Does poor sleep quality affect skin ageing?

BACKGROUND: Sleep is important for growth and renewal of multiple physiological systems. The effects of chronic poor sleep quality on human skin function and visible signs of ageing have not been elucidated. AIM: To evaluate the effect of chronic poor sleep quality on measures of skin health and ageing. Self-perceived satisfaction with appearance was also assessed. METHODS: 60 healthy caucasian women, who were categorized as poor quality sleepers [Pittsburg Sleep Quality Index (PSQI) > 5, sleep duration ≤ 5 h] or good quality sleepers (PSQI ≤ 5, sleep duration 7-9 h). A validated clinical tool, SCINEXA(TM) , was used to assess intrinsic and extrinsic skin ageing. Dark under-eye circles were evaluated using standardized photos. Measurement of in vivo transepidermal water loss (TEWL) was used to assess recovery of the skin barrier after tape stripping. Subjects were exposed to simulated solar ultraviolet light, and recovery from erythema was monitored. Subjects also completed a questionnaire evaluating self-perception of attractiveness. RESULTS: Good sleepers had significantly lower intrinsic skin ageing scores by SCINEXA(TM) . At baseline, poor sleepers had significantly higher levels of TEWL. At 72 h after tape stripping, good sleepers had 30% greater barrier recovery compared with poor sleepers. At 24 h after exposure to ultraviolet light, good sleepers had significantly better recovery from erythema. Good sleepers also reported a significantly better perception of their appearance and physical attractiveness compared with poor sleepers. CONCLUSIONS: This study indicates that chronic poor sleep quality is associated with increased signs of intrinsic ageing, diminished skin barrier function and lower satisfaction with appearance.

Morphological characterization of solar lentigines by in vivo reflectance confocal microscopy: a longitudinal approach.

Solar lentigines are benign hyperpigmented skin lesions. Despite their widespread distribution, knowledge on the mechanisms of development is largely unknown. A clinical study was designed in which solar lentigines were characterized using various non-invasive clinical techniques. A subset of solar lentigines was followed over a 5-year time period. One hundred and twenty-eight solar lentigines were evaluated using in vivo reflectance confocal microscopy (RCM) for the evaluation of the length and density of their dermal papillae as well as the deformation of the alignment pattern of hyperrefractive basal cells. Skin colour, colour contrast, the size of the solar lentigo, epidermal proliferation rate, melanin and haemoglobin content were quantified. RCM imaging of solar lentigines revealed a profound structural deformation of the dermal papillae, as the alignment pattern of hyperrefractive basal cells shifted from a circle in non-lesional skin to an irregular non-circular shape in solar lentigines. There was a rise in the number of dermal papillae, and these dermal papillae were significantly longer. Solar lentigines had increased melanin and haemoglobin levels and a higher rate of epidermal proliferation. For a subset of nineteen solar lentigines, a longitudinal study was set-up in which these measurements were repeated 5 years after the first evaluation. The deformation and the number of the hyperrefractive dermal papillary rings increased significantly over the 5-year time span. The size of the lesion increased, and the skin colour became darker. RCM is a useful non-invasive clinical tool for the characterization of solar lentigines, in particular the compressive deformation of the dermal papillae. This deformation became more severe over a time period of 5 years. To our knowledge, this is the first time that the in vivo time-dependent progression of solar lentigines was supported by RCM images, contributing to an improved understanding of the formation and progression of solar lentigines.

Localization of DNA damage and its role in altered antigen-presenting cell function in ultraviolet-irradiated mice.

Prior ultraviolet (UV) irradiation of the site of application of hapten on murine skin reduces contact sensitization, impairs the ability of dendritic cells in the draining lymph nodes (DLN) to present antigen, and leads to development of hapten-specific suppressor T lymphocytes. We tested the hypothesis that UV-induced DNA damage plays a role in the impaired antigen-presenting activity of DLN cells. First, we assessed the location and persistence of cells containing DNA damage. A monoclonal antibody specific for cyclobutyl pyrimidine dimers (CPD) was used to identify UV-damaged cells in the skin and DLN of C3H mice exposed to UV radiation. Cells containing CPD were present in the epidermis, dermis, and DLN and persisted, particularly in the dermis, for at least 4 d after UV irradiation. When fluorescein isothiocyanate (FITC) was applied to UV-exposed skin, the DLN contained cells that were Ia+, FITC+, and CPD+; such cells from mice sensitized 3 d after UV irradiation exhibited reduced antigen-presenting function in vivo. We then assessed the role of DNA damage in UV-induced modulation of antigen-presenting cell (APC) function by using a novel method of increasing DNA repair in mouse skin in vivo. Liposomes containing T4 endonuclease V (T4N5) were applied to the site of UV exposure immediately after irradiation. This treatment prevented the impairment in APC function and reduced the number of CPD+ cells in the DLN of UV-irradiated mice. Treatment of unirradiated skin with T4N5 in liposomes or treatment of UV-irradiated skin with liposomes containing heat-inactivated T4N5 did not restore immune function. These studies demonstrate that cutaneous immune cells sustain DNA damage in vivo that persists for several days, and that FITC sensitization causes the migration of these to the DLN, which exhibits impaired APC function. Further, they support the hypothesis that DNA damage is an essential initiator of one or more of the steps involved in impaired APC function after UV irradiation.

Permeabilization of ultraviolet-irradiated Chinese hamster cells with polyethylene glycol and introduction of ultraviolet endonuclease from Micrococcus luteus.

Chinese hamster V-79 cells were made permeable by treatment with polyethylene glycol and then incubated with a Micrococcus luteus extract containing ultraviolet-specific endonuclease activity. This treatment introduced nicks in irradiated, but not in unirradiated, deoxyribonucleic acid. The nicks remained open for at least 3 h; there was no loss of endonuclease-sensitive sites, and no excision of dimers as measured by chromatography was detected. In addition, there was no increase in ultraviolet resistance in treated cells. This suggests that the absence of a significant amount of excision repair in rodent cells is due to the lack of both incision and excision capacity.

Detection of messenger RNA from O6-methylguanine-DNA methyltransferase gene MGMT in human normal and tumor tissues.

BACKGROUND: The level of the DNA repair protein O6-methylguanine-DNA methyltransferase is an important determinant of the response of tumor cells in culture to alkylating nitrosoureas. In these cells, the abundance of messenger RNA (mRNA) is directly correlated with repair activity. PURPOSE: Our purpose was to compare transferase mRNA levels with the repair activity in primary human tumors. METHODS: Human transferase mRNA was measured in tissue samples from brain tumors, normal lung, lung tumors, ovarian tumors, and normal human liver by use of an RNA protection assay with an antisense probe prepared from the cloned gene. RESULTS: Normal and tumor tissue samples from the same patient had similar transferase activity levels, but transferase expression varied widely among tissue samples from different patients. Brain and lung samples, on average, had transferase mRNA levels closer to those in liver samples than their transferase activity levels. In two cases, tissue samples which were transferase deficient by the activity assays were found to lack transferase mRNA. CONCLUSIONS: Deficiencies in transferase activity are due to reduced or absent mRNA transcription or processing. In brain and lung, there may be post-transcriptional control of mRNA expression. The wide interindividual variation in transferase expression is also seen at the transcription level. IMPLICATIONS: These are among the first measures of transferase mRNA expression in primary human tissue. More samples should be examined to extend these observations.

Expression of the O6-methylguanine-DNA methyltransferase gene MGMT in MER+ and MER- human tumor cells.

DNA probes prepared from human O6-methylguanine-DNA methyltransferase complementary DNA were hybridized to mRNA isolated from human liver and fifteen human tumor cell lines proficient (Mer+) or deficient (Mer-) in transferase activity. Liver and Mer+ cells contained levels of transferase-specific mRNA that correlated with their transferase activity levels, whereas Mer- cells contained undetectable amounts of transferase mRNA. The mRNA levels were not induced in human cells by treatments that induce other DNA damage-inducible genes. These results demonstrate that in human cells the transferase gene is constitutively expressed, that its expression is related to activity levels, and that in Mer- tumor cells the expression of the transferase gene is probably blocked at the level of mRNA production.

Inactivation of O6-methylguanine-DNA methyltransferase and sensitization of human tumor cells to killing by chloroethylnitrosourea by O6-methylguanine as a free base.

Human fibroblasts and tumor cells with constitutive levels of the DNA repair protein O6-methylguanine-DNA methyltransferase were incubated with mM concentrations of the free base O6-methylguanine for up to 24 h. This treatment depleted the cells of their transferase activity, and sensitized the cells to killing by the antineoplastic drug 1-[2-chloroethyl]-1-nitrosourea. Cells constitutively lacking the methyltransferase were not sensitized to cell killing. Cell free extracts incubated with O6-methylguanine also lost methyltransferase activity. Other alkylpurines, such as O6-methylguanosine, S6-methylthioguanine, O6-ethylguanine, and 3-methyladenine, did not have this effect on extracts of human tumor cells, while O6-methylguanosine and O6-methylguanine inactivated purified methyltransferase from Escherichia coli. The data suggest that the free base O6-methylguanine is probably a substrate for the methyltransferase. Calculation of the second order rate constants for free base versus O6-methylguanine in DNA, and experiments in which the free base was mixed with DNA containing O6-methylguanine before reaction with methyltransferase, indicated that the base in DNA is about 4 X 10(7) better as a substrate than is the free base. These results demonstrate that DNA repair capacity of tumor cells can be diminished without DNA damage, and suggest a method for increasing the efficiency of chemotherapy.

Pyrimidine dimer removal enhanced by DNA repair liposomes reduces the incidence of UV skin cancer in mice.

UV exposure has been linked to skin cancer in humans by epidemiology and the rare genetic disease xeroderma pigmentosum. However, UV produces multiple photoproducts in DNA, and their relative contribution is uncertain. An enzyme which specifically repairs cyclobutane pyrimidine dimers in DNA, T4 endonuclease V, was encapsulated in liposomes for topical delivery into mouse and human skin. In both species, liposomes applied after UV exposure localized in the epidermis and stimulated the removal of cyclobutane pyrimidine dimers. UV-irradiated mice treated with these liposomes had a dose-dependent decrease in the incidence of squamous cell carcinoma compared to controls. The results demonstrate that unrepaired cyclobutane pyrimidine dimers in DNA are a direct cause of cancer in mammalian skin.

O6-methylguanine-DNA methyltransferase in human normal and tumor tissue from brain, lung, and ovary.

The resistance of human tumor strains in culture to cell killing by alkylating nitrosoureas is correlated with their levels of the DNA repair activity O6-methylguanine-DNA methyltransferase. Strains with the Mer- phenotype have no activity and are extremely sensitive. However, the relationship between the sensitivity of human tumors in vivo and transferase levels is not known, and even the existence of Mer- human tumors in vivo has been questioned. In this study 73 human tumor and normal tissue samples from brain, lung, and ovary were assayed for transferase levels and methylpurine glycosylase activity. For each organ, transferase levels varied over 100-fold, and Mer- tumors were detected in each group. There was no correlation between transferase and glycosylase levels, indicating that the absence of transferase in some tumor samples was not an artifact due to necrosis or inactivation of enzymes in the extract.

Retrospective study of the correlation between the DNA repair protein alkyltransferase and survival of brain tumor patients treated with carmustine.

We tested the hypothesis that the level of the DNA repair protein O6-alkylguanine-DNA alkyltransferase in brain tumors was correlated with resistance to carmustine (BCNU) chemotherapy. Alkyltransferase levels in individual cells in sections from 167 primary brain tumors treated with BCNU were quantitated with an immunofluorescence assay using monoclonal antibodies against human alkyltransferase. Patients with high levels of alkyltransferase had shorter time to treatment failure (P = 0.05) and death (P = 0.004) and a death rate 1.7 times greater than patients with low alkyltransferase levels. Furthermore, the size of the subpopulation of cells with high levels of alkyltransferase was correlated directly with drug resistance. For all tumors the variables most closely correlated with survival, in order of importance, were age, tumor grade, and alkyltransferase levels. For glioblastoma multiforme, survival was more strongly correlated with alkyltransferase levels than with age. These results should encourage prospective studies to evaluate alkyltransferase levels as a method, for identifying brain tumor patients with the best likelihood of response to BCNU chemotherapy.

Correlation of tumor O6 methylguanine-DNA methyltransferase levels with survival of malignant astrocytoma patients treated with bis-chloroethylnitrosourea: a Southwest Oncology Group study.

PURPOSE: Prior studies show that increased levels of the DNA repair protein O6 methylguanine-DNA methyltransferase (MGMT), also referred to as O6-alkylguanine-DNA alkyltransferase (AGT) correlate with the resistance of glioma cell lines to nitrosoureas. The observed nitrosourea sensitivity of MGMT-deficient lines (methyl excision repair negative [MER-]) and those repair-proficient lines pretreated with MGMT-specific inhibitors (eg, O6 benzylguanine) has raised the possibility that tumor MGMT levels may be an important predictor of survival in patients with gliomas. PATIENTS AND METHODS: We correlated the MGMT level in malignant astrocytoma tissues, obtained from patients treated with radiotherapy and bis-chloroethylnitrosourea (BCNU) on a prior prospective trial (Southwest Oncology Group [SWOG] 8737), with overall and failure-free survival. RESULTS: Of 64 assessable patients with malignant astrocytoma (63% glioblastoma, 37% anaplastic astrocytoma), 64% had high (> 60,000 molecules/nucleus) MGMT levels. The overall median survival for patients with high versus low MGMT levels was 8 and 29 months, respectively (P=.0002), and median failure-free survival 3 and 6 months, respectively (P=.008). Subset analysis by histology (high v low MGMT levels) for anaplastic astrocytoma was 14 versus 62 months (n=24) and for glioblastoma was 7 versus 12 months (n=40). The overall hazards ratio (risk for death) for high versus low MGMT levels was 3.41; in young patients, the hazards ratio was higher (age 18 to 40 years, 4.19; age 41 to 60 years, 3.08) but became equal by MGMT level at age older than 60 years (1.11). Multivariate analysis showed that MGMT was independent of other known prognostic factors (age, performance status, histology). CONCLUSION: The MGMT level in tumor tissue specimens may be a predictive marker of survival in patients with malignant astrocytoma that is independent of other previously described prognostic variables.

Quantitation of DNA repair capacities of human tumor cells by estimation of transfer of DNA adducts to repair proteins.

The repair of O6-methylguanine produced in DNA by alkylating agents is accomplished by a unique lesion reversal mechanism which recognizes the methyl group and transfers it to itself in a suicide reaction. Much of what we know about the importance of O6-methylguanine-DNA methyltransferase repair in human cells comes from the study of Mer- tumor cell strains which are deficient in transferase activity. The human transferase has a preference for repair of methyl groups, but will also act on other substrates. Assays for transferase activity detect either the loss of O6-methylguanine from DNA or the appearance of methylated protein. A new assay detects the recovery of a restriction site in a synthetic polymer following demethylation. Inhibition of transferase activity can be produced in cells by several methods.

O6-alkylguanine-DNA alkyltransferase in cutaneous T-cell lymphoma: implications for treatment with alkylating agents.

Mycosis fungoides is a low-grade cutaneous T-cell lymphoma. Early treatment often involves the use of topical chemotherapy such as mechlorethamine or carmustine although single-agent oral chemotherapy with alkylators is common for advanced disease. Recently, in a Phase I study of the new alkylating agent temozolomide, two mycosis fungoides patients experienced a complete response. The mechanism of resistance to alkylating drugs such as temozolomide is thought to be due to the presence in tumor cells of the DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGT). The protein mediates a reaction with the O6-position of guanine in DNA, removing the lesion and leaving guanine intact. We, therefore, examined the levels of AGT in CD4+ T lymphocytes obtained by negative antibody selection from the blood of noncancerous individuals and mycosis fungoides patients, and in paraffin-embedded sections from mycosis fungoides patch, plaque, or tumor lesions and cells from involved lymph nodes. AGT protein levels were measured by quantitative immunofluorescence microscopy using a monoclonal antibody against human AGT. Using this approach, the mean level of our positive control (AGT-expressing cells) was 84,807 molecules/nucleus; values below 5,000 molecules/nucleus are considered very low. The mean AGT level in CD4+ T lymphocytes from noncancerous and cancerous individuals was 18,618 (n = 12) and 8,593 (n = 5), respectively. The mean fraction of outliers in circulating CD4+ T lymphocytes from mycosis fungoides patients was statistically significantly lower than T cells in lymph nodes. AGT molecules/nucleus from lymph node biopsies from 8 of 10 patients showed low (< 10,000 molecules/nucleus) or undetectable levels (n = 5) of AGT. The mean AGT level from samples of mycosis fungoides patch/plaque and tumor was also low at 221 (n = 4) and 2,363 (n = 6), respectively. Surprisingly, Hut78, a mycosis fungoides T-cell lymphoma cell line, was positive for AGT activity (median: 77,700 molecules/nucleus), and Hut102--another mycosis fungoides cell line--was low (median: 5,990 molecules/nucleus). Because AGT is a primary means of cell resistance to alkylating agents, the low level of AGT in neoplastic T lymphocytes from patients with mycosis fungoides suggests that treatment with alkylating agents producing O6-alkylguanine adducts, such as carmustine or temozolomide, may produce improved clinical outcomes.

Effects of sunscreens and a DNA excision repair enzyme on ultraviolet radiation-induced inflammation, immune suppression, and cyclobutane pyrimidine dimer formation in mice.

Exposure of skin to ultraviolet (UV) radiation inhibits the induction of delayed-type hypersensitivity (DTH) responses initiated at a distant, unirradiated site. Recent studies attributed this form of immune suppression to DNA damage in the form of cyclobutane pyrimidine dimers (CPD). In the present study, we investigated the protective defects of sunscreens on UV-induced systemic suppression of DTH to Candida albicans, inflammation, and DNA damage. The photoprotective effects of sunscreen preparations containing 8% octyl-N-dimethyl-p-aminobenzoate, 7.5% 2-ethylhexyl-p-methoxycinnamate, or 6% benzophenone-3 were studied in C3H mice exposed to a single dose of 500 mJ/cm2 UVB radiation from FS40 sunlamps. Inflammation was determined by the amount of skin edema at the site of UV irradiation, and DNA damage was assessed by measuring the frequency of endonuclease-sensitive sites in the epidermis. Application of the sunscreens before UV irradiation gave 75-97% protection against UV-induced edema, 67-91% protection against formation of CPD, but only 30-54% protection against suppression of DTH. In contrast, the topical application of liposomes containing a CPD-specific DNA repair enzyme immediately after UV irradiation resulted in 82% protection against suppression of DTH, but at best, 39% protection against skin edema. These findings demonstrate that sunscreens give less protection against UV-induced immune suppression than against skin edema and CPD formation. Furthermore, they suggest that less DNA damage is required to cause UV-induced immune suppression than to cause sunburn.

Enhanced unscheduled DNA synthesis in UV-irradiated human skin explants treated with T4N5 liposomes.

Epidermal keratinocytes cultured from explants of skin cancer patients, including biopsies from xeroderma pigmentosum patients, were ultraviolet light-irradiated and DNA repair synthesis was measured. Repair capacity was much lower in xeroderma pigmentosum patients than in normal patients. The extent of DNA repair replication did not decline with the age of the normal patient. Treatment with T4N5 liposomes containing a DNA repair enzyme enhanced repair synthesis in both normal and xeroderma pigmentosum keratinocytes in an irradiation- and liposome-dose dependent manner. These results provide no evidence that aging people or skin cancer patients are predisposed to cutaneous malignancy by a DNA repair deficiency, but do demonstrate that T4N5 liposomes enhance DNA repair in the keratinocytes of the susceptible xeroderma pigmentosum and skin cancer population.

Encapsulation of the UV-DNA repair enzyme T4 endonuclease V in liposomes and delivery to human cells.

T4 endonuclease V, a pyrimidine-dimer-specific DNA repair enzyme, was encapsulated in liposomes under mild conditions. The encapsulated enzyme was active, and when applied to ultraviolet (UV)-irradiated human cells in culture, the liposomes increased incision of UV-irradiated cellular DNA, enhanced DNA repair replication, and enhanced survival of UV-irradiated cells. This method is a first step in a new approach for topical application of DNA repair enzymes to human skin to prevent skin cancer.

Sunscreens and T4N5 liposomes differ in their ability to protect against ultraviolet-induced sunburn cell formation, alterations of dendritic epidermal cells, and local suppression of contact hypersensitivity.

Exposure of skin to ultraviolet (UV) radiation can lead to diverse biologic effects, including inflammation, sunburn cell formation, alterations of cutaneous immune cells, and impaired induction of contact hypersensitivity responses. The molecular mechanisms of these UV-induced effects are not completely understood. We investigated the ability of sunscreens and liposomes containing the DNA excision repair enzyme T4 endonuclease V to prevent these effects of UV radiation. The use of T4N5 liposomes, which increase the repair of cyclobutyl pyrimidine dimers, provides an approach for assessing the role of DNA damage in the effects of UV radiation on the skin. Exposing C3H mice to 500 mJ/cm2 UVB radiation from FS40 sunlamps resulted in skin edema, sunburn cell formation, and morphologic alterations and decreased numbers of Langerhans cells and Thy-1+ dendritic epidermal T cells. In addition, the induction of contact hypersensitivity after application of 2,4-dinitrofluorobenzene on UV-irradiated skin was diminished by 80%. Applying sunscreens containing octyl-N-dimethyl-p-aminobenzoate, 2-ethylhexyl-p-methoxycinnamate, or benzophenone-3 before this dose of UV irradiation gave nearly complete protection against all of these effects of UV irradiation. In contrast, topical application of T4N5 liposomes after UV irradiation had no effect on UV-induced skin edema and only partially protected against sunburn cell formation and local suppression of contact hypersensitivity, although its ability to protect against alterations in dendritic immune cells was comparable to that of the sunscreens. These results suggest that DNA damage is involved in only some of the local effects of UV radiation on the skin. In addition, T4N5 liposomes may be a useful adjunct to sunscreens because they can reduce some of the deleterious effects of UV radiation on skin even after a sunburn has been initiated.

Treatment of human melanocytes and S91 melanoma cells with the DNA repair enzyme T4 endonuclease V enhances melanogenesis after ultraviolet irradiation.

Tanning is a protective response of ultraviolet (UV)-irradiated skin that decreases damage from subsequent sun exposures by increasing the epidermal content of melanin, a brown-black pigment that absorbs light energy throughout the UV and visible portions of the electromagnetic spectrum. The melanin pigment is made by epidermal melanocytes and transferred to surrounding keratinocytes. The action spectrum, time course, and histologic features of tanning are well studied, but the initiating molecular events are unknown. Previous work has shown that T4 endonuclease V, a prokaryotic DNA repair enzyme that catalyzes the first and rate-limiting step in repair of UV-induced pyrimidine dimers, delivered in carrier liposomes (T4N5), enhances repair of UV-induced DNA damage in cultured human cells and protects against photocarcinogenesis in an animal model. We now report that T4N5 treatment enhances UV-induced melanogenesis, as measured by melanin content, tyrosinase activity, 14C-dopa incorporation, and visual assessment in both S91 murine melanoma cells and human melanocytes. T4N5 treatment also increases cell yields following UV irradiation. These data suggest that tanning can be stimulated through enhanced DNA repair.

Cyclobutane pyrimidine dimers in UV-DNA induce release of soluble mediators that activate the human immunodeficiency virus promoter.

Ultraviolet (UV) irradiation of human cells induced expression of a stably maintained fusion gene consisting of the human immunodeficiency virus long terminal repeat promoter controlling the bacterial chloramphenicol acetyltransferase gene. Two experiments demonstrated that DNA damage can initiate induction: UV induction was greater in DNA repair-deficient cells from a xeroderma pigmentosum patient than in repair-proficient cells, and transfection of UV-irradiated DNA into unirradiated cells activated gene expression. Increased repair of cyclobutane pyrimidine dimers by T4 endonuclease V abrogated viral gene activation, suggesting that dimers in DNA are one signal leading to increased gene expression. This signal was spread from UV-irradiated cells to unirradiated cells by co-cultivation, implicating the release of soluble factors. Irradiation of cells from DNA repair-deficiency diseases resulted in greater release of soluble factors than irradiation of cells from unaffected individuals. These results suggest that UV-induced cyclobutane pyrimidine dimers can activate the human immunodeficiency virus promoter at least in part by a signal-transduction pathway that includes secretion of soluble mediators.

Localization of liposomes containing a DNA repair enzyme in murine skin.

T4N5 liposomes, which contain the DNA repair enzyme T4 endonuclease V, were applied to mouse skin in vivo and added to cultured murine keratinocytes in vitro. The fate of the liposome membrane was followed using a fluorescent, lipophilic dye, and the fate of the enzyme was traced by immunogold labeling, followed by brightfield, fluorescence, or transmission electron microscopy. In vivo, T4N5 liposomes penetrated the stratum corneum, localized in epidermis and appendages of the skin, and were found inside basal keratinocytes. The enzyme was found inside keratinocytes treated in vitro and in the epidermis, hair follicles, and sebaceous glands of topically treated skin. Ultrastructural studies demonstrated the presence of liposomes in the cytoplasm of cells in the epidermis often concentrated in a perinuclear location. The enzyme was present in both nucleus and cytoplasm of keratinocytes and Langerhans cells. Liposomes were found in cells of the lymph nodes draining the site of contact sensitization, in association with topically applied antigen. The results demonstrate that liposomes can deliver encapsulated proteins into cells of the skin in vivo and provide insight into how liposome-enhanced DNA repair reduces UV-induced skin cancer and systemic immunosuppression in mice.