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• W2061229162 abstract "The distribution and persistence of cyclobutane pyrimidine dimers were investigated in mouse skin after chronic and acute exposures to ultraviolet-B radiation. We found that DNA damage accumulated in response to chronic irradiation and persisted in a unique set of epidermal cells located at the basal layer. Treatment with a tumor promoter caused the heavily damaged epidermal cells to divide and p53-immunopositive clusters to form within 24 h suggesting that these cells may be progenitors of the mutant p53 clusters associated with actinic keratoses and squamous cell carcinomas. In contrast to low fluence chronic irradiation, daily treatment with a higher fluence of ultraviolet-B produced extensive hyperplasia and considerably reduced penetration of photodamage. Exposure of chronically irradiated skin to an acute “sunburn dose” of ultraviolet-B also produced significant epidermal hyperplasia and resulted in complete loss of heavily damaged basal cells within 4 d postirradiation. The occurrence and distribution of cyclobutane dimers in human skin correlated well with putative sunlight exposure and resembled that observed in ultraviolet-B-irradiated mice. Heavily damaged basal cells were observed at various sites, including those receiving sporadic sunlight exposure, suggesting that these cells may play an important role in carcinoma formation in humans. The distribution and persistence of cyclobutane pyrimidine dimers were investigated in mouse skin after chronic and acute exposures to ultraviolet-B radiation. We found that DNA damage accumulated in response to chronic irradiation and persisted in a unique set of epidermal cells located at the basal layer. Treatment with a tumor promoter caused the heavily damaged epidermal cells to divide and p53-immunopositive clusters to form within 24 h suggesting that these cells may be progenitors of the mutant p53 clusters associated with actinic keratoses and squamous cell carcinomas. In contrast to low fluence chronic irradiation, daily treatment with a higher fluence of ultraviolet-B produced extensive hyperplasia and considerably reduced penetration of photodamage. Exposure of chronically irradiated skin to an acute “sunburn dose” of ultraviolet-B also produced significant epidermal hyperplasia and resulted in complete loss of heavily damaged basal cells within 4 d postirradiation. The occurrence and distribution of cyclobutane dimers in human skin correlated well with putative sunlight exposure and resembled that observed in ultraviolet-B-irradiated mice. Heavily damaged basal cells were observed at various sites, including those receiving sporadic sunlight exposure, suggesting that these cells may play an important role in carcinoma formation in humans. cyclobutane pyrimidine dimer Sunlight is a potent and ubiquitous carcinogen responsible for much of the skin cancer in the human population today with skin tumors in man accounting for about 30% of all new cancers reported annually (Cleaver and Mitchell, 1996Cleaver J.E. Mitchell D.L. Ultraviolet radiation carcinogenesis.in: Holland J.F. Frei III., E. Bast Jr., R.C. Kufe D.W. Morton D.L. Weichselbaum R.R. Cancer Medicine. 4th edn. Williams & Wilkins, Baltimore1996: 307-318Google Scholar). More than 1,000,000 cutaneous malignancies are diagnosed each year in the U.S.A. alone (Miller and Weinstock, 1994Miller D.L. Weinstock M.A. Nonmelanoma skin cancer in the United States: incidence.J Am Acad Dermatol. 1994; 30: 774-778Abstract Full Text PDF PubMed Scopus (877) Google Scholar) of which ≈30,000 are malignant melanomas (Skolnick et al., 1994Skolnick M.H. Cannon-Albright L.A. Kamb A. Genetic predisposition to melanoma.Eur J Cancer. 1994; 30A: 1991Abstract Full Text PDF PubMed Scopus (20) Google Scholar). Of the remaining malignancies basal cell carcinomas occur at about 4-fold greater frequency than squamous cell carcinomas. Much of the human population is routinely exposed to low levels of solar ultraviolet-B radiation (UVB), the dose depending on geographic location, custom, occupation, and recreational behavior. Indeed, casual exposure to direct sunlight in the mid-U.S. latitudes is not trivial and may result in the accumulation of a mean lethal dose to unprotected human cells within approximately 30 min (Scotto et al., 1983Scotto J. Fears T.R. Fraumeni J.F. Incidence of nonmelanoma skin cancer in the United States.US Department of Health and Human Services. 1983: 2433Google Scholar). Recent increases in melanoma and carcinoma correlate with ozone depletion (de Gruijl and van der Leun, 1993de Gruijl F.R. van der Leun J.C. Influence of ozone depletion on the incidence of skin cancer: quantitative prediction.in: Young A.R. Bjorn L.O. Moan J. Nultsch W. Environmental UV Photobiology. Plenum Press, New York1993: 89-109Crossref Google Scholar) and calculations suggest that a 1% depletion in stratospheric ozone will increase nonmelanoma skin cancer by about 2.0% (Urbach, 1997Urbach F. Ultraviolet radiation and skin cancer of humans.J Photochem Photobiol B. 1997; 40: 3-7Crossref PubMed Scopus (113) Google Scholar). Hence, chronic low level UV exposure is an important consideration in studies directed toward determining the etiology and epidemiology of sunlight-induced skin cancer and in targeting high-risk populations. Evidence strongly suggests that dimerizations between adjacent pyrimidine bases by the direct absorption of UVB radiation (290–320 nm) are the predominant premutagenic events responsible for the initiation of human basal and squamous cell carcinomas (Brash et al., 1991Brash D.E. Rudolph J.A. Simon J.A. et al.A role for sunlight in skin cancer: UV-induced p53 mutations in squamous cell carcinoma.Proc Natl Acad Sci USA. 1991; 88: 10124-10128Crossref PubMed Scopus (1636) Google Scholar). Transition mutations arising chiefly at the 3′ base of a cytosine-thymine dipyrimidine have been found in the p53 tumor suppressor gene of 50% of human basal cell carcinomas (Ziegler et al., 1993Ziegler A.M. Leffell D.J. Kunala S. et al.Mutation hotspots due to sunlight in the p53 gene of nonmelanoma skin cancers.Proc Natl Acad Sci USA. 1993; 90: 4216Crossref PubMed Scopus (640) Google Scholar) and in the ras proto-oncogene in murine tumors at a much lower rate (Ananthaswamy and Pierceall, 1990Ananthaswamy H.N. Pierceall W.E. Molecular mechanisms of ultraviolet radiation carcinogenesis.Photochem Photobiol. 1990; 52: 1119Crossref PubMed Scopus (322) Google Scholar). The C µT and CC µTT tandem double transition mutations are considered the “signature mutations” of solar UVB radiation. The predominant photoproducts occurring at these sites are the cyclobutane pyrimidine dimer (CPD), pyrimidine(6–4)pyrimidone dimer, and the Dewar photoisomer of the (6–4) photoproduct. The relative contribution of these lesions to mutation induction, tumor initiation, and tumor promotion is the subject of ongoing investigations. An important consideration of photocarcinogenesis is the effect that chronic sunlight exposure may have on the formation and accumulation of DNA photodamage in the skin. Vink and coworkers (Vink et al., 1991Vink A.A. Berg R.J.W. de Gruijl F.R. Roza L. Baan R.A. Induction, repair, and accumulation of thymine dimers in the skin of UVB-irradiated hairless mice.Carcinogenesis. 1991; 12: 861-864Crossref PubMed Scopus (45) Google Scholar;Vink et al., 1993Vink A.A. Berg R.J. de Gruijl F.R. Lohman P.H. Roza L. Baan R.A. Detection of thymine dimers in suprabasal and basal cells of chronically UV-B exposed hairless mice.J Invest Dermatol. 1993; 100: 795-799Crossref PubMed Scopus (24) Google Scholar) analyzed the induction, repair, and accumulation of CPD in the skin of UVB-irradiated hairless mice using immunohistochemistry and found that the accumulation in epidermal cells reached a maximum level after three exposures (total dose of 4.5 kJ per m2) with the photoproduct content in suprabasal cells exceeding that in basal cells. After the third exposure, CPD levels decreased and the level of damage in basal cells prior to exposure (i.e., 24 h after the previous exposure) was at background. More recently,de Gruijl and Berg, 1998de Gruijl F.R. Berg R.J. In situ molecular dosimetry ad tumor risk: UV-iduced DA damage and tumor latency time.Photochem Photobiol. 1998; 68: 555-560PubMed Google Scholar showed that the CPD frequency in the epidermis of chronically irradiated mice decreased with increasing hyperplasia and became stationary with time, and that the level of persistent damage correlated with the daily UVB fluence and was inversely related to tumor latency. In a recent report (Mitchell et al., 1999Mitchell D.L. Greinert R. de Gruijl F.R. et al.Effects of chronic low dose ultraviolet-B radiation on DNA damage and repair in mouse skin.Cancer Res. 1999; 59: 2875-2884PubMed Google Scholar), we treated Skh-1 hairless mice with daily doses of suberythemal UVB for 40 d and analyzed the amount and distribution of DNA photodamage and DNA repair using radioimmunoassays and immunohistochemistry. We found that DNA damage accumulated in mouse skin as a result of chronic UVB irradiation and that this damage persisted in the dermis and epidermis for several weeks after the chronic treatment was terminated. Although the persistent damage was evenly distributed throughout the dermis, it remained in the epidermis as a small number of heavily damaged basal cells. In this study, we investigated the fate of the heavily damaged basal cells in mouse skin and determined whether similar cells could occur in sun-exposed human skin. Female hairless mice (Skh:Hr1) (Charles River), 5–6 wk old at the start of the experiment, were housed under yellow lights and periodically irradiated under a bank of eight 100 W TL01 fluorescent lamps (Philips) emitting predominantly UVB light [88% UVB (290–320); 6% UVAII (315–340); 6% UVAI (340–400)] with a peak wavelength at ≈313 nm. The lights were filtered through UVT cast acrylic (Polycast Technology, Stamford, CT), which excludes stray light below 280 nm. Under these conditions the minimal erythemal dose (MED) was estimated to be ≈3.5 kJ per m2. The fluence rate was measured with an IL1400A radiometer/photometer coupled to an SEL240/UVB-1/TD detector (International Light, Newburyport, MA). Animals were exposed to UVR in an irradiation chamber of our design and manufacture to maximize incident fluence uniformity (StarchArt, Smithville, TX) (for details seeMitchell et al., 1999Mitchell D.L. Greinert R. de Gruijl F.R. et al.Effects of chronic low dose ultraviolet-B radiation on DNA damage and repair in mouse skin.Cancer Res. 1999; 59: 2875-2884PubMed Google Scholar). The average fluence rate at the level of the dorsum (≈20 cm) was 3.8 J per m2 per s and the total incident dose for each treatment was determined by integrating the fluence over the time of exposure. Animals were irradiated at approximately 24 h intervals (daily) with either 0.5 kJ per m2 (0.1–0.2 × MED) or 2 kJ per m2 (0.5–0.75 × MED) UVB radiation. For tumor promotion experiments, dorsa were coated with 100 μg per ml 12-O-tetradecanoylphorbol-13-acetate (TPA) diluted in acetone using a cotton-tipped applicator. Control mice received acetone alone. After CO2 asphyxiation two 1.5 × 2 cm2 sections of skin were excised from the dorsa of four mice, one from the anterior and one from the posterior. Three to six 5 mm × 1.5 cm strips were cut from each section, fixed in 70% ethanol, and paraffin-embedded, and 4 μm sections were cut from each. Sections were extracted from paraffin blocks by incubation at 60°C for 1 h followed by four immersions in xylol for 5 min each, rehydrated by 2 min incubations in 100%, 96%, and 70% EtOH, washed in phosphate-buffered saline (PBS) for 5 min, denatured in 0.1 N NaOH/70% EtOH for 3 min, dehydrated for 1 min each in 70%, 90%, and 100% EtOH, air dried, and incubated with proteinase K (10 μg per ml) at 37°C for 10 min. After five washes for 5 min each in PBS sections were incubated with 5% goat serum for 30 min at 23°C, rewashed five times for 5 min in PBS, and incubated overnight at 4°C with monoclonal antibody specific for CPD (Kamiya Biomedical) diluted 1:1000 in PBS. Sections were then washed five times for 5 min each in PBS and incubated with goat antimouse IgG conjugated with fluorescein isothiocyanate (FITC) (Dianova) diluted 1:100 in PBS. After five additional 5 min washes in PBS slides were mounted with “antifade” [2.3% DABCO (Sigma) in 90% glycerol in 20 mM Tris, pH 8.0] and covered. Sections were visualized using a CCD camera (Fa. Kappa) coupled to a Leica DM fluorescence microscope. Human skin biopsies were routinely performed on dermatologic patients at Dermatologisches Zentrum, Buxtehude, Germany, and immediately frozen in liquid nitrogen at -196°C. Sections (4 μm) were cut from frozen tissue using a cryostat and after slide preparation were sequentially fixed for 5 min in methanol and acetone at -20°C. After fixation the slides were stored at -20°C. The CPD staining protocol was essentially as described above for mouse skin sections. For apoptosis sections were fixed for 10 min in acetone/3% H2O2, rinsed three times for 2 min each with PBS/0.05% Tween 20, preincubated for 20 min at 23°C with PBS/Tween containing 10% goat serum, and incubated for 60 min at 23°C with a 1:100 dilution of a polyclonal rabbit antih/m caspase 3 (active) (R&D Systems, Cat. # AF835) in a humidified chamber. Subsequent to primary antibody treatment a biotin-streptavidin kit (LAB-SA from Zymed) was used for diaminobenzamide (DAB) visualization of caspase-3-positive cells. For p53 analysis, sections were deparaffinized and placed immediately into 0.3% H2O2 in methanol for 20 min, washed three times in PBS/albumin for 5 min each, and incubated with a rabbit polyclonal antiserum against mouse p53 protein (CM-5 from Novacastra Laboratories) diluted 1:35 in PBS/albumin. All incubations were carried out at 23°C. CM-5 antiserum recognizes both mutant and wild-type p53 protein. The CM-5-positive cells were visualized using a biotin-strepatavidin-DAB system as described above. For each set of experimental conditions (i.e., acetone control, 24 and 72 h post-TPA) four adjacent 4 μm sections were prepared separated from an additional group of four sections by 20 μm; 10 such areas were prepared from duplicate mice and each section was examined by three individuals. Section lengths were measured using a reticle and the average number of UV-damaged cells per millimeter of interfollicular epidermis was determined. For p53 quantification, we scored alternate cross-sections (to those used for CPD analyses) and averaged the single cell and cluster frequencies per unit length of section. Mean and standard error of the mean (SEM) were determined for each group and differences between groups were calculated using one-way ANOVA (SigmaStat, SPSS). Immunofluorescence microscopy was used to quantify relative CPD frequencies in individual dermal and epidermal cell nuclei in chronically irradiated mouse skin. Daily irradiation with low UVB doses resulted in greater numbers of FITC-positive cells and a progressive increase in the average fluorescence per nucleus (Mitchell et al., 1999Mitchell D.L. Greinert R. de Gruijl F.R. et al.Effects of chronic low dose ultraviolet-B radiation on DNA damage and repair in mouse skin.Cancer Res. 1999; 59: 2875-2884PubMed Google Scholar). Negligible fluorescence was observed in skin cell nuclei from unirradiated mice. CPD-associated fluorescence was detectable and uniformly distributed in the epidermis after 10 d of chronic irradiations but thereafter showed an increasing nonrandom distribution. In samples biopsied at 20 d and later, the epidermis showed a somewhat uniform fluorescence field punctuated by heavily damaged cells located primarily in the basal layer. At 40 d after the last treatment heavily damaged nuclei were still evident although fluorescence in the remaining cells was observed at background levels. The induction of CPD in mouse skin biopsied immediately after a single acute exposure to 6 kJ per m2 UVB radiation is shown in Figure 1 (a). The distribution was similar to that observed at 24 h after chronic high dose treatments (panel B); i.e., high levels of DNA damage were observed throughout the epidermis and dermis. Significant epidermal hyperplasia was seen 24 h after the acute exposure and few FITC-positive cells were evident in the epidermis at 4 d postirradiation (data not shown). When the 6 kJ per m2 acute dose was administered to mice with heavily damaged basal cells (i.e., 60 d after a 60 d course of chronic low dose irradiation) negligible damage remained in the epidermis at 4 d postirradiation (data not shown). In Figure 1 (b) CPD fluorescence is shown in mouse skin biopsied 24 h after a total accumulated dose of 10 kJ per m2 delivered at 2.0 kJ per m2 per d for 5 d. CPD-specific immunofluorescence was significant and variable throughout the epidermis with particularly high fluorescence (and background) evident in the dermis. In contrast to low daily exposures (see Figure 1d), we observed immediate proliferation (i.e., within 24–48 h) in the epidermis after exposure to 2.0 kJ per m2 per d and distribution of heavily damaged cells throughout Figure 1b. The thickening of the epidermis and accumulation of anucleated cells in the stratum corneum reduced the expected dose to the epidermis and dermis (compare intensities and frequencies of immunopositive epidermal nuclei in Figures 1b and d). Radioimmunoassay data of these same samples showed significantly greater attenuation of photoproduct formation in the skin of mice exposed to the higher daily doses of UVB (Mitchell et al, in press). Effects of high dose chronic irradiations were transient with normal morphology and negligible CPD levels measured in the epidermis and dermis at 60 d after the chronic treatment ended Figure 1c. We observed no persistent heavily damaged cells (i.e., at 60 d) in skin in response to the chronic high dose irradiations. The effects of a low chronic dose regimen (i.e., 0.5 kJ per m2 per d) on CPD formation are shown in Figure 1(d). Epidermal thickness was comparable to that seen in normal skin and immediately after a single high dose of UVB Figure 1a with variable CPD immunofluorescence observed throughout the epidermis and dermis. Cells displaying particularly high fluorescence were observed periodically in the basal layer (upper left of Figure 1d). The effects of tumor promotion on the heavily damaged cells remaining after 40 d chronic UVB were studied using TPA. The average frequency of heavily damaged basal cells per millimeter in the acetone control was 1.75 (SEM = 0.22) (shown in Figure 2(a), at 200× magnification). At 24 h after TPA treatment we observed ≈3-fold increase in epidermal thickness and much reduced levels of heavily damaged cells; i.e., 0.12 (SEM = 0.04) damaged cells per mm in the basal layer and 0.38 (SEM = 0.07) per mm in the suprabasal epidermis. At 72 h after TPA treatment we detected no FITC-positive cells in the epidermis. The mean values determined for the different experimental conditions were significantly different. Immunopositive doublets were observed at 24 h at the basal layer and are shown at 400× and 1000× magnification in Figures 2(b) and (c), respectively. In Figure 2(d), immunopositive cells in the suprabasal epidermis and associated with a hair follicle are shown at 1000× magnification. No differences in the frequency of activated caspase-3-positive (i.e., apoptotic) cells were observed in the control and TPA-treated epidermis at 24 h (data not shown). These data suggest that by 24 h after TPA treatment ≈70% of the damage-retaining cells had divided sufficiently (or repaired sufficient damage) to reduce the CPD frequencies to background. Skin sections excised 24 h after TPA treatment from duplicate mice were stained with antibodies that bind wild-type and mutant mouse p53 protein Figure 3. We found p53-positive cells occurring individually and in clusters with comparable frequencies. The great majority of the clusters contained p53-positive cells that appeared to be laterally contiguous along the basal layer (as shown in Figure 3), although some quasi-ovoid clusters were noted in the suprabasal epidermis. We found 0.02 (SEM = 0.01) p53 clusters per mm in the control and 0.27 (SEM = 0.04) and 0.29 (SEM = 0.07) clusters per mm at 24 and 72 h post-TPA treatment, respectively. The differences in the mean values of the acetone and TPA treated groups are significant (p < 0.001). Individual cells staining positively for p53 occurred at 0.16 and 0.22 cells per mm at 24 and 72 h, respectively. There was an average of seven p53-positive cells per cluster. The distribution of clusters was nonuniform; whereas some sections contained no clusters, others had considerably more than the average. We were interested to see if damage also persisted after a single exposure to a “sunburn” dose of UVB and what effect this treatment would have on the levels of persistent damage that accumulated during chronic low dose irradiation. CPD were visualized at 0, 4, and 60 d after irradiation with 6 kJ per m2 UVB (2–3 × MED) in control mice (i.e., receiving no prior exposure) and in mice chronically exposed to UVB light. In Figure 1(a), extensive damage is evident (in all cells) in the skin of mice biopsied immediately after acute irradiation. In contrast, we detected no immunofluorescent cells 4 d after the high acute dose in control mice receiving no chronic irradiation or in chronically irradiated mice with heavily damaged basal cells (data not shown). In addition to loss of fluorescence we observed significant epidermal hyperplasia in these mice. These data suggest that a single “sunburn” dose of UVB may serve as a promotion event with results very similar to those seen after TPA treatment. The occurrence of basal cells containing persistent photodamage was not limited to mouse skin as similar cells were observed in human epidermis. In Figure 4, CPD are shown in biopsies from patients treated at Dermatologisches Zentrum in Buxtehude, Germany, for various skin disorders. Background levels of fluorescence not associated with cell nuclei were seen in unexposed skin biopsies from the groin area of a 47-y-old female patient with acne inversa Figure 4a particularly along the basement membrane Figures 4a and b. In contrast, skin from sun-exposed regions of two patients showed extensive FITC-positive staining in the epidermis and dermis. In Figure 4(b) a biopsy from the forehead of a 79-y-old male patient with basal cell carcinoma showed heavily damaged cells at all levels of the epidermis. This section was taken from unaffected tissue adjacent to an excised tumor. In Figure 4(c), skin from the forehead of a 67-y-old male patient admitted for wrinkle surgery showed a somewhat different distribution of damage with fluorescent nuclei restricted to the surface and basal layers of the skin. The paucity of intraepidermal damage, compared to the patient shown in Figure 4(b), may reflect intermittent exposure to sunlight with persistent lesions located at the basal layer and nascent damage limited to the upper layers of the epidermis (i.e., stratum granulosum). In Figure 4(d), a biopsy from the groin region of a 40-y-old female patient with malignant melanoma is shown. The section was taken during dissection of a lymph node metastasis and would have received periodic sunlight exposure during sunbathing. It is probable that the DNA damage evident in the FITC-positive basal cells represents persistent damage that was induced at some considerable time prior to the biopsy. We investigated the formation and fate of DNA damage in the epidermis of mice chronically irradiated with multiple suberythemal doses of UVB radiation, exposed to a single erythemal dose of UVB light, or exposed to a single erythemal dose subsequent to chronic irradiation. We found that significant amounts of photodamage accumulated in a small number of epidermal cells after chronic low dose irradiation and subsequent treatment with TPA or a “sunburn dose” of UVB resulted in complete loss of damage from the epidermis. In addition, we could detect no persistent DNA damage in epidermal cells after chronic irradiation that enhanced cell proliferation (i.e., daily exposure to 2 kJ per m2 UVB). It is noteworthy that both erythemal (i.e., 6 kJ per m2) and suberythemal (i.e., 2 kJ per m2) UVB exposures induced significant hyperplasia. The observation that certain basal cells retain high levels of DNA damage for several weeks after UV exposure indicates that these cells are noncycling. Lack of staining by antihuman Melan-A, Clone A103 (DAKO), specific for melanocytes and melanoma cells indicated that these cells were not melanocytes. Indeed, the behavior of these cells is very similar to the slowly cycling, carcinogen-retaining basal cells found in the central regions of the epidermal proliferative units described by Morris and coworkers (Morris et al., 1986Morris R.J. Fischer S.M. Slaga T.J. Evidence that a slowly cycling subpopulation of adult murine epidermal cells retains carcinogen.Cancer Res. 1986; 46: 3061-3066PubMed Google Scholar,Morris et al., 1997Morris R.J. Coulter K. Tryson K. Steinberg S.R. Evidence that cutaneous carcinogen-initiated epithelial cells from mice are quiescent rather than actively cycling.Cancer Res. 1997; 57: 3436-3443PubMed Google Scholar). Of particular interest is the observation that treatment of the carcinogen-label-retaining cells with TPA induces proliferation of the labeled cells within 24 h with concomitant diffusion of the carcinogen in the basal nuclei. In our experiments, topical treatment with TPA not only produced significant loss of the CPD fluorescence signal within 24 h but induced formation of p53-positive cells and clusters Figure 5. It is suggested that the slowly cycling, repair-deficient cells are stem cells (Morris et al., 1986Morris R.J. Fischer S.M. Slaga T.J. Evidence that a slowly cycling subpopulation of adult murine epidermal cells retains carcinogen.Cancer Res. 1986; 46: 3061-3066PubMed Google Scholar,Morris et al., 1997Morris R.J. Coulter K. Tryson K. Steinberg S.R. Evidence that cutaneous carcinogen-initiated epithelial cells from mice are quiescent rather than actively cycling.Cancer Res. 1997; 57: 3436-3443PubMed Google Scholar). Recent data indicate that hair follicule stem cells may represent a major source of keratinocyte stem cells in mouse skin (Taylor et al., 2000Taylor G. Lehrer M.S. Jensen P.J. Sun T.T. Lavker R.M. Involvement of follicular stem cells in forming not only the follicle but also the epidermis.Cell. 2000; 102: 451-461Abstract Full Text Full Text PDF PubMed Scopus (913) Google Scholar). Taylor et al.'s experiments show that these upper follicular keratinocytes, located in the bulge region, emigrate into the epidermis in response to a penetrating wound. In Figure 2, panel D, we observed cells with intermediate immunofluorescent signals associated with the upper portion of a hair follicle. Such cells may represent heavily damaged stem cells emigrating from the hair follicle in response to TPA-induced hyperplasia. The ability of a subpopulation of basal cells to accumulate CPD suggests that they are repair-deficient and quiescent. Global genome repair of CPD in mouse epidermis is biphasic with some of the damage (i.e., 10%-30%) rapidly removed within the first 24–48 h postirradiation and most of the lesions still present 72–120 h later (Bowden et al., 1975Bowden G.T. Trosko J.E. Shapas B.G. Boutwell R.K. Excision of pyrimidine dimers from epidermal DNA and nonsemiconservative epidermal DNA synthesis following ultraviolet irradiation of mouse skin.Cancer Res. 1975; 35: 3599-3607PubMed Google Scholar;Ley et al., 1977Ley R.D. Sedita A. Grube D.D. Fry R.J. Induction and persistence of pyrimidine dimers in the epidermal DNA of two strains of hairless mice.Cancer Res. 1977; 37: 3243-3248PubMed Google Scholar;Cooke and Johnson, 1978Cooke A. Johnson B.E. Dose response, wavelength dependence and rate of excision of ultraviolet radiation-induced pyrimidine dimers in mouse skin DNA.Biochim Biophys Acta. 1978; 517: 24-30Crossref PubMed Scopus (29) Google Scholar;Johnson, 1978Johnson B.E. Formation of thymine containing dimers in skin exposed to ultraviolet radiation.Bull Cancer. 1978; 65: 283-297PubMed Google Scholar;Qin et al., 1995Qin X. Zhang S. Oda H. et al.Quantitative detection of ultraviolet-induced photoproducts in mouse skin by immunohistochemistry.Jpn J Cancer Res. 1995; 86: 1041-1048Crossref PubMed Scopus (22) Google Scholar). Biphasic transcription-coupled repair in the hairless mouse shows 60% of the CPD removed from active genes during the first 4 h postirradiation and no further removal up to 24 h (Ruven et al., 1993Ruven H.J. Berg R.J. Seelen C.M. Dekkers J.A. Lohman P.H. Mullenders L.H. van Zeeland A.A. Ultraviolet-induced cyclobutane pyrimidine dimers are selectively removed from transcriptionally active genes in the epidermis of the hairless mouse.Cancer Res. 1993; 53: 1642-1645PubMed Google Scholar). Hence, it is possible that in the absence of cell division a considerable amount of damage may accumulate in genomic and transcriptionally active DNA sequences. DNA damage and mutation hotspots in the p53 tumor suppressor gene (Ziegler et al., 1993Ziegler A.M. Leffell D.J. Kunala S. et al.Mutation hotspots due to sunlight in the p53 gene of nonmelanoma skin cancers.Proc Natl Acad Sci USA. 1993; 90: 4216Crossref PubMed Scopus (640) Google Scholar) and ras oncogene (Ananthaswamy and Pierceall, 1990Ananthaswamy H.N. Pierceall W.E. Molecular mechanisms of ultraviolet radiation carcinogenesis.Photochem Photobiol. 1990; 52: 1119Crossref PubMed Scopus (322) Google Scholar) in squamous and basal cell carcinomas may represent such sites of persistent damage. Current models for photocarcinogenesis suggest that chronic UV confers a selective advantage on cells with a dysfunctional p53 gene and that reduced apoptosis and increased clonal expansion of these damaged cells leads to formation of p53 clusters, actinic keratoses, and, ultimately, carcinomas (Berg et al., 1996Berg R.J. van Kranen H.J. Rebel H.G. et al.Early p53 alterations in mouse skin carcinogenesis by UVB radiation: immunohistochemical detection of mutant p53 protein in clusters of preneoplastic epidermal cells.Proc Natl Acad Sci USA. 1996; 93: 274-278Crossref PubMed Scopus (189) Google Scholar;Jonason et al., 1996Jonason A.S. Kunala S. Price G.J. et al.Frequent clones of p53-mutated keratinocytes in normal human skin.Proc Natl Acad Sci USA. 1996; 93: 14025-14029Crossref PubMed Scopus (523) Google Scholar). With the removal of the UV stress, the selective advantage is lost and the damaged cells regress (i.e., produce fewer mutant p53 clusters). Our data suggest that cessation of a chronic UVB stress may result in removal of a transiently amplifying cell compartment from the epidermis but leave a subpopulation of latent neoplastic cells that become activated by a proliferation event (i.e., promotion). In response to promotion it is probable that the majority of the cells die (e.g., via apoptosis) or enter a truncated proliferative pathway leading to cell regression. Loss of CPD-associated immunofluorescence in these cells suggests cell death, dilution through cell division, or recovery of a latent DNA repair capacity. The number of p53-immunopositive clusters observed after TPA treatment was ≈25% of the number of heavily damaged cells lost. Such a high mutation frequency may arise from the high CPD frequencies observed in the damage-retaining cells. As we observed no increase in the number of activated caspase-3-positive cells 24 h after TPA treatment compared to the control it is doubtful that apoptosis played a significant role in removal of the damaged cells. From these results we suggest that a single exposure to a “proliferative” dose of UVB may act as a promoter, causing most (if not all) of the heavily damaged cells located at the basal layer to enter the proliferative compartment. This event may serve to clear the skin of latent neoplastic cells and at the same time increase the immediate risk for preneoplastic lesion formation. Studies correlating skin tumor induction and the frequency of heavily damaged cells should shed some light on the biologic significance of this unique but important class of basal stem cells. Our data have important implications for assessing the risks of solar UV exposure. Mice treated with 0.5 kJ per m2 UVB showed negligible observable effects on skin morphology; those treated with 2 kJ per m2 showed profound hyperplasia soon after the start of the chronic protocol. Hence, it is possible that a “threshold” dose may exist between these two doses under which cells are initiated and beyond which cells are both initiated and promoted. In parallel studies we have found that extensive hyperplasia greatly attenuates the amount of UVB penetration in the epidermis (Mitchell et al, in press). Hence, a chronic dose of UVB beyond the hypothetical “proliferative threshold” would be more effective as a cancer promoter than initiator. This work was supported by American Cancer Society Grant RPG-97–094–01-CNE, National Institute of Environmental Health Science Center Grant ES 07784, and Bundesminister für Umwelt, Naturschutz und Reaktorsicherheit, Bonn." @default.
• W2061229162 created "2016-06-24" @default.
• W2061229162 creator A5009379231 @default.
• W2061229162 creator A5060082494 @default.
• W2061229162 creator A5070440074 @default.
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• W2061229162 date "2001-09-01" @default.
• W2061229162 modified "2023-10-03" @default.
• W2061229162 title "Identification of a Non-Dividing Subpopulation of Mouse and Human Epidermal Cells Exhibiting High Levels of Persistent Ultraviolet Photodamage" @default.
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