Telomerase and differentiation in multicellular organisms: turn it off, turn it on, and turn it off again

Telomerase is a ribonucleoprotein complex that catalyses the addition of TTAGGG repeats onto telomeres, repetitive DNA structures found at the ends of linear chromosomes. The majority of human somatic tissues do not display telomerase activity and undergo telomeric shortening with consecutive divisions. This telomeric shortening results in replicative senescence in vitro and likely in vivo. Telomerase activity is present in the vast majority of tumors, preventing telomeric shortening and thereby enabling indefinite cell divisions. Telomerase activity is regulated throughout human development, undergoing silencing in almost all organ systems from embryogenesis onwards. However, regulated telomerase activity is seen in basal/stem cell compartments of highly regenerative tissues, such as those of the immune system, skin, and intestine. Avian species display telomerase repression and telomeric shortening similar to that seen in humans. However, rodents retain telomerase-competency throughout their lifespan and have not been shown to display division-dependent telomere shortening. The regulation of telomerase activity in plants is less well understood, although early indications suggest ubiquitous competency. The aim of this review is to present current data regarding developmental regulation of telomerase in humans, mice, chickens and flowering plants. Differentiation, quiescence and telomerase activity regulation will then be addressed in three human representative tissue systems; blood, skin, and intestine. We will also highlight similarities, differences and misconceptions in the developing field of telomere and telomerase biology.     

Stem cells in asexual reproduction of the colonial ascidian <Emphasis Type="Italic">Botryllus tubaratus</Emphasis> (Tunicata: Ascidiacea)

Histological, cytochemical and ultrastructure research on the budding of the colonial ascidian Botryllus tuberatus aimed at searching for stem cells was performed. A dense mass of undifferentiated cells and the connection of the outer epidermal and inner atrial epithelia were revealed for the first time in the early buds of B. tuberatus. Undifferentiated cells revealed in the early buds and vascular system of the colony had morphological features of the stem and primary germ cells of metazoans. Intensive expression of alkaline phosphatase, the cytochemical marker of embryonal stem and primary germ cells of vertebrate animals, was revealed in developing buds and in some cells of the hematocyte population. Based on the literature and the author’s data it is hypothesized that the self-renewing pool of stem cells of the colonial ascidian B. tuberatus is the cellular basis of its reproductive strategy, including both sexual and asexual reproduction.     

Identification of the endostyle as a stem cell niche in a colonial chordate

Stem cell populations exist in "niches" that hold them and regulate their fate decisions. Identification and characterization of these niches is essential for understanding stem cell maintenance and tissue regeneration. Here we report on the identification of a novel stem cell niche in Botryllus schlosseri, a colonial urochordate with high stem cell-mediated developmental activities. Using in vivo cell labeling, engraftment, confocal microscopy, and time-lapse imaging, we have identified cells with stemness capabilities in the anterior ventral region of the Botryllus' endostyle. These cells proliferate and migrate to regenerating organs in developing buds and buds of chimeric partners but do not contribute to the germ line. When cells are transplanted from the endostyle region, they contribute to tissue development and induce long-term chimerism in allogeneic tissues. In contrast, cells from other Botryllus' regions do not show comparable stemness capabilities. Cumulatively, these results define the Botryllus' endostyle region as an adult somatic stem cell niche.     

Development patterns of telomerase activity in barley and maize

Eukaryotic chromosomes terminate with specialized structures called telomeres. Maintenance of chromosomal ends in most eukaryotes studied to date requires a specialized enzyme, telomerase. Telomerase has been shown to be developmentally regulated in man and a few other multicellular organisms, while it is constitutively expressed in unicellular eukaryotes. Recently, we demonstrated telomerase activity in plant extracts using the PCR-based TRAP (Telomeric Repeat Amplification Protocol) assay developed for human cells. Here we report telomerase activities in two grass species, barley and maize, using a modified, semi-quantitative TRAP assay. Telomerase was highly active in very young immature embryos and gradually declined during embryo development. The endosperm telomerase activity was detectable, but significantly lower than in the embryo and declined during kernel development with no detectable activity in later stages. Telomerase activity in dissected maize embryo axis was several orders of magnitude higher than in the scutellum. Telomerase activity was not detected in a range of differentiated tissues including those with active meristems such as root tips as well as the internode and leaf base. The role of telomerase repression during differentiation and the relationship between chromosome healing and telomerase activity is discussed.     

Lgr5 intestinal stem cells have high telomerase activity and randomly segregate their chromosomes

Somatic cells have been proposed to be limited in the number of cell divisions they can undergo. This is thought to be a mechanism by which stem cells retain their integrity preventing disease. However, we have recently discovered intestinal crypt stem cells that persist for the lifetime of a mouse, yet divide every day. We now demonstrate biochemically that primary isolated Lgr5+ve stem cells contain significant telomerase activity. Telomerase activity rapidly decreases in the undifferentiated progeny of these stem cells and is entirely lost in differentiated villus cells. Conversely, asymmetric segregation of chromosomes has been proposed as a mechanism for stem cells to protect their genomes against damage. We determined the average cell cycle length of Lgr5+ve stem cells at 21.5 h and find that Lgr5+ve intestinal stem cells randomly segregate newly synthesized DNA strands, opposing the 'immortal strand' hypothesis.     

Developmental pattern of telomerase expression in the sand scallop, Euvola ziczac

Abstract. Telomerase is a ribonucleoprotein that can maintain telomeres, the repetitive sequences of DNA found at the end of eukaryotic chromosomes, and confer long-term proliferative capacity on cells. Telomerase expression is essential during periods of intense cell division such as the early developmental process. In later development, some species retain telomerase activity while others repress telomerase activity in what is thought to be a tumor-protective mechanism. Despite the importance of telomerase expression in both development and neoplastic disease, no studies to date have characterized its expression in bivalves. We present the first report of telomerase expression in a bivalve species, the sand scallop, Euvola ziczac. Telomerase activity was detected throughout the early stages of development and in all adult tissues examined. Analysis of DNA isolated from adult tissues indicated long telomeres, with terminal restriction fragment lengths >20 kb in both somatic and germ tissues. Ubiquitous telomerase expression throughout development and into adulthood would suggest a lack of telomere-related senescence and suggests that these scallops do not use telomerase repression as a mechanism to suppress the formation of neoplasm.     

Suppression of programmed cell death regulates the cyclical degeneration of organs in a colonial urochordate

The survival of animal tissues and organs is controlled through both activation and suppression of programmed cell death. In the colonial urochordate Botryllus schlosseri, the entire parental generation of zooids in a colony synchronously dies every week as the asexually derived generation of buds reaches functional maturity. This process, called takeover, involves massive programmed cell death (PCD) of zooid organs via apoptosis followed by programmed removal of cell corpses by blood phagocytes within approximately 1 day. We have previously reported that developing buds in conjunction with circulating phagocytes are key effectors of zooid resorption and macromolecular recycling during takeover, and as such engineer the reconstitution of a functional asexual generation every week [Lauzon, R.J., Ishizuka, K.J., Weissman, I.L., 2002. Cyclical generation and degeneration of organs in a colonial urochordate involves crosstalk between old and new: a model for development and regeneration. Dev. Biol. 249, 333-348]. Here, we demonstrate that zooid lifespan during cyclic blastogenesis is regulated by two independent signals: a bud-independent signal that activates zooid PCD and a bud-dependent, survival signal that acts in short-range fashion via the colonial vasculature. As zooids represent a transient, mass-produced commodity during Botryllus asexual development, PCD regulation in this animal via both activation and suppression enables it to remove and recycle its constituent zooids earlier when intra-colony resources are low, while maintaining the functional filter-feeding state when resources are adequate. We propose that this crosstalk mechanism between bud and parent optimizes survival of a B. schlosseri colony with each round of cyclic blastogenesis.     

Vasa expression in a colonial ascidian, Botrylloides violaceus

Evolution of solitary or colonial life histories in tunicates is accompanied by dramatic developmental changes that affect morphology and reproduction. We compared vasa expression in a solitary ascidian and a closely related colonial ascidian, in an effort to uncover developmental mechanisms important during the evolution of these contrasting life histories, including the ability to reproduce by budding. In this study, we explored the origin of germ cells in new buds developing by asexual reproduction in a colonial ascidian, Botrylloides violaceus and compared it to the source of germ cells in a solitary ascidian Boltenia villosa. We studied expression by in situ hybridization of vasa, a DEAD box RNA helicase gene found in germ cells across the metazoans. In B. villosa, bv-vasa mRNA was expressed in putative germ cells and oocytes of adult gonads, and was sequestered into a posterior lineage during embryogenesis. In mature colonies of the ascidian B. violaceus, bot-vasa mRNA was expressed in putative spermatogonia, in oocytes of zooids, and in some circulating cells in the zooids and differentiating buds. We propose that expression of vasa in cells other than gonadal germ cells of zooids in a colonial ascidian may serve as a source of germ-line stem cells in the colony.     

Role of Vasa, Piwi, and Myc-expressing coelomic cells in gonad regeneration of the colonial tunicate, Botryllus primigenus

In the colonial tunicate, Botryllus primigenus Oka, gonads consist of indifferent germline precursor cells, the primordial testis and ovary, and mature gonads, of which the immature gonad components can be reconstructed de novo in vascular buds that arise from the common vascular system, although the mechanism is uncertain. In this study, we investigated how and what kinds of cells regenerated the gonad components. We found that few Vasa-positive cells in the hemocoel entered the growing vascular bud, where their number increased, and finally developed exclusively into female germ cells. Simultaneously, small cell aggregates consisting of Vasa(-) and Vasa(±) cells appeared de novo in the lateral body cavity of developing vascular buds. Double fluorescent in situ hybridization showed that these cell aggregates were both Piwi- and Myc-positive. They could form germline precursor cells and a primordial testis and ovary that strongly expressed Vasa. Myc knockdown by RNA interference conspicuously lowered Piwi expression and resulted in the loss of germline precursor cells without affecting Vasa(+) oocyte formation. Myc may contribute to gonad tissue formation via Piwi maintenance. When human recombinant BMP 4 was injected in the test vessel, coelomic Piwi(+) cells were induced to express Vasa in the blood. We conclude, therefore, that in vascular buds of B. primigenus, female germ cells can develop from homing Vasa(+) cells in the blood, and that other gonad components can arise from coelomic Vasa(-)/Piwi(+)/Myc(+) cells.     

Adenovirus-mediated suicide SCLC gene therapy using the increased activity of the hTERT promoter by the MMRE and 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 or stem cells. The aim of this study was to use increased telomerase promoter activity in small-cell lung cancer (SCLC) gene therapy. The hTERT promoter and Myc-Max response elements (MMRE) in pGL3-Control vector containing SV40 enhancer resulted in strong expression of the luciferase gene only in telomerase positive and myc overexpressing SCLC cell line but not in normal human cell line. To investigate the possibility of the utilization of the MMRE, hTERT promoter, and SV40 enhancer in targeted SCLC gene therapy, adenovirus vector expressing HSV-TK controlled by the MMRE, hTERT promoter, and SV40 enhancer for the induction of telomerase positive and myc-overexpressing cancer specific cell death was constructed. SCLC cells infected with Ad-MMRE-hT-TK-enh were significantly suppressed and induced apoptosis more than those of Ad-hT-TK or Ad-hT-TK-enh infected cells. Telomerase and c-myc are activated in 60 approximately 80% of SCLC, so the increased activity of telomerase promoter can be used for targeted SCLC gene therapy. These results show that the MMRE, hTERT promoter, and SV40 enhancer can be used in SCLC targeted cancer gene therapy.     

Cyclical generation and degeneration of organs in a colonial urochordate involves crosstalk between old and new: a model for development and regeneration

Botryllus schlosseri is a colonial marine urochordate in which all adult organisms (called zooids) in a colony die synchronously by apoptosis (programmed cell death) in cyclical fashion. During this death phase called takeover, cell corpses within the dying organism are engulfed by circulating phagocytic cells. The "old" zooids and their organs are resorbed within 24-36 h (programmed cell removal). This process coincides temporally with the growth of asexually derived primary buds, that harbor a small number of undifferentiated cells, into mature zooids containing functional organs and tissues with the same body plan as adult zooids from which they budded. Within these colonies, all zooids share a ramifying network of extracorporeal blood vessels embedded in a gelatinous tunic. The underlying mechanisms regulating programmed cell death and programmed cell removal in this organism are unknown. In this study, we extirpated buds or zooids from B. schlosseri colonies in order to investigate the interplay that exists between buds, zooids, and the vascular system during takeover. Our findings indicate that, in the complete absence of buds (budectomy), organs from adult zooids underwent programmed cell death but were markedly impaired in their ability to be resorbed despite engulfment of zooid-derived cell corpses by phagocytes. However, when buds were removed from only half of the flower-shaped systems of zooids in a colony (hemibudectomy), the budectomized zooids were completely resorbed within 36-48 h following onset of programmed cell death. Furthermore, if hemibudectomies were carried out by using small colonies, leaving only a single functional bud, zooids from the old generation were also resorbed, albeit delayed to 48-60 h following onset of programmed cell death. This bud eventually reached functional maturity, but grew significantly larger in size than any control zooid, and exhibited hyperplasia. This finding strongly suggested that components of the dying zooid viscera could be reutilized by the developing buds, possibly as part of a colony-wide recycling mechanism. In order to test this hypothesis, zooids were surgically removed (zooidectomy) at the onset of takeover, and bud growth was quantitatively determined. In these zooidectomized colonies, bud growth was severely curtailed. In most solitary, long-lived animals, organs and tissues are maintained by processes of continual death and removal of aging cells counterbalanced by regeneration with stem and progenitor cells. In the colonial tunicate B. schlosseri, the same kinds of processes ensure the longevity of the colony (an animal) by cycles of death and regeneration of its constituent zooids (also animals).     

Ontogeny of telomerase in chicken: impact of downregulation on pre- and postnatal telomere length in vivo

Telomeres are the termini of linear chromosomes composed of tandem repeats of a conserved DNA sequence. Telomerase provides a mechanism for proliferating cells to offset telomeric sequence erosion by synthesizing new repeats onto the end of each parental DNA strand. Reduced or absent telomerase activity can lead to telomere shortening and genome instability. Telomeres and telomerase have not previously been characterized during ontogeny of any avian species. In the present study, telomerase activity in the chicken model was examined from early differentiation embryos through to adulthood. Telomerase activity was detected in all early embryos (preblastula through neurula) and in tissues throughout organogenesis. Subsequently, telomerase was downregulated in the majority of somatic tissues, either pre- or postnatally. A subset of tissues, such as intestine, immune and reproductive organs, exhibited constitutive activity. The impact of telomerase downregulation on telomere length was investigated and a telomere reduction of 3.2 kb in somatic tissues compared with germ line was observed in 5-year-old adults. The present results suggest that the telomere clock function is a conserved feature of avians as well as mammals. Knowledge regarding the relationships among telomerase regulation, proliferation/senescence profiles and differentiation status will be useful for numerous applications of chicken cells.     

Anti-apoptotic role of telomerase in pheochromocytoma cells

Telomerase is a protein-RNA enzyme complex that adds a six-base DNA sequence (TTAGGG) to the ends of chromosomes and thereby prevents their shortening. Reduced telomerase activity is associated with cell differentiation and accelerated cellular senescence, whereas increased telomerase activity is associated with cell transformation and immortalization. Because many types of cancer have been associated with reduced apoptosis, whereas cell differentiation and senescence have been associated with increased apoptosis, we tested the hypothesis that telomerase activity is mechanistically involved in the regulation of apoptosis. Levels of telomerase activity in cultured pheochromocytoma cells decreased prior to cell death in cells undergoing apoptosis. Treatment of cells with the oligodeoxynucleotide TTAGGG or with 3,3'-diethyloxadicarbocyanine, agents that inhibit telomerase activity in a concentration-dependent manner, significantly enhanced mitochondrial dysfunction and apoptosis induced by staurosporine, Fe2+ (an oxidative insult), and amyloid beta-peptide (a cytotoxic peptide linked to neuronal apoptosis in Alzheimer's disease). Overexpression of Bcl-2 and the caspase inhibitor zVAD-fmk protected cells against apoptosis in the presence of telomerase inhibitors, suggesting a site of action of telomerase prior to caspase activation and mitochondrial dysfunction. Telomerase activity decreased in cells during the process of nerve growth factor-induced differentiation, and such differentiated cells exhibited increased sensitivity to apoptosis. Our data establish a role for telomerase in suppressing apoptotic signaling cascades and suggest a mechanism whereby telomerase may suppress cellular senescence and promote tumor formation.     

Immunolocalization of the telomerase‐1 component in cells of the regenerating tail,testis, and intestine of lizards

Using an antibody against a lizard telomerase‐1 component the presence of telomerase has been detected in regenerating lizard tails where numerous cells are proliferating. Immunoblots showed telomerase positive bands at 75–80 kDa in normal tissues and at 50, 75, and 90 kDa in those regenerating. Immunofluorescence and ultrastructural immunolocalization showed telomerase‐immunoreactivity in sparCe (few/diluted) mesenchymal cells of the blastema, early regenerating muscles, perichondrium of the cartilaginous tube, ependyma of the spinal cord, and in the regenerating epidermis. Clusters of gold particles were detected in condensing chromosomes of few mesenchymal and epithelial cells in the regenerating tail, but a low to undetectable labeling in interphase cells. Telomerase‐immunoreactivity was intense in the nucleus and sparCe (few/diluted) in the cytoplasm of spermatogonia and spermatocytes and drastically decreased in early spermatids where some nuclear labeling remains. Some intense immunoreactivity was seen in few cells near the basal membrane of intestinal enterocytes or in leukocytes (likely lymphocytes) of the intestine mucosa. In spermatogonia, spermatids and in enterocytes part of the nuclear labeling formed cluster of gold particles in dense areas identified as Cajal Bodies, suggesting that telomerase is a marker for these stem cells. This therefore suggests that also the sparCe (few/diluted) telomerase positive cells detected in the regenerating tail may represent sparCe (few/diluted) stem cells localized in regenerating tissues where transit amplifying cells are instead preponderant to allow for tail growth. This observation supports previous studies indicating that few stem cells are present in the stump after tail amputation and give rise to transit amplifying cells for tail regeneration. J. Morphol. 276:748–758, 2015. © 2015 Wiley Periodicals, Inc.     

Telomerase activity could be used as a marker for neoplastic transformation in gastric adenocarcinoma: but it does not have a prognostic significance

Telomerase activity is responsible for telomere maintenance and is believed to be crucial in most immortal cells and cancer cells; however, its clinicopathological significance in gastric cancer remains to be clarified. The aim of the present study was to assess whether malignant progression of gastric adenocarcinoma correlates with telomerase activity. We also investigated the correlation between telomerase activity and histopathological findings. We examined telomerase activity in tumor specimens and adjacent normal tissues from 43 patients with gastric adenocarcinoma. Telomerase activity was measured quantitatively by the TRAPEZE Gel Based Telomerase Detection Kit. Approximately 98% of the tumor tissues were telomerase positive, but telomerase activity was detected not only in tumor tissues but also in normal gastric mucosa. Although telomerase activity was found to be higher in tumor samples than normal tissue for each subject, we could not find a general cut-off level for telomerase activity in gastric adenocarcinoma. In addition, telomerase activity was not correlated with tumor invasion, lymph node involvement and histological stage. Our results support the idea that telomerase reactivation is a common event in gastric adenocarcinoma and it is not related to histopathological parameters. Since it is difficult to set a cut-off level for this type of cancer, we suggest that the prognostic utility of telomerase assay has not yet reached the clinic in terms of predicting outcome for patients with gastric adenocarcinoma. For the assessment of gastric carcinoma, telomerase activity should be evaluated in both tumor and normal tissues, because normal gastric mucosa samples show appreciable telomerase activity.     

Stem cells are units of natural selection in a colonial ascidian

Stem cells are highly conserved biological units of development and regeneration. Here we formally demonstrate that stem cell lineages are also legitimate units of natural selection. In a colonial ascidian, Botryllus schlosseri, vascular fusion between genetically distinct individuals results in cellular parasitism of somatic tissues, gametes, or both. We show that genetic hierarchies of somatic and gametic parasitism following fusion can be replicated by transplanting cells between colonies. We prospectively isolate a population of multipotent, self-renewing stem cells that retain their competitive phenotype upon transplantation. Their single-cell contribution to either somatic or germline fates, but not to both, is consistent with separate lineages of somatic and germline stem cells or pluripotent stem cells that differentiate according to the niche in which they land. Since fusion is restricted to individuals that share a fusion/histocompatibility allele, these data suggest that histocompatibility genes in Botryllus evolved to protect the body from parasitic stem cells usurping asexual or sexual inheritance.