Roles of Aag, Alkbh2, and Alkbh3 in the Repair of Carboxymethylated and Ethylated Thymidine Lesions.

Environmental and endogenous genotoxic agents can result in a variety of alkylated and carboxymethylated DNA lesions, including N3-ethylthymidine (N3-EtdT), O(2)-EtdT, and O(4)-EtdT as well as N3-carboxymethylthymidine (N3-CMdT) and O(4)-CMdT. By using nonreplicative double-stranded vectors harboring a site-specifically incorporated DNA lesion, we assessed the potential roles of alkyladenine DNA glycosylase (Aag); alkylation repair protein B homologue 2 (Alkbh2); or Alkbh3 in modulating the effects of N3-EtdT, O(2)-EtdT, O(4)-EtdT, N3-CMdT, or O(4)-CMdT on DNA transcription in mammalian cells. We found that the depletion of Aag did not significantly change the transcriptional inhibitory or mutagenic properties of all five examined lesions, suggesting a negligible role of Aag in the repair of these DNA adducts in mammalian cells. In addition, our results revealed that N3-EtdT, but not other lesions, could be repaired by Alkbh2 and Alkbh3 in mammalian cells. Furthermore, we demonstrated the direct reversal of N3-EtdT by purified human Alkbh2 protein in vitro. These findings provided important new insights into the repair of the carboxymethylated and alkylated thymidine lesions in mammalian cells.

DNA repair in human pluripotent stem cells is distinct from that in non-pluripotent human cells.

The potential for human disease treatment using human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells (iPSCs), also carries the risk of added genomic instability. Genomic instability is most often linked to DNA repair deficiencies, which indicates that screening/characterization of possible repair deficiencies in pluripotent human stem cells should be a necessary step prior to their clinical and research use. In this study, a comparison of DNA repair pathways in pluripotent cells, as compared to those in non-pluripotent cells, demonstrated that DNA repair capacities of pluripotent cell lines were more heterogeneous than those of differentiated lines examined and were generally greater. Although pluripotent cells had high DNA repair capacities for nucleotide excision repair, we show that ultraviolet radiation at low fluxes induced an apoptotic response in these cells, while differentiated cells lacked response to this stimulus, and note that pluripotent cells had a similar apoptotic response to alkylating agent damage. This sensitivity of pluripotent cells to damage is notable since viable pluripotent cells exhibit less ultraviolet light-induced DNA damage than do differentiated cells that receive the same flux. In addition, the importance of screening pluripotent cells for DNA repair defects was highlighted by an iPSC line that demonstrated a normal spectral karyotype, but showed both microsatellite instability and reduced DNA repair capacities in three out of four DNA repair pathways examined. Together, these results demonstrate a need to evaluate DNA repair capacities in pluripotent cell lines, in order to characterize their genomic stability, prior to their pre-clinical and clinical use.

Alkbh2 protects against lethality and mutation in primary mouse embryonic fibroblasts.

Alkylating agents modify DNA and RNA forming adducts that disrupt replication and transcription, trigger cell cycle checkpoints and/or initiate apoptosis. If left unrepaired, some of the damage can be cytotoxic and/or mutagenic. In Escherichia coli, the alkylation repair protein B (AlkB) provides one form of resistance to alkylating agents by eliminating mainly 1-methyladenine and 3-methylcytosine, thereby increasing survival and preventing mutation. To examine the biological role of the mammalian AlkB homologs Alkbh2 and Alkbh3, which both have similar enzymatic activities to that of AlkB, we evaluated the survival and mutagenesis of primary Big Blue mouse embryonic fibroblasts (MEFs) that had targeted deletions in the Alkbh2 or Alkbh3 genes. Both Alkbh2- and Alkbh3-deficient MEFs were ∼2-fold more sensitive to methyl methanesulfonate (MMS) induced cytotoxicity compared to the wild type control cells. Spontaneous mutant frequencies were similar for the wild type, Alkbh2-/- and Alkbh3-/- MEFs (average--1.3×10(-5)). However, despite the similar survival of the two mutant MEFs after MMS treatment, only the Alkbh2-deficient MEFs showed a statistically significant increase in mutant frequency compared to wild type MEFs after MMS treatment. Therefore, although both Alkbh2 and Alkbh3 can protect against MMS-induced cell death, only Alkbh2 shows statistically significant protection of MEF DNA against mutations following treatment with this exogenous methylating agent.

Calcineurin inhibitors reduce nuclear localization of transcription factor NFAT in UV-irradiated keratinocytes and reduce DNA repair.

Calcineurin inhibitors are drugs used to suppress the immune system by blocking the nuclear localization of the NFAT transcription factor. Systemic use of these drugs is essential to organ transplantation, but comes at the cost of elevated rates of skin cancer. They have been used topically in atopic dermatitis and other skin diseases on the assumption that they avoid the cancer risk by localized use. The results here show that in skin cells and artificial models of human skin, calcineurin inhibitors block UV-induced nuclear localization of NFAT, and significantly reduce repair of cyclobutane pyrimidine dimers induced in DNA. In addition they inhibit apoptosis of UV-irradiated cells. The effect of blocking nuclear localization of NFAT and inhibiting DNA repair should be considered in judging the risk of topical use of calcineurin inhibitors.

Bicyclic monoterpene diols stimulate release of nitric oxide from skin cells, increase microcirculation, and elevate skin temperature.

Bicyclic monoterpene diols (BMTd) stimulate nitric oxide synthesis in melanoma and neuronal cells, representing cell types arising from embryonic neural crest tissue. This study shows that an equimolar mixture of the BMTd's 2,3-cis/exo-pinanediol and 2,3-cis/exo-camphanediol stimulates nitric oxide synthesis in epithelial cells of the skin, specifically normal human epidermal keratinocytes (NHEK) and normal human microvascular endothelial cells (HMVEC). A 1 mM mixture increased nitric oxide 3-fold in HMVEC in the first 24 h after treatment, and a 2 mM mixture produced an equivalent increase in NHEK. We hypothesized that an increase in nitric oxide in skin would lead to an increase in microcirculation, thereby increasing skin temperature. We found that twice daily application of 1mM BMTd lotion significantly increased arm skin temperature by 0.5 degrees C in 14 days compared to placebo, while a 2 mM mixture significantly increased skin temperature by 0.3 degrees C in 7 days (P < or = 0.05; ANOVA). A single application of a 2 mM BMTd mixture applied 30 min before a 30 min cold challenge (6 degrees C), maintained facial skin temperature 1.4 degrees C above untreated control sites (P < or = 0.05; ANOVA). We also tested whether BMTd treatment would benefit people with dark circles under their eyes. Twenty-six panelists with dark undereye circles completed 2-week, twice daily application of a lotion containing the 1mM mixture to one eye while the other eye was untreated. Seven of 26 subjects showed a reduction of darkness of undereye circles (P < or = 0.05; paired t test). Application of 2 mM BMTd lotion to lips resulted in a significant increase in their redness, as measured by the erythema index (P < or = 0.05; ANOVA). These results show that a mixture of BMTd's increases nitric oxide, and application to skin increases microcirculation and skin temperature.

Anti-inflammatory activity in skin by biomimetic of Evodia rutaecarpa extract from traditional Chinese medicine.

BACKGROUND: Wu-Zhu-Yu, is an extract prepared from the small berry fruit of Evodia rutaecarpa and is reported to have anti-inflammatory and anti-nociceptic activity. Methyl nicotinate (MN) is known to induce the release of PGD(2) resulting in localized erythema within 30 min after topical application to human skin. OBJECTIVE: The purpose of this study was to determine if a defined biomimetic mixture of components of Evodia fruit extract inhibit inflammation in human cells and skin. METHODS: In order to control the potency of the test article, we prepared a defined biomimetic mixture of synthetic and natural forms of the active components of Evodia fruit extract, containing rutaecarpine, dehydroevodiamine, and evodin. This was tested for anti-inflammatory activity in UVB-irradiated cultured cells and in the MN model of micro-inflammation in human skin. RESULTS: This Evodia biomimetic mixture was a potent inhibitor of UVB-induced PGE(2) released by keratinocytes in culture. We found that MN also induces release of nitric oxide from cultured keratinocytes and microvascular endothelial cells. Twice daily application of 0.1-1% Evodia biomimetic mixture for 2 weeks significantly inhibited erythema after a MN challenge. A single application of 1% Evodia biomimetic mixture also significantly inhibited MN-induced erythema when applied at 60 min before, or within 5 min after MN exposure. The Evodia biomimetic mixture was significantly more effective at inhibiting erythema than bisabolol, the active component of chamomile. CONCLUSIONS: These results demonstrate that compounds found in E. rutaecarpa (including the indole quinazoline alkaloids) have powerful anti-inflammatory activity when applied topically to human skin.

Calcineurin inhibitors decrease DNA repair and apoptosis in human keratinocytes following ultraviolet B irradiation.

The use of calcineurin inhibitors in solid organ transplantation results in an increased risk of skin cancer. We examined the effect of these drugs on DNA repair in normal human keratinocytes after ultraviolet B (UVB) irradiation. We found that both cyclosporine A (CsA) and ascomycin inhibited removal of cyclobutane pyrimidine dimers, and that they also inhibited UVB-induced apoptosis. We also observed that UVB induced nuclear localization of the transcription factor nuclear factor of activated T-cells (NFAT), and that this was blocked by CsA and ascomycin. These data suggest that the increased risk of skin cancer observed in organ-transplant patients may be as a result of not only systemic immune suppression but also the local inhibition of DNA repair and apoptosis in skin by calcineurin inhibitors. These findings may have implications for the use of topical calcineurin inhibitors in sun-exposed skin and eyes.