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(The FASEB Journal. 2001;15:1014-1020.)
© 2001 FASEB

Telomerase expression prevents replicative senescence but does not fully reset mRNA expression patterns in Werner syndrome cell strains

DONGHEE CHOI*1, PETER S. WHITTIER*, JUNKO OSHIMA{dagger} and WALTER D. FUNK*

* Geron Corporation, Menlo Park, California 94025, USA, and
{dagger} Department of Pathology, University of Washington, Seattle, Washington 98195, USA

1Correspondence: Geron Corporation, 230 Constitution Drive, Menlo Park, CA 94025, USA. E-mail: dchoi{at}geron.com


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Reduced replicative capacity is a consistent characteristic of cells derived from patients with Werner syndrome. This premature senescence is phenotypically similar to replicative senescence observed in normal cell strains and includes altered cell morphology and gene expression patterns. Telomeres shorten with in vitro passaging of both WRN and normal cell strains; however, the rate of shortening has been reported to be faster in WRN cell strains, and the length of telomeres in senescent WRN cells appears to be longer than that observed in normal strains, leading to the suggestion that senescence in WRN cell strains may not be exclusively associated with telomere effects. We report here that the telomere restriction fragment length in senescent WRN fibroblasts cultures is within the size range observed for normal fibroblasts strains and that the expression of a telomerase transgene in WRN cell strains results in lengthened telomeres and replicative immortalization, thus indicating that telomere effects are the predominant trigger of premature senescence in WRN cells. Microarray analyses showed that mRNA expression patterns induced in senescent WRN cells appeared similar to those in normal strains and that hTERT expression could prevent the induction of most of these genes. However, substantial differences in expression were seen in comparisons of early-passage and telomerase-immortalized derivative lines, indicating that telomerase expression does not prevent the phenotypic drift, or destabilized genotype, resulting from the WRN defect.—Choi, D., Whittier, P. S., Oshima, J., Funk, W. D. Telomerase expression prevents replicative senescence but does not fully reset mRNA expression patterns in Werner syndrome cell strains.


Key Words:


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
WERNER (WRN) SYNDROME IS an autosomal recessive genetic disease resulting from mutations within the WRN helicase gene (1) . The disease causes many clinical features associated with aging in humans, including skin atrophy, graying of the hair, arteriosclerosis, and various cancers (2) . Studies of Wrn-/- knockout mice have revealed cellular phenotypes similar to those described for human WRN syndrome lines, although a corresponding organismal aging phenotype has not been observed in mice (3 , 4) . Mutations at the human WRN locus generally introduce nonsense codons (5) , thus truncating the WRN protein and preventing nuclear localization (6) . The WRN protein catalyzes recQ-type 3'->5'-helicase activity (7) and also displays 3'->5'-exonuclease activity (8 9 10) . Interactions of pWRN with DNA replication and transcription complexes have been reported (11 12 13) .

The means by which the biochemical deficiencies resulting from WRN mutations are translated into the pathophysiology of the syndrome are not yet defined. WRN cells display genomic instability and a mutator phenotype, resulting in an increased frequency of variegated chromosomal abnormalities (14 15 16) . Sensitivity to DNA damage induced by treatment with chemicals such as 4-nitroquinoline-1-oxide has been reported (17) , although UV repair mechanisms appear normal in WRN cells (18) , suggesting that deficiencies in DNA maintenance are selective. A hallmark cellular phenotype of WRN fibroblasts is a reduced replicative potential compared with age-matched controls (19) . As in normal fibroblasts cultured in vitro, telomeres shorten with replicative passage, although the absolute length of telomeres in senescent WRN cultures has been reported to be longer than that seen in senescent cultures of normal cells, suggesting that senescence in WRN cells may be induced by effectors other than shortened telomeres (20) . The parallels seen between accelerated tissue aging in WRN patients with accelerated in vitro aging of derived cells indicate that replicative senescence may play an important role in WRN pathologies. Recent studies have documented the immortalization of WRN fibroblast strains by the expression of an hTERT transgene (21 , 22) . In this study, we have examined the gene expression patterns observed in early- and late-passage cultures of WRN fibroblasts and contrasted these with derivative cultures that express a telomerase transgene.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture
Primary dermal fibroblast strains derived from WRN syndrome patients were obtained from the American Type Culture Collection (Manassas, Va.). All cell cultures were performed in humidified incubators (37°C) with 5% CO2, using Dulbecco’s modified Eagle’s medium (DMEM)/M199 medium (Life Technologies, Gaithersburg, Md.) with 10% fetal bovine serum (FBS). Population doublings (PD) were calculated based on cell counts at each passage. Primary strains were kept in continuous passage until cell numbers failed to double after 3 wk.

WRN transcript sequence analysis
RNA from cell strain AGO3141B was prepared as described earlier (23) . Reverse transcriptase-polymerase chain reaction (RT-PCR) and DNA sequence analysis of the WRN transcript was performed as previously described (24) .

hTERT transduction
pBABE and pBABE-hTERT retroviral preparations were a kind gift of Woodring Wright (University of Texas Medical Center at Dallas). Actively growing WRN strains were infected with virus at PD 13 (AGO3141B), PD 8.9 (AGO0780G), and PD 13.5 (AGO5229B). Three days after infection, cells were switched to selective medium containing 0.3 µg/ml puromycin.

Telomere length analysis
Mean telomere restriction fragment (TRF) lengths for WRN cell cultures were determined by Southern blotting as described earlier (25) .

Senescence-associated ß-galactosidase (SAß-gal)
Assays were performed as previously described (26) on WRN cell cultures cultured in DMEM plus 10% FBS.

Microarray analysis
The microarray system and methodologies used in this study have been described previously (23) . A complete description of the composition and performance of this system can be viewed at (http://www.geron.com/pubsupplement/wrn.html). All analyses were performed under contract with Incyte Microarray Services (Fremont, Calif.). Poly(A)+ mRNA was prepared from subconfluent cultures using OligoTex cartridges (Qiagen, Valencia, Calif.). The RNA was quantified by A260 measurements, assessed by agarose gel electorphoresis. Conversion of mRNA into either a Cy5- or a Cy3-labeled cDNA probe and competitive hybridizations of probes were performed substantially as described earlier (27) , and bound signal was quantified by fluorescence measurement. Signals that scored with a signal to background level <2.5 in both channels were not considered. The total Cy5 signal across all elements was normalized to the total Cy3 signal, and differential expression ratios were then calculated. Each experimental pairing of mRNAs was performed in duplicate, and the results present the average of the two measurements (see website for details). Genes whose expression levels changed by a mean average of 2.5-fold or more are presented in this report. The identities of all genes indicated in this report were confirmed by DNA sequence analysis of the corresponding cDNAs.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cell culture
We chose three WRN cell strains for our study. AGO0780G carries the C->T homozygous alteration at the nucleotide 1339, which results in the stop codon mutation at amino acid 389: CGA(Glu)->TGA(Stp) (5) . The WRN status of cell strain AGO5299B has not yet been determined; however, WRN protein is undetectable when assessed by Western analysis or immunostaining (28) . The status of strain AGO3141B was examined by RT-PCR sequencing of the WRN locus (24) and indicates that the presence of a novel homozygous C->T transition at nucleotide 2433, introducing a nonsense codon at amino acid 748. This result was confirmed by the PCR sequencing of the corresponding exon 19.

hTERT transduction
All three confirmed WRN strains showed typically abbreviated replicative potential, with each displaying nearly complete growth arrest by PD 20–30 (Fig. 1 ). Transduction of these strains with a control retrovirus did not alter their replicative life span, but transduction with hTERT-expressing retrovirus resulted in robust, exponential growth rates. Telomerase activity assays (TRAP) indicated that all three hTERT-transduced lines displayed abundant telomerase activity, whereas no activity was observed in control or parental strains (data not shown). The telomerase-expressing cultures showed continuous growth well past the normal senescence point of the parental strains and showed no sign of slowing or decrease for the duration of the study.



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  		Figure 1. Growth curves of parental WRN strains and hTERT-expressing derivatives. Actively dividing WRN strains ({square}) were transduced with hTERT-pBABE retrovirus ({blacksquare}) or control pBABE retrovirus ({circ}). Population doublings are shown.

We next analyzed telomere lengths in two of the WRN strains and their telomerase-expressing derivatives. Telomere lengths shortened with replicative passage by ~0.6 kbp and 1.6 kbp for strains AGO3141B and AGO0780G, respectively (Fig. 2 ). Our analyses did not include sufficient time points for these cultures to be able to predict rates of shortening accurately; however, the lengths of telomeres in these late-passage WRN strains did not appear to be significantly different from those reported for normal fibroblast strains (23 , 25 , 29) . Telomere lengths in hTERT-transduced AGO3141B cultures initially have similar TRF lengths as PD length-matched control cultures, and only with additional passaging did mean telomere lengths increase. In both hTERT-derived strains, TRF lengths continued to increase, resulting in later stage cultures with TRF lengths that were twice the length of the original WRN parental cultures.



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  		Figure 2. Telomere dynamics of WRN strains. Genomic DNA was analyzed for TRF size by Southern blotting. Results for two WRN strains and hTERT-expressing derivatives are shown.

Gene expression patterns
As a preliminary assessment, we first analyzed the SAß-gal activity of early- and late-passage WRN cells, and in hTERT-transduced derivatives. As seen in Fig. 3 , SAß-gal activity was absent from both early-passage and hTERT-transduced cultures of AGO0780G but was abundant in late-passage nontransduced cultures. Similar results were obtained with the equivalent groupings of cell strains AGO3141B and AGO5229B (data not shown).



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  		Figure 3. SAß-gal staining of early- and late-passage WRN cells and an hTERT-expressing derivative. Cultures were maintained in 10% FBS and were stained for this standard marker of senescence (Dimri et al., 1995).

We previously described our development of a midscale cDNA microarray and its application in studying senescence-associated gene expression patterns in normal cell strains (23) . This array surveys ~1000 independent genes and samples most of the major signaling and biochemical circuitries of human cells. Here, we used this same array to assess differences in mRNA expression patterns in early- and late-passage WRN lines and hTERT-expressing derivatives. All three possible pairings for each of the cultures were performed (early passage vs. late passage, early passage vs. hTERT expressing, late passage vs. hTERT expressing), allowing us to cross-check the consistency of the array system.

For initial consideration, we compared early and senescent passages of WRN cultures (Fig. 4 , dark bars). The mRNA expression patterns induced at senescence in WRN cells varied between independent cell strains and overlapped with those observed in normal senescent human fibroblast strains. In particular, senescence in WRN strains induced the expression of degradative proteases such as stromelysin-1, stromelysin-2, collagenase, and cathepsin O; immune and inflammatory response markers such as intercellular adhesion molecule-1 (ICAM-1), monocyte chemoattractant protein-1 (MCP-1), and interleukin 6 (IL-6); and growth factor modulators such as insulin-like growth factor (IGF) binding proteins 2 and 5 and stanniocalcin. In addition to these markers of fibroblast senescence, a pronounced induction of the mismatch repair homologue PMS3 was seen in two of the three lines, along with the induction of a SCG10, a homologue of the phosphoprotein stathmin. As well, senescence in two of the three lines induced significant expression of prostaglandin D synthase. mRNAs that were down-regulated by senescence in WRN cells include matrix structural proteins, such as the collagens and elastin, and hepatoma-derived growth factor. In one of the cell strains, AGO5229B, we did not observe down-regulation of any genes beyond our threshold cut-off of 2.5-fold.



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  		Figure 4. Microarray analysis of senescence-associated gene expression patterns in WRN cell strains. Genes whose levels were increased at senescence (positive values) or decreased at senescence (negative values) are indicated for strains AGO3141B (A), AGO0780G (B), and AGO5229B (C). Black bars indicate genes whose values changed between early- and late-passage nontransduced WRN cells. White bars indicate the expression levels for these same senesence-associated genes when early-passage and hTERT-expressing cells were compared.

A comparison of the expression of these same genes in hTERT-expressing WRN cell derivatives is presented (Fig. 4 , white bars). For many of the genes whose expression levels were increased at senescence, the overall effect of hTERT expression was to maintain expression at levels comparable to those seen in early-passage WRN cells. In two of the WRN strains, AGO0780G and AGO5229B, the induction of high levels of matrix proteases and inflammation-associated markers was prevented almost completely by hTERT expression, and similar effects were seen for the WRN-associated markers PMS3 and SCG10. For strain AGO3141B, the prevention of senescence-associated gene expression was less complete, and high levels of cathepsin O, rho8, and p18 persisted in hTERT-expressing cells.

For genes whose expression is reduced at senescence, hTERT expression had relatively little effect in preventing this process. For strain AGO3141B, hTERT expression did not affect the down-regulation of genes for structural proteins such as collagen IV, keratin 1 or 18, and tenascin; similar results were seen in strain AGO0780G, in which hTERT expression did not prevent the loss of expression of elastin and collagens. In neither strain did hTERT expression prevent the down-regulation at senescence of the gene for hepatoma-derived growth factor, a gene also observed to be down-regulated in normal fibroblasts at senescence (23) .

A direct comparison of early-passage WRN cells with hTERT-derived strains is presented in Table 1 and confirms the results observed for senescence-associated gene expression patterns. For instance, hTERT-expressing AGO3141B cells expressed higher levels of cathepsin O and decreased levels of collagens compared with early-passage AGO3141B cells. These comparisons reveal differences in expression in addition to those associated with WRN senescence. Compared with hTERT-expressing derivatives, early-passage AGO3141B cells overexpressed inflammation-associated mRNAs, such as MCP-1, Gro-{alpha}, and IL-6, and matrix-degrading stromelysin and tissue plasminogen activator. Interestingly, early-passage AGO3141B cells also expressed higher levels of a series of collagens and fibronectin than did hTERT-derived cells. Inversely, hTERT-derived AGO3141B cells expressed higher levels of a scattered variety of mRNAs, including transforming growth factor ß receptor 3, PPAR receptor, and the hyaluronic acid receptor CD44. Comparisons of AGO0780G cells showed higher levels of stromelysin and elastin in early-passage cells than in hTERT-expressing derivatives, whereas higher levels of the early serum response genes EGRP1 and c-fos were seen in hTERT-derivatized cells.


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  		Table 1. Comparison of gene expression between early-passage WRN cells and hTERT-expressing derivativesa


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The onset of cellular senescence can be triggered by multiple cellular events, including replicative passage in vitro, oxidative stressors, expression of activated oncogenes of cell cycle inhibitors, and many others. Our results here and those reported recently (21 , 22) clearly demonstrate that the premature replicative senescence observed in WRN fibroblasts can be thwarted by the expression of telomerase and thus implicates telomeric shortening as the principal trigger of senescence of WRN cells passaged in vitro. By what means might the WRN deficit trigger premature senescence? Because these studies strongly indicate that telomerase is capable of conferring replicative immortality, we propose that the telomere is susceptible to generalized DNA damage in repair-defective cell strains such as WRN, and as such, telomere degradation is accelerated, resulting in the premature tripping of senescence-inducing circuits. Such heightened susceptibility of the telomere to DNA-damaging agents has also been reported after the application of oxidative stressors to normal cells, conditions under which telomeres shorten at an accelerated pace (30 , 31) .

The results from mRNA expression analysis of WRN cells at senescence reinforce previous findings that the response of individual cell strains varies significantly. Although all three WRN strains investigated here arrest with similar morphologies and express a common biochemical marker, SAß-gal activity, the cadre of markers induced by senescence shares only partial overlap in the WRN lines. Similar variability has been observed in the replicative capacity of WRN strains, even among different strains developed from the same donor sample (32) . The overlap between senescence-induced expression profiles of WRN cells with those of senescent cultures of normal cells is substantial and has been predicted by previous studies of WRN cultures (33) . These include induced expression of proinflammatory genes, matrix-degrading activities, and reduced expression of matrix structural genes, all of which could contribute to the associated pathologies of the syndrome.

The induction of a homologue of a DNA mismatch repair enzyme homologue PMS3 seen in two of the WRN lines has not been reported for other normal human cell strains at senescence and also appears to correlate with the induced expression of SCG10, a homologue of the signal-transducing protein stathmin. Whether these effects are linked to the known mutator phenotype of WRN cells is unknown. Extracts from WRN cells are deficient in mismatch repair activity (34) , and thus, the accumulation of chromosomal abnormalities observed in WRN cells passaged in vitro may eventually result in the induction of repair-associated activities. However, the induction of both the PMS3 homologue and SCG10 is prevented by the expression of telomerase, suggesting that telomeric effects, and not simply random accumulation of DNA damage throughout the chromosome, contribute to the induction of these two genes.

Premature replicative senescence is a defining phenotype of primary WRN cells and thus the immortalization of these cells by telomerase has led to the suggestion that telomerase may be useful in preventing pathologies associated with the disease (21) .

In all three WRN strains examined here, telomere expression prevented the induction of only some of those genes associated with senescence. In normal diploid fibroblasts, telomerase expression prevents the induction of senescence-associated genes almost completely (35) . This difference in gene expression between telomerized WRN and normal cell strains may reflect the unstable genomic phenotype of WRN strains, because genes whose expression is changing as a result of mutation, rather than as a result of a senescence arrest, might not be affected by telomerase expression.

To what extent does telomerase affect other forms of molecular pathology in WRN cells? WRN cells accumulate numerous forms of chromosomal abnormalities, including translocations (15 , 36) , deletions (37) , and aneuploidy (38) . Karyotype analysis of early-passage (PD14) WRN strain AGO0780G revealed substantial abnormality, including aneuploidy, telomeric associations, and deletions. Telomerase expression had no obvious effect on the disarranged karyotype of this strain, indicating the preservation of the mutator phenotype in these immortalized cultures (S. Pathak, W.D.F., unpublished results). Similar results have also been reported for other telomerase-immortalized WRN strains, indicating that fundamental molecular defects are preserved by telomerization (22) . In addition, the expression of telomerase did not change the resistance of the same WRN strain toward 4-nitroquinoline (D. Shelton and W. D. F., unpublished results), again suggesting that this susceptibility of WRN cells is not related to replicative potential. In WRN cells expressing a wild-type WRN transgene, sensitivity to 4-nitroquinoline was also not suppressed, suggesting that certain pathologies of WRN cells are secondary to the primary genetic mutation (39) .

At present, there is little evidence to suggest which deficits in WRN cells are responsible for the associated pathologies of the syndrome, or the extent to which primary or secondary mutational effects contribute. For instance, karyotypic instability or increased susceptibility to DNA damage may contribute to the early-onset cancers observed in WRN patients, whereas a decreased replicative capacity and premature senescence may result in regenerative impairment and thus may contribute to skin aging or cardiovascular disease. Furthermore, accelerated telomere loss may also contribute to genetic instability and tumor progression, whereas telomere-independent mutational changes could exacerbate replicative senescence-associated changes in tissue degeneration. Our results here suggest that in addition to preventing early-onset replicative senescence, telomerase activation can prevent the induction of senescence-associated gene expression patterns in WRN cells but fails to prevent the phenotypic drift associated with long-term cultivation of WRN cells.


   ACKNOWLEDGMENTS
 
We thank Dawne Shelton for assistance with the microarray analyses, Sen Pathak (The University of Texas M.D. Anderson Cancer Center, Houston, Tex.) for karyotype analysis, and Calvin Harley for critical reading of the manuscript. DNA sequence analysis of strain AGO3141B was supported by a grant to J. Oshima from the National Institutes of Health (RO1 AG14446).

Received for publication March 16, 2000. Revision received September 15, 2000.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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