Overexpression of Telomerase Confers Growth Advantage, Stress Resistance, and Enhanced Differentiation of ESCs Toward the Hematopoietic Line
http://www.100md.com
《干细胞学杂志》
a Department of Biological Sciences, University of Durham, Durham DH1 3LE, United Kingdom;
b Institute for Ageing and Health, University of Newcastle upon Tyne NE6 4BE, United Kingdom;
c Institute of Human Genetics, University of Newcastle upon Tyne NE1 3BZ, United Kingdom
Key Words. ESCs ? Telomerase ? Hematopoietic stem cells ? Oxidative stress ? Cell proliferation ? Cell cycle ? Apoptosis
Correspondence: Majlinda Lako, Ph.D., Institute of Human Genetics, International Centre for Life, Central Parkway, Newcastle upon Tyne NE1 3BZ, U.K. Telephone: 00-44-191-241-8688; Fax: 00-44-191-241-8666; e-mail: Majlinda.Lako@ncl.ac.uk
ABSTRACT
Embryonic stem cells (ESCs) are derived from the inner cell mass of blastocysts, and under appropriate culture conditions they can be maintained indefinitely in culture while preserving their pluripotency and genomic stability. There are only very small numbers of ESCs that give rise to all tissues of the embryo proper. This small size of the population means that ESCs need to be equipped with efficient mechanisms of preventing and repairing DNA damage. In fact, murine ESCs differ from their more differentiated counterparts by high levels of antioxidant defense and good DNA strand break repair capacity . Furthermore, telomerase activity decreases during differentiation of mouse and human ESCs, mostly because of transcriptional downregulation of the telomerase reverse transcriptase, Tert . Currently, it is not known whether the downregulation of telomerase during differentiation of ESCs influences the ability of the derived progeny to cope with DNA damage, oxidative stress, and the differentiation process itself. This is an important issue in regenerative medicine, given the current efforts to selectively differentiate human ESCs into various precursors for use in cell-replacement therapies. In the study reported here, we wanted to investigate whether the continuous expression of telomerase during differentiation can help to transfer some of the excellent properties of ESCs to ESC-derived progeny.
The telomerase holoenzyme core encompasses a reverse transcriptase (TERT) that catalyzes the addition of new repeats and a structural RNA component (TR) containing the template region that binds the telomere repeats . Telomeric DNA consists of regions composed of conserved tandem hexanucleotide repeats with a protruding G-rich overhang and is replicated by the ribonucleoprotein telomerase. Most human somatic cells show very little or no telomerase activity, and their chromosome ends shorten at each cell division, thereby limiting the replicative lifespan and leading eventually to senescence . Immortalized human cells and cancer cells, however, exhibit stable telomere length upon prolonged propagation in culture and typically have detectable telomerase activity . In addition to maintaining telomere length, telomerase may also prevent the loss of G-rich single-stranded overhangs that participate, together with specific telomere-binding proteins, in forming the T-loop structure . Erosion of the telomeric overhang can be a cause or a consequence of the collapse of the T-loop structure and the uncapping of the telomere end that eventually signals DNA damage and activates the senescence pathway .
In addition to detection in cancer cells and in artificially immortalized cells, telomerase activity has been found in various tissues with self-renewal capacity, including the basal layer of the epidermis , intestinal crypt cells , germ line cells , and the hematopoietic system , and at lower levels in cycling primary presenescent fibroblasts . This suggests that proper maintenance of telomere length and structure is required for the self-renewal function. Supportive evidence comes from mice lacking telomerase activity which exhibit functional impairment in organs composed of highly proliferative cells in late generations .
Telomerase knock-down experiments in human tumor cells and overexpression studies in somatic cells have identified telomerase as a "survival enzyme" that not only allows long-term unlimited growth but also improves cellular resistance against a wide variety of stressors and cytotoxic agents. In many cases, this survival function appears unrelated to maintenance of telomere length . Possible mechanisms include length-independent stabilization of the telomere cap or induction of stress defense genes via unknown pathways .
Telomerase activity has also been implicated in the maintenance of stem cell function and self-renewal, especially in the hematopoietic system. Primitive hematopoietic stem cells (HSCs) are typically quiescent and display low levels of telomerase activity, which is upregulated upon mitogenic stimulation . Telomerase-deficient HSCs show a reduced long-term repopulating capacity and increased genomic instability compared with wild-type cells . In patients with dyskeratosis congenita, mutations in different genes compromise the function of telomerase. This leads to enhanced telomere shortening and functional deficiencies in tissues such as gut, skin, and bone marrow, all of which depend on renewal from stem cells .
Current knowledge of TERT regulation and telomerase activity in normal development is limited. ESCs afford a good model system because they can be propagated indefinitely in culture and differentiated into various cell types, recapitulating many aspects of early embryonic development . In the mouse, telomerase activity is important for ESC growth. Deletions leading to loss of either the telomerase RNA or its reverse transcriptase result in progressive loss of telomeres, genomic instability, aneuploidy, telomeric fusions, and eventual reduced growth rate . Recent work has indicated that human ESCs express the TERT gene and show high levels of telomerase activity; however, upon differentiation, the levels of TERT and telomerase activity decrease with the emergence of a maturing population of cells . It is not clear from these studies whether or not the down-regulation of telomerase activity is necessary for the differentiation to proceed normally. Here, we stably overexpressed Tert in murine ESCs and investigated the effects of continuous telomerase expression on cell proliferation and apoptosis, differentiation toward hematopoietic lineage, and resistance to oxidative stress.
MATERIALS AND METHODS
Overexpression of Tert in Murine ESCs
We have shown before that telomerase is downregulated by more than one order of magnitude upon differentiation of murine ESCs to EBs . To investigate the effects of continued telomerase expression during the differentiation of ESCs, we stably transfected a construct containing the full-length cDNA of Tert under the control of MPSV LTR, resulting in significant overexpression of Tert as measured by real-time RT-PCR using primers directed against the mouse Tert-coding sequence, which amplify both endogenous and exogenous Tert mRNA. One single clone (tert-1) and one pooled clone (tert-2) were selected for further analysis. To measure telomerase activity, we performed TRAP assays under linear conditions of product amplification, which allowed for semi-quantitative analysis of TRAP activity levels. A significant increase in telomerase activity was observed in Tert-transfected cells (Fig. 1A). Although tert-1 ESC and tert-2 ESC clones over-expressed Tert mRNA at different levels, the telomerase activity levels were similar to each other. Most probably, an 11-fold over-expression of Tert as found in tert-2 ESCs is already sufficient to saturate the formation of active telomerase complexes. During differentiation, telomerase activity decreased in control ESCs but was maintained in the Tert-transfected cells (Fig. 1B).
Figure 1. Overexpression of Tert results in maintenance of telomerase activity during the differentiation of embryonic stem (ES) cells. (A): Telomerase activity was measured in a dilution series by semiquantitative TRAP ELISA in control (wild-type) ES cells and Tert-overexpressing ESCs. The results represent the mean ± SEM from three independent experiments. Asterisks denote a statistically significant difference (Student t-test) versus wild-type: *p < .05; **p < .01. (B): Telomerase activity from 10 ng of cell extract was measured using the TRAP-ELISA assay. Telomerase-specific products were determined by the presence of a 6-bp incremental DNA ladder on a 12% polyacrylamide gel stained with ethidium bromide, which indicates the addition of the 6-bp repeats (TTAGGG) to the synthetic primer by telomerase. Abbreviation: EB, embryoid body.
To investigate whether continuous overexpression of Tert in ESCs contributes to gross genomic instability, we carried out G banding on Tert-transfected and wild-type ESCs at three different population doublings (Table 2). Observation of 30 metaphases for tert-1 ESCs and tert-2 ESCs showed no deviations from the normal karyotype at PD130. Increasing aneuploidy was observed in metaphases obtained at PD190 and PD300 for both wild-type and Tert-transfected ESCs. It is well known that murine ESCs in prolonged culture become severely aneuploid . Chi-square analysis (p < .01) revealed that Tert overexpression did not increase the severity or frequency of these aberrations.
Table 2. Frequency of aneuploidy in control and Tert-overexpressing embryonic stem cells
Using pulsed field gel electrophoresis we could not detect any changes in average telomere length or telomere length distribution (data not shown). Flow-FISH is more sensitive for the detection of small length differences in the highly heterogeneous telomeres from mice . We found that telomere fluorescence intensity measured by Flow-FISH did not change significantly during the differentiation of either wild-type or Tert-overexpressing ESCs into day 6 EBs (data not shown). There was some variation in G-rich overhang length between Tert-overexpressing ESCs, possibly due to clonal selection. Importantly, the length of G-rich single-stranded overhangs decreased during the differentiation of wild-type ESCs to EBs, while no significant change was observed during the differentiation of the Tert-overexpressing ESCs (Fig. 2; supplementary online Fig. 2).
Figure 2. Expression of Tert maintains G-rich single-stranded overhangs during differentiation of embryonic stem cells (ESCs). Overhang length was measured as hybridization intensity ratio native versus denatured gel . Overhang length of wild-type (control) ESCs was set at 1. The data are mean ± SEM from four independent experiments. Embryoid bodies (EBs) are from day 6 of the differentiation protocol (*p < .05 ). Abbreviation: ns, not significant.
Tert-transfected ESCs grew faster than the control (Fig 3A). This appeared to be due to faster transit of Tert-transfected cells through the G2 and M phases of the cell cycle and, accordingly, higher fractions of cells in S phase (Fig. 3B). The doubling time for tert-1 ESCs is 16.6 hours and for tert-2 is 16.3 hours, compared with the control clone which is 23.2 hours. In view of this result, it is possible that the G2/M checkpoint might have been activated. It is equally possible that the cells simply transit G2/M phase more rapidly because of their intact telomeres.
Figure 3. Continued expression of Tert during differentiation of embryonic stem (ES) cells results in accelerated cell proliferation and faster transit through G2/M. (A): Cell growth is shown as cumulative population doublings (PDs) versus time. PDs were set to 0 at the beginning of the experiment. (B): Frequencies of wild-type (control) and Tert-overexpressing ES cells in different cell cycle phases, as measured by propidium iodide staining and flow cytometry. The data are mean ± SEM from three independent experiments. Asterisks denote a statistically significant difference (Student t-test) versus wild-type: *p < .001.
We next investigated the effect of Tert overexpression on a poptosis. Cell death was induced by adding a protein kinase C inhibitor, bisindolylmaleimide II . We found that overexpression of Tert in ESCs conferred a higher resistance to inhibitor-induced apoptosis (Fig. 4A). Frequencies of both spontaneous cell death and inhibitor-induced apoptosis were higher in day 6 wild-type EBs than in wild-type ESCs. Overexpression of Tert protected day-6 EBs from spontaneous apoptosis. However, bisindolylma-leimide II induced close to 100% apoptosis in day-6 EBs, and this could not be rescued by Tert overexpression (Fig. 4B).
Figure 4. Improved apoptosis resistance in Tert-overexpressing cells. (A): Wild-type (control) and Tert-overexpressing embryonic stem (ES) cells were treated with the protein kinase C inhibitor bisindolyl-maleimide I in the indicated concentrations. The frequency (in %) of apoptotic cells was measured by annexin V staining. Data are mean ± SEM from three independent experiments. (B): Apoptosis was induced as indicated above in day-6 embryoid bodies (EBs) derived from control and Tert-transfected cells. Asterisks denote a statistically significant difference (Student t-test) versus wild-type: *p < .01; **p < .001; ***p < .0001.
Murine ESCs have highly efficient antioxidant defense mechanisms, but this protective capacity is progressively lost during differentiation into EBs . To investigate whether continued expression of Tert would interfere with this loss of antioxidant defense during differentiation, we first measured intracellular levels of peroxides by DCF-DA staining . Differentiation of wild-type ESCs into EBs resulted in an approximately fivefold increase in cellular DCF fluorescence at day 6, in accordance with our earlier results . In contrast, peroxide levels increased significantly less during differentiation of both Tert-overexpressing ESCs (Fig. 5A).
Figure 5. Tert-overexpressing cells maintain better stress defense capabilities than wild-type (control) during differentiation. (A): Intracellular peroxide content was measured by 2',7-dichlorofluorescin staining of embryonic stem (ES) cells and embryoid bodies (EBs) at day 0 (d0), day 2 (d2), day 4 (d4), and day 6 (d6) of the differentiation protocol. Data are mean ± SD from two independent experiments with triplicate measurements each. Fluorescence values measured in wild-type day-6 EBs were set at 100% in both experiments. The slopes of the regression lines for both Tert cells are significantly smaller than wild-type (p < .05) but not significantly different from each other (ANOVA). (B): Reverse transcription polymerase chain reaction analysis of expression of 15 candidate stress response genes in wild-type (control) and tert-2 ES cells and day-4 EBs. Aliquots of each reaction were equalized for the internal control Gapdh and were run on 2% agarose gels containing ethidium bromide. Genotypes and differentiation states are indicated. Abbreviations: AO, genes with functions in antioxidant defense; DDR, DNA damage–repair gene; HSP, chaperone (heat-shock protein); HY, gene regulated in response to hypoxia; IC, internal control; NC, negative control; TF, transcription factor.
Next, we compared the expression of a number of candidate genes involved in oxidative stress resistance. Before we had identified 15 stress-response candidate genes whose expression changed during the differentiation of wild-type ESCs, including 9 genes with possible roles in antioxidant defense, 3 chaperone genes, 1 DNA damage–repair gene, 1 regulated in response to hypoxia, and 1 transcriptional repressor for checkpoint control genes . Investigating the same set of candidate genes, we found that the expression changes of 5 out of 15 genes (Pdha2, Gpx4, Gpx3, Hif3, and Prdx2) were similar during the differentiation of Tert-overexpressing and control ESCs (Fig. 5B). In contrast, the expression of 10 out of these 15 candidate genes (Mortalin, Hspa1b, Bmi1, Gpx2, Sod2, Txnip, Ercc4, Hspa1a, Tgr, and Gsta3) was maintained by forced telomerase expression in day 4 EB cells while downregulated in control day 4 EBs (Fig. 5B). Together with the functional data shown above, this suggests that telomerase overexpression can maintain stress defense capacities during differentiation at levels more similar to undifferentiated ESCs.
To investigate whether continued Tert expression influences the efficiency of differentiation toward hematopoietic lineages, we measured the hematopoietic commitment of wild-type and Tert-overexpressing EBs. First, we estimated the total frequencies of colony-forming cells (CFCs) within preparations of EBs at day-0 to day-6 stages of the EB differentiation protocol by progenitor colony assays (CFU-GEMM). Formation of myeloid cell colonies was significantly more frequent from Tert-overexpressing EBs from day 2 onward (Fig. 6A). We then compared the abilities of day-3.5 wild-type and tert-2 EBs to differentiate along different hematopoietic lineages. Day-3.5 EBs were disrupted to single cells, and the ability to form colonies was measured under combinations of growth factors and cytokines that stimulate the differentiation toward myeloid and erythroid lineages. This showed that Tert-2 EBs gave rise to significantly higher numbers of mixed colonies (CFU-GEMM) and colonies derived from more committed progenitors (CFU-GM and BFU-E) than wild-type EBs (Fig. 6B; supplementary online Fig. 1).
Figure 6. Continued Tert expression results in enhanced differentiation of embryonic stem (ES) cells toward the hematopoietic lineage. (A): Embryoid bodies (EBs) were allowed to differentiate in the absence of leukemia inhibitory factor (LIF), and hematopoietic commitment was assessed by counting the proportion of EBs producing colonies of myeloid lineage in the CFU-GEMM assay. The data are M ± SEM from three experiments with triplicate measurements each. Asterisk denotes a statistically significant difference (Student t-test) versus wild-type: *p < .05. (B): Day-3.5 EBs from control and tert-2 transfected cells were disrupted to single cells, and their hematopoietic commitment was assessed by counting the number of colonies per 100,000 cells in a CFU-GEMM assay. The data are M ± SEM from three experiments with triplicate measurements each. Asterisks denote a statistically significant difference (Student t-test) versus wild-type: *p < .01; **p < .001; ***p < .0001. (C): Flow cytometry analysis of day-3.5 EBs derived from control and tert-2 transfected ESCs for the expression of Flk-1. The data shown are representative of three independent experiments. (D): Some 10,000 Flk-1+ cells were sorted from day-3.5 EBs derived from tert-2 and control ES cells and tested for hematopoietic activity in a CFU-GEMM assay. The data are M ± SEM from two experiments with triplicate measurements each. Asterisk denotes a statistically significant difference (Student t-test) versus wild-type: *p < .01.
These results suggested that the number of progenitor cells in the EBs derived from Tert-transfected ESCs is higher because of either faster proliferation or better survival in culture. To investigate this, we stained day-3.5 EBs with Flk-1 antibody. Flk-1 expression defines a population of cells within EBs that retain the capacity for becoming either hematopoietic or endothelial cells, depending on Scl expression, and is suggestive of hemangioblast precursors during the differentiation of ESCs . A higher percentage of Flk-1+ cells was found in Tert-overexpressing cells (Fig. 6C). We then sorted 10,000 Flk-1+ cells from day-3.5 EBs derived from wild-type and Tert-transfected cells and subjected them to CFU-GEMM assays. We obtained higher numbers of colonies from Tert-transfected cells (Fig. 6D), suggesting that overexpression of Tert results in an increase in the number of hematopoietic progenitor cells obtained during differentiation of ESCs. This is also supported by staining of day 6 EBs with myeloid (Gr-1, Mac-1), erythroid (Ter-119), and lymphoid lineage markers (B220). All of these markers were expressed in a higher fraction of tert-2 EBs than in wild-type EBs (Table 3).
Table 3. Day-6 embryoid bodies derived from tert-2 and control embryonic stem cells were disrupted to single cells and stained with antibodies against Gr-1, Mac-1, Ter-119, and B220
To understand the molecular basis of the effects of Tert over-expression, we performed a comparative gene expression analysis of Tert-transfected and control ESCs using the Affymetrix Mouse 430A target array, which includes approximately 14,000 known mouse genes. Of these, 275 genes were identified, using a 2.8-fold change in expression between the samples as the cut-off criterion. We used the Affymetrix Gene Ontology mining tool to select 10 candidate genes known to be involved in cell-cycle regulation, response to oxidative stress and DNA damage, and hematopoietic differentiation (Table 4). Differential expression was confirmed for all of these genes by RT-PCR. Moreover, RT-PCR also confirmed that the differential expression pattern that existed between wild-type and Tert-overexpressing ESCs was maintained during differentiation into EBs at least until day 4 (Fig. 7).
Table 4. Overexpression of Tert in murine embryonic stem cells (ESCs) results in changes in expression of genes known to be involved in oxidative stress, cell cycle regulation, and hematopoietic differentiation
Figure 7. Overexpression of Tert results in changes in expression of genes involved in cellular proliferation, DNA damage repair, oxidative stress, and hematopoietic differentiation. Reverse transcription polymerase chain reaction analysis of expression of 10 candidate genes in embryonic stem (ES) cells and day-4 embryoid bodies (EBs). Aliquots of each reaction were equalized for the internal control Gapdh and were run on 2% agarose gels containing ethidium bromide. Abbreviations: AO, antioxidant and stress defense genes; CC, genes with functions in cell-cycle regulation; DDR, DNA damage–repair gene; HD, factors relevant in differentiation and hematopiesis; IC, internal control; NC, negative control.
Most prominently, genes positively involved in cell cycle progression such as Ccnd1 (cyclin D1), Cdc6, and AurkB were upregulated, while the cell-cycle inhibitor p21 was downregulated in tert-2 ESCs in accordance with the faster proliferation of ESCs as a result of increased telomerase activity (Fig. 3A). Two genes with roles in detoxification of peroxidation products (Aldh3AI, Mgst1) were found to be upregulated as a result of Tert overexpression. This corresponds well with the lower peroxide levels in Tert-over-expressing cells (Fig. 5A). Together with the upregulation of an additional DNA damage repair gene (Rad51), this finding is well in accordance with the downregulation of the two important cell-cycle checkpoint arrest genes p21 and Gadd45a. p21 is known to be upregulated in telomere-dependent cellular senescence , and Gadd45 was also shown to be upregulated in response to telomere dysfunction before . Finally, we observed upregulation of Nov, a gene involved in regulation of differentiation, and of c-myb, a key hematopoietic transcription factor that is involved in regulation of proliferation and commitment within the hematopoietic hierarchy . c-myb expression is even more increased in day 4 EBs derived from the differentiation of Tert-transfected ESCs (Fig. 7). This could be indicative of an increased proliferation of hematopoietic progenitor cells within the EBs compared with those derived from control ESCs.
DISCUSSION
We thank scientists at Geron Corporation for providing us with the full-length cDNA of the murine Tert gene, Dr. Mauro Santibanez-Koref for help with the statistical analysis, Jaclyn C. Barel and Karen A. Thompson for help with cytogenetics analysis. This work was supported by Life Knowledge Park (M.L. and G.S.), The Biotechnology and Biological Sciences Research Council (L.L. and N.H.), Leukemia Research Foundation (NH), Newcastle Health Charity (G.S.), and Research into Ageing (T.v.Z.). L.A. and G.S. contributed equally to this work.
REFERENCES
Saretzki G, Armstrong L, Leake A et al. Stress defense in murine embryonic stem cells is superior to that of various differentiated murine cells. STEM CELLS 2004;22:962–971.
Armstrong L, Lako M, Cairns PM et al. mTert expression correlates with telomerase activity during the differentiation of murine embryonic stem cells. Mech Dev 2000;97:107–116.
Lebkowski JS, Gold J, Xu C et al. Human embryonic stem cells: culture, differentiation, and genetic modification for regenerative medicine applications. Cancer J 2001;7(suppl 2):S83–93.
Armstrong L, Lako M, van Herpe I et al. A role for nucleoprotein Zap3 in the reduction of telomerase activity during embryonic stem cell differentiation. Mech Dev 2004;121:1509–1522.
Greider CW, Blackburn EH. Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell 1985;43:405–413.
Lingner J, Hughes TR, Shevchenko A et al. Reverse transcriptase motifs in the catalytic subunit of telomerase. Science 1997;276:561–567.
Harley CB, Futcher AB, Greider CW. Telomeres shorten during ageing of human fibroblasts. Nature 1990;345:458–466.
Kim NW, Piatyszek MA, Prowse KR et al. Specific association of human telomerase activity with immortal cells and cancer. Science 1994;266:2011–2015.
Griffith JD, Comeau L, Rosenfield S et al. Mammalian telomeres end in a large duplex loop. Cell 1999;97:503–514.
Stewart SA, Ben-Porath I, Carey VJ et al. Erosion of the telomeric single-strand overhang at replicative senescence. Nat Genet 2003;33:492–496.
Masutomi K, Yu EY, Khurts S et al. Telomerase maintains telomere structure in normal human cells. Cell 2003;114:241–253.
Harle-Bachor C, Boukamp P. Telomerase activity in the regenerative basal layer of the epidermis in human skin and in immortal and carcinoma-derived skin keratinocytes. Proc Natl Acad Sci U S A 1006;93:6476–6481.
Hiayma K, Tatsumoto N, Shay JW et al. Telomerase activity in human intestine. Int J Oncol 1996;9:453–458.
Wright WE, Piatyszek MA, Rainey WE et al. Telomerase activity in human germ line and embryonic tissues and cells. Dev Genet 1996;18:173–179.
Hiyama K, Hirai Y, Kyoizumi S et al. Activation of telomerase in human lymphocytes and hematopoietic progenitor cells. J Immunol 1995;155:3711–3715.
Broccoli D, Young JW, DeLange T. Telomerase activity in normal and malignant hematopoietic cells. Proc Natl Acad Sci U S A 1995;9:9082–9086.
Lee HW, Blasco MA, Gottlieb GJ et al. Essential role of mouse telomerase in highly proliferative organs. Nature 1998;392:569–574.
Blasco MA, Lee HW, Hande MP et al. Telomere shortening and tumor formation by mouse cells lacking telomerase RNA. Cell 1997;91:25–34.
Saretzki G, Ludwig A, von Zglinicki T et al. Ribozyme-mediated telomerase inhibition induces immediate cell loss but not telomere shortening in ovarian cancer cells. Cancer Gene Ther 2001;8:827–34.
Sharma GG, Gupta A, Wang H et al. hTERT associates with human telomeres and enhances genomic stability and DNA repair. Oncogene 2003;22:131–146.
von Zglinicki T. Telomeres and replicative senescence: is it only length that counts? Cancer Lett 2001;168:111–116.
Engelhardt M, Kumar R, Albanell J et al. Telomerase regulation, cell cycle, and telomere stability in primitive hematopoietic cells. Blood 1997;90:182–193.
Yui J, Chiu CP, Lansdorp PM. Telomerase activity in candidate stem cells from fetal liver and adult bone marrow. Blood 1998;91:3255–3262.
Samper E, Fernandez P, Eguia R et al. Long-term repopulating ability of telomerase deficient murine hematopoietic stem cells. Blood 2002;99:2767–2775.
Heiss NS, Knight SW, Vulliamy TJ et al. X-linked dyskeratosis congenita is caused by mutations in a highly conserved gene with putative nucleolar functions. Nat Genet 1998;19:32–38.
Vulliamy T, Marrone A, Goldman F et al. The RNA component of telomerase is mutated in autosomal dominant dyskeratosis congenital. Nature 2001;413:432–435.
Montanaro L, Tazzari PL, Derenzini M. Enhanced telomere shortening in transformed lymphoblasts from patients with X linked dyskeratosis. J Clin Pathol 2003;56:583–586.
Lavon N, Benvenisty N. Differentiation and genetic manipulation of human embryonic stem cells and the analysis of the cardiovascular system. Trends Cardiovasc Med 2003;13:47–52.
Niida H, Matsumoto T, Satoh H et al. Severe growth defect in mouse cells lacking the telomerase RNA component. Nat Genet 1998;19:203–206.
Liu Y, Snow BE, Hande MP et al. The telomerase reverse transcriptase is limiting and necessary for telomerase function in vivo. Curr Biol 2000;10:1459–1462.
Tzukerman M, Shachaf C, Ravel Y et al. Identification of a novel transcription factor binding element involved in the regulation by differentiation of the human telomerase (hTERT) promoter. Mol Biol Cell 2000;11:4381–4391.
Quinlan LR, Faherty S, Kane MT. Phospholipase and protein kinase C involvement in mouse embryonic stem-cell proliferation and apoptosis. Reproduction 2003;126:121–131.
Rufer N, Dragowska W, Thornbury G et al. Telomere length dynamics in human lymphocyte subpopulations measured by flow cytometry. Nat Biotechnol 1998;16:743–747.
Petersen S, Saretzki G, von Zglinicki T. Preferential accumulation of single-stranded regions in telomeres of human fibroblasts. Exp Cell Res 1998;25:152–160.
Sitte N, Saretzki G, von Zglinicki T. Accelerated telomere shortening in fibroblasts after extended periods of confluency. Free Radic Biol Med 1998;24:885–893.
Keys B, Serra V, Saretzki G et al. Telomere shortening in human fibroblasts is not dependent on the size of the telomeric-33-overhang. Aging Cell 2004;3:103–109.
Longo L, Bygrave A, Grosveld FG et al. The chromosome make-up of mouse embryonic stem cells is predictive of somatic and germ cell chimaerism. Transgenic Res 1997;6:321–328.
Canela A, Martin-Caballero J, Flores JM et al. Constitutive expression of Tert in thymocytes leads to increased incidence and dissemination of T-cell lymphoma in Lck-Tert mice. Mol Cell Biol 2004;24:4275–4293.
von Zglinicki T, Pilger R, Sitte N. Accumulation of single-strand breaks is the major cause of telomere shortening in human fibroblasts. Free Radic Biol Med 2000;28:64–67.
Chung YS, Zhang WJ, Arentson E. Lineage analysis of the hemangioblast as defined by FLK1 and SCL expression. Dev 2002;129:5511–5520.
Magalhaes JP, Chainiaux F, Remacle J et al. Stress induced premature senescence in BJ and hTert-BJ1 human foreskin fibroblasts. FEBS Lett 2002;523:157–162.
Zhang X, Ma L, Enkemann SA et al. Role of Gadd45 in the density dependent G1 arrest induced by p27kip1. Oncogene 2003;22:4166–4174.
Sakamoto K, Yamaguchi S, Ando R et al. The nephroblastoma overexpressed gene (NOV/ccn3) protein associates with Notch1 extracellular domain and inhibits myoblast differentiation via Notch signaling pathway. J Biol Chem 2002;277: 29399–29405.
Emambokus N, Vegiopoulos A, Harman B et al. Progression through key stages of haematopoiesis is dependent on distinct threshold levels of c-Myb. EMBO J 2003;22:4478–4488.
Dean M, Cleveland JL, Rapp UR et al. Role of c-myc in the abrogation of IL-3 dependence of myeloid FDC-P1 cells. Oncog Res 1987;1:279–296.
Blasco MA. Telomerase beyond telomeres. Nat Rev Cancer 2002;2:627–633.
Ludwig A, Saretzki G, Holm PS et al. Ribozyme cleavage of telomerase mRNA sensitizes breast epithelial cells to inhibitors of topoisomerase. Cancer Res 2001;61:3053–3061.
Lorenz M, Saretzki G, Sitte N et al. BJ fibroblasts display high antioxidant capacity and slow telomere shortening independent of hTERT transfection. Free Radic Biol Med 2001;31:824–831.
Jeyapalan J, Leake A, Ahmed S et al. The role of telomeres in Etoposide-induced tumor cell death. Cell Cycle 2004;3:1169–1176.
Gonzalez-Suarez E, Samper E, Ramirez A et al. Increased epidermal tumours and increased skin wound healing in transgenic mice overexpressing the catalytic subunit of telomerase mTert in basal keratinocytes. EMBO J 2001;20:2619–2630.
Oh H, Taffet GE, Youker KA et al. Telomerase reverse transcriptase promotes cardiac muscle cell proliferation, hypertrophy, and survival. Proc Natl Acad Sci U S A 2001;98:10308–10313.
Simonsen JL, Rosada C, Serakinci N et al. Telomerase expression extends the proliferative lifespan and maintains the osteogenic potential of human bone marrow stromal cells. Nat Biotechnol 2002;20:592–596.
Sh S, Gronthos S, Chen S et al. Bone formation by human postnatal bone marrow stromal cells is enhanced by telomerase expression. Nat Biotechnol 2002;20:587–591.
Murasawa S, Llevadot J, Silver M et al. Constitutive human telomerase reverse transcriptase expression enhances regenerative properties of endothelial progenitor cells. Circulation 2002;106:1133–1139.
Blackburn EH, Chan S, Chang J et al. Molecular manifestations and molecular determinants of telomere capping. Cold Spring Harb Symp Quant Biol 2000;65:253–263.
Karlseder J, Broccoli D, Dai Y et al. p53- and ATM-dependent apoptosis induced by telomeres lacking TRF2. Science 1999;283:1321–1325.
de Lange T. Telomeres and senescence: ending the debate. Science 1998;279:334–335.
d’Adda di Fagagna F, Reaper PM, Clay-Farrace L et al. A DNA damage checkpoint response in telomere-initiated senescence. Nature 2003;426:194–198.
Herbig U, Jobking WA, Chen BP et al. Telomere shortening triggers senescence of human cells through a pathway involving ATM, p53, and p21 (Cip1) but not p16/Ink4a. Mol Cell 2004;14:501–513.
Saretzki G, Sitte N, Merkel U et al. Telomere shortening triggers a p53-dependent cell cycle arrest via accumulation of G-rich single stranded DNA fragments. Oncogene 1999;18:5148–5158.
von Zglinicki T. The role of oxidative stress in telomere length regulation and replicative senescence. Ann N Y Acad Sci 2000;908:99–110.
Wong KK, Chang S, Weiler SR et al. Telomere dysfunction impairs DNA repair and enhances sensitivity to ionizing radiation. Nat Genet 2000;26:85–88.
Goytisolo FA, Samper E, Martin-Caballero J et al. Short telomeres result in organismal hypersensitivity to ionizing radiation in mammals. J Exp Med 2000;192:1625–1636.
Damm K, Hemmann U, Garin-Chesa P et al. A highly selective telomerase inhibitor limiting human cancer cell proliferation. EMBO J 2001;20:6958–6968.
Smith LL, Coller HA, Roberts JM. Telomerase modulates expression of growth controlling genes and enhances cell proliferation. Nat Cell Biol 2003;5:474–479.
Xiang H, Wang J, Mao Y et al. Human telomerase accelerates growth of lens epithelial cells through regulation of genes mediating RB/E2F pathway. Oncogene 2002;21:3784–3791.
Kang SS, Kwon T, Kwon DY et al. Akt protein kinase enhances human telomerase activity through phosphorylation of telomerase reverse transcriptase subunit. J Biol Chem 1999;274:13085–13090.
Kang KW, Ryu JH, Kim SG. Activation of phosphatidylinositol 3-kinase by oxidative stress leads to the induction of microsomal epoxide hydrolase in H4IIE cells. Toxicol Lett 2001;121:191–197.
Gonzalez-Suarez E, Flores JM, Blasco MA. Cooperation between p53 mutation and high telomerase transgenic expression in spontaneous cancer development. Mol Cell Biol 2002;22:7291–7301.
Serakinci N, Guldberg P, Burns JS et al. Adult human mesenchymal stem cell as a target for neoplastic transformation. Oncogene 2004;23:5095–5098.
Sherr CJ. Cancer Cell Cycles. Science 1996;274:1672–1677.
Illenye S, Heintz NH. Mouse Cdc6 is associated with the mitotic spindle apparatus. Genomics 2004;83:66–75.
Adams RR, Carmena M, Earnshaw WC. Chromosomal passengers and the (aurora) ABC of mitosis. Trends Cell Biol 2001;11:49–54.
Kumari A, Schultz N, Helleday T. p53 protects from replication associated DNA double strand breaks in mammalian cells. Oncogene 2004;23:2324–2329.
Pappa A, Chen C, Koutalos Y et al. Aldh3a1 protects human corneal epithelial cells from ultraviolet and 4-hydroxy-2-noneal-induced oxidative damage. Free Radic Biol Med 2003;34:1178–1189.
Kelner MJ, Bagnell RD, Montoya MA et al. Structural organization of the microsomal glutathione S- transferase gene (MGST1) on chromosome 12p13.1–13.2. J Biol Chem 2000;275:13000–13006.(L. Armstronga,c, G. Saret)
b Institute for Ageing and Health, University of Newcastle upon Tyne NE6 4BE, United Kingdom;
c Institute of Human Genetics, University of Newcastle upon Tyne NE1 3BZ, United Kingdom
Key Words. ESCs ? Telomerase ? Hematopoietic stem cells ? Oxidative stress ? Cell proliferation ? Cell cycle ? Apoptosis
Correspondence: Majlinda Lako, Ph.D., Institute of Human Genetics, International Centre for Life, Central Parkway, Newcastle upon Tyne NE1 3BZ, U.K. Telephone: 00-44-191-241-8688; Fax: 00-44-191-241-8666; e-mail: Majlinda.Lako@ncl.ac.uk
ABSTRACT
Embryonic stem cells (ESCs) are derived from the inner cell mass of blastocysts, and under appropriate culture conditions they can be maintained indefinitely in culture while preserving their pluripotency and genomic stability. There are only very small numbers of ESCs that give rise to all tissues of the embryo proper. This small size of the population means that ESCs need to be equipped with efficient mechanisms of preventing and repairing DNA damage. In fact, murine ESCs differ from their more differentiated counterparts by high levels of antioxidant defense and good DNA strand break repair capacity . Furthermore, telomerase activity decreases during differentiation of mouse and human ESCs, mostly because of transcriptional downregulation of the telomerase reverse transcriptase, Tert . Currently, it is not known whether the downregulation of telomerase during differentiation of ESCs influences the ability of the derived progeny to cope with DNA damage, oxidative stress, and the differentiation process itself. This is an important issue in regenerative medicine, given the current efforts to selectively differentiate human ESCs into various precursors for use in cell-replacement therapies. In the study reported here, we wanted to investigate whether the continuous expression of telomerase during differentiation can help to transfer some of the excellent properties of ESCs to ESC-derived progeny.
The telomerase holoenzyme core encompasses a reverse transcriptase (TERT) that catalyzes the addition of new repeats and a structural RNA component (TR) containing the template region that binds the telomere repeats . Telomeric DNA consists of regions composed of conserved tandem hexanucleotide repeats with a protruding G-rich overhang and is replicated by the ribonucleoprotein telomerase. Most human somatic cells show very little or no telomerase activity, and their chromosome ends shorten at each cell division, thereby limiting the replicative lifespan and leading eventually to senescence . Immortalized human cells and cancer cells, however, exhibit stable telomere length upon prolonged propagation in culture and typically have detectable telomerase activity . In addition to maintaining telomere length, telomerase may also prevent the loss of G-rich single-stranded overhangs that participate, together with specific telomere-binding proteins, in forming the T-loop structure . Erosion of the telomeric overhang can be a cause or a consequence of the collapse of the T-loop structure and the uncapping of the telomere end that eventually signals DNA damage and activates the senescence pathway .
In addition to detection in cancer cells and in artificially immortalized cells, telomerase activity has been found in various tissues with self-renewal capacity, including the basal layer of the epidermis , intestinal crypt cells , germ line cells , and the hematopoietic system , and at lower levels in cycling primary presenescent fibroblasts . This suggests that proper maintenance of telomere length and structure is required for the self-renewal function. Supportive evidence comes from mice lacking telomerase activity which exhibit functional impairment in organs composed of highly proliferative cells in late generations .
Telomerase knock-down experiments in human tumor cells and overexpression studies in somatic cells have identified telomerase as a "survival enzyme" that not only allows long-term unlimited growth but also improves cellular resistance against a wide variety of stressors and cytotoxic agents. In many cases, this survival function appears unrelated to maintenance of telomere length . Possible mechanisms include length-independent stabilization of the telomere cap or induction of stress defense genes via unknown pathways .
Telomerase activity has also been implicated in the maintenance of stem cell function and self-renewal, especially in the hematopoietic system. Primitive hematopoietic stem cells (HSCs) are typically quiescent and display low levels of telomerase activity, which is upregulated upon mitogenic stimulation . Telomerase-deficient HSCs show a reduced long-term repopulating capacity and increased genomic instability compared with wild-type cells . In patients with dyskeratosis congenita, mutations in different genes compromise the function of telomerase. This leads to enhanced telomere shortening and functional deficiencies in tissues such as gut, skin, and bone marrow, all of which depend on renewal from stem cells .
Current knowledge of TERT regulation and telomerase activity in normal development is limited. ESCs afford a good model system because they can be propagated indefinitely in culture and differentiated into various cell types, recapitulating many aspects of early embryonic development . In the mouse, telomerase activity is important for ESC growth. Deletions leading to loss of either the telomerase RNA or its reverse transcriptase result in progressive loss of telomeres, genomic instability, aneuploidy, telomeric fusions, and eventual reduced growth rate . Recent work has indicated that human ESCs express the TERT gene and show high levels of telomerase activity; however, upon differentiation, the levels of TERT and telomerase activity decrease with the emergence of a maturing population of cells . It is not clear from these studies whether or not the down-regulation of telomerase activity is necessary for the differentiation to proceed normally. Here, we stably overexpressed Tert in murine ESCs and investigated the effects of continuous telomerase expression on cell proliferation and apoptosis, differentiation toward hematopoietic lineage, and resistance to oxidative stress.
MATERIALS AND METHODS
Overexpression of Tert in Murine ESCs
We have shown before that telomerase is downregulated by more than one order of magnitude upon differentiation of murine ESCs to EBs . To investigate the effects of continued telomerase expression during the differentiation of ESCs, we stably transfected a construct containing the full-length cDNA of Tert under the control of MPSV LTR, resulting in significant overexpression of Tert as measured by real-time RT-PCR using primers directed against the mouse Tert-coding sequence, which amplify both endogenous and exogenous Tert mRNA. One single clone (tert-1) and one pooled clone (tert-2) were selected for further analysis. To measure telomerase activity, we performed TRAP assays under linear conditions of product amplification, which allowed for semi-quantitative analysis of TRAP activity levels. A significant increase in telomerase activity was observed in Tert-transfected cells (Fig. 1A). Although tert-1 ESC and tert-2 ESC clones over-expressed Tert mRNA at different levels, the telomerase activity levels were similar to each other. Most probably, an 11-fold over-expression of Tert as found in tert-2 ESCs is already sufficient to saturate the formation of active telomerase complexes. During differentiation, telomerase activity decreased in control ESCs but was maintained in the Tert-transfected cells (Fig. 1B).
Figure 1. Overexpression of Tert results in maintenance of telomerase activity during the differentiation of embryonic stem (ES) cells. (A): Telomerase activity was measured in a dilution series by semiquantitative TRAP ELISA in control (wild-type) ES cells and Tert-overexpressing ESCs. The results represent the mean ± SEM from three independent experiments. Asterisks denote a statistically significant difference (Student t-test) versus wild-type: *p < .05; **p < .01. (B): Telomerase activity from 10 ng of cell extract was measured using the TRAP-ELISA assay. Telomerase-specific products were determined by the presence of a 6-bp incremental DNA ladder on a 12% polyacrylamide gel stained with ethidium bromide, which indicates the addition of the 6-bp repeats (TTAGGG) to the synthetic primer by telomerase. Abbreviation: EB, embryoid body.
To investigate whether continuous overexpression of Tert in ESCs contributes to gross genomic instability, we carried out G banding on Tert-transfected and wild-type ESCs at three different population doublings (Table 2). Observation of 30 metaphases for tert-1 ESCs and tert-2 ESCs showed no deviations from the normal karyotype at PD130. Increasing aneuploidy was observed in metaphases obtained at PD190 and PD300 for both wild-type and Tert-transfected ESCs. It is well known that murine ESCs in prolonged culture become severely aneuploid . Chi-square analysis (p < .01) revealed that Tert overexpression did not increase the severity or frequency of these aberrations.
Table 2. Frequency of aneuploidy in control and Tert-overexpressing embryonic stem cells
Using pulsed field gel electrophoresis we could not detect any changes in average telomere length or telomere length distribution (data not shown). Flow-FISH is more sensitive for the detection of small length differences in the highly heterogeneous telomeres from mice . We found that telomere fluorescence intensity measured by Flow-FISH did not change significantly during the differentiation of either wild-type or Tert-overexpressing ESCs into day 6 EBs (data not shown). There was some variation in G-rich overhang length between Tert-overexpressing ESCs, possibly due to clonal selection. Importantly, the length of G-rich single-stranded overhangs decreased during the differentiation of wild-type ESCs to EBs, while no significant change was observed during the differentiation of the Tert-overexpressing ESCs (Fig. 2; supplementary online Fig. 2).
Figure 2. Expression of Tert maintains G-rich single-stranded overhangs during differentiation of embryonic stem cells (ESCs). Overhang length was measured as hybridization intensity ratio native versus denatured gel . Overhang length of wild-type (control) ESCs was set at 1. The data are mean ± SEM from four independent experiments. Embryoid bodies (EBs) are from day 6 of the differentiation protocol (*p < .05 ). Abbreviation: ns, not significant.
Tert-transfected ESCs grew faster than the control (Fig 3A). This appeared to be due to faster transit of Tert-transfected cells through the G2 and M phases of the cell cycle and, accordingly, higher fractions of cells in S phase (Fig. 3B). The doubling time for tert-1 ESCs is 16.6 hours and for tert-2 is 16.3 hours, compared with the control clone which is 23.2 hours. In view of this result, it is possible that the G2/M checkpoint might have been activated. It is equally possible that the cells simply transit G2/M phase more rapidly because of their intact telomeres.
Figure 3. Continued expression of Tert during differentiation of embryonic stem (ES) cells results in accelerated cell proliferation and faster transit through G2/M. (A): Cell growth is shown as cumulative population doublings (PDs) versus time. PDs were set to 0 at the beginning of the experiment. (B): Frequencies of wild-type (control) and Tert-overexpressing ES cells in different cell cycle phases, as measured by propidium iodide staining and flow cytometry. The data are mean ± SEM from three independent experiments. Asterisks denote a statistically significant difference (Student t-test) versus wild-type: *p < .001.
We next investigated the effect of Tert overexpression on a poptosis. Cell death was induced by adding a protein kinase C inhibitor, bisindolylmaleimide II . We found that overexpression of Tert in ESCs conferred a higher resistance to inhibitor-induced apoptosis (Fig. 4A). Frequencies of both spontaneous cell death and inhibitor-induced apoptosis were higher in day 6 wild-type EBs than in wild-type ESCs. Overexpression of Tert protected day-6 EBs from spontaneous apoptosis. However, bisindolylma-leimide II induced close to 100% apoptosis in day-6 EBs, and this could not be rescued by Tert overexpression (Fig. 4B).
Figure 4. Improved apoptosis resistance in Tert-overexpressing cells. (A): Wild-type (control) and Tert-overexpressing embryonic stem (ES) cells were treated with the protein kinase C inhibitor bisindolyl-maleimide I in the indicated concentrations. The frequency (in %) of apoptotic cells was measured by annexin V staining. Data are mean ± SEM from three independent experiments. (B): Apoptosis was induced as indicated above in day-6 embryoid bodies (EBs) derived from control and Tert-transfected cells. Asterisks denote a statistically significant difference (Student t-test) versus wild-type: *p < .01; **p < .001; ***p < .0001.
Murine ESCs have highly efficient antioxidant defense mechanisms, but this protective capacity is progressively lost during differentiation into EBs . To investigate whether continued expression of Tert would interfere with this loss of antioxidant defense during differentiation, we first measured intracellular levels of peroxides by DCF-DA staining . Differentiation of wild-type ESCs into EBs resulted in an approximately fivefold increase in cellular DCF fluorescence at day 6, in accordance with our earlier results . In contrast, peroxide levels increased significantly less during differentiation of both Tert-overexpressing ESCs (Fig. 5A).
Figure 5. Tert-overexpressing cells maintain better stress defense capabilities than wild-type (control) during differentiation. (A): Intracellular peroxide content was measured by 2',7-dichlorofluorescin staining of embryonic stem (ES) cells and embryoid bodies (EBs) at day 0 (d0), day 2 (d2), day 4 (d4), and day 6 (d6) of the differentiation protocol. Data are mean ± SD from two independent experiments with triplicate measurements each. Fluorescence values measured in wild-type day-6 EBs were set at 100% in both experiments. The slopes of the regression lines for both Tert cells are significantly smaller than wild-type (p < .05) but not significantly different from each other (ANOVA). (B): Reverse transcription polymerase chain reaction analysis of expression of 15 candidate stress response genes in wild-type (control) and tert-2 ES cells and day-4 EBs. Aliquots of each reaction were equalized for the internal control Gapdh and were run on 2% agarose gels containing ethidium bromide. Genotypes and differentiation states are indicated. Abbreviations: AO, genes with functions in antioxidant defense; DDR, DNA damage–repair gene; HSP, chaperone (heat-shock protein); HY, gene regulated in response to hypoxia; IC, internal control; NC, negative control; TF, transcription factor.
Next, we compared the expression of a number of candidate genes involved in oxidative stress resistance. Before we had identified 15 stress-response candidate genes whose expression changed during the differentiation of wild-type ESCs, including 9 genes with possible roles in antioxidant defense, 3 chaperone genes, 1 DNA damage–repair gene, 1 regulated in response to hypoxia, and 1 transcriptional repressor for checkpoint control genes . Investigating the same set of candidate genes, we found that the expression changes of 5 out of 15 genes (Pdha2, Gpx4, Gpx3, Hif3, and Prdx2) were similar during the differentiation of Tert-overexpressing and control ESCs (Fig. 5B). In contrast, the expression of 10 out of these 15 candidate genes (Mortalin, Hspa1b, Bmi1, Gpx2, Sod2, Txnip, Ercc4, Hspa1a, Tgr, and Gsta3) was maintained by forced telomerase expression in day 4 EB cells while downregulated in control day 4 EBs (Fig. 5B). Together with the functional data shown above, this suggests that telomerase overexpression can maintain stress defense capacities during differentiation at levels more similar to undifferentiated ESCs.
To investigate whether continued Tert expression influences the efficiency of differentiation toward hematopoietic lineages, we measured the hematopoietic commitment of wild-type and Tert-overexpressing EBs. First, we estimated the total frequencies of colony-forming cells (CFCs) within preparations of EBs at day-0 to day-6 stages of the EB differentiation protocol by progenitor colony assays (CFU-GEMM). Formation of myeloid cell colonies was significantly more frequent from Tert-overexpressing EBs from day 2 onward (Fig. 6A). We then compared the abilities of day-3.5 wild-type and tert-2 EBs to differentiate along different hematopoietic lineages. Day-3.5 EBs were disrupted to single cells, and the ability to form colonies was measured under combinations of growth factors and cytokines that stimulate the differentiation toward myeloid and erythroid lineages. This showed that Tert-2 EBs gave rise to significantly higher numbers of mixed colonies (CFU-GEMM) and colonies derived from more committed progenitors (CFU-GM and BFU-E) than wild-type EBs (Fig. 6B; supplementary online Fig. 1).
Figure 6. Continued Tert expression results in enhanced differentiation of embryonic stem (ES) cells toward the hematopoietic lineage. (A): Embryoid bodies (EBs) were allowed to differentiate in the absence of leukemia inhibitory factor (LIF), and hematopoietic commitment was assessed by counting the proportion of EBs producing colonies of myeloid lineage in the CFU-GEMM assay. The data are M ± SEM from three experiments with triplicate measurements each. Asterisk denotes a statistically significant difference (Student t-test) versus wild-type: *p < .05. (B): Day-3.5 EBs from control and tert-2 transfected cells were disrupted to single cells, and their hematopoietic commitment was assessed by counting the number of colonies per 100,000 cells in a CFU-GEMM assay. The data are M ± SEM from three experiments with triplicate measurements each. Asterisks denote a statistically significant difference (Student t-test) versus wild-type: *p < .01; **p < .001; ***p < .0001. (C): Flow cytometry analysis of day-3.5 EBs derived from control and tert-2 transfected ESCs for the expression of Flk-1. The data shown are representative of three independent experiments. (D): Some 10,000 Flk-1+ cells were sorted from day-3.5 EBs derived from tert-2 and control ES cells and tested for hematopoietic activity in a CFU-GEMM assay. The data are M ± SEM from two experiments with triplicate measurements each. Asterisk denotes a statistically significant difference (Student t-test) versus wild-type: *p < .01.
These results suggested that the number of progenitor cells in the EBs derived from Tert-transfected ESCs is higher because of either faster proliferation or better survival in culture. To investigate this, we stained day-3.5 EBs with Flk-1 antibody. Flk-1 expression defines a population of cells within EBs that retain the capacity for becoming either hematopoietic or endothelial cells, depending on Scl expression, and is suggestive of hemangioblast precursors during the differentiation of ESCs . A higher percentage of Flk-1+ cells was found in Tert-overexpressing cells (Fig. 6C). We then sorted 10,000 Flk-1+ cells from day-3.5 EBs derived from wild-type and Tert-transfected cells and subjected them to CFU-GEMM assays. We obtained higher numbers of colonies from Tert-transfected cells (Fig. 6D), suggesting that overexpression of Tert results in an increase in the number of hematopoietic progenitor cells obtained during differentiation of ESCs. This is also supported by staining of day 6 EBs with myeloid (Gr-1, Mac-1), erythroid (Ter-119), and lymphoid lineage markers (B220). All of these markers were expressed in a higher fraction of tert-2 EBs than in wild-type EBs (Table 3).
Table 3. Day-6 embryoid bodies derived from tert-2 and control embryonic stem cells were disrupted to single cells and stained with antibodies against Gr-1, Mac-1, Ter-119, and B220
To understand the molecular basis of the effects of Tert over-expression, we performed a comparative gene expression analysis of Tert-transfected and control ESCs using the Affymetrix Mouse 430A target array, which includes approximately 14,000 known mouse genes. Of these, 275 genes were identified, using a 2.8-fold change in expression between the samples as the cut-off criterion. We used the Affymetrix Gene Ontology mining tool to select 10 candidate genes known to be involved in cell-cycle regulation, response to oxidative stress and DNA damage, and hematopoietic differentiation (Table 4). Differential expression was confirmed for all of these genes by RT-PCR. Moreover, RT-PCR also confirmed that the differential expression pattern that existed between wild-type and Tert-overexpressing ESCs was maintained during differentiation into EBs at least until day 4 (Fig. 7).
Table 4. Overexpression of Tert in murine embryonic stem cells (ESCs) results in changes in expression of genes known to be involved in oxidative stress, cell cycle regulation, and hematopoietic differentiation
Figure 7. Overexpression of Tert results in changes in expression of genes involved in cellular proliferation, DNA damage repair, oxidative stress, and hematopoietic differentiation. Reverse transcription polymerase chain reaction analysis of expression of 10 candidate genes in embryonic stem (ES) cells and day-4 embryoid bodies (EBs). Aliquots of each reaction were equalized for the internal control Gapdh and were run on 2% agarose gels containing ethidium bromide. Abbreviations: AO, antioxidant and stress defense genes; CC, genes with functions in cell-cycle regulation; DDR, DNA damage–repair gene; HD, factors relevant in differentiation and hematopiesis; IC, internal control; NC, negative control.
Most prominently, genes positively involved in cell cycle progression such as Ccnd1 (cyclin D1), Cdc6, and AurkB were upregulated, while the cell-cycle inhibitor p21 was downregulated in tert-2 ESCs in accordance with the faster proliferation of ESCs as a result of increased telomerase activity (Fig. 3A). Two genes with roles in detoxification of peroxidation products (Aldh3AI, Mgst1) were found to be upregulated as a result of Tert overexpression. This corresponds well with the lower peroxide levels in Tert-over-expressing cells (Fig. 5A). Together with the upregulation of an additional DNA damage repair gene (Rad51), this finding is well in accordance with the downregulation of the two important cell-cycle checkpoint arrest genes p21 and Gadd45a. p21 is known to be upregulated in telomere-dependent cellular senescence , and Gadd45 was also shown to be upregulated in response to telomere dysfunction before . Finally, we observed upregulation of Nov, a gene involved in regulation of differentiation, and of c-myb, a key hematopoietic transcription factor that is involved in regulation of proliferation and commitment within the hematopoietic hierarchy . c-myb expression is even more increased in day 4 EBs derived from the differentiation of Tert-transfected ESCs (Fig. 7). This could be indicative of an increased proliferation of hematopoietic progenitor cells within the EBs compared with those derived from control ESCs.
DISCUSSION
We thank scientists at Geron Corporation for providing us with the full-length cDNA of the murine Tert gene, Dr. Mauro Santibanez-Koref for help with the statistical analysis, Jaclyn C. Barel and Karen A. Thompson for help with cytogenetics analysis. This work was supported by Life Knowledge Park (M.L. and G.S.), The Biotechnology and Biological Sciences Research Council (L.L. and N.H.), Leukemia Research Foundation (NH), Newcastle Health Charity (G.S.), and Research into Ageing (T.v.Z.). L.A. and G.S. contributed equally to this work.
REFERENCES
Saretzki G, Armstrong L, Leake A et al. Stress defense in murine embryonic stem cells is superior to that of various differentiated murine cells. STEM CELLS 2004;22:962–971.
Armstrong L, Lako M, Cairns PM et al. mTert expression correlates with telomerase activity during the differentiation of murine embryonic stem cells. Mech Dev 2000;97:107–116.
Lebkowski JS, Gold J, Xu C et al. Human embryonic stem cells: culture, differentiation, and genetic modification for regenerative medicine applications. Cancer J 2001;7(suppl 2):S83–93.
Armstrong L, Lako M, van Herpe I et al. A role for nucleoprotein Zap3 in the reduction of telomerase activity during embryonic stem cell differentiation. Mech Dev 2004;121:1509–1522.
Greider CW, Blackburn EH. Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell 1985;43:405–413.
Lingner J, Hughes TR, Shevchenko A et al. Reverse transcriptase motifs in the catalytic subunit of telomerase. Science 1997;276:561–567.
Harley CB, Futcher AB, Greider CW. Telomeres shorten during ageing of human fibroblasts. Nature 1990;345:458–466.
Kim NW, Piatyszek MA, Prowse KR et al. Specific association of human telomerase activity with immortal cells and cancer. Science 1994;266:2011–2015.
Griffith JD, Comeau L, Rosenfield S et al. Mammalian telomeres end in a large duplex loop. Cell 1999;97:503–514.
Stewart SA, Ben-Porath I, Carey VJ et al. Erosion of the telomeric single-strand overhang at replicative senescence. Nat Genet 2003;33:492–496.
Masutomi K, Yu EY, Khurts S et al. Telomerase maintains telomere structure in normal human cells. Cell 2003;114:241–253.
Harle-Bachor C, Boukamp P. Telomerase activity in the regenerative basal layer of the epidermis in human skin and in immortal and carcinoma-derived skin keratinocytes. Proc Natl Acad Sci U S A 1006;93:6476–6481.
Hiayma K, Tatsumoto N, Shay JW et al. Telomerase activity in human intestine. Int J Oncol 1996;9:453–458.
Wright WE, Piatyszek MA, Rainey WE et al. Telomerase activity in human germ line and embryonic tissues and cells. Dev Genet 1996;18:173–179.
Hiyama K, Hirai Y, Kyoizumi S et al. Activation of telomerase in human lymphocytes and hematopoietic progenitor cells. J Immunol 1995;155:3711–3715.
Broccoli D, Young JW, DeLange T. Telomerase activity in normal and malignant hematopoietic cells. Proc Natl Acad Sci U S A 1995;9:9082–9086.
Lee HW, Blasco MA, Gottlieb GJ et al. Essential role of mouse telomerase in highly proliferative organs. Nature 1998;392:569–574.
Blasco MA, Lee HW, Hande MP et al. Telomere shortening and tumor formation by mouse cells lacking telomerase RNA. Cell 1997;91:25–34.
Saretzki G, Ludwig A, von Zglinicki T et al. Ribozyme-mediated telomerase inhibition induces immediate cell loss but not telomere shortening in ovarian cancer cells. Cancer Gene Ther 2001;8:827–34.
Sharma GG, Gupta A, Wang H et al. hTERT associates with human telomeres and enhances genomic stability and DNA repair. Oncogene 2003;22:131–146.
von Zglinicki T. Telomeres and replicative senescence: is it only length that counts? Cancer Lett 2001;168:111–116.
Engelhardt M, Kumar R, Albanell J et al. Telomerase regulation, cell cycle, and telomere stability in primitive hematopoietic cells. Blood 1997;90:182–193.
Yui J, Chiu CP, Lansdorp PM. Telomerase activity in candidate stem cells from fetal liver and adult bone marrow. Blood 1998;91:3255–3262.
Samper E, Fernandez P, Eguia R et al. Long-term repopulating ability of telomerase deficient murine hematopoietic stem cells. Blood 2002;99:2767–2775.
Heiss NS, Knight SW, Vulliamy TJ et al. X-linked dyskeratosis congenita is caused by mutations in a highly conserved gene with putative nucleolar functions. Nat Genet 1998;19:32–38.
Vulliamy T, Marrone A, Goldman F et al. The RNA component of telomerase is mutated in autosomal dominant dyskeratosis congenital. Nature 2001;413:432–435.
Montanaro L, Tazzari PL, Derenzini M. Enhanced telomere shortening in transformed lymphoblasts from patients with X linked dyskeratosis. J Clin Pathol 2003;56:583–586.
Lavon N, Benvenisty N. Differentiation and genetic manipulation of human embryonic stem cells and the analysis of the cardiovascular system. Trends Cardiovasc Med 2003;13:47–52.
Niida H, Matsumoto T, Satoh H et al. Severe growth defect in mouse cells lacking the telomerase RNA component. Nat Genet 1998;19:203–206.
Liu Y, Snow BE, Hande MP et al. The telomerase reverse transcriptase is limiting and necessary for telomerase function in vivo. Curr Biol 2000;10:1459–1462.
Tzukerman M, Shachaf C, Ravel Y et al. Identification of a novel transcription factor binding element involved in the regulation by differentiation of the human telomerase (hTERT) promoter. Mol Biol Cell 2000;11:4381–4391.
Quinlan LR, Faherty S, Kane MT. Phospholipase and protein kinase C involvement in mouse embryonic stem-cell proliferation and apoptosis. Reproduction 2003;126:121–131.
Rufer N, Dragowska W, Thornbury G et al. Telomere length dynamics in human lymphocyte subpopulations measured by flow cytometry. Nat Biotechnol 1998;16:743–747.
Petersen S, Saretzki G, von Zglinicki T. Preferential accumulation of single-stranded regions in telomeres of human fibroblasts. Exp Cell Res 1998;25:152–160.
Sitte N, Saretzki G, von Zglinicki T. Accelerated telomere shortening in fibroblasts after extended periods of confluency. Free Radic Biol Med 1998;24:885–893.
Keys B, Serra V, Saretzki G et al. Telomere shortening in human fibroblasts is not dependent on the size of the telomeric-33-overhang. Aging Cell 2004;3:103–109.
Longo L, Bygrave A, Grosveld FG et al. The chromosome make-up of mouse embryonic stem cells is predictive of somatic and germ cell chimaerism. Transgenic Res 1997;6:321–328.
Canela A, Martin-Caballero J, Flores JM et al. Constitutive expression of Tert in thymocytes leads to increased incidence and dissemination of T-cell lymphoma in Lck-Tert mice. Mol Cell Biol 2004;24:4275–4293.
von Zglinicki T, Pilger R, Sitte N. Accumulation of single-strand breaks is the major cause of telomere shortening in human fibroblasts. Free Radic Biol Med 2000;28:64–67.
Chung YS, Zhang WJ, Arentson E. Lineage analysis of the hemangioblast as defined by FLK1 and SCL expression. Dev 2002;129:5511–5520.
Magalhaes JP, Chainiaux F, Remacle J et al. Stress induced premature senescence in BJ and hTert-BJ1 human foreskin fibroblasts. FEBS Lett 2002;523:157–162.
Zhang X, Ma L, Enkemann SA et al. Role of Gadd45 in the density dependent G1 arrest induced by p27kip1. Oncogene 2003;22:4166–4174.
Sakamoto K, Yamaguchi S, Ando R et al. The nephroblastoma overexpressed gene (NOV/ccn3) protein associates with Notch1 extracellular domain and inhibits myoblast differentiation via Notch signaling pathway. J Biol Chem 2002;277: 29399–29405.
Emambokus N, Vegiopoulos A, Harman B et al. Progression through key stages of haematopoiesis is dependent on distinct threshold levels of c-Myb. EMBO J 2003;22:4478–4488.
Dean M, Cleveland JL, Rapp UR et al. Role of c-myc in the abrogation of IL-3 dependence of myeloid FDC-P1 cells. Oncog Res 1987;1:279–296.
Blasco MA. Telomerase beyond telomeres. Nat Rev Cancer 2002;2:627–633.
Ludwig A, Saretzki G, Holm PS et al. Ribozyme cleavage of telomerase mRNA sensitizes breast epithelial cells to inhibitors of topoisomerase. Cancer Res 2001;61:3053–3061.
Lorenz M, Saretzki G, Sitte N et al. BJ fibroblasts display high antioxidant capacity and slow telomere shortening independent of hTERT transfection. Free Radic Biol Med 2001;31:824–831.
Jeyapalan J, Leake A, Ahmed S et al. The role of telomeres in Etoposide-induced tumor cell death. Cell Cycle 2004;3:1169–1176.
Gonzalez-Suarez E, Samper E, Ramirez A et al. Increased epidermal tumours and increased skin wound healing in transgenic mice overexpressing the catalytic subunit of telomerase mTert in basal keratinocytes. EMBO J 2001;20:2619–2630.
Oh H, Taffet GE, Youker KA et al. Telomerase reverse transcriptase promotes cardiac muscle cell proliferation, hypertrophy, and survival. Proc Natl Acad Sci U S A 2001;98:10308–10313.
Simonsen JL, Rosada C, Serakinci N et al. Telomerase expression extends the proliferative lifespan and maintains the osteogenic potential of human bone marrow stromal cells. Nat Biotechnol 2002;20:592–596.
Sh S, Gronthos S, Chen S et al. Bone formation by human postnatal bone marrow stromal cells is enhanced by telomerase expression. Nat Biotechnol 2002;20:587–591.
Murasawa S, Llevadot J, Silver M et al. Constitutive human telomerase reverse transcriptase expression enhances regenerative properties of endothelial progenitor cells. Circulation 2002;106:1133–1139.
Blackburn EH, Chan S, Chang J et al. Molecular manifestations and molecular determinants of telomere capping. Cold Spring Harb Symp Quant Biol 2000;65:253–263.
Karlseder J, Broccoli D, Dai Y et al. p53- and ATM-dependent apoptosis induced by telomeres lacking TRF2. Science 1999;283:1321–1325.
de Lange T. Telomeres and senescence: ending the debate. Science 1998;279:334–335.
d’Adda di Fagagna F, Reaper PM, Clay-Farrace L et al. A DNA damage checkpoint response in telomere-initiated senescence. Nature 2003;426:194–198.
Herbig U, Jobking WA, Chen BP et al. Telomere shortening triggers senescence of human cells through a pathway involving ATM, p53, and p21 (Cip1) but not p16/Ink4a. Mol Cell 2004;14:501–513.
Saretzki G, Sitte N, Merkel U et al. Telomere shortening triggers a p53-dependent cell cycle arrest via accumulation of G-rich single stranded DNA fragments. Oncogene 1999;18:5148–5158.
von Zglinicki T. The role of oxidative stress in telomere length regulation and replicative senescence. Ann N Y Acad Sci 2000;908:99–110.
Wong KK, Chang S, Weiler SR et al. Telomere dysfunction impairs DNA repair and enhances sensitivity to ionizing radiation. Nat Genet 2000;26:85–88.
Goytisolo FA, Samper E, Martin-Caballero J et al. Short telomeres result in organismal hypersensitivity to ionizing radiation in mammals. J Exp Med 2000;192:1625–1636.
Damm K, Hemmann U, Garin-Chesa P et al. A highly selective telomerase inhibitor limiting human cancer cell proliferation. EMBO J 2001;20:6958–6968.
Smith LL, Coller HA, Roberts JM. Telomerase modulates expression of growth controlling genes and enhances cell proliferation. Nat Cell Biol 2003;5:474–479.
Xiang H, Wang J, Mao Y et al. Human telomerase accelerates growth of lens epithelial cells through regulation of genes mediating RB/E2F pathway. Oncogene 2002;21:3784–3791.
Kang SS, Kwon T, Kwon DY et al. Akt protein kinase enhances human telomerase activity through phosphorylation of telomerase reverse transcriptase subunit. J Biol Chem 1999;274:13085–13090.
Kang KW, Ryu JH, Kim SG. Activation of phosphatidylinositol 3-kinase by oxidative stress leads to the induction of microsomal epoxide hydrolase in H4IIE cells. Toxicol Lett 2001;121:191–197.
Gonzalez-Suarez E, Flores JM, Blasco MA. Cooperation between p53 mutation and high telomerase transgenic expression in spontaneous cancer development. Mol Cell Biol 2002;22:7291–7301.
Serakinci N, Guldberg P, Burns JS et al. Adult human mesenchymal stem cell as a target for neoplastic transformation. Oncogene 2004;23:5095–5098.
Sherr CJ. Cancer Cell Cycles. Science 1996;274:1672–1677.
Illenye S, Heintz NH. Mouse Cdc6 is associated with the mitotic spindle apparatus. Genomics 2004;83:66–75.
Adams RR, Carmena M, Earnshaw WC. Chromosomal passengers and the (aurora) ABC of mitosis. Trends Cell Biol 2001;11:49–54.
Kumari A, Schultz N, Helleday T. p53 protects from replication associated DNA double strand breaks in mammalian cells. Oncogene 2004;23:2324–2329.
Pappa A, Chen C, Koutalos Y et al. Aldh3a1 protects human corneal epithelial cells from ultraviolet and 4-hydroxy-2-noneal-induced oxidative damage. Free Radic Biol Med 2003;34:1178–1189.
Kelner MJ, Bagnell RD, Montoya MA et al. Structural organization of the microsomal glutathione S- transferase gene (MGST1) on chromosome 12p13.1–13.2. J Biol Chem 2000;275:13000–13006.(L. Armstronga,c, G. Saret)