AGE-DEPENDENT TELOMERE SHORTENING IS SLOWED …with a Millipore filter Molcut II, and an aliquot (50...

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Life Sciences, Vol. 63, No. 11, pp. 935448,1998 &pyright 0 1998 Ekvier Science Inc.

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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


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

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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.


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


A PDL

ITAS _

Control Asc2P 130pM


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.


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.


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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AGE-DEPENDENT TELOMERE SHORTENING IS SLOWED …with a Millipore filter Molcut II, and an aliquot (50...

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