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ELSEVIER
Life Sciences, Vol. 63, No. 11, pp. 935448,1998 &pyright 0 1998 Ekvier Science Inc.
Printed in the USA. All rights reserved 0024-3205/S% $19.00 + .LXI
PII SOO24-3205(98)00351-S
AGE-DEPENDENT TELOMERE SHORTENING IS SLOWED DOWN
BY ENRICHMENT OF INTRACELLULAR VITAMIN C VIA
SUPPRESSION OF OXIDATIVE STRESS
Kayo Furumoto’, Eiji Inoue’, Norio Nagao’, Eiso Hiyama’, and Nobuhiko Miwa’
‘Department of Cell Biochemistry, Hiroshima Prefectural University School of Biosciences,
Shobara, Hiroshima 727-0023: 2Department of General Medicine, Hiroshima University School
of Medicine, Kasumi, Minami-ku, Hiroshima 734, Japan
(Received in final form June 30, 1998)
Summary
Telomeres in eukaryotic somatic cells are destined to the age-dependent
shortening which has not been demonstrated to correlate to direct lesion of
telomeric DNA by reactive oxygen intermediates (ROI): still less explicable is
the inhibitory effect of ROI-scavenging on telomere shortening. Here, we
succeeded in artificial slowdown of age-dependent telomere shortening to 52-
62% of the untreated control, in human vascular endothelial cells, by addition of
the oxidation-resistant type of ascorbic acid (Asc), Asc-2-O-phosphate (Asc2P),
which concurrently achieved both extension of cellular life-span and prevention
of cell size enlargement indicative of cellular senescence. The results are
attributable to a 3.9-fold more marked enrichment of intracellular Asc (Asc,,) by
addition of AsQP, subsequently dephosphorylated before or during
transmembrane influx, than by addition of Asc itself, and also attributed to
diminution of intracellular ROI to 53% of the control level by Asc2P;
telomerase activity was at a trace level and underwent an age-dependent decline,
which was significantly decelerated by AsQP. Thus, age-dependent telomere-
shortening can be decelerated by suppression of intracellular oxidative stress
and/or by telomerase retention, both of which are achieved by enriched Asc,, but
not by extracellular Asc overwhelmingly more abundant than Asc,,.
Key Words: telomeres, telomerase, cell aging, life-span extension, oxidative stress, vitamin C transport, ascorbic acid-2-O-phosphate
DNA sequences at ends of eukaryotic chromosomes, called telomeres, are destined to shorten
during ageing of somatic cells (1). This is considered to be principally due to imperfection of
DNA replication (2). There may be other telomere-shortening triggers, among which direct lesion of telomeric DNA such as induced by ultraviolet light (3) or hyperoxia (4) is considered to be the most influential, although not shown to be related to age-dependent telomere
Corresponding author: Nobuhiko Miwa, Department of Cell Biochemistry, Hiroshima Prefectural University School of Biosciences, 562 Nanatsuka, Shobara, Hiroshima 727-0023, Japan. Tel +81-8247-4-1754, Fax +81-8247-4-0191, E-mail: miwa-nob@,bio.hiroshima-
pu.ac.jp
936 Vitamin C Prevents Telomere Shortening Vol. 63, No. 11, 1998
shortening. They are artificial agents which exogenously or transiently generate a pathogenic
level of ROI, whereas effects of a physiologic level of ROI endogenously and continuously
generated during normal aerobic metabolism on telomere-shortening are unknown. still less
explicit is the inhibitory effect of ROI-scavenging on telomere shortening.
In addition, it has not been elucidated whether retention of telomere length may elongate cellular
life-span or not. The cumulative frequency of cell division, named as a population doubling
level (PDL), is maximum when normal somatic cells exhaust the finite replicative capacity (5).
The loss of doubling potential of mortal cells may be partly due to telomere shortening below a
length of the permissive limit assumably through chromosomal instability (6) To investigate
effects of scavenging of intracellular ROI on both telomeric DNA and cellular replicative
capacity, we intended to grow human umbilical vein endothelial (HUVE) cells as representative
of nontransformed vascular cells which play a crucial role in mammalian senescence (7).
As an intracellular ROI-scavenger exogenously added to HUVE cells, we have focused ascorbic
acid (Asc), which is known to diminish humoral ROI most efficiently out of diverse antioxidant
biomolecules such as SH groups, c’-tocopherol, bilirubin and urate naturally contained in human
plasma (8). We firstly tried to serially subcultivate HUVE cells in the presence of Asc, and
failed in both the artificial slowdown of age-dependent telomere shortening and extension of
cellular life-span assumedly owing to lability of Asc So we examined whether age-dependent
telomere shortening can be prevented by the oxidation-resistant derivative of vitamin C, Asc2P,
that is phosphorylated at the 2,3-enediol moiety of an Asc molecule (9). Here, we showed
that Asc2P, but not Asc itself, is an ROI-scavenger precursor that succeeded in the artificial
slowdown of age-dependent telomere shortening.
Methods
Cell culture. HUVE cells (Kurabo Co., Osaka) being mycoplasma-free were grown in
Humedia-EB2 medium (Kurabo) supplemented with fetal bovine senrm (2%) human
recombinant epidermal growth factor (10 n@ml), human recombinant basic fibroblastic growth
factor (5 n,g/ml), hydrocortisone (I ug/ml), heparin (IO &ml), gentamycin (50 ug/ml) and
amphotericin B (50 ng/ml) (Complete Humedia) in a humidified atmosphere of 5% COz/95% air
at 37 “C, and collected at 90% confluence Cells were fed with or without Asc2P of 130 uM
successively upon every culture passage Simultaneously, other cells were successively
treated with HZ02 of 0. I or 1 uM being uncytotoxic. Cells were enumerated upon every
passage with a Coulter counter for substratum-attaching cells Spontaneously detaching cells
were as few as below the detectable limit except for H202-treated cells of terminal passage.
PDL is regarded as zero for culture starting immediately after the primary culture of human
umbilical vein, and calculated to increase according to the equation:
logI {(the number of collected cells)/(the number of seeded cells)}
CeN size &tribution. HUVE cells fed with or without Asc (Sigma) or Asc2P (Showa Denko
Co., Tokyo) were rinsed, trypsinized and then analyzed with a Coulter counter ZM equipped
with a channelyzer model 256 with calibration using PDVB latex particles (Becton Dickinson)
Vol. 63, No. 11, 1998 Vitamin C Prevents Telomere Shortening 937
of 5.1 and 13.7 urn in diameters.
Intracellular ascorbic acid (Asc,,J contents. Cells of a known number in PBS received freeze-
thawing twice and were crushed with a Potter-type teflon homogenizer for 30 set on the ice.
The cell homogenate was centrifuged at 5 “C, and supemate thus separated was stored on the
ice. Cell debris pellet separatively obtained was suspended in PBS and centrifuged again.
Supernate thus obtained was combined with supemate previously stored, and determined for
protein content with DC protein assay kit (Bio-Rad). Combined supernates were filtrated
with a Millipore filter Molcut II, and an aliquot (50 ~1) was injected on an octadecylsilica gel-
prepacked column Shodex ODSpak F-4llA of 4.6 x 150 mm (ShowaDenko Co.) connected in
a series circuit of a Gilson HPLC system 305, followed by development with mobile phase
solution consisting of 0.1 M sodium acetate, 0.02% EDTA and 0.017% n-octylamine at 40 “C
and a flow rate of 1.5 mlimin. Asc and Asc2P were detected with a coulometric ECD (electro-
chemical detector) Coulochem II (ESA Co., Bedford) at -200 mV/150 mV or with a Gilson UV
detector 115 at 2541265 nm. Authentic Asc or Asc2P mixed with extract from unadded cells
was similarly treated till injection on an HPLC column, and was detected as both a
chromatographic peak area and a retention time similar to those of Asc or Asc2P dissolved in a
Mini-Q ultrapure water.
Determination of telomere length by Southern blots. Genomic DNA was extracted with
nucleic acid extraction kit IsoQuick (ORCA Research Inc.) from IO6 cells of each passage
collected when reaching 90% confluence, and quantified by fluorometry using Hoechst 33258
(Sigma) and NM Image analysis for agarose minigel electrophorogram. Extracted DNA was
completely digested with the restriction enzyme Hinf I (TaKaRa, Kyoto) to produce terminal
restriction fragments (TRFs) as previously described (10). A portion (2 u&ne) was loaded
onto a 0.8% agarose gel, and electrophoresed at 35 V/cm for 20 hr together with 1 kb DNA
Ladder (Gibco BRL) and lambda DNA/Hind III digest (Nippon Gene, Tokyo) as size markers.
DNA was depurinated by soaking gels in 0.2 N NaOH/ 0.6 M NaCl for 25 min, and transferred
to a nitrocellulose membrane Opt&ran BA-S 85 (Schleicher & Schuel). DNA was
prehybridized with denatured salmon sperm DNA (Wako) at 65 “C, and hybridized in 10 x
Denhart solution, 1 M NaCl, 50 mM Tris-HCl @H 7.4) 10 mM EDTA, 0.1% SDS and 50
ug/rnl denatured salmon sperm DNA at 50 “C with 5’-end [32P]-labelled (TTAGGG),
(TaKaRa). Membranes were washed in 4 x SSC/O.l% SDS at 55 “C and underwent
autoradiogaphy with a Kodak X-ray film Scientific Imaging Film, followed by densitometry
with a Pharmacia laser densitometer UltroScan XL. Additionally, TRFs of each manner-
treated cells of several randomly selected passages (including PDL zero) were simultaneously
analyzed by Southern blots on the same single agarose gel, resulting in TRF lengths similar to
those estimated from separate gels.
PCR-based assay for telomerase activity. Cells of each passage collected and frozen were
assessed for telomerase activity by TRAP method (11). Briefly, lo5 cells were lysed in 10
mM Tris-HCl buffer (pH 7.5) containing 0.5% CHAPS, 0.1 mM AEBSF, 1 mM EGTA, 5
mM-mercaptoethanol, 10% glycerol and 1 mM MgC12, followed by preparation of 5 x lo3 cell-
equivalent extracts. A portion (2 ~1) was mixed with 20 mM Tris-HCl buffer (pH 8.3)
containing 0.1 @ml TS primer sequence 5’-AATCCGTCGAGCAGAGTT-3’ (ToYoBo), 50
938 Vitamin C Prevents Telomere Shortening Vol. 63, No. 11, 1998
uM dNTPs and 150 kBq [d-“*P]dCTP (Amersham) as three kinds of telomerase substrates
besides 5 x lo-” g//ml internal telomerase assay standard (ITAS), 0.5 uM T4 gene 32 protein
(Boehringer Mannheim), 2 U/ml Taq polymerase, 0.05% Tween-20, 1 mM EGTA, 1 5 mM
MS12 and 68 mM KCI. Telomerase reaction was conducted at 37 “C for 30 min and
terminated by inactivation at 90 “C for 3 min. RNase (Boehringer-Mannheim)-pretreated cell
lysate was similarly treated as the blank to confirm the activity specificity for telomerase, a
ribonucleoprotein. Simultaneously PCR amplification was hot-started by melting of wax
barrier previously formed in the tube to isolate the upper from the lower compartment
containing 0.1 ug/ml CX primer. 5’-(CCCTTA)3 CCCTAA-3’ (ToYoBo) PCR amplification
was repeated by 3 1 cycles using an Astec thermal cycler PC-800 with setting 94 “C for 40 set,
50 “C for 40 set and 72 “C for 50 set as one cycle. PCR products of 20 ulilane were loaded
onto a 10% polyacrylamide gel, and electrophoresed at 300 V/cm in 0.5 x TBE, followed by
autoradiography and densitometry.
Intracellular oxidative stress. HUVE cells seeded at 2500-10000 cells/cm* were grown on a
96-well microplate in the presence or absence of Asc2P or H202 for 18 hr, were rinsed three
times with phenol red-free and serum-free MEM and replaced by the medium containing 10
uM CDCFH (Molecular Probes, Eugene, OR) (12). After 15 min incubation, the fluorescence
intensity was measured with a fluorescence plate reader CytoFluor 2350 (Millipore, Betford,
Mass). The excitation and emission wave lengths used were 480 nm and 530 nm, respectively.
Fluorescence of the oxidative form of CDCFH increased in a manner dependent on cell numbers
and incubation times for viable cells, but not for methanol-killed cells similarly treated as the
blank.
Results
Enriching of intraceCIular Ax. HUVE cells in culture were administered with Asc or the
oxidation-resistant vitamin C derivative Asc2P of 60 to 200 uM nearly equal to Asc
concentrations in normal human plasma (13), and assessed for the intracellular Asc (Asc,,)
content by HPLC (Fig. 1A). More marked enrichment of Asc,, was achieved by Asc2P than
by intact Asc itself This is assumably due to instability of Asc in the aqueous solution,
resulting in irreversible oxidative degradation via dehydroascorbic acid to 2,3-diketogulonic acid.
Addition of Asc2P at the optimal dose of 130 uM enriched intracellular Asc 3.9-fold more
markedly than that of intact Asc of the same dose did. AsQP was dephosphorylated to free
Asc before or during uptake into HUVE cells so promptly as to be scarcely detectable as an
intact Asc2P form within cells as shown by HPLC of cell extracts.
Extension of cellular life-span. To examine effects of ROI or its scavenger on both telomeres
and cellular doubling potential under conditions as physiologic as possible, we serially
subcultivated HUVE cells until spontaneous stoppage of cell division in the absence or
presence of AsQP of 130 uM or hydrogen peroxide (H202) of 0.1 or 1 uM corresponding to
uncytotoxic concentrations much lower than H202 concentrations (>25 uM) inhibiting the cell
growth under the same conditions. Untreated cells rapidly grew at PDLs as early as below 14,
but progressively decelerated the growth and finally ceased it even under well-nourished
A
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Fig
.1.
Eff
ects
of
vi
tam
in
C
and
its
deri
vativ
e on
ce
llula
r ca
paci
ties
of
hum
an
vasc
ular
en
doth
elia
l
(HU
VE
) ce
lls.
(A)
Intr
acel
lula
r as
corb
ic
acid
(A
sc)
cont
ents
in
H
UV
E
cells
exog
enou
sly
fed
with
or
with
out
Asc
or
th
e ox
idat
ion-
resi
stan
t
type
of
vi
tam
in
C,
AsQ
P,
as
estim
ated
by
H
PLC
us
ing
a
coul
omet
ric
EC
D
and
a U
V
dete
ctor
. (B
) D
epen
denc
e of
leve
l (c
umul
ativ
e nu
mbe
r)
of
popu
latio
n do
ublin
gs
(PD
Ls)
on
cultu
re
time
for
each
pas
sage
of
cells
ser
ially
su
bcul
tivat
ed
in t
he
pres
ence
or
abs
ence
of
AsQ
P of
130
uM
or H
z02
of 0
.1 o
r 1
PM.
(C)
Cha
nges
of
m
ean
cell
size
duri
ng
cultu
re
as
anal
yzed
w
ith
a ch
anne
lizer
. (D
) C
ell
size
dist
ribu
tion
for
typi
cal
PDL
of
cells
. D
ata
are
typi
cal
of
thre
e in
depe
nden
t ex
peri
men
ts
cond
ucte
d w
ith
dish
es
in
dupl
icat
e fo
r ce
lls
subj
ect
to
each
tre
atm
ent,
SD
of w
hich
is
indi
cate
d by
the
bar
.
940 Vitamin C Prevents Telomere Shortening Vol. 63, No. 11, 1998
23.1-
12.2-
10.2-
8.-I-
6.1-
4.1-
0.1 fllvl Hz02 1 ,dvl H202
Vol. 63, No. 11, 1998 Vitamin C Prevents Telomere Shortening 941
---o----Asc2P 130 pM I __
---~---t-l202 0.1 /JM
--a----H202 1 PM
0 10 20 30 40 Population Doubling Level
Fig.2.
Dependence of telomere length on PDLs of HUVE cells serially subcultivated in
the presence or absence of Asc2P or H202. (A) Telomere length was
determined by Southern blots using an [d-32P]-labeled (TTAGGG)4
oligonucleotide probe for terminal restriction fragments (TRFs) of genomic
DNA extracted from each passage of cells fed with or without AsQP of 130
uM or H,OZ of 0.1 or 1 PM. (B) Mean TRF length is estimated as a center
of mass and expressed in kb f SE based on the equation (3): X (MWi x ODi)/ C
(ODi) where ODi is the densitometric output and MWi is the length of the
DNA at position i. Data are typical of three independent experiments, each of
which consisted of Southern blots in triplicate or quardruplicate, SD of which is
indicated by the vertical bar. Data shown are typical of two independent
experiments conducted with dishes in duplicate for cells of each treatment
group.
942 Vitamin C Prevents Telomere Shortening Vol. 63, No. 11, 1998
conditions when PDL reached the maximum of 26-28 regardless of addition of H202 of
uncytotoxic concentrations {Fig. 1B). In contrast, addition of Asc2P accelerated the cell
growth more rapidly, and enabled the retention of both the full replicative capacity and young
cell morphology (not shown) even at PDL 26-28 when control cells grew no longer (Fig. IB).
AsQP-added cells markedly enhanced the maximum PDL to 42, being higher by 14- 16 than that
of control cells, showing that Asc2P transcended beyond a barrier of the longest life-span
proper to the cells. On the other hand, antioxidants such as intact Asc, &tocopherol and N-
acetylcysteine have failed in marked and reproducible longevity extension of normal mammalian
cells in culture in contrast to success by non-antioxidants (14). Moreover, an increase in
maximum lifespan in mammals IN vwo has been shown to be rather negatively related to total
Asc contents quantified for both extracellular Asc and Asc,, in tissues or plasma (I 5).
Longevity extension has not been accomplished by a great deal of extracellular Asc labile to
oxidative degradation into injurious reductones via dehydroascorbic acid (15); it is accomplished
by enriching of Asc,,, which is converted from exogenous AsQP generating only a scarce
amount of intact Asc in the extracellular space as marginally detected with a coulometric ECD
in HPLC (16).
Suppression of ceN enlargement. AsQP exerted also inhibitory effects on PDL-dependent
increment in the cell size as analyzed with a channehzer (Fig. IC, D). Cell enlargement,
known as one of symptoms of cell senescence (17) occurred from 13.4-14.1 pm to more than
15.0 pm in diameter at a culture stage as early as PDL 11-14 preceding the outset (PDL 29-38)
of slowdown of cell division for control cells. In contrast, AsQP-added cells retained cell
sizes as small as 12 9-14.2 pm during longer passages until PDL 32, and were thereafter
enlarged drastically at the terminal passage. Thus, enriching of intracellular Asc substantiated
by Asc2P is shown to retain smallness in cell size indicative of young cell morphology.
Inhibition of age-dependent telomere shortening. DNA extracted from HUVE cells of each
culture passage was digested with the restrictionenzyme Hw!~ I to produce TRFs consisting of
both entire telomeric and partial subtelomeric regions, and analyzed by Southern blots using a
telomeric oligonucleotide (TTAGGG)4 probe (Fig. 2A) (10). Telomeres in Asc2P-unadded
cells cultured at 0, 0.1 or 1 uM Hz02 shortened by 0.16-O. 17 kilobases (kb) per PDL on the
average during serial subcultivation, and finally reached the minimum TRF length of 7.4-7 5 kb
(Fig. 2B) when cellular replicative capacity disappeared (Fig. IB) Telomere-shortening rate
was almost steady against advancement of culture passages. Age-dependent telomere
shortening was also suggested to be scarcely affected by an uncytotoxic amount of exogenous
H202, which could be scavenged assumably by cellular inherent antioxidants. In contrast,
telomere-shortening rate in AsQP-added ceils was 0.09-O. 10 kb/PDL being as slow as 52-62%
of that of unadded cells. The slowdown of telomere-shortening was distinct at the later stage
more than PDL 15. At PDLs of 26-28 when telomere length in control cells reached the
minimum, Asc2P-added cells retained TRFs or telomeres 1.3-1.6 kb longer than those of
unadded cells. AsQP-added cells at the final passage showed a TRF length of 7.0 kb, which
nearly accords with that (7.2-7.8 kb) of unadded cells of the final passage, suggesting that the
shortest length of telomeres necessary for continuation of cell division is settled independently
of either the maximal PDL or addition of the anti-oxidant or pro-oxidant. Thus, Asc2P
achieved a marked slowdown of age-dependent telomere-shortening, which may be at least
Vol. 63, No. 11, 1998 Vitamin C Prevents Telomere Shortening 943
A PDL
ITAS _
Control Asc2P 130pM
944 Vitamin C Prevents Telomere Shortening Vol. 63, No. 11, 1998
Population Doubling Level
Control AsczP 130fl M
_
ii irl
Fig.&
Dependence of telomerase activity on PDLs of HUVE ceils serially
subcultivated in the presence or absence of Asc2P or HI02 Telomerase
activity in 5000 cells was measured by TRAP method (11) (A), was quantified
by a density ratio of DNA ladder versus lSO-bp ITAS (internal telomerase
assay standard) (B), and expressed as a functron of PDL (C) Addition of
ribonuclease (RNase) to telomerase reaction mixture caused disappearance of the
activity Data shown are typical of two independent experiments conducted
with dishes in duplicate for cells of each treatment group
Vol. 63, No. 11, 1998 Vitamin C Prevents Telomere Shortening
6 6000
5000
4000
3000
2000
IO00
0
/ .
/
I-+-
,_
._
-9
Control 130 PM 0.1 PM 1 IJM
AsdP “202
Fig.4.
Levels of intracellular gross oxidative stress in HUVE cells serially subcultivated
in the presence or absence of AsQP or HZ02 as assayed by fluorometry using
CDCFH, a membrane-permeable dye precursor (12). Asc2P- or HzOz-added
or control cells were loaded with CDCFH, and underwent fluorography (A) and
fluorometry (B). A scale in the fluorograph indicates 20 pm. Fluorometric
data are typical of three independent experiments conducted with triplicate
wells containing cells of each treatment group, SD of which is indicated by the
vertical bar.
946 Vitamin C Prevents Telomere Shortening Vol. 63, No. 11, 1998
partly responsible for prolongation of the maximal life-span by the vitamin C derivative (Fig
IS).
Suppression of age-dependent decline in telomerase activity. Telomerase, an intracellular
reverse transcriptase that elongates or retains telomeres, was measured for each passage of
H202- or Asc2P-added HUVE cells by a hypersensitive method based on PCR-amplification of
telomeric repeats (11) and densitometry (Fig. 3A & B). Telomerase activity of HUVE cells
were detectable: this is rare as normal human non-regenerative somatic cells, although less
obvious than that of embryonic, stem or tumor cells (18). The activity declined age-
dependently, followed by eventual falling below the detectable lower threshold. Asc2P-added
cells exhibited telomerase activity significantly higher than that of AsQP-unadministered cells
of the same PDL (Fig. 3C) in terms of the relative activity versus the positive control for PCR
amplification (ITAS in Fig. 3A). Telomerase activity in HUVE cells is also considered to be
of a trace, but enough to satisfactorily explain the fact that the life-span was extended by I4- I6
population doublings upon Asc2P addition (Fig. IB).
Suppression of intracellular oxidative stress. Another causative candidate putatively enabling
the slowdown of telomere shortening may be efficient scavenging of ROI endogenously
generated in cells, because, inversely, acceleration of telomere-shortening is induced by
normobaric hyperoxia (40% oxygen) in human fibroblasts (6). Accordingly, ROI within Asc2P-
or H20z-added cells was quantified by fluorometry using CDCFH indicative of a gross amount
of intracellular oxidative stress (I 2). CDCFH taken up into cells is esterolysed to be
membrane-impermeable, and oxidized to be highly fluorescent primarily by HZOZ, hydroxyl
radicals and diverse peroxides (12). Control cells became notably fluorescent after loading
with CDCFH, whereas Asc2P-added cells looked dark (Fig 4A), suggesting that intracellular
ROI generated during normal aerobic metabolism was efficiently scavenged by the antioxidant.
The fluorometry showed that AsQP-added cells contained an intracellular amount of oxidized
CDCFH-attributable ROI as small as 53% of that of unadded cells (Fig. 4B). The results
suggest that ROI-scavenging is attributable to enriching of Asc,, (Fig. 1 A), being in contrast to a
conventional view that ROI can be scavenged by total vitamin C in humors or tissues which
substantially deserves the extracellular Asc that is overwhelmingly more abundant than Asc,,,
In addition, treatment with H20, of uncytotoxic concentrations (Fig. IB) and the resultant
increase in intracellular ROI to 120-124% levels versus those of untreated cells cannot
accelerate age-dependent telomere-shortening (Fig. 2A,B), sugCrestinp the telomere-maintenance
mechanism against exogenous oxidative stress to an appreciable extent.
Discussion
Age-dependent telomere shortening can be decelerated (Fig. 2B) by artificial scavenging of an
appreciable portion out of all the endogenous ROI amount, but is not accelerated by artificial
increasing of intracellular ROI within an uncytotoxic level. The decelerated telomere
shortening is attibutable to an appreciable decrease in intracellular ROI (Figs. 4A & B), resulting
in both reduced lesion in telomeric DNA and moderated lowering of telomerase activity (Figs.
Vol. 63, No. 11, 1998 Vitamin C Prevents Telomere Shortening 947
3A-C). Radiation to normal human fibroblasts with ultraviolet light is shown to damage
telomeric DNA, but not shown to shorten it (3). Normobaric hyperoxia treatment of
fibroblasts is shown to shorten telomeric DNA after Sl nuclease treatment, but not shown to
be related to cell ageing (4). Thus, any ROI-induced DNA damages have not been shown to
accelerate telomere-shortening under natural conditions; still less explicable are effects of the
inhibition of either DNA damages or ROI generation on deceleration of telomere shortening.
Another study is known for artificial telomere-elongation that is actualized by feeding of G-rich
oligonucleotides to telomerase-abundant immortal hybrid cells (19); it is attributable to direct
telomere-elongation, but not to such prevention of telomere-shortening as conformed for
telomerase-scarce mortal cells in the present study. Furthermore, normal cells transfected
with a telomerase gene extend the life-span and retain the telomere length in appearance (20);
this will not be due to prevention of age-dependent telomere shortening, but due to
compensation for the shortened telomere length, possibly resulting in malignant transformation
due to overexpression of telomerase. In contrast, maintenance of cellular normality is attained
concurrently with life-span extension in the present study.
Acknowledgements
We thank Takashi Iwai and Yuko Yamada for their technical assistance. The present study
was supported in part by Grant-in-Aid for Biochemical Research from Nagase Science and
Technolom Foundation to N.M., and by Grant-in-Aid for Exploratory Research from the
Ministry of Education, Science and Culture of Japan to N.M.
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(1995).
4. T.VON ZGLINICKI, G. SARETZKI, W.D CKE and C.LOTZE, Exptl.Cell Res. 220
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