Identification of a novel transcription factor binding element involved in the regulation by differentiation of the human telomerase (hTERT) promoter.

Three different cell differentiation experimental model systems (human embryonic stem cells, mouse F9 cells, and human HL-60 promyelocytic cells) were used to determine the relationship between the reduction in telomerase activity after differentiation and the regulation of the promoter for the hTERT gene. Promoter constructs of three different lengths were subcloned into the PGL3-basic luciferase reporter vector. In all three experimental systems, all three promoter constructs drove high levels of reporter activity in the nondifferentiated state, with a marked and time-dependent reduction after the induction of differentiation. In all cases, the smallest core promoter construct (283 nt upstream of the ATG) gave the highest activity. Electrophoretic mobility shift assays revealed transcription factor binding to two E-box domains within the core promoter. There was also a marked time-dependent reduction in this binding with differentiation. In addition, a distinct and novel element was identified within the core promoter, which also underwent time-dependent reduction in transcription factor binding with differentiation. Site-directed mutagenesis of this novel element revealed a correlation between transcription factor binding and promoter activity. Taken together, the results indicate that regulation of overall telomerase activity with differentiation is mediated at least in part at the level of the TERT promoter and provides new information regarding details of the regulatory interactions that are involved in this process.

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title: Chromosome first aid
abstract: Most people take the stability of chromosomes for granted. Some may imagine metaphase chromosomes lined up like a butterfly collection on a page, reassuringly in order and unchanging. However, evidence that has been accumulating for over half a century indicates that genome stability is a dynamic, not a static, process. In the 1940s natural chromosomes ends, or telomeres, were shown to be specialized structures inherently different from broken chromosome ends. McClintock demonstrated in maize that telomeres are required for the stable maintenance of linear chromosomes. Random ends generated by breakage fuse to generate dicentric chromosomes that undergo repeated cycles of breakage and fusion. Occasionally, during this cycle of breakage-fusion-bridge, chromosomes would apparently heal; their characteristic stickiness would disappear, and they would be well behaved in subsequent divisions (reviewed in Blackburn and Szostak, 1984). In addition to sporadic healing events, McClintock observed developmentally regulated, tissue-specific chromosome healing. She found that the fate of a single chromatid broken at meiosis depends on both the tissue and the time in the cell cycle at which the broken end is present. When a single broken chromatid is generated at meiosis, rounds of breakage-fusion-bridge occur in the following mitotic divisions. After fertilization, the breakage cycle continues in the endosperm tissue, while in the zygotic tissue the broken end is healed. The healed chromosome is stably propagated throughout subsequent plant development. These observations suggested that an activity that heals broken ends is present in zygotic tissue but absent in the endosperm (McClintock, 1939, 1942). In the past ten years there has been an explosive increase in our understanding of the molecular structure and function of telomeres. However, only very recently has the process of chromosome healing, initially documented over 50 years ago, been understood at the molecular level. Telomere Structure and Synthesis Telomeric sequences, first characterized in ciliates, are remarkably conserved throughout evolution. Functional telomeres consist of many tandem repeats of simple G-rich sequences. In Tetrahymena there are approximately 70 (TTGGGG) repeats, while in humans there are more than 600 (TTAGGG) repeats found at all chromosome ends. The number of repeats on any given chromosome varies, giving telomeric restriction fragments a characteristic fuzzy appearance on Southern blots. Because most DNA polymerases are not able to replicate completely the molecular end of a DNA molecule, the mechanism of telomere replication was the topic of speculation for many years (reviewed in Blackburn and Szostak, 1984). However, the discovery of the enzyme telomerase in Tetrahymena presented a simple explanation for both the structure and Minireview

title: Telomere end-replication problem and cell aging.
abstract: Since DNA polymerase requires a labile primer to initiate unidirectional 5'-3' synthesis, some bases at the 3' end of each template strand are not copied unless special mechanisms bypass this "end-replication" problem. Immortal eukaryotic cells, including transformed human cells, apparently use telomerase, an enzyme that elongates telomeres, to overcome incomplete end-replication. However, telomerase has not been detected in normal somatic cells, and these cells lose telomeres with age. Therefore, to better understand the consequences of incomplete replication, we modeled this process for a population of dividing cells. The analysis suggests four things. First, if single-stranded overhangs generated by incomplete replication are not degraded, then mean telomere length decreases by 0.25 of a deletion event per generation. If overhangs are degraded, the rate doubles. Data showing a decrease of about 50 base-pairs per generation in fibroblasts suggest that a full deletion event is 100 to 200 base-pairs. Second, if cells senesce after 80 doublings in vitro, mean telomere length decreases about 4000 base-pairs, but one or more telomeres in each cell will lose significantly more telomeric DNA. A checkpoint for regulation of cell growth may be signalled at that point. Third, variation in telomere length predicted by the model is consistent with the abrupt decline in dividing cells at senescence. Finally, variation in length of terminal restriction fragments is not fully explained by incomplete replication, suggesting significant interchromosomal variation in the length of telomeric or subtelomeric repeats. This analysis, together with assumptions allowing dominance of telomerase inactivation, suggests that telomere loss could explain cell cycle exit in human fibroblasts.

title: Telomeres shorten during ageing of human fibroblasts
abstract: THE terminus of a DNA helix has been called its Achilles' heel1. Thus to prevent possible incomplete replication2 and instability3,4 of the termini of linear DNA, eukaryotic chromosomes end in characteristic repetitive DNA sequences within specialized structures called telomeres5. In immortal cells, loss of telomeric DNA due to degradation or incomplete replication is apparently balanced by telomere elongation610, which may involve de novo synthesis of additional repeats by a novel DNA polymerase called telomerase1114. Such a polymerase has been recently detected in HeLa cells15. It has been proposed that the finite doubling capacity of normal mammalian cells is due to a loss of telomeric DNA and eventual deletion of essential sequences1,16,17. In yeast, the est1 mutation causes gradual loss of telomeric DNA and eventual cell death mimicking senescence in higher eukaryotic cells17. Here, we show that the amount and length of telomeric DNA in human fibroblasts does in fact decrease as a function of serial passage during ageing in vitro and possibly in vivo. It is not known whether this loss of DNA has a causal role in senescence.

title: Telomere loss: mitotic clock or genetic time bomb?
abstract: The Holy Grail of gerontologists investigating cellular senescence is the mechanism responsible for the finite proliferative capacity of somatic cells. In 1973, Olovnikov proposed that cells lose a small amount of DNA following each round of replication due to the inability of DNA polymerase to fully replicate chromosome ends (telomeres) and that eventually a critical deletion causes cell death. Recent observations showing that telomeres of human somatic cells act as a mitotic clock, shortening with age both in vitro and in vivo in a replication dependent manner, support this theory's premise. In addition, since telomeres stabilize chromosome ends against recombination, their loss could explain the increased frequency of dicentric chromosomes observed in late passage (senescent) fibroblasts and provide a checkpoint for regulated cell cycle exit. Sperm telomeres are longer than somatic telomeres and are maintained with age, suggesting that germ line cells may express telomerase, the ribonucleoprotein enzyme known to maintain telomere length in immortal unicellular eukaryotes. As predicted, telomerase activity has been found in immortal, transformed human cells and tumour cell lines, but not in normal somatic cells. Telomerase activation may be a late, obligate event in immortalization since many transformed cells and tumour tissues have critically short telomeres. Thus, telomere length and telomerase activity appear to be markers of the replicative history and proliferative potential of cells; the intriguing possibility remains that telomere loss is a genetic time bomb and hence causally involved in cell senescence and immortalization.

title: Telomere length predicts replicative capacity of human fibroblasts.
abstract: When human fibroblasts from different donors are grown in vitro, only a small fraction of the variation in their finite replicative capacity is explained by the chronological age of the donor. Because we had previously shown that telomeres, the terminal guanine-rich sequences of chromosomes, shorten throughout the life-span of cultured cells, we wished to determine whether variation in initial telomere length would account for the unexplained variation in replicative capacity. Analysis of cells from 31 donors (aged 0-93 yr) indicated relatively weak correlations between proliferative ability and donor age (m = -0.2 doubling per yr; r = -0.42; P = 0.02) and between telomeric DNA and donor age (m = -15 base pairs per yr; r = -0.43; P = 0.02). However, there was a striking correlation, valid over the entire age range of the donors, between replicative capacity and initial telomere length (m = 10 doublings per kilobase pair; r = 0.76; P = 0.004), indicating that cell strains with shorter telomeres underwent significantly fewer doublings than those with longer telomeres. These observations suggest that telomere length is a biomarker of somatic cell aging in humans and are consistent with a causal role for telomere loss in this process. We also found that fibroblasts from Hutchinson-Gilford progeria donors had short telomeres, consistent with their reduced division potential in vitro. In contrast, telomeres from sperm DNA did not decrease with age of the donor, suggesting that a mechanism for maintaining telomere length, such as telomerase expression, may be active in germ-line tissue.

title: Specific association of human telomerase activity with immortal cells and cancer.
abstract: Synthesis of DNA at chromosome ends by telomerase may be necessary for indefinite proliferation of human cells. A highly sensitive assay for measuring telomerase activity was developed. In cultured cells representing 18 different human tissues, 98 of 100 immortal and none of 22 mortal populations were positive for telomerase. Similarly, 90 of 101 biopsies representing 12 human tumor types and none of 50 normal somatic tissues were positive. Normal ovaries and testes were positive, but benign tumors such as fibroids were negative. Thus, telomerase appears to be stringently repressed in normal human somatic tissues but reactivated in cancer, where immortal cells are likely required to maintain tumor growth.

title: Telomerase and cancer: revisiting the telomere hypothesis.
abstract: Telomerase is a ribonucleoprotein DNA polymerase that elongates telomeres in eukaryotes. The telomere hypothesis implicates short telomere length and telomerase activation as critical players in cellular immortalization and cancer. In this review, we refine the original telomere hypothesis to address the results of recent studies on telomerase and telomere length regulation.

title: Evidence for a mitotic clock in human hematopoietic stem cells: loss of telomeric DNA with age.
abstract: The proliferative life-span of the stem cells that sustain hematopoiesis throughout life is not known. It has been proposed that the sequential loss of telomeric DNA from the ends of human chromosomes with each somatic cell division eventually reaches a critical point that triggers cellular senescence. We now show that candidate human stem cells with a CD34+CD38lo phenotype that were purified from adult bone marrow have shorter telomeres than cells from fetal liver or umbilical cord blood. We also found that cells produced in cytokine-supplemented cultures of purified precursor cells show a proliferation-associated loss of telomeric DNA. These findings strongly suggest that the proliferative potential of most, if not all, hematopoietic stem cells is limited and decreases with age, a concept that has widespread implications for models of normal and abnormal hematopoiesis as well as gene therapy.

title: Tales of tails: regulation of telomere length and telomerase activity during lymphocyte development, differentiation, activation, and aging
abstract: Telomerase activity and the regulation of telomere length are factors which have been implicated in the control of cellular replication. These variables have been examined during human lymphocyte development, differentiation, activation, and aging. It was found that telomere length of peripheral blood CD4+ T cells decreases with age as well as with differentiation from naive to memory cells in vivo, and decreases with cell division in vitro. These results provide evidence that telomere length correlates with lymphocyte replicative history and residual replicative potential. In contrast, telomere length appears to increase during tonsil B‐cell differentiation and germinal center (GC) formation in vivo. It was also found that telomerase activity is highly regulated during T‐cell development and B‐cell differentiation in vivo, with high levels of telomerase activity expressed in thymocytes and GC B cells, and low levels of telomerase activity in resting mature peripheral blood lymphocytes. Finally, resting lymphocytes retain the ability to upregulate telomerase activity upon activation, and this capacity does not appear to decline with age. Although the precise role of telomerase in lymphocyte function remains to be elucidated, telomerase may contribute to protection from telomere shortening in T and B lymphocytes, and may thus play a critical role in lymphocyte development, differentiation and activation. The future study of study telomerase and its regulation of telomere length may enhance our understanding of bow the replicative lifespan is regulated in lymphocytes.

title: Constitutive and regulated expression of telomerase reverse transcriptase (hTERT) in human lymphocytes.
abstract: Human telomerase consists of two essential components, telomerase RNA template (hTER) and telomerase reverse transcriptase (hTERT), and functions to synthesize telomere repeats that serve to protect the integrity of chromosomes and to prolong the replicative life span of cells. Telomerase activity is expressed selectively in germ-line and malignant tumor cells but not in most normal human somatic cells. As a notable exception, telomerase is expressed in human lymphocytes during development, differentiation, and activation. Recent studies have suggested that regulation of telomerase is determined by transcription of hTERT but not hTER. The highly regulated expression of telomerase in lymphocytes provides an opportunity to analyze the contribution of transcriptional regulation of hTERT and hTER. We report here an analysis of hTERT expression by Northern and in situ hybridization. It was found that hTERT mRNA is expressed at detectable levels in all subsets of human lymphocytes isolated from thymus, tonsil, and peripheral blood, regardless of the status of telomerase activity. hTERT expression is regulated as a function of lineage development, differentiation, and activation. Strikingly, however, telomerase activity in these cells is not correlated strictly with the levels of hTERT and hTER transcripts. The absence of correlation between telomerase activity and hTERT mRNA could not be attributed to the presence of hTERT splice variants or to detectable inhibitors of telomerase activity. Thus, transcriptional regulation of hTERT is not sufficient to account for telomerase activity in human lymphocytes, indicating a likely role of posttranscriptional factors in the control of enzyme function.

title: Developmental regulation of telomerase activity in human fetal tissues during gestation.
abstract: Telomerase is a ribonucleoprotein that adds hexanucleotide repeats (telomeres) to the ends of linear chromosomes, compensating for the loss of telomeric DNA which occurs with DNA replication. In humans, telomerase has been previously detected in germ-line tissues, blastocysts, 16-20 week old fetal tissue, and most cancers, but not in mature sperm or ova, or in most normal somatic tissues. It has been hypothesized that telomerase is suppressed during somatic development and reactivated in malignancy. To test the hypothesis that telomerase is suppressed during somatic development, human fetal tissues of 8-21 weeks gestational age were assayed for telomerase activity. All tissues expressed telomerase at the earliest ages examined. Lung, liver, spleen, and testis maintained telomerase activity through the latest age assayed, namely 21 weeks. Brain and kidney telomerase activity was present up to the 16th week and was undetectable thereafter. Heart tissue did not display activity beyond the 12th week. Lysates of heart, brain, and kidney without telomerase activity did not inhibit the activity of known telomerase-positive cells, suggesting that suppression of telomerase activity during gestational development is due to a lack of active telomerase rather than to the presence of an inhibitor. These findings demonstrate tissue-specific and developmental regulation of telomerase in the human fetus, suggesting an important role for this ribonucleoprotein in human fetal tissue differentiation and development.

title: Synchrony in telomere length of the human fetus
abstract: Telomere length, measured by terminal restriction fragments, was examined in tissues from human fetuses of gestational ages estimated as 15–19 weeks. The length of telomeres was similar in most fetal tissues. However, there were significant variations in telomere length among fetuses, with no apparent relationship between gestational age and telomere length. We conclude that synchrony in telomere length exists among tissues of the human fetus. This synchrony is apparently lost during extrauterine life.

title: Differentiation of immortal cells inhibits telomerase activity.
abstract: Telomerase, a ribonucleic acid-protein complex, adds hexameric repeats of 5'-TTAGGG-3' to the ends of mammalian chromosomal DNA (telomeres) to compensate for the progressive loss that occurs with successive rounds of DNA replication. Although somatic cells do not express telomerase, germ cells and immortalized cells, including neoplastic cells, express this activity. To determine whether the phenotypic differentiation of immortalized cells is linked to the regulation of telomerase activity, terminal differentiation was induced in leukemic cell lines by diverse agents. A pronounced downregulation of telomerase activity was produced as a consequence of the differentiated status. The differentiation-inducing agents did not directly inhibit telomerase activity, suggesting that the inhibition of telomerase activity is in response to induction of differentiation. The loss of telomerase activity was not due to the production of an inhibitor, since extracts from differentiated cells did not cause inhibition of telomerase activity. By using additional cell lineages including epithelial and embryonal stem cells, down-regulation of telomerase activity was found to be a general response to the induction of differentiation. These findings provide the first direct link between telomerase activity and terminal differentiation and may provide a model to study regulation of telomerase activity.

title: Lack of cell cycle regulation of telomerase activity in human cells.
abstract: Conflicting reports have appeared concerning the cell cycle regulation of telomerase activity and its possible repression during quiescence and cell differentiation. We have reexamined these issues in an attempt to uncover the basis for the discrepancies. Variations in extracted telomerase activity during the cell cycle are not observed in cells sorted on the basis of DNA content. Variations are observed in cells synchronized using some biochemical cell cycle inhibitors, but only with those agents where cellular toxicity is evident. A progressive decline in telomerase activity is observed in cells whose growth rate is reduced from seven to eight population doublings per week to one to two doublings per week. Telomerase is largely absent in cells that truly exit the cell cycle and do not divide over the 7-day period. Although it is not necessary for all cell types to regulate telomerase in the same way, we conclude that in the immortal cultured cell lines examined, extracted telomerase activity does not change significantly during progression through the stages of the cell cycle. Telomerase activity generally correlates with growth rate and is repressed in cells that exit the cell cycle and become quiescent.

title: Repression of the telomerase catalytic subunit by a gene on human chromosome 3 that induces cellular senescence
abstract: The cellular senescence program is controlled by multiple genetic pathways, one of which involves the regulation of telomerase and telomere shortening. The introduction of a normal human chromosome 3 into the human renal cell carcinoma cell line RCC23 caused repression of telomerase activity, progressive shortening of telomeres, and restoration of the cellular senescence program. We attributed the repression of telomerase activity to the marked downregulation of the gene encoding the catalytic subunit of telomerase (hEST2/hTRT) but not another protein component (TP1/TLP1) or the RNA component of telomerase. These results suggest that a senescence‐inducing gene on chromosome 3 controls hEST2/hTRT gene expression either directly or indirectly and support the notion that hEST2/hTRT is the major determinant of telomerase enzymatic activity in human cells.  1998.  This article is a US Government work and, as such, is in the public domain in the United States of America.

title: Evidence for a putative telomerase repressor gene in the 3p14.2–p21.1 region
abstract: Telomeres, which are the repeated sequences located on both ends of chromosomes in eukaryotes, are known to shorten with each cell division, and their eventual loss is thought to result in cellular senescence. Unlike normal somatic cells, most tumor cells show activation of telomerase, a ribonucleoprotein enzyme that stably maintains telomere length by addition of the sequences of TTAGGG repeats to telomeres. The KC12 cell line derived from a renal cell carcinoma in a patient with von Hippel‐Lindau disease showed telomerase activity and loss of heterozygosity on the short arm of chromosome 3. Introduction of a normal human chromosome 3 into KC12 cells by microcell fusion induced cellular senescence, accompanied by suppression of telomerase activity and shortening of telomere length. Microcell hybrids that escaped from cellular senescence maintained telomere length and telomerase activity similar to those of the parental KC12 cells. We previously showed a similar suppression of telomerase activity by introduction of chromosome 3 into another renal cell carcinoma cell line, RCC23. The putative telomerase repressor gene was mapped to chromosome region 3p14.2–p21.1 by deletion mapping of KC12 + chromosome 3 revertants that escaped from cellular senescence and by transfer of subchromosomal fragments of chromosome 3 into RCC23 cells. Genes Chromosomes Cancer 23:123–133, 1998.

title: Telomerase activation by hTRT in human normal fibroblasts and hepatocellular carcinomas
abstract: Telomerase is a specialized type of reverse transcriptase which catalyzes the synthesis and extension of telomeric DNA (for review, see ref.1). This enzyme is highly active in most cancer cells, but is inactive in most somatic cells2. This striking observation led to the suggestion that telomerase might be important for the continued growth3 or progression4 of cancer cells. However, little is known about the molecular mechanism of telomerase activation in cancer cells. Human telomerase reverse transcriptase (hTRT) has recently been identified as a putative human telomerase catalytic subunit5,6. We transfected the gene encoding hTRT into telomerase-negative human normal fibroblast cells and demonstrated that expression of wild-type hTRT induces telomerase activity, whereas hTRT mutants containing mutations in regions conserved among other reverse transcriptases did not. Hepatocellular carcinoma (2O samples) and non-cancerous liver tissues (19 samples) were examined for telomerase activity and expression of hTRT, the human telomerase RNA component (hTR; encoded by TER)7 and the human telomerase-associated protein (HTLP1; encoded by 7EP7)8,9. A significant correlation between hTRT expression and telomerase activity was observed. These results indicate that the hTRT protein is the catalytic subunit of human telomerase, and that it plays a key role in the activation of telomerase in cancer cells.

title: Telomerase activation. One step on the road to cancer?
abstract: Ever since the discovery that telomeres are short in cancer cells and telomerase is activated in immortal cells, telomerase has been an oncogene wannabe. Oncogenes have been the glamour genes of molecular biology for 20 years, garnering flashy headlines and name recognition. More recently, tumor-suppressor genes have joined oncogenes on center stage. Recent evidence has shown that MYC upregulates the catalytic subunit of telomerase, TERT, and that TERT cooperates with HPV E7 in cell immortalization. This evidence now supports the placement of telomerase among the cancer gene elite.

title: The human telomerase catalytic subunit hTERT: organization of the gene and characterization of the promoter.
abstract: Telomerase, the enzyme that synthesizes telomeric DNA, is not expressed in most human somatic cells but is activated with in vitro immortalization and during tumorigenesis, and repressed by cell differentiation. Of the two components of the core enzyme, the catalytic protein hTERT is limiting for activity. To investigate mechanisms of hTERT gene regulation, we have cloned genomic sequences encompassing the complete hTERT transcription unit. The hTERT gene consists of 16 exons and 15 introns spanning approximately 35 kb. Transient transfections of immortal human cells with potential regulatory 5' sequences linked to a reporter, combined with deletion analysis of these sequences, indicated that elements responsible for promoter activity are contained within a region extending from 330 bp upstream of the ATG to the second exon of the gene. Assays in different cell types have shown that the hTERT promoter is inactive in normal and in transformed pre-immortal cells, but, like telomerase, it is activated with cell immortalization. Sequence analysis revealed that the hTERT promoter is GC-rich, lacks TATA and CAAT boxes but contains binding sites for several transcription factors that may be involved in its regulation. The abundance of these sites suggests the possibility that hTERT expression may be subject to multiple levels of control and be regulated by different factors in different cellular contexts.

title: Genomic organization and promoter characterization of the gene encoding the human telomerase reverse transcriptase (hTERT).
abstract: The enzyme telomerase plays a crucial role in cellular proliferation and tumorigenesis. By adding hexameric repeats to chromosome ends, it prevents telomeric loss and, thus, entry into senescence. Recent data suggest that expression of the human telomerase reverse transcriptase subunit (hTERT) represents the limiting factor for telomerase activity. To gain an insight into the mechanisms regulating hTERT expression, we have determined the complete genomic organization of the hTERT gene and isolated the 5'- and 3'- flanking region. The hTERT gene encompasses more than 37kb and consists of 16 exons. We show that all hTERT insertion and deletion variants described so far most likely result from the usage of alternative splice consensus sequences in intron or exon regions. Furthermore, we identified a new hTERT splice variant. Analysis of the DNA sequence surrounding the putative transcriptional start region revealed a TATA-less promoter located in a CpG island. A promoter fragment spanning the first 1100bp upstream of the initiating ATG start codon exhibited high-level activity in HEK-293 cells. Several consensus binding sites for the transcription factor Sp1 as well as a c-Myc binding site were identified in this promoter region. Altogether, these results provide the basis for more detailed studies on the regulation of telomerase activity in normal and cancer cells, and may lead to the development of new cancer therapies.

title: Telomerase reverse transcriptase gene is a direct target of c-Myc but is not functionally equivalent in cellular transformation
abstract: The telomerase reverse transcriptase component (TERT) is not expressed in most primary somatic human cells and tissues, but is upregulated in the majority of immortalized cell lines and tumors. Here, we identify the c-Myc transcription factor as a direct mediator of telomerase activation in primary human fibroblasts through its ability to specifically induce TERT gene expression. Through the use of a hormone inducible form of c-Myc (c-Myc-ER), we demonstrate that Myc-induced activation of the hTERT promoter requires an evolutionarily conserved E-box and that c-Myc-ER-induced accumulation of hTERT mRNA takes place in the absence of de novo protein synthesis. These findings demonstrate that the TERT gene is a direct transcriptional target of c-Myc. Since telomerase activation frequently correlates with immortalization and telomerase functions to stabilize telomers in cycling cells, we tested whether Myc-induced activation of TERT gene expression represents an important mechanism through which c-Myc acts to immortalize cells. Employing the rat embryo fibroblast cooperation assay, we show that TERT is unable to substitute for c-Myc in the transformation of primary rodent fibroblasts, suggesting that the transforming activities of Myc extend beyond its ability to activate TERT gene expression and hence telomerase activity.

title: Sp1 cooperates with c-Myc to activate transcription of the human telomerase reverse transcriptase gene (hTERT).
abstract: Telomerase activation is thought to be a critical step in cellular immortalization and carcinogenesis. The human telomerase catalytic subunit (hTERT) is a rate limiting determinant of the enzymatic activity of human telomerase. In the previous study, we identified the proximal 181 bp core promoter responsible for transcriptional activity of the hTERT gene. To identify the regulatory factors of transcription, transient expression assays were performed using hTERT promoter reporter plasmids. Serial deletion assays of the core promoter revealed that the 5'-region containing the E-box, which binds Myc/Max, as well as the 3'-region containing the GC-box, which binds Sp1, are essential for transactivation. The mutations introduced in the E-box or GC-box significantly decreased transcriptional activity of the promoter. Overexpression of Myc/Max or Sp1 led to significant activation of transcription in a cell type-specific manner, while Mad/Max introduction repressed it. However, the effects of Myc/Max on transactivation were marginal when Sp1 sites were mutated. Western blot analysis using various cell lines revealed a positive correlation between c-Myc and Sp1 expression and transcriptional activity of hTERT. Using fibroblast lineages in different stages of transformation, we found that c-Myc and Sp1 were induced to a dramatic extent when cells overcame replicative senescence and obtained immortal characteristics, in association with telomerase activation. These findings suggest that c-Myc and Sp1 cooperatively function as the major determinants of hTERT expression, and that the switching functions of Myc/Max and Mad/Max might also play roles in telomerase regulation.

title: In vivo and in vitro analyses of Myc for differential promoter activities of the human telomerase (hTERT) gene in normal and tumor cells.
abstract: 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.

title: Direct activation of TERT transcription by c-MYC
abstract: The MYC proto-oncogene encodes a ubiquitous transcription factor (c–MYC) involved in the control of cell proliferation and differentiation. Deregulated expression of c–MYC caused by gene amplification, retroviral insertion, or chromosomal translocation is associated with tumorigenesis. The function of c–MYC and its role in tumorigenesis are poorly understood because few c–MYC targets have been identified. Here we show that c–MYC has a direct role in induction of the activity of telomerase, the ribonucleoprotein complex expressed in proliferating and transformed cells, in which it preserves chromosome integrity by maintaining telomere length. c–MYC activates telomerase by inducing expression of its catalytic subunit, telomerase reverse transcriptase (TERT). Telomerase complex activity is dependent on TERT, a specialized type of reverse transcriptase. TERT and c–MYC are expressed in normal and transformed proliferating cells, downregulated in quiescent and terminally differentiated cells, and can both induce immortalization when constitutively expressed in transfected cells. Consistent with the recently reported association between MYC overexpression and induction of telomerase activity, we find here that the TERT promoter contains numerous c–MYC–binding sites that mediate TERT transcriptional activation. c–MYC–induced TERT expression is rapid and independent of cell proliferation and additional protein synthesis, consistent with direct transcriptional activation of TERT. Our results indicate that TERT is a target of c–MYC activity and identify a pathway linking cell proliferation and chromosome integrity in normal and neoplastic cells.

title: Formation of functional tissue from transplanted adrenocortical cells expressing telomerase reverse transcriptase
abstract: We report the first use of human telomerase reverse transcriptase (hTERT) expression in experimental xenotransplantation. Previously, we showed that bovine adrenocortical cells can be transplanted into severe combined immunodeficient (SCID) mice, and that these cells form functional tissue that replaces the animals' own adrenal glands. We cotransfected primary bovine adrenocortical cells with plasmids encoding hTERT, SV40 T antigen, neo, and green fluorescent protein. These clones do not undergo loss of telomeric DNA and appear to be immortalized. Two clones were transplanted beneath the kidney capsule of SCID mice. Animals that received cell transplants survived indefinitely despite adrenalectomy. The mouse glucocorticoid, corticosterone, was replaced by the bovine glucocorticoid, cortisol, in the plasma of these animals. The tissue formed from the transplanted cells resembled that formed by transplantation of cells that were not genetically modified and was similar to normal bovine adrenal cortex. The proliferation rate in tissues formed from these clones was low and there were no indications of malignant transformation.

title: Telomerase activity in human germline and embryonic tissues and cells.
abstract: Telomerase is a ribonucleoprotein that synthesizes telomere repeats onto chromosome ends and is involved in maintaining telomere length in germline tissues and in immortal and cancer cells. In the present study, the temporal regulation of expression of telomerase activity was examined in human germline and somatic tissues and cells during development. Telomerase activity was detected in fetal, newborn, and adult testes and ovaries, but not in mature spermatozoa or oocytes. Blastocysts expressed high levels of telomerase activity as did most human somatic tissues at 16-20 weeks of development with the exception of human brain tissue. This activity could no longer be detected in the somatic tissues examined from the neonatal period onward. Neither placenta nor cultured fetal amniocytes contained detectable telomerase activity. Fetal tissues explanted into primary cell culture showed a dramatic decline in telomerase activity which became undetectable after the first passage in vitro. Elucidation of the regulatory pathways involved in the repression of telomerase activity during development may lead to the ability to manipulate telomerase levels and explore the consequences both for cellular aging and for the survival of cancer cells.

title: Expression of the RNA component of telomerase during human development and differentiation.
abstract: We used a radioactive in situ method to study expression of the RNA component of human telomerase (hTR) during normal human development and differentiation using archival tissues. In embryonic tissues, the highest and most uniform expression was present in undifferentiated neuroepithelium. Expression was stronger in immature epithelium than in accompanying immature mesenchyme. Differentiation of most tissues was accompanied by decreased or absent expression. Except for testis and adrenal, the adult pattern of expression was present by the 10th postnatal week. In adult tissues, high expression was present in the testis (primary spermatocytes and Sertoli cells), moderate expression was present in lymphoid follicles (germinal centers), and weak expression was present in epithelia (regenerative cells) but was absent in the nervous system and mesenchymal derived tissues. Expression in adult tissues was predominantly limited to dividing cells, although certain differentiated postmitotic cells expressed the hTR. Our studies demonstrate the complex interrelationship of hTR expression with human development, differentiation, and cell division.

title: Telomerase activity in human development is regulated by human telomerase reverse transcriptase (hTERT) transcription and by alternate splicing of hTERT transcripts.
abstract: The correlation between telomerase activity, telomere lengths, and cellular replicative capacity has led to the theory that maintenance of telomere lengths by telomerase acts as a molecular clock to control replicative capacity and senescence. Regulation of this molecular clock may have applications in the treatment of cell aging and tumorigenesis, although little is presently known about the regulation of telomerase activity. To investigate possible mechanisms of regulation, we examined telomerase activity and the expression of the human telomerase RNA subunit and the human telomerase reverse transcriptase protein (hTERT) during human fetal heart, liver, and kidney development. The human telomerase RNA subunit is expressed in all three tissues at all gestational ages examined. hTERT expression correlates with telomerase activity in the liver, where both are expressed at all ages surveyed, and in the heart, where both are present until the 11th gestational week but not thereafter. However, although telomerase activity in the kidney is suppressed after the 15th gestational week, the hTERT transcript can be detected until at least the 21st week. Reverse transcription-PCR using primers within the reverse transcriptase domain of hTERT show the presence of multiple alternately spliced transcripts in these tissues, corresponding to full-length message as well as spliced messages with critical reverse transcriptase motifs deleted. Of note, telomerase activity in the kidney is only present at those gestational ages when full-length hTERT message is expressed (until approximately week 15), with spliced transcripts continuing to be expressed at later stages of development. The tissue-specific and gestational-age dependent expression of hTERT mRNA seen in human development suggests the presence of at least two regulatory mechanisms controlling the activity of telomerase: transcriptional control of the hTERT gene and alternate splicing of hTERT transcripts.

title: Experimental elongation of telomeres extends the lifespan of immortal x normal cell hybrids.
abstract: Hybrids between immortal cells that express telomerase and normal cells that lack telomerase have a limited lifespan. We demonstrate that telomerase is repressed in such hybrids. Treatment of immortal human cell lines with certain oligonucleotides resulted in telomere elongation. We took advantage of this observation to test the hypothesis that elongation of telomeres would extend the lifespan of cells in culture. An immortal human cell line was treated with an oligonucleotide to lengthen its telomeres and then was fused with mortal cells. The lifespan of these hybrid cells was longer than that of the hybrids in which telomeres had not been elongated. These observations provide the first direct evidence supporting the hypothesis that telomere length determines proliferative capacity of human cells.

title: Telomerase repressor sequences on chromosome 3 and induction of permanent growth arrest in human breast cancer cells.
abstract: Activation of the enzyme telomerase, which has been associated with cellular immortality, may constitute a key step in the development of human cancer. Telomerase is repressed in most normal human somatic cells. This study was conducted, using a genetic complementation approach, with the aim of identifying and mapping the genes responsible for repressing telomerase and, simultaneously, to establish the effect of experimentally induced telomerase repression on human tumor cell growth.



Individual human chromosomes isolated from normal diploid cells and tagged with bacterial antibiotic resistance genes (for later selection) were introduced into cells of the human breast carcinoma cell line 21NT by means of microcell transfer. Selected hybrid clones were screened for telomerase activity by use of the polymerase chain reaction-based telomere repeat amplification protocol (TRAP) assay, and the proliferative fate of the hybrid clones was determined. Regions of the introduced chromosomes associated with telomerase repression were mapped using segregant hybrids and a deletion analysis that employed microsatellite DNA markers.



Strong repression of telomerase was observed following transfer of human chromosome 3 into 21NT cells but not after transfer of chromosomes 8, 12, or 20. The vast majority of hybrid clones with repressed telomerase entered permanent growth arrest after 10-18 population doublings. Deletion analysis of nonrepressed segregant monochromosome 3 hybrids indicated two regions on the short arm of chromosome 3 (3p21.3-p22 and 3p12-21.1) where telomerase regulator genes may be located.



Telomerase in human breast cancer cells is efficiently repressed by a gene or genes on normal human chromosome 3p, and this repression is associated with permanent growth arrest of the tumor cells.

title: Developmental and tissue-specific regulation of mouse telomerase and telomere length.
abstract: Telomere shortening and telomerase activation in human somatic cells have been implicated in cell immortalization and cellular senescence. To further study the role of telomerase in immortalization, we assayed telomere length and telomerase activity in primary mouse fibroblasts, in spontaneously immortalized cell clones, and in mouse tissues. In the primary cell cultures, telomere length decreased with increased cell doublings and telomerase activity was not detected. In contrast, in spontaneously immortalized clones, telomeres were maintained at a stable length and telomerase activity was present. To determine if telomere shortening occurs in vivo, we assayed for telomerase and telomere length in tissues from mice of different ages. Telomere length was similar among different tissues within a newborn mouse, whereas telomere length differed between tissues in an adult mouse. These findings suggest that there is tissue-specific regulation of mouse telomerase during development and aging in vivo. In contrast to human tissues, most mouse tissues had active telomerase. The presence of telomerase in these tissues may reflect the ease of immortalization of primary mouse cells relative to human cells in culture.