Epidermal cell kinetics of pig skin in vivo following UVB irradiation: Apoptosis induced by UVB is enhanced in hyperproliferative skin condition

Journal of Dermatological Science 18 (1998) 43 – 53

Epidermal cell kinetics of pig skin in vivo following UVB irradiation: Apoptosis induced by UVB is enhanced in hyperproliferative skin condition Naoko Kawagishi a, Yoshio Hashimoto b, Hidetoshi Takahashi b, Akemi Ishida-Yamamoto b, Hajime Iizuka b,* b

a Di6ision of Dermatology, Kitami Kobayashi Hospital, Kitami, Japan Department of Dermatology, Asahikawa Medical College, 3 -11 Nishikagura, Asahikawa 078, Japan

Received 8 December 1997; received in revised form 23 February 1998; accepted 26 February 1998

Abstract We investigated the effects of ultraviolet B (UVB) irradiation on pig epidermal sunburn cell (apoptotic cell) formation. Expression of p53 tumor suppressor gene product, p21 (WAF1/CIP1), and proliferating cell nuclear antigen (PCNA) was also determined immunohistochemically. Apoptotic cells appeared at 12 h and reached a peak at 48 h following 2 MED-UVB irradiation. The formation of sunburn cells was confirmed by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) method. p53-positive cells, and p21-positive cells appeared at 6 h, and 12 h, respectively, following the UVB-irradiation. The peak of p53-positive cells was observed at 24 h, and that of p21-positive cells was at 48 h. No expression of TUNEL-, p53-, or p21-positive cells was detected in non-irradiated epidermis. The increase in PCNA-positive cells was observed at 24 h and reached its peak at 96 h following the UVB-irradiation. Flow cytometric analyses indicated a decrease in S-phase cells at 24 h, that was followed by their increase at 96 h. Cells in G2/M phase were also considerably decreased at 6 h and 48 h following the UVB-irradiation, and was followed by their increase thereafter. The [3H]thymidine uptake and mitotic counts remained low up until 48 h, and then both parameters increased reaching their peaks at 72 – 96 h. Effects of UVB irradiation were also determined in tape stripping-induced hyperproliferative epidermis. The numbers of UVB-induced apoptotic cells and PCNA positive cells were markedly enhanced in the tape stripping-treated epidermis, while the numbers of p53- and p21-positive cells were not significantly altered. No induction of apoptosis, p53, or p21 was observed by tape stripping alone. Our results indicate that UVB irradiation induces G1 arrest, prolonged S, and G2/M block of epidermal keratinocytes as well as apoptosis. These processes provide a G1 check point and the elimination of possibly hazardous cells carrying DNA damage, respectively. Our results also indicate Abbre6iations: ACs, apoptotic cells; AEC, 3-amino-9-ethylcarbazole; CDK, cyclin-dependent kinase; FCM, flow cytometry; MED, minimal erythema dose; PBS, phosphate buffered saline; PCNA, proliferating cell nuclear antigen; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling; UVB, ultraviolet light B. * Corresponding author. Tel: + 81 166 652111, extn. 2522; fax: + 81 166 657751. 0923-1811/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0923-1811(98)00024-3

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that the UVB-induced apoptotic process is enhanced in hyperproliferative skin condition suggesting that apoptosis is closely associated with cell cycle progression. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: UVB; Apoptosis; p53; p21; PCNA

1. Introduction UVB irradiation is known to produce numerous effects on keratinocytes, among which is sunburn cell formation [1,2]. The sunburn cells are keratinocytes showing eosinophilic cytoplasm with or without remnants of shrunken and condensed nuclei in hematoxylin and eosin staining. They are regarded as a specific type of apoptosis, that is triggered by DNA damage; UVB is primarily absorbed in the epidermis [3], causing molecular damage to DNA including UV-induced pyrimidine dimers and 6-4 photoproducts [4,5]. Apoptosis is usually accompanied by DNA fragmentation, that can be detected histochemically with TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling) method [6] or as a characteristic ladder of nucleosome-sized fragments on agarose gels [7]. Several lines of evidence indicate that apoptosis is a process intimately linked to the cell cycle [8,9]; apoptosis is almost exclusively found in proliferating tissues, and many oncogenes that promote cell cycle progression induce apoptosis [10]. Sunburn cell formation also depends on cell cycle, where proliferating cells, especially S phase cells, appear to be responsible [2]. Still cell proliferation is prohibited in the apoptosis, suggesting that defective cell cycle progression is intimately linked to the apoptotic process. p53 tumor suppressor gene plays a major role in controlling cell cycle after DNA damage [11,12]. p53 will either cause a delay in G1 providing time for DNA-damaged cells to repair any point mutations [12] or trigger apoptosis eliminating the cells as a potential source of persistent somatic mutations [13]. The G1 arrest seems to depend on p21 (WAF1/CIP1), a p53 target gene containing a p53 transcriptional regulatory motif [14,15]. Recent evidence indicates that UVB irradiation induces both p53 and p21 in human epidermis [16 – 18].

Besides the sunburn cell formation, UVB irradiation is known to induce epidermal hyperproliferation [19–21]. This is preceded by a hypoproliferative phase, indicating that an inhibitory control of cell cycle is also operative. In the present study, we investigated the UVB-induced apoptosis in pig epidermis especially in terms of cell cycle controlling mechanism that depends on p53. Because tape-stripping also induces epidermal hyperproliferation without DNA damage [22,23], we further analyzed the UVB-induced apoptotic process in the tape stripping-induced hyperproliferative skin condition.

2. Materials and method

2.1. UVB irradiation Domestic pigs and hairless micropigs (ASR) weighing about 5 kg were anesthetized i.p. with Nembutal (30 mg/kg). After anesthesia, both sides of the backs of the pigs were washed and shaved. One side was irradiated with Toshiba-Eizai Dermaray equipment (DMR-1, Tokyo, Japan). The other side was used as the control. The equipment produced UVB irradiation at 280–370 nm with the main emission at 295–315 nm and peak emission at 305 nm. In tape stripping experiments, one side of the back skin was repeatedly stripped with pressure-sensitive tape (Nitiban brand cellophane tape) until discrete glistening of the skin appeared. Usually, 40–50 strippings were required for the complete removal of the horny layer, that was histologically confirmed. At 24 h following the tape stripping, when thymidine incorporation shows its peak [23], the stripped side was irradiated with the Dermaray equipment. The minimal erythema dose (MED), determined on three pigs at 24 h after UVB irradiation, was 1259 10 mJ cm2. Since variations between pigs were minimal, a dose of 125 mJ/cm2

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was used as 1 × MED in the present experiments. Following UVB irradiation, pigs were anesthetized at an appropriate time and the skin specimens were obtained from the irradiated and non-irradiated sites using a Castroviejo Keratome (set at 0.3 mm) and a 6-mm punch. To determine the UVB dose effect, 1, 2 and 3 MED-UVB was irradiated and skin specimens were obtained 48 h after the UVB irradiation.

2.2. ACs counting and mitotic counts Six-millimeter punch biopsies were taken, processed routinely, and stained with hematoxylin and eosin. The number of apoptotic cells in the epidermis was counted and expressed as number of cells per cm [24]. The following criteria were used to define apoptotic cells: (1) location in the living epidermal layers, not in the dermis, or stratum corneum; (2) a dense, irregular nucleus, smaller and darker than the nuclei of adjacent keratinocytes; (3) a hypereosinophilic cytoplasm as compared to cytoplasm of adjacent keratinocytes. The average number of ACs per section and mitotic counts were calculated.

2.3. Immunohistochemical staining Skin specimens were fixed in 10% formalin for 1 – 3 days, embedded in paraffin, and cut in consecutive sections 5 mm thick. The sections were deparaffinized in xylene and rehydrated in a series of graded alcohols. The sections were treated with 0.3% hydrogen peroxide solution to exhaust endogenous peroxidase activity. After microwave treatment, the sections were preincubated in 10% rabbit serum in phosphate buffered saline (PBS), and monoclonal antibodies were applied to the sections: DO-7 (Dako; code M7001), which reacts with wild-type and mutant forms of human p53 protein, dilution 1:50; 6B6 (Pharmingen; catalog number 15091A), which reacts with human senescent cell-derived inhibitor 1 (Sdi1), also known as wild type p53-activated fragment 1 (WAF1), Cdkinteracting protein 1 (CIP-1), p21-, and p53-regulated inhibitor of Cdks (Pic1), dilution 1:50; PC10 (Dako; code M879), which reacts with PCNA from all vertebrate species and with proliferating cells in

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a wide range of normal tissues, dilution 1:50. The sections were washed in PBS, followed by application of the Histofine streptavidin-biotin (SAB)PO(M) Kit (Nichirei, Tokyo, Japan). Biotinylated rabbit anti-mouse antibody was used as secondary antibody, and the immunoreaction was visualized by avidine/biotin complex, with 0.03% hydrogen peroxide as substrate and diaminobenzidine (DAB) or 3-amino-9-ethylcarbazole (AEC) as a chromogen. Counter staining was not performed. The numbers of p53-, p21- and PCNA-positive cells were counted and described as number of cells per cm [24].

2.4. Obser6ation of apoptosis in UVB irradiated pig skin by TUNEL The TUNEL method described by Gavrieli et al. [6] was adopted with minor modifications. Paraffin sections (5 mm) were deparaffinized by heating at 60°C for 1 h, rehydrated, and incubated with proteinase K (10 mg/ml) to strip away nuclear proteins. After a 2-min washing in ultrapure water (twice), the slides were covered with 2% hydrogen peroxide for 5 min to inactivate endogenous peroxidase. After rinsing with PBS, TUNEL staining was performed with the apoptosis staining kit, ApopTag™ (Oncor, Gaithersburg, MD). Control slides were incubated without TdT (terminal deoxynucleotidyl transferase) enzyme. The slides were then treated with a solution containing 20 mg of AEC (Sigma, St. Louis, MO) dissolved in 5 ml of N,N-dimethylformamide, 95 ml of 0.1 mol/l acetate buffer at pH 5.2 and 0.1 ml of 30% hydrogen peroxide for 5 min. Counter hematoxylin staining for nuclei was performed. The slides were immersed in running tap water for 10 min and mounted with glycerin jelly without dehydration.

2.5. DNA-Flow cytometry Skin slices were cut into 5 × 5 mm squares, washed three times in RPMI 1640 medium, and floated with their keratin layers up in a fresh RPMI 1640 medium which contained 1000 units/ml dispase (Godo Shusei, Tokyo, Japan). After incubation for 30 min at 37°C in 5% CO2 in air, pure epidermal sheets were peeled from the dermis with

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sharp forceps. The epidermal pieces were minced with scissors and washed twice in PBS (pH 7.4). The minced pieces were centrifuged at 500× g for 5 min and the precipitates were gently fixed in cold ethanol (70% v/v) at − 70°C for 24 – 48 h. The fixed pieces were washed in PBS at 4°C and treated with 0.05% pepsin (Nakarai, Kyoto, Japan) for 15 min at 37°C in a shaking water bath. The isolated cells were washed and incubated in PBS containing RNase (1500 units/ml; Worthington, Freehold, USA) at 37°C for 15 min. The cells were then stained with propidium iodide (100 mg/ml; Sigma, St. Louis, MO) at 4°C for 30 min. The relative DNA content of 104 nuclei was analyzed by EpicsElite (Coulter, Nikaki, Tokyo) equipped with an argon ion laser excited at 488 nm. Pig lymphocytes were used as an external standard for G0/G1 phase DNA content. The fraction of cells in the G0/G1, S, and G2/M phases was analyzed by Multicycle program.

2.6. Thymidine incorporation The keratome-biopsied skin specimens were cut into 5×5 mm squares, washed three times in RPMI 1640 medium, and then floated, with their keratin layers up on fresh RPMI 1640 medium containing [3H]thymidine (1 mCi/ml) at 37°C for 2 h in an atmosphere of 5% CO2 in air. After incubation, the skin squares were washed in saline, and then floated with their keratin layers up, in 2 M NaBr. After the NaBr treatment at 37°C for 2 h, pure epidermal sheets were peeled from the dermis with sharp forceps and washed three times in saline. Individual epidermal tissues were placed in 10×76 mm test tubes containing 200 ml 0.5 N NaOH, tightly capped, and placed in a 95°C water bath for 30 min. At the end of the boiling, test tubes were cooled on ice and vortexed vigorously, and aliquots of the solution were taken for counting the radioactivity [25]. The NaBr treatment allowed the unincorporated thymidine nucleotides to leak out, leaving the thymidine incorporated into DNA.

2.7. Statistics Statistical significance of the data obtained was

evaluated by Student’s t-test using an unpaired analysis.

3. Results

3.1. UVB-dependent induction of apoptosis, p53, and p21 Apoptotic cells (ACs) appeared within 12 h in the lower epidermis especially on the basal cell layer. The peak of ACs was observed at 48 h post UVB (Fig. 1A), when ACs were mainly located in middle or upper layers of the epidermis. Within 72 h, most of the apoptotic cells disappeared. Within 48 h post UVB, many TUNEL-labeled keratinocytes were detectable in the epidermis (Fig. 1B). No TUNELlabeled cells were found in non-irradiated control epidermis. A small number of p53-positive cells appeared within 6 h following the UVB irradiation (Fig. 2A). They were observed in the basal and lower epidermal cell layers. Then the p53-positive cells markedly increased with its peak at 24 h and 48 h, when positive cells were located all over the epidermis (Fig. 2B). The number of p53-positive cells declined within 72 h and mostly disappeared by 96 h post UVB. A significant number of p21-positive cells was detected within 12 h following the UVB irradiation (Fig. 3A), reaching its peak at 48 h, when the number of ACs also reached its peak (Fig. 1A). The p21-positive cells were mainly located in the basal or lower epidermal layer at 24 h, but in the upper layer at 48 h (Fig. 3B). The number of p21 positive cells declined at 96 h and disappeared by 120 h post UVB (Fig. 3A). No expression of p53 or p21 was detected in non-irradiated pig epidermis. The number of ACs and p53 positive cells increased with the irradiation dose (Fig. 4A,B). The number of p21 positive cells also increased in 2 MED compared with 1 MED. In 3 MED, however, the number of p21 positive cells considerably decreased compared with 2 MED (Fig. 4C). Combined eosin staining and p53 immunostaining indicated that apoptotic cells and p53-positive cells were not the same; there were many p53-positive keratinocytes without the features of apoptosis

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Fig. 1. Apoptosis following 2 MED-UVB irradiation. Pig skin was irradiated and the apoptotic cells (sunburn cells) were counted at the indicated time (A). Many TUNEL-positive cells (arrowheads) were detected at 48 h following the 2 MED-UVB irradiation (B).

(Fig. 5). The combined eosin staining and p21 immunostaining also indicated that the apoptotic cells and the p21-positive cells were not the same (data not shown).

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Fig. 2. p53-positive cells following UVB irradiation. Following 2 MED-UVB-irradiation, p53-positive cells were counted (A). Many p53-positive cells are observed at 48 h following the irradiation (B). Dots indicate the dermo – epidermal junction.

3.2. UVB-induced alteration of cell cycle progression The thymidine incorporation and mitotic counts remained low up until 48 h following the

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Fig. 3. p21-positive cells following UVB irradiation. Following 2 MED-UVB-irradiation, p21 Cip1/WAF1-positive cells were counted (A). Many p21-positive cells are observed at 48 h following the irradiation (B). Dots indicate the dermo–epidermal junction.

Fig. 4. Dose response of UVB-irradiation. Pig skin was irradiated with 1 – 3 MED-UVB irradiation and apoptotic cells (A), p53-positive cells (B), and p21-positive cells (C) were counted at 48 h following the irradiation. *P B0.01 compared with 1 MED. Essentially no apoptotic cells, p53-, or p21-positive cells were detected in the control epidermis (no UVB-irradiation).

UVB irradiation, then both parameters rapidly increased (Fig. 6). The peak of mitotic counts was at 72 h and the peak of thymidine incorpo-

ration was at 96 h. They returned to basal levels by 168 h. PCNA, which is required for both DNA repli-

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cation and repair [31], was also induced by UVB irradiation. The increase was detected at 24 h, and reached its peak at 72 – 96 h (Fig. 7). DNA-flow cytometric analyses indicated that the proportion of cells in S phase was considerably decreased at 24 h following the UVB irradiation (Fig. 8). In 48 h, the decrease in S phase cells disappeared, that was accompanied by a decrease in cells in G2/M phase, suggesting that cells in S phase failed to proceed into G2/M phase until 24–48 h post UVB. The peak of S phase was observed at 96 h. It returned to basal levels by 168 h. Consistent with a decrease in mitotic counts up until 48 h (Fig. 6), the proportion of G2/M phase cells showed a considerable decrease at 6 h and at 48 h following the UVB irradiation (Fig. 8). This was followed by a gradual increase in G2/M proportion reaching its peak at 96 h. This was concomitant with the peak of S phase cells. It returned to basal levels by 168 h (Fig. 8).

3.3. Effect of tape stripping on UVBinduced apoptosis, p53, and p21 expression Tape stripping is known to induce epidermal hyperproliferation. UVB irradiation was performed at 24 h following the stripping, when thymidine incorporation shows its peak [28]. The numbers of UVB-induced ACs and PCNA-

Fig. 6. Thymidine incorporation (A) and mitotic counts (B) following 2 MED-UVB irradiation. *P B0.05 compared with control; **PB 0.01 compared with control.

positive cells were significantly higher in the tape-stripping-treated epidermis (Fig. 9A,B). However, there were no significant differences in p53- and p21-positive cells (data not shown). There was no expression of p53 or p21 in the tape-stripped site without the UVB irradiation. No apoptotic cells were detected in the tapestripped site without the UVB irradiation, either.

4. Discussion Fig. 5. Apoptotic (sunburn) cells (arrowheads) and p53-positive cells (asterisks) are distinguishable from each other. Following 2 MED-UVB irradiation, sunburn cells and p53-positive cells were visualized histologically.

We have shown that UVB irradiation induces apoptosis of pig epidermal keratinocytes in vivo (Fig. 1). This was UVB-dose-dependent and was accompanied by a sequential expression of p53

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Fig. 7. PCNA-positive cells following UVB irradiation. Following 2 MED UVB-irradiation, PCNA-positive cells were counted.

and p21CIP1/WAF1 (Figs. 2 – 4). Consistent with p53-dependent nature of p21 gene expression [18,19], the peak of p53 was followed by that of p21 with a delay of about 24 h, that is at 48 h following the irradiation, when the apoptotic cell number revealed its maximum (Figs. 1 – 3). Although these results suggest a close relation of p53 and p21 in inducing apoptosis, the precise contribution of these proteins remains to be determined. Immunohistochemical analyses disclosed that the apoptotic cells and p53- (or p21)-positive cells were not necessarily the same (Fig. 5). It has

Fig. 8. Changes in the proportion of cells in S-phase () and G2/M phase (“) following 2 MED-UVB irradiation. Data are presented as % of control (non-irradiated) epidermis at each time point following the irradiation. *PB 0.05 compared with control; **PB0.01 compared with control.

Fig. 9. Combined effects of tape stripping and UVB irradiation. At 24 h following tape-stripping, 2 MED-UVB irradiation was performed on pig skin. At an appropriate time following the UVB irradiation, apoptotic cells (A) and PCNApositive cells (B) were counted. A: 48 h following the irradiation; B: 24 h following the irradiation. Essentially no apoptotic cells were detected in the control (no tape-stripping, no UVB irradiation) epidermis. PCNA-positive cells were around 50/ cm in the control epidermis. *P B0.01 compared with UVBtreatment alone.

been known that while p53 is significantly involved in apoptosis [27,28], simple restoration of p53 activity in p53-deficient cell lines is insufficient to trigger apoptosis; rather p53 appears to render cells more sensitive to induction of apoptosis by DNA damage [27,29]. Regarding p21, recent evidence indicates that p21 is dispensable for apoptosis [30], suggesting that other p53-inducible genes such as bax are more closely related [31]. Further, p53 is not the sole inducer of p21 expression [32,33]; p21 has been reported to participate

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in terminal differentiation by a mechanism independent of p53 [34,35]. In the present study, while apoptotic cells and p53-positive cells increased with the irradiation dose, p21-positive cells rather decreased at higher irradiation dose (Fig. 4). Functional analyses of other p53-inducible genes, that include bax, gadd45, mdm2, would be required to clarify the nature of p53-dependent apoptosis of keratinocytes [36 – 38]. UVB irradiation has been known to induce keratinocyte proliferation, that is preceded by an initial inhibitory phase [19 – 21]. Consistent with previous reports [39,40], UVB irradiation inhibited cell cycle progression at G1 as well as S and G2/M phases of the cell cycle up until 48 h following the irradiation (Figs. 6 and 8). Interestingly, in contrast to the G1 arrest, the UVB-induced prolonged S phase and G2/M block are reported to be independent of p53 [39,40]. This explains the initial inhibition of mitosis following the UVB irradiation (Fig. 6B), that is observed without the induction of p53 or p21 (Figs. 2 and 3). p53 inhibits G1/S transition through p21, a universal inhibitor of CDK [8,11,39]. p21 also directly binds and inhibits PCNA, a subunit required by DNA polymerase d [41]. p21, thus, by inhibiting CDK and PCNA essential for G1/S transition, provides time for cells to repair DNA damage following the UVB irradiation. This is consistent with the recent finding that UVB irradiation on neonatal rat keratinocytes resulted in a rise in p53 protein levels, in association with induction of p21 [42]. Further, a rise in p21CDK2 inhibitory activity paralleled the loss of G1 cyclin-CDK activity, supporting a role for p21 in the UVB-induced G1 check point. It has been reported that while p53 is induced by DNA damage irrespective of the cell cycle [43], only cells in S phase progress to the apoptotic pathway following the UVB irradiation [2,42]. Thus the cells that escaped from G1 check point or the cells that are already in the S phase at the time of UVB irradiation would progress into the apoptotic pathway; this explains the finding that p53- or p21-positive cells are not necessarily the same as the apoptotic cells (Fig. 5), and consistent with the finding that the numbers of p53- or p21-positive cells are

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considerably more than that of the apoptotic cells (Figs. 1–3). Besides DNA replication, PCNA is known to be involved in DNA repair [26]. While p21 directly binds to PCNA, inhibiting replicative DNA synthesis [41], it does not interfere with the PCNA-dependent DNA excision repair [44,45]. This explains our finding that G1 arrest was detected at 24 h following the irradiation, despite a significant induction of PCNA (Figs. 6 and 7). It seems likely that PCNA functions in DNA repair synthesis during the early phase post-UVB irradiation, where the p53-dependent induction of p21 plays an essential role for the G1 check point. The peak of PCNA-positive cells was observed at 72– 96 h, at the peak of the cell proliferation monitored by thymidine uptake, suggesting its role in DNA replication at this stage post-UVB irradiation. Our results indicate that tape stripping-induced hyperproliferative epidermis shows an increased apoptotic response. Both UVB-induced apoptosis and PCNA-positive cells were markedly increased following the tape-stripping (Fig. 9A,B). This is consistent with the recent finding that cell proliferative activity is closely associated with apoptotic response [46]. Interestingly, the augmentation of apoptosis by tape stripping was not accompanied by that of p53- and p21-positive cell number. This is consistent with the fact that p53 is induced irrespective of the cell cycle [43], and that only a fraction of UVB-induced p53-positive cells enters the apoptotic pathway, reinforcing the view that keratinocytes are sensitive to apoptosis in a specific phase of the cell cycle. Without an alteration of p53-positive cell number, no alteration of p21positive cells would be expected. This again is consistent with the view that p53- or p21-positive cells are not necessarily the same as the apoptotic cells (Fig. 5). Our results indicate that UVB irradiation induces both cell cycle arrest and apoptosis. These provide a G1 check point as well as an elimination mechanism of possibly hazardous cells carrying DNA damage, respectively. Consistent with the previous results [2], apoptotic sunburn cell formation was closely associated with cell cycle progression and the tape stripping-induced hyper-

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proliferation augmented the apoptotic response. It might be suggested that the hyperproliferative epidermis such as psoriasis is more susceptible to UVB-induced apoptosis, providing another rationale for UVB therapy for psoriasis. This is not defying the role of UVB irradiation on the psoriatic intraepidermal T lymphocytes [47], that appear to be more susceptible to UVB than the keratinocytes.

Acknowledgements This study was supported in part by grants 08457233 (HI), 08770618 (HT), 08670940 (AI-Y) and 08670939 (YH) from Ministry of Education, Science, Sports and Culture of Japan and from the Ministry of Health and Welfare, Japan.

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