This page has been archived and is no longer updated
Essential role of mouse telomerase in highly proliferative organs
Author: H. Lee
Keywords
Keywords for this Article
Add keywords to your Content
Save
|
Cancel
Share
|
Cancel
Revoke
|
Cancel
Rate & Certify
Rate Me...
Rate Me
!
Comment
Save
|
Cancel
Flag Inappropriate
The Content is
Objectionable
Explicit
Offensive
Inaccurate
Comment
Flag Content
|
Cancel
Delete Content
Reason
Delete
|
Cancel
Close
Full Screen
"Nature � Macmillan Publishers Ltd 1998 8 NATURE | VOL 392 | 9 APRIL 1998 569 articles Essential role of mouse telomerase in highly proliferative organs Han-Woong Lee*??, Maria A. Blasco?�k, Geoffrey J. Gottlieb�, James W. Horner II*, Carol W. Greider?� & Ronald A. DePinho* * Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461, USA � Cold Spring Harbor Laboratory, PO Box 100, Cold Spring Harbor, New York 11724, USA kDepartment of Immunology and Oncology, Centro Nacional de Biotecnologia/CSIC, Universidad Autonoma Madrid, Campus Cantoblanco, Madrid E-28049, Spain � Quest Diagnostics Incorporated, One Malcolm Avenue, Teterboro, New Jersey 07608, USA ? These authors contributed equally to this work. ........................................................................................................................................................................................................................................................ We have investigated the role of the enzyme telomerase in highly proliferative organs in successive generations of mice lacking telomerase RNA. Late-generation animals exhibited defective spermatogenesis, with increased programmed cell death (apoptosis) and decreased proliferation in the testis. The proliferative capacity of haematopoietic cells in the bone marrow and spleen was also compromised. These progressively adverse effects coincided with substantial erosion of telomeres (the termini of eukaryotic chromosomes) and fusion and loss of chromosomes. These findings indicate an essential role for telomerase, and hence telomeres, in the maintenance of genomic integrity and in the long-term viability of high-renewal organ systems. Telomeres are guanine-rich, simple repeat sequences that constitute the physical termini of eukaryotic chromosomes 1 . Early work 2,3 indicated that these terminal structures may be important in chromosome function 4 . Synthesis and maintenance of telomeric repeats are mediated by a specialized ribonucleoprotein complex known as telomerase 5,6 . The telomerase holoenzyme consists of an essential RNA template 6,7 and protein components, including one with similarity to reverse transcriptases 8,13 . In the absence of telomerase, the failure of DNA polymerase to fully synthesize DNA termini leads to chromosomal shortening with each round of replication. As most somatic human tissues and primary cells possess low or undetectable telomerase activity, continual organ renewal in vivo or passage of cells in culture leads to a steady decline in telomere length 14,15 . Such a reduction in telomere length may play a role in replicative senescence, as constitutive telomerase activity can extend the proliferative capacity of normal human cells 16 . Viral oncoproteins or mutational events can drive senescent cultures through additional rounds of replica- tion with continued telomere loss until ?crisis? ensues?an event highlighted by marked genetic instability and cell death. From this crisis period, immortal cells emerge that have reactivated telomerase and maintain stable telomere structures 17?19 . The reactivation of telomerase during cellular immortalization matches well with what occurs during tumour progression in mice and humans. In particular, a marked upregulation of telomerase activity occurs in .90% of malignant samples compared with normal adjacent tissue 19 . These data led to the speculation that telomerase may be required for the efficient growth of neoplastic cells and that telomerase inhibition could represent a new thera- peutic approach for cancer 14,15 . We have recently shown that primary mouse cells lacking the mouse telomerase RNA (mTR) gene, and thus lacking telomerase activity, can still spontaneously become immortalized and generate a fully transformed tumorigenic phenotype after viral oncogenesis 20 , indicating that telomerase activity may not be limiting for tumorigenesis in the mouse. The idea that telomeres play an essential role in normal cellular growth and survival has been substantiated in unicellular eukaryo- tes. In yeast, deletion of the telomerase RNA gene leads to telomere shortening and, after a generational lag period, to loss of cell viability 21,22 . The lag period presumably comprises cell divisions during which telomeres shorten before they reach a critical length that is the threshold for chromosome instability and cell death. Elimination of a telomeric end from a yeast chromosome causes a RAD9-mediated cell-cycle arrest and chromosomal loss, indicating that telomeres may function to help cells distinguish intact chro- mosomes from broken DNA molecules and to maintain genomic stability 23,24 . In mammals, an in vivo role for telomerase and telomeres in normal cellular growth and long-term maintenance of organ systems has not yet been examined. On the basis of the phenotype of telomerase-null yeast and the great increase in chromosomal aberrations and aneuploidy in mTR - /- cells 20?22 , we expected that telomerase deficiency would have its greatest impact on highly proliferative organ systems. Here we demonstrate progressive adverse effects of telomerase deficiency on the reproductive and haematopoietic systems. These effects on cell renewal, coupled with telomere-length reductions and abnormal cytogenetic profiles, establish a role for telomerase in normal cellular homeostasis at the biological and genomic levels. Early-generation telomerase-deficient mice We reported previously a decline in telomere length with each successive generation of the mTR - /- mouse 20 . Here we performed a phenotypic analysis of each generation, and paid particular atten- tion to organ systems with high proliferative activity. Studies of an ageing population of the first-generation (G 1 ) mTR - /- mice (from heterozygous intercrosses) did not reveal adverse effects on the overall clinical condition (lifespan, motor behaviour/activity and weight gain) through 20 months of life (data not shown). Similarly, the haematopoietic organ histology and peripheral white and red blood cell counts, as well as the capacity of haematopoietic pro- genitor cells to grow and differentiate in vitro and of mature immunocytes to respond to mitogenic or infectious stimuli, were not significantly different from those of controls (see below). In addition, other organs with a more moderate level of renewal activity, such as intestine and skin, exhibited normal morphology and rates of proliferation and apoptosis (data not shown). These results indicate that telomerase activity per se is not essential in organogenesis or postnatal organ function or maintenance. It is possible that stressful conditions leading to increased cell turnover ? Present addresses: Department of Biochemistry and Molecular Biology, Ilchun Institute for Molecular Medicine, Seoul National University College of Medicine, Seoul 110-790, Korea (H.-W.L.); Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA (C.W.G.). Nature � Macmillan Publishers Ltd 1998 8 (for example, phlebotomy, chronic infection, skin wounding, peta- tectomy) might be able to elicit an age-dependent phenotype in old mTR - /- mice. Preliminary analysis indicates that some G 1 mTR - /- animals in two independent colonies exhibit erosive dermatitis, which might indicate a defect in dermal stem cells (H.W.L. and R.A.D., and E. H. Herrera, J. M. C. Caballero and M.A.B., unpub- lished observations). At present the limited sample size does not allow us to assign this phenotype unambiguously to the mTR deficiency, although studies are continuing. On the basis of previous studies in lower organisms that show a temporal lag between telomere loss and diminished cell viability, we suspected that substantial telomere loss would be required before a compromised clinical condition became apparent. Thus, as described 20 , we have produced additional generations (G 2 , G 3 , G 4 , G 5 and G 6 ) of mTR - /- mice from successive matings of homozygous- null intercrosses. Avoidance of inbreeding in the later generations was achieved through regular cousin-mating schemes 25 of offspring produced from 20 different G 1 mTR - /- matings; these G 1 mating pairs were in turn derived from 10 different mTR +/- intercrosses. Reproductive system Reproductive function is highly dependent on proper germ-cell expansion and development. All matings proved to be productive up to the fifth generation. However, a statistically significant decline in litter size became apparent in the G 4 intercrosses (Fig. 1a; G 4 P � 0:021 compared with G 1 ). No offspring were produced from eight different G 6 intercrosses. Both sexes appear to be affected, as only one in eight G 6 males and one in eight G 6 females produced a litter when mated to a wild-type partner. Male reproductive system. As the number of spermatocyte articles 570 NATURE | VOL 392 | 9 APRIL 1998 Figure 1 Effects of lack of telomerase on the mouse reproductive system. a, Impaired reproductive function in late-generation mTR-deficient mice. Males aged 6?12 weeks and females aged 4?12 weeks of the same generation of telomerase-deficient mice were mated over a period of 2 months or longer; n indicates the number of mating pairs examined. The P values of each generation were calculated compared with G 1 (G 2 , P � 0:063; G 3 , P � 0:070; G 4 , P � 0:021; and G 5 , P � 0:004). b, Testicular atrophy and testes weights. Scale bar, 5 mm. c, Uterine horns. Grossly dissected uterine horns (u) and ovaries (arrows) (scale bar, 5mm) and haematoxylin and eosin (H&E) staining (magnification � 50). d, Ovarian structure and function. Oestrus-matched ovaries are shown at � 50 magnification (left). E, endometrium; M, myometrium; GF, Graafian follicle; CL, corpora lutea. The graphs show a weight comparison of ovaries, average egg yields following natural mating with a wild-type stud male, and the percentage of in vitro development of pre-implantation embryos. BC, blastocyst. Figure 2 Proliferation and apoptosis in the male germ-cell compartment. a, Histologyof WTandG 6 testis.Top:sectionsatlow magnification(� 50)throughWT testes and representative G 6 testes show the diminished cellularity in the G 6 testes although there are some tubules undergoing nearly normal spermatogen- esis (left edge of G 6 section). At higher magnification (� 200 (bottom)), all stages of spermatogenesis are well represented in WT seminiferous tubules, whereas most G 6 tubules lack spermatogenesis yet retain Sertoli cells. In the G 6 testes, the apparent local accumulation of Leydig cells in the interstitium is secondary to contraction of the seminiferous tubules and an overall decrease in testis volume. Sn, Sertoli cell; L, Leydig cell; Sg, spermatogonia; Sc, primary spermatocyte; ES, early spermatid; LS, late spermatid. b, Comparative BrdU incorporation and TUNEL assays. Relative to WT samples, there was an approximate reduction in BrdU-positive nuclei (arrows) of three- to fivefold for G 3 mice and greater than 20- to 100-fold for G 6 mice. In the TUNEL (TdT) assay, G 3 seminiferous tubules and G 6 tubules with adequate cellularity for comparative analysis possessed many TUNEL-stained germ-cell nuclei (arrows), whereas only an occasional apoptotic cell was detected in the WT samples. Magnification � 100. Nature � Macmillan Publishers Ltd 1998 8 divisions from zygote to mature sperm has been estimated to be .60, whereas oocytes undergo arrest and await maturation after only 25 cell divisions 26 , we suspected that telomerase deficiency would be seen initially and most adversely in the male germ-cell compart- ment. On gross inspection, the size and weight of G 6 testes were found to be reduced by ,80% compared with age-matched wild-type or G 3 testes, whereas other organs (such as brain) or accessory reproductive organs (such as seminal vesicles) in G 6 animals were proportional to the overall body size (Fig. 1b). At the histological level, testes of wild-type through to G 4 mice possessed well-developed seminiferous tubules with a full complement of support cells (Sertoli cells) and germ cells representing all stages of spermatogenesis, similar to what is seen in wild-type age-matched controls (Fig. 2a). By the fifth generation, germ cells became depleted (data not shown) and, by the sixth generation, most seminiferous tubules showed a striking absence of spermatogenesis yet maintained an apparently adequate representation of the less proliferative Sertoli and Leydig cells (Fig. 2a). Thus spermatogenic cells, not support/ accessory cells, are particularly affected by telomerase deficiency in the G 6 mTR - /- mice. As sexual behaviour and seminal-vesicle development and structure are highly dependent on adequate levels of androgens 27 , the observations that male sexual performance (plugging efficiency) and seminal-vesicle size and structure were normal strongly indicate that germ-cell depletion is not a simple consequence of low serum testosterone levels (data not shown). Progressive hypospermia could result from a checkpoint-arrest response and/or diminished viability (apoptosis) as late-generation germ cells lose telomere sequences and experience genomic instabil- ity. To investigate these possibilities, we monitored the rates of proliferation and apoptosis in wild-type, G 3 , G 5 and G 6 testes. In situ assays of bromodeoxyuridine (BrdU) incorporation revealed abun- dant S-phase activity in many seminiferous tubules of the wild-type testes (Fig. 2b). In the histologically normal G 3 testes, a greater than fivefold decrease in the number of BrdU-positive nuclei was noted (Fig. 2b). This proliferative decline was more pronounced in the G 5 and G 6 testes, even in tubules with moderate cellularity (Fig. 2b; G 5 data not shown). Moreover, a marked increase in apoptosis (mea- sured with TdT-mediated dUTP nick end labelling (TUNEL)) was detected in the G 3 and G 6 testes compared with wild-type controls (Fig. 2b; compare G 3 and G 6 with wild-type). Thus, at least two processes are responsible for progressive germ-cell depletion: decreased proliferation and increased apoptosis. Female reproductive system. On gross examination, oestrus- staged G 6 uterine horns exhibited minimal to significant reductions in length and diameter compared with age- and stage-matched wild-type controls (Fig. 1c). G 6 ovaries also varied considerably in size and, on average, weighed markedly less than wild-type controls (Fig. 1d; wild-type 5:4 6 2:5 mg, G 6 2:6 6 2:0 mg; P � 0:0212). Detailed histological analysis of G 6 uterine horns showed normal endometrial thickness, integrity and cellularity; however, myo- metrial thickness was reduced and atrophy of the smooth muscle cells was increased in the smaller and more contracted G 6 uterine horns (Fig. 1c). Although histological examination of the ovaries confirmed the reduced size of the G 6 ovary, both wild-type and G 6 ovaries showed a full spectrum of follicular development including Graafian follicles and corpora lutea (Fig. 1d). To assess physiological aspects of reproduction, the plugging frequency and egg yields were measured after natural matings of G 6 or wild-type females with wild-type males. Under identical temporal and environmental conditions, 11 of 11 wild-type and 8 of 10 G 6 females were plugged and produced an average of 9.8 (6 3.8) and 3.9 (62.9) fertilized eggs per female, respectively (Fig. 1d, P � 0:0175). We then examined the capacity of these embryos to progress through early embryogenesis after culturing in vitro. Although all (15 of 15) wild-type/wild-type F 1 embryos developed to the blastocyst stage (Fig. 1d, P � 0:0061), only 46.7% (7 of 15) of G 7 /wild-type F 1 embryos developed to the morula stage and none progressed to the blastocyst stage. To compare reproductive-tract function, wild-type fertilized eggs were implanted into the oviducts of G 6 or wild-type pseudopregnant females. Only 2 of 7 G 6 females were able to support the full-term development of these embryos compared with 8 of 10 wild-type females. Thus, although the comparable plugging efficiencies indicate normal hormonal regula- tion and end-organ responsiveness, infertility probably results from several independent factors. These include a decrease in the number of oocytes upon ovulation, poor early developmental progression of G 7 wild-type F 1 embryos, and possible compromise in uterine structure/function. Haematopoietic system Most haematopoietic cells have a lifespan considerably shorter than that of other somatic cells, and these cells are continually replen- articles NATURE | VOL 392 | 9 APRIL 1998 571 Figure 3 Effects of the lack of telomerase on haematopoietic cells. a, Peripheral blood counts for red blood cells (RBC) and white blood cells (WBC). Peripheral blood was subjected to the Coulter STK/S counter analysis for the complete blood count. b, Haematopoietic colony assays. Formation of bone-marrow- derived colonies was assessed in vitro using cells obtained from WTand from G 6 femurs. Representative colonies of CFU-GEMM and CFU-GM are shown (magnification � 10). HPP-CFC colonies are scored on the basis of size (see Methods). c, Ratios of numbers of knockout (KO) to WT colonies. P values were calculated from the comparison between numbers of WT and homozygous-null mice of each generation; n is the number of mice that were used for each experiment. Nature � Macmillan Publishers Ltd 1998 8 ished by a self-renewing populations of stem cells 28 . A histological survey of adult bone marrow, spleen and thymus revealed normal organ size, architecture and cellularity through to the sixth genera- tion (data not shown). A complete peripheral blood count and profile of sex- and age-matched mice did not show any differences in all genotypes through successive generations of telomerase defi- ciency (Fig. 3a). Moreover, no differences were observed in the percentages of lymphocytes, neutrophils, basophils, eosinophils and monocytes. Fluorescence-activated cell sorting (FACS) analysis of splenocytes showed identical cell-type profiles as measured by antibodies against T cells (antibodies against CD4, D8, and CD3), B cells (anti-B220), lymphoid cells (anti-CD45), macrophages (anti-F4/80), and erythrocytes (anti-TER-119) (data not shown). Similarly, FACS analyses of bone-marrow cells showed identical profiles using antibodies against D45, F4/80, TER-119 and CD34 (stem cell) (data not shown). To test the immune response of telomerase-deficient mice, six wild-type and six G 5 naive mice were infected intravenously with a sublethal innoculum of Listeria monocytogenes. The two groups showed similar survival and recovery rates. Upon rechallenge with a dose that would be lethal to naive mice, all mice survived, thus demonstrating that gross function of the immune response and memory is not affected. Mature haematopoietic organ structure and function seem to be well compensated in mTR-deficient mice. As relatively few haematopoietic stem cells (HSCs) sustain long- term haematopoiesis, reductions in HSC numbers or growth/ survival potential might not be reflected in peripheral blood count measurements. Thus we used in vitro haematopoietic colony-forming unit (CFU) assays to determine whether the early progenitor-cell compartment was compromised. Bone-marrow cell suspensions derived from G 1 and G 3 mTR - /- mice were comparable to WT suspensions in their ability to generate CFU-granulocyte, monocyte (CFU-GM), CFU-granulocyte, erythrocyte, monocyte, megakaryocyte (CFU-GEMM) and high-proliferative-potential colony-forming cell (HPP-CFC) colonies (Fig. 3c). In contrast, multiple CFU assays comparing wild-type, G 1 , G 3 and G 6 progenitor- cell profiles showed a statistically significant decrease in the total number of CFU-GM (P � 0:0028), CFU-GEMM (P � 0:0011) and HPP-CFC (P � 0:0005) colonies in the G 6 samples (Fig. 3b, c). These results indicate that the long-term renewal of haematopoietic stem cells is compromised upon telomere loss. Nevertheless, the stochastic growth behaviour of these cells may allow the generation of a reserve pool that is still capable of populating the peripheral haematopoietic compartment. Lymphocyte mitogenic response Telomerase activity is low or undetectable in normal human B and T cells and increases dramatically after in vitro mitogenic stimulation 29,30 . This transient increase in telomerase activity indi- cates that somatic activation of telomerase may delay replicative senescence and facilitate mitogen-induced clonal expansion 31 . To determine the effects of telomerase deficiency on the ability of mature lymphocytes to proliferate, we exposed splenocytes, isolated from wild-type through to G 6 mTR - /- mice, mitogens that mainly activate T cells (phorbol myristate acetate (PMA) plus ionomycin (Ion), or anti-CD3/CD28 antibodies, or a more general T-cell- mitogenic activator (Concanavalin A (Con A)). For the three mitogenic stimuli, splenocytes derived from wild-type or G 1 through to G 4 mTR - /- mice showed almost identical degrees of uptake of 3 H-labelled thymidine, although a decrease first became apparent in an occasional G 4 mouse (data not shown). In contrast, G 5 (four mice) and G 6 (three mice) stimulated mTR - / - splenocytes exhibited a statistically significant decrease in incorporation of 3 H-labelled thymidine compared with wild-type (seven mice) con- trols (Fig. 4a). Incorporation of 3 H-labelled thymidine in G 6 mTR - /- splenocytes was ,32% with Con A, ,38% with PMA/Ion, and ,39% with anti-CD3/CD28 antibodies relative to wild-type controls (Fig. 4a). To determine the basis for the decreased incorporation of 3 H- labelled thymidine, we assayed the cell-cycle profile and the percen- tage of apoptosis after PMA/Ion stimulation of wild-type and G 6 splenocytes. Although wild-type and G 6 cell-cycle profiles were nearly identical after stimulation, a marked increase in the percen- tage of late apoptotic cells (annexin-positive and propidium iodide (PI)-positive) was observed in the G 6 splenocytes; this increase was inversely proportional to the decrease in incorporation of 3 H-labelled thymidine (Fig. 4b). The normal mitogenic response of earlier generation mTR - /- splenocytes and the pronounced and progressive effect on the later generations indicates that, although telomerase does not play a direct role in splenic lym- phocyte activation and expansion, telomere shortening and chromosomal aberrations in late generations may lead to induction of apoptosis, possibly through either DNA-damage-response path- ways or significant chromosome loss or rearrangement after mitogenic stimulation. Chromosome analysis To examine whether the impaired mitogenic (pro-apoptotic) response of cultured lymphocytes could be related to significant telomere shortening and genomic instability, PMA/Ion- or Con A- activated splenocytes from two wild-type and six G 6 mTR - /- mice were treated with colcemid and karyotyped by G-banding. The percentage of activated cells with an abnormal chromosome count (aneuploidy) was 16.7% (7 of 42) for mTR +/+ and 37.7% (53 of 144) for G 6 samples (Table 1a). In contrast, control metaphase spreads of articles 572 NATURE | VOL 392 | 9 APRIL 1998 Figure 4 Response of lymphocytes to lack of telomerase. a, Splenocyte mitogenic response. Splenocytes from WT, G 5 and G 6 homozygous-null animals were mitogenically activated and their uptake of 3 H-labelled thymidine was measured. The y-axis represents the percentage of fold-activation of mTR- deficient splenocytes by indicated mitogens (x-axis) compared with activation of WT splenocytes. Mitogenic activation of WT splenocytes was set at 100%. Error bars indicate standard deviations from quadruplicated samples of four G 5 mice and three G 6 mice. Black bars show the data from G 5 mice and grey bars indicate data from G 6 mice. b, Cell-cycle profile and annexin/PI-stained FACS profiles. Cell-cycle profiles are shown at the left. Percentages of the early and late stages of apoptotic cells are shown at the right. There were approximately 30% more G 6 apoptotic splenocytes (annexin-positive) than WT apoptotic splenocytes 48h after activation by PMA/Ion. Nature � Macmillan Publishers Ltd 1998 8 mTR +/+ mouse embryonic fibroblast (MEF) cells showed no (0 of 17) aneuploidy (data not shown); the high baseline aneuploidy in activated mTR +/+ splenocytes is not understood. When splenocyte metaphase spreads were scored for abnormal chromosome struc- tures, 3.6% (2 of 55) of wild-type and 21.1% (40 of 190) of G 6 samples had chromosomes with abnormal G-banding patterns. We found end-to-end fusions that seem to be of the Robertsonian type 32,33 . To identify the specific chromosomes involved in the fusions, we conducted chromosomal G-banding on randomly selected G 6 metaphases and on the sole wild-type metaphases exhibiting fusion. In seven informative cases for the activated G 6 splenocytes, chromosomes 3, 13 and 14 were involved in the fusion events. This differed from the fusions in the two wild-type samples, where chromosomes 1 and X were involved (Table 1b). Although the frequent involvement of chromosome 3 in the G 6 samples (5 out of 7 fusions; Table 1b) is intriguing and could relate to the unusually short telomeres of its short (p) arm 34 , the biological significance of this is unknown. Studies of human tumours and ageing cells did not show correlations between telomere length and frequency of involvement in fusion events for a given chromosome 35 . Finally, analysis of telomere length in several tissues by Southern blotting did not show any significant differences between the different generations or among tissues of the same generation (data not shown). In contrast, fluorescence in situ hybridization (FISH) using a probe for telomere repeat DNA generated fewer than four end signals in only a few chromosomes (,7.5%) in the wild- type metaphase spreads, whereas far more chromosomes in G 6 mTR - /- cells exhibited fewer than four signals (,58.75%) (data not shown). As all Robertsonian fusions in the mTR - /- sample failed to generate a signal at their junctions, these fusion events seem to be caused by the loss of telomere function at these chromosomes. Discussion In dividing cells, telomerase is essential in telomere maintenance and, in its absence, significant erosion of telomeres is thought to signal replicative senescence and eventually lead to a crisis period highlighted by genetic instability and loss of cell viability. The presence of telomerase activity in germ cells and highly proliferative stem-cell compartments, and its upregulation in activated T lym- phocytes, indicate that telomerase may be required for the pro- liferation and long-term viability of such cells. Here we demonstrate that telomerase deficiency leads to a depletion of male germ cells, diminished haematopoietic colony formation, and impaired mito- gen-induced proliferation of primary splenocytes. In the testes, the significant decline in BrdU incorporation and increase in TUNEL-positive nuclei suggests that telomere short- ening leads to both checkpoint and apoptotic responses in vivo. In T cells, mitogenic stimulation of cells with short telomeres led to cytogenetic abnormalities and increased apoptosis. These results indicate that telomerase is essential for the long-term maintenance of diverse cell types with high proliferation profiles, and suggest that alterations in telomere structure dramatically affect the number, growth potential and/or survival of renewing cell populations. Marked aneuploidy and Robertsonian fusions were observed with high frequency in activated splenocytes derived from G 6 mTR - /- mice, a finding similar to that reported for late passage mTR - /- MEF culture 20 . These chromosomal anomalies were associated with a striking absence in telomere-repeat signal on a pronounced number of chromosome ends, further substantiating the concept that telomeres are vital in maintaining chromosomal integrity and genomic stability in normal cells in vivo. The delay of several generations in emergence of a phenotype parallels the lag period observed in telomerase-deficient yeast cells, in which viability was compromised only after many cell divisions. The long latency in the appearance of defects in the mouse may simply relate to the long telomere lengths of the laboratory mouse strain Mus musculus 37 . Once significant telomere loss is reached, some cell types seem to be more adversely affected than others. The basis for the more prominent phenotype in the haematopoietic and germ cells could relate to a higher level of cell turnover and resultant greater telomere loss; however, other highly proliferative organs such as the intestine appear to be unaffected. Some cell types might be intrinsically less tolerant of genetic instability, perhaps reflecting a strategy to ensure faithful transmission of genetic information in the case of sperm cells or to guard against haematopoietic malig- nancies over a lifetime of intense proliferation. A detailed compar- ison of telomere lengths and karyotypes among these various cell types will be necessary to better resolve this issue. Telomerase deficiency ultimately produced a reduction in pro- liferation and an increase in apoptosis in the mouse male germ cells. Such a response as the reduction of telomeres to ?critical length? in cultured human cells correlates with cessation of proliferation and maintenance of a viable senescent state. Human cells will remain in this state unless this checkpoint is commandeered by viral onco- proteins or genetic mutation. The difference between mice and humans may be related to the fact that the rodent cells find it easier to escape from the slow-growth ?senescent? phase than do human cells, the growth of which is tightly regulated. Thus the mouse cells continue more readily towards crisis. These studies demonstrate that, although telomerase activity per se does not play a direct biological role in mammalian development, telomerase is vital in cell survival and organ homeostasis through maintenance of adequate telomere structure during cell division. The mouse model described here provides a system with which to understand the complexities of telomere dynamics and how they relate to cell physiology. These studies provide new insights into basic telomere biology and help us anticipate the systemic effects of telomerase inhibitors in cancer therapy. The maintenance of normal peripheral blood cell counts, immune competence and gastro- intestinal function in the telomerase-deficient mouse indicates that diverse stem-cell compartments may sustain organ function after systemic administration of telomerase inhibitors. The high articles NATURE | VOL 392 | 9 APRIL 1998 573 Table 1 Cytogenic analysis a Number of chromosomes per metaphase Number of metaphases WT G 6 ............................................................................................................................................................................. .40 1 12 40 (normal) 35 91 39 1 26 38 1 7 37 1 4 36 1 0 35 1 1 34 0 1 33 0 3 32 1 0 31 0 2 ............................................................................................................................................................................. Total number of metaphases examined 42 155 ............................................................................................................................................................................. Aneuploidy (%) 16.7 37.7 ............................................................................................................................................................................. b Sample number Chromosome involved in fusions WT G 6 ............................................................................................................................................................................. 1 t(1;1) t(3;3) 2 t(X;#) t(3;3) 3 t(3;3) 4 t(3;13) 5 t(3;14) 6 t(13;13) 7 t(14;14) ............................................................................................................................................................................. a, Metaphase spreads derived from activated WT and G 6 splenocytes were examined for abnormal chromosome number (aneuploidy). Percentages of aneuploidy were calculated by the number of metaphases carrying abnormal chromosome number (not 40) divided by thetotalnumberofmetaphasesexamined.b,Chromosomenumbersthatparticipatedinthe fusions were identified by G-banding and listed. All fusions seemed to be the Robertsonian type and the short (p) arms were involved in all cases. #, not identifiable. Nature � Macmillan Publishers Ltd 1998 8 proliferative index of most cancer cells compared with the more sporadic cycling of normal stem-cell populations suggests that telomerase inhibition will be well tolerated in clinical settings. M . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methods Mice. The genetic background of the mice (WW6/C57BL/6J) is identical to that of our previous work 20 . Age- and sex-matched animals were used in all experiments, with ages ranging from 6 to 14 weeks old. For analysis of female reproductive system, females 6?14 weeks old of the indicated genotypes were mated with CBA/B6 F 1 stud males. Fertilized eggs were cultured in KSOM media (Specialty). Haematopoieticcolonyassay. Murine colony assay was performed according to the manufacturer?s recommendation (StemCell Technologies). The colony counts for CFU-GM were performed on days 7?10 and for CFU-GEMM and HPP-CFC on days 12?14. HPP-CFC was defined as those colonies measuring 0.5 mm on day 12?14 (ref. 38). Incorporation of 3 H-labelled thymidine and FACS analyses. Splenocytes were resuspended in RPMI1640/10% FBS/0.55 mM b-mercaptoethanol. The mitogens used (Sigma or Pharmingen) were as follows: PMA (20 nM), ionomycin (1 mM), Con A (4mg ml - 1 ), hamster anti-mouse CD3e (10 ng ml - 1 ) and hamster anti-mouse CD28 (10 ng ml - 1 ). After a pulse of 3 H-labelled thymidine, the cells were collected at 48 h following the addition of mitogen. The incorporated c.p.m. was determined by a liquid scintillation counter. We processed samples in quadruplicate and calculated averages, standard deviations and P values. To analyse haematopoietic subpopulations by FACS, cells from each organ were incubated with antibodies (Pharmingen or Caltag) and analysed by FACScan (Becton Dickinson Immunocytometry Systems; LYSIS II). The antibodies were CD45-PE, IgM-PE, CD4-PE, CD3-fluorescein isothiocyanate (FITC), B220-FITC, CD8-ITC and MAC-1-FITC. Cell-cycle analysis and apoptosis assay. Splenocytes were activated by PMA/Ion for 48 h. Cell-cycle and apoptosis assays were then used. For cell-cycle analysis we modified the method in ref. 39. For apoptosis assays, we used annexin-V-Fluos (Boehringer) to detect the apoptotic cell population accord- ing to the manufacturer?s recommendation. For tissue sections, in situ apoptosis and BrdU incorporation assays were performed as described 40 . Cytogenetic and FISH analyses. Metaphase spreads of splenocytes were prepared and processed for G-banding by standard methods 41 following activation by Con A or PMA/Ion and colcemid treatment. For FISH analysis, hybridization of FITC-conjugated (TTAGGG) n (Oncor) was performed according to the manufacturer?s recommendations. Received 29 September 1997; accepted 17 February 1998. 1. Greider, C. W. Telomere length regulation. Annu. Rev. Biochem. 65, 337?365 (1996). 2. Muller, H. J. The remaking of chromosomes. Collect. Net 8, 182?195 (1938). 3. McClintock, B. The stability of broken ends of chromosomes in Zea mays. Genetics 26, 234?282 (1941). 4. Greider, C. W. Chromosome first aid. Cell 67, 645?647 (1991). 5. Greider, C. W. & Blackburn, E. H. Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell 43, 405?413 (1985). 6. Greider, C. W. & Blackburn, E. H. A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeat synthesis. Nature 337, 331?337 (1989). 7. Feng, J. et al. The RNA component of human telomerase. Science 269, 1236?1241 (1995). 8. Lingner, J. et al. Reverse transcriptase motifs in the catalytic subunit of telomerase. Science 276, 561? 567 (1997). 9. Harrington, L. et al. Human telomerase contains evolutionarily conserved catalytic and structural subunits. Genes Dev. 11, 3109?3115 (1997). 10. Kilian, A. et al. Isolation of a candidate human telomerase catalytic subunit gene, which reveals complex splicing patterns on different cell types. Hum. Mol. Genet. 6, 2011?2019 (1997). 11. Nakayama, J. et al. Telomerase activation by hTRT in human normal fibroblasts and hepatocellular carcinomas. Nature Genet. 18, 65?68 (1998). 12. Meyerson, M. et al. HEST2, the putative human telomerase catalytic subunit gene, is upregulated in tumor cells and during immortalization. Cell 90, 785?795 (1997). 13. Nakamura, T. M. et al. Telomerase catalytic subunit homologs from fission yeast and human. Science 277, 955?959 (1997). 14. Harley, C. B., Futcher, A. B. & Greider, C. W. Telomeres shorten during aging of human fibroblasts. Nature 345, 458?460 (1990). 15. Hastie, N. D. et al. Telomere reduction in human colorectal carcinoma and with ageing. Nature 346, 866?868 (1990). 16. Bodnar, A. G. et al. Extension of life-span by introduction of telomerase into normal human cells. Science 279, 349?352 (1998). 17. Counter, C. M. et al. Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity. EMBO J. 11, 1921?1929 (1992). 18. Prowse, K. R. & Greider, C. W. Developmental and tissue-specific regulation of mouse telomerase and telomere length. Proc. Natl Acad. Sci. USA 92, 4818?4822 (1995). 19. Kim, N. W. et al. Specific association of human telomerase activity with immortal cells and cancer. Science 266, 2011?2015 (1994). 20. Blasco, M. A. et al. Telomere shortening and tumor formation by mouse cells lacking telomerase RNA. Cell 91, 25?34 (1997). 21. Singer, M. S. & Gottschling, D. E. TLC1: template RNA component of Saccharomyces cerevisiae telomerase. Science 266, 403?409 (1994). 22. McEachern, M. J. & Blackburn, E. H. Runaway telomere elongation caused by telomerase RNA gene mutations. Nature 376, 403?409 (1995). 23. Haber, J. E. & Thorburn, P. C. Healing of broken linear dicentric chromosomes in yeast. Genetics 106, 207?226 (1984). 24. Sandell, L. L. & Zakian, V. A. Loss of a yeast telomere: arrest, recovery, and chromosome loss. Cell 75, 729?739 (1993). 25. Wright, S. Systems of mating. Genetics 6, 111?178 (1921). 26. Drost, J. B. & Lee, W. R. Biological basis of germline mutation: comparisons of spontaneous germline mutation rates among Drosophila, mouse, and human. Environ. Mol. Mutagen. 25 (suppl. 26), 48?64 (1995). 27. Shima, H., Motomu, T., Young, P. & Cunha, G. R. Postnatal growth of mouse seminal vesicle is dependent on 5a-dihydrotestosterone. Endocrinology 127, 3222?3233 (1990). 28. Erslev, A. J. & Lichtman, M. A. in Hematology 4th edn 37?47 (McGraw-Hill, 1993). 29. Buchkovich, K. J. & Greider, C. W. Telomerase regulation during entry into the cell cycle in normal human T cells. Mol. Biol. Cell 7, 1443?1454 (1996). 30. Weng, N.-P., Levine, B. L., June, C. H. & Hodes, R. J. Regulated expression of telomerase activity in human T lymphocyte development and activation. J. Exp. Med. 183, 2471?2479 (1996). 31. Bodnar, A. .G., Kim, N. W., Effros, R. B. & Chiu, C. P. Mechanism of telomerase induction during T cell activation. Exp. Cell Res. 228, 58?64 (1996). 32. Garagna, S. et al. Robertsonian metacentrics of the house mouse lose telomeric sequences but retain some minor satellite DNA in the pericentromeric area. Chromosoma 103, 685?692 (1995). 33. Nanda, I., Schneider-Rasp, S., Winking, H. & Schmid, M. Loss of telomeric sites in the chromosomes of Mus musculus domesticus (Rodentia: Muridae) during Robertsonian rearrangement. Chromosome Res. 3, 399?409 (1995). 34. Zijlmans, J. M. et al. Telomeres in the mouse have large inter-chromosomal variations in the number of T 2 AG 3 repeats. Proc. Natl Acad. Sci. USA 94, 7423?7428 (1997). 35. Saltman, D., Morgan, R., Cleary, M. L. & de Lange, T. Telomeric structure in cells with chromosome end associations. Chromosoma 102, 121 (1993). 36. McEachern, M. J. & Blackburn, E. H. Cap-prevented recombination between terminal telomeric repeat arrays (telomere CAPR) maintains telomeres in Kluyveromyces lactis lacking telomerase. Genes Dev. 10, 1822?1834 (1996). 37. Kipling, D. & Cooke, H. J. Hypervariable ultra-long telomeres in mice. Nature 347, 400?402 (1990). 38. Watt, S. M. & Visser, J. W. M. Recent advances in the growth and isolation of primitive human haemopoietic progenitor cells. Cell Prolif. 25, 263?297 (1992). 39. Vindelov, L. L., Christensen, I. J. & Nissen, N. L. A detergent-trypsin method for the preparation of nuclei for flow cytometric DNA analysis. Cytometry 3, 323?327 (1983). 40. Morgenbesser, S. D. et al. P53-dependent apoptosis produced by Rb-deficiency in the developing mouse lens. Nature 371, 72?74 (1994). 41. Cannizzaro, L. A. & Shi, G. Fluorescent in situ hybridization (FISH) for DNA probes in the interphase and metaphase stages of the cell cycle. Methods Mol. Biol. 75, 313?322 (1997). Acknowledgements. We thank L. Cannizzaro, M. Zohouri, D. Gebhard, R. Mazzaccaro, B. Furman, K. E. Cedeno-Baier, L. Husted, A. Silverman, N. Squillante, H. Hou and J. Lauridsen for advice and assistance; N. Schreiber-Agus, R. Greenberg, L. Chin, J. Pollard, E. R. Stanley and R. Kucherlapati for critical reading of the manuscript and advice; and E. Herrera and J. M. Caballero for sharing the dermatitis data collected in the National Center of Biotechnology (Madrid) before publication. Work in the lab of R.A.D. was supported by the NIH, AHA Grant-in-Aid and a Liver Center Initiative. R.A.D. is a recipient of the Irma T. Hirschl Career Scientist Award. R.A.D. and C.W.G. are also supported by Cancer Core grants. Work in the lab of C.W.G. was supported by the NIH and by Geron Corporaiton. M.A.B. was supported by the Leukemia Society of America, a grant from the Ministry of Education of Spain, and the Department of Immunology and Oncology (founded by the Spanish research Council and Pharmacia-Upjohn). H.W.L. was supported by an NIH postdoctoral training grant. Correspondence and requests for materials should be addressed to R.A.D. or C.W.G. articles 574 NATURE | VOL 392 | 9 APRIL 1998 "
Add Content to Group
|
Bookmark
|
Keywords
|
Flag Inappropriate
share
Close
Digg
Facebook
MySpace
Google+
Comments
Close
Please Post Your Comment
*
The Comment you have entered exceeds the maximum length.
Submit
|
Cancel
*
Required
Comments
Please Post Your Comment
No comments yet.
Save Note
Note
View
Public
Private
Friends & Groups
Friends
Groups
Save
|
Cancel
|
Delete
Please provide your notes.
Next
|
Prev
|
Close
|
Edit
|
Delete
Genetics
Gene Inheritance and Transmission
Gene Expression and Regulation
Nucleic Acid Structure and Function
Chromosomes and Cytogenetics
Evolutionary Genetics
Population and Quantitative Genetics
Genomics
Genes and Disease
Genetics and Society
Cell Biology
Cell Origins and Metabolism
Proteins and Gene Expression
Subcellular Compartments
Cell Communication
Cell Cycle and Cell Division
Scientific Communication
Career Planning
Loading ...
Scitable Chat
Register
|
Sign In
Visual Browse
Close
Comments
CloseComments
Please Post Your Comment