[CANCER RESEARCH 59, 4591– 4602, September 15, 1999] Time Course for Early Adaptive Responses to Ultraviolet B Light in the Epidermis of SKH-1 Mice 1 Yao-Ping Lu, You-Rong Lou, Patricia Yen, David Mitchell, Mou-Tuan Huang, and Allan H. Conney 2 Laboratory for Cancer Research, Department of Chemical Biology, College of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854-8020 [Y-P. L., Y-R. L., P. Y., M-T. H., A. H. C.], and University of Texas M. D. Anderson Cancer Center, Smithville, Texas 78957 [D. M.] ABSTRACT Hairless SKH-1 mice were exposed once to UVB light (180 mJ/cm 2 ), and mechanistically important early adaptive responses in the epidermis were evaluated by immunohistochemical and morphological methods. Interrelationships in the time course for these UVB-induced responses were examined. The number of epidermal cells with DNA strand breaks (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling- positive cells) or with thymine dimers increased to maximal levels within 30 min after UVB. The number of cells with DNA strand breaks located specifically in the basal layer of the epidermis was increased substantially by 3–30 min after UVB and gradually increased further over the next 5.5 hours. DNA strand breaks specifically in the basal layer of the epidermis were increased maximally at 6 h after UVB. The number of epidermal cells with DNA strand breaks or thymine dimers decreased markedly between 12 and 36 h. Pyrimidine (6-4) pyrimidone photodimers (6-4 photoproducts) in isolated epidermal DNA were increased immediately after irradiation of the mice with UVB and decreased markedly during the next 6 h. Exposure to UVB caused a rapid 8-fold increase in the number of epidermal cells with the DNA mismatch repair protein, MSH2 (within 30 – 60 min), and the level of MSH2-positive cells remained elevated for at least 48 h. These observations suggest a possible role of MSH2 in the repair of UVB-induced DNA damage. The number of epidermal cells with wild-type p53 protein started to increase at 1 h after UVB exposure and reached maximal levels by 8 –12 h. The number of p53-positive cells fell markedly between 24 and 48 h. The time course for UVB-induced increases in the number of p53-positive cells was paralleled very closely by the time course for UVB-induced increases in the number of cells with p21(WAF1/CIP1), increases in morphologically distinct apoptotic sunburn cells, and decreases in the number of epidermal cells with bromodeoxyuridine (BrdUrd) incorpora- tion into DNA. Although the start of UVB-induced increases in the num- ber of p21(WAF1/CIP1)-positive cells was similar to that for the increase in p53-positive cells and very high levels of p21(WAF1/CIP1)-positive cells were observed at 8 –12 h, maximal increases in p21(WAF1/CIP1)-positive cells were not achieved until 24 h after UVB irradiation (12 h after the peak value for p53). Myeloperoxidase-positive epidermal cells started to increase by 30 min after UVB exposure, and maximal numbers of my- eloperoxidase-positive epidermal cells were observed at 2 h after UVB (18-fold higher than in nonirradiated control mice). An increased level of epidermal peroxidase enzyme activity in the epidermis was also observed from 1 to 24 h after exposure of the mice to UVB. Although neutrophil infiltration into the epidermis was not seen after exposure to UVB, neu- trophil infiltration into the dermis (inflammatory response) was observed from 4 to 144 h after UVB exposure. In contrast to the marked inhibitory effect of UVB on BrdUrd incorporation into the DNA of epidermal cells observed at 8 –12 h after UVB irradiation (>90% inhibition), BrdUrd incorporation into the DNA of epidermal cells was markedly increased (30-fold increase in the number of BrdUrd-positive cells) at 48 h after UVB exposure, and increases in epidermal cell layers and epidermal thickness (hyperplasia) were also observed. These later effects were asso- ciated with regeneration of the damaged epidermis. INTRODUCTION Sunlight-induced, nonmelanoma skin cancer is a major cancer in the United States and in other temperate parts of the world (1–3). UVB light (280 –320 nm) and to a much lesser extent UVA (320 – 400 nm) are responsible for sunlight-induced cancers (4, 5). Molecular studies on mutations observed in the p53 tumor suppressor gene in human cancers have also implicated UV light as a major cause of human skin cancer (6 –9). UVB irradiation is believed to exert its carcinogenic and cytotoxic actions mainly through the direct forma- tion of cyclobutane pyrimidine dimers (thymine dimers) and pyrimi- dine (6-4) pyrimidone photodimers (6-4 photoproducts) in DNA, but UVB exposure also results in the formation of reactive oxygen species that damage DNA and non-DNA cellular targets (10 –15). To maintain genetic integrity after DNA damage, several cellular responses are activated, including mechanisms for removal of DNA damage, cell cycle delay, and apoptosis. The p53 tumor suppressor gene has an important role in protecting cells from DNA-damaging agents (16 –22). DNA damage triggers a rapid increase in the level of cellular wild-type p53 protein, which shuts off cell replication and DNA synthesis, thereby allowing more time for DNA repair and/or apoptosis. This block of the cell cycle by increased levels of wild-type p53 protein prevents the replication of damaged DNA templates. The increased level of p53 protein after DNA damage is also associated with enhanced programmed cell death (apoptosis), presumably in those cells that are too damaged for adequate DNA repair (23–27). Several studies have shown a transient stimulatory effect of UV light on the level of wild-type p53 in cultured cells and in mouse and human epidermis (18, 28 –32). The kind of DNA damage required to enhance p53 levels was investigated in cultured cells by Nelson and Kastan (33), who concluded that DNA strand breaks were necessary to stimulate the formation of increased p53 levels. An important function of p53 protein is to act as a transcription factor by binding to a p53-specific DNA consensus sequence in responsive genes (26, 34). p21(WAF1/CIP1), gadd 45, and mdm-2 genes contain a p53 binding site, and the expression of these genes is responsive to wild-type p53 protein but not to mutant p53 protein (35–38). Accordingly, UVB-induced increases in the level of wild- type p53 protein would be expected to increase the synthesis of p21(WAF1/CIP1), GADD 45, and MDM-2 proteins. Increases in p21(WAF1/CIP1) and GADD 45 inhibit the cell cycle, whereas an increase in MDM-2 inhibits p53 function and enhances its degrada- tion. Interestingly, recent studies showed that increased MDM-2 pro- tein is mediated by a p53-dependent increase in p300 (39) and that ARF inhibits the action of MDM-2 (40). Recent studies have also shown that a DNA-dependent protein kinase is activated after DNA damage, and this kinase is required for p53 sequence-specific DNA binding and expression of p21(WAF1/CIP1) (41). In addition, phos- phorylation of p53 by DNA-protein kinase that is induced by ionizing radiation prevents MDM-2 from inhibiting p53-dependent transacti- vation (42). These results indicate that DNA-protein kinase both activates p53 binding to DNA and blocks p53 inactivation by MDM2. Received 11/3/98; accepted 7/21/99. 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 accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by NIH Grant CA49756. A. H. C. is the William M. and Myrtle W. Garbe Professor of Cancer and Leukemia Research. 2 To whom requests for reprints should be addressed, at Laboratory for Cancer Research, Department of Chemical Biology, College of Pharmacy, Rutgers, The State University of New Jersey, 164 Frelinghuysen Road, Piscataway, NJ 08854-8020. Phone: (732) 445-4940; Fax: (732) 445-0687; E-mail: aconney@rci.rutgers.edu. 4591 on July 17, 2015. © 1999 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from