Chemoprevention of basal cell carcinomas in the ptc1+/- mouse--green and black tea.

Skin cancers are a rising menace as their incidence increases, attributed in part to increasing ultraviolet radiation exposure. This increasing problem has stimulated efforts to devise useful preventive approaches. The uncertain efficacy of exhortations to avoid sun exposure and to use protective clothing and sunscreens to reduce damage when exposed argue for the development of an oral chemopreventive agent. Bickers and others have studied the effects and mechanisms of tea and of its putative active components on inhibition of skin cancer in experimental models. To continue this work, we have studied the effects of oral green tea and black tea on a new model of ultraviolet-induced skin carcinogenesis - the development of basal cell carcinomas in ptc1+/- mice. To our surprise, we have found that tea preparations which others have used to prevent squamous cell carcinoma formation in mice fail to inhibit basal cell carcinogenesis in our model, suggesting that prevention of this cancer may require special, tumor-specific approaches.

A role of PDGFRalpha in basal cell carcinoma proliferation.

Activation of the hedgehog pathway, through the loss of patched (PTC) or the activation of smoothened (SMO), occurs frequently in basal cell carcinoma (BCC), the most common human cancer. However, the molecular basis of this neoplastic effect is not understood. The downstream molecule Gli1 is known to mediate the biological effect of the pathway and is itself up-regulated in all BCCs. Gli1 can drive the production of BCCs in the mouse when overexpressed in the epidermis. Here we show that Gli1 can activate platelet-derived growth factor receptor alpha (PDGFRalpha) in C3H10T(1/2) cells. Functional up-regulation of PDGFRalpha by Gli1 is accompanied by activation of the ras-ERK pathway, a pathway associated with cell proliferation. The relevance of this mechanism in vivo is supported by a high level expression of PDGFRalpha in BCCs of mice and humans. In the murine BCC cell line ASZ001, in which both copies of the PTC gene are inactivated, DNA synthesis and cell proliferation can be slowed by re-expression of PTC, which down-regulates PDGFRalpha expression, or by downstream inhibition of PDGFRalpha with neutralizing antibodies. Therefore, we conclude that increased expression of PDGFRalpha may be an important mechanism by which mutations in the hedgehog pathway cause BCCs.

Ultraviolet B(UVB)-induced cox-2 expression in murine skin: an immunohistochemical study.

Cyclooxygenase (COX) is the rate-limiting enzyme in the production of prostaglandins from arachidonic acid. This enzyme exists in at least two isoforms, COX-1 and COX-2. COX-1 is constitutively expressed in most tissues and plays various physiological roles. However, COX-2 expression is induced by a variety of agents, which include pro-inflammatory agents and mitogens. Evidence exists to indicate that increased expression of COX-2 occurs in several types of epithelial neoplasms. In this study, we show the effect of chronic exposure of murine skin to carcinogenic UVB on cutaneous COX-2 expression. SKH-1 mice were irradiated with 180 mJ/cm(2) UVB daily for five days a week for periods ranging from 1 to 20 weeks. Nontumor bearing skin areas of irradiated mice, skin of age-matched controls and benign papillomas and malignant tumors were assessed immunohistochemically for COX-2 expression in these mice. No epidermal staining occurred in any of the non-UVB-treated controls throughout the experiment. Epidermal COX-2 expression only occurred in UVB-irradiated mice. After 1 and 5 weeks of irradiation, patchy epidermal staining mostly confined to the granular layer and stratum corneum was observed. At week 9, staining intensity had increased, particularly in the granular layer. At week 13, staining was uniformly seen in all epidermal layers with particular prominence in the basal cell layer underlying areas of visible epidermal hyperplasia. It is of interest that the most intense staining was seen in the perinuclear region of keratinocytes and at the plasma membrane. At week 20, COX-2 staining was predominant in the granular layer, although in some tissue sections, the entire epidermis was positive. In benign papillomas, staining was confined to the superficial layers of the epidermis and in squamous cell carcinomas (SCCs), patchy staining in the granular and spinous layers predominated. In general, COX-2 expression was more intense in well-differentiated SCCs than in papillomas. In summary, our results indicate that COX-2 serves as an early marker of epidermal UVB exposure and its expression increases in benign papillomas and in SCCs. These results suggest that pharmacological intervention using specific COX-2 inhibitors could have anticarcinogenic effects in UVB-induced human skin cancer.

Ultraviolet and ionizing radiation enhance the growth of BCCs and trichoblastomas in patched heterozygous knockout mice.

Basal cell carcinomas, the commonest human skin cancers, consistently have abnormalities of the hedgehog signaling pathway and often have PTCH gene mutations. We report here that Ptch+/- mice develop primordial follicular neoplasms resembling human trichoblastomas, and that exposure to ultraviolet radiation or ionizing radiation results in an increase in the number and size of these tumors and a shift in their histologic features so that they more closely resemble human basal cell carcinoma. The mouse basal cell carcinomas and trichoblastoma-like tumors resemble human basal cell carcinomas in their loss of normal hemidesmosomal components, presence of p53 mutations, frequent loss of the normal remaining Ptch allele, and activation of hedgehog target gene transcription. The Ptch mutant mice provide the first mouse model, to our knowledge, of ultraviolet and ionizing radiation-induced basal cell carcinoma-like tumors, and also demonstrate that Ptch inactivation and hedgehog target gene activation are essential for basal cell carcinoma tumorigenesis.

Ultraviolet radiation mutagenesis of hedgehog pathway genes in basal cell carcinomas.

The identification of mutations in Hedgehog (HH) pathway genes in some basal cell carcinomas (BCC) and the detection of HH pathway dysregulation in almost all BCC confirms the importance of this developmental regulatory pathway in human BCC tumorigenesis. Moreover, the occurrence of UVB signature mutations in key HH pathway genes in BCC provides the first genetic evidence that UV radiation (UVR) may be the principal mutagen involved in BCC tumorigenesis. We review herein current advances in the understanding of the role of the HH pathway in BCC tumorigenesis including transgenic and knock-out animal models of HH pathway dysregulation. Furthermore, we summarize abnormalities in other tumor suppressors and oncogenes including ras and p53 and evidence for interactions between these regulatory genes and the HH pathway.

Identification of mutations in the human PATCHED gene in sporadic basal cell carcinomas and in patients with the basal cell nevus syndrome.

Mutations in PATCHED (PTC), the human homolog of the Drosophila patched gene, have been identified in most exons of the gene in patients with the basal cell nevus syndrome and in sporadic basal cell carcinomas. We have screened the 23 PTC exons for mutations using single strand conformation polymorphism analysis of DNA from 86 basal cell nevus syndrome probands, 26 sporadic basal cell carcinomas, and seven basal cell nevus syndrome-associated basal cell carcinomas. This screen identified mutations located in eight exons in 13 of the basal cell nevus syndrome patients and in three of the tumors. The most common mutations were frameshifts resulting in premature chain termination. These results provide further evidence for the crucial role of PTC as a tumor suppressor in human keratinocytes.

Glucocorticoids accelerate fetal maturation of the epidermal permeability barrier in the rat.

The cutaneous permeability barrier to systemic water loss is mediated by hydrophobic lipids forming membrane bilayers within the intercellular domains of the stratum corneum (SC). The barrier emerges during day 20 of gestation in the fetal rat and is correlated with increasing SC thickness and increasing SC lipid content, the appearance of well-formed lamellar bodies in the epidermis, and the presence of lamellar unit structures throughout the SC. Because glucocorticoids accelerate lung lamellar body and surfactant maturation in man and experimental animals, these studies were undertaken to determine whether maternal glucocorticoid treatment accelerates maturation of the epidermal lamellar body secretory system. Maternal rats were injected with betamethasone or saline (control) on days 16-18, and pups were delivered prematurely on day 19. Whereas control pups exhibited immature barriers to transepidermal water loss (8.16 +/- 0.52 mg/cm2 per h), glucocorticoid-treated pups exhibited competent barriers (0.74 +/- 0.14 mg/cm2 per h; P < 0.001). Glucocorticoid treatment also: (a) accelerated maturation of lamellar body and SC membrane ultrastructure; (b) increased SC total lipid content twofold; and (c) increased cholesterol and polar ceramide content three- to sixfold. Thus, glucocorticoids accelerate the functional, morphological, and lipid biochemical maturation of the permeability barrier in the fetal rat.

Preservation of permeability barrier ontogenesis in the intrauterine growth-retarded fetal rat.

The epidermal permeability barrier is provided by intercellular lipids forming multiple membrane bilayers in the stratum corneum. In the fetal rat, the barrier to transepidermal water loss forms during the 20th d of gestation and is accompanied by 1) increasing stratum corneum thickness; 2) increasing stratum corneum lipid content, particularly nonpolar ceramide and cholesterol content; and 3) the formation of lamellar unit structures throughout the stratum corneum interstices. In this report, we demonstrate that among pups of 20 d gestational age increasing barrier competence is correlated with increasing fetal weight. It has been previously demonstrated that fetal rats subjected to intrauterine growth retardation (IUGR) exhibit a thinner stratum corneum and decreased content of differentiation-specific epidermal structural proteins. To determine whether IUGR fetal rats also exhibit immaturity of barrier function and the barrier membrane system, maternal rats underwent unilateral uterine vessel ligation on d 17 or 18 of gestation and IUGR and control littermates were harvested on d 20, 21, or 22 of gestation for determination of transepidermal water loss. Despite significant somatic growth retardation and a thinner stratum corneum, barrier function in IUGR fetal rats did not significantly differ from that in control littermates at any gestational age. In both IUGR and control fetal rat epidermis at 21 d gestational age, lipids were deposited in a membrane pattern as visualized by nile red fluorescence microscopy and formed lamellar unit membrane structures throughout the stratum corneum intercellular domains as observed by electron microscopy.(ABSTRACT TRUNCATED AT 250 WORDS)

Ontogeny of the epidermal barrier to water loss in the rat: correlation of function with stratum corneum structure and lipid content.

The mammalian epidermal permeability barrier is provided by highly hydrophobic lipids forming multiple membrane bilayers within the extracellular domains of the outer, cornified cell layers. To characterize the critical events associated with barrier maturation, we correlated the emergence of a competent barrier to transepidermal water loss with development of the lamellar body secretory system, the organization of stratum corneum membrane bilayers, and the lipid composition of these membranes in the perinatal rat. Whereas pups of 19 d estimated gestational age had no measurable barrier (transepidermal water loss greater than 10 mg/cm2/h), by 21 d the barrier was well established (mean transepidermal water loss 0.41 mg/cm2/h). Development of a functional barrier correlated with increasing thickness of the stratum corneum, as well as with development of a membrane pattern of lipid deposition, visualized with the hydrophobic fluorescent probe nile red. At 19 d estimated gestational age, the stratum corneum intercellular domains exhibited an abundance of secreted lamellar body contents, but they were not organized into basic bilayer unit structures. Lamellar unit structures became evident by 20 d and extended throughout the stratum corneum interstices by 22 d (term). The quantity of lipid in isolated stratum corneum increased significantly between 19 and 20 d (34.08 versus 50.08 mean micrograms lipid/cm2, respectively; p less than 0.02) and still further between 20 and 21 d estimated gestational age (74.49 micrograms lipid/cm2; p less than 0.001). This increase was due to progressive accumulation of neutral lipids, particularly cholesterol, as well as nonpolar ceramides, as shown by thin-layer chromatography/scanning densitometry. These studies imply that in the development of cutaneous barrier function in the fetal rat both the generation of sufficient quantities of hydrophobic lipids and the organization of these lipids into bilayer unit structures are required.