
above, indicates that interaction of these epigenetic modifications of DNA and histones also plays a role in regulating the
hTERT transcription (Figure 1). However, status of histone
acetylation may also regulate the hTERT transcription independently of DNA methylation: treatment with TSA of normal
human cells (including normal human fibroblasts confirmed to
have the unmethylated hTERT promoter in refs 88 and 89)
activated the hTERT promoter activity and induced hTERT
mRNA expression (95±98). This activation was associated
with hyperacetylation of histones at the hTERT promoter
(Figure 1). The histone hyperacetylation at the hTERT promoter was also observed with the natural hTERT upregulation in
activated T lymphocytes (97). Thus, chromatin structure associated with the hypoacetylated state of histones seems important for repressing the transcription from the unmethylated
hTERT promoter in normal human cells.
Activators and repressors of the hTERT transcription may
function by controlling chromatin structure at the hTERT gene
locus. Indeed, a few factors have thus far been linked to
regulation of the chromatin structure. Although the Sp1 transcription factor has been identified as a potential activator of
the hTERT transcription based on its ability to activate the
hTERT promoter activity when `overexpressed' in `cancer'
cells (as described above; ref. 24), Hou et al. (97) and Won
et al. (98) have recently provided evidence that the `endogenous' Sp1 is a critical player in the hTERT repression in `normal' fibroblasts and T lymphocytes. In these cells the
endogenous Sp1, as well as its relative Sp3, physically interacts with HDAC and recruits it to the hTERT promoter, resulting in the deacetylation of nucleosomal histones and probably
leading to the repressed chromatin structure at the hTERT gene
(97,98). The dual activity of Sp1 as an activator or a repressor
may be explained by the finding that the Sp1 is also found in a
complex containing the co-activator protein p300 with histone
acetyltransferase (HAT) activity (99,100). Whether Sp1
recruits HDAC or HAT activity to the hTERT promoter may
depend on cellular contexts and/or experimental conditions
(e.g. availability of other proteins interacting with Sp1, normal
versus cancer cells, or endogenous versus over-expressed
Sp1). Further investigations of this issue are essential to understand the chromatin structure-mediated regulation of the
hTERT transcription. The E-box binding activator c-Myc and
repressor Mad1, which compete with each other for the common binding partner Max, also regulate the hTERT transcription through modulating the acetylation status of nucleosomal
histones at the hTERT promoter in proliferating versus differentiated HL60 leukemic cells (31), although it is still to be
Transcriptional regulation of the telomerase hTERT
examined whether the same mode of action by c-Myc and
Mad1 plays a role in transition from the repression in normal
cells to the activation in cancer cells. The endogenous c-Myc/
Max complex binding to the hTERT promoter in the proliferating HL60 cells was associated with the acetylated histones
and the hTERT expression (and therefore telomerase activity),
whereas the complex was replaced by the endogenous Mad1/
Max complex in the differentiated HL60 cells, with the histones deacetylated and the hTERT repressed (31). This is
consistent with the ability of Mad1/Max to recruit a HDACcontaining co-repressor complex to target genes (101). c-Myc
is known to interact with the transformation±transactivation
domain-associated protein (TRRAP), which is part of the
SAGA complex containing a hGCN5 HAT (102). Nikiforov
et al. (103) have recently shown that the recruitment of
TRRAP and its associated HAT activity are required for activation of the hTERT transcription in normal human fibroblasts
by c-Myc over-expression. The transcriptional activation of
hTERT by a viral protein (adenovirus type 12 E1A mutant)
also involves suppression of HDAC activity and recruitment of
the p300/CBP complex with HAT activity (75).
Multiple mechanisms to regulate the hTERT gene
transcription
A variety of mechanisms exist to control transcription of the
hTERT gene, leading to repression or activation of telomerase
activity in normal and cancer cells, respectively (summarized
in Figure 1). Those include the activation by cellular and viral
oncogenic factors, as well as the repression by tumor suppressor proteins and pathways, as described in this article. A
growing number of protein factors are being added to the list
of putative hTERT regulators, including growth factors (e.g.
epidermal growth factor) (34), protein kinases involved in the
mitogen-activated protein kinase signaling pathway (34,104)
and the stress-activated protein kinase signaling pathway
(105), and an oncogenic, Polycomb group repressor Bmi-1
(106), which appear to control the hTERT transcription
through the regulation of known or unknown proteins binding
to the hTERT promoter. It should be also noted that the hTERT
gene promoter is a target of hormonal carcinogenesis in
humans. The estrogen receptor a (ERa) binds to a putative
estrogen response element within the hTERT promoter and
activates the hTERT transcription in a ligand-dependent manner (107±109) (Figure 1). The ERa-induced hTERT activation
is inhibited by progesterone or tamoxifen (104,109,110), in
part explaining their anti-estrogenic effect in treatment of
estrogen-dependent tumors such as breast cancers. The agents
that control telomerase activity through down-regulation
of the hTERT expression include a chemopreventive agent
N-(4-hydroxyphenyl)retinamide (111), a herbal medicinal
complex with anticancer activity (112), and a sphingolipid
C6 -ceramide (113).
The studies in this area have thus far focused mainly on the
promoter region-mediated control of the hTERT transcription.
It is possible that, however, mechanisms involving some other
specific regions or a large chromosomal domain at the hTERT
gene locus also exist. The cloning of the whole, functional
copy of the hTERT gene, covering from 24 kb upstream to 28
kb downstream, in a single BAC (bacterial artificial chromosome) (114) provides a good material containing all putative
regulatory elements, including ones in the introns and 50 and 30
flanking regions. An interesting feature of the hTERT gene is
its chromosomal localization in close proximity to the telomere of chromosome arm 5p (115,116). Our recent finding
(114) suggests that the gene is located 2 Mb away from the
telomere, not as terminal as thought previously (115,116). It is
still interesting to examine whether telomeric heterochromatin
plays a role in the hTERT repression through a mechanism
called `telomere position effect' (117) and whether the critically short telomere at the end of replicative capacity of normal human cells (at `crisis' stage, from which a rare population
of telomerase-expressing, immortal cells emerges) releases the
hTERT gene from the `telomere position effect' and leads to
the induction of, or the competence for, the hTERT expression.
Conclusion and perspectives
A growing number of findings have defined the hTERT gene as
an important target for a variety of cellular and viral oncogenic
mechanisms. Some of the candidate regulators may represent
true `physiological' and `endogenous' regulators to repress or
activate the hTERT transcription, but others may not. Much of
our current knowledge about the hTERT transcriptional regulation is based on the analyses of over-expressed and/or recombinant proteins rather than the endogenous ones. As shown for
c-Myc, physiological target genes of a transcription factor can
be different from genes affected when it is over-expressed
(118). Cells used in experimental studies are not always appropriate to test a biological significance of candidate hTERT
regulators in the telomerase activation during human carcinogenesis: for example, study on a candidate activator should
include telomerase-negative, normal and/or pre-malignant
cells, rather than only using cancer cells whose telomerase
activity is already sufficient for stable telomere maintenance;
and activity of a candidate repressor should be tested in cancer
cells that have a verified defect in the candidate factor (e.g.
inactivating mutation or loss of expression). In order to examine whether an endogenously expressed protein (rather than
the exogenously over-expressed one) is responsible for regulating the hTERT transcription, inhibition of candidate activators and repressors, e.g. by RNA interference (32), should be
carried out in hTERT-expressing cancer cells and hTERTnegative normal/pre-malignant cells, respectively. Binding to
the endogenous hTERT regulatory sequences by chromatin
immunoprecipitation assay (31,98) would validate results
from EMSA or other in vitro DNA binding experiments and
transient transfection-based reporter gene assays. After this
initial stage of study on the hTERT transcriptional regulation,
it is now time to focus on endogenous factors of physiological
and pathological significance and to establish functional links
among apparently fragmentary findings thus far obtained.
We propose that an hTERT transcriptional regulator with
biological relevance to physiology of normal cells and pathology of cancer cells should: (i) exhibit activity at physiological
levels; (ii) act on the promoter or regulatory region of the
endogenous hTERT gene; and (iii) show alterations during
transition from normal to cancer cells in the factor or its
regulatory pathway. To our best knowledge, none of the factors described in this article have met all of these criteria.
Further biochemical and functional analyses of the known
candidates for the hTERT activators and repressors are
required, as well as searches for endogenous proteins that
bind the DNA elements critical to hTERT activation or repression, including an unidentified MT-box-binding factor in ovarian cancers (119,120) and an unidentified E-box-binding
1173
I.Horikawa and J.C.Barrett
factor involved in the hTERT repression by chromosome 3
transfer (27). Characterization of such regulators should clarify the critical mechanisms of telomerase regulation in normal
versus cancer cells, and possibly during development and
differentiation in humans, and may provide a basis for a
novel, telomerase-based strategy for anticancer therapy.
Acknowledgements
We thank Drs Ettore Appella, Sharlyn Mazur, Dimiter Dimitrov, Xiaodong
Xiao, Vladimir Larionov, Sun-Hee Leem (National Cancer Institute), Cindy
Afshari, LouAnn Cable, Theodora Devereux (National Institute of
Environmental Health Sciences), Richard Schlegel, Tim Veldman
(Georgetown University Lombardi Cancer Center), Hak-Zoo Kim, Joonho
Choe (Korea Advanced Institute of Science and Technology), HansChristophe Kirch, Bertram Opalka (Universitatsklinikum Essen), Mitsuo
Oshimura and Hiromi Tanaka (Tottori University) for fruitful collaborations.
We also thank Drs Lewis Roberts, Matthew Ferber and David Smith (Mayo
Clinic) for sharing their unpublished data.
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Received March 11, 2003; revised April 28, 2003; accepted May 4, 2003