Transcriptional regulation of the telomerase hTERT gene as a target for cellular and viral oncogenic mechanisms
Izumi Horikawa1 and
J. Carl Barrett
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
1 To whom correspondence should be addressed Email: horikawi{at}mail.nih.gov
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Abstract
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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-2'-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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Introduction
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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 (1114). Although various steps at post-transcriptional and post-translational 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 (1113,16). 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 physiological regulators, whose alteration during carcinogenesis plays a causative role in hTERT activation in human cancer cells.
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Known transcription factors as the candidate hTERT transcriptional activators
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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 5'-regulatory region (promoter) of the gene (1722). 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 helixloophelix 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 over-expressed 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 (2729). On the other hand, Xiao et al. (30) recently showed, by means of a PCR-based DNAprotein 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 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.

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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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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.
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Known transcription factors as the candidate hTERT transcriptional repressors
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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 E2F-1 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-ß/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-ß/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-ß 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 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.
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Investigation of endogenous mechanisms for telomerase repression: a telomerase repressor gene on chromosome 3 acts on the hTERT promoter
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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 (5563), 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 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 telomerase-negative 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 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.
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Viral activation of the hTERT gene in trans
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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 pre-cancerous 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 (latency-associated 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).
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Viral activation of the hTERT gene in cis
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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 trans-activation 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 510%) 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 co-operates 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 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).
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DNA methylation- and histone acetylation-mediated regulation of the hTERT expression
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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 tumor-associated 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-2'-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 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 (9598). 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 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 HDAC-containing co-repressor complex to target genes (101). c-Myc is known to interact with the transformationtransactivation 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
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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
(ER
) binds to a putative estrogen response element within the hTERT promoter and activates the hTERT transcription in a ligand-dependent manner (107109) (Figure 1). The ER
-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 5' and 3' 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
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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 hTERT-negative 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 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.
 |
Acknowledgments
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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), Hans-Christophe Kirch, Bertram Opalka (Universitätsklinikum 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.