hTERT Gene Promoter Research Articles

Down-regulation of hTERT expression plays an important role in 15-deoxy-Δ12,14-prostaglandin J2-induced apoptosis in cancer cells

The cyclopentenone prostaglandin 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) has been shown to possess antineoplastic activity in human cancers of various origins. However, the mechanism of the antineoplastic activity of 15d-PGJ2 remains to be completely elucidated. It has been reported that inhibiting the expression of human telomerase reverse transcriptase (hTERT), a major determinant of telomerase activity, induces rapid apoptosis in cancer cells. In this study, we investigated the effect of 15d-PGJ2 on hTERT expression. Treatment with 30 microM 15d-PGJ2 for 72 h induced apoptosis in the colon cancer cells LS180. 15d-PGJ2 treatment decreased hTERT protein expression in a dose-dependent manner. Down-regulation of hTERT expression by hTERT-specific small inhibitory RNA induced apoptosis. These results indicate that the down-regulation of hTERT expression by 15d-PGJ2 plays an important role in its proapoptotic properties. Since 15d-PGJ2 reduced hTERT mRNA expression, we examined the effect of 15d-PGJ2 on the DNA-binding activity of c-Myc, specificity protein 1 (Sp1) and estrogen receptor (ER) to the hTERT gene promoter using an electrophoretic mobility shift assay. 15d-PGJ2 attenuated the DNA-binding of all three transcriptional factors. Further, we observed that 15d-PGJ2 inhibited the DNA binding of these factors by different mechanisms; suppressed c-Myc mRNA expression, enhanced Sp1 protein degradation via the ubiquitin-proteasome pathway and inhibited ERbeta phosphorylation at serine residues. We conclude that hTERT down-regulation by 15d-PGJ2 plays an important role in its proapoptotic properties. Furthermore, 15d-PGJ2 inhibits the transcriptional activity of c-Myc, Sp1 and ER by three different mechanisms and results in the transcriptional repression of the hTERT gene.

Retraction

Human telomerase reverse transcriptase (hTERT) is central to maintain telomeres for continuous cell proliferation, but it remains unknown how extracellular cues regulate telomerase maintenance of telomeres. Here we report that the cytokine bone morphogenetic protein-7 (BMP7) induces Smad3 phosphorylation, nuclear translocation, and hTERT gene repression. BMP7 induces Smad3-dependent telomerase inhibition in a time- and concentration-dependent manner in breast cancer cells. Chronic exposure of breast cancer cells to BMP7 results in short telomeres, cell senescence, and apoptosis. Mutation of BMPRII receptor, but not TGFbetaRII, ACTRIIA, or ACTRIIB, blocked BMP7-induced repression of the hTERT gene promoter activity, leading to increased telomerase activity, lengthened telomeres, and continued cell proliferation. Expression of hTERT inhibits BMP7-induced breast cancer cell senescence and apoptosis. Thus, our data suggest that BMP7 induces breast cancer cell aging and death by a mechanism involving inhibition of telomerase activity and telomere maintenance via BMPRII receptor- and Smad3-mediated repression of the hTERT gene.

Ets2 Maintains hTERT Gene Expression and Breast Cancer Cell Proliferation by Interacting with c-Myc

Human telomerase reverse transcriptase (hTERT) underlies cancer cell immortalization, and the expression of hTERT is regulated strictly at the gene transcription. Here, we report that transcription factor Ets2 is required for hTERT gene expression and breast cancer cell proliferation. Silencing Ets2 induces a decrease of hTERT gene expression and increase in human breast cancer cell death. Reconstitution with recombinant hTERT rescues the apoptosis induced by Ets2 depression. In vitro and in vivo analyses show that Ets2 binds to the EtsA and EtsB DNA motifs on the hTERT gene promoter. Mutation of either Ets2 binding site reduces the hTERT promoter transcriptional activity. Moreover, Ets2 forms a complex with c-Myc as demonstrated by co-immunoprecipitation and glutathione S-transferase pulldown assays. Immunological depletion of Ets2, or mutation of the EtsA DNA motif, disables c-Myc binding to the E-box, whereas removal of c-Myc or mutation of the E-box also compromises Ets2 binding to EtsA. Thus, hTERT gene expression is maintained by a mechanism involving Ets2 interactions with the c-Myc transcription factor and the hTERT gene promoter, a protein-DNA complex critical for hTERT gene expression and breast cancer cell proliferation.

Construction of a Regulatable Cancer-Specific Adenoviral Expression System Using Human Telomerase Reverse Transcriptase Gene Promoter

We constructed a novel cancer-specific regulatable adenoviral expression system comprising two vectors: one expressing rtTA, a reverse tetracycline transactivator regulated by the human telomerase reverse transcriptase (hTERT) gene promoter, the other expressing the target gene regulated by the tetracycline response element (TRE). rtTA transactivates target gene expression in the presence of doxycycline. Using these vectors, we constructed an adenoviral expression system, the Tel-On system, which enables highly efficient target gene transduction. This system enabled efficient and regulatable cancer-specific gene expression, and can be used in targeted cancer gene therapy.

Radio-activation of hTERT promoter in larynx squamous carcinoma cells: An ‘indirected-activator’ strategy in radio-gene-therapy

The usefulness of human telomerase reverse transcriptase (hTERT) gene promoter has been proposed in cancer-targeted gene therapy. However, this promoter may not be strong enough to achieve therapeutic levels of transgene expression. In this study, we tested an 'indirected-activator' strategy that utilizes radiation to increase the activity of the hTERT gene promoter. We demonstrated that hTERT may participate in the process of DNA repair induced by irradiation. We found that Zidovudine (AZT, an hTERT inhibitor) can decrease the telomerase activity in human HEp-2 larynx squamous carcinoma cells and lower the survival fraction of HEp-2 cells exposed to radiation. In HEp-2 cells exposed to 6 Gy-radiation, the hTERT promoter showed 2.9-fold higher activity compared with unirradiated cells. Importantly, an increased expression of enzyme horseradish peroxidase (HRP) controlled by the hTERT promoter was found in the transfected cells after irradiation, which coincided with a higher killing rate for HEp-2 cells after prodrug indole-3-acetic acid (IAA; converted by HRP into a cytotoxin) incubation combined with irradiation or not. Our observations suggest that hTERT promoter-mediated gene therapy could be improved in combination with radiotherapy, which may be due to cellular DNA damage responses.

Mechanisms of Action of TGF‐β in Cancer

Transforming growth factor-beta (TGF-beta) induces cell differentiation and suppresses cell proliferation, but the mechanisms underlying the actions of TGF-beta remain to be fully elucidated. Recent studies suggest that TGF-beta suppresses neoplastic cell development by employing Smad3 protein to repress the gene of human telomerase reverse transcriptase (hTERT). In human breast cancer cells, TGF-beta induces rapid phosphorylation and subsequent entry of Smad3 into the nucleus. In the nucleus, Smad3 binds to the hTERT gene promoter directly and inhibits hTERT gene transcription activity. By interacting with c-myc, Smad3 also represses the c-myc gene. Thus, TGF-beta prevents continuous cell proliferation by switching off telomerase activity through Smad3 repression of the hTERT gene and the action of c-myc. Modulating the interface between Smad3 and the hTERT gene, and the potential feedback loop from telomeres to Smad3 via Smurf2, may represent a novel approach to regulate cell lifespan of proliferation.

Transforming growth factor-β inhibits telomerase through SMAD3 and E2F transcription factors

Cancer arises from multiple genetic changes within the cell, among which constitutive telomerase activity and attainment of immortality are central. Expression of hTERT, the protein component of telomerase, is increased in most cancer cells. Transforming growth factor-β (TGFβ), a potent tumor suppressor, has been reported to regulate hTERT expression. We found that TGFβ represses hTERT expression in normal and cancer cells and that this effect is mediated through Smad3 but also requires Erk1/2, p38 kinase and histone deacetylase activity. Furthermore, we identified four critical E2F transcription factor binding sites within the hTERT gene promoter that confer the TGFβ response. Finally, using the E2F-1 knockout model, we showed that loss of E2F-1 abolishes TGFβ inhibition of telomerase expression. These findings highlight the prominent role of TGFβ in regulating telomerase expression and identify Smad3 and E2F-1 as critical mediators of TGFβ effects in both normal and cancer cells.

Novel Functional Single Nucleotide Polymorphisms in the Latent Transforming Growth Factor-β Binding Protein-1L Promoter: Effect on Latent Transforming Growth Factor-β Binding Protein-1L Expression Level and Possible Prognostic Significance in Ovarian Cancer

Latent transforming growth factor (TGF)binding proteins (LTBPs) play important roles in the secretion and activation of TGF. We previously reported that LTBP-1L is overexpressed in some patients with ovarian cancer. To clarify the molecular mechanism of LTBP-1L regulation, we analyzed DNA sequences in the promoter region of LTBP-1L and identified two novel single nucleotide polymorphisms, 202G/C and 20A/C. While the alleles with 202C and 20C were initially reported, our data demonstrated that 202G and 20A are common in both ovarian cancer patients and healthy patients in the Japanese population. Luciferase reporter assays revealed that the G-A haplotype induced transcriptional activation in a Sp1-dependent manner. Electrophoretic mobility shift assays showed that increased binding affinity of Sp1 to the promoter with 202G and 20A. Interestingly , ovarian cancer patients (n 42) with G-A/G-A homozygous genotype had increased expression of LTBP-1 and apparently poorer survival than those with other genotypes (P 0.02). These findings suggest that the single nucleotide polymorphisms 202G/C and 20A/C on the LTBP-1L promoter may affect the clinical outcome of ovarian cancer patients , probably via up-regulating protein expression. Further studies using a larger number of samples will definitively determine the correlation between LTBP-1 haplotype and clinical behavior of ovarian cancer. (J Mol Diagn 2006, 8:342–350; DOI: 10.2353/jmoldx.2006.050133) Transforming growth factor (TGF)is a potent growth inhibitor for most cell types, including epithelial cells. The secretion of TGFby tumor cells may contribute to tumor suppression by autocrine growth inhibition, but on the other hand, it may also promote tumor progression by stimulating tumor invasion and angiogenesis, and inhibiting immune response. TGFis synthesized as latent high-molecular weight complexes, composed of TGF, the NH2-terminal part of the TGFprecursor, and the latent TGFbinding protein (LTBP). LTBP is a glycoprotein with a molecular weight of more than 190 kd; it possesses 16 to 18 epidermal growth factor (EGF)-like domains and several repeats of a unique motif containing eight cysteine residues. Four isoforms of LTBP have been found in mammalian species (LTBP-1 to LTBP4). LTBP-1 binds to TGF1 through one of the eight cysteine motifs and facilitates assembly and secretion and activation of TGF1. Immunoelectronmicroscopic observations indicate that LTBP-1 is one of the extracellular microfibrillar components; it targets TGF1 to extracellular structures and participates in the activation of latent TGF1, perhaps by concentrating latent TGF1 on the cell surface where activation occurs. LTBP-1S (short) and LTBP-1L (long) are derived from independent promoters and alternative splicing between codons 145 and 146 of LTBP-1S. LTBP-1L has a N-terminal extension of 346 amino acids that is not found in the LTBP-1S. The N-terminal extension contains an EGF-like domain that facilitates matrix incorporation, and LTBP-1L has been confirmed to associate more efficiently than LTBP-1S with the extracellular matrix.

Ets2 binding site single nucleotide polymorphism at the hTERT gene promoter – effect on telomerase expression and telomere length maintenance in non-small cell lung cancer

The aim of this study was to elucidate the occurrence of DNA sequence changes in the promoter region of hTERT gene, and its effect on telomerase expression and telomere length maintenance in non-small cell lung cancer (NSCLC). Between January 2002 and December 2003, 66 NSCLC patients were studied. The expression of hTERT, telomerase activity (TA), and c-Myc were examined, and the terminal restriction fragment length (TRFL) was measured. A t/n-TRFLR was obtained by dividing the TRFL of the tumour tissue by TRFL of the paired normal tissue. PCR products were sequenced and compared with known hTERT gene promoter sequence for a length of 716 bp upstream of the transcription starting code. The changes of any known sequence and/or c-Myc expression with their impact on telomerase activity and TRFL maintenance were measured. Positive hTERT, TA and c-Myc expression was observed in 43 (65.2%), 39 (59.1%) and 59 (89.4%) of the tumour tissue samples, respectively. Except for one patient who had C/C (in normal tissue) homozygotes to T/C (in tumour tissue) heterozygotes point mutation, a novel single nucleotide polymorphism (SNP) −245kb upstream ( Ets2 binding site) of the hTERT gene was observed in all normal and tumour tissues, including C/C in 9, T/C in 35, and T/T in 22 of the tumour tissues. The TA of C/C homozygotes was lower than that of T/T homozygotes (P = 0.0331), while the t/n-TRFLR of C/C homozygotes was higher than that of T/T homozygotes (P = 0.0621). The latter was even more obvious when c-Myc were positive (P = 0.0185). Our data shows that T/T homozygotes have a lower t/n-TRFLR, but a stronger TA expression, suggesting that the studied Ets2 binding site is a positive regulator of hTERT gene. SNP may interfere with Ets2 binding and lower TA expression in T/C heterozygotes and C/C homozygotes.

Hypoxic regulation of telomerase gene expression by transcriptional and post-transcriptional mechanisms

Basal telomerase activity is dependent on expression of the hTERT and hTR genes and upregulation of telomerase gene expression is associated with tumour development. It is therefore possible that signal transduction pathways involved in tumour development and features of the tumour environment itself may influence telomerase gene regulation. The majority of solid tumours contain regions of hypoxia and it has recently been demonstrated that hypoxia can increase telomerase activity by mechanisms that are still poorly defined. Here, we show that hypoxia induces the transcriptional activity of both hTR and hTERT gene promoters. While endogenous hTR expression is regulated at the transcriptional level, hTERT is subject to regulation by alternative splicing under hypoxic conditions, which involves a switch in the splice pattern in favour of the active variant. Furthermore, analysis of the chromatin landscape of the telomerase promoters reveals dynamic recruitment of a transcriptional complex involving the hypoxia-inducible factor-1 transcription factor, p300, RNA polymerase II and TFIIB, to both promoters during hypoxia, which traffics along and remains associated with the hTERT gene as transcription proceeds. These studies show that hTERT and hTR are subject to similar controls under hypoxia and highlight the rapid and dynamic regulation of the telomerase genes in vivo.

The hTERT and hTERC Telomerase Gene Promoters Are Activated by the Second Exon of the Adenoviral Protein, E1 A, Identifying the Transcriptional Corepressor CtBP as a Potential Rearessor of Both Genes

Telomerase plays a role in the unlimited replicative capacity of the majority of cancer cells and provides a potential anticancer target. The regulation of telomerase is complex but transcriptional control of its two essential components, hTERC (RNA component) and hTERT (reverse transcriptase component), is of major importance. To investigate this further, we have used the adenoviral protein, E1A, as a tool to probe potential pathways involved in the control of telomerase transcription. The second exon of the adenoviral protein E1A activates both telomerase gene promoters in transient transfections. The corepressor, C terminal binding protein, is one of only two proteins known to bind to this region, and we propose that E1A activates both promoters by sequestering CtBP, thereby relieving repression. Activation by exon 2 of E1A involves the SP1 sites in both promoters, and consistent with this, SP1 and CtBP interact in coimmunoprecipitation studies. Modulation of the chromatin environment has been implicated in the regulation of hTERT transcription and appears to involve the SP1 sites. CtBP can be found within a histone-modifying complex and it is possible that a CtBP complex, associating with the SP1 sites, represses transcription from the telomerase promoters by modifying chromatin structure.

Adenovirus-mediated HSV-TK gene therapy using the human telomerase promoter induced apoptosis of small cell lung cancer cell line

Telomerase is a ribonucleoprotein complex the function of which is to add telomeric repeats (TTAGGG)n to chromosomal ends, and it is known to play an important role in cellular immortalization. Telomerase is highly active in most tumor cells, but not in normal cells. As such, it may have possible applications in cancer gene therapy. Telomerase consists of two essential components, telomerase RNA template (hTR) and catalytic subunit (hTERT). hTERT is expressed only in cells and tissues positive for telomerase activity, i.e., tumor and fetal cells. We tested the possibility of the utilization of the hTERT promoter in targeted cancer gene therapy. We cloned the hTERT promoter in the place of the CMV promoter and sub-cloned HSV-TK gene to be controlled by hTERT gene promoter in adenovirus shuttle plasmid. Then we constructed recombinant adenovirus Ad-hT-TK, and infected them into a normal and a small cell lung cancer cell line. Through these experiments, we identified the selective tumor specific cell death by Ad-hT-TK. Furthermore, cell death detection ELISA and FACS analysis suggest that the reduced viability is mediated through the induction of apoptosis, indicating that this approach may be a useful method for suppressing cancer growth in targeted cancer gene therapy. These results show that Ad-hT-TK could be used for SCLC gene therapy.

Combination of caspase transfer using the human telomerase reverse transcriptase promoter and conventional therapies for malignant glioma cells

Recently, we have reported the therapeutic efficacy of delivering initiator caspase (caspase-8) or executioner active caspase (rev-caspase-6) to telomerase-positive malignant glioma cells using the human telomerase reverse transcriptase (hTERT) gene promoter system (hTERT/caspase-8 or hTERT/rev-caspase-6). In the present study, we investigated if conventional treatments for malignant gliomas augment the efficacy of the hTERT/caspase therapy. First, we demonstrated that hTERT/rev-caspase-6 exhibited a greater ability to induce apoptosis in malignant glioma U87-MG and U373-MG cells than hTERT/caspase-8. Next, as conventional treatments to combine with hTERT/rev-caspase-6, apoptosis-inducing agents [cisplatin (CDDP), paclitaxel (PTX), and BCNU] and non-apoptosis-inducing therapies [temozolomide (TMZ) and gamma-irradiation (IR)] were used. Combination of hTERT/rev-caspase-6 gene therapy with PTX yielded a dose-dependent additive effect, while CDDP and BCNU had additive effect only when tumor cells were treated at IC75 of each agent. A decline in the combination effect of CDDP and BCNU at IC50 was due to decreased activity of telomerase in treated tumor cells prior to the hTERT/rev-caspase-6 transfer. On the other hand, TMZ or IR had no significant additive effect on induction of apoptosis. These results suggest that agents, which induce apoptosis without inhibiting telomerase activity are a promising counterpart to combine with hTERT/rev-caspase-6 therapy for the management of malignant gliomas.

Telomerase and c-myc expression in hepatocellular carcinomas

Background. Telomerase is activated in the majority of cancer tissues and immortalized cell lines. The hTERT (human telomerase reverse transcriptase—major component of telomerase) gene promoter has been cloned and contains many c-myc binding sites that mediate hTERT transcriptional activation. Thus far, the role of hTERT in tumorigenesis in hepatocellular carcinoma (HCC) has been little studied using RNA in situ hybridization. The relationship between c-myc and telomerase in human HCC tissue is undetermined. Materials and methods. The telomerase activity was assayed using TRAP in specimens from 23 HCC patients, hTERTmRNA was detected using in situ hybridization from 57 HCC patients. The immunohistochemistry for c-myc and DNA sequence for hTERT promoter, and tumour differentiation in relation to hTERT and c-myc expression were determined in 57 specimens. Result. hTERTmRNA was found in 47/57 (82.5%) HCC specimens using in situ hybridization. The hTERT expression paralleled telomerase activity, but hTERTmRNA regulation was not significantly associated with c-myc level ( P<0.954). The DNA sequence analysis of the hTERT promoter in specimens from 17 HCC revealed 15 cases of nucleotide transition (T→C) over 5′-end of distal E-box and one case of nucleotide transversion (G→C) over 5′-end of proximal E-box. Neither the hTERT expression ( P<0.890) nor c-myc level ( P<0.348) were related to HCC differentiation. Conclusions. The hTERT expression paralleled telomerase activity. The telomerase activity in HCC was not only regulated by c-myc. Another pathways might contribute to hTERT and telomerase activity regulation. The lack of telomerase activity in specimens from 17.4% of HCC cases might indicate an alternative pathway for maintaining telomere length. Furthermore, both the telomerase activity and c-myc had no significant role in HCC differentiation.

Activity of the human telomerase catalytic subunit (hTERT) gene promoter could be increased by the SV40 enhancer.

Telomerase is a ribonucleoprotein complex of which the function is to add telomeric repeats to chromosomal ends. Telomerase consists of two essential components, the telomerase RNA template (hTR) and the catalytic subunit (hTERT). hTERT is expressed only in cells and tissues positive for telomerase activity, i.e., tumor and fetal cells. The aim of this study is to test the increased telomerase promoter activity for cancer gene therapy in adenovirus vector. We cloned the hTERT promoter in place of the SV40 promoter in the pGL3-contol vector to be increased by the SV40 enhancer sequences, resulting in strong expression of luc+ only in telomerase positive cancer cells. Then we transfected the constructed plasmid into a normal human cell line and several cancer cell lines. Through these experiments, we identified the selective and increased expression of the luciferase gene controlled by the hTERT promoter and the SV40 enhancer in the telomerase positive cancer cell lines. To investigate the possibility of utilizing the hTERT promoter and the SV40 enhancer in targeted cancer gene therapy, we constructed an adenovirus vector expressing HSV-TK controlled by the hTERT promoter and the SV40 enhancer for the induction of specific telomerase positive cancer cell death. NSCLC cells infected by Ad-hT-TK-enh were more significantly suppressed and induced apoptosis than those infected by Ad-hT-TK. Telomerase is activated in 80 approximately 90% of cancers, so adenovirus with increasing telomerase promoter activity might be used for targeted cancer gene therapy using suicide genes. These results show that the hTERT promoter and the SV40 enhancer might be used for targeted cancer gene therapy.

Demethylation of the human telomerase catalytic subunit ( hTERT) gene promoter reduced hTERT expression and telomerase activity and shortened telomeres

Telomerase is the ribonucleoproteic complex involved in maintaining telomere size. It is expressed in germ and stem cells but not in normal somatic cells. In most tumors, telomerase is reactivated. In humans, telomerase activity is tightly regulated by expression of the hTERT gene. In a previous study, we found a direct correlation between methylation of the hTERT promoter and hTERT gene expression. In order to demonstrate this correlation, demethylation experiments were performed with the demethylating agent 5aza-2′-deoxycytidine (5azadC). Three telomerase-positive tumor cell lines (Lan-1, HeLa, and Co115), presenting a hypermethylated hTERT promoter, were treated with different doses and types of treatment for a long period. Analysis of methylation revealed a final hTERT promoter demethylation up to 95%. Quantification of hTERT mRNA showed that transcription was strongly repressed during drug exposure. In contrast, expression of c-Myc, an activator of hTERT promoter, was barely down-regulated or increased by the treatment. Using a TRAP assay, telomerase activity was semiquantified in all experiments. It strongly decreased or was suppressed after two to four passages. Finally, telomere length was measured by Southern blot. Their averages were not modified, but ranges concentrated around the mean. Thus, it is likely that hTERT promoter hypermethylation would be necessary for its expression.

Adenovirus-mediated suicide gene therapy using the human telomerase catalytic subunit (hTERT) gene promoter induced apoptosis of ovarian cancer cell line.

Telomerase is a ribonucleoprotein complex the function of which is to add telomeric repeats (TTAGGG)(n) to chromosomal ends, and it is known to play an important role in cellular immortalization. Telomerase is highly active in most tumor cells, yet not in normal cells. As such, it may have possible applications in cancer gene therapy. Telomerase consists of two essential components, telomerase RNA template (hTR) and catalytic subunit (hTERT). hTERT is expressed only in cells and tissues positive for telomerase activity, i.e., tumor and fetal cells. We here tested the possibility of the utilization of the hTERT promoter in targeted cancer gene therapy. We cloned the hTERT promoter in the replace of the CMV promoter and sub-cloned HSV-TK gene to be controlled by hTERT gene promoter in adenovirus shuttle plasmid. Then we constructed recombinant adenovirus Ad-hT-TK, and infected them into normal and human gynecological cancer cell lines. Through these experiments, we identified the selective tumor specific cell death by Ad-hT-TK. Furthermore, FACS analysis and TUNEL assay suggests that the reduced viability is mediated through the induction of apoptosis, indicating that this approach may be a useful method for suppressing cancer growth in targeted cancer gene therapy. These results show that Ad-hT-TK could be used for gynecological cancer gene therapy.

Identification and characterization of negative regulatory elements of the human telomerase catalytic subunit (hTERT) gene promoter: possible role of MZF-2 in transcriptional repression of hTERT.

Human telomerase reverse transcriptase (hTERT) is a catalytic subunit of human telomerase and is a critical determinant of the enzymatic activity of telomerase. Expression of hTERT is known to be regulated mainly at the transcriptional level. In the present study, using transient expression assays, we identified a 400 bp silencer of the hTERT promoter between -776 and -378 upstream of the proximal core promoter. The inhibitory effects of this silencer were enhanced with cellular differentiation. A computer-assisted homology search identified multiple binding motifs for myeloid-specific zinc finger protein 2 (MZF-2) within this region. Mutation introduced in these sites resulted in significant activation of hTERT transcription. Gel shift assays demonstrated that MZF-2 proteins specifically bound to these sites. Overexpression of MZF-2 in cells led to down-regulation of hTERT transcription as well as telomerase activity. These findings suggest that the 400 bp region upstream of the hTERT core promoter that we identified functions as a negative regulatory region and that MZF-2 may be an effector of negative regulation of hTERT.

In Vivo and in Vitro Analyses of Myc for Differential Promoter Activities of the Human Telomerase (hTERT) Gene in Normal and Tumor Cells

Induction of the catalytic subunit of human telomerase, hTERT, plays a critical role in the activation of telomerase during tumorigenesis. Here, we isolate the hTERT gene promoter and define the functional promoter region, which is inactive in normal cells but active in tumor cells. Myc directly interacts with the hTERT promoter and activates its transcription both in vivo and in vitro. Activation or repression of Myc can alter hTERT promoter activities in normal or tumor cells. Furthermore, we detect high levels of Myc protein in tumor cells compared with normal cells. The ability of Myc to modulate the activity of the hTERT promoter and, hence, the telomerase enzyme may contribute to its ability to promote tumor formation.