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Proc Natl Acad Sci U S A. Oct 4, 2005; 102(40): 14398–14403.
Published online Sep 19, 2005. doi:  10.1073/pnas.0504161102
PMCID: PMC1242297
Medical Sciences

Dissociation of telomerase activity and telomere length maintenance in primitive human hematopoietic cells

Abstract

Primitive human hematopoietic cells have low endogenous telomerase activity, yet telomeres are not maintained. In contrast, ectopic telomerase expression in fibroblasts and other cells leads to telomere length maintenance or elongation. It is unclear whether this disparity can be attributed to telomerase level or stems from fundamentally different telomere biology. Here, we show that telomerase overexpression does not prevent proliferation-associated telomere shortening in human hematopoietic cells, pointing to the existence of cell type-specific differences in telomere dynamics. Furthermore, we observed eventual stabilization of telomere length without detectable changes in telomerase activity during establishment of two leukemic cell lines from normal cord blood cells, indicating that additional cooperating events are required for telomere maintenance in immortalized human hematopoietic cells.

Keywords: hematopoiesis, leukemogenesis

Telomeres are specialized structures that protect chromosome ends from recombination and fusion and from triggering cellular DNA damage checkpoint responses. Human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase, uses a template provided by the telomerase RNA component (1) to add telomeric DNA to chromosome ends (2). Most normal human somatic cells do not detectably express telomerase, but telomerase activity can be reconstituted by expression of the hTERT subunit, which in fibroblasts and other cell types leads to immortalization and maintenance or elongation of telomeres (3-6). In contrast, normal primitive human hematopoietic cells have low telomerase activity (7, 8) which is transiently up-regulated in response to cytokine-induced proliferation and cell-cycle entry (9-11). However, telomerase activity in hematopoietic stem cells (HSC) is unable to prevent age- and proliferation-associated loss of telomeric DNA (10, 12, 13). This observation has fueled speculation that increased and sustained expression of telomerase may stabilize telomere length and extend the replicative lifespan of HSC, as it does in other somatic cell types. However, recent studies have shown that regulation of telomeres and telomerase is complex, with involvement of several telomere-associated proteins (14, 15). Furthermore, control of telomere dynamics in mortal somatic cells such as fibroblasts may differ from that in long-lived self-renewing cells such as HSC, which constitutively express basal levels of telomerase. Thus, it is not clear whether telomerase overexpression alone would be sufficient to alter telomere dynamics and extend proliferation in hematopoietic cells. Elevated levels of telomerase in HSC from mice transgenic for murine TERT are associated with stabilization of telomere length, albeit without a concomitant increase in serial transplantation capacity (16). However, there are fundamental differences in telomere biology between mice and humans (17); thus, the role of telomerase in human hematopoiesis cannot simply be inferred from murine studies but must be studied directly in human cells.

One of the barriers that virtually all human cancers must overcome is the mortality limit imposed by the erosion of telomeres that occurs with each round of cell division (18). Solid tumors arising in telomerase-negative tissues typically circumvent this barrier by inactivation of checkpoints triggered by loss of telomere function and eventually by activation of telomerase expression. Although acute myelogenous leukemia (AML) is thought to originate in telomerase-expressing HSC (19, 20), telomeres in AML blasts are generally shorter than those in normal cells (21, 22), suggesting that as in normal HSC, telomerase in leukemic stem cells does not prevent proliferation-associated loss of telomeric DNA. Nevertheless, high levels of telomerase activity are present in >70% of AML cases (23). These observations raise questions about the role of telomerase in leukemic development. Up-regulation of telomerase expression may occur late in leukemogenesis under the selective pressure of compromised cell viability caused by critically short telomeres, implying that a level of telomerase activity higher than the basal levels found in HSC is needed to maintain telomeres. Alternatively, increased telomerase activity may be present early in leukemogenesis, possibly providing (pre)leukemic stem cells with a growth advantage or simply reflecting the high proportion of actively proliferating leukemic progenitors. Early telomerase up-regulation would imply that telomeres in leukemic cells shorten with proliferation despite high telomerase activity and suggests that other regulatory factors are involved in the eventual stabilization and maintenance of telomeres at a short length.

Here, we show that elevated telomerase activity does not prevent telomere shortening in human hematopoietic cells during extended proliferation. Furthermore, we demonstrate that eventual stabilization of telomere length occurs without a change in telomerase activity levels, supporting a requirement for additional events that cooperate with telomerase overexpression in immortalized hematopoietic cells. Our findings point to more complex regulation of telomere length dynamics in human hematopoietic cells compared with other somatic cell types and have implications for the role and timing of telomerase up-regulation during human leukemogenesis.

Methods

Sample Collection and Purification. Samples of cord blood (CB) were obtained according to procedures approved by the institutional review boards of the University Health Network and Trillium Hospital (Mississauga, Ontario). Samples were collected, purified, and stored as described (24).

Translocation Liposarcoma (TLS)-ETS-Related Gene (ERG) Retroviral Infection Protocol. PG13-murine stem cell virus (MSCV)-TLS-ERG or PG13-MSCV-Neo retroviral supernatant was harvested from packaging cell lines as described (25). Lineage-depleted (Lin-) CB cells were infected as described (24). Gene transfer efficiency ranged from 6% to 15% for TLS-ERG and from 12% to 43% for Neo.

hTERT Lentiviral Infection Protocol. hTERT cDNA (26) was cloned into a third-generation lentiviral vector backbone (27) containing an internal ribosome entry site-pac selection cassette. A control vector contained the GFP gene in place of hTERT. Viral supernatants pseudotyped with the vesicular stomatitis virus G protein were generated by transient transfection of 293T cells as described (28) and concentrated by ultracentrifugation. The functional titers of hTERT and GFP vectors as determined by infection of HeLa cells were 1.0-1.9 × 108 and 3.7-16 × 108 infectious particles per ml, respectively. Infections were carried out at a multiplicity of infection of 40-100. Gene transfer efficiency into Lin- CB cells ranged from 1% to 24% for hTERT and from 3% to 39% for GFP and into TLS-ERG-transduced cells from 4% to 64% for hTERT and from 19% to 100% for GFP.

Liquid Culture. Infected cells were cultured in Iscove's Modified Dulbecco's Medium containing 15% FCS, 20 ng/ml stem cell factor, and 2 ng/ml IL-3 (Amgen Biologicals). Retrovirus- and lentivirus-infected cells were selected in 1,500 ng/ml G418 or 250-400 ng/ml puromycin, respectively. Cells were harvested once or twice per week and reseeded at 4 × 105 cells per ml.

Measurement of Telomerase Activity and Telomere Length. To measure telomerase activity, a modified version of the telomeric repeat amplification protocol (TRAP), TRAP-eze telomerase detection kit (Intergen, Purchase, NY), was used. Briefly, cells were lysed in a buffer containing a 0.5% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) and protein extract from 50-10,000 cells was used for each reaction. Extract from HeLa cells was used as a positive control. Extension of the TS primer by telomerase was performed for 30 min at 25°C, and the products generated were amplified by 30 cycles of PCR at 94°C for 30 s, 50°C for 30 s, and 72°C for 90 s in the presence of 32P by using the TS and RP primers. Amplified products were resolved on a 12% polyacrylamide gel, visualized by a phosphorimaging system, and analyzed with imagequant software (Amersham Pharmacia). Protein titrations were performed to ensure the TRAP was in the linear range of the assay. Relative telomerase activity was determined by measuring the ratio of total product generated/internal control for each sample within a gel.

To assess telomere length in cells, flow-FISH analysis of cells hybridized with a telomere-specific FITC-conjugated peptide nucleic acid probe was performed as described (29, 30). Mean telomere length was calculated as described (30) by using cow thymocytes as an internal standard. Samples were performed in duplicate, and SD is indicated by error bars. Where cow thymocytes were not used, telomere length is expressed in arbitrary telomere fluorescence units.

Flow Cytometry. Flow cytometric analysis was performed with a FACSCalibur (Becton Dickinson). Cells were stained with FITC-conjugated anti-CD15, phycoerythrin-conjugated anti-CD14 or anti-CD38 (all Becton Dickinson), and PC5-conjugated anti-CD33 or anti-CD34 (Coulter) antibodies. Isotype controls were stained with mouse IgG conjugated to the appropriate fluorochrome. GFP fluorescence was detected by using detector channel FL1 calibrated to the FITC emission profile.

Results

Telomeres of Normal Human Hematopoietic Cells Do Not Shorten During Short-Term Culture. To assess the effect of telomerase overexpression in human hematopoietic cells, we constructed lentivirus vectors expressing either hTERT or GFP. To confirm that hTERT was functional when expressed from the lentivirus construct, we infected simian virus 40-transformed HA-5 cells with hTERT or GFP. GFP-transduced HA-5 cells underwent crisis as expected, whereas hTERT-transduced cells were immortalized and exhibited telomere elongation (data not shown).

Next, we infected primary CD34+ Lin- cells from five CB samples with hTERT or GFP lentivirus. After transduction, cells were carried in suspension cultures under conditions that promote myeloid differentiation. We observed a persistent increase in telomerase activity in hTERT-infected cells to levels comparable to those of HeLa cells (Fig. 1A). However, this increase did not result in a notable change in mean telomere length compared with control cells; indeed, telomere length remained stable in both control and hTERT-transduced cells for the duration of culture (Fig. 1B). The stability of telomere length in hematopoietic cells during short-term culture has also been reported by others (31).

Fig. 1.
Absence of telomere shortening during short-term culture of normal human hematopoietic cells. (A) Telomerase activity of GFP- and hTERT-transduced Lin- CB cells assessed by the telomeric repeat amplification protocol. Cumulative days in culture are indicated. ...

hTERT Overexpression Does Not Prevent Telomere Shortening During Extended Culture of Hematopoietic Cells Expressing TLS-ERG. Because of the short proliferative lifespan of normal CB cells under the culture conditions used, long-term telomere dynamics could not be examined. We therefore used a TLS-ERG-initiated leukemogenesis model developed in the Dick laboratory (25) to assess the effects of hTERT overexpression in hematopoietic cells during extended proliferation. In this system, TLS-ERG, a fusion oncogene associated with AML (32, 33), alters the erythroid and myeloid differentiation of Lin- CB cells and increases the proliferative and self-renewal capacity of myeloid progenitors (25). Despite increased self-renewal, TLS-ERG-expressing cells are not immortal and decline after proliferating in culture for 3-6 months [up to 20 population doublings (PD) versus 5 for controls] (Fig. 2A). As in control cells, telomerase activity is very low or undetectable in TLS-ERG-expressing cells after 3-4 weeks in culture (Fig. 2B), and telomere shortening occurs during extended culture (Fig. 2C). This model system thus allows investigation of the effects of hTERT overexpression in hematopoietic cells under conditions in which telomere shortening is observed.

Fig. 2.
TLS-ERG-expressing Lin- CB cells have low telomerase activity and exhibit telomere shortening. (A) Proliferation of Neo-transduced ([open triangle]) and TLS-ERG-transduced (○) cells from a representative experiment. (B) Telomerase activity of Neo- and ...

After variable times in culture (3-11 weeks), we infected TLS-ERG-transduced cells with either hTERT or GFP lentivirus (eight experiments with six CB samples). Despite significantly elevated telomerase activity that persisted for the duration of culture (Fig. 3A), the telomeres of TLS-ERG-expressing cells transduced with hTERT continued to shorten with proliferation to the same extent as those of control cells (Fig. 3B). These findings demonstrate that hTERT overexpression does not prevent telomere shortening in hematopoietic cells, even when provided with a growth advantage by oncogene expression.

Fig. 3.
hTERT overexpression does not prevent telomere shortening in TLS-ERG-expressing CB cells during extended culture. (A) Telomerase activity of cells transduced with Neo, TLS-ERG, GFP, or hTERT as indicated. Cumulative days in culture and PD are indicated ...

Eventual Stabilization of Telomere Length in hTERT-Transduced Hematopoietic Cells Occurs Without a Detectable Change in Telomerase Activity. In most cases, hTERT expression in TLS-ERG-expressing CB cells did not increase growth potential. In one experiment, however, TLS-ERG-expressing cells transduced with hTERT (T2a cells) continued to proliferate beyond the point at which the parent TLS-ERG cells or those transduced with GFP declined (data not shown). T2a cells were continuously cultured for >30 months, expanding beyond 220 PD; we consider these cells to be immortalized. As with other cells transduced with TLS-ERG and hTERT, hTERT overexpression in T2a cells did not induce further phenotypic changes. In particular, doubling time and immunophenotypic and morphologic profiles of T2a cells were similar to those of cells expressing TLS-ERG alone (Fig. 4A). The extended lifespan of T2a cells allowed us to examine telomere dynamics in hematopoietic cells over a longer period of culture. Interestingly, telomeres of T2a cells continued to shorten for >50 PD to a minimum length of 2.2 kb (Fig. 4B) despite persistently elevated telomerase activity (Fig. 4C).

Fig. 4.
Eventual stabilization of telomere length in hTERT-transduced hematopoietic cells without a concomitant change in telomerase activity. (A) (Upper) Flow cytometric analysis showing expression profiles of CD14, CD15, CD33, CD34, and CD38, and May-Grünwald-Giemsa-stained ...

Although T2a cells did not initially exhibit phenotypic alterations, after 18 months of continuous culture (≈53 PD) we observed a period of several weeks during which net proliferation continued, yet significant numbers of nonviable cells were found on morphologic examination of cytospin preparations. After this period, the growth rate of T2a cells accelerated and cell viability returned to normal. Subsequent analysis of T2a cells showed new genetic alterations, including trisomy 8 and a t(1;14) translocation (34), indicating that these cells had undergone clonal evolution. Interestingly, growth acceleration was accompanied by maintenance of telomeres at a somewhat elongated length, between 3.6 and 3.8 kb (Fig. 4B), even though the level of telomerase activity did not significantly increase over this period (Fig. 4C). Overall, these findings provide further evidence of a dissociation between telomerase activity and telomere length maintenance in human hematopoietic cells and suggest that additional genetic or epigenetic alterations independent of telomerase are required for telomere maintenance in immortalized hematopoietic cells.

Absence of Telomere Length Maintenance in TLS-ERG-Expressing Cells with Elevated Endogenous Telomerase Activity. We (34) have reported the establishment of a human leukemic cell line (termed TEX cells) originating from normal human hematopoietic cells transduced with TLS-ERG. TEX cells up-regulated endogenous telomerase activity in the course of clonal evolution, leading to immortalization. This cell line provided a tool to assess whether the lack of telomere length maintenance in TLS-ERG-transduced cells expressing exogenous hTERT was caused by specific aspects of viral expression in hematopoietic cells. Telomerase activity in TEX cells declined to very low levels by 3-4 weeks after transduction with TLS-ERG, as is typical of TLS-ERG-transduced cells, but was significantly elevated by 140 days of culture and continued to increase over the next 5 months (Fig. 5). The increase in telomerase activity in the bulk TEX culture over time is suggestive of continual selection for a subset of cells expressing high telomerase activity. Regardless of the underlying mechanism, the acquisition of elevated endogenous telomerase activity by TEX cells did not prevent telomere shortening over the first 8 months of culture (>60 PD) (Fig. 5B). Furthermore, similar to what was observed with T2a cells, telomeres in TEX cells eventually stabilized at a length of 2.4-2.8 kb (Fig. 5B and Table 1). Overall, these findings exclude issues arising from viral expression as a basis for the inability of hTERT overexpression to prevent telomere shortening in human hematopoietic cells and further support a requirement for additional cooperating events in the maintenance of telomere length in immortalized hematopoietic cells.

Fig. 5.
Absence of telomere length maintenance in TLS-ERG-expressing cells with elevated levels of endogenous telomerase activity. (A) Telomerase activity of TEX cells after cumulative days in culture indicated. Protein extract from 50-5,000 cells was used where ...
Table 1.
Telomere length of TEX cells after prolonged culture assessed by flow-FISH

Discussion

Here, we show that elevated levels of telomerase activity do not prevent proliferation-associated telomere shortening in hematopoietic cells. This observation contrasts with results obtained in other types of human somatic cells (3-6, 35, 36) and demonstrates that there are cell type-specific differences in telomere length regulation. Furthermore, the dissociation between telomerase activity and telomere length maintenance, together with the observed selection for elevated telomerase activity during clonal evolution of TEX cells, suggests that telomerase dysregulation can occur in the early stages of leukemogenesis.

We have clearly shown that elevated telomerase activity in TLS-ERG-expressing CB cells, caused either by viral overexpression or up-regulation of endogenous enzyme, does not prevent loss of telomeric DNA during extended proliferation. These results indicate that telomerase overexpression alone is unable to overcome the stringent mechanisms regulating telomere length in primitive human hematopoietic cells. In contrast to our findings, numerous studies have shown that ectopic expression of hTERT in human fibroblasts and other somatic cells that lack detectable telomerase activity is sufficient to immortalize and also generally to maintain or elongate telomeres (3-6, 35, 36). In long-lived species such as human, repression of telomerase activity in normal somatic cells may have evolved as a tumor suppressor mechanism, limiting proliferation through telomere attrition (37). However, highly regenerative tissues require telomerase to extend renewal capacity, as suggested by the bone marrow failure of patients with partial telomerase deficiency (38, 39). In tissues that renew throughout life, one would expect that strict regulation of telomerase function is required to meet lifelong proliferative requirements while at the same time preventing cancer development. Such mechanisms may not be in place in cells such as fibroblasts, which proliferate only under unusual conditions, as during wound healing. This fundamental biological difference may explain the differing effects of hTERT overexpression on telomere length dynamics in primitive human hematopoietic cells and fibroblasts.

Another example of the dissociation between telomerase activity and telomere length in hematopoietic cells was recently reported in hTERT-transduced CD4+ T lymphocytes (40). However, in contrast to our findings, the lifespan of CD4+ T cells is readily extended upon overexpression of hTERT alone, suggesting that telomere-related barriers to proliferation in mature lymphocytes are less stringent than in primitive hematopoietic cells, despite the fact that the former retain some stem cell properties such as self-renewal. Interestingly, immortalized subclones of hTERT-transduced CD8+ T lymphocytes do maintain their telomeres (41), demonstrating that the effect of hTERT overexpression differs even between different lymphoid lineages. Collectively, these observations emphasize the difficulty in extrapolating findings across different cell types and underscore the need to examine telomere biology within the cell population of interest.

Our findings of telomere shortening in hTERT-transduced primitive human hematopoietic cells contrast with those reported in mice, in which elevated telomerase activity in HSC leads to stabilization of telomere length (16). Short-lived animals such as mice do not have the same requirements for rigorous tumor suppression mechanisms as longer-lived organisms and would not gain any teleologic advantage to offset the antiproliferative effects arising from strict repression of telomerase function. The ability of telomerase to maintain telomeres in murine but not human hematopoietic cells is likely linked to less stringent telomerase regulation in the former (42) and is consistent with other reported differences in telomere biology between the two species (17). These observations underscore the importance of using human cells in the investigation of the role played by telomerase in both normal and leukemic hematopoiesis.

Our observations dispel a commonly held assumption regarding the timing of telomerase dysregulation in the leukemogenic process. In AML blasts, telomeres are generally shorter than those of normal cells (21, 22), which has been thought to reflect up-regulation of telomerase expression only in the later stages of leukemogenesis after telomeres have shortened because of leukemic proliferation. Our finding of dissociation between telomerase activity and telomere length maintenance in hematopoietic cells indicates that telomerase up-regulation can be an earlier step in the leukemogenic program. This concept is supported by the selection for elevated telomerase activity early in the evolution of TEX cells. Indeed, telomerase may be providing a growth advantage to hematopoietic cells early in leukemogenesis in a manner not dependent on telomere maintenance. However, as our experiments involved measurements of average telomere length, we cannot exclude the possibility that telomerase in hematopoietic cells is preferentially maintaining critically short telomeres, thereby protecting cells from reduced viability caused by genomic instability (43, 44). Nevertheless, the mechanisms underlying telomerase dysregulation during leukemogenesis are likely to differ from those in the development of solid tumors that originate from somatic cell types in which telomerase overexpression does lead to telomere length maintenance.

Over the course of 18 months of continuous culture, telomeres in T2a cells eventually shortened to a point at which cell viability appeared to be compromised, suggestive of a crisis-like barrier to continued proliferation despite the elevated levels of telomerase activity present. The ensuing acceleration in growth rate, coinciding with improved cell viability and elongation and subsequent maintenance of telomere length, is indicative of clonal evolution, with acquisition of secondary changes leading to improved maintenance of telomeres. In the absence of detectable changes in the level of telomerase activity, the eventual stabilization of telomere length points to alterations in telomere-associated proteins leading to better utilization of the telomerase present in the cells, possibly through improved recruitment of the enzyme to the telomere. Human orthologs to yeast Est1 and Cdc13, which mediate access of telomerase to telomeres (45), have recently been identified (46, 47) and may play a similar role in telomere stabilization in human cells. The additional genetic changes in postcrisis T2a cells (34) provide a starting point for investigating the molecular basis of telomere length regulation in human hematopoietic cells. Overall, our data demonstrate that increased telomerase activity alone is insufficient to maintain telomere length and support a requirement for additional events that cooperate with telomerase overexpression to allow the optimal growth of immortalized human hematopoietic cells. Insight into the underlying mechanisms may provide additional targets for therapy that are specific to the leukemogenic process.

Acknowledgments

We thank Irma Vulto for assistance with flow-FISH and members of the Dick laboratory for critical comments on the manuscript. This work was supported by a Canadian Institutes of Health Research studentship (to J.K.W.), a Canadian Institutes of Health Research fellowship (to J.C.Y.W.), National Cancer Institute of Canada grants (to J.E.D. and L.H.), a Canadian Cancer Society grant (to J.E.D.), Canadian Institutes of Health Research grants (to J.E.D. and P.M.L.), National Institutes of Health grants (to P.M.L. and L.H.), and a Canada Research Chair grant (to J.E.D.).

Notes

Author contributions: J.C.Y.W., J.K.W., P.M.L., L.H., and J.E.D. designed research; J.C.Y.W., J.K.W., and N.E. performed research; J.C.Y.W. and J.K.W. analyzed data; and J.C.Y.W., J.K.W., P.M.L., L.H., and J.E.D. wrote the paper.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: hTERT, human telomerase reverse transcriptase; HSC, hematopoietic stem cells; AML, acute myelogenous leukemia; CB, cord blood; TLS, translocation liposarcoma; ERG, ETS-related gene; Lin-, lineage-depleted; PD, population doublings; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate.

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