2028 Chin Med J 2007;120(22):2028-2035

Original article
Aminoguanidine delays the replicative senescence of human
diploid fibroblasts
WANG Pei-chang, ZHANG Jian, ZHANG Zong-yu and TONG Tan-jun

Keywords: aminoguanidine; replicative senescence; human diploid fibroblast;

advanced glycation end products; comet assay

Background The accumulation of free radicals and advanced glycation end products (AGEs) in cell plays a very
important role in replicative senescence. Aminoguanidine (AG) has potential antioxidant effects and decreases AGE
levels. This study aimed to investigate its effect on replicative senescence in vitro.
Methods The effects of aminoguanidine on morphology, replicative lifespan, cell growth and proliferation, AGEs, DNA
damage, DNA repair ability and telomere length were observed in human fetal lung diploid fibroblasts (2BS).
Results Aminoguanidine maintained the non-senescent phenotype of 2BS cells even at late population doubling (PD)
and increased cumulative population doublings by at least 17-21 PDs. Aminoguanidine also improved the potentials of
growth and proliferation of 2BS cells as detected by the MTT assay. The AGE levels of late PD cells grown from early PD
in DMEM containing aminiguanidine decreased significantly compared with those of late PD control cells and were similar
to those of young control cells. In addition, the cells pretreated with aminoguanidine had a significant reduction in DNA
strand breaks when they were exposed to 200 μmol/L H2O2 for 5 minutes which indicated that the compound had a
strong potential to protect genomic DNA against oxidative stress. And most of the cells exposed to 100 μmol/L H2O2 had
much shorter comet tails and smaller tail areas after incubation with aminoguanidine-supplemented DMEM, which
indicated that the compound strongly improved the DNA repair abilities of 2BS cells. Moreover, PD55 cells grown from
PD28 in 2 mmol/L or 4 mmol/L aminoguanidine retain telomere lengths of 7.94 kb or 8.12 kb, which was 0.83 kb or 1.11
kb longer than that of the control cells.
Conclusion Aminoguanidine delays replicative senescence of 2BS cells and the senescence-delaying effect of
aminoguanidine appear to be due to its many biological properties including its potential for proliferation improvement, its
inhibitory effect of AGE formation, antioxidant effect, improvement of DNA repair ability and the slowdown of telomere
shortening.
Chin Med J 2007;120(22):2028-2035

F ree radicals (FR) and reactive oxygen species (ROS)

accumulate in cells with replicative senescence,
which speeds up protein oxidative modification, oxidative
DNA damage, telomere shortening, senescence-
associated gene expression and the formation of advanced
glycation end products (AGEs).1-4 Many different
sublethal oxidative stresses including hyperoxia,
hydrogen peroxide (H2O2) and tert-butylhydroperoxide
also induce senescence-like growth arrest or premature
senescence characterized by biomarkers of cellular
senescence.5-7 These facts strongly support the FR theory
of senescence and suggest that antioxidant
supplementation may delay replicative senescence
according to their efficacy in removing FR and ROS,
which has been demonstrated in previous studies.8-10
Aminoguanidine (AG) has potential antioxidant effects
and its supplementation reduces the concentration of lipid
peroxides in primary culture. It is also an inhibitor of
diamine oxidase and nitric oxide synthase and it inhibits
the formation of AGEs. 11-13 In previous studies, the
compound was shown to protect against the aging of the
immune system, skin and other tissues.14,15 Moreover, 4
mmol/L or 2 mmol/L aminoguanidine supplementation
significantly decelerated the rate of decline in saturation
density and the decrease in the frequency of diploid cells
in cell lines from senescence-accelerated mice (SAM
mice).16 The aim of this study was to investigate the
effect of aminoguanidine on replicative senescence and
its mechanism using human fetal lung diploid fibroblasts
(2BS). Cumulative population doublings (CPDs) is used
to measure the lifespan of cultured cells. Several markers
related to replicative senescence, including the potential
of cell proliferation, AGEs, DNA repair ability and
telomere lengths, were also studied to provide a better
knowledge of the mechanism of delayed senescence by
aminoguanidine. Carnosine was used as the control.
Department of Medical Laboratory, Xuanwu Hospital of Capital
Medical University, Beijing 100053, China (Wang PC and Zhang J)
Aging Research Center, Medical and Health Center of Peking
University, Beijing 100083, China (Zhang ZY and Tong TJ)
Correspondence to: Dr. WANG Pei-chang, Department of Medical
Laboratory, Xuanwu Hospital, Capital Medical University, Beijing
100053, China (Tel: 86-10-83198688. Fax: 86-10-83154745.
Email: peichangwang@yahoo.com)
This work was supported by the grants from the National Natural
Science Foundation of China (No. 30672469) and the Beijing
Natural Science Foundation (No. 7062030).
Chinese Medical Journal 2007; 120(22):2028-2035
METHODS
2BS Cells
2BS cells were previously isolated from female lung
fibroblast tissue and had been fully characterized.17,18 The
2BS cell line was originally established at the National
Institute of Biological Products (Beijing, China). The
2BS cells were considered to be young at PD30 or earlier
and to be fully senescent at PD55 or later.
Cell culture
The cells were grown in Dulbecco’s Modification of
Eagle’s Minimum Essential Medium (DMEM, Life
Technologies Inc., Grand Island, NY, USA) supplemented
with 10% fetal calf serum and with 60 μg/ml penicillin
and 100 μg/ml streptomycin in an incubator at 37°C with
5% CO2. The CPDs were calculated as log2 (D/D0), where
D and D0 are defined as the density of cells at the time of
harvesting and seeding, respectively.
MTT assay
The method of Chiba was used.19 In brief, the cells at
PD28 were seeded into flat-bottomed 96-well microplates
at a density of 2.5 × 103 cells/0.2 ml per well. After 20
hours, when the cells reached a sub-confluent state, the
cells were transferred to grow in a special culture medium
containing various concentrations of aminoguanidine
(Sigma, St. Louis, MO, USA), in an incubator at 37°C
with 5% CO2 up to 108 hours. The absorbance values of
each well were determined spectrophotometrically at 490
nm using a Microplate Reader (BIO-TEK, Rockville, MA,
USA).
Determination of AGE
AGEs from 2BS cells were determined with a modified
method of Yan.20 The collected cells were broken up with
three cycles of sonication (JY92-Ⅱ, Ningbo, China) for 5
seconds and extracted with 1.0 ml ethanol:diethyl ether
(3:1 (v/v)). The supernatants were prepared and then
assessed for fluorescence which was monitored on an
F-3000 Hitachi spectrofluorimeter. Values for relative
fluorescence were expressed in arbitrary units correcting
for solvent volumes and the protein content of the sample.
Protein contents were determined by a modified Lowry
assay. Scans of both the excitation and the emission
characteristics were performed in 340 nm and 430 nm,
respectively.
Comet assay
The single-cell gel electrophoresis (SCGE, also called
comet assay) technique was performed as described
previously with minor modifications.21 In brief, fully
frosted microscopic slides were covered with 110 μl of
0.5% normal melting agarose (NMA), then 70 μl of 1%
lower melting agarose (LMA) containing 5×104 2BS cells
was rapidly pipetted onto the first agarose layer. The
slides were immersed in freshly prepared cold lysing
solution (2.5 mol/L NaCl, 100 mmol/L Na2EDTA, 10
mmol/L Tris, pH 10, 1% sodium sarcosinate) with 1%
Triton X-100 for 40 minutes at 4°C in the dark.
2029
Electrophoresis was conducted at 4°C for 25 minutes
using 15 V (1 V/cm). The image analysis system
(Q550CW, Leica, Manheim, Germany) was used. The
percentage of comet tail area (tail area/total area) and the
tail length (from the center of head to the end of the tail)
were analyzed in 100 cells for one slide.
Determination of telomere length by southern blots
DNA extracted from 2BS cells was completely digested
with the restriction enzyme EcoR1 to produce terminal
restriction fragments (TRFs). The digested DNA was
loaded onto a 1.0% agarose gel and electrophoresed at 40
V for 8 hours together with λDNA/Hind Ⅲ digest as a
size marker. DNA was depurinated by soaking gels in
0.25 mol/L HCl for 10 minutes, denatured in 0.2 mol/L
NaOH-0.6 mol/L NaCl for 25 minutes then transferred to
a nitrocellulose membrane, NYTRAN (Schleicher &
Schuell, Inc., NH). DNA was prehybridized with
denatured salmon sperm DNA (ssDNA) at 50°C for 8
hours and hybridized in a solution (5×Denhardts, 5×SSC,
0.1% SDS, 100 μg/ml ssDNA, 20 mmol/L NaH2PO4, pH
7.4) at 50°C with 5-end [32P]-labeled (TTAGGG)4 for 36
hours. Membranes were washed in 4×SSC/0.1% SDS at
56°C and underwent autoradiography with Kodak X-ray
film. The system of Image Master VDS (Pharmacia
Biosci. Inc., Sweden) was used to take the pictures and
the Leica Image analysis system was used to analyze the
length of telomeres. The telomere-shortening rate was
calculated as (L28–Ln)/(n–28), where Ln was the
telomere length of 2BS cells in PDn.
Statistical analysis
Values were expressed as mean ± standard deviation (SD).
The significance of differences between experimental
groups was analyzed by Student t test using SAS software.
A P value less than 0.05 was considered statistically
significant.
RESULTS
Effect of aminoguanidine on cell morphology
The results are shown in Figs.1 and 2. The control cells in
late PD showed characteristics of senescence with
accumulation of granular cytoplasmic inclusions.
Compared with control cells, the cells grown in 2 mmol/L
or 4 mmol/L aminiguanidine had a flat, spread-out
appearance with very uniform spacing. Evan at late PD
the cells continually retained unaltered morphology, a
refractive cytoplasm with thin and long projections and a
regular array, like young control cells. The phenotypic
characteristics of aminoguanidine-treated cells is similar
to that of carnosine-cultured cells, as described
previously.8,9 When these cells were transferred to normal
DMEM a senescent phenotype appeared after one week
of incubation. However if the normal cells at late PD
were treated by aminoguanidine, 2 mmol/L or 4 mmol/L,
the senescent phenotype of the cells could not be reversed
completely and the culture could not become confluent
like cells in early PD. These results suggested that
aminoguanidine had a strong effect on maintaining a
2030 Chin Med J 2007;120(22):2028-2035

Fig. 1. Alteration of 2BS cell morphology in response to long-term growth in DMEM medium supplemented with aminoguanidine or
carnosine (original magnification×100). A Control cells at PD55 demonstrating a senescent phenotype; B, C and D represent cells at
PD55 grown from PD28 in DMEM containing 20 mmol/L carnosine, 2 mmol/L aminoguanidine and 4 mmol/L aminoguanidine
respectively.

Fig. 2. 2BS cells morphology of cells grown in DMEM supplemented with aminoguanidine, and the effect of removing aminoguanidine
(original magnification×100). A and C represent cells at PD56 grown from PD28 in DMEM containing 2 mmol/L and 4 mmol/L
aminoguanidine; B and D represent cells of A or C after splitting and transferring to normal DMEM for 7 days.

Table 1. The life span of 2BS cells in CPDs, based on the actual additional 11 PDs). The effect of 8 mmol/L
number of cells harvested and seeded (n=3) aminoguanidine was similar to that of 2 mmol/L or 4
Treatment Time of transfer to CPDs Average PDs
mmol/L aminoguanidine, while 1 mmol/L aminoguanidine
special medium per week
Control – 54.2±3.4 1.7±0.1
seemed to be less effective in delaying the replicative
AG 2 mmol/L PD 28 71.6±6.8* 2.6±0.2 senescence of 2BS cells. In comparison to the control
AG 4 mmol/L PD 28 75.3±6.6* 2.7±0.2 cells, the cells with aminoguanidine treatment had a much
Carnosine 20 mmol/L PD 28 65.7±5.2* 2.3±0.1 higher growth rate. The 2BS cells grown in 2 mmol/L or
Cells were grown from PD28 in DMEM supplemented with aminoguanidine or 4 mmol/L aminoguanidine were split 2.65 times or 2.74
carnosine. The cultured cells were split in ratios of 1:2 or 1:4 when the
confluence of the culture was over 85%. Single cell suspensions were counted
times per week on an average while the control cells were
with a coulter counter. CPDs were calculated as log2(D/D0), where D and D0 split only 1.75 times per week.
were defined as the density of cells at the time of harvesting and at seeding,
respectively. The last culture was defined as the subculture that could not
Optimum concentrations of aminoguanidine and
become confluent in 3 weeks. *P<0.05, vs control group.
their effects on the potentials of growth and
non-senescent phenotype of late PD 2BS cells, whereas, it proliferation of 2BS cells
has no potential to reverse the senescent phenotype of the The effects of different concentrations of aminoguanidine
cells. on the potential for growth and proliferation of 2BS cells
were measured with the MTT assay, as shown in Fig. 3.
Effect of aminoguanidine on cumulative population The absorbance values of PD28 cells grown in 2 mmol/L,
doublings or greater, aminoguanidine for 60 hours were significantly
As shown in Table 1, aminoguanidine at 2 mmol/L or 4 higher than that of the cells grown in normal DMEM,
mmol/L delayed replicative senescence of 2BS cells whereas, there was no significant difference in the
significantly by at least 17–21 PDs, which appears to be absorbance values between control cells and the cells
better than 20 mmol/L carnosine (only increasing by an treated with 1 mmol/L aminoguanidine (P>0.05).
Chinese Medical Journal 2007; 120(22):2028-2035
Fig. 3. Effects of various concentrations of aminoguanidine on
proliferation of 2BS cells in the MTT assay. PD28 2BS cells
were seeded in 96-well microplates at a density of 2.5×103
cells/0.2 ml per well. After the cells had grown at 37°C with 5%
CO2 for 20 hours, the cells were transferred to special DMEM
containing various concentrations of aminoguanidine for 60
hours. Cell proliferation was determined by measuring
absorbance values at 490 nm. DMSO was used as a reagent
blank. The MTT assays were performed independently. Each
point represents the mean of six experiments. *P<0.01 vs control
group.
Fig. 4. Time course of the effect of aminoguanidine on
proliferation of 2BS cells in the MTT assay. PD28 2BS cells
were seeded in 96-well microplates at a density of 2.5×103
cells/0.2 ml per well, then cultured with DMEM containing 2
mmol/L or 4 mmol/L aminoguanidine for 12, 36, 60, 84 and 108
hours. Cell proliferation was determined by MTT assay. Each
point represents the mean of six experiments.
The phenomenon of 2 mmol/L or 4 mmol/L
aminoguanidine increasing the absorbance of PD28 2BS
cells can be obviously observed after 36 hours of
incubation with the compound. Moreover, the absorbance
values of cells incubated with 2 mmol/L or 4 mmol/L
aminoguanidine were significantly higher than those
without aminoguanidine treatment at 60, 84 and 108
hours (P<0.01). The results above indicate indirectly that
aminoguanidine could be helpful for the growth and
proliferation of 2BS cells.
AGE levels of 2BS cells grown in aminoguanidine
The reduction of AGE levels of 2BS cells grown in 2
mmol/L or 4 mmol/L aminoguanidine is summarized in
Table 2. 2BS cells following long-term growth in
2031
aminoguanidine had much lower levels of AGEs
compared with control cells in the senescent phase
(P<0.01). Moreover, the AGE levels in the late PD 2BS
cells grown from PD28 in aminoguanidine were similar
to those of early PD 2BS cells. Two mmol/L or 4 mmol/L
aminoguanidine appeared to be better than 20 mmol/L
carnosine in decreasing the AGE levels of the cells. These
facts indicated that aminoguanidine could inhibit the
formation of AGEs or speed up AGE degradation in 2BS
cells.
Table 2. The AGE levels of 2BS cells grown from PD28 in DMEM
supplemented with aminoguanidine or carnosine
Treatment PD AGEs
Control 28 2.85±0.24
Control 55 9.68±1.61
Aminoguanidine 2 mmol/L 56 3.98±0.47*
Aminoguanidine 4 mmol/L 56 3.12±0.41*
Carnosine 20 mmol/L 56 5.23±0.65*
Data were obtained from three independent experiments and
analyzed by SAS statistics software. *P<0.05, vs PD55 control.
Aminoguanidine protected genomic DNA against
oxidative damage induced by H2O2
PD28 2BS cells, pretreated with different concentrations
of aminoguanidine, were exposed to 200 μmol/L H2O2 for
5 minutes in the dark (Table 3). The DNA damage of the
cells was measured with the comet assay. The cells
pretreated with aminoguanidine had much shorter comet
tails and smaller comet tail areas compared with the cells
without aminoguanidine treatment. The comet tail lengths
and comet tail areas of the cells were negatively related
with the pretreatment concentration of aminoguanidine.
Moreover, the cells pretreated with 8 mmol/L
aminoguanidine had no obvious comet tail. These facts
suggested that aminoguanidine had a strong potential to
protect the genomic DNA of 2BS cells against oxidative
stress.
Table 3. The extent of the 200 µmol/L H2O2-induced DNA damage
of the PD28 2BS cells pretreated with different concentrations of
aminoguanidine for 12 hours
Aminoguanidine (mmol/L) Tail length (μm) Tail area/total area(%)
0 122.42±6.21 92.12±7.25
1 91.58±6.55 80.71±7.62
2 40.57±3.36 37.36±4.75
4 14.88±2.05 11.49±1.24
8 11.53±1.97 7.03±1.45
10 5.50±1.65 4.66±1.54
Aminoguanidine improved DNA repair ability of 2BS
cells
PD31 2BS cells were damaged by 100 μmol/L H2O2 for 5
minutes in the dark, and then the cells were continually
cultured with DMEM containing different concentrations
of aminoguanidine. The DNA damage of the cells was
measured with the comet assay. The results are shown in
Fig. 6 and Table 4. The comet tail lengths and tail areas of
the cells post-treated with aminoguanidine decreased
significantly compared with those of the cells post-treated
with normal DMEM and, were negatively correlated with
the aminoguanidine concentration. Most of the cells
2032 Chin Med J 2007;120(22):2028-2035

Fig. 5. The protective effects of different concentrations of aminoguanidine on genomic DNA of PD 28 2BS cells against oxidative stress
induced by H2O2 (original magnification×600). PD 28 2BS cells were cultured with DMEM containing different concentrations of
aminoguanidine for 12 hours. The cells were then exposed to 200 µmol/L H2O2 for 5 minutes at 4℃ in the dark. DNA damage was
analyzed using the comet assay. A, B, C and D represent 2BS cells pretreated with 0 mmol/L, 2 mmol/L, 4 mmol/L or 8 mmol/L
aminoguanidine, respectively.

Fig. 6. The effect of aminoguanidine on the repair abilities for DNA damage in 2BS cells using the comet assay. PD31 2BS cells were
damaged by 100 μmol/L H2O2 in the dark then cultured continually with DMEM containing 0 mmol/L (A),2 mmol/L (B), 4 mmol/L (C)
or 8 mmol/L (D) aminoguanidine for 1 hour.

Table 4. The effect of HDTIC aminoguanidine on the repair ability Table 5. Effect of aminoguanidine on telomere lengths
of oxidative DNA damage in 2BS cells using the comet assay in 2BS cells (n=3)
Aminoguanidine Tail length (µm) Tail area/total area (%) Treatment PD 28 (kb) PD 55 (kb) Telomere-shortening
0 109.31±13.26 84.07±10.01 rate (bp/PD)
1 36.91±4.30 34.40±3.82 Control 8.93±0.54 7.01±0.43 71.1
2 30.56±3.57 28.36±3.59 AG 2 mmol/L 8.93±0.54 7.94±0.47 36.7*
4 21.19±2.84 18.44±2.08 AG 4 mmol/L 8.93±0.54 8.12±0.53 30.0
8 9.93±1.42 9.95±1.72 The telomere lengths of 2BS cells grown from PD 28 in 2 mmol/L or 4 mmol/L
10 6.87±1.45 5.89±1.80 aminoguanidine were analyzed. Mean TRF length was estimated as a center of
PD31 2BS cells were damaged by 100 µmol/L H2O2 for 5 minutes in the dark mass based on the equation: Σ(MWi×ODi)/Σ(ODi), where ODi is the
then cultured continually with DMEM supplemented with aminoguanidine for 1 densitometric output and MWi is the length of the DNA at position i. Data are
hour. The DNA damages of the cells were observed using the comet assay. typical of three independent experiments. *P<0.01, vs control.

post-treated with 4 mmol/L aminoguanidine had no DISCUSSION

obvious comet tails. These results indicated that
aminoguanidine improved the DNA repair ability of 2BS
cells.
Telomere lengths of 2BS cells cultured with
aminoguanidine
The telomere lengths of the 2BS cells treated with
aminoguanidine are shown in Fig. 7 and Table 5. At PD55,
when telomere length of control cells reaches the minimum
(7.01 kb), the cells grown from PD28 in 2 mmol/L or 4
mmol/L aminoguanidine retained telomeres of 7.94 kb and
8.12 kb. Quantitatively, the telomeres of 2BS cells grown
in 2 mmol/L or 4 mmol/L aminoguanidine were shorten by
36.7 bp/PD and 30 bp/PD, respectively, while, the
telomere-shortening rate of the control cells was 71.1 bp
per PD. These results showed that aminoguanidine had a
strong potential of slowing down the shortening rate of
telomere of 2BS cells.
Human diploid fibroblasts can divide for only a limited
number of times in vitro, a phenomenon known as
replicative senescence.22 Senescent cells have charac-
teristic morphological changes, including irregularities in
the size and shape, a failure to line up in parallel arrays,
debris in the medium, accumulation of granular material
in the cytoplasm and even loss of the potential to be
confluent. Although many kinds of factors, such as
senescent-associated genes, AGEs, DNA damage and
repair ability, telomere shortening etc, are responsible for
cellular senescence, the accumulation of free radicals and
reactive oxygen species may play a key role in replicative
senescence. 4-7 In fact, several antioxidants, including
carnosine, have been demonstrated previously to delay
senescence and to maintain a non-senescent phenotype of
diploid fibroblasts.8-10 Aminoguanidine, a scavenger of
active oxygen free radicals, inhibits diamine oxidase and
nitric oxide synthase.11-13 In this study we observed that 2
Chinese Medical Journal 2007; 120(22):2028-2035
Fig. 7. Telomere lengths in 2BS cells grown from PD 28 in 2
mmol/L or 4 mmol/L aminoguanidine. Genomic DNA was
digested completely with EcoR1. Telomere length was
determined by southern blots using [γ-32p]-labeled (TTAGGG)4
oligonucleotide probe for terminal restriction fragments (TRFs)
of genomic DNA. Lanes 1-4 represent the telomere lengths of
control cells at PD55, 4 mmol/L AG-treated cells at PD55, 2
mmol/L AG-treated cells at PD55 and 20 mmol/L
carnosine-treated cells at PD55, respectively.
mmol/L or 4 mmol/L aminoguanidine maintained a much
more normal morphology of 2BS cells even as they
reached the end of their lifespan. However, these cells
showed a senescent phenotype within a week when they
were transferred to the DMEM containing no
aminoguanidine. If the normal cells at late PD were
treated by aminoguanidine, the senescent phenotype of
the cells could not be reversed completely and the culture
could not become confluent like cells at early PD. These
results suggest that the compound maintains the
non-senescent phenotype but has no potential to reverse
the senescent phenotype of 2BS cells.
Cumulative population doublings (CPDs) represent
replicative life-spans of cells. Aminoguanidine
significantly delayed the replicative senescence of 2BS
cells by at least 17-21 PDs, which appears to be better
than carnosine, which only delayed replicative
senescence an additional 6-10 PDs. In comparison to
control cells the cells incubated with aminoguanidine
proliferate fast, which was measured by the split times of
the culture per week. These data indicate that
aminoguanidine significantly delay the replicative
senescence of 2BS cells.
Many studies have claimed that the number of
non-dividing cells increased exponentially during
proliferation of normal cells, which directly caused a
failure to become confluent at the end of the culture in
vitro.23,24 The results of MTT assay showed that the
absorbance values of 2BS cells incubated with
aminoguanidine increased significantly when compared
with that of control cells, which indicates that the
compound may promote cell division or prevent the entry
2033
of cells from M or S phase into the G0 phase of the cell
cycle. Aminoguanidine improvment of cell growth and
proliferation may be related to the fast rate of growth of
the subculture of the 2BS cells grown in the compound
and, partly, explain how the compound prevents the onset
of senescence of the cells.
Although the mechanism of replicative senescence is very
complicated, the accumulation of damaged macro-
molecules, including AGEs, plays an important role in
ageing.25 AGEs are generated by glycation, whereby
reduced sugars react with protein amino groups. The
highly cross-linked AGEs are often deleterious to cells
because they can provoke a hyperoxic response in cells.26
Moreover, the accumulation of aberrant protein molecules
with carbonyl groups is a common molecular signal of
ageing.27 Our study found that the AGE levels of 2BS
cells cultured with aminoguanidine decreased
significantly even at late PD, which is similar to previous
studies.28 These results suggest that aminoguanidine
block the formation of AGEs or speed up the
decomposition of AGEs. The reduction of AGEs by
aminoguanidine is related with the prevention of the onset
of the senescent phenotype and it maintains the dividing
state of the cells.
In fact, AGEs and their precursors usually contain
reactive carbonyl groups, which can be generated by the
actions of oxygen FR and related species.20,29 FR and
ROS seem to be essential for the initiation of AGE
formation and, to be responsible for the speed-up of the
formation of deleterious material.30 Effective removal of
these FR and ROS may block the formation of AGEs.
Aminoguanidine has antioxidative potential and inhibits
diamine oxidase and nitric oxide synthase. We
hypothesize that aminoguanidine inhibits AGE formation
by removing FR and ROS.
Accumulation of single-strand breaks is the major cause
of telomere shortening in human fibroblasts.5 DNA
damage located in DNA binding domains blocks
transcription factors from binding to the damaged domain
and can also cause cell growth arrest through the induced
expression of p53.7,31 The decline of replicative activity is
analogous to a checkpoint response to accumulated
chromosomal damage. Our study found that 2BS cells
pretreated with aminoguanidine had much shorter comet
tails after exposure to 200 μmol/L H2O2 for 5 minutes,
which suggest that the compound has a strong potential to
protect genomic DNA from oxidative stress. Moreover,
most of the cells incubated with H2O2 have a reduction of
DNA damage when the cells are continually cultured with
DMEM containing aminoguanidine, which indicates that
the compound improves the DNA repair ability of 2BS
cells. These facts suggest that the cells grown long-term
in aminoguanidine maintain the integrity of their genomic
DNA, which may be one of the important mechanisms of
aminoguanidine delaying replicative senescence.
2034
Telomeres in human fibroblasts are shorten by about
30-200 bp with each cell division. This shortening has
long been suspected, and recently proven, as a biological
clock in proliferating fibroblasts and, eventually, as an
important trigger of replicative senescence.32
Quantitatively, stress-mediated telomere damage
contributes most to telomere shortening under standard
conditions.33 Therefore, it is expected that
aminoguanidine slows down the telomere shortening rate
of 2BS cells through attenuating DNA damage induced
by oxidative stress; which is demonstrated in this study.
In summary, aminoguanidine has the obvious effect of
delaying senescence and preventing the onset of the
senescent morphology of 2BS cells. The results of our
study show that aminoguanidine improves cell growth
and proliferation and decreases the AGE levels of 2BS
cells. In addition, the compound has a strong potential to
prevent DNA single-strand breaks induced by oxidative
stress and to improve the DNA repair ability.
Aminoguanidine also slows down the telomere shortening
rates of 2BS cells. We favor the view that the antioxidant
activity of aminoguanidine appears to be due to its
inhibitory effect of AGE formation and its potential to
protect genomic DNA against oxidative stress. The
biological properties of aminoguanidine, including its
potential for proliferation improvement, inhibitory effect
on AGE formation, antioxidant effects, improvement of
DNA repair ability and the slowdown of telomere
shortening, contribute to its senescence-delaying activity.
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(Received July 14, 2007)
Edited by SHEN Xi-bin