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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.06-5964fjev1 20/11/1895    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Arad, S. Articles by Gilchrest, B. A. Search for Related Content PubMed PubMed Citation Articles by Arad, S. Articles by Gilchrest, B. A.

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

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

SPECIFIC AIMS

We have shown that DNA oligonucleotides substantially homologous to the telomere 3'-overhang sequence (T-oligos) increase DNA repair capacity (DRC) in cultured human cells and decrease UV-induced mutagenesis and photocarcinogenesis in mice. We sought to investigate the protective effects of T-oligos in adult human skin explants and determine whether T-oligo-mediated DNA damage-like protective responses are inducible in human skin.

PRINCIPAL FINDINGS

1. T-oligo pretreatment accelerates the removal of CPDs in human skin
To determine whether T-oligo treatment accelerates the removal of cyclobutane pyrimidine dimers (CPDs), the major UV-induced DNA photoproduct, we pretreated human explants with diluent or T-oligo for 24 h, then UV-irradiated once and processed tissue harvested at various times post-UV 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 post-UV (Fig. 1 A). In explants pretreated with T-oligo or diluent, then UV-irradiated, CPDs decreased with time. However, CPD removal rates were strikingly faster 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. 1A, B ). By 48 h, 68 ± 14% vs. 25 ± 8% of CPDs (+) nuclei remained in diluent-treated vs. T-oligo-treated explants (P<0.05); and 72 h, >60% vs. fewer than 20% of initially CPD (+) nuclei remained (P<0.05) (Fig. 1A, B ). These findings are consistent with our previous reports that T-oligos accelerate the removal of UV-induced photoproducts in human cells in vitro and murine skin in vivo.

View larger version (27K): [in this window] [in a new window]   Figure 1. T-oligo accelerates the removal of CPDs. 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. Skin was harvested at various times post-UV and was immunostained with anti-CPD antibodies. B) Quantification of CPD (+). CPD (+) nuclei were counted in at least 3–5 randomly selected areas of 100 µm of linear epidermis in explants from 10 donors/treatment condition for each time point. 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. T-oligo-treated (open bars) epidermis. C, control; D, diluent; T, T-oligo.

2. 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. To determine whether T-oligos up-regulate melanogenic enzymes and increase pigmentation in human skin, we treated human skin explants with diluent, T-oligos, or 30 mJ/cm² UVB, then harvested explants at various times for 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 number of Tyr/Mel5 double (+) cells in diluent-treated explants was constant throughout the experiment (Fig. 2 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. 2A, B ), indicating rapid and persistent increases in the expression of melanogenic enzymes.

View larger version (23K): [in this window] [in a new window]   Figure 2. T-oligos and UVB comparably up-regulate expression of melanogenic proteins and increase epidermal pigmentation in human explants. 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 dermo-epidermal junction as visualized by light microscopy. B) Quantification of double Tyr/Mel5 (+) nuclei. C) Adjacent sections of the same explants shown in Fig. 2 A were processed for Fontana-Masson silver staining. D) Quantification of epidermal melanin content. Epidermal melanin content was quantified by selecting an epidermal area (excluding stratum corneum) and obtaining computer assisted/calculated data for percent black (black represents epidermal melanin) in at least 3–5 randomly selected areas of 100 µm of linear epidermis in explants from 3 donors/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. Interestingly, as early as 24 h, T-oligo and UV-treated explants showed readily detectable and comparable increases in epidermal pigmentation, mainly in the basal layer, accompanied by the formation of prominent nuclear caps in basal and suprabasal layer keratinocytes (Fig. 2C ). This tanning response was quantified (as percent epidermal area occupied by melanin) 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 (P<0.001) moderately in T-oligo- and UVB-treated skin for up to 120 h (Fig. 2D ). These data indicate that a single T-oligo treatment induces pigmentation (tanning) in human skin explants comparable to a moderate UVB exposure.

CONCLUSIONS AND SIGNIFICANCE

Human skin is constantly subjected to environmental DNA damage, probably the most common form of which is UV exposure. 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, as well as increased melanogenesis (tanning), responses that help protect against subsequent UV exposures. Compromised DNA damage responses contribute to age-related increased skin cancer risk in certain diseases and likely also during normal aging.

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-like responses, presumably by mimicking a physiological signal generated during the course of DNA damage or its repair, and in any case mimicking the cellular response to telomere loop disruption and overhang exposure, a presumptive DNA damage signal.

The major UV-induced photoproducts are CPDs, predominantly thymine dimers, that account for >75% of all UV-induced DNA lesions. 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 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 (see full-length version), the accelerated removal of photoproducts in T-oligo-treated adult human skin explants documents a capacity for inducible DNA damage responses. 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.

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. DNA damage or its repair contributes substantially to the UV-induced tanning response. It is known that tanned skin has a higher threshold for sunburn and that skin able to tan well is far more resistant to photodamage than skin that tans poorly. Tyrosinase, the rate-limiting enzyme in melanogenesis, and the tyrosinase-related protein TRP-1 are transcriptionally regulated by p53, known to be activated by T-oligos as well as by UV irradiation. 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 are associated with 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 (Fig. 3 ). These responses include up-regulation of DNA repair rate, increased melanin pigmentation, and a decrease in epidermal proliferation (see full-length version) during the period of post-UV DNA repair.

View larger version (59K): [in this window] [in a new window]   Figure 3. Schematic representation of T-oligo-mediated photoprotective effects in human skin. I) Before UV irradiation, T-oligo induces epidermal pigmentation (an effect without prior DNA damage). II) Immediately after UV irradiation in control explants, all keratinocytes, including those in the basal layer with its presumptive stem cells (shown in green), are substantially UV damaged (shown in yellow), whereas in T-oligo-treated explants, basal and suprabasal ketatinocytes are largely protected from the same initial UV damage by T-oligo-induced pigmentation. III) 24–48 h after UV irradiation in control-treated explants, after the UV-induced transient growth arrest, epidermal keratinocytes, including those in the basal layer, begin to proliferate (shown in red) before the removal of DNA damage is completed, whereas UV-induced growth arrest is prolonged in T-oligo-treated skin and there is more rapid removal of UV-induced photoproducts (CPDs). IV) 72 h after UV irradiation in control-treated explants, many epidermal keratinocytes, including those in the basal layer, proliferate (shown in red), a phenomenon known as UV-induced "hyperprolifertive rebound" while the UV-induced DNA damage persists, whereas UV-induced growth arrest is prolonged in T-oligo-treated explants and removal of UV-induced photoproducts (CPDs) is substantially complete.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5964fje

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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.06-5964fjev1 20/11/1895    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Arad, S. Articles by Gilchrest, B. A. Search for Related Content PubMed PubMed Citation Articles by Arad, S. Articles by Gilchrest, B. A.