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Human Molecular Genetics Pages 1137-1141  

Reconstitution of wild-type or mutant telomerase activity in telomerase-negative immortal human cells
Introduction
Results
   hTERT restores telomerase activity in hTER-positive ALT cells
   Reconstitution of wild-type or mutant telomerase activity in hTER-negative ALT cells
Discussion
Materials And Methods
   Cells
   Plasmids and viruses
   Telomerase assay
   Northern and western blots
Acknowledgements
References

Reconstitution of wild-type or mutant telomerase activity in telomerase-negative immortal human cells

Reconstitution of wild-type or mutant telomerase activity in telomerase-negative immortal human cells

Jianping Wen, Yu-Sheng Cong, Silvia Bacchetti*

Cancer Research Group, Department of Pathology, McMaster University Medical Center, 1200 Main Street West, Hamilton, Ontario L8N 3Z5, Canada

Received February 13, 1998; Revised and Accepted April 2, 1998

Telomere shortening in human somatic cells and telomere maintenance in most human immortal cell lines and tumours correlate respectively with the absence and presence of telomerase, the enzyme that synthesizes telomeric DNA de novo. However, ~30% of in vitro immortalized human cell lines do not express this enzyme and maintain telomeres by an alternative pathway (ALT) that may also operate in some tumours. Human telomerase is a reverse transcriptase comprising minimally an RNA subunit (hTER) and a catalytic protein moiety (hTERT). Normal somatic cells retain expression of hTER but not of hTERT, and can be converted to a telomerase-positive phenotype by ectopic expression of the catalytic protein. We similarly have restored enzymatic activity to those ALT cell lines that retain hTER expression. We also report that in those ALT cells that are hTER negative, reintroduction of both hTER and hTERT is necessary and sufficient for conversion to telomerase positivity. Moreover, transfection of these cells with hTERT in conjunction with hTERs with a mutated template results in the expression of an enzyme with altered specificity. Reconstitution of telomerase activity in ALT cells, particularly an activity capable of synthesizing mutant telomeric DNA, may be exploited for the study of the ALT mechanism and its interaction with the telomerase-dependent pathway, and for assessing the effects of mutant telomeres on cell viability.

INTRODUCTION

Telomeres, the essential terminal structures of eukaryotic chromosomes, consist of sequence-specific DNA-binding proteins and of G-rich short DNA sequences (TTAGGG in vertebrates) organized in long tandem arrays (for a review, see ref. 1). Telomeric DNA is not fully replicated by conventional DNA polymerases (2,3) but can be synthesized by the specialized reverse transcriptase telomerase, a ribonucleoprotein whose RNA moiety contains a template domain for de novo addition of deoxynucleotides to the G-rich strand (4,5). Telomerase activity is thus able to overcome the end replication problem and to maintain telomere length at an equilibrium (reviewed in ref. 6).

In recent years, telomere length has been implicated in the regulation of cell lifespan (see refs 7,8). In the human system, telomere length is maintained in cells with inherent or acquired unlimited proliferative capacity (germline cells and immortalized or tumour cells, respectively), which generally express telomerase activity. Conversely, in somatic cells, where telomerase activity is undetectable or expressed at a very low level, telomeres progressively shorten with ageing in vivo or with proliferation in vitro. Experimental evidence has shown further that abrogation of telomerase activity in immortal human cells (9) and, most recently, its restoration in normal cells (10) result respectively in growth arrest or extension of lifespan. These results have established a causal link between telomere maintenance and the proliferative potential of the cell. The presence of telomerase activity in the majority of established human cell lines and tumours (11), in turn, identifies telomerase-dependent DNA synthesis as the most common means of telomere maintenance.

However, a substantial proportion of human cell lines that have been immortalized in vitro do not express telomerase but utilize an alternative mechanism (denoted ALT) to maintain their telomeres (12). Characteristically, these telomeres are much longer than those present in telomerase-positive lines (20-50 versus 3-4 kbp) and more heterogeneous. Based on these parameters, human tumours that may use the ALT mechanism for telomere maintenance have also been identified (13). In previous work, we have shown that lack of telomerase activity in ALT cell lines is not due to the absence of the essential template RNA (hTER) of the enzyme (14). Indeed, most ALT lines retain expression of this subunit. Moreover, reintroduction of wild-type hTER in that subset of ALT lines that lack it, does not restore enzymatic activity. Recently, a second essential component of telomerase, the protein moiety involved in catalysis [hEST2/hTERT/TCS1/TP2 recently renamed hTERT (15)] has been cloned (16-22). Expression of the hTERT mRNA correlates very strictly with telomerase activity in immortalized cell lines and tumours (16), pointing to a key regulatory role for this subunit. Most importantly, ectopic expression of the protein is sufficient to restore telomerase activity in human somatic cells (10,22,23), demonstrating that in these cells the catalytic subunit is the only missing component of the holoenzyme.

In the present study, we have investigated the requirements for expression of telomerase activity in ALT cells. We report that, as is the case for normal cells, ALT cells that retain expression of hTER require only hTERT to acquire telomerase activity. On the other hand, hTERT alone is insufficient to convert to telomerase-positive those ALT cells that fail to express hTER, and in this case reintroduction of both subunits is necessary and sufficient for acquisition of this phenotype. We have exploited this latter finding to reconstitute wild-type (wt) and mutant telomerase activity in these cells by transfection with hTERT and different forms of hTER.

RESULTS

hTERT restores telomerase activity in hTER-positive ALT cells

GM847 is a telomerase-negative immortal cell line that maintains telomeres via the ALT mechanism (12). The cells express the template RNA of telomerase, hTER, but fail to express the mRNA encoding hTERT, the catalytic subunit of the enzyme (14,18). To investigate whether reintroduction of this protein could restore telomerase activity to ALT cells, GM847 cells were transiently transfected with pCI-neo-hTERT-HA which encodes hTERT under control of the human cytomegalovirus (HCMV) promoter (23). Telomerase-negative WI-38 normal fibroblasts were transfected similarly as a control. Cell extracts were then assayed for telomerase activity (Fig. 1). As expected, no ladder of telomerase products was produced in reactions with extracts from untrasfected cells or cells transfected with a control plasmid vector. Transfection of the plasmid encoding hTERT, on the other hand, resulted in the appearance of a ladder of products typical of telomerase activity in both cell types. These results demonstrate that lack of hTERT expression is solely responsible for the lack of telomerase activity in hTER-positive ALT cells.


Figure 1. Telomerase activity in hTER-positive ALT cells and normal cell strains following transfection of hTERT. GM847 and WI-38 cell monolayers were transfected by lipofectamine with the pCI-neo-hTERT-HA plasmid (hTERT lanes) or a control plasmid (vector lanes), or were left untransfected (`no DNA' lanes). Cell extracts, prepared at 24 h after transfection, were assayed for wt telomerase using 5 µg of protein per assay. Lanes labelled `blank' or `293' refer to control assays in the absence of cell protein or with 0.5 µg of protein from telomerase-positive 293 cells.

Reconstitution of wild-type or mutant telomerase activity in hTER-negative ALT cells

Among ALT cell lines, WI-38 VA13/2RA and SUSM-1 do not express hTER although they retain a wt gene (14). Lack of expression of this telomerase subunit, however, is not solely responsible for the lack of enzymatic activity since cells infected with recombinant adenoviruses encoding wt hTER remain telomerase negative even while expressing abundant amounts of the RNA component (14). To investigate the requirements for telomerase activity in this system, and the possibility that re-expression of hTERT may lead to up-regulation of the endogenous hTER, VA13/2RA and SUSM-1 cells were transiently transfected with plasmids encoding hTERT or hTER, separately or in combination. As shown in Figure 2, assays of cell extracts demonstrated that neither subunit alone was proficient in restoring telomerase activity to these cells, whereas the combination of both subunits clearly did. Enzymatic activity was detectable as early as 24 h after transfection and persisted without significant decrease for at least 96 h (data not shown). Lack of telomerase activity in cells transfected with a single subunit was not due to lack of expression since hTERT protein (Fig. 3A) and hTER (Fig. 3B) were easily detected in cells receiving one or both components.


Figure 2. Restoration of telomerase activity in hTER-negative ALT cells requires hTER and hTERT. WI-38VA13/2RA and SUSM-1 were transfected by the lipofectamine method with pBSU2-33 (encoding wt hTER), or pCI-neo-hTERT-HA or both plasmids. Cells were harvested and assayed for wt telomerase activity as in Figure 1. Control assays (`293', `blank', `no DNA' lanes) are as in Figure 1.


Figure 3. Analysis of hTERT and hTER expression. Transfection was as in Figure 2. After 24 h, duplicate cultures were harvested for preparation of protein extracts or isolation of RNA. (A) Western blot analysis: expression of the hTERT protein in untransfected (no DNA) cells or in cells transfected with pBSU2-33 (hTER), pCI-neo-hTERT-HA (hTERT) or both (hTER + hTERT) was detected with antibody 3F10 against the HA tag and chemiluminescence reagents. Forty µg of cell protein were loaded in each lane. (B) Northern blot analysis: detection of hTER in cells transfected as in (A) was with a digoxigenin-labelled RNA probe and chemiluminescence reagents. All lanes were loaded with 5 µg of total RNA. RNA (1 µg) from untransfected 293 cells was analysed in parallel as control. 18S RNA served as loading control.

In recent studies, we have expressed a template mutant of hTER in telomerase-positive immortal cells in order to redirect the enzyme towards the synthesis of mutant telomeric sequences that may impair cell proliferation (24). Although mutant telomeres were generated in the cells, the co-existence of wt and mutant telomerase activity resulted in a subtle and variable phenotype in terms of cell growth and viability, probably because of the formation of hybrid telomeric ends containing both wt and mutant sequences. Our findings that the endogenous hTER in VA13/2RA and SUSM-1 cells remains silent even in the presence of the hTERT protein, while exogenous hTER can be expressed readily (14, and this work), renders these cells virtually nullizygous for the wt gene and thus suitable hosts for the expression of template mutants of hTER and of mutant telomerase activity.

To assess whether mutant telomerase activity could be expressed in this system, clonal populations stably expressing the hTERT protein were generated by transfection of VA13/2RA cells with pCI-neo-hTERT-HA and selection with G418. When assayed for wt telomerase activity, hTERT-positive clones (e.g. clone C3, Fig. 4B) were found to be negative (data not shown, but see Fig. 4A, lanes labelled `wt'), indicating that even constitutive expression of hTERT does not activate expression of the endogenous wt hTER. Clone C3 was then infected with adenovirus vectors encoding hTER mutant 34 (AdU2-34) or 37 (Ad37) and cell extracts were assayed for the corresponding mutant telomerase activity (Fig. 4A) using specific primers (9,24). While no activity was detected in uninfected C3 cells, mutant activity was clearly present in cells infected with either virus. The lack of mutant activity in uninfected 293 cells (which express wt telomerase) when assayed with the MuA reverse primer, and of wt activity in C3 cells infected with AdU2-34 or Ad37 when assayed with the ACT reverse primer, attest to the specificity of the reaction conditions. Of note is the fact that, although the endogenous hTER remains silent in C3 cells, expression of hTER 37, which is under control of the same promoter, was readily achieved (Fig. 4C).


Figure 4. Mutant telomerase activity in ALT cells expressing wt hTERT and mutant hTERs. (A) C3 is a clone of WI38VA13/2RA cells that stably expresses the hTERT protein. C3 cells, uninfected or infected with 50 p.f.u./cell of AdU2-34 or Ad37 were assayed for mutant or wt telomerase activity using specific reverse primers [MuA, (TCAC3)4 and ACT, respectively]. As control, 293 cells uninfected or infected with 10 p.f.u./cell of either virus were assayed for mutant activity. In all cases, cell extracts were prepared 48 h after infection and assays were performed with 1 µg of protein. (B) Expression of the hTERT protein in stably transfected C3 cells was detected with antibody 3F10 and chemiluminescence. The parental WI38VA13/2RA cell line was also assayed as control. Proteins were extracted 48 h after infection and 40 µg were loaded in each lane. (C) Expression of mutants hTER 34 or 37 in C3 cells uninfected or infected with AdU2-34 or Ad37 was detected with an RNA probe and chemiluminescence reagents. Total RNA was isolated 48 h after infection and 5 µg were loaded in each lane. 18S RNA served as loading control.

DISCUSSION

We have shown that telomerase-negative immortal human cells that maintain telomeres by the ALT pathway can be converted to a telomerase-positive phenotype by transfection of appropriate components of the enzyme. In GM847 cells, an example of ALT lines which retain expression of hTER, we were able to restore enzymatic activity by reintroduction of only the catalytic subunit of telomerase, hTERT. Similar results have been obtained recently by others (23). This finding indicates that GM847 cells, like telomerase-negative human cell strains (10,22,23, and this work), express all other components necessary for telomerase function. In a subset of ALT cell lines, however, lack of telomerase activity is accompanied by shut-off of both the hTERT and the hTER subunits of the enzyme, and ectopic expression of either subunit alone was insufficient to restore activity. Again, any additional components that may be required for a functional telomerase evidently persist in these cells, since we were able to convert them to a telomerase-positive phenotype by reintroduction of hTERT and hTER. Moreover, the lack of endogenous hTER in these cells allowed us to induce them to express exclusively mutant telomerase by transduction of hTERs with mutations in the template domain.

Telomerase-independent mechanisms for maintenance of telomeres, such as the ALT process, have been detected and characterized in other eukaryotes. Drosophila utilizes transposition of repetitive elements for telomere preservation (reviewed in ref. 25). In Saccharomyces, as in human cells, synthesis by telomerase is the common mode for restoration of telomeric DNA. However, mutants that are unable to express the enzyme due to deletion of the template RNA gene TLC1 (26) can activate a recombinational pathway that results in an increase of telomeric sequences (27). The dynamics of telomeres in ALT cells, a see-saw pattern of erosion and rapid increase in the length of telomeric DNA tracts, has suggested that recombination may similarly be responsible for the ALT mechanism (12,28). Like telomerase-positive human cells immortalized in vitro, ALT cell lines are derived from a variety of human tissues in which telomerase presumably was repressed during development through down-regulation of hTERT expression (16,17). ALT cells are unable to reactivate the enzyme even under the selective pressure that operates when transformed cells enter a proliferative crisis with critically short telomeres, and which leads to telomerase expression in most immortalized lines (12,29, and see refs 7,8). In this respect as well, ALT cells resemble the TLC1 deletion mutants of Saccharomyces. The status of the hTERT gene in ALT cells is unknown, but the fact that most of these cells retain expression of the hTER gene, yet have failed to reactivate the enzyme, is compatible with mutation or deletion of hTERT. This may also be the case for ALT cells that lack expression of hTER, although here a second block to telomerase reactivation has clearly occurred. The presence of a wt hTER gene in VA13/2RA and SUSM-1 cells suggests a block at the level of transcription. On the other hand, as shown here, ectopic expression of hTER is not impeded even in the case of hTER 37 whose transcription is under control of the same promoter. Whether stable expression of this hTER can be achieved in these cells remains to be investigated.

We are interested in understanding the ALT mechanism of telomere maintenance and in studying the effects of mutant telomeres on cell growth and viability. Our results on the reconstitution of wt or mutant telomerase activity in ALT cells may provide a means of addressing both questions. Long-term re-expression of telomerase through the stable transfection of GM847 cells with hTERT (23) may reveal whether co-existence of both mechanisms of telomere maintenance can be tolerated by the cells or whether abrogation of one or the other pathway ultimately would occur. Most importantly, the possibility of tagging the telomeres of ALT cells with mutant sequences via the transient expression of mutant telomerase should allow us to follow the behaviour of telomeres subject to ALT. Conversely, stable expression of mutant telomerase and substitution of wt telomeres with mutant telomeres over time can be used for the screening of different hTER mutants for their effects on cell proliferation.

MATERIALS AND METHODS

Cells

The human lung fibroblast strain WI-38 and the WI-38 VA13/2RA cell line, derived from WI-38 by immortalization by SV40, were obtained from the American Type Culture Collection and were grown in [alpha]-MEM with 10% fetal calf serum (FCS). GM847, a line of SV40 immortalized skin fibroblasts was a gift of Dr O. Pereira-Smith and was also grown in [alpha]-MEM with 10% FCS. SUSM-1, derived from liver fibroblasts by immortalization with 4-nitroquinoline (30), was obtained from Dr M. Namba and grown in Dulbecco's modified Eagle's medium (DMEM) plus 10% FCS. The 293 cell line, that constitutively expresses the E1 region of adenovirus type 5 (Ad5) (31), was grown in MEM-F11 with 10% FCS.

Plasmids and viruses

pCI-neo-hTERT-HA (23), a gift of Dr R. Weinberg, contains the hTERT cDNA with an in-frame haemagglutinin (HA) epitope tag at the 3[prime] end, and the neomycin resistance gene. Expression of hTERT in this plasmid is under control of the HCMV immediate early promoter and the SV40 polyadenylation signal. pBSU2-33 and pBSU2-34 encode, respectively, wt (specifying TTAGGG telomeric sequences) and mutant MuA or 34 hTER [specifying TTTGGG telomeric sequences (9,24)] both under control of the human U2 small nuclear RNA promoter. pGRN37 encodes mutant MuE or 37 hTER, specifying TGAGGG telomeric sequences, under control of the genomic hTER promoter (S. Weinrich, personal communication; 32). p[Delta]E1Sp1 encodes Ad5 sequences from map units 0 to 16.1, with a deletion in the E1 region (map units 1.0-9.8) (33). pJM17 and pBHG10 encode the balance of the Ad5 genome from 3.7 map units to the extreme right end with, in the case of pBHG10, a deletion of the E3 region (map units 78.3-85.8) (33,34). All plasmids were purified by centrifugation in caesium chloride-ethidium bromide density gradients. Transient transfection of plasmids into human cells was by lipofectamine (Gibco BRL) while stable transfection was by DNA-calcium phosphate co-precipitation (35). Recombinant adenoviruses were obtained by subcloning hTER constructs into p[Delta]E1Sp1, followed by co-transfection of each vector with pJM17 or pBHG10 into the complementing 293 cells (L. Marusic, M. Anton and S. Bacchetti, unpublished data; 33).Viral plaques were isolated and expanded, and recombinant viruses identified by restriction analysis of the viral genome. Viral stocks were grown and titred on 293 cells.

Telomerase assay

Cell extracts were prepared by detergent lysis, and enzymatic activity was detected by the PCR-based telomere repeat amplification protocol (TRAP) (36) as previously described (24). Forward and reverse primers and amplification conditions for detection of wt (TS and ACT) and mutant 34 (TS and MuA) telomerase activity have also been described (24). For detection of mutant 37 telomerase activity, the forward and reverse primers used were: 5[prime]-GGAACGTCCTGCATCTGAG-3[prime] and 5[prime]- (TCAC3)4-3[prime] respectively, and amplification was carried out as for wt activity (M. Anton and S. Bacchetti, unpublished data).

Northern and western blots

For detection of hTER, total RNA was extracted with Trizol (Gibco BRL), electrophoresed on 1.2% agarose-formaldehyde gels and transferred to nylon membranes (Boehringer Mannheim). Hybridization was with a digoxigenin-labelled RNA probe and detection was by chemiluminescence, using Boehringer Mannheim reagents and protocols (24). For detection of hTERT expression, cell lysates were resolved by SDS-PAGE and transferred to PVDF membranes. Immunodetection of the protein was with rat monoclonal antibody 3F10 against the HA tag (Boehringer Mannheim) and chemiluminescence (NEN Dupont).

ACKNOWLEDGEMENTS

We are grateful to Geron Corp. for providing us with the hTER mutants, and to L. Marusic and M. Anton for the recombinant adenovirus vectors. We thank Frank L. Graham for critical comments on the manuscript, Vivian Leong for help with the telomerase assays and Ping Wang for technical assistance and for help with the preparation of figures. This work was supported by a grant from the National Cancer Institute of Canada (NCIC). S.B. is a Terry Fox Cancer Research Scientist of the NCIC.

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*To whom correspondence should be addressed. Tel: +1 905 525 9140 ext. 22296; Fax: +1 905 546 9940; Email: bacchett@fhs.mcmaster.ca

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