Motoki Nakamura

Necessary and Sufficient Role for a Mitosis Skip in Senescence Induction

Senescence is a state of permanent growth arrest and is a pivotal part of the antitumorigenic barrier in vivo. Although the tumor suppressor activities of p53 and pRb family proteins are essential for the induction of senescence, molecular mechanisms by which these proteins induce senescence are still not clear. Using time-lapse live-cell imaging, we demonstrate here that normal human diploid fibroblasts (HDFs) exposed to various senescence-inducing stimuli undergo a mitosis skip before entry into permanent cell-cycle arrest. This mitosis skip is mediated by both p53-dependent premature activation of APC/C(Cdh1) and pRb family protein-dependent transcriptional suppression of mitotic regulators. Importantly, mitotic skipping is necessary and sufficient for senescence induction. p16 is only required for maintenance of senescence. Analysis of human nevi also suggested the role of mitosis skip in in vivo senescence. Our findings provide decisive evidence for the molecular basis underlying the induction and maintenance of cellular senescence.

Role of the aryl hydrocarbon receptor in tobacco smoke extract–induced matrix metalloproteinase‐1 expression

Findings from large epidemiologic studies indicate that there is a link between smoking and extrinsic skin ageing. We previously reported that matrix metalloproteinases (MMPs) mediate connective tissue damage in skin exposed to tobacco smoke extracts. Tobacco smoke contains more than 3800 constituents, including numerous water‐insoluble polycyclic aromatic hydrocarbons (PAHs) that trigger aryl hydrocarbon receptor (AhR) signalling pathways. To analyse the molecular mechanisms involved in tobacco smoke–induced skin ageing, we exposed primary human fibroblasts and keratinocytes to tobacco smoke extracts. Hexane‐ and water‐soluble tobacco smoke extracts significantly induced MMP‐1 mRNA in both human cultured fibroblasts and keratinocytes in a dose‐dependent manner. To clarify the involvement of the AhR pathway, we used a stable AhR‐knockdown HaCaT cell line. AhR knockdown abolished the increased transcription of the AhR‐dependent genes CYP1A1/CYP1B1 and MMP‐1 induced by either of the tobacco smoke extracts. Furthermore, the tobacco smoke extracts induced 7‐ethoxyresorufin‐O‐deethylase activity, which was almost completely abolished by AhR knockdown. Likewise, treating fibroblasts with AhR pathway inhibitors, that is, the flavonoids 3‐methoxy‐4‐nitroflavone and α‐naphthoflavone, blocked the expression of CYP1B1 and MMP‐1. These findings suggest that the tobacco smoke extracts induce MMP‐1 expression in human fibroblasts and keratinocytes via activation of the AhR pathway. Thus, the AhR pathway may be pathogenetically involved in extrinsic skin ageing.

Environment-induced lentigines: formation of solar lentigines beyond ultraviolet radiation

There is no doubt that ultraviolet radiation (UVR) contributes to the generation of acquired lentigines in human skin, as indicated by the term solar lentigo. A growing number of recent epidemiological and mechanistic studies, however, strongly suggest that in addition to UVR, other environmental factors contribute to lentigines’ formation as well. We therefore here introduce the term ‘environment‐induced lentigo’ (EIL) to refer to acquired pigment spots of human skin. In this view point, we (i) summarize the existing evidence to support a role of environmental toxicants other than UVR in the pathogenesis of EILs, (ii) we argue that activation of aryl hydrocarbon receptor (AHR) signalling by UVR and environmental toxicants is critically involved in triggering and sustaining a crosstalk between melanocytes, keratinocytes and fibroblasts, which then causes the development and persistence of EILs in human skin, and (iii) we discuss clinical implications for the prevention and treatment of EILs resulting from this concept.

Tobacco smoke-induced skin pigmentation is mediated by the aryl hydrocarbon receptor.

It is widely recognized that tobacco smoke causes skin pigmentation. No studies, however, have directly evaluated the mechanisms of the changes in smokers skin pigmentation. In this study, when cultured with water‐soluble tobacco smoke extract, the human epidermal melanocytes grew to a large size and produced more melanins. We evaluated melanocyte activation by quantifying microphthalmia‐associated transcription factor (MITF) expression by real‐time polymerase chain reaction. MITF expression was significantly and dose‐dependently increased by exposure to tobacco smoke extract. The Wnt/β‐catenin signalling pathway seemed to mediate the tobacco smoke extract–induced melanocyte activation. Immunocytochemical studies revealed that the activated melanocytes actively expressed aryl hydrocarbon receptors (AhR) around the nuclear membrane. The tobacco smoke extract–induced MITF activation was inhibited by RNA silencing of the AhR. This study provides the evidence that tobacco smoke enhances pigmentation in vitro and that the increase in pigmentation may involve β‐catenin‐ and AhR‐mediated mechanisms inside human melanocytes.

Efficacy of 1‐mm minigrafts in treating vitiligo depends on patient age, disease site and vitiligo subtype

Vitiligo vulgaris is a refractory skin disease. Treatment modalities include topical steroids, phototherapy, suction blister roof grafts and cellular grafting techniques. Adverse effects may occur, however, and some cases remain unresponsive to treatment. To evaluate the efficacy of small (1‐mm) punch minigraft therapy in relation to patient age, disease site, disease duration and vitiligo subtype. We used a recently developed disposable 1.0‐mm punch apparatus to perform minigraft therapy in 20 patients with either generalized (n = 4), segmental (n = 9) or limited (n = 7) vitiligo, and evaluated the area and rate of repigmentation in relation to patient age, disease site, disease duration and vitiligo subtype. The area of repigmentation was significantly greater in patients with segmental vitiligo (n = 9) than in those with generalized vitiligo (n = 4). Repigmentation covered a broader area and occurred more quickly in patients under 15 years of age than in those over 20 years of age (n = 9). Disease duration did not affect the repigmentation rate. The results of the present study suggest that 1‐mm minigrafts are effective for treating patients with vitiligo. Better results occurred in patients under 15 years of age, patients with facial grafts, and patients with segmental and limited subtypes.

Establishment of suction blister roof grafting by injection of local anesthesia beneath the epidermis : Less painful and more rapid formation of blisters

The number of patients suffering from diabetic foot ulcers [1] and rheumatic ulcers [2] is increasing rapidly. Vitiligo is an acquired depigmented skin disorder that results from the destruction of melanocytes with unknown etiology. Suction blisters were first used for the transplantation of viable epidermis in vitiligo lesions and chronic wounds [3], followed by a simplified modification to form blisters [4]. We have previously reported the usefulness of epidermal sheet grafts to treat intractable skin ulcers caused by autoimmune diseases [2], diabetes mellitus [1] or other diseases [5,6] through their unique actions that differ from conventional skin grafts that include dermal components [7,8]. The technique to use syringes and three-way stopcocks to form suction blisters is comparatively simple and conservative, but it has two major problems. One is the time required to make blisters; Gupta and Kumar reported that as long as 78, 98 and 123 min are required to generate suction blisters when 5, 10 and 20 ml syringes are used, respectively [9], which is one reason why this simple technique is not used more commonly to treat chronic wounds. The other problem is the pain caused by the procedure; negative pressure at donor sites sometimes causes pain, which imposes a great burden on the patient. The original method [3] and its modifications [4,9] do not use local anesthesia except for a case report [10], which did not emphasize the impor-

NB-UVB irradiation increases filaggrin expression in a three-dimensional human skin model

Ultraviolet (UV) B irradiation affects the immune system and is ed for therapeutic purposes. The mechanisms underlying the fects of UVB phototherapy are thought to include the induction of NB-UVB, and broadband UVB (BB-UVB); both Toshiba Medical Supply, Tochigi, Japan) to irradiate the cells. At 24 h after UVB irradiation, total RNA was extracted using an RNeasy mini kit (Qiagen, Hilden, Germany), and examined FLG, IVL and LOR mRNA expression. At 72 h after UVB irradiation, the skin model was fixed in 20% buffered formalin and embedded in paraffin. Tissue sections were deparaffinized and rehydrated by sequential immersion in xylene followed by graded descending co in m bo an PB tis co w re co se

Monotherapy for vitiligo using a 308‐nm xenon‐chloride excimer laser: Colorimetric assessment of factors that influence treatment efficacy

Dear Editor, The excimer laser selectively treats a small area without unnecessarily exposing normal skin to radiation. Several studies have confirmed the efficacy of excimer laser monotherapy or combination therapy for vitiligo, but their efficacy for recalcitrant vitiligo is unclear. The excimer laser is a more potent inducer of T-cell apoptosis than narrowband (NB) ultraviolet (UV)-B, and many cytokines are associated with melanocyte migration and DNA synthesis stimulation in melanocytes. We previously reported that endothelin-1, an intrinsic melanogen or mitogen for human melanocytes in UV-B-induced pigmentation, is induced more prominently by excimer laser than other UV-B light sources. In the present study, we evaluated the efficacy of 308-nm excimer laser monotherapy in 45 patients with 157 vitiligo lesions for up to 50 treatment sessions, which is a greater number of treatments than that given in previous studies. In addition, we assessed the minimal erythema dose (MED) and skin color and determined the factors that contribute to a positive response to excimer laser treatment. None of the patients received any form of phototherapy in the 4 weeks before study entry. Lesions were grouped: group 1, face and neck; group 2, acral; and group 3, other. Vitiligo patches were treated twice weekly with a 308-nm xenon-chloride excimer laser (XTRAC Ultra Laser; Photomedex, San Diego, CA, USA). Percentage of pigmentation of the treated areas was scored 0–5 as follows: 0, 0%; 1, 1–5%; 2, 6–25%; 3, 26–50%; 4, 51–75%; and 5, 76–100%. The end-point was 50 treatments or when the repigmentation score was 5. The initial treatment was 1 MED. In this study, the skin color of the vitiligo patch (>8 mm in diameter) was measured using the non-invasive colorimetric method with a chromameter (CM-200; Konica Minolta, Osaka, Japan). In colorimetric study in Asians, the melanin index was correlated negatively with L* (lightness) value, and positively with a* (redness) and b* (darkness) values. The erythema index has a positive correlation with a* value. Skin color measurement was performed on the largest macule in the treated lesions. For measurement of skin color of treated vitiligo, cases that showed only follicular pigmentation were omitted. Some repigmentation occurred within the first 10 treatment sessions in 99 of the 154 lesions (65%). Repigmentation was observed in 100% of the patients in groups 1 and 3. The mean total treatment doses were 12.6 J/cm (group 1), 31.9 J/cm (group 2) and 20.07 J/cm (group 3). Representative cases are shown in Figure 1. Although the repigmentation scores in (a)

Case of annular-shaped giant basal cell carcinoma.

Dear Editor, A 68-year-old Japanese male patient was referred to our hospital because of a tumor on his left chest. It initially appeared as a small, brown macule and began to grow gradually over 20 years. The tumor was 11 cm · 7 cm, and comprised a red plaque and a surrounding brown outer edge, with an erythematous area in-between. Dermoscopy revealed multiple blue-gray globules with arborizing telangiectasia in the red plaque (Fig. 1). Computed tomography showed the tumor partly extending into the subcutaneous fat layer, but no abnormality was seen in other areas. Serum squamous cell carcinoma antigen levels were normal. Preliminary biopsy study revealed basal cell carcinoma (BCC). We diagnosed the lesion as BCC limited to the left thoracic wall skin and subcutaneous fat layer. Surgical excision of the tumor and split-thickness skin grafting were performed. We planned the excision to be 2 cm from the edge of the red plaque and 1 cm from the brown outer edge (Fig. 2a). Histopathological examination showed nests of basophilic cells in the red plaque (Fig. 2b). The islands of tumor cells comprised peripheral palisading cells, revealing a nodular type of BCC (Fig. 2e). The erythematous area had no evident tumor cells (Fig. 2d). The

A multilayered polyurethane foam technique for skin graft immobilization.

BACKGROUND Several techniques are applicable for skin graft immobilization. Although the sponge dressing is a popular technique, pressure failure near the center of the graft is a weakness of the technique that can result in engraftment failure. OBJECTIVE To evaluate the efficacy of a new skin graft immobilization technique using multilayered polyurethane foam in vivo and in vitro. METHODS AND MATERIALS Twenty‐six patients underwent a full‐thickness skin graft. Multiple layers of a hydrocellular polyurethane foam dressing were used for skin graft immobilization. In addition, we created an in vitro skin graft model that allowed us to estimate immobilization pressure at the center and edges of skin grafts of various sizes. RESULTS Overall mean graft survival was 88.9%. In the head and neck region (19 patients), mean graft survival was 93.6%. Based on the in vitro outcomes, this technique supplies effective pressure (<30 mmHg) to the center region of the skin graft. CONCLUSIONS This multilayered polyurethane foam dressing is simple, safe, and effective for skin graft immobilization. The authors have indicated no significant interest with commercial supporters.