Daniel B. Yarosh

Effect of topically applied T4 endonuclease V in liposomes on skin cancer in xeroderma pigmentosum: a randomised study

Summary Background In patients with xeroderma pigmentosum the frequency of all forms of skin cancer is higher than in the general population, owing to a genetic defect in DNA repair. The bacterial DNA repair enzyme, T4 endonuclease V, delivered intracellularly, increases the rate of repair of sunlight-induced DNA damage in human cells. We tested the ability of this enzyme in a liposomal delivery vehicle applied topically (T4N5 liposome lotion) to lower the rate of new skin cancers in patients with xeroderma pigmentosum. Methods 30 patients were enrolled in this prospective, multicentre, double-blind study. Patients were randomly assigned T4N5 liposome lotion or a placebo liposome lotion, to be applied daily for 1 year. At 3-monthly visits, new actinic keratoses and basal-cell carcinomas were identified and removed. Analyses were by intention to treat. Findings 20 patients were assigned T4N5 liposome lotion and ten placebo lotion; one placebo-group patient withdrew before treatment and one withdrew with progressive disease at 9 months. The annualised rate of new actinic keratoses was 8·2 among the patients assigned T4N5 liposome lotion and 25·9 among those assigned placebo (difference 17·7 [95% CI 11·8–26·5]; p=0·004 by Poisson modelling). For basal-cell carcinoma, the annualised rates of new lesions were 3·8 in the treatment group and 5·4 in the placebo group (difference 1·6 [0·38–2·82]). No significant adverse effects were found among any of the patients. Interpretation DNA damage has an important role in the development of skin cancer and precancerous skin lesions. The topical application of DNA repair enzymes to sun-damaged skin of patients with xeroderma pigmentosum lowered the rate of development of two forms of these lesions during a year of treatment.

UV-induced DNA damage initiates release of MMP-1 in human skin.

Abstract:  Destruction of collagen is a hallmark of photoaging. The major enzyme responsible for collagen 1 digestion, matrix metalloproteinase‐1 (MMP‐1), is induced by exposure to sunlight. To study the molecular trigger for this induction, human skin was ultraviolet‐B (UVB)‐irradiated and treated with liposome‐encapsulated DNA repair enzymes. The photolyase‐mediated DNA repair of epidermal UV damage was associated with a reduction of MMP‐1 mRNA and protein expression in both the epidermal and dermal compartments of the skin. The role of the epidermal cells in MMP‐1 induction in the fibroblasts was examined when human epidermal keratinocytes were irradiated with UVB and their media were transferred to unirradiated human dermal fibroblasts. Transfer of media from irradiated keratinocytes to unirradiated fibroblasts enhanced MMP‐1 mRNA and protein. Thus, UV damage to keratinocytes of the epidermis may participate in the destruction of collagen in the dermis by release of soluble mediators that signal fibroblasts to release MMP‐1. The MMP‐1 induction was reduced when the keratinocytes were treated with DNA repair enzymes T4 endonuclease V or UV endonuclease prior to transfer of the media to fibroblasts. This implies that UVB, which deposits most of its energy on the chromatin of the epidermal keratinocytes and to a lesser extent in the upper dermis, has a significant role in photoaging. DNA damage in the keratinocytes initiates one of the signals for MMP‐1 release, and enhancing DNA repair can reduce MMP‐1 expression in human skin cells and tissue.

UV-DNA Damage in Mouse and Human Cells Induces the Expression of Tumor Necrosis Factor α

Ultraviolet light induces the expression of tumor necrosis factor α (TNFα) in many mammalian cells. We have examined the signal for this induction in a human DNA repair‐deficient cell line carrying a transgene composed of the murine TNF regulatory sequences fused to the chloramphenicol acetyltransferase (CAT) structural gene. When compared by fluence, UVC was a more efficient inducer of CAT than was UVB, but they were equivalent inducers when compared by the frequency of cyclobutyl pyrimidine dimers produced by each source. Further, treatment of UV‐irradiated cells with the prokaryotic DNA repair enzyme T4 endonuclease V in‐creased the level of repair of dimers and concomitantly reduced CAT gene expression. Membrane‐bound TNFα expression was increased by UV and reduced by repair of dimers. Finally, in the TNFcat transgene system, DNA damage directly to the cell with the transgene was required as cocultivation of unirradiated TNFcat cells with UV‐irradiated cells did not increase CAT activity. These results show that DNA damage is a signal for the induction of TNFa gene expression in mouse and human cells.

The Role of O6-Methylguanine in Human Cell Killing, Sister Chromatid Exchange Induction and Mutagenesis: A Review

SUMMARY O6-methylguanine (O6mG) produced in DNA by such SN1 methylating agents as N-methyl-N-nitrososurea and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) has been suggested by some to be the lesion that leads to certain biological endpoints in mammalian cells: cell killing, sister chromatid exchange (SCE) production, mutagenesis and cellular transformation. Other evidence is interpreted as inconsistent with this point of view. The finding of Karran & Williams (1985) that O6mG delivered to cells in culture resulted in the depletion of the activity of the protein responsible for repair of O6mG in DNA (O6mG-DNA methyltransferase, O6MT) provided a tool for the assessment of the role of O6mG in producing biological endpoints. In this paper we review much of the literature on human cells pertinent to this question. In addition we present our survival data obtained using the depletion technique of Karran & Williams as well as data supporting a model invoking a mismatch and excision response to O6mG proposed by Sklar & Strauss (1980). Although data linking O6mG to causation are inconclusive, it is premature to conclude that O6mG is not a lesion lethal to certain cultured cells.

Intracellular localization and intercellular heterogeneity of the human DNA repair protein O 6 -methylguanine-DNA methyltransferase

Abstract O6-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that removes alkyl adducts from DNA and may be important in tumor resistance to alkylation chemotherapy. MGMT was visualized in human cells and tumor tissues with monoclonal antibodies against MGMT and immunofluorescence microscopy, and fluorescent signals were quantified by digital image analysis. MGMT was found both in the cytoplasm and the nucleus, and in either locale the protein reacts with alkylated DNA bases and becomes inactivated and lost from the cell. Cell lines in culture and xenografts showed a broad normal distribution of nuclear MGMT levels, but human brain tumors often showed a skewed distribution, with a significant fraction of cells with high levels of MGMT. O6-Benzylguanine, a suicide substrate inactivator for MGMT activity, reduced MGMT in human cells and in a mouse xenograft to levels undetectable by antibody assay 1 h post-treatment. In melanoma specimens taken from a patient 3 h post-treatment with temozolomide, MGMT levels were reduced by 70%. This quantitative immunofluorescence assay can be used to monitor MGMT and its depletion in human tumors to improve the use of alkylating agents in cancer chemotherapy.

Enzyme therapy of xeroderma pigmentosum: safety and efficacy testing of T4N5 liposome lotion containing a prokaryotic DNA repair enzyme

Xeroderma pigmentosum (XP) is a rare genetic disease in which patients are defective in DNA repair and are extremely sensitive to solar UV radiation exposure. A new treatment approach was tested in these patients, in which a prokaryotic DNA repair enzyme specific for UV‐induced DNA damage was delivered into the skin by means of topically applied liposomes to supplement the deficient activity. Acute and chronic safety testing in both mice and humans showed neither adverse reactions nor significant changes in serum chemistry or in skin histology. The skin of XP patients treated with the DNA repair liposomes had fewer cyclobutylpyrimidine dimers in DNA and showed less erythema than did control sites. The results encourage further clinical testing of this new enzyme therapy approach.

Measurement of UVB-induced DNA damage and its consequences in models of immunosuppression

Exposure to UVB results in formation of cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts in DNA. These can be quantified by a variety of techniques including alkaline gel electrophoresis, ELISAs, Southwestern blotting, and immunohistochemistry. Damage to DNA results in activation of damage response pathways, as indicated by Western blotting using antibodies specific for p53 and breast cancer-associated gene 1 (BRCA1) phosphorylation. The signal from DNA damage to activation of these response pathways appears to be mediated by FKBP12-rapamycin-associated protein (FRAP), since these phosphorylation events are blocked by rapamycin. UVB-induced DNA damage also leads to induction of immunosuppressive cytokines including tumor necrosis factor alpha (TNF-alpha) and interleukin (IL)-10 in skin. Induction of TNF-alpha by UVB is readily detectable in cultured normal human epidermal keratinocytes (NHEKs) using ELISA, while induction of IL-10 is readily detectable in cultured mouse keratinocytes but not in NHEKs. Induction of DNA damage by liposome-encapsulated HindIII results in induction of immunosuppressive responses similar to UVB. Clinical testing shows that liposome-encapsulated T4 endonuclease V or photolyase stimulates repair of CPDs in the skin of human subjects, and prevents UVB-induced immunosuppression. Stimulation of repair and prevention of immunosuppression have been linked to prevention of skin cancer by liposome-encapsulated T4 endonuclease V in repair-deficient xeroderma pigmentosum patients.

Regulation of TNFα production and release in human and mouse keratinocytes and mouse skin after UV‐B irradiation

TNFα is a primary cytokine responsible for inflammatory and immunosuppressive responses in skin. After UV‐B irradiation of cultured human keratinocytes, we found that TNFα was released into the media, as monitored by ELISA, and was bound to cells, as observed by immunofluorescence microscopy. The release of TNFα into cell culture supernatant during the 24 h after UV‐B irradiation was augmented by the addition of IL‐1α to the cells. Further, we found this secretion was unaffected by rapamycin, and therefore independent of FRAP DNA‐protein kinase mediated signal transduction. However, UV‐B also induced expression of membrane‐bound TNFα, and this was dependent on FRAP signaling. In wild type mice, TNFα bound to skin increased immediately after irradiation, declined at 6 h, and then rose again at 12 h before falling by 24 h. This pattern of induction was confirmed by RT‐PCR of TNFα mRNA message in cultured epidermal cells. Induction of membrane‐bound TNFα was also found in c‐fos gene knockout mice deficient in the AP‐1 transcription factor, suggesting that, although AP‐1 containing c‐fos signaling is required for some UV responses, AP‐1 containing c‐fos is not required for this TNFα activation. However, in homozygous p53 knockout mice the basal level of TNFα bound to the epidermis was greatly elevated without UV irradiation. This level declined and remained constant following irradiation. This implies that p53 directly or indirectly represses TNFα gene expression and that modification of p53 mRNA stability or phosphorylation of p53 protein after UV may be responsible for TNFα induction in the membrane. Overexpression of the immunosuppressive cytokine TNFα in this locale may contribute to the carcinogen‐susceptibility of p53 knockout mice.

Liposomes in investigative dermatology

Liposomes are microscopic spheres, usually composed of amphiphilic phospholipids. They may be useful without skin penetration if they simply protect or sequester compounds that would otherwise be unstable in the formulation. Liposomes that remain on the skin surface are useful as light‐absorbers, agents to deliver color or sunscreens, or as depots for timed‐release. Liposomes that penetrate the stratum corneum have the potential to interact with living tissue. Topically applied liposomes can either mix with the stratum corneum lipid matrix or penetrate the stratum corneum by exploiting the lipid‐water interface of the intercellular matrix. There are at least four major routes of entry into the skin: pores, hair follicles, columnular spaces and the lipid:water matrix between squames. A major force driving liposome penetration is the water gradient, and flexible liposomes are best able to exploit these delivery opportunities. Some liposomes release their contents extracellularly. Topical application of photosensitizers may be enhanced by encapsulation in liposomes. Higher and longer‐lasting drug concentrations may be produced in localized areas of skin, particularly at disease sites where the stratum corneum and the skin barrier function are disrupted. The liposome membrane should be designed to capture lipophilic drugs in the membrane or hydrophilic drugs in the interior. Other types of liposomes can be engineered to be taken up by cells. Once inside cells, the lysosomal sac and clatherin‐coated pit are the dead‐end destinations for liposomes unless an escape path has been engineered into the liposome. A novel method has been developed to allow delivery into cells of the skin, by escape from the lysosomal sac. These liposomes have been used to topical deliver active DNA repair enzymes from liposomes into epidermal cells and to enhance DNA repair of UV‐irradiated skin. From these studies a tremendous amount has been learned about the relationship of DNA damage and skin cancer. Both mutations and immunosuppression appear to be essential to skin cancer and both are induced by DNA damage. DNA damage produces immediate effects by inducing the expression of cytokines, which means that DNA damage can induce signaling in neighboring, undamaged cells. The repair of only a fraction of the DNA damage has a disproportionate effect on the biological responses, clearly demonstrating that not all DNA damage is equivalent. This technology demonstrates that biologically active proteins can be delivered into the cells of skin, and opens up a new field of correcting or enhancing skin cell metabolism to improve human health.

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.