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Divisions of Carcinogenesis and Differentiation [R. F., H. L., P. B.] and Genetic Alterations in Carcinogenesis [M. H.], German Cancer Research Center, D-69120 Heidelberg, Germany, and DAKO A/S, DK-2600 Glostrup, Denmark [K. V. N.]
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ABSTRACT |
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7 kb at senescence (after 60 population
doublings). Aging of fibroblasts from both infant and adult donors was
not accompanied by chromosomal abnormalities but was correlated with
increased telomere repeat-binding factor 2 expression at both the
protein and transcriptional level. |
Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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Many immortal cell lines and tumor cells, on the other hand, have developed mechanisms to avoid telomere erosion by activation or up-regulation of telomerase activity, the ribonucleoprotein complex that is able to de novo add telomeric sequences (for review, see Ref. 5 ). In addition, some of the immortal cell lines and a small fraction of tumor cells have developed an alternative and as yet unknown pathway to maintain telomeres (6) . These cells lack detectable telomerase activity but nevertheless are characterized by long telomeres.
Olovnikov (7) was the first to propose that progressive shortening of chromosomes in dividing somatic cells eventually leads to irreversible cell cycle exit. This telomere hypothesis of cellular senescence is now widely accepted; however, detailed kinetics of telomere erosion in normal human fibroblasts from infant and adult donors have not been established. We demonstrate here that the rate of telomere erosion varies between these two donor groups and that aging of human fibroblasts in vitro is compatible with maintaining chromosomal integrity. We further show that the level of TRF-2 is up-regulated with aging, and we suggest that this up-regulation is closely linked to the aging process.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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ß-Gal Staining.
Cells were plated on glass coverslips and grown to 60–80%
confluence. Staining and fixation were performed as described
previously (8)
.
Southern Blotting.
Genomic DNA was isolated by using DNAzol reagent (Life Technologies,
Inc., Eggenstein, Germany). To determine the terminal restriction
fragment lengths, 5 µg of DNA were digested with RsaI at
37°C overnight. The digested products were subjected to
electrophoresis on a 0.7% agarose gel, blotted on a positively charged
nylon membrane (Boehringer Mannheim, Mannheim,
Germany), and neutralized [1.5 M
NaCl/0.5 M Tris-HCl and 10
mM EDTA (pH 7.2)] for 30 min at room
temperature, and the DNA was cross-linked to the membrane by UV
light (Stratalinker; Stratagene, Heidelberg, Germany). The membranes
were prehybridized (5x SSC, 0.1% lauroylsarcosine, 0.02% SDS, and
1% 10x blocking reagent; Boehringer Mannheim) at 68°C for 30 min,
hybridized for 12–18 h at 68°C with 20 ng/ml digoxygenin-labeled
telomeric human DNA probe (Oncor Appligene, Heidelberg, Germany),
washed in 4x SSC and 1% SDS for 10 min, washed in 2x SSC and 0.1%
SDS for 5 min, and washed twice in 0.1x SSC and 0.1% SDS.
Detection was performed using the CDP-Star kit (Boehringer Mannheim).
In Situ Hybridization.
Fibroblasts and HaCaT cells were harvested from cultures after
vinblastine (80 ng/ml) incubation for 50 min. After hypotonic
swelling in 0.075 M KCl buffer for 13 min at 37°C, cells
were fixed and stored in methanol/acetic acid according to standard
procedures. Hybridization was performed with the DAKO Cy3 telomere PNA
FISH kit (K5326; DAKO, Glostrup, Denmark), essentially following the
protocol of Lansdorp et al. (9)
.
Quantitative Image Analysis.
Digital images were recorded with a charge-coupled device camera
(Photometrics 1400, München, Germany) on an Axiophot fluorescence
microscope (Zeiss, Oberkochem, Germany) using a constant 7-s exposure
time. If telomeres could not be detected under these conditions, their
presence was verified by using an extended exposure time. Chromosomes
and telomeres were identified through segmentation of the
4',6-diamidino-2-phenylindole and Cy3 images, respectively. Both images
were combined and corrected for pixel shifts. The Qwin computer program
(Leica, Bensheim, Germany) was used for image analysis. Using
standardized settings, integrated fluorescence intensity was calculated
for each telomere and recorded as a graph with increasing intensity
values on the X axis and number of signals on the
Y axis. The integrated fluorescence intensity of individual
telomeres is expressed in arbitrary units, and all intensity values
were ranged into 30 intensity classes.
Indirect Immunofluorescence.
Cells grown on coverslips were fixed and stained with rabbit antibodies
against TRF-1 and TRF-2 (at a dilution of 1:500; generously provided by
T. de Lange; Rockefeller University, New York, NY) as described
previously (10)
. The secondary Cy3-labeled antirabbit
antibody (Jackson Immunoresearch Laboratory, Dianova, Hamburg,
Germany) was used at a dilution of 1:500 for 20 min at 37°C, followed
by 20 min at room temperature. Nuclei were viewed under a Leica
microscope (Leitz, Wetzlar, Germany) equipped with epifluorescence
optics using a x100 objective.
Western Blot Analysis.
Cells were grown to semiconfluence, washed, and trypsinized. To measure
protein level on the basis of equivalent cell numbers,
106 cells were lysed in 100 µl of 2x Laemmli
buffer, denatured for 5 min at 95°C, and kept frozen at -20°C
until use. When measured on the basis of equivalent protein
concentrations, 2–4 x 106 cells
were lysed in 100 µl of radioimmunoprecipitation assay buffer [10
µl of 1 M Tris (pH 7.2); 300 µl of 0.5 M NaCl;
10 µl of Triton X-100; 10 µl of 10% SDS; 10 µl of 10% sodium
desoxycholate; 50 µl of 100 mM EDTA; 610 µl of
H2O; 1 µl each of aprotinin, leupeptin, and
pepstatin (1 µg/µl); and 5 µl each of vanadate (100
mM) and Pefa-bloc (1 µg/liter)], and the protein
concentration was determined by using BCA Protein Assay Reagent A
(Pierce, Rockford, IL). Either 20-µl aliquots or 20 µg of
protein in a total volume of 30 µl of 1x Laemmli buffer,
respectively, were separated by SDS-PAGE and blotted onto
nitrocellulose membranes. Filters were blocked for 2 h in 10%
low-fat milk (in PBS with 0.1% Tween 20) and incubated with rabbit
anti-TRF-1 and TRF-2, respectively (at a dilution of 1:1,000) for
3 h at room temperature. Filters were washed with blocking buffer,
incubated with a horseradish peroxidase-conjugated goat antirabbit
antibody (1:10,000; Jackson Immunotech, Hamburg, Germany) in blocking
buffer for 1 h at room temperature, washed, and developed by using
enhanced chemiluminescence reagents (Amersham Buchler, Braunschweig,
Germany).
RT-PCR.
RNA was isolated from early and late passage fibroblasts by using
QIAshredders and the RNeasy Qiagen Minikit (Qiagen GmbH, Hilden,
Germany). RT-PCR was performed using the Omniscript RT-Kit and PCR with
Hotstar Taq polymerase (both from Qiagen). TRF-1, TRF-2, and ß-actin
were amplified for 37 cycles (95°C, 1 min; 55°C, 1 min; 72°C, 1
min). Primer sequences for TRF-1, giving rise to a 421-bp fragment,
were 5'-TGTGCGGATGGTAGGGATGC-3' and 5'-GGGCTGATTCCAAGGGTGTA-3', primer
sequences for TRF-2 (636 bp) were 5'-AGTCAATCGCTGGGTGCTCA-3' and
5'-CCTGGTGCTGGCTGTTTATC-3', and primer sequences for ß-actin (244 bp)
were 5'-GAAGTGTGACGTGGACATC-3' and 5'-CAAGCAGGAGTATGACGAGT-3'.
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Results and Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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Several reports indicated that telomeres are stable in
telomerase-positive cell lines (for review, see Ref. 12
).
To confirm this in our laboratory, we first determined the telomere
status of the immortal telomerase-positive HaCaT skin keratinocyte cell
line (13
, 14) . Q-FISH analysis demonstrated a rather
narrow signal distribution pattern, with most signals being of low
intensity (Fig. 1A
) and corresponding to a mean TRFL of 4–5 kb as determined
by Southern blot analysis (data not shown). This distribution was
characteristic for all metaphases within a given population as well as
after cloning and long-term passaging (see Fig. 1A
). Fetal
human fibroblasts, on the other hand, reportedly exhibit long
telomeres. Also, our investigation of a secondary culture of fetal
human fibroblasts by Q-FISH analysis revealed that the majority of
telomere signals were of high intensity. In addition, these cells were
characterized by a wide range of signal intensities (Fig. 1B
), demonstrating a much more heterogeneous distribution of
telomere lengths than seen in the HaCaT cells.
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), and terminal growth arrest. Previous
studies suggest that telomeres shorten in normal human somatic cells
with continuous passaging at a rate of 50–200 bp with each population
doubling, implying an approximately linear loss of telomere sequences
during aging in vitro (Ref. 4
and the
references therein).
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35 and 42 population doublings). Q-FISH analysis of
early-passage cells revealed a broad distribution of signal intensities
(Figs. 1C
and 2C
). With passaging, the number of
high-intensity signals steadily decreased (Fig. 2D
), and the
number of undetectable telomeres increased from 18 in early
passages to >40 in passages close to senescence (see Fig. 1C
). Thus, the number of very short telomeres, which were
only detectable when the exposure time was extended, increased
from about 11% to 22%. This pattern of quite uniform telomere erosion
was highly reproducible, as shown by repeat experiments with cells from
the same fibroblast strain as well as experiments with fibroblasts from
the second donor and confirmed by Southern blot analysis. The final
mean TRFL was estimated to be 5 kb (data not shown).
Reduced Telomere Loss in Foreskin Fibroblasts from Young Donors.
Whereas fibroblasts from adult donors exhibited a rather short life
span in vitro, the fibroblasts from the two infant donors,
VH7 and VH12, could be maintained in a proliferatively active
state for >20 passages (>65 population doublings). Q-FISH
analysis of these cells revealed a heterogeneous telomere profile for
earlypassage cells similar to that observed for fibroblasts from
adult donors (Fig. 1D
). However, during passaging, an
unexpected "stable" telomere profile was seen (see Fig. 1D
and Fig. 2 and F
). The shift of
the major signal peak as well as the increase in very low signal
intensities was modest, and the number of undetectable telomeres
increased only moderately from <10 in early-passage metaphases to
20 close to senescence, leaving 90% of the telomeres visible
under standard conditions that allowed the detection of all HaCaT
telomeres and 80% of telomeres of the fibroblasts from adult donors
(Fig. 2 and F
). In agreement with these
results, Southern blot analysis revealed only a minor loss of telomere
DNA, leaving a final mean TRFL of 7 kb (data not shown). This
difference in visible telomeres from infant and adult donors is
compatible with the results of a previous study in which lymphocytes
from young donors and adult donors were compared using the primed
in situ labeling method (15)
. In
addition, it was shown by Frenck et al. (16)
that the rate of telomere loss varied with age in hematopoietic cells.
They reported a rapid loss in very young children, a plateau between
the age of 4 years and adulthood, and gradual loss again later in life.
Together, these findings lend support to their hypothesis that at
different stages of maturation in vivo, the cells may differ
quantitatively or qualitatively. With regard to dermal fibroblasts, we
suggest that during infancy, i.e., a phase of rapid growth,
factor(s) are active that are able to minimize telomere erosion,
whereas these factor(s) are ineffective (down-regulated or inhibited)
in adults. Whereas this is easily detectable under "forced"
proliferation in vitro, it may not be as evident in the
dermis in vivo because proliferation of fibroblasts is
minimal in adults.
TRF-2 Is Up-Regulated with Aging in Vitro.
In the search for possible candidates involved in telomere
stabilization in fibroblasts from young donors, we first examined and
were able to exclude reactivation of telomerase activity at any point
during fibroblast cultivation by using the telomeric repeat
amplification protocol assay (data not shown). Next we investigated the
two double-stranded TTAGGG repeat-binding proteins, TRF-1 and TRF-2,
because they are believed to be involved in telomere stabilization
(10
, 17 , 18)
. Whereas TRF-1 is presumed to be a negative
regulator of telomere length, TRF-2 is suggested to play a key role in
protecting the chromosome ends from end-to-end fusions. Punctated
nuclear staining was easily detectable for TRF-1 at all passage levels
by immunohistochemistry (Fig. 3A
). TRF-2 staining, on the other hand, was faint in early
passages (Fig. 3B
) but was significantly increased in later
passage cells (Fig. 3C
), and the increase appeared to
parallel the pattern of telomere erosion. This increase was also
verified by Western blot analysis. TRF-2, present as a distinct band of
Mr 56,000, steadily increased
up to about 15-fold in late-passage fibroblasts as compared with
early-passage fibroblasts from adult and infant donors, and this
was irrespective of whether protein loading was normalized for cell
number or protein concentration (Fig. 4A
). The increase in protein was also accompanied by an
increase in TRF-2 transcription, as verified by semiquantitative RT-PCR
analysis (Fig. 4B
). Whereas the level of TRF-1 transcription
increased only slightly, if at all, in late-passage versus
early-passage cells, a clear increase was seen for TRF-2 expression,
suggesting transcriptional up-regulation of TRF-2 with aging.
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). In summary, during the process of
replicative senescence, the level of TRF-2 protein increases in human
skin fibroblasts and does not appear to be displaced from the telomeres
as suggested by the characteristic punctated pattern also seen in
interphase nuclei of late-passage fibroblasts when cells are stained
with an antibody against TRF-2 (see Fig. 3C
).
It is generally believed that senescence is accompanied by an increased
number of chromosome aberrations (dicentric and ring
chromosomes, and sister chromatid fusions; for review, see Ref.
21
). It has recently been shown that loss of TRF-2 is
responsible for such chromosomal changes (10
, 22)
. We
therefore hypothesized that due to the increasing amount of TRF-2 in
the aging fibroblasts, such aberrations would not occur. In fact, when
we screened for aberrant chromosomes, we detected chromosomal changes
only rarely in metaphases from normal cells. All metaphases from
late-passage fibroblasts analyzed here were numerically and
structurally normal (see Fig. 2 and F
).
To determine the initial functional consequences of TRF-2
up-regulation, we sought to decrease the level of TRF-2 protein by
treating late-passage VH12 fibroblasts (passage 18 represents 60
population doublings) with TRF-2 antisense oligonucleotides. After 4
weeks of treatment, the level of TRF-2 protein was reduced by 20–40%,
whereas the level of p53 remained relatively unchanged (Fig. 5C
). In these cultures, we found an approximately 25%
reduction in ß-gal (pH 6.0) staining and a slight increase in cell
number (data not shown). The most obvious change was in cell
morphology. Big, odd-shaped cells characteristic of aging cultures and
present in all controls (Fig. 5A
) were rare; instead,
elongated, more actively proliferating cells were observed (Fig. 5B
). Q-FISH analysis of the telomeres from antisense
oligonucleotide-treated or control cells (either treated with control
oligonucleotides or untreated) revealed the same telomere size
distribution in all cultures (Fig. 5D
). Thus, in contrast to
the recent finding that complete inhibition of TRF-2 by a dominant
negative TRF-2 construct caused telomeric fusions and rapid growth
arrest or even apoptosis in certain cell types (10
, 22)
, a
slight reduction of TRF-2 in aging fibroblasts appeared rather to
protract the aging process. Future studies will aim to more precisely
characterize the role and function of TRF-2 in this process.
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Acknowledgments |
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FOOTNOTES |
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1 Supported in part by Deutsche
Forschungsgemeinschaft Grant Bo 1246/4-2 (to P. B.) and European
Union Grant QLRT-1999–01341 (to P. B.). R. F. and H. L.
contributed equally to this work. 
2 R. Figueroa and P. Boukamp contributed equally
to this work.
3 To whom requests for reprints should be
addressed, at Division of Carcinogenesis and Differentiation, German
Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg,
Germany. Phone: 49-6221-424516; Fax: 49-6221-424551; E-mail: P.Boukamp{at}DKFZ-Heidelberg.de
4 The abbreviations used are: TRF, telomere
repeat-binding factor; TRFL, telomere restriction fragment length; PNA,
peptide nucleic acid; FISH, fluorescence in situ
hybridization; Q-FISH, quantitative FISH; RT-PCR, reverse
transcription-PCR; ß-gal, ß-galactosidase.
Received 6/ 1/99. Accepted 4/14/00.
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