Shinichi Inohara

Expression of Cyclin D1 and p53 Protein in Various Malignant Skin Tumors

BACKGROUND Overexpression of cyclin D1 and p53 protein has been reported in many types of malignant tumors. OBJECTIVES We investigated whether cyclin D1 was detected immunohistochemically in various types of malignant tumors of the skin, comparable with p53 protein. METHODS Immunohistochemical staining of cyclin D1 and p53 protein was applied to squamous cell carcinoma, malignant melanoma and malignant fibrous histiocytoma and various kinds of benign skin tumors. RESULTS Cyclin D1 was positive only in malignant tumors at the same incidence as p53 protein. CONCLUSION Cyclin D1 immunohistochemical staining may be a malignant marker for various skin tumors.

Coexpression of p21Waf1/Cip1 and p53 in sun‐exposed normal epidermis, but not in neoplastic epidermis

Summary Wild‐type p53 accumulation induced by DNA damaging agents such as ultraviolet (UV) radiation. γ‐irradiation and drugs, may arrest the cell cycle until DNA damage is repaired. p21Waf1/Cip1 is a cyclin‐dependent kinase (CDK) inhibitor induced by wild‐type p53. CDK is activated by cyclin and progresses the cell cycle. On the other hand. CDK inhibitors inhibit CDK activity to arrest the cell cycle. Thus, p21Waf1/Cip1 is thought to mediate the signal of p53 induced by DNA damaging agents to arrest the cell cycle. p21Waf1/Cip1 is induced by wild‐type, but not mutant p53. To investigate p21Waf1/Cip1 regulation by p53 in epidermis in vivo, immunohistochemical staining of p21Waf1/Cip1 and p53 were conducted in chronically sun‐exposed normal epidermis and in neoplastic epidermis. p21Waf1/Cip1 expression was found to be coincident with the p53‐positive regions or not coincident with the p53‐positive regions in chronically sun‐exposed normal epidermis, whereas there was only low or undetectable p21Waf1/Cip1 expression in any regions including the p53‐positive regions of solar keratosis and squamous cell carcinoma of the skin. This suggests that wild‐type p53 and p21Waf1/Cip1 may play a part in chronically sun‐exposed normal epidermis response to UV exposure, whereas p21Waf1/Cip1 cannot be induced by mutated p53 in solar keratosis and squamous cell carcinoma of the skin.

Effects of protein kinase C activators on mouse skin in vivo

Tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) is known to have many biological effects on epidermal cells [14]. TPA activates protein kinase C directly both in vivo and in vitro [3], and protein kinase C is the receptor of TPA [5, 8]. Therefore, it is possible that biological effects of TPA on epidermis are mediated by protein kinase C, which is also known to be activated by diacylglycerol generated from phosphoinositides of cell membrane [9]. It has been reported that synthetic diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG), as well as TPA, is able to penetrate cell membrane and activate protein kinase C in intact cells [1, 4, 9]. OAG has some biological effects on epidermal cells as does TPA [7, 12]. We investigated marcoand microscopic changes that could be induced by topical application of OAG, as compared with application of TPA. Dorsal skin (6 cm z) of Balb/c and Swiss-Webster mice aged 10 weeks were shaved with an electric shaver so as to avoid injury. TPA (20 gg) and OAG (40 rag) were each dissolved in 100 ~tl acetone. Each 100-1~1 acetone solution was applied with a microsyringe onto the shaved back skin of the mice. In consideration of previous reports [7, 11] these concentrations of TPA and OAG could be thought to be effective biologically on epidermal cells; 100 lal of acetone only was used as control. Five mice were used per group. The mice were treated with each solution once weekly for 3 weeks. They were killed 1 week after the last treatment, and the skins were taken and stained with HE. The hair regrowth patterns in each group of Balb/c mice are shown in Fig. 1 a c . Hair regrowth

Immunohistological identification of protein kinase C isozymes in normal and psoriatic epidermis.

Protein kinase C (PKC), the Ca 2 + and phospholipiddependent protein kinase, is activated by diacylglycerol, which is generated from inositol phospholipids of plasma membrane, and it is recognized that PKC functions as a transducer in the transmembrane signal transduction system [7]. PKC is also known to be activated by tumor promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA), and it is a receptor protein of TPA [7]. Since it is well known that TPA has striking effects on epidermal proliferation and differentiation, it is possible that PKC has an important role in the regulation of epidermal function [5, 8]. It has been reported that PKC is present in human epidermis [1] and that its activity is lower in psoriatic epidermis than in epidermis from a normal healthy person [3]. Therefore, abnormal proliferation and differentiation of psoriatic epidermis may be relevant to alterations in PKC-mediated processes due to decreased PKC activity. Recently, at least three different isozymes types I (7), II (/~), and III (c 0 of PKC have been identified biochemically [6]. In the present study, localization of PKC isozymes was examined in normal epidermis from a healthy person as well as in both involved and uninvolved epidermis from a psoriatic person using immunohistological study. Skin biopsy specimens were obtained from trunks of three healthy and three psoriatic persons aged 4 0 6 0 years old. The biopsy specimens were immediately snap-frozen in liquid nitrogen, and stored at 7 0 ~ until used. Serial 5-gin

Actin filament and desmosome formation in cultured human keratinocytes

Cytoskeletal systems have an essential role in the maintenance of cell shape. It has also been reported that they have important roles in various cell functions, such as growth, differentiation, and cell to cell contact. The presence of actin in keratinocytes has been confirmed in previous reports [4, 5], as has the presence of other cytoskeletal proteins (keratin and tubulin). The culture of keratinocytes at a reduced concentration of Ca 2 + prevents desmosome formation. Raising the concentration of Ca 2 + in the medium induces desmosome formation [2]. Recently it was reported that actin filaments developed sooner than desmoplakin (one of the desmosomal proteins) and keratin filaments developed along the cell membrane of mouse keratinocytes after the addition of Ca 2+ into the low Ca 2+ medium [1]. These findings indicate that actin filaments may play a crucial role in desmosome formation in cultured mouse keratinocytes. Using phase-contrast, immunofluorescence, and electron microscopic studies, we investigated whether cytochalasin B (CB), which induces the disruption of actin filaments, affected Ca 2 +-induced desmosome formation in cultured human keratinocytes. Human keratinocytes were obtained from foreskin and subcultured in serum-free modified MCDB 153 supplemented with epidermal growth factor (10 ng/ml), insulin (5 gg/ml), hydrocortisone (0.5 gg/ml), and bovine pituitary extracts (150 tag/ml). The calcium concentration was 0.15 raM. The distribution of actin filaments and desmoplakin was examined in keratinocytes cultured in low Ca 2 + medium and after 24 h culture following the addition of 1.2 mM Ca 2 +. In order to observe the effects of CB on CaZ+-induced desmosome formation, cells grown in low Ca 2 + medium were exposed to CB for 2 h before the addition of Ca 2 +. CB was dissolved in DMSO

Immunohistochemical detection of cyclin D and cyclin A in human hyperproliferative epidermis.

Cyclin was first identified in sea urchin eggs as a protein that is synthesized prior to repeated cell division alter fertilization, and it disappears rapidly on completion of cell division [3]. Cyclin, when combined with cdc2, activates cdc2 kinase which then phosphorylates various proteins to regulate the cell cycle [10]. Such a mechanism is common to all eukaryotic cells [ 11 ]. Subtypes exist for both cyclin and cdc2 in mammalian cells and the advance of the cell cycle is controlled by the combination of these subtypes [13]. Five cyclin subtypes (A to E) have so far been identified. These cell cycle proteins are thought to be abnormal in carcinoma cells. It has been suggested that cyclin A and cyclin D may be oncogenic proteins. In one case of hepatocellular carcinoma, it was reported that the hepatitis B virus gene is inserted into the intron of the cyclin A gene [14]. The cyclin D gene was first reported as a gene (PRAD1) that is activated by chromosome inversion in cells of parathyroid tumours [9]. At about the same time, it was reported as a gene (CYL) that is induced in the G1 phase by stimulation of the growth factor specific to macrophages [8] and also as a human gene (cyclin D gene) that can rescue G1 phase-deficient yeast cells [7, 16]. Furthermore, it has been reported that the cyclin D gene is overexpressed by chromosome translocation or gene amplification in B cell lymphoma [15], squamous cell carcinoma of the head and neck [6], mammary carcinoma [6] and oesophageal cancer [5]. These findings indicate that cyclin D may be an oncogenic cyclin. In the present study, we examined the immunohistochemical localization of cyclin D and cyclin A in normal human epidermis and various epidermal hyperproliferative diseases including psoriasis vulgaris, seborrhoeic

Diacylglycerol Mimics Phorbol Ester Induction of Morphological Changes in Low Ca2+ Regulated Epidermal Cells

It is known that tumor‐promoting phorbol ester, 12‐O‐tetradecanoylphorbol‐13‐acetate, induces morphological changes in mouse epidermal cells cultured in low Ca2+ medium. In the present study, we found on phase‐contrast microscopic examination that synthetic diacylglycerol, l‐oleoyl‐2‐acetylglycerol, induced similar morphological features. Ultrastructural examination revealed that both materials used for treatment induced morphological changes, such as cell‐to‐cell contact. These two materials are known to directly activate protein kinase C in intact cells. On the other hand, cyclic AMP‐dependent protein kinase activating agents induced no morphological changes in the same culture system. Data obtained in this experiment suggest that the morphological changes brought about by 1‐oleoyl‐2‐acetylglycerol in this report may occur via the action of diacylglycerol on the protein kinase C system.

Cell cycle regulators in the keratinocyte (cyclin-cdk)

Abstract It has recently become clear that cyclin‐dependent kinase (cdk) complex regulates the cell cycle by phosphorylating Rb protein, a tumor suppressor protein. It is likely that this complex is a target of various growth factors and anti‐growth factors (UV TGF‐β etc.) in keratinocyte (KC). It has also been suggested that abnormalities in the cell cycle regulating mechanism such as increased activity of cyclin‐cdk due to mutation of p53, a tumor suppressor gene, and overexpression of cyclin D may be concerned with carcinogenesis of KC. Thus, recent studies indicate that the cyclin‐cdk complex is a common target of proliferation and carcinogenesis in KC.

IMMUNOHISTOLOGIC LOCALIZATION OF RAS P21 IN NORMAL, HYPERPLASTIC, AND NEOPLASTIC EPIDERMIS

Background. Ras p21, a ras oncogene product, plays an important role in tumorigenesis, proliferation, and differentiation in various tissues and cells.

Flow Cytometric Analysis of Protein Kinase C in Lymph Node Cells during the Induction Phase of Contact Hypersensitivity Reaction

On the fourth day after a single painting of 2,4,6‐trinitro‐chlorobenzene on the abdominal skin, inguinal lymph nodes were removed, and a single‐cell suspension was prepared. The cells were analyzed flow cytometrically, using monoclonal anti‐protein kinase C antibody. It was found that the number of lymph node cells in which protein kinase C was detected on the cell surface was significantly increased over that in non‐treated mice (p<0.01). On the basis of our results and discussions in the literature, it is thought that protein kinase C is related to the initiation of the contact hypersensitivity reaction.