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Published as doi: 10.1096/fj.06-5964fje.
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(The FASEB Journal. 2006;20:1895-1897.)
© 2006 FASEB

T-oligos augment UV-induced protective responses in human skin

Simin Arad, Nellie Konnikov, David A. Goukassian1 and Barbara A. Gilchrest1

Department of Dermatology, Boston University School of Medicine, Boston, Massachusetts, USA

1Correspondence: Department of Dermatology, Boston University School of Medicine, 609 Albany St., Boston, MA 02118, USA. E-mail: dgoukass{at}bu.edu; bgilchre{at}bu.edu

ABSTRACT

We have shown that DNA oligonucleotides substantially homologous to the telomere 3-prime overhang sequence (T-oligos) increase DNA repair capacity (DRC) in cultured human cells and decrease UV-induced mutation rate and photocarcinogenesis in mouse skin. To investigate the protective effects of T-oligos in intact human skin, paired skin explants obtained from adult donors were treated with T-oligos or diluent alone for 24 h, then UVB- or sham-irradiated, and processed after 6, 24, 48, 72, and 96 h for histological analysis. After UV irradiation apoptotic epidermal cells were comparable in diluent- and T-oligo-treated skin. Proliferating (Ki67+) cells were sparse in sham-irradiated skin and for 24 h after UV in both diluent- and T-oligo-treated specimens. However, compared to diluent controls, at 48 and 72 h T-oligos significantly inhibited UV-induced rebound hyperproliferation. Maximum and comparable cyclobutane pyrimidine dimers (CPDs) were detected immediately after UV irradiation in diluent- and T-oligo-treated skin, but CPDs were strikingly reduced in T-oligo- vs. diluent-treated skin at 24, 48, and 72 h. Total and activated p53 protein was increased in T-oligo- vs. diluent-pretreated skin at the time of irradiation, and up to 3-fold increases persisted for 24 h post-UV. Over 5 days, UV irradiation and T-oligo comparably increased expression of melanogenic proteins and each increased epidermal melanin content 3- to 5-fold, with distinct nuclear capping in many keratinocytes. In combination, these findings predict that T-oligo treatment will increase melanogenesis, prolong epidermal arrest, and increase DNA repair rate after UV irradiation, thus decreasing the severity of acute and chronic photodamage in human skin. Moreover, the data document an inducible SOS-like response consisting of increased melanogenesis and increased DNA repair capacity in human skin following UV-induced damage that is also produced by T-oligos in the absence of initial damage.—Arad, S., Konnikov, N., Goukassian, D. A., Gilchrest, B. A. T-oligos augment UV-induced protective responses in human skin.


Key Words: NER • oligonucleotides • melanin content • CPD

MORE THAN 1.2 MILLION new cases of skin cancer are reported yearly in the U.S. (1 , 2) . Cyclobutane pyrimidine dimers (CPD) and 6–4 pyrimidine-pyrimidone photoproducts (6–4 PP) are the major forms of UV damage leading to mutagenesis and carcinogenesis (3 , 4) and nucleotide excision repair (NER) is the skin’s major defense mechanism (5) .

The p53 tumor suppressor protein, the so-called guardian of genome (6) , plays a critical role in NER by transactivating genes that are involved in DNA damage recognition or repair, cell cycle arrest, or, in case of severe DNA damage, programmed cell death (apoptosis) (6 , 7) . Presumably for this reason, p53 is the most commonly mutated gene in human malignancies (8 , 9) , and the mutational hot spots for p53 in skin cancers are mainly at sites that are substrates for CPD formation (3 , 10) .

In addition to baseline NER, there is an inducible component of DNA repair, first recognized in bacteria (11 , 12) and termed the SOS response, by Radman in 1975 (13) . Several reports have demonstrated that NER is also inducible in mammalian cells (14) . In our laboratory, inducible protective responses were initially documented after treatment of cells or intact guinea pig skin with thymidine dinucleotides (pTT) that mimic UV effects (15) , presumably by substituting for the nuclear DNA damage signal in the absence of actual DNA damage (16) . These effects were specific for pTT and were not observed after treatment with the complementary sequence pAA (15 16 17 18) . Ultimately, it becomes apparent that multiple oligonucleotides known to concentrate rapidly in the nucleus (19 , 20) , possibly as a consequence of an active uptake mechanism dependent on 5' phosphorylation (19) , are even more effective than pTT in stimulating these protective responses (19 20 21) and that efficacy depends at least crudely on their length and percent telomere homology. Sequences complementary or unrelated to TTAGGG repeats were again found to be inactive. pTT, representing one-third (33%) of the TTAGGG sequence, was thus ~40% as effective on a molar basis as a 9 base 55% telomere homologue, with maximally effective doses of 100 µM and 40 µM, respectively (21 22 23) .

The SOS-like response in human cells is mediated in part by the p53 protein after initiation of DNA damage signaling (16 , 22 , 24) . The magnitude of the induced response is substantial, doubling or tripling levels of proteins involved in cell cycle regulation, apoptosis, and DNA repair as well as rates of repair both in vitro and in murine skin in vivo (16 , 22 , 23) .

In addition to the responses described above in cultured cells, pTT and other telomere homologue oligonucleotides (T-oligos) induce transient reversible cell cycle arrest (16 , 25 , 26) as well as the melanocyte-specific response of increased melanogenesis both in vitro and in vivo (15) , a p53-mediated response (27 , 28) to multiple forms of DNA damage (29) , of which UV is the best known. Like inducible NER, delayed tanning has the effect of decreasing DNA damage from subsequent exposures, in this case by absorbing UV photons (or binding DNA-damaging chemicals or reactive oxygen species, or ROS) before they interact with nucleoside bases (30) .

We report here that T-oligo-mediated, DNA damage-like protective responses are inducible in human skin ex vivo. T-oligos prepare the epidermis to better resist and repair DNA damage due to subsequent UV irradiation as early as 24 h after a single dosing. In combination with previous work, these results predict that T-oligo treatment will reduce acute and chronic photodamage, as well as the risk of photocarcinogenesis in human subjects.

MATERIALS AND METHODS

Human skin explants
Fragments of normal human skin from healthy donors (aged 56±15 year, mean ±SD) were brought to the laboratory within 30 min after excision during plastic surgical procedures. Tissue from a total of 18 Caucasian donors were used in our studies and, based at least on the appearance of skin when brought to the laboratory, the donors were at least moderately fair-skinned. Hence, our donors are not representative of the general population, but rather are more representative of the vulnerable cancer-prone population of fair-skinned individuals. After removing subcutaneous fat and deep dermis, the skin was cut into 5 x 5 mm squares and placed in 60 mm tissue culture dishes (4 to 6 explants per dish). Typically, one donor sample of skin yielded 12–18 explants and comparisons over time and between the two T-oligos were made among these single-donor skin fragments. Paired dishes of skin explants were incubated in either medium alone (DME/10% calf serum: KBM-2 with growth factors, 50:50 vol:vol) or medium supplemented with 100 µM pTT or 40 µM pGAGTATGAG (p9mer) for 24 h, as described (21) . The skin explants were then irradiated as described below. One set was sham-irradiated as a negative control. For each treatment group, one explant was harvested immediately after UVB irradiation. All dishes were then refed with fresh medium lacking T-oligos and explants were harvested at various times up to 96–120 after UVB irradiation. Harvested skin was snap-frozen at –80°C in OCT medium for later processing.

UVB irradiation
After 24 h of incubation in medium containing pTT, p9mer, or medium only, skin explants were placed in PBS and irradiated through the plastic culture dish cover using a solar simulator (Spectral Energy Corporation, Westwood, NJ, USA). The irradiance of the 1kW xenon arc lamp (XMN-1000–21; Optical Radiation Corp., Azuza, CA, USA) was adjusted to 5 x 10–5 W/cm2, and dishes were exposed to 30 mJ/cm2 as measured with a research radiometer fitted with a UV probe at 285 ± 5 nm (model IL1700 A; International Light, Newburyport, MA, USA) (21) , a protocol that exposes the explants to a spectrum of light closely resembling terrestrial sunlight (31) . Sham-irradiated skin explants were handled identically except that they were shielded with aluminum foil during irradiation. After irradiation, all skin explants were given fresh medium lacking T-oligos.

Oligonucleotides
Previous experiments have shown that 100 µM pTT and 40 µM p9mer are roughly bioequivalent concentrations for the elicitation of UV-mimetic responses (16 , 22) , including p53 up-regulation and activation (21 , 22) . T-oligos (pTT and p9mer) were synthesized with phosphodiester linkage by the Midland Certified Reagent Company (Midland, TX, USA) and diluted in H2O to form a 2 mM stock. T-oligos were prepared by chemical synthesis using the O-cyanoethyl phosphoramite chemistry. After removal of the protective groups, the product was purified by gel filtration chromatography. The degree of chemical purity was always higher than 92% and as high as 98% for pTT-and 9mer, with the dephosphorylated form as the major contaminant for both T-oligos. Consistent results were obtained with multiple batches of pTT. The stock solution was diluted in culture medium to 100 µM or 40 µM, respectively, and added to dishes with skin explants for use in experiments. Skin explants were provided T-oligos only once in 24 h prior to UVB or sham irradiation, then harvested at intervals according to the design of the specific experiment. All experiments were conducted using both pTT and p9mer and gave identical results with either T-oligo.

Immunohistology, immunofluorescence, and melanin staining
Five micrometer sections of frozen tissue were fixed on slides with ice-cold methanol-acetone (1:1) for 10 min at –20°C, then air dried. For immunofluorescent detection of CPDs, acetone-fixed sections were incubated with CPD-specific antibodies (kind gift from Professor Toshio Mori, Nara Medical University, Kashihara, Nara, Japan) overnight, then with FITC-labeled secondary antibodies (Jackson Immunoresearch, West Grove, PA, USA) to visualize nuclei sections stained with TopRo-3, then mounted with Vectashield mounting media (21) . Note that CPD antibodies recognize all possible dipyrimidine sequences (TT, TC, CT, and CC), as demonstrated by Prof. Toshio Mori and as referenced by the current manufacturer (Medical & Biological Laboratories, Woburn, MA, USA). All CPD-stained slides were analyzed using a Zeiss confocal microscope (Axiovert 100, Zeiss, Germany) and were photographed and analyzed using a digital image system (Pixera, San Diego, CA, USA). For each time point and treatment condition, we analyzed three randomly selected visual fields of ~100 linear µm of CPD-stained epidermis from each of 10 donors using computer-assisted image analysis, as described (21) . The average number of CPD (+) nuclei at time 0 for each condition was set at 100%, and the percent remaining CPDs were recalculated as for each time point (23) .

Sections immunostained for p53total (anti-p53 DO-7 DakoCytomation, Carpinteria, CA, USA) and p53Ser-15 (antiphospho-p53Ser-15 Cell Signaling Technology, Beverly, MA, USA) were processed as described previously (21) . Briefly sections were blocked in 10% goat normal serum in Tris-buffered saline (TBS) for 15 min at room temperature, then incubated with primary antibody (Ab) overnight at 4°C. Sections were then washed in TBS and incubated with the appropriate FITC-labeled secondary Ab at 37°C for 45 min. Finally, sections were mounted with Vectashield mounting medium containing 4',6-diamidino-2-phenylindole (DAPI) to visualize nuclei and stored at –20°C.

To evaluate the effect of T-oligos on epidermal proliferation, skin sections were stained with the proliferative marker Ki67 (DakoCytomation, Carpinteria, CA, USA). Cryosections were first incubated with mouse monoclonal antibody (mAb) against human Ki67 overnight at room temperature, then the 3-amino-9-ethyl carbazole (AEC) detection system (LabVision, Fremont, CA, USA) was used to visualize the Ab staining, as described in the manufacturer’s protocol.

For melanocyte identification, we processed skin sections treated with diluent vs. T-oligos for double immunostaining with monoclonal antibodies against two melonogenic markers: rate-limiting enzyme in melanin production-tyrosinase (Vector Laboratories, Burlingame, CA, USA) and tyrosinase-related protein 1(Trp1 or Mel-5) (Signet Laboratories, Dedham, MA, USA), followed by incubation with FITC-labeled rabbit antimouse IgG (for tyrosinase) and TRITC-labeled goat anti-rabbit IgG (for Mel-5) for 45 min (both from Jackson Immunoresearch), as described (32) . In all staining protocols, negative controls for immunostaining were performed by omitting primary antibodies.

A multicolor fluorescence microscope and a digital image analysis system (both Nikon) were used for preparation of images and quantification of p53total, p53Ser-15, Ki67, and tyrosinase/Mel-5 double (+) nuclei. For each time point and treatment condition, we analyzed 3–5 randomly selected visual fields of ~100 linear µm of p53total, p53Ser-15, Ki67, and for epidermal staining with tyrosinase, and Mel 5 antibodies. At least three donors were studied for each marker and quantification performed as described previously (21 , 23) .

To determine melanin content and intracellular distribution after T-oligo treatment, 5 µm sections were stained with Fontana-Masson silver stain as described (33) and changes in epidermal melanin content were quantified using NIH Image J (version 1.3) Image Analysis software package. An investigator "blinded" to the treatment groups evaluated the immunostaining and melanin content and distribution in all specimens.

Statistical analysis
Differences in the rate of removal of UV-induced CPDs, in the number of p53total, p53Ser-15 and Ki67 (+) nuclei, and the number of tyrosinase/Mel5 (+) epidermal melanocytes in T-oligo-treated vs. control skin explants were subjected to ANOVA post hoc analysis, as performed by the StatView program (SAS Institute, Cary, NC, USA). The results were considered statistically significant when P < 0.05.

RESULTS

Skin explants retain normal appearance and physiological responses through 5 days
First we evaluated viability and UV responsiveness of the human skin explants in the test system. Untreated unirradiated explants were normal in appearance and unchanged over 5 days (data not shown). After 24 h in organ culture, some skin explants were UVB-irradiated in PBS with 30 mJ/cm2, skin was harvested at various times up to 96 h post-UV (120 h in culture), then processed for routine histology and evaluation of apoptosis (TUNEL staining) and UV-induced photoproducts (CPDs) (Fig. 1 A–C). Histological evaluation showed that the irradiated skin architecture was intact up to at least 96 h post-UV and 120 h in organ culture (Fig. 1A ). TUNEL staining revealed occasional TUNEL (+) epidermal cells in all explants with no apparent increase over 96 h post-UV (Fig. 1B ). Evaluation of CPD staining showed that human adult skin in organ culture efficiently removed these photoproducts, which were maximal at time 0 (immediately after UV), were gradually removed through 72 h post-UV, and were virtually gone by 96 h (Fig. 1C ), consistent with removal rates reported for intact skin of normal volunteers (30 , 34) and indicating preservation of normal DNA repair in our organ-culture model.


Figure 1
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  		Figure 1. Viability and functionality of human skin ex vivo. Human skin organ cultures were prepared as described in Materials and Methods. After 24 h in the culture media, explants were UVB-irradiated (30 mJ/cm2), then harvested at various times up to 96 h post-UV (120 h in the culture) and processed for H&E staining, TUNEL staining, and detection of major UV-induced photoproducts (CPDs). A) H&E staining showed a normal histological architecture of human skin in organ culture up to 96/120 h after UV/in culture, respectively. B) TUNEL staining showed occasional TUNEL (+) cells (stained green) in untreated samples and no significant increase after 120 h in culture and 96 h post-UV. C) Note the gradual decrease in CPD (+) nuclei (red nuclear staining) over a period of 2 days post-UV and no CPD immunostaining by 96 h, an indication of complete removal of UV-induced CPDs. The dotted white and black lines indicate the dermoepidermal junction x200 magnification in all panels.

T-oligo treatment up-regulates and activates p53 in human skin
To determine whether T-oligos can induce p53 protein level and serine-15 phosphorylation indicative of p53 activation (35) in vivo, we processed T-oligo- vs. diluent-treated or untreated skin sections for both p53total and p53Ser-15 immunofluorescent staining. As expected (21) , in unirradiated skin there was minimal detectable p53 at the time of irradiation, although T-oligo-treated sections had significantly more compared to diluent-treated skin p53 total positive (+) nuclei (3.0±0.8 vs. 1.0±0.3, per 100 µm, P<0.03) (Fig. 2 A, B), confirming prior in vitro and in vivo work (21 , 23) . Further, in UV-irradiated samples, by 6 h the number of p53total (+) nuclei in T-oligo-treated samples was significantly increased above diluent controls (15.0±1 vs. 10.0±0.6, P<0.001), and >30-fold above sham-irradiated samples (Fig. 2A, B ). Thereafter, the number of p53 (+) cells decreased progressively in all groups but remained significantly (P<0.001) higher in T-oligo vs. diluent pretreated explants for at least 48 h (Fig 2A, B ). p53 expression in the untreated sham-irradiated explants remained very low and statistically less than either UVB-irradiated group, as expected after 24 h (Fig 2A, B ).


Figure 2
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  		Figure 2. T-oligo treatment increases constitutive and UVB-induced p53total and p53Ser-15 levels in human skin. A) Skin section (5 µm) were reacted with an Ab to total p53 (DO-1) and examined under a fluorescent microscope. There is no change in low p53 expression in untreated nonirradiated explants over the 72 h experiment. T-oligo (pTT 100 µM; p9mer data not shown) increased constitutive and UV-induced levels of p53total expression, as shown by the number of positive p53total (+) nuclei (green fluorescence) in the epidermis (upper panel). Compared to diluent, T-oligo treatment increased UV-induced p53total expression 6 h after UVB, and the number of p53total (+) nuclei remained twice as high and far greater than in sham-irradiated control explants. B) Quantitative analysis of p53total (+) nuclei as determined by blindly examining at least 3–5 fields (x400) and averaging. Values are expressed per 100 µm of epidermal length and represent explants prepared from three donors. To confirm the validity of quantifying p53total (+) nuclei per 100 µm of epidermal length, we also quantified p53total (+) nuclei per number of nuclei (DAPI) (+) cells (data not shown). No difference in the relative expression of p53total (+) nuclei was found using either quantification method (data not shown). C) Adjacent sections of the same explants shown in Fig. 2A were reacted with a fluorescently tagged Ab to phospho-p53Ser-15, then analyzed as described for Fig. 2B . T-oligo (pTT 100 µM; p9mer data not shown) treatment variably and minimally increased constitutive levels of p53Ser-15 expression (upper panel), with the number of p53Ser-15 (+) nuclei in the epidermis consistently below 0.7 per 100 µm of epidermal length in all groups (P=NS, not significant). After UV irradiation, the number of p53Ser-15 (+) nuclei increased dramatically compared with sham-irradiated controls within 6 h and through 24 h for both T-oligo- and diluent-treated explants. D) Quantitative analysis of p53Ser-15 (+) nuclei (3 donors/treatment condition) revealed a roughly >20- to 35-fold increase (compared to sham-irradiated) in p53Ser-15 above preirradiation levels for both diluent-treated and T-oligo-treated explants, with T-oligo-treated explants showing ~25 and 35% higher numbers of (+) nuclei at 6 and 24 h, respectively, a significant difference (P<0.003 and P<0.0001) at both times. C, control; D, diluent; T, T-oligo; NS, not significant. Data presented in graphs is mean ±SE.

After 24 h, T-oligo treatment minimally increased the number of nuclei with detectable p53Ser-15 levels, which remained below 1% (Fig. 2C, D ). However, compared with diluent-treated UV-irradiated skin, there was a modest but highly statistically significant increase in the number of p53Ser-15 (+) nuclei in T-oligo-treated UV-irradiated skin samples at 6 and 24 h (6 h, 13.8±0. 6 vs. 10±0.8, P<0.003; and 24 h, 12.2±0.6 vs. 8.4±0.9, T-oligo vs. diluent, P<0.0001) as well as striking and highly significant increases above sham-irradiated untreated samples in both groups (Fig. 2C, D ). These data establish that T-oligo pretreatment increases both expression and activation level of p53 in UV-irradiated skin during the period of maximal DNA repair.

T-oligo treatment inhibits UV-induced hyperproliferation in human skin
UV irradiation causes a dose-dependent arrest of epidermal proliferation, followed by a period of hyperplasia (36) . To determine whether T-oligo pretreatment can inhibit this UV-induced hyperproliferation, we evaluated the number of Ki67 (+) nuclei in diluent- vs. T-oligo-pretreated, then UV-irradiated explants. In untreated sham-irradiated skin, the number of Ki67 (+) nuclei was low and constant over the 120 h duration of the experiment (Fig. 3 A, B), as expected. No significant difference in the number of Ki67 (+) nuclei was detected in diluent- vs. T-oligo-treated skin at the time of irradiation (Fig. 3A ). However, by 48 h in diluent-treated UV-irradiated explants, the number of Ki67 (+) nuclei increased (P<0.05) to >3-fold the number in T-oligo-treated explants, and by 72 h post-UV almost the entire basal layer of the epidermis was Ki67 (+) (Fig. 3A ), 4-fold the number in T-oligo-treated explants (Fig. 3B ). Thus, T-oligo pretreatment inhibited UV-induced hyperproliferation in human skin explants almost completely.


Figure 3
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  		Figure 3. T-oligo pretreatment inhibits UV-induced hyperproliferation in human skin. Adjacent sections of the same explants shown in Fig. 2A, C were immunostained with Ki67, a marker of proliferation, then analyzed as described in Materials and Methods. In untreated and unirradiated (>96 h) and diluent and/or T-oligo-treated (pTT 100 µM; p9mer data not shown) skin for 24 h, constitutive levels of Ki67 were not changed (A, upper panel) with the number of Ki67 (+) nuclei (red-stained) in the epidermis consistently below 3 per 100 µm of epidermal length in all groups (P=NS, not significant). After UV irradiation, the number of Ki67 (+) nuclei decreased insignificantly compared with sham-irradiated controls within 24 h, through 48 h for T-oligo-, and within 24 h for diluent-treated explants. Compared to T-oligo, in diluent-treated skin by 48 h (and more dramatically by 72 h) post-UV, the number of Ki67 (+) nuclei was increased and was mostly localized in the basal and suprabasal layers. B) Quantitative analysis of Ki67 (+) nuclei (4 donors/treatment condition) performed as for Fig. 2B, D revealed a roughly >2-fold (48 h) and 6-fold (72 h) increase (compared to sham-irradiated) in Ki67 (+) nuclei above preirradiation levels for diluent-treated but not for T-oligo-treated explants (P<0.05). C, control; D, diluent; T, T-oligo; NS, not significant. Data presented in graphs is mean ±SE, x400 in all panels.

Severely UV-damaged epidermal keratinocytes are also known to undergo apoptosis (37) and, as demonstrated above and in previous studies (16 , 21 , 23) , T-oligos activate p53, a mediator of apoptosis in such cells. We therefore stained sections from the same skin samples processed for Ki67 staining for evidence of apoptosis [terminal deoxynucleotidyl transferase-dUTP terminal nick-end labeling (TUNEL)]. Sham-irradiated explants showed no TUNEL staining, as expected. All other explants (24, 48, and 72 h post-UV) only rarely showed TUNEL-positive presumptively apoptotic cells; no difference between diluent and T-oligo-treated explants was observed (data not shown). Thus, the decreased number of proliferating epidermal cells in T-oligo-treated skin was not due to loss of viable cells.

T-oligo treatment accelerates the removal of CPDs in human skin
To determine whether T-oligo treatment accelerates the removal of CPDs, the major UV-induced DNA photoproduct, we treated human explants as described in Materials and Methods and processed tissue for immunohistology using CPD-specific antibodies. We observed no CPD-positive (+) nuclei in unirradiated explants and the highest number of CPDs, comparable in T-oligo- and diluent-pretreated explants, at time 0, immediately after UV (Fig. 4 A). In explants pretreated with T-oligo or diluent, then UV-irradiated, CPDs decreased with time, as expected, except there was a slight increase at 4 h as noted by our group and others earlier (38) , possibly due to alterations in chromatin structure as part of the DNA repair process that facilitates Ab binding to photoproducts. However, CPD removal rates were strikingly better in T-oligo-treated explants. After 24 h in diluent-treated epidermis, 82 ± 12% of the initial CPD (+) nuclei remained whereas only 45 ± 7% of initial CPD (+) nuclei remained in T-oligo-treated epidermis (P<0.05) (Fig. 4A, B ). By 48 h, 68 ± 14% vs. 25 ± 8% of CPDs (+) nuclei remained in diluent-treated vs. T-oligo-treated explants (P<0.05). By 72 h, in diluent-treated explants >60% of CPDs (+) nuclei remained while < 20% of the initial number of CPD (+) nuclei remained in T-oligo-treated explants (P<0.05) (Fig. 4A, B ). These findings are consistent with our previous reports that T-oligos accelerate the removal of UV-induced photoproducts in human cells in vitro (16 , 22) and murine skin in vivo (23) . Taken together with the T-oligo-mediated inhibition of UV-induced hyperproliferation (Fig. 3A, B ) and the lack of effect on epidermal apoptosis, these findings suggest that the effect of T-oligo was largely the consequence of more rapid DNA repair rather than of greater loss of damaged cells through apoptosis or the dilution of CPD-bearing cells by greater proliferation of surrounding less damaged cells in T-oligo-treated skin.


Figure 4
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  		Figure 4. T-oligo accelerates the removal of CPDs in adult human skin after UV irradiation. A) Representative images of skin explants from a 77-year-old donor were pretreated with diluent or T-oligo for 24 h, followed by UVB irradiation, then stained immunofluorescently with antibodies against CPDs, major UV-induced photoproducts, then analyzed as described in Materials and Methods. There is no immunostaining in nonirradiated skin explants, a similar number of CPD (+) nuclei immediately after UVB (time 0), indicating that T-oligos do not act as a sunscreen. In the skin of this elderly donor, compared to control diluent-treated UV-irradiated skin, in T-oligo-treated skin explants there were half as many remaining CPDs by 24 h and virtually no CPDs left by 72 h, whereas significantly higher number of CPDs remained in diluent-treated skin at 72 h post-UV. B) Quantification of CPD (+) nuclei in UV-irradiated skin. All samples were coded, then evaluated by a single "blinded" observer to eliminate bias and interobserver variability. CPD (+) nuclei were counted in at least 3–5 randomly selected areas of 100 µm of linear epidermis in explants from 10 donors per treatment condition for each time point. 320 images were analyzed. The number of initial CPDs (time 0) was similar in all groups and designated as 100%. CPD (+) nuclei after 4, 24, 48, and 72 h are shown for diluent-treated (gray bars) vs. pTT-treated (open bars) epidermis. C, control; D, diluent; T, T-oligo; x680 in all panels.

T-oligo treatment up-regulates the level of melanogenic proteins and induces epidermal pigmentation in human skin
We have previously shown that T-oligos up-regulate tyrosinase, the rate-limiting enzyme in melanogenesis, and increase melanin production in murine and human melanocytes in vitro and guinea pig and murine skin in vivo (15 , 19 , 27 , 29) . To determine whether T-oligos up-regulate melanogenic enzymes and increase pigmentation in human skin, we treated human skin explants with diluent (as negative control), T-oligos, or 30 mJ/cm2 UVB (as positive control), then harvested skin explants at various times up to 120 h. Sections were double stained with antityrosinase (Tyr) and anti-Mel5 (Trp1) antibodies and processed for Fontana-Masson staining to evaluate epidermal melanin content and distribution. The relative intensity of Tyr and Mel5 staining varied considerably among positive cells, but virtually all cells positive for one marker were positive for the other, and no consistent differences were noted by group or over time. Therefore, only the number of double (+) cells was analyzed. The number of Tyr/Mel5 double (+) cells in diluent-treated explants was constant throughout the experiment (Fig. 5 A, B). However, compared to diluent-treated sham-irradiated explants, the number of Tyr/Mel5 (+) cells doubled in T-oligo-treated explants and nearly tripled in UV-treated explants within 24 h (3.2±0.4 vs. 6.4±0.8 vs. 8.1±1.6, P<0.04 and P<0.001, between diluent and T-oligo, diluent and UVB, respectively; P=NS between T-oligo and UVB). These significant increases in the number of Tyr/Mel5 (+) cells persisted through 120 h after a single T-oligo treatment or single UVB exposure (Fig. 5A, B ), indicating rapid and persistent increases in the expression of melanogenic enzymes.


Figure 5
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  		Figure 5. T-oligos and UVB comparably up-regulate expression of melanogenic proteins tyrosinase and Trp1 (Mel5) and comparably increase pigmentation in human explants. Human skin explants were treated with diluent or T-oligo and UVB-irradiated with 30 mJ/cm2 and harvested at various times for up to 120 h. Then skin samples were processed for double immunovisualization of tyrosinase and Mel5 expression. A) Double Tyr/Mel5 (+) cells [yellow stained; yellow color represents colocalization of green, tyrosinase (+); and red, Mel5 (+) cells] in the basal layer, representing epidermal melanocytes. The dotted white line indicates the dermoepidermal junction as visualized by light microscopy. x400 magnification in all panels. B) Quantification of double Tyr/Mel5 (+) nuclei in diluent/T-oligo-treated and UV-irradiated skin. All samples were coded, then evaluated by a single blinded observer to eliminate bias and interobserver variability. Tyr/Mel5 (+) nuclei were counted in at least 3–5 randomly selected areas of 100 µm of linear epidermis in explants from 5 donors per treatment condition for each time point. [Note that the number of double Tyr/Mel 5(+) nuclei in diluent-treated explants varied insignificantly throughout 5-day experiment and was always lower than 4.3 + 0.8, the highest number of Tyr/Mel5(+) nuclei at 120 h.] C) Adjacent sections of the same explants shown in Fig. 5A were processed for Fontana-Masson silver staining to evaluate melanin content and cellular distribution. T-oligo and UV treatment comparably increased melanin content in basal and suprabasal layer. Note a distinct nuclear capping in basal layer keratinocytes at 24 h and also suprabasal cells at 120 h after T-oligo and UVB but not diluent treatments. Sections were then counterstained with eosin to enhance visualization of the cells. x1000 in all panels. D) Quantification of epidermal melanin content (using NIH Image-J image analyzing software package) in diluent/T-oligo-treated and UV-irradiated skin. All samples were coded, then evaluated by a single "blinded" observer to eliminate bias and interobserver variability. Epidermal melanin content was quantified by selecting an epidermal area (excluding stratum corneum) and obtaining computer assisted/calculated data for % black (black represents epidermal melanin) in at least 3–5 randomly selected areas of 100 µm of linear epidermis in explants from 3 donors per treatment condition for each time point. D, diluent; T, T-oligo; U, UVB.

Fontana-Masson staining revealed modest pigmentation of the control diluent-treated explants (Fig. 5C ), consistent with the donors’ relatively fair skin. As early as 24 h, T-oligo and UV-treated explants showed readily detectable comparable increases in epidermal pigmentation, mainly in the basal layer, accompanied by the formation of prominent nuclear caps in basal and suprabasal layer keratinocytes. This tanning response (percent epidermal area occupied by melanin) was quantified using NIH Image-J software. Compared to diluent-treated, sham-irradiated explants, epidermal melanin content more than doubled in T-oligo- and UVB-treated explants by 24 h (3.1±0.7 vs. 8.0±0.7 vs. 7.6±1.3, P<0.006 and P<0.02, between diluent and T-oligo, diluent, and UVB, respectively; P=NS between T-oligo and UVB), was unchanged in diluent-treated explants over time, and continued to increase moderately in T-oligo- and UVB-treated skin up to 120 h (P<0.001, P<0.0001, respectively), the last time of examination (Fig. 5D ). These data indicate that a single T-oligo treatment induces pigmentation (tanning) in human skin explants comparable to moderate UVB exposure.

DISCUSSION

Human skin is constantly subjected to environmental DNA damage, of which probably the most common form is the UV exposure (39) . Human skin responds to UV irradiation with repair of the DNA damage, transient cell cycle arrest, and programmed cell death (apoptosis) of severely damaged cells (5 , 14 , 40 41 42) , as well as increased melanogenesis (tanning), responses that help protect against subsequent UV exposures. The tumor suppressor and transcription factor p53 and p53-regulated genes contribute to these responses in human cells through modulation of NER, cell cycle arrest, and apoptosis (16 , 41 , 43 , 44) ; compromised DNA damage responses appear to contribute to age-related increases in skin cancer risk (38 , 45 46 47 48 49) .

We have shown that thymidine dinucleotide, pTT, and other DNA oligonucleotides partially or totally homologous to the 3' telomere overhang (T-oligos) induce protective DNA damage responses (15 , 16 , 19 20 21 22 23 , 25) , presumably by mimicking a physiological signal generated during the course of DNA damage or its repair (16 , 20) , and in any case mimicking the cellular response to telomere loop disruption and overhang exposure (20 , 25 , 50) , a presumptive DNA damage signal (51) . In the present study, using adult human skin explants, T-oligos induced and activated p53 within 24 h after a single treatment and significantly enhanced p53 increases after UV exposure, consistent with previous in vitro and vivo studies (16 , 21 22 23) .

Among the UV-induced effects on human skin are transient cell cycle arrest (52) (16–36 h, depending on UV dose) that presumably allows time for DNA to repair before cell division resumes, and subsequent epidermal hyperproliferation, the so-called UV-induced "proliferative rebound" (53) that compensates for the period of growth arrest and leads to an increase in epidermal thickness that may be viewed as protective in that it increases the path length for photons to reach presumptive stem cells in the epidermal basal layers during subsequent UV exposures. However, replication of DNA in preparation for cell division prior to completion of DNA repair poses a risk of mutation. Notably, in the present experiments, >15% of cells in control explants were Ki67 (+) 72 h after UV irradiation (Fig. 3) , at a time when more than 60% of initial CPD (+) nuclei remained heavily damaged, as indicated by continued nuclear positivity, including many in the basal layer (Fig. 4) . In contrast, in T-oligo-treated explants,72 h after UV exposure only 5% of epidermal cells were Ki67 (+) and fewer than 20% of initially CPD (+) nuclei remained positive. The combined effects of prolonged cell cycle arrest, blunted proliferative rebound, and increased DNA repair rate after UV exposure would be expected to lower mutation rate, as indeed was observed after T-oligo applications to chronically UV-irradiated hairless mice (23) .

The major UV-induced photoproducts are CPDs, predominantly thymine dimers, that account for >75% of all UV-induced DNA lesions, and pyrimidine (6–4) pyrimidone photoproducts [6–4) PPs] (34 , 54) . We previously demonstrated minimal removal of thymine dimers from adult-derived fibroblasts within 24 h after UV irradiation (38) , as well as accelerated removal of thymine dimers by T-oligo pretreatment (22) . We therefore examined the effect of T-oligo pretreatment on the removal rate of CPDs in UV-irradiated skin explants from adult donors. As expected, T-oligo-pretreated explants removed UV-induced DNA damage (as shown by CPD immunostaining) far more rapidly then did diluent-treated control explants. Taken together with the prolongation of UV-induced epidermal growth arrest and inhibition of rebound hyperproliferation, the accelerated removal of photoproducts in T-oligo-treated adult human skin explants documents a capacity for inducible DNA damage responses consistent with earlier finding in cultured human fibroblasts (22) . The biological importance of this inducible DNA repair capacity is suggested by the fact that far smaller percent differences in constitutive repair rates, as measured by a host-cell reactivation assay, are reported to distinguish young adult from old adult donor cells and cancer-prone from normal subjects (55) .

Tanning or UV-induced melanogenesis is another photoprotective response of human skin in which the amount of epidermal melanin increases gradually over several days, rendering skin less vulnerable to subsequent UV damage (56) . DNA damage or its repair contributes substantially to the UV-induced tanning response (14 , 29) . After sufficient sun exposure for 2–5 days, there is an increase in tyrosinase gene expression and activity, followed by increased production and distribution of melanin in the epidermis, manifested clinically as a suntan (14 , 57 , 58) . It is known that tanned skin has higher threshold for sunburn and that skin able to tan well is far more resistant to photodamage than skin that tans poorly (59 , 60) . Tyrosinase, the rate-limiting enzyme in melanogenesis, and the tyrosinase-related protein TRP-1 are transcriptionally regulated by p53 (27 , 28) , known to be activated by pTT as well as by UV irradiation (16 , 21) . In the current study we demonstrate that T-oligo increases both tyrosinase and TRP-1 levels in skin explants over the same time course and to the same extent as moderate UVB exposure. Moreover, these increases in tyrosinase and TRP-1 levels result in marked increases in epidermal melanin content and accumulation of melanin in so-called "nuclear caps," further supporting the proposition that tanning is correctly viewed as a DNA damage response and that exogenously provided T-oligos are recognized by epidermal melanocytes in human skin explants as a physiological signal for tanning.

In summary, our present results demonstrate T-oligo-inducible SOS-like DNA damage responses in human skin. These responses include up-regulation of DNA repair rate, increased melanin pigmentation, and a decrease in epidermal proliferation during the period DNA repair following UV irradiation. These results strongly suggest that topical application of T-oligos may reduce UV-induced mutagenesis and decrease the carcinogenic risk in sun-exposed human skin.

ACKNOWLEDGMENTS

We are grateful to Dr. Gregory Galico for providing adult skin for organ cultures; to Dr. Toshio Mori for providing CPD antibodies; and to Daniella Adrien for assistance in the preparation of the manuscript. This work was supported by grants from the Herzog Foundation and NIH-ROI CA 10515–02 (to B.A.G.).

Received for publication February 24, 2006. Accepted for publication April 17, 2006.

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