Submitted September 21, 1997.
In this review, I summarize the most recent progress in the studies on mammalian telomerase, especially focusing on the molecular aspects. Possible regulation mechanisms of telomerase activity in mammalian cells are discussed.
KEY WORDS: telomerase, telomere, end replication problem, regulation.
Mammalian Telomerase RNA Templates
The human telomerase RNA template gene was cloned by a PCR-based subtraction strategy , and the mouse counterpart was isolated by cross-hybridization of the gene . The significance of the expression level of the RNA template genes involved in the regulation of telomerase activity is controversial [5, 6].
Tetrahymena Telomerase Protein Component Genes
Due to the necessity of forming many telomeres de novo during macronuclei development, ciliates contain a relatively large amount of telomerase and have provided good sources for biochemical studies. Indeed, Blackburn and Greider first identified and characterized telomerase activity using Tetrahymena macronuclei . In 1995, Greider and her colleagues succeeded in a large scale preparation of the Tetrahymena telomerase and determined the amino acid sequences of peptides putatively derived from the enzyme [8, 9]. Two Tetrahymena genes, namely p80 and p95, were cloned, and each was found to produce a protein having an apparent molecular weight indicated by the name of the gene . It was shown that the p80 protein binds to the Tetrahymena template RNA, while the p95 protein binds to the telomeric primers that are used as the substrates for the elongation reactions. Moreover, the predicted amino acid sequences of p95 showed a weak homology with conserved motifs found in DNA and RNA polymerases. Therefore, it was speculated that the p80 and p95 proteins might be the regulatory and catalytic components of the Tetrahymena telomerase, respectively. To verify that a set of proteins constitute the telomerase, it is necessary to carry out a reconstitution experiment to show that a mixture of the purified recombinant components forms an active enzyme. A successive experiment using the recombinant p80 and p95 proteins and the in vitro transcribed Tetrahymena template RNA has not yet been reported. Therefore, it is not clear at present whether p80 and p95 are essential telomerase proteins, or whether they are the only protein components of Tetrahymena telomerase.
Mammalian Telomerase-Associated Genes
In 1997, mammalian p80-related genes were independently identified by our group and Harrington et al. using similar approaches with different species. In late 1995, we noted a rat EST (Expression Sequence Tag) clone with a nucleotide sequence showing a limited homology to the Tetrahymena p80 gene deposited in the GenBank EST database. We cloned the full length cDNA and determined the nucleotide sequence, coming to the conclusion that this gene, named TLP1 (Telomerase Protein 1), represents the rat homolog of the Tetrahymena p80 gene . The predicted protein product of the TLP1 gene was 2629 amino acids, which was much larger than that of the p80 gene (719 amino acids). Regions with homology to p80 are limited to the N-terminal third of the product. In addition to the p80-homologous region, a nucleotide binding domain (Walker's motif) and WD-40 repeats, which presumably function in protein--protein interaction, constitute the central and the C-terminus, respectively. Therefore, the TLP1 protein was considered to be a multifunctional protein, having additional roles that are not possessed by the p80 protein. The predicted amino acid sequence of the TLP1 protein does not have any motif that has been found in DNA/RNA polymerases, as in the case of the Tetrahymena p80. Thus, we suggested that the cloned TLP1 might have a role in the regulation of telomerase activity, but is not the catalytic component of the mammalian telomerase.
One reason that made the biochemical study of mammalian telomerase difficult was the lack of a sensitive and quantitative method to detect the weak mammalian telomerase activity. A simple and sensitive PCR-based TRAP (Telomeric Repeat Amplification Protocol) had been reported . However, the method was qualitative, or semi-quantitative at most, and was not useful for biochemical studies. We independently developed another PCR-based method, the Stretch PCR Assay, to estimate relative titers of telomerase and have shown that the method is useful for clinical diagnoses as well as basic research . Using this method, we showed that the TLP1 protein was co-purified with the telomerase activity in a number of purification steps using different modes of separation. Moreover, the anti-TLP1 antibody significantly immunoprecipitated the telomerase activity from cell extracts. From these observations, we concluded that the TLP1 protein is a telomerase-associated protein.
The TLP1 gene is ubiquitously expressed in almost all cells, including telomerase-negative cells. Thus, the expression level of the TLP1 gene does not regulate telomerase activity. A pulse chase experiment revealed that the TLP1 protein is translated as a nascent protein with an apparent molecular weight of 240 kD (p240) and that p240 is processed to another protein with an apparent molecular weight of 230 kD (p230) in telomerase-positive cells. This modification is moderately rapid; the half life of the reaction was estimated to be one hour or less. However, the conversion from p240 to p230 was not detected in cells that do not show telomerase activity. Therefore, we proposed that the modification of the TLP1 product might have a role in the regulation of telomerase activity. Since TLP1 is expressed in telomerase-negative cells, p240 may have additional roles, such as the maintenance of telomeric DNA--protein structures in both telomerase-negative and -positive cells, besides its telomerase related functions.
Harrington et al. independently identified the mouse and human p80-homolog, the TP1 genes, by a homology search for p80 with a collection of mouse ESTs . Using the "three-hybrid" method, they showed that the mouse TP1 product binds to the mouse telomerase template RNA, a property shared by the original Tetrahymena p80 protein.
These two studies clearly indicated that p80-homologs are widely conserved among the eukaryotes from ciliates to humans. However, it is of interest that a budding yeast p80-homolog has not been found by a homology search with the published total genomic sequences of Saccharomyces cerevisiae. The telomerase activity in budding yeast has been well characterized . Therefore, the lack of a p80-related gene in budding yeast suggests that the p80-homolog genes might not be an essential component of telomerase, such as a catalytic subunit, but might have species-specific regulatory roles. TLP1 and TP1 are identical genes identified in different species and hereafter will be referred to as the TLP1/TP1 gene.
Mammalian Telomerase Catalytic Components
In 1996, Lingner and Cech reported the preparation of a highly purified telomerase from the ciliate Euplotes. Subsequently, they obtained amino acid sequences from the peptides derived from the sample and cloned a gene called p123, encoding a component of the Euplotes telomerase . Significantly, the predicted amino acid sequence from the p123 cDNA contains motifs conserved among the various types of reverse transcriptases derived from retroviruses and retrotransposons. Since telomerase is a kind of reverse transcriptase, synthesizing telomeric DNA using the intrinsic template RNA, this observation suggested that the gene they had cloned actually encoded the catalytic subunit of the Euplotes telomerase.
At the same time, Lundblad's group identified and cloned a collection of budding yeast genes, EST1-4 (Ever Shorter Telomeres), the mutants of which showed progressive shortening of telomeres . Nucleotide sequencing of the genes revealed that EST2 has significant homology with p123. The EST2 amino acid sequence contains all the motifs conserved among the reverse transcriptases and p123. Moreover, there are additional amino acid regions conserved only within p123 and EST2, but not with other types of reverse transcriptase. In vitro mutagenesis of the conserved amino acid and replacement of the EST2 gene with the mutated genes abolished telomerase activity. Therefore, it was concluded that the p123 and EST2 genes encode the catalytic subunit of the telomerases in Euplotes and budding yeast, respectively .
The rate of increase of information provided by the human genome sequencing project is enormous. At the same time as the p123/EST2 paper was published, a human EST clone having homology with p123 was deposited in the GenBank EST database. The cloning of the full length cDNA of this human gene was independently reported by Cech's group and Weinberg's group [17, 18]. The genes are now called TRT (Telomerase Reverse Transcriptase). The expression of the human TRT (hTRT) was shown to correlate with the telomerase activity in telomerase-negative and -positive cells. Moreover, when the human leukemic cell line HL60 was induced to differentiate and undergo apoptosis by retinoic acid treatment, TRT expression was significantly down-regulated with a simultaneous decline in telomerase activity . Nakamura and Cech also identified the fission yeast Schizosaccharomyces pombe TRT gene (SpTRT) . Therefore, the TRT genes are conserved among three highly divergent eukaryotes, yeasts, ciliates, and metazoans.
Compositions of Mammalian Telomerase
The recent studies summarized above clearly indicated that the telomerase catalytic component is encoded by the TRT genes. One of the most interesting questions is whether the essential telomerase activity is performed by a bipartite complex of TRTs and the template RNAs. The answer to this question will be obtained before long. Biochemical purification of the human and mouse telomerase suggested that mammalian telomerases are extracted as a complex having an approximate molecular weight of 1000 kD (Nakayama et al., unpublished). Obviously, the predicted molecular weight of a complex formed by hTRT (molecular weight of about 130 kD) and the human RNA template (about 450 nucleotides) is far less than the observed value if the telomerase were composed of one molecule of each component. One possibility is that the essential catalytic activity is performed by the TRT/RNA core complex, but telomerase-associated proteins, such as the TLP1/TP1 protein, associate with the core complex in vivo to modulate activity in response to various stimuli, as will be described below.
Telomerase Regulation in Normal Somatic Cells
Telomerase was found to be active in germ cells and in most cases of cancer cells . However, the following studies indicated that some specialized normal somatic cells also possess telomerase activity. More significantly, the telomerase activity in these cells seems to be highly regulated to be expressed at specific stages. In some cases, telomerase is regulated by extra-cellular signals, such as growth factors. Three types of normal somatic cells where telomerase activity was studied in detail will be described here.
Hematopoietic Cells. Hematopoietic cells are the earliest example of somatic cells where telomerase activity was detected . Hiyama et al. fractionated human bone marrow cells and identified significant telomerase activity in the CD34+CD38+ committed hematopoietic progenitor cells, but not in the more primitive CD34+CD38-/low cells . Since the CD34+CD38-/low cells proliferate significantly less than the CD34+CD38+ cells, they suggested that telomerase activity in human hematopoietic cells might increase upon proliferation and differentiation. Lansdorp and his group extended this study. A subset of hematopoietic cells characterized by the expression of CD34+CD71lowCD45RAlow surface markers is the most primitive type of human hematopoietic cells fractionated by currently available techniques. These cells subsequently differentiate into CD34+CD71+ and CD34- cells after induction by growth factors. CD34+CD71lowCD45RAlow cells are mostly quiescent and the cells proliferate only after they are committed into CD34+CD71+ cells. They found that the most primitive and quiescent CD34+CD71lowCD45RAlow cells, and further committed but still growing CD34- cells did not show any telomerase activity . However, the proliferating early progenitor CD34+CD71+ cells showed a transient but significant level of activity. Therefore, it seems that there is a transient surge in telomerase activity at a specific early stage during the differentiation of hematopoietic cells. In another study reported by Engelhardt et al., human CD34+ cells were cultured and expanded in vitro in the presence of cytokines. A combination of kit-ligand, IL-3, IL-6, erythropoietin, and G-CSF significantly stimulated the proliferation of CD34+ cells. In parallel with the growth stimulation by these cytokines, the telomerase activity in CD34+ cells was also stimulated . The kinetics of the cytokine-induced telomerase stimulation was examined. The effect first appeared at 48 h after the initiation of the cytokine treatment and was at its maximum after about one week. The relative telomerase titer then declined while cells were still growing. These studies had an important implication--being in a most primitive state by itself does not render cells telomerase-positive. Similarly, rapidly growing cells do not necessarily express telomerase activity. Rather, it seems that telomerase is activated when primitive cells are induced to proliferate.
The implications of the transient expression of telomerase activity in these primitive progenitor cells are unknown. It was shown that the telomere lengths of the hematopoietic cells decrease with age in vivo  and with prolonged culture in vitro . Therefore, the transient increase of telomerase activity at the specific differentiation stage of hematopoiesis does not seem to ensure the long term maintenance of telomere length in hematopoietic progenitor cells.
Lymphocytes. T- and B-lymphocytes are known to be activated by several stimuli to induce telomerase activity [20, 24-26]. Buchkovich and Greider found that mature T-lymphocytes activated either by PHA (phytohemagglutinin) or receptor cross-linking by the anti-CD3 and anti-CD28 antibodies significantly up-regulated telomerase activity . They carried out a careful analysis to determine the effect of cell-cycling on this telomerase activation. When rapamycin was added to PHA-treated cells, telomerase was not activated. Rapamycin inhibits cells from proceeding from G1 to S-phase. Therefore, the G1 to S-phase transition was thought to be a key to telomerase activation. In contrast, hydroxyurea, an agent inhibiting DNA polymerase alpha and thus DNA synthesis, did not have any effect on telomerase activation by PHA, suggesting that DNA synthesis by itself is not necessary for the activation. Igarashi and Sakaguchi examined the telomerase-inducing effects of different T-lymphocyte stimuli . They independently found that T-cells proliferated as well as induced telomerase activity when treated with either PHA or anti-CD3 antibody. When T-cells were first exposed to PHA for 12 h, then incubated with IL-2 in the absence of PHA, the cells proliferated to the same extent as cells which were treated by PHA throughout the experiment. However, in contrast to a continued exposure to PHA, a short exposure to PHA followed by IL-2-treatment did not induce any significant level of telomerase activity. They concluded that different stimuli resulting in T-cell proliferation had different effects on telomerase induction.
Human B-cells are also induced to produce telomerase activity by the cross-linking of the B-cell receptor (BCR) by treatment of the cells with anti-IgM beads . Incubation of the cells with soluble anti-IgM monoclonal antibody failed to stimulate the telomerase activity. Anti-IgM beads are known to stimulate B-cell proliferation and differentiation efficiently, presumably by multi-valent cross-linking of BCR. In contrast, a soluble anti-IgM antibody is less efficient at stimulating B-cell proliferation and requires additional signals, such as IL-2, IL-4, and IL-13 cytokines, for maximal stimulation. In parallel with the growth stimulation effects, the soluble anti-IgM antibody induced telomerase activity only when a co-stimulatory cytokine was applied, but not when cytokine was absent . These observations suggested that signals leading to efficient B-cell proliferation resulted in telomerase activation. However, as shown in T-cells, different stimuli leading to the same level of B-cell proliferation showed variable levels of telomerase activation. Treatment with the anti-IgM antibody and IL-2 induced a significantly lower level of telomerase compared to that induced by the anti-IgM antibody and IL-4, although these two procedures stimulated B-cell growth to a comparative level. Again, as demonstrated in T-cells by the same authors, telomerase is not induced simply when cells proliferate; different mitogenic signals seem to have distinct effects on telomerase induction. In the case of both B- and T-cells, phorbol dibutylate (PDB) and Ca2+ ionophore treatment (PDB/Ca) is known to bypass the TCR (T-cell receptor)- or BCR-mediated signal transductions and to result in G0 to G1 transition. PDB/Ca was shown to stimulate telomerase activity very efficiently, suggesting that TCR- or BCR-mediated signaling is an essential component of telomerase induction in T- and B-cells, respectively, and that telomerase is induced only after cells are committed in proceeding from G0 to G1 [25, 26].
As in the case of hematopoietic cells, the implications of the inducible telomerase activity in T- and B-cells remain to be studied. The in vitro studies described above showed that the induced telomerase activity appeared transiently after stimulation, and the activity eventually declined even when the cells still keep growing. It is not known whether telomerase activation is always coupled with lymphocyte divisions in vivo.
Two classes of T-cells, naive and memory T-cells, are present in the peripheral lymphoid organs. Naive T-cells have their own antigen-specific receptors (TCR), but are not stimulated by exogenous antigens. When naive T-cells are stimulated by a combination of the antigen and a "co-stimulatory signal" mediated by CD28 molecules, the naive cells proliferate and differentiate into armed memory T-cells (reviewed in ). During the second challenge by the antigen, memory T-cells can be stimulated to proliferate without the need of the co-stimulatory signals. The telomere lengths of naive and memory T-cells derived from healthy donors of different ages were examined . Both naive and memory T-cells showed reduced TRF (Terminal Restriction Fragment) length with higher donor ages. Also, the TRFs of memory T-cells were consistently shorter than those of naive T-cells by about 1.4 kb. These results suggested that the telomerase activity in lymphocytes activated upon cell division might not prevent the telomere shortening which occurs during aging or the clonal expansion which occurs when the memory T-cells develop from naive T-cells in vivo.
Endometrium. Uterine endometrium has gained attention as another normal somatic organ expressing regulated telomerase activity [29, 30]. Pre-menopausal endometrial tissues undergo a regular cycle of menstruation, alternating between the proliferative phase and the secreting phase. The proliferative phase is stimulated by the action of estrogen secreted by ovaries and characterized by active proliferation of endometrial gland tissues. The peak proliferative activity induced by the peak level of estrogen occurs at the late proliferative stage and is followed by a rapid decline of estrogen level at the time of ovulation. In the following secretory phase, progesterone is the major sex steroid hormone released by the ovaries. In response to progesterone, the endometrial tissues stop proliferating and show active secretory activity. When progesterone secretion is withdrawn, the endometrial tissues break down and menstruation occurs. In the post-menopausal uterine, the menstrual cycle does not occur, as shown by a static endometrial tissue with low proliferation activity. Telomerase activity was found to be precisely regulated during the menstrual cycle [29, 30]. The activity was highest at the late proliferative phase, when the tissues proliferate most extensively. The activity declined after the tissues entered the secretory phase when proliferating activity had declined. No telomerase activity was found with post-menopausal endometrial tissues. Therefore, the telomerase activity in the endometrium is tightly linked to the proliferating activity and it seems to be induced by estrogen.
Telomerase Activity during the Cell Cycle
As it is known that telomerase is regulated by the proliferating activity of cells, it is particularly interesting to ask if telomerase activity is expressed at a specific phase of the cell cycle. However, the available data are somewhat controversial. By blocking the cell cycle of human tumor cell lines with blocking agents, it was shown that telomerase is most active during G1/S phase, whereas it is most inactive at G2/M phase . In contrast, when HT1080 and HL60 cells at different phases of the cell cycle were fractionated by flow cytometry, no significant variation was observed . Similarly, when telomerase activity was stimulated by treating human T-cells with the anti-CD3 monoclonal antibody, a dependence of the activity upon a specific phase of the cell cycle was not observed . In summary, it seems that once the cells are cycling, telomerase activity is not significantly restricted to any specific phase of the cell cycle. However, as noted from the different effects of rapamycin and hydroxyurea upon the telomerase activity of PHA-treated T-cells, the transition from G0 to G1 phase might be important for the initial induction of telomerase activity in quiescent telomerase-negative cells.
Possible Regulation Mechanisms of Mammalian Telomerase
The analyses of telomerase activity in somatic cells which have been summarized in the previous sections suggest three different levels of regulation for telomerase activity. First, telomerase has never been detected to be active in most types of terminally differentiated normal somatic cells, such as fibroblast cells, irrespective of whether they are quiescent or proliferate. Therefore, cell type specificity seems to be the primary determinant of telomerase activity. Many telomerase-positive normal cells, such as germ cells, embryonic tissue cells , hematopoietic progenitor cells, epidermal skin cells [35, 36], and follicular hair cells , are characterized as containing "stem cells". Stem cells are defined by having both the potential to renew, and the ability to differentiate into the multiple types of cells which compose the tissues. By applying the flow cytometry single cell sorting technique to hematopoietic cells, Morrison et al. proposed that telomerase activity is tightly linked to the self-renewal potential of the cells . However, the activation of naive T-cells to memory T-cells is obviously not a self-renewing but a differentiating process; it is not known whether the "telomerase as an enzyme of self-renewing cells" hypothesis is correct in all cases. It will be important to analyze the details of the specific stages when telomerase is activated in various tissues.
The second determinant which influences telomerase activity is the proliferating activity of cells. The most primitive stem cells in many tissues are considered to be quiescent or proliferating very slowly. A population of cells produced from the primitive stem cells and restricted to a narrower range of differentiation potentials grows rapidly. As clearly indicated with the hematopoietic primitive progenitor cells, telomerase seems to be active specifically with these "primitive and proliferating" cells. Future studies to test this hypothesis in other types of tissue cells is necessary.
The third potential determinant is the phase-specific mechanism to up- and down-regulate the activity during cell cycle, although this possibility needs to be examined more extensively.
Telomerase synthesizes telomeric DNA not only at the native telomere ends, but also at broken non-telomeric chromosomal ends, albeit the efficiency of this process is not high . This de novo addition of telomeric DNA by telomerase may result in the healing of broken and aberrant chromosomes, leading to cancer development by the deletion of anti-oncogenes . Therefore, telomerase activity should be precisely regulated in order to minimize the risk of cancers. The expression level of the TRT genes is clearly the primary determinant to regulate the activity, since the TRT proteins represent the catalytic cores of the telomerase. However, given the multiple mechanisms by which telomerase is regulated as summarized here, other mechanisms modulating telomerase activity might also contribute to determining which cells are telomerase active. Modulations through telomerase-associated proteins and RNA template genes are candidates for the fine tuning of telomerase activity.
The author is grateful to Drs. E. A. Kamei and H. Nakauchi for critical reading of and comments on the manuscript. The excellent secretarial work of M. Fukuda is also acknowledged.
This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports, and Culture of Japan, a Grant-in-Aid of Special Coordination Funds for Promoting Science and Technology from the Science and Technology Agency of Japan, and a Grant-in-Aid from the Mitsubishi Foundation.
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