[CANCER RESEARCH 52, 4227-4231. August 1. 1992] Pyrimidine Dimer Removal Enhanced by DNA Repair Liposomes Reduces the Incidence of UV Skin Cancer in Mice1 Daniel Yarosh,2 Lori Green Alas, Vivien Yee, Andrew Oberyszyn,3 Jeannie Tsimis Kibitel, David Mitchell,4 Rebecca Rosenstein, Alan Spinowitz, and Marc Citron Applied Genetics Inc., Freeport, New York 11520 (D. Y., L. G. A., V. Y., A. O., J. T. K.]; Laboratory of Radiobiology, University of California, San Francisco, California 94143 [D. M.J; Eye Research Institute, Boston, Massachusetts 02114 [R. R./; and Department of Hematology/Oncology, Long Island Jewish Medical Center. New Hyde Park, New York 11042 [M. C.¡ ABSTRACT (10). Purified T4 endonuclease V, encapsulated in liposomes and delivered to UV-irradiated cells in culture, increased CPD UV exposure has been linked to skin cancer in humans by epidemi removal and DNA repair synthesis and enhanced survival in ology and the rare genetic disease xeroderma pigmentosum. However, repair-deficient cells (11-13). The liposomes were more effi UV produces multiple photoproducts in DNA, and their relative contri bution is uncertain. An enzyme which specifically repairs cyclobutane cient than cell permeabilization or denV gene transfection (14). pyrimidine dimers in DNA, T4 endonuclease V, was encapsulated in Liposome encapsulation of drugs has been used for the top liposomes for topical delivery into mouse and human skin. In both ical application of agents to reduce systemic toxicity (15). We species, liposomes applied after UV exposure localized in the epidermis show here that liposome-encapsulated T4 endonuclease V pen and stimulated the removal of cyclobutane pyrimidine dimers. UV-irra- etrates and localizes in mouse and human skin, increases the diated mice treated with these liposomes had a dose-dependent decrease specific removal of CPD from DNA, and reduces the incidence in the incidence of squamous cell carcinoma compared to controls. The of skin cancer in UV-irradiated mice. results demonstrate that unrepaired cyclobutane pyrimidine dimers in DNA are a direct cause of cancer in mammalian skin. MATERIALS AND METHODS INTRODUCTION T4N5 Liposomes. T4N5 liposomes were prepared by encapsulating UV-induced carcinogenesis is important both as a model sys purified T4 endonuclease V in liposomes composed of phosphatidyl tem for human cancer and for practical human health reasons: choline, phosphatidyl ethanolamine, oleic acid, and cholesterol the etiological agent (UV light) is well defined, and its dosim- hemisuccinate (2:2:1:5 molar ratio) by the detergent dialysis method (12). The concentration of the entrapped enzyme was determined by etry can be experimentally controlled; few types of DNA lesions enzyme-linked immunosorbent assay (11) and is expressed as MgT4 are produced; the target tissue (skin) is readily accessible, and endonuclease V/ml of lotion or cell culture media. The encapsulated the pathology of the malignant disease is defined; and most activity was assayed by the nicking of UV-supercoiled DNA with and importantly, skin cancer is a very real disease for the rapidly without dissolution of the liposome (11). Control liposomes contained growing number of people who develop it each year (1). boiled (enzymatically inactive) T4 endonuclease V (12). For the topical UV produces two major genotoxic lesions in DNA, CPD,5 treatment of mouse and human skin, the liposomes were mixed into a and (6-4) photoproducts, and several minor ones (2). In the 1% hydrogel (Hypan SS201; Kingston Hydrogels, Dayton, NJ) made human genetic disease xeroderma pigmentosum, patients have with phosphate-buffered saline. In the first photocarcinogenesis exper a biochemical defect in the DNA repair of both CPD and (6-4) iment, 5% dextran was substituted for 1% hydrogel. The liposome photoproducts (3) and develop skin cancers on sun-exposed dosage was adjusted by the amount of liposomes added to the lotion. sites at greatly increased frequencies (4). Sorting out the relative Cyclobutane Pyrimidine Dimer and (6-4) Photoproduct Assay. The contribution of these DNA lesions to human skin cancer has frequency of CPD in skin DNA was measured by the alkaline agarose not been simple. Photoreactivation of CPD reduced cancer in gel assay (16). The epidermis was isolated by trypsin digestion of ex the thymus offish (5) and the skin of marsupials (6), but these cised skin, and the DNA was purified by protease digestion, phenol extraction, and ethanol precipitation. The DNA was then treated with animals are distantly related to humans, and photoreactivation of CPD also increased the removal of (6-4) photoproducts in purified T4 endonuclease V to produce breaks at all CPD sites, and the single strands were separated by alkaline agarose gel electrophoresis. frog cells (7). Mutations have been identified which originated Photographic negatives of the ethidium bromide-stained gels were an in C-Cdipyrimidines within the raíoncogene (8) &ndp53 tumor alyzed by computerized densitometry. which calculated the change in suppressor gene (9) in human skin cancers, but this provides the average molecular length of DNA resulting from breaks at CPD only circumstantial evidence for the initial DNA lesion. sites. Mouse 10T'/2 fibroblasts were labeled with [l4C)thymidine prior Discrimination between these two major lesions can be ac to UV irradiation and incubated with liposomes in 4% fetal calf serum, complished by making use of the specificity of a DNA repair and the removal of CPD and (6-4) photoproducts from UV-irradiated enzyme. T4 endonuclease V, the product of the phage T4 den\ DNA was measured by radioimmunoassay (17). gene, initiates the removal of CPD from DNA by a lesion- Human Skin Organ Culture. Human breast skin was obtained im mediately after surgery for mastectomy, cut into 1-cm2 pieces, and specific glycosylase and apyrimidinic endonuclease activity incubated dermal side down surrounded by Dulbecco's modified Eagle's medium with 10% fetal calf serum and 2 ^g/ml hydrocortisone. The Received 12/18/91; accepted 5/12/92. exposed epidermis was irradiated with UV-B from two FS20 sunlamp The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accord bulbs, calibrated with a UXV digital radiometer and UV31 probe (Ul ance with 18 U.S.C. Section 1734 solely to indicate this fact. tra-Violet Products, San Gabriel, CA). The T4N5 liposome lotion was 1Supported in part by National Cancer Institute Small Business Innovative Research Grant 2-R44-CA52401-01 to Applied Genetics Inc. applied with a moist cotton swab, and after incubation the skin was 2To whom requests for reprints should be addressed, at Applied Genetics Inc.. fixed in 4% formaldehyde, paraffin embedded, and sectioned at 10 pm. 205 Buffalo Avenue. Freeport, NY 11520. The sections on slides were dewaxed, rehydrated, and incubated with ' Present address: Wise Hall, Ohio Stale University. Columbus, OH 43210. anti-endonuclease V polyclonal antibodies 14C-labeled by reductive me- 4 Present address: University of Texas, M. D. Anderson Science Park. Research thylation (18). The slides were then coated with nuclear track emulsion, Division. P. O. Box 389. Smithville. TX 78957. 5The abbreviations used are: CPD. cyclobutane pyrimidine dimers; UV-B. ul exposed, developed, and photographed using a Nikon diaphot micro traviolet B. scope at X300. 4227 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1992 American Association for Cancer Research. PYRIMIDINE DIMER REPAIR REDUCES CANCER Animal Care. SKH-1 hairless mice, 6-8 weeks old, were obtained tumor >1 mm, and the mean time to first tumor was calculated from from Charles River Laboratories (Wilmington, MA), housed in plastic the Weibull survival distribution. cages with wire mesh covers, and fed standard Purina food pellets and water ad libitum, in compliance with the NIH Policy on Humane Care and Use of Laboratory Animals. The 12 h/day lighting was by fluores RESULTS cent bulbs filtered with amber Toob-Gards (Malconite, Clifton, NJ) to eliminate photoreactivating light. T4N5 Liposome Penetration and Localization in Skin. Ap Skin Penetration and Histopathology. The animals were treated plication of T4N5 liposomes containing T4 endonuclease V to with T4N5 liposomes in 1% hydrogel lotion and then returned to mammalian skin results in rapid penetration and localization in Charles River Laboratories. There they were sacrificed, the dorsal skin the epidermis. Normal human skin obtained within l h of mas was section and stained, and the slides were interpreted by a veterinary tectomy was cultured for 2 h and then treated topically with pathologist who was unaware of the treatment. T4N5 liposomes were T4N5 liposomes in a 1% hydrogel lotion. Penetration was de also prepared using [3H]oleic acid ( 10 Ci/mmol; New England Nuclear) tected in skin sections by autoradiography using l4C-polyclonal and applied to mouse skin at 2 x IO8 dpm/ml. At various times after treatment the mice were sacrificed, and the treated skin was fixed, antibodies to T4 endonuclease V (Fig. 1). Nonspecific binding embedded in paraffin, and sectioned parallel to the dermal plane. The was not observed in untreated skin (Fig. 1, A and B). Ten min radioactivity in each section was measured by liquid scintillation count after treatment, low levels of antibody binding were detected in ing. the epidermis (Fig. 1, C and D), and by l h the binding of Serum Chemistry. The animals were treated topically with T4N5 antibody in the epidermal layers is easily seen (Fig. 1, £and F). liposomes in 1% hydrogel 5 days/week for 3 weeks, or received one i.v. Similar results were obtained using mouse skin treated with injection of T4N5 liposomes alone. The animals were sacrificed, and T4N5 liposomes and ¡mmunofluorescent microscopy, which serum, separated by centrifugation, was sent to Tufts Veterinary Diag nostic Laboratory' (N. Grafton, MA), for automated processing with a also indicated that hair follicles were a significant pathway of Hitachi 737 Chemistry Analyzer.