２２回日本研究皮膚科学会総会・抄録

２２回日本研究皮膚科学会総会・抄録

-30- Apoptosis 9/4 Room-C
INDUCTION OF APOPTOSIS BY HEAT STRESS IN GUINEA PIG SKIN.
Makoto Nagata, Ryo Shibagaki, Hideya Takenaka, Saburo Kishimoto, Hirokazu Yasuno.
Department of Dermatology, Kyoto Prefectual University of Medicine, Kyoto, Japan.
Unwanted cells are eliminated by apoptosis, when cells are damaged. In this study, apoptosis was examined in the experimental burn wounds under some severities of heat stress by TUNEL staining. Burn wounds, 1cm in diameter, were made on thighs of guinea pigs by electorical iron for 5-60 min at 43℃, 5-60 min at 45℃, 60 min at 55℃, and 3 sec at 180℃, respectively. Skin samples were excised at day2 after burn. Apoptosis was seen through epidermis to lower dermis in central region of the wound at 43℃, 5-20 min. Necrosis was seen in central region of the wound but apoptosis was seen in peripheral region of the wound at the other conditions. Intermediate damage(43℃, 5-20 min) induces apoptosis, that seems to be second burn, and more damage induces necrosis in guinea pig skin.

-31- Apoptosis 9/4 Room-C
THE EFFECTS OF COOLING ON APOPTOSIS SEEN IN SECOND-DEGREE BURNED SKIN OF GUINEA PIGS.
Ryo Shibagaki, Saburo Kishimoto, Hideya Takenaka, Makoto Nagata, Hirokazu Yasuno.
Department of Dermatology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
The local application of cold to burned areas is recommended as first-aid emergency management. The aim of the present study was to analyze the effects of topical cooling on the number of apoptotic cells in early stage of burns. A second-degree burn was made on the skin of guinea pigs by a heated iron at 43℃for 5min. The animals were divided into 5 groupes. Four groups were treated with application of cold water (4℃) immediately after burn for 5min., 10min., 20min. and 30min., respectively. Specimens were excised at 48 hours postburn, and processed for TUNEL method. We counted apoptotic index (number of TUNEL-positive cells / total number of cells) both in the epidermis and anagen hair follicles, and evaluated these values statistically among each group. In terms of the hair bulb of anagen hairs, apoptotic index in 30 min.-treated group was significant compared with those in the control group. Our findings demonstrated that topical cooling on burned surface decreased apoptotic cells of the hair bulb, and supported the clinical efficacy for cooling in burned wound.

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ACTIVE FORM OF VITAMIN D3 INDUCES APOPTOSIS IN SEBORRHEIC KERATOSIS.
Yoshihiko Mitsuhashi, Takehiko Aoki, Hiroshi Sugiki, Yutaka Hozumi and Sigeo Kondo.
Department of Dermatology, Yamagata University School of Medicine, Yamagata, Japan.
We found that a long term application of active vitamin D3 (tacalcitol) cleared seborrheic keratosis in about half case of the patients. To elucidate the pathomechanism, we carried out an organ culture experiment. Resected seborrheic keratoses were used as materials for rotation organ culture. In culture medium, 0, 10-8 , 10-7,10-6 or 10-5 M of tacalcitol was added. After the culture for 3days, the materials were offered for histopathological obser- vation with H & E staining and immunopathological observation. In higher concentrations of tacalcitol (10-5 M or 10-6M), we found degenerative changes of the tumor. The immunopathological examination to detect Nick ends of fragmented DNA revealed numerous positive cells when tacalcitol was added in any concentrations, in spite of only sporadic positive cells in the control group. Our results suggest that avtive vitamin D3 may induce apoptosis in seborrheic keratosis.

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INTERLEUKIN-1b-CONVERTING ENZYME (ICE) IS INVOLVED IN ULTRAVIOLET B-INDUCED APOPTOSIS OF SVHK CELLS.
Hidetoshi Takahashi, Hajime Iizuka.
Department of Dermatology, Asahikawa Medical College, Asahikawa, Japan.
Interleukin-1β-converting enzyme (ICE) and CPP32 are cysteine proteinases, that are involved in apoptotic process in various cell systems. We investigated the effects of ICE on ultraviolet B(UVB) induced-apoptosis in SV40 transformed human keratinocytes (SVHK cells). The ICE inhibitor (Z-Val-Ala-Asp-CH2F) and CPP32 inhibitor (Z-Asp-Glu-Val-Asp-CH2F) blocked the apoptotic cell death caused by UVB irradiation. The addition of both ICE inhibitor and CPP32 inhibitor to the incubation medium resulted in neither an additive nor a synergistic suppression of UVB-induced apoptosis. Reverse transcription and polymerase chain reaction (RT-PCR) analysis indicated that SVHK cells expressed ICE-α, and β mRNAs. UVB irradiation increased the mRNA of both isoforms and Western blot analysis confirmed that UVB increased an active form of ICE protein, p20, that is generated by autoproteolytic cleavage of inactive 45kDa proenzyme derived from both mRNAs. Transfection of ICE expression vector into SVHK cells resulted in apoptosis in a dose dependent manner and UVB-irradiation further augmented the ICE expression vector-induced apoptosis. These results indicate that ICE plays an important role on UVB-induced apoptosis of SVHK cells.

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P53 MAY NOT BE A PREREQUISITE FOR THE UVB INDUCED APOPTOSIS IN HUMAN KERATINOCYTES in vitro.
Fumio Washio, Toshinori Bito, Masato Ueda, Masamitsu Ichihashi.
Department of Dermatology, Kobe University School of Medicine, Kobe, Japan.
One of the morphological features of sun-exposed epidermis is the 'sunburn cell', which is presumed to be an apoptotic cell. The role of the p53 tumor suppressor protein in apoptosis has attracted much attention recently, since p53 knock-out mice were shown to form much less number of the sunburn cells . To further know the molecular mechanisms of UVB-induced apoptosis in skin, we analyzed the apoptotic phenotypes and the expression of apoptosis related molecules in human keratinocyte cell lines. Both HaCaT and HSC1 cells which carry mutant p53 showed DNA ladder formation and nuclear condensation, the characteristics of apoptosis, after UVB exposure. p53 and p21 protein expression did not alter by UVB exposure in these cells, while Bcl-2 expression appears to be down-regulated. In contrast, normal human keratinocytes were resistant to the induction of apoptosis by UVB although p53 protein expression was induced. These data may indicate that p53 may not be a prerequisite for UVB-induced apoptosis of human keratinocytes.

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CERAMIDE-INDUCED APOPTOSIS IN A CULTURED CELL LINE, HSC-1 DERIVED FROM HUMAN EPIDERMIS.
Hiroshi Sugiki, Yutaka Hozumi, Yoshihiko Mitsuhashi, Shigeo Kondo.
Department of Dermatology, Yamagata University School of Medicine, Yamagata, Japan.
It is known that ceramide is involved in the cell proliferation, differentiation, and induction of apoptosis in the leukemic cells etc. In this study, we have examined whether ceramide inhibits the cell proliferation and induces apoptosis in the epidermal cell-derived cells as well. An established cell-line, HSC-1 derived from human epidermis was treated with C2-ceramide or vehicle alone as control. The morphological changes of the ceramide treated cells were observed, the alamar Blue Assay as a cell viability test was performed, and DNA electrophoresis was done. Morphological study revealed the remarkable cytoplasmic and nuclear shrinkage and chromatin condensation in the ceramide treated cells; the alamar Blue Assay showed ceramide inhibited the cell proliferation in a dose-dependent manner; and DNA ladder was found in the ceramide treated cells. These results show that C2-ceramide inhibits the cell proliferation and induces apoptosis in HSC-1 derived from the epidermis.