ORIGINAL CONTRIBUTION
Gene expression profiling reveals new protective roles for vitamin C in human skin cells
Tiago L. Duarte1,2*, Marcus S. Cooke1,3, George D. D. Jones1
1
Radiation & Oxidative Stress Group, Department of Cancer Studies & Molecular Medicine,
University of Leicester, Leicester, LE1 7RH, U.K.
2
Iron Genes & Immune System Group, IBMC – Instituto de Biologia Molecular e Celular,
Universidade do Porto, 4150-180 Porto, Portugal.
3
Department of Genetics, University of Leicester, Leicester, LE1 7RH, U.K.
*To whom correspondence should be addressed. Iron Genes & Immune System Group,
Instituto de Biologia Molecular e Celular, Rua do Campo Alegre 823, 4150-180 Porto,
Portugal. Tel.: +351 226074956. Fax: +351 226098480. E-mail address: tduarte@ibmc.up.pt
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Abstract:
The skin is a protective barrier against external insults and any lesion must be rapidly and
efficiently repaired. Dermal fibroblasts are the major source of extracellular connective tissue
matrix and play an important role in wound healing. Vitamin C is an important water-soluble
free radical scavenger and an essential co-factor for collagen synthesis by dermal fibroblasts,
and consequently may contribute to the maintenance of a healthy skin. Using microarray
analysis, we investigated the effects of long-term exposure to a stable vitamin C derivative,
ascorbic acid 2-phosphate (AA2P), in contact-inhibited populations of primary human dermal
fibroblasts. When comparing with ‘scorbutic’ cells, exposure to AA2P increased the
expression of genes associated with DNA replication and repair, and with G2/M phase of the
cell cycle. Consistent with the gene expression changes, AA2P increased the mitogenic
stimulation of quiescent fibroblasts by serum factors and cell motility in the context of wound
healing. Furthermore, AA2P-treated fibroblasts showed faster repair of oxidatively damaged
DNA bases. We propose that vitamin C may protect the skin by promoting fibroblast
proliferation, migration and replication-associated base excision repair of potentially
mutagenic DNA lesions, and we discuss the putative involvement of hypoxia inducible
transcription factor-1 (HIF-1) and collagen receptor-related signalling pathways.
Keywords: Ascorbic acid; Fibroblast; Gene expression; DNA repair; 8-oxoguanine; Wound
healing; Cell proliferation; Cell migration.
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Introduction
Human skin consists of two tissue layers, namely a keratinized stratified epidermis and an
underlying thick layer of collagen-rich dermal connective tissue. The skin serves as a
protective barrier against external insults and therefore any lesion must be rapidly and
efficiently repaired. Wound healing is a complex process consisting of several consecutive
and overlapping phases: inflammation, cell proliferation and migration, extracellular matrix
deposition and wound contraction [1]. Dermal fibroblasts are the major source of extracellular
connective tissue matrix and they play an important role in wound healing. They are recruited
to the wound area by inflammatory cells, invade lesions and play an important role in
promoting re-epithelialization and re-establishing tissue integrity. Consequently, cultured
fibroblasts have been incorporated into various tissue-engineered products that are used in the
clinics for skin regeneration [2]. Fibroblasts that are found at sites of lesions proliferate more
and more actively secrete extracellular matrix components such as collagens and fibronectin
[1].
Solar ultraviolet radiation is a genotoxic agent and considered to be the principal cause
of skin carcinogenesis. UVB is directly absorbed by DNA bases, leading to damage, such as
pyrimidine dimers, but has limited penetrance into the epidermis. In contrast, UVA radiation
is known to reach the dermal layer, where damage is formed indirectly, either by energy
transfer reactions, or the formation of reactive oxygen species (ROS) [3]. Singlet oxygen,
formed by the excitation of cellular photosensitisers and transfer of energy to molecular
oxygen, reacts with DNA molecule forming, almost exclusively, 7,8-dihydro-8-oxo-guanine
(8-oxoGua) [4], which is mutagenic [5]. Vitamin C is an important free radical scavenger and
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therefore it may protect the skin against ROS-mediated damage. It is also a co-factor for
prolyl and lysyl hydroxylation, required for collagen maturation [6] and ceramide synthesis
[7]. Whilst vitamin C can stimulate the differentiation of several mesenchymal cell types
(reviewed by [8]), it seems to be associated with a higher proliferation rate in skin fibroblasts.
Recently, it was shown that the addition of a stable vitamin C derivative, magnesium Lascorbic acid 2-phosphate (AA2P), to the cell culture medium led to an increase in dermal
fibroblast number and a better organization of the basement membrane zone in an in vitro
human reconstructed skin model [9].
We previously shown that vitamin C modulates intracellular iron metabolism and
affects the expression of iron-related genes in fibroblasts from human skin [10]. In the present
work, we aimed at elucidating additional role(s) of vitamin C in these cells by performing a
genome-wide expression profiling analysis of vitamin C-replete versus ‘scorbutic’ cells (i.e.
cells depleted of vitamin C by serial propagation in culture without the addition of the
vitamin). Dermal fibroblasts do not exhibit a rapid proliferating activity in vivo, except when
exposed to certain stimuli, e.g. during wounding. Therefore, a system where cells enter a nondividing state but still keep the ability to divide when exposed to several stimuli would
represent a better model of these cells in vivo. In vitro, this can be achieved according to two
well-established drug-independent methods: serum deprivation and cell contact inhibition
[11]. We made primary human fibroblasts quiescent by growing them to a post-confluence
density and incubated them with ascorbic acid (AA) or AA2P for up to 5 days, with daily
repletion, to study the basal gene expression profiles associated with a long-term exposure to
vitamin C. We have previously shown that, whilst AA is unstable and its auto-oxidation
generates H2O2 in culture media, the amount of H2O2 produced by physiological
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concentrations of AA ( 100 µM) is not toxic to confluent fibroblast monolayers. AA2P, on
the other hand, does not generate H2O2 in cell culture media [12]. We hypothesised that with
the above-mentioned approach we would gain new insights on the participation of vitamin C
in important processes in human skin, such as wound healing or the repair of oxidative DNA
lesions in skin cells.
Materials and Methods
Reagents
Minimal Essential Medium with Earle’s salts and non-essential amino acids, foetal bovine
serum (FBS) and Glutamax-I were purchased from Invitrogen (Paisley, UK). Magnesium salt
of AA2P (C6H6O9P 3/2 Mg; Figure 1) was obtained from Wako Pure Chemical Industries
(Neuss, Germany). All other chemicals and reagents were purchased from Sigma-Aldrich
(Poole, UK) unless otherwise stated.
Cell culture conditions
GM5659 primary fibroblasts established from a skin biopsy of an apparently healthy donor
were obtained from the NIGMS Human Genetic Cell Repository (Coriell Institute for Medical
Research, New Jersey, USA). Cells were grown as a monolayer culture in Nunclon culture
flasks at 37 °C in a humidified atmosphere containing 5 % CO2. Fibroblasts were grown in
Minimal Essential Medium with 2 mM Glutamax-I and 10 % FBS. Cells were passaged when
nearly confluent and all experiments were performed at passage numbers 14-17. Cells were
grown to confluence prior to the experiments.
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Millimolar solutions of AA and AA2P were freshly prepared and sterilised by passing
through a 0.22 µm membrane immediately prior to the experiments. Solutions were then
diluted in pre-warmed medium to obtain the desired concentrations. Post-confluent fibroblasts
were supplemented with 100 µM AA or AA2P added fresh every day for a period of up to 5
days. Control cells received medium alone.
Gene expression profiling
Total-RNA was extracted using TRI Reagent, according to the manufacturer’s instructions. A
second RNA cleanup was performed using the RNeasy Mini kit (QIAGEN, Crawley, UK).
RNA concentration was determined by measuring the absorbance at 260 nm and integrity was
investigated in the Agilent 2100 Bioanalyzer using the Eukaryote Total RNA Nano assay
(Agilent Technologies, Palo Alto, California, USA). Total RNA (6 µg) was reverse
transcribed into double stranded cDNA with an oligo-dT primer using the One-cycle cDNA
Synthesis Kit (Affymetrix, Santa Clara, California, USA) and cleaned up using the Affymetrix
Sample Cleanup Module. Synthesis of biotin labelled cRNA was performed by in vitro
transcription using the Affymetrix IVT labelling kit, followed by cRNA cleanup with the
Affymetrix Sample Cleanup Module. Size distribution of the labelled transcripts was
investigated in the Agilent 2100 Bioanalyzer. Fragmented, biotin-labeled cRNAs (20 µg) were
hybridised to GeneChip Affymetrix Human U133 Plus 2.0 arrays for 16 hr at 45 °C with
rotation at 60 rpm. Probe array washing and staining were performed according to Affymetrix
EukGE-WS2v5 Gene Chip protocol in the Fluidics Station 400. Probe arrays were scanned at
570 nm using an Affymetrix GeneChip scanner and the fluorescence intensity of the scanned
image registered in CEL intensity files. DNA-Chip Analyser (dChip) software [13] was used
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to automatically select probes, detect outliers/artefacts, normalise the fluorescence intensity
over multiple arrays and calculate model-based expression values from the differences
between perfect match and mismatch probes in the cell intensity files. The data have been
deposited in NCBI’s Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/)
and are accessible through GEO Series accession number GSE11919. In addition, dChip
software was used to filter a set of AA- and AA2P-responsive genes for which the expression
levels were significantly altered in the triplicate experiments (t-test, P<0.01), with an average
fold change ≥2 or ≤-2 when comparing with the control groups and a difference in average
fluorescence intensity >50.
Real-time RT-PCR
Real-time reverse transcription-PCR (RT-PCR) was performed on the MX4000
spectrofluorometric thermal cycler (Stratagene, Amsterdam, Netherlands) as described
previously [10]. The amplification protocol consisted of denaturation at 95 °C for 4 min and
40 cycles of 95 °C for 30 sec, annealing temperature for 1 minute and 72 °C for 30 sec.
Amplification conditions and primer sequences are listed in Table 1. The quantity of each
transcript was estimated against the respective standard curve and normalised against the
quantity of the endogenous control gene, Hypoxanthine phosphoribosyltransferase 1
(HPRT1). The final gene expression value in each treated sample was subsequently
determined as a ratio of the gene expression in the control sample.
Cell cycle analysis
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Cells were fixed in 70 % ethanol at approximately 1 × 106 cells/ml and kept at 4 °C for a
minimum of 24 hr. Prior to analysis, cells were centrifuged at 600 × g for 10 min and
resuspended in phosphate-buffered saline with 0.1 mg/ml RNase A and 5 µg/ml propidium
iodide. Following incubation at room temperature for 30 min, 10,000 cells were acquired in a
FACScan flow cytometer (Becton Dickinson, Oxford, UK). The proportion of cells in the
different phases of the cell cycle was calculated from the histogram of number of cells per
propidium iodide fluorescence intensity with ModFitLT v2.0 software (Verity Software House,
Topsham, Maine, USA).
Cell viability assay
Cell viability was measured by analysis of propidium iodide uptake and cell size by flow
cytometry, exactly as described previously [10].
Wound healing assay
The migratory behaviour of control and AA2P-treated cells was assessed in an in vitro wound
healing assay. Fibroblasts were grown to confluence and incubated with 100 µM AA2P added
fresh every day for a period of 3 days. Control cells received medium alone. An artificial
wound was created with a 200 µl micropipette tip. After washing each culture to remove debris,
cells were further incubated with AA2P or growth medium for 24 hr. Fibroblast migration was
assessed by measuring the distance between wound edges, by means of an inverted microscope.
DNA repair assay
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Cells were incubated with phosphate-buffered saline containing 0.4 µM Ro 19-8022 (Roche,
Basel, Switzerland) and irradiated on ice at 35 cm distance from a 500 W halogen lamp to induce
oxidative modification of DNA via 1O2. The repair of 8-oxoGua was subsequently monitored
using the human 8-oxoguanine DNA glycosylase 1 (hOGG1) comet assay (hOGG1 comet) [14]
with substantial modifications. After trypsinisation, cells were suspended in 0.6 % low melting
point agarose. Eighty microliters of the agarose gel (containing approximately 2 × 104 cells) were
dispensed onto glass microscope slides previously coated with 1 % normal melting point agarose.
The agarose was allowed to set on ice under a coverslip and the slides left overnight in ice-cold
lysis buffer (100 mM disodium EDTA, 2.5 M NaCl, 10 mM Tris-HCl, pH 10 containing 1 %
triton X-100 added fresh). Slides were washed once with distilled water and immersed in two
changes of enzyme digestion buffer [40 mM HEPES, 0.1 M KCl, 0.5 mM EDTA and 0.2 mg/mL
bovine serum albumin (pH 8.0)], for 5 min each time, at room temperature. In preliminary
experiments, hOGG1 (New England Biolabs, Hitchin, UK) was added to the gel (50 µL/gel) at
different dilutions, to achieve optimal concentration. Gels were covered with a cover slip and
incubated in a humidified chamber at 37 ºC for 45 min. The cover slips were removed and the
slides were placed in a horizontal electrophoresis tank. From this step onwards, the assay was
performed exactly as described [14]. DNA damage was expressed as the percentage of DNA in
the comet tails.
Statistical analysis
Data are expressed as mean values ± standard deviation for at least 3 independent samples.
Statistical evaluation was performed using Minitab software (Minitab Ltd, Coventry, UK).
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Differences among multiple groups were compared by one-way analysis of variance with posthoc Tukey’s test. Unless otherwise stated, statistical significance was assumed at P < 0.05.
Results
Gene expression profiling
Our previous work confirmed that primary dermal fibroblasts become depleted of vitamin C
(i.e. ‘scorbutic’) following serial propagation in vitamin C-free medium. Incubating
fibroblasts with the stable vitamin C derivative AA2P, however, leads to a dose- and timedependent intracellular accumulation of AA, with a concentration plateau being reached
between 12 and 24 hr of incubation [10]. In the current work, we incubated confluent
fibroblasts with either 100 µM AA2P or AA added fresh every day at 24-hour intervals for a
period of up to 5 days, to maintain a constant level of intracellular AA repletion. Using this
model, we did not see a reduction in cell viability when incubating cells with AA2P or AA at
20-500 µM (cell viability >96 %), which agrees with our previous findings [12]. Visual
inspection of the cultures revealed that, whilst control cells remained a tightly packed, 2dimensional monolayer, with only minimal cell overlap after 5 days of incubation in growth
medium, cells incubated with AA2P or AA exhibited post-confluent growth, as indicated by a
much higher cell density and extensive cell overlap (data not shown). The ability of vitamin C
to induce post-confluent growth of skin fibroblasts has been previously described [15-17].
To identify genes whose expression was altered by continued exposure to vitamin C,
we grew GM5659 human dermal fibroblasts to confluence and subsequently incubated them
with 100 M AA2P for 5 days with daily repletion, as mentioned above. Control cells were
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given fresh medium without AA2P. Affymetrix high-density oligonucleotide DNA microarray
analysis was employed to investigate the genome-wide expression profiles of treated and
control (scorbutic) cells. The expression of 294 genes and expressed sequence tags was
significantly modulated by AA2P with at least 2-fold induction or repression in 3 independent
experiments (P<0.01). Genes were grouped according to the Gene Ontology functional
categories (Table 2 and supplementary table 1). AA2P induced about 20 genes that promote
the passage through the different phases of the cell cycle by regulating the transitions of G1 to
S phase and G2 to M phase, including several cyclins and cyclin-dependent kinases. Also upregulated was a cluster of genes that are essential components of the cellular DNA replication
machinery, such as primase, DNA polymerase alpha 1, DNA topoisomerase 2 alpha (TOP2A)
and replication factors. Some of the genes in this group are also known to be involved in DNA
repair, like RAD51 homolog, flap structure-specific endonuclease 1 (FEN1) or nei
endonuclease VIII-like 3 (NEIL3). Furthermore, AA2P increased the expression of nearly 40
genes that are associated with the mitotic phase of the cell cycle, including genes encoding
chromosome segregation and spindle associated proteins. Altogether, these results agree with
the ability of AA2P to promote the proliferation of contact-inhibited fibroblasts. This may also
explain the fact that AA2P increased the expression of genes involved in lipid and steroid
biosynthesis, including a key regulator of cholesterol synthesis, the low-density lipoprotein
receptor (LDLR). In fact, cells that are undergoing mitotic division have a higher demand of
free cholesterol and fatty acids for the synthesis of new membranes, and hence express more
LDLR than quiescent cells [18-19].
On the other hand, AA2P repressed the expression of genes involved in glucose
metabolism, including the glucose transporter 1 (GLUT1), which is involved in the cellular
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uptake of the oxidised form of AA, dehydroascorbate, . Furthermore, AA2P caused a 2-fold
down-regulation of a gene involved in iron uptake, transferrin receptor (TFRC), as previously
described [10], and reduced the expression of 4 members of a family of negative regulators of
transcription, inhibitor of DNA binding 1-4.
In addition, AA2P modulated the expression of genes involved in extracellular matrix
remodelling and cell adhesion, as well as genes encoding cytoskeleton proteins. A
comprehensive list of all significant expression changes is presented in supplementary table 1,
which also contains results from AA-incubated fibroblasts. The expression profiles of
fibroblasts incubated with 100 µM AA were almost identical to those obtained with AA2P
(supplementary table 1), which is not surprising given the fact that both vitamin C derivatives
yield similar intracellular ascorbate levels [10], and that the pro-oxidant effect of AA in
culture media is minimised when cells are at a high density [12].
The microarray results were validated by confirming the expression of genes involved
in G2 to M phase transition (cyclin B1, CCNB1), DNA replication/repair (TOP2A, FEN1,
NEIL3), lipid metabolism (LDLR), iron metabolism (TFRC) and glucose metabolism
(GLUT1) by real-time quantitative RT-PCR. This independent technique confirmed that all
selected genes were significantly modulated by AA or AA2P in the same fashion as in the
microarray analysis (Figure 2). If at all different, the expression changes had slightly higher
amplitude in the RT-PCR analysis, which is probably due to the higher sensitivity of the
assay.
Cell cycle re-entry
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Our gene expression profiling results showed that AA and AA2P modulate cell cycle
progression and thus stimulate the post-confluent growth of contact-inhibited fibroblasts. In
vivo, skin fibroblasts are only exposed to serum growth factors during the process of wound
healing, where resting fibroblasts are required to proliferate. Therefore, we wondered whether
AA2P would stimulate quiescent fibroblasts to re-enter the cell cycle when exposed to a
mitogenic stimulus. For this purpose, nearly confluent fibroblasts were made quiescent by
serum starvation (0.5 % FBS) for 48 hr. This protocol arrests cells at the G0/G1 phase of the
cell cycle, but these cells can be stimulated to re-enter the cell cycle by exposure to serum (10
% FBS) [20]. As depicted in Table 3, over 96 % of the serum starved cells were at the G0/G1
phase of the cell cycle. Culture medium was subsequently replaced with fresh low-serum
medium (0.5 % FBS) or complete growth medium (10 % FBS) and cells were further
incubated for 24 hr. As expected, cells that were returned to low-serum medium for further 24
hr remained arrested at G0/G1. In contrast, a proportion of the cells that were returned to
complete growth medium re-entered the cell cycle, as indicated by an increase in the
percentage of cells in S and G2/M phases. The addition of AA2P with low-serum medium did
not change cell cycle arrest at G0/G1. But the addition of AA2P to growth medium increased
the number of cells undergoing division. In particular, AA2P caused a significant, 2-fold
increase in the number of cells undergoing DNA synthesis (P<0.001). We can thus conclude
that the presence of AA2P in the medium is not enough to modulate fibroblast cell cycle
progression, but it stimulates the cell cycle re-entry of quiescent fibroblasts in the presence of
serum.
Cell motility
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During the early stages of the physiological response to a wound, not only are fibroblasts
required to proliferate, they’re also the first cell type to migrate and invade the wound. Cell
motility/migration is a process that involves the coordinated expression of several motility
factors and extracellular matrix proteases. In this study, AA2P increased the expression of the
urokinase-type plasminogen activator, a protease that plays an important role in cellular
migration and is vital during the initial phases of wound healing [21] (Table 2). Likewise,
AA2P up-regulated the hyaluronan-mediated motility receptor, which is required for fibroblast
migration in the context of wound healing [22], and interleukin 6, which also promotes cell
motility and matrix remodelling during wound healing [23]. We could thus hypothesise that
vitamin C may have yet another role in fibroblast activation during wound healing by
enhancing cell migration. We investigated the effect of AA2P on cellular migration using a
standard in vitro wound healing assay [24]. Post-confluent fibroblasts were incubated in the
presence or absence of 100 µM AA2P for 3 days, when an artificial wound was created by
gently scratching the cell monolayer. Cells were returned to growth medium with or without
AA2P and wound closure was assessed 24 hr later. As expected, fibroblasts migrated as a
loosely connected cell population. However, wound invasion occurred at substantially
different rates in control (scorbutic) and AA2P-treated cells (Figure 3). Whilst control cells
migrated slowly towards the wound area, treated cells show full invasion of the wound within
24 hr post insult. Results clearly show that AA2P (100 μM) increased the number of cells that
migrated into the wound area, which is indicative of enhanced healing.
DNA repair
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We have shown here that AA2P induces the expression of a cluster of genes that are essential
for DNA replication during S phase of the cell cycle (Table 2). Some of these genes, however,
also play important roles in DNA repair. For example, RAD51 is involved in DNA unwinding
during replication but also in double-strand break repair via homologous recombination.
Histone H2A.X is also required for double-strand break repair [25]. Other genes, up-regulated
by AA2P, which are associated with base excision repair (BER) include FEN1, replication
factor C and NEIL3. Tissue injury is often associated with oxidative damage and indeed most
products of base oxidation are thought to be repaired by BER [26]. In addition, it has been
suggested that vitamin C may promote DNA repair in vivo [27]. In this study, we investigated
whether continuous exposure to AA2P could render skin fibroblasts more capable of
processing base lesions resulting from DNA oxidation. We induced oxidative damage to DNA
by exposing cells to the polar photosensitizer Ro19-8022 in the presence of light. This system
is a generator of singlet oxygen, leading to the formation of oxidised purines, predominantly
8-oxoGua, in much higher yield than single-strand breaks or sites of base loss [28]. To
monitor the formation and subsequent removal of oxidised bases from genomic DNA, we
employed the alkaline comet assay in conjunction with the hOGG1 repair glycosylase. OGG1
removes oxidation products of guanine from DNA, mainly 8-oxoG and, to a much lower
extent, ring opened guanine, i.e., formamidopyrimidine [29]. In the method, hOGG1 acts at
the sites of oxidised bases, leaving apurinic/apyrimidinic sites that are converted into breaks in
the assay. Smith et al. [14] have used the assay before and reported high specificity towards
oxidative DNA damage. The DNA repair assay was implemented here with several
modifications, compared with the protocol of Smith et al [14]. To ensure that we were only
measuring the lesions of interest, rather than non-specific breakage, we established the
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optimal reaction conditions by titration, varying concentration of enzyme and time of
incubation. An incubation time of 45 min at 37 ºC was needed to ensure full enzyme digestion
(data not shown). Preliminary experiments have also shown that hOGG1 cutting of samples
treated with Ro19-8022 and light increased with increasing amounts of the enzyme until a
plateau was reached at about 0.16 U of the enzyme (Figure 4A). No adventitious cutting was
observed in untreated samples, which confirms assay specificity (Figure 4A). We have used the
assay conditions above (digestion time and amount of enzyme) in subsequent experiments and
obtained a linear dose-response on the enzyme digestion of samples exposed to visible light in
the presence of the photosensitizer for increasing times (Figure 4B). A treatment with visible
light in the presence of the photosensitizer for 30 min was enough to induce an amount of DNA
damage that was within the dynamic range of the assay (Figure 4B). Minimal levels of damage
were present in the samples where no hOGG1 treatment was employed, confirming that this
treatment specifically generates oxidatively damaged DNA that is recognised by hOGG1 (Figure
4B).
In the DNA repair experiments, we incubated post-confluent fibroblasts with growth
medium in the presence or absence of 100 µM AA2P for 3 days before exposing them to
visible light in the presence of the photosensitizer. It is worth noting that the treatment with
Ro19-8022 plus light was not toxic to fibroblasts. Cells were exposed to Ro19-8022 plus light
and cell viability was assessed 48 hr later by the propidium iodide uptake method, as indicated
in Materials and Methods. Irrespective of AA2P pre-incubation, the viability of cells exposed
to Ro19-8022 plus light was higher than 95 %. Following treatment with Ro19-8022 plus
light, fibroblasts were incubated for up to 9 hr and the repair of 8-oxoGua was monitored at
regular intervals. Whilst the amounts of damage detected in AA2P-treated and in scorbutic
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cells immediately post exposure to Ro19-8022 plus light were identical, the repair kinetics
was much faster in the former. In fact, most damage persisted in scorbutic cells at the end of
the 9-hour period post-insult, whereas cells that were pre-incubated with AA2P had removed
most of the damage within the same time period (Figure 4C). We can thus conclude that
AA2P stimulates the repair of 8-oxoGua lesions in contact-inhibited dermal fibroblasts.
Discussion
This is the first report of the genome-wide effects of vitamin C on gene expression in primary
dermal fibroblasts. Non-dividing populations of fibroblasts were employed here as they may
represent a better model of dermal cells in vivo than actively growing cells. Moreover, instead
of looking at the immediate effects of a single addition of vitamin C to cell medium, we
focused on the effects of sustained exposure. The most striking effect of both vitamin C
derivatives in the gene expression profiling experiments was the induction of a large set of
genes that are involved in DNA replication, or in the control or execution of the progression
through the G2/M phases of the mitotic cell cycle, thus stimulating cell proliferation. This
confirms previous reports that both AA [16-17] and AA2P [15] promote post-confluent
proliferation of primary fibroblasts. But our results suggest that vitamin C may have a broader
range of actions that are relevant to the process of wound healing. We showed that AA2P
increased the percentage of quiescent fibroblasts that were stimulated to synthesise DNA and
proliferate after serum stimulation. In vivo, dermal fibroblasts are exposed to serum during the
re-colonisation of a wound. Growth factors released at a wound site can act as mitogens or as
chemotactic factors for dermal fibroblasts. In response, fibroblasts begin to proliferate and
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eventually migrate into the wound clot. This response is not immediate though, the ratelimiting step in this phase of wound contraction being the time required for fibroblasts to
emerge from quiescence [30]. In this respect, our results suggest that vitamin C may favour
wound healing by stimulating re-entry of quiescent fibroblasts into the cell cycle and by
promoting cell migration. Indeed, fibroblasts incubated in the presence of AA2P expressed
higher levels of genes involved in cell motility and matrix remodelling during wound healing
such as urokinase-type plasminogen activator, hyaluronan-mediated motility receptor and
interleukin 6, and showed markedly faster wound invasion in vitro. Studies in humans and in
guinea pigs have shown that vitamin C deficiency impairs wound healing and repletion of the
vitamin corrects the problem [31-32]. Recently, studies in mice showed that vitamin C
enhances the healing of wounds caused by gamma-irradiation, namely by increasing the
degree of wound contraction and reducing mean wound healing time [33]. Our work suggests
that vitamin C may have an important role in wound healing by stimulating the activation of
quiescent fibroblasts and fibroblast wound invasion.
Notably, we also report that AA2P-treated fibroblasts repair 8-oxoGua lesions faster
than scorbutic cells. BER proteins are known to associate with DNA replication proteins in
cycling cells, allowing recruitment of the former at DNA base lesions and strand breaks
occurring at replication forks and thereby ensuring fidelity of genomic replication in
proliferating cells [34]. Indeed, the long patch-BER subpathway utilises the DNA replication
machinery, including proliferating cell nuclear antigen, replication factor C and FEN1. The
latter plays a central role by cleaving a single-stranded DNA flap of 5-7 deoxynucleotides that
are displaced during repair synthesis by nuclear replicative DNA polymerases Polδ/ε
(reviewed in [26]). Here we show that these factors are co-expressed in cycling cells and
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present evidence for preferential repair of oxidised bases in replicating cells. Although we
cannot exclude the possibility that the intracellular ascorbate remaining during the postincubation period may somehow enhance the induction of DNA repair enzymes secondary to
the exposure to photosensitizer+light, our data suggest that vitamin C, by stimulating cell
cycle progression, may increase the cellular ability to perform BER.
It is widely considered that hOGG1 is the principal repair protein responsible for the
removal of 8-oxoGua [35]. However, in our experiments hOGG1 mRNA expression was not
affected by either vitamin C derivative. Nevertheless, it is known that mammalian cells have
back-up systems for repairing the lesion, involving another family of oxidative lesion-specific
DNA glycosylases [36-37]. We show that AA2P and AA increased the expression of NEIL3,
a recently discovered DNA glycosylase that belongs to the Fpg/Nei family of oxidised basespecific DNA glycosylases [38-39]. Although the glycosylase activity of NEIL3 has not been
established yet [40], Morland et al. [39] showed that it could excise Fapy lesions.
Whilst oxidised base-specific glycosylases such as hOGG1 and NTH1 excise base
lesions only from duplex DNA, members of the NEIL family preferentially excise their
substrates from single-stranded DNA or unpaired sequences in bubble DNA structures, which
suggests that NEILs are preferentially involved in BER of oxidized bases during transcription
or DNA replication [41]. The specific activation of NEIL1 is specifically activated during Sphase [42] and binds to the proliferating cell nuclear antigen [43], but no such information
exists for NEIL3. However, NEIL3 co-localises with replication protein A in the cell nucleus
[39]. We have not established whether NEIL3 is implicated in the enhancement of DNA repair
kinetics by AA2P, but our expression results support the idea that the gene is preferentially
up-regulated in cycling cells and may associate with the replication machinery to remove
19
oxidised lesions from single-stranded DNA. We propose that the importance of this, with
regard to wound healing, might be in providing fibroblast, in proximity to the wound, greater
protection from ‘collateral’ ROS-induced DNA damage, arising from the activity of
inflammatory cells.
It is worth noting that fibroblast proliferation can also be induced in vivo in response to
cytokines and growth factors released from inflammatory cells, potentially leading to tissue
fibrosis. We have previously proposed that vitamin C may enhance oxidative tissue injury in
conditions associated with elevated production of ROS (e.g. during inflammation) [10], which
could potentially result in higher production of inflammatory agents. Here we show that
vitamin C stimulates fibroblast proliferation. Hence, it would be interesting to determine
whether AA supplementation could aggravate tissue fibrosis in vivo.
Our work shows that vitamin C can activate intracellular signalling cascades that
regulate fibroblast proliferation and motility. Presumably, the same mechanism explains the
increased DNA repair capacity, as discussed. A proliferative response is generally mediated
by the binding of growth factors to specific transmembrane tyrosine kinase receptors. The
identification of the particular signal transduction pathway(s) sensitive to vitamin C warrants
further investigation. However, it is possible that the effects reported herein are related with
its ability to stimulate collagen synthesis. Dermal fibroblasts express two unrelated
transmembrane receptors, namely α1β1 integrin receptor [44] and discoidin domain receptor 2
(DDR2) [45], which can transduce signals in response to collagen. Whilst DDRs possess
intrinsic tyrosine kinase activity, integrins activate cytoplasmic protein tyrosine kinases of the
Src- and focal adhesion kinase (Fak)-family [46]. Both DDRs and integrin receptors thus
respond to collagen binding by initiating intracellular signals that synergize with those of
20
growth factors in the activation of protein tyrosine kinases and result in the downstream
modulation of cytoskeletal tension and cell migration, as well as cell proliferation [46-47].
Vitamin C is necessary for the intracellular hydroxylation of prolyl and lysyl residues during
collagen biosynthesis and therefore AA2P, which increases collagen deposition in skin
fibroblasts [15], may activate either DDRs or integrin receptors (or indeed both) and
downstream intracellular signalling pathways that modulate fibroblast proliferation and
migration.
It is unlikely, however, that such signalling pathways could explain all the gene
expression effects described in the present study, some of which may relate to baseline
physiological roles of vitamin C in cells, rather than to its involvement in wound healing. As
discussed, the main effect of vitamin C depletion is the inactivation of prolyl hydroxylase
reactions, resulting in insufficiently hydroxylated collagen and subsequently scurvy. These
reactions are catalysed by members of the family of Fe2+- and 2-oxoglutarate-dependent
dioxygenases that are also responsible for the hydroxylation of prolyl residues in the α subunit
of the hypoxia-inducible transcription factor-1 (HIF-1), which prompts HIF-1 for destruction
by the proteasome [48]. Ascorbate, which we have previously shown to increase intracellular
catalytic iron (i.e. Fe2+) [10], is required for optimal activity of these enzymes, presumably by
keeping the iron centre in the reduced state. Accordingly, it was recently shown that human
skin fibroblasts and other cell types cultured in vitamin C-deficient media have a basal level of
HIF-1 protein that is abolished when the medium is supplemented with physiological
concentrations of ascorbate [49]. In our experiments, cells were grown to post-confluence,
which induces a mild hypoxia [50]. According to our microarray results, dermal fibroblasts do
not express some of the most studied HIF-1 targets, such as nitric oxide synthase or
21
erythropoietin. However, for several other genes that were previously shown to be upregulated during hypoxia in a HIF-1-dependent manner, the expression levels were
significantly lower in AA- or AA2P-treated cells when comparing with vitamin C-depleted
cells: vascular endothelial growth factor, TFRC, endothelin 1, stanniocalcin 1, several genes
involved in glucose and energy metabolism (GLUT1, hexokinase 2, aldolase A,
phosphoglycerate kinase 1, enolase 2, pyruvate dehydrogenase kinase 1), and two of the key
enzymes in collagen synthesis, prolyl 4-hydroxylase-α 1 and lysyl-hydroxylase [51,52]. Our
results thus support the idea that ascorbate plays an important role in primary cells by limiting
HIF-1 signalling and inhibiting the response to hypoxic stress [49].
In summary, whilst the activation of specific signalling pathways remains to be
elucidated, our results present new evidence that vitamin C repletion in skin cells is required
for efficient wound healing and replication-associated repair of potentially mutagenic products
of DNA oxidation.
22
Acknowledgements
We are grateful to Dr. E. Moiseeva (University of Leicester) for collecting the wound repair
assay images. We acknowledge two anonymous reviewers for helpful suggestions, in
particular regarding the involvement of HIF-1 signalling. This work was supported by funding
from the Fundação para a Ciência e a Tecnologia, Portugal, and the European Social Fund,
Third Framework Programme to T. L. D. Comet assay studies in the laboratory of GDDJ are
supported by grants from Cancer Research UK (Ref C13560/A46) and the Hope Foundation.
M.S.C. and G.D.D.J. are partners of ECNIS (Environmental Cancer Risk, Nutrition and
Individual Susceptibility), a network of excellence operating within the European Union 6th
Framework Program, Priority 5:"Food Quality and Safety" (Contract No 513943).
23
List of Abbreviations
8-oxoGua, 7,8-dihydro-8-oxo-guanine
AA, ascorbic acid
AA2P, ascorbic acid 2-phosphate
BER, base excision repair
CCNB1, cyclin B1
DDR, discoidin domain receptor
FBS, foetal bovine serum
FEN1, flap structure-specific endonuclease 1
GLUT1, glucose transporter 1
HIF-1, hypoxia-inducible transcription factor-1
hOGG1, human 8-oxoguanine DNA glycosylase 1
LDLR, low-density lipoprotein receptor
NEIL3, nei endonuclease VIII-like 3
ROS, reactive oxygen species
TFRC, transferrin receptor
TOP2A, DNA topoisomerase 2 alpha
24
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30
Table 1. Real-time PCR conditions.
Gene
Name
HPRT1
CCNB1
TOP2A
LDLR
FEN1
NEIL3
TFRC
GLUT1
Annealing
Temp.
(°C)
57
55
55
57
57
57
57
57
MgCl2
(mM)
Forward Primer Sequence (5’-3’)
Reverse Primer Sequence (5’-3’)
2.5
2
2
3
2
2
3.5
2
GCAGACTTTGCTTTCCTTGGTCAG
ATGATGTGGATGCAGAAGATGGA
TCAAGCCCTCCTGCTACACATTTC
GCAAGTGGCTTTCAACACACAACAG
CCCATTATACCTCCTTCACCCCAGA
GGAAGTCGGAAGAGCACTGGACCT
TCTGACACGTCTGCCTACCCATTCG
TGCAACGGCTTAGACTTCGACTCA
GTCTGGCTTATATCCAACACTTCGTG
TCTGACTGCTTGCTCTTCCTCAA
TTTGCTGCTGTCTTCTTCACTGTCA
GGGTTTGGCTTAGAGATTGGTGGATG
TAGAATCCTCTCTCCACCCCGTGA
TCAAGCAAGCATCACATGCCTTAGA
TATGATGGTTCACTCACGGAGCTTCG
TCTCTGGGTAACAGGGATCAAACAGA
31
Product
Size
(bp)
103
104
94
85
81
94
84
100
Table 2. Genes involved in cell proliferation, differentiation or migration with significant
differential expression in response to 100 µM AA2P in contact-inhibited dermal fibroblasts*.
Probe set ID†
Gene ID‡
Gene name
Fold Change
P-value
Regulation of cell cycle
204170_s_at
1164
203214_x_at
983
202870_s_at
991
201853_s_at
994
205167_s_at
995
204126_s_at
8318
204510_at
8317
221436_s_at
83461
224753_at
113130
224428_s_at
83879
221520_s_at
55143
203418_at
890
214710_s_at
891
202705_at
9133
204826_at
899
209714_s_at
1033
228033_at
144455
219990_at
79733
238756_at
283431
CDC28 protein kinase regulatory subunit 2
cell division cycle 2
cell division cycle 20
cell division cycle 25B
cell division cycle 25C
cell division cycle 45L
cell division cycle 7
cell division cycle A3
cell division cycle A5
cell division cycle A7
cell division cycle A8
cyclin A2
cyclin B1
cyclin B2
cyclin F
cyclin-dependent kinase inhibitor 3
E2F transcription factor 7
E2F transcription factor 8
growth arrest-specific 2 like 3
2.4
2.6
2.8
2.1
2.9
2.9
2.4
3.0
2.2
2.2
2.5
2.3
2.8
2.8
2.7
2.7
2.9
3.0
2.6
0.001
0.001
0.002
0.000
0.002
0.004
0.010
0.000
0.002
0.003
0.000
0.001
0.000
0.001
0.003
0.001
0.002
0.002
0.002
DNA replication and/or repair
228868_x_at
81620
204768_s_at
2237
218350_s_at
51053
205436_s_at
3014
202107_s_at
4171
222036_s_at
4173
216237_s_at
4174
219502_at
55247
204835_at
5422
205053_at
5557
205024_s_at
5888
204128_s_at
5983
203022_at
10535
209773_s_at
6241
202338_at
7083
1554696_s_at
7298
201292_at
7153
DNA replication factor
flap structure-specific endonuclease 1
geminin, DNA replication inhibitor
H2A histone family, member X
MCM2 minichromosome maintenance deficient 2
MCM4 minichromosome maintenance deficient 4
MCM5 minichromosome maintenance deficient 5
nei endonuclease VIII-like 3
polymerase (DNA directed), alpha 1
primase, polypeptide 1, 49kDa
RAD51 homolog (RecA homolog)
replication factor C (activator 1) 3, 38kDa
ribonuclease H2, subunit A
ribonucleotide reductase M2 polypeptide
thymidine kinase 1, soluble
thymidylate synthetase
topoisomerase (DNA) II alpha 170kDa
2.5
2.0
2.0
2.1
2.2
2.1
2.8
2.9
2.0
2.2
2.5
2.1
2.4
3.4
2.4
3.2
2.3
0.005
0.000
0.004
0.000
0.000
0.000
0.000
0.005
0.008
0.008
0.002
0.009
0.005
0.000
0.000
0.000
0.000
2.6
2.7
0.001
0.000
M phase of the mitotic cell cycle
219918_s_at
259266
asp (abnormal spindle)-like
208079_s_at
6790
aurora kinase A
32
209464_at
212949_at
215509_s_at
aurora kinase B
barren homolog 1
BUB1 budding uninhibited by benzimidazoles 1
homolog
BUB1 budding uninhibited by benzimidazoles 1
homolog beta
cell division cycle associated 1
centromere protein A
centromere protein E, 312kDa
centromere protein F, 350/400ka (mitosin)
centromere protein K
centromere protein M
centromere protein N
centrosomal protein 55kDa
chromosome condensation protein G
G-2 and S-phase expressed 1
kinesin family member 11
kinesin family member 14
kinesin family member 18A
kinesin family member 20A
kinesin family member 23
kinesin family member 2C
kinesin family member 4A
kinesin family member C1
kinetochore associated 1
kinetochore associated 2
MAD2 mitotic arrest deficient-like 1
M-phase phosphoprotein 1
NIMA (never in mitosis gene a)-related kinase 2
nucleolar and spindle associated protein 1
NudE nuclear distribution gene E homolog 1
PDZ binding kinase
polo-like kinase 4
protein regulator of cytokinesis 1
spindle pole body component 24 homolog
spindle pole body component 25 homolog
TPX2, microtubule-associated, homolog
TTK protein kinase
3.2
3.6
3.3
0.001
0.004
0.001
2.5
0.000
3.2
3.3
3.0
2.9
2.5
3.5
2.3
2.4
3.1
2.2
2.4
2.5
2.3
2.3
2.8
3.0
3.0
2.2
2.1
4.2
2.5
2.6
2.7
2.5
2.0
2.7
3.2
2.7
3.5
3.1
3.1
2.6
0.001
0.001
0.002
0.001
0.002
0.001
0.007
0.005
0.000
0.004
0.008
0.001
0.002
0.001
0.000
0.010
0.001
0.006
0.001
0.000
0.000
0.001
0.004
0.000
0.001
0.002
0.007
0.000
0.000
0.006
0.000
0.006
Lipid and steroid metabolism
200862_at
1718
208962_s_at
3992
201626_at
3638
202068_s_at
3949
200831_s_at
6319
24-dehydrocholesterol reductase
fatty acid desaturase 1
insulin induced gene 1
low-density lipoprotein receptor
stearoyl-CoA desaturase (delta-9-desaturase)
2.1
2.0
3.8
2.6
2.4
0.003
0.002
0.004
0.001
0.003
Glucose metabolism
238996_x_at
226
201313_at
2026
201250_s_at
6513
aldolase A, fructose-bisphosphate
enolase 2 (gamma, neuronal)
facilitated glucose transporter 1
-2.5
-3.2
-2.9
0.000
0.004
0.003
203755_at
223381_at
204962_s_at
205046_at
207828_s_at
222848_at
218741_at
219555_s_at
218542_at
218662_s_at
204318_s_at
204444_at
206364_at
221258_s_at
218755_at
204709_s_at
211519_s_at
218355_at
209680_s_at
206316_s_at
204162_at
1554768_a_at
205235_s_at
204641_at
218039_at
227249_at
219148_at
204886_at
218009_s_at
235572_at
209891_at
210052_s_at
204822_at
9212
23397
699
701
83540
1058
1062
1063
64105
79019
55839
55165
64151
51512
3832
9928
81930
10112
9493
11004
24137
3833
9735
10403
4085
9585
4751
51203
54820
55872
10733
9055
147841
57405
22974
7272
33
202934_at
200738_s_at
226452_at
200822_x_at
3099
5230
5163
7167
hexokinase 2
phosphoglycerate kinase 1
pyruvate dehydrogenase kinase, isozyme 1
triosephosphate isomerase 1
Negative regulation of transcription
202672_s_at
467
activating transcription factor 3
204998_s_at
22809
activating transcription factor 6
208937_s_at
3397
inhibitor of DNA binding 1
201565_s_at
3398
inhibitor of DNA binding 2
207826_s_at
3399
inhibitor of DNA binding 3
209292_at
3400
inhibitor of DNA binding 4
202364_at
4601
max interactor 1
Extracellular matrix/Cell adhesion or migration
229271_x_at
1301
collagen, type XI, alpha 1
207977_s_at
1805
dermatopontin
227265_at
10875
fibrinogen-like 2
209709_s_at
3161
hyaluronan-mediated motility receptor
205207_at
3569
interleukin 6
210150_s_at
3911
laminin, alpha 5
211668_s_at
5328
plasminogen activator, urokinase
202620_s_at
5352
procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2
207543_s_at
5033
procollagen-proline, 2-oxoglutarate 4-dioxygenase,
alpha 1
218638_s_at
10417
spondin 2, extracellular matrix protein
203083_at
7058
thrombospondin 2
203868_s_at
7412
vascular cell adhesion molecule 1
212171_x_at
7422
vascular endothelial growth factor
Cytoskeleton
205132_at
222608_s_at
210334_x_at
203764_at
215116_s_at
222802_at
238621_at
201596_x_at
209016_s_at
203276_at
216952_s_at
237206_at
222077_s_at
222513_s_at
204288_s_at
219888_at
203145_at
217714_x_at
238688_at
70
54443
332
9787
1759
1906
342184
3875
3855
4001
84823
93649
29127
10580
8470
6676
10615
3925
7168
actin, alpha, cardiac muscle 1
anillin, actin binding protein
baculoviral IAP repeat-containing 5 (survivin)
discs, large homolog 7
dynamin 1
endothelin 1
formin 1
keratin 18
keratin 7
lamin B1
lamin B2
myocardin
Rac GTPase activating protein 1
sorbin and SH3 domain containing 1
sorbin and SH3 domain containing 2
sperm associated antigen 4
sperm associated antigen 5
stathmin 1/oncoprotein 18
tropomyosin 1 (alpha)
34
-3.9
-2.0
-3.8
-2.0
0.000
0.000
0.000
0.000
2.0
-2.2
-16.8
-3.1
-3.9
-3.2
-2.2
0.003
0.010
0.005
0.001
0.001
0.001
0.001
2.5
2.2
-5.9
2.2
2.0
-2.3
2.2
-2.0
-3.1
0.000
0.000
0.003
0.001
0.005
0.004
0.004
0.002
0.000
2.9
2.2
2.3
-2.2
0.000
0.000
0.001
0.000
-4.3
2.4
2.5
2.7
2.1
-2.2
-3.4
-2.5
-2.2
2.9
2.3
-3.2
2.1
-2.6
-2.4
-3.0
2.5
2.2
-2.3
0.000
0.000
0.001
0.001
0.003
0.010
0.003
0.005
0.003
0.003
0.006
0.002
0.000
0.005
0.001
0.002
0.000
0.000
0.001
*See supplementary table 1 for full list of results. †Probe set ID according to Affymetrix.
‡Gene
ID according to Entrez Gene.
35
Table 3. Effect of AA2P on cell cycle re-entry in serum starved fibroblasts
Time post serum starvation
Cell cycle distribution*
G0/G1
S
G2/M
0 hr
24 hr low-serum medium
24 hr low-serum medium + AA2P (100 µM)
24 hr growth medium
24 hr growth medium + AA2P (100 µM)
96.2 ± 0.2
95.8 ± 0.2
94.2 ± 0.2
87.5 ± 0.3
79.1 ± 0.8
3.3 ± 0.2
3.5 ± 0.1
4.8 ± 0.1
6.6 ± 0.1
8.2 ± 0.2
0.6 ± 0.0
0.8 ± 0.2
1.0 ± 0.1
6.0 ± 0.4
12.7 ± 0.7 ***
*Results are the mean ± S.D. from 3 independent experiments. ***, P < 0.001 versus the
proportion of cells in S phase in cells returned to growth medium for 24 hr.
36
Figure 1. Molecular structure of L-ascorbic acid 2-phosphate, magnesium salt.
Figure 2. Validation of the expression of target genes by RT-PCR. Confluent GM5659
fibroblasts were incubated in complete growth medium alone or in the presence of 100 µM
AA or AA2P added fresh every day for 5 days. T-RNA was extracted and the relative steadystate mRNA levels of cyclin B1 (CCNB1), DNA topoisomerase 2A (TOP2A), flap
endonuclease 1 (FEN1), nei endonuclease VIII-like 3 (NEIL3), low-density lipoprotein
receptor (LDLR), transferrin receptor (TFRC) and glucose transporter 1 (GLUT1) were
measured by real-time RT-PCR as described in Materials and Methods. Results are the mean
± S.D. from 3 separate determinations. All results are P<0.005 versus control.
Figure 3. Effect of AA2P on cellular migration towards the wound area. Fibroblasts plated on
12-well plates were grown to confluence and further incubated with growth medium alone
(plates A-B) or in the presence of 100 µM AA2P (plates C-D) for 3 days. Post-confluent cells
were wounded by gently scratching using a micropipette tip and incubated in fresh growth
medium (plates A-B) or in medium containing AA2P (plates C-D) for 24 hr. The arrow mark
represents the wound edge. Results are representative of 2 independent experiments, each
containing 16 individual replicates.
Figure 4. Effect of AA2P on the repair of oxidative DNA base lesions. A. Cells were either
incubated with phosphate-buffered saline containing 0.4 µM Ro19-8022 and visible light for 30
min or sham-irradiated. Cells were harvested immediately and kept on ice. DNA damage was
measured with the alkaline comet assay in conjunction with increasing amounts of hOGG1
37
enzyme, as described in Materials and Methods. Results are the mean ± S.E.M. of 100 cells
pooled from duplicate slides. B. Cells were exposed to Ro19-8022 and visible light for 0, 15, 30
and 45 min. DNA damage was measured with the alkaline comet assay in conjunction with
hOGG1 enzyme, as described in Materials and Methods. Enzyme was used at 0.16 U in 50 µL of
enzyme buffer. Control samples received enzyme buffer only. Results are the mean ± S.E.M. of
100 cells pooled from duplicate slides. C. Confluent fibroblasts were incubated with growth
medium in the presence or absence of 100 µM AA2P for 3 days before exposure to Ro19-8022
plus visible light, as described in Materials and Methods. Following treatment, cells were
returned to growth medium and the repair of 8-oxoGua was monitored at 0, 3, 6 and 9 hr with
the hOGG1 alkaline comet assay. Results are the mean ± S.D. from 3 independent
experiments.
38