An in vivo RNAi screen identifies SALL1 as a tumor suppressor in human breast cancer with a role in CDH1 regulation.

The gold standard for determining the tumorigenic potential of human cancer cells is a xenotransplantation into immunodeficient mice. Higher tumorigenicity of cells is associated with earlier tumor onset. Here, we used xenotransplantation to assess the tumorigenic potential of human breast cancer cells following RNA interference-mediated inhibition of over 5000 genes. We identify 16 candidate tumor suppressors, one of which is the zinc-finger transcription factor SALL1. Analyzing this particular molecule in more detail, we show that inhibition of SALL1 correlates with reduced levels of CDH1, an important contributor to epithelial-to-mesenchymal transition. Furthermore, SALL1 expression led to an increased migration and more than twice as many cells expressing a cancer stem cell signature. Also, SALL1 expression correlates with the survival of breast cancer patients. These findings cast new light on a gene that has previously been described to be relevant during embryogenesis, but not carcinogenesis.

Localization of cyclo-oxygenase-2 in human recurrent colorectal cancer.

AIM: The aim of this paper is to examine COX-2 expression in human recurrent colorectal carcinoma tissues using immunohistochemistry and quantative real-time PCR (qPCR). METHODS: Colon and rectal specimens were obtained from 26 patients with recurrent colorectal carcinomas. We examined COX-2 expression in human recurrent colorectal carcinoma tissues using immunohistochemistry and quantative real-time PCR (qPCR). RESULTS: In recurrent colorectal cancer a strong cytoplasmic and perinuclear staining of COX-2 was found. Moderate to strong immunosignals were detected in almost all of the carcinomas. We observed a strong specific staining of COX-2 in vascular endothelium. COX-2 immunoreactivity was also detected in stromal cells such as mononuclear cells, fibroblasts, and smooth muscle cells. The real-time PCR analyses demonstrated marked overexpression of the COX-2 gene in the cancer mucosa in concert with the immunohistochemistry data. CONCLUSION: We investigated COX-2 expression at the level of its protein as well as its messenger RNA in a series of recurrent colorectal cancers. These observations give additional information about the possibility that COX-2 could be involved in tumor promotion during colorectal cancer progression.

What are cyclooxygenases and lipoxygenases doing in the driver's seat of carcinogenesis?

Substantial evidence supports a functional role for cyclooxygenase- and lipoxygenase-catalyzed arachidonic and linoleic acid metabolism in cancer development. Genetic intervention studies firmly established cause-effect relations for cyclooxygenase-2, but cyclooxygenase-1 may also be involved. In addition, pharmacologic cyclooxygenase inhibition was found to suppress carcinogenesis in both experimental mouse models and several cancers in humans. Arachidonic acid-derived eicosanoid or linoleic acid-derived hydro[peroxy]fatty acid signaling are likely to be involved impacting fundamental biologic phenomena as diverse as cell growth, cell survival, angiogenesis, cell invasion, metastatic potential and immunomodulation. However, long chain unsaturated fatty acid oxidation reactions indicate antipodal functions of distinct lipoxygenase isoforms in carcinogenesis, i.e., the 5- and platelet-type 12-lipoxygenase exhibit procarcinogenic activities, while 15-lipoxygenase-1 and 15-lipoxygenase-2 may suppress carcinogenesis.

Differential protein expression in the epidermis of wild-type and COX-2 transgenic mice.

Cyclooxygenases (COX) 1 and 2 are the key enzymes of prostaglandin biosynthesis. Like in many tissues, in adult skin COX-1 is a constitutive 'housekeeping' enzyme, while COX-2 is induced transiently in stress situations such as tissue damage and regeneration. In human skin carcinomas and corresponding early-stage cancer lesions, permanent COX-2 expression and activation is a consistent feature. Knockout and various transgenic approaches and pharmacologic studies show strong evidence for a cause-and-effect relationship between the aberrant COX-2 activation and tumor formation. In skin epidermis, keratin 5 promoter-driven overexpression of COX-2 caused hyperplasia and dysplasia, and sensitized skin for carcinogenesis. Therefore, this model offers the unique possibility of identifying COX-2-dependent and prostaglandin-mediated molecular pathways leading to the formation and malignant progression of early-stage cancer lesions.

PAF-receptor in inflammatory versus non inflammatory human epidermis, cell cultures and embryonal cells.

OBJECTIVE: PAF-R (platelet activating factor-receptor) has been found on human keratinocytes to bind PAF, a proinflammatory phospholipid. We aimed to study PAF-R in a range of dermal cell lines and in samples of normal and psoriatic human skin to learn about its further role in humans. METHODS: PAF-R was labeled immunocytochemically, histochemically and additionally studied with western blotting in human keratinocytes, human fibroblasts, embryonal keratinocytes, tumor cell lines and samples of normal and psoriatic human skin. RESULTS: Keratinocytes from adult and embryonal epidermis of the 20th week of pregnancy showed a low level of labeling for PAF-R, but 3 +/- 0.05% of plantar keratinocytes from adults were positive as were 4.2 +/- 0.05% of embryonal plantar keratinocytes from the 21st week of pregnancy. In fibroblasts from adult and embryonal epidermis the protein was expressed at low levels. Western blotting revealed PAF-R positive bands at 67 k.Da in normal human skin and psoriasis, in psoriasis additionally at 45 k.Da. A 68 k.Da band was found in the colon cancer line HT 29 (control), and HaCaT cells, in embryonal keratinocytes additionally at 116 k.Da. CONCLUSIONS: PAF-R seems not to be important for embryonal or adult fibroblasts. In embryonal keratinocytes it is turned on after the 21st pregnancy week in a few cells seen as a band of 67 k.Da and at 116 k.Da, the latter is not found in adult keratinocytes. An additional 45 k.Da band of PAF-R was found in psoriasis that might stand for a truncated receptor. In the epithelial tumor cell line HaCaT and the HT29 colon cancer cell line PAF-R characterizes the anti-apoptotic effect of this receptor propagating tumor proliferation.

Abnormal differentiation of epidermis in transgenic mice constitutively expressing cyclooxygenase-2 in skin.

In prostanoid biosynthesis, the first two steps are catalyzed by cyclooxygenases (COX). In mice and humans, deregulated expression of COX-2, but not of COX-1, is characteristic of epithelial tumors, including squamous cell carcinomas of skin. To explore the function of COX-2 in epidermis, a keratin 5 promoter was used to direct COX-2 expression to the basal cells of interfollicular epidermis and the pilosebaceous appendage of transgenic mouse skin. COX-2 overexpression in the expected locations, resulting in increased prostaglandin levels in epidermis and plasma, correlated with a pronounced skin phenotype. Heterozygous transgenic mice exhibited a reduced hair follicle density. Moreover, postnatally hair follicle morphogenesis and thinning of interfollicular dorsal epidermis were delayed. Adult transgenics showed a body-site-dependent sparse coat of greasy hair, the latter caused by sebaceous gland hyperplasia and increased epicutaneous sebum levels. In tail skin, hyperplasia of scale epidermis reflecting an increased number of viable and cornified cell layers was observed. Hyperplasia was a result of a disturbed program of epidermal differentiation rather than an increased proliferation rate, as reflected by the strong suppression of keratin 10, involucrin, and loricrin expression in suprabasal cells. Further pathological signs were loss of cell polarity, mainly of basal keratinocytes, epidermal invaginations into the dermis, and formation of horn perls. Invaginating hyperplastic lobes were surrounded by CD31-positive vessels. These results demonstrate a causal relationship between transgenic COX-2 expression in basal keratinocytes and epidermal hyperplasia as well as dysplastic features at discrete body sites.

Aberrant cyclooxygenase isozyme expression in human intrahepatic cholangiocarcinoma.

METHODS: Cellular localisation of the cyclooxygenase (COX) isozymes COX-1 and COX-2 was analysed in 24 cholangiocarcinomas, including 17 matched tissues originating from non-tumorous liver tissue adjacent to tumours and seven biopsies of normal human liver, by immunohistochemistry using isozyme selective antibodies. RESULTS: In normal liver, constitutive expression of COX-2 protein was a characteristic feature of hepatocytes whereas no COX-2 immunosignal was detectable in normal bile duct epithelium, Kupffer, and endothelial cells. In cholangiocarcinoma cells, COX-2 protein was strongly expressed at high frequency. The intensity, percentage of positive cells, and pattern of COX-2 expression were found to be independent of the stage of tumour differentiation. In hepatocytes of matched non-tumorous tissue, COX-2 expression was unaltered. In contrast, strong COX-1 expression was frequently localised to Kupffer cells, endothelial cells, and occasionally to hepatocytes, but not to bile duct epithelial cells. In approximately half of moderately and poorly differentiated but not well differentiated cholangiocarcinomas, weak to moderate COX-1 staining was found in tumour cells while COX-1 expression in Kupffer cells was much more pronounced. CONCLUSION: Aberrant COX-2 expression occurs during the early stage while COX-1 over expression seems to be related to later stages of cholangiocarcinogenesis.

A causal relationship between unscheduled eicosanoid signaling and tumor development: cancer chemoprevention by inhibitors of arachidonic acid metabolism.

Cancer results from disturbances of cellular signal transduction and data processing at the genetic and epigenetic level. In the early phase of the disease these disturbances are mainly caused by environmental toxic agents, i.e. genotoxic and non-genotoxic carcinogens, whereas endogenous agents derived from dys-regulated metabolic reactions may take over this role at later stages, thereby leading to a state of 'genetic instability' and 'growth autonomy'. Among these metabolic reactions becoming dys-regulated in the course of tumorigenesis, eicosanoid biosynthesis from arachidonic acid seems to play a particular role. A steadily increasing body of evidence indicates a causal relationship between cancer development and an abnormal overexpression of eicosanoid-forming enzymes, i.e. cyclooxygenases and lipoxygenases, in a wide variety of human and animal tumors. This overexpression seems to result from disturbances of cellular signaling cascades such as the Ras-Raf-MAPkinase cascade due to oncogenic gene mutations. Presently, research is focussed on the proinflammatory enzyme cyclooxygenase-2 (COX-2) the pathological overexpression of which has been found to be related to key events of tumor promotion such as cellular hyperproliferation, inhibition of programmed cell death, and tumor angiogenesis. In the mouse skin model of multistage carcinogenesis COX-2-derived prostaglandin F(2alpha) has been indentified as an endogenous tumor promoter. Moreover, genotoxic byproducts of both cylooxygenase and lipoxygenase-catalyzed arachidonic acid metabolism (such as active oxygen species, free radicals etc.) are suspected to contribute to 'genetic instability' and thus to malignant progression of tumor cells. The enzymes of eicosanoid biosynthesis rank therefore among the most attractive targets for cancer chernoprevention. In fact, both nonsteroidal antiinflammatory drugs, i.e. non-specific COX inhibitors, and isozyme-specific COX-2 inhibitors have been shown to inhibit experimental and human cancer development, in the latter case in particular in the large bowel. Beside their role as indicators of neoplastic development eicosanoids may be also used as reporters of skin irritation. Based to this concept an in vitro test system for skin toxicity has been developed in which the release of arachidonic acid and interleukin-1alpha, i.e. two key mediators of acute inflammation, from a human keratinocyte cell line is measured. The excellent correlation found between this mediator release and the effects of various chemical irritants on human skin in vivo indicates that, in the near future, this and related methods may help to do without animal experiments in toxicological testing.

Production of sophorolipids from whey. II. Product composition, surface active properties, cytotoxicity and stability against hydrolases by enzymatic treatment.

Sophorolipids, obtained by a two-stage process starting from deproteinized whey concentrate using Cryptococcus curvatus ATCC 20509 and Candida bombicola ATCC 22214, were compared to products from one-stage processes, using different lipidic compounds as substrates. Results showed that above all carbon source and not cultivation conditions had a distinct influence on the composition of the crude product mixture and therefore on the physicochemical and biological properties of the sophorolipids, such as, for example, surface activity, cytotoxicity and stability against hydrolases. The results were completed by corresponding data for purified mono- and diacetylated (17-hydroxyoctadecenoic)-1',4"-lactonized sophorolipids. Crude sophorolipid mixtures showed moderate to good surface active properties (SFTmin 39 mN m-1, CMC 130 mg l-1), water solubilities (2-3 g l-1) and low cytotoxicities (LC50 300-700 mg l-1). In contrast, purified sophorolipids were more surface active (SFTmin 36 mN m-1, CMC 10 mg l-1), less water soluble (max. 70 mg l-1) and showed stronger cytotoxic effects (LC50 15 mg l-1). Incubation of crude sophorolipid mixtures with different hydrolases demonstrated that treatment with commercially available lipases such as from Candida rugosa and Mucor miehei distinctly reduced the surface active properties of the sophorolipids, while treatment with porcine liver esterase and glycosidases had no effect.

Prostaglandin-H-synthase isozyme expression in normal and neoplastic human skin.

Expression of prostaglandin-H-synthase (PGHS) isozymes was analyzed in 50 biopsies of normal human skin and of pre-malignant and malignant skin lesions, by means of quantitative RT-PCR, immunoprecipitation and Western blotting, as well as immunohistochemistry. Normal skin constitutively expressed PGHS-1 in all cell layers of the epidermis, in endothelial cells of small blood vessels and in sweat-gland epithelium. PGHS-2 expression was very low and restricted to a few keratinocytes of the interfollicular and follicular epidermis. Steady-state concentrations of PGHS-1 and PGHS-2 mRNA were similar in normal skin and in basal-cell carcinomas, but PGHS-1 mRNA was reduced and PGHS-2 mRNA was elevated in actinic keratoses, squamous-cell carcinomas and keratoacanthomas. PGHS-1 protein was detected in all tumor biopsies, being occasionally increased in basal-cell carcinomas. High amounts of PGHS-2 protein were found in actinic keratoses, squamous-cell carcinomas and keratoacanthomas, but not in basal-cell carcinomas. Four malignant melanomas included in this study contained PGHS-1 but no PGHS-2 protein. Immunohistochemical analysis of the biopsies identified keratinocytes, in addition to cells of inflammatory infiltrates and of dendritic morphology, as the major PGHS-expressing cell types. PGHS-2-specific signals were spread throughout the epidermal part of actinic keratoses and squamous-cell carcinomas. These data suggest that constitutive up-regulation of PGHS-2 expression is a consistent pre-malignant event in squamous-cell cancer development in man, as it is in animal models of skin carcinogenesis. Thus, pre-cancerous lesions such as actinic keratoses present a likely target for chemoprevention of skin cancer by selective PGHS-2 inhibitors.

Metabolic targets of cancer chemoprevention: interruption of tumor development by inhibitors of arachidonic acid metabolism.

Tumor promotion is understood as a process that favors the clonal outgrowth of single mutated (initiated) cells to premalignant lesions through co-mitogenic and anti-apoptotic effects. This process can be evoked by repeated induction of a regenerative tissue response as achieved either by irritation and wounding or by agents (tumor promoters) that interact with the corresponding pathways of cellular signaling. Metabolic processes regulated by such pathways and essential for tumor development are potential targets of cancer chemoprevention. Examples are provided by the expression of ornithine decarboxylase and the activation of eicosanoid formation from arachidonic acid. Arachidonic acid metabolism is a particularly attractive and important target of chemopreventive measures. Its induction is a characteristic response to tissue damage and irritation and an apparently critical event in epithelial tumor promotion. Inhibitors of eicosanoid formation, such as nonsteroidal anti-inflammatory drugs, rank among the most powerful chemopreventive agents in animal models and have been shown to halve the incidence of colorectal cancer in man. Recently, the role of cyclooxygenase-2 (COX-2)-catalyzed prostaglandin synthesis has been the subject of much attention. COX-2 is a typical 'emergency enzyme', since in most tissues it is transiently induced only in the course of repair and defense reactions. In epithelial neoplasia, i.e. in skin and colorectal tumors, the enzyme is constitutively overexpressed along different molecular pathways, and it seems to be critically involved in tumor promotion. Consequently, specific COX-2 inhibitors have been shown to exhibit considerable cancer chemopreventive potential. The putative role of other pathways of arachidonic acid metabolism in tumor promotion and malignant progression is presently under investigation.

Cellular localization of cyclo-oxygenase isozymes in Crohn's disease and colorectal cancer.

Deregulation of cyclo-oxygenase isozyme expression has been shown to be a consistent feature of inflammatory bowel diseases and colorectal cancer in humans. This study investigated the cellular localization of aberrant cyclo-oxygenase expression in normal and diseased colon. Biopsies of seven normal colonic tissues, eight tissue samples from patients suffering from Crohn's disease, five polyps from patients with familiar adenomatous polyposis coli, and ten sporadic adenocarcinomas were analyzed using isozyme-selective immunoprecipitation, western blotting, and immunohistochemistry. Cyclo-oxygenase-1 expression was demonstrated in normal human colon, Crohn's disease, and colorectal tumors. In normal colon and also in adenomatous polyps, cyclo-oxygenase-1 specific immunosignals were localized to epithelial cells of the upper part of the crypts and endocrine cells of the lower part. In Crohn's disease cyclo-oxygenase-1 expression was restricted to cells of the inflammatory infiltrate. While barely detectable in normal colon, cyclo-oxygenase-2 protein was strongly increased in epithelial cells located in the uppermost part of the crypts, in surface epithelial cells, and in mononuclear cells of the lamina propria of Crohn's disease. The constitutive overexpression of cyclo-oxygenase-2 protein observed in the majority of the adenomatous polyps and all adenocarcinomas was attributed to both epithelial and interstitial cells in that the latter predominated in adenomas, and epithelial cells were the prevailing cyclo-oxygenase-2 expressing cell type in adenocarcinomas. In conclusion, both autocrine and paracrine effects of aberrant cyclooxygenase-2 expression may contribute to the development of Crohn's disease and colonic tumor development.

Arachidonic acid metabolism as a reporter of skin irritancy and target of cancer chemoprevention.

Keratinocytes respond to skin irritation and injury by cytokine release and a rapid but transient activation of arachidonic acid metabolism along both the cyclooxygenase and lipoxygenase pathways. In the first part of this article results are reviewed indicating that the release of pro-inflammatory mediators such as eicosanoids and interleukin-1 from keratinocytes provides a suitable in vitro parameter of irritancy. Based on this response an assay system has been established which may partially replace animal tests such as the Draize test. A permanent overactivation of arachidonic acid metabolism appears to be a driving force of tumor development in both experimental animals and man. Inhibition of the enzymes involved (such as cyclooxygenases by nonsteroidal antiinflammatory drugs) provides, therefore, a powerful and promising measure of cancer chemoprevention. The state of the art in this rapidly developing field is briefly reviewed in the second part of this article.

Localization of prostaglandin H synthase isoenzymes in murine epidermal tumors: suppression of skin tumor promotion by inhibition of prostaglandin H synthase-2.

The growth factor- and phorbol ester-inducible prostaglandin H synthase (PGHS)-2 has been found to be constitutively overexpressed in epidermal tumors generated by the initiation-promotion protocol in murine skin, whereas the expression of PGHS-1 does not change under these conditions. In this paper we report the intra-tumor distribution of the aberrantly expressed PGHS-2 and the cancer chemopreventive activity of a specific PGHS-2 inhibitor. By immunohistochemical methods using isoenzyme-specific antibodies, we found that the PGHS-1 protein was expressed in keratinocytes and Langerhans cells dispersed throughout the epithelial part of papillomas and squamous cell carcinomas and in inflammatory infiltrates occasionally seen in these tumors. A uniform pattern of PGHS-2 expression was observed in the basal keratinocytes of papillomas and in the follicular keratinocytes of carcinomas. In addition, Langerhans cells as well as tumor-associated inflammatory infiltrates exhibited PGHS-2-specific immunoreactivity. PGHS-2-catalyzed prostaglandin synthesis stimulated by the phorbol ester 12-O-tetradecanoylphorbol-13 acetate (TPA) in mouse epidermis in vivo was dose-dependently suppressed by topical administration of SC-58125, a specific PGHS-2 inhibitor. TPA-induced edema formation, epidermal DNA synthesis, and mitotic activity were not impaired by SC-58125 applied at a dose that inhibited TPA-induced prostaglandin E2 synthesis. However, the repetitive epicutaneous administration of SC-58125 substantially and significantly suppressed papilloma development. Malignant progression of papillomas was slightly retarded by the drug. These results indicate that aberrant expression of PGHS-2 in epidermal tumors may be a relevant target for prevention of epidermal cancer development in experimental animals and that the PGHS-2-specific inhibitor SC-58125, which is a potent inhibitor of tumor promotion in mouse skin, may be important for cancer chemoprevention in humans as well.

Localization of prostaglandin-H synthase-1 and -2 in mouse skin: implications for cutaneous function.

Prostaglandin-H synthase (PGHS)-1 and -2 expression in mouse skin and in keratinocytes in culture was determined using immunohistochemistry and Western blot analysis. In normal skin PGHS-1 immunoreactivity was found in individual keratinocytes present in the interfollicular epidermis and the upper part of the hair follicle. PGHS-2 immunostaining was detected in very few individual basal cells of the interfollicular epidermis and of the hair follicle. Upon induction by TPA of an inflammatory epidermal hyperplasia (regenerative hyperplasia) the number of PGHS-2-expressing keratinocytes scattered throughout the basal but not the suprabasal compartment of the interfollicular epidermis was found to be increased while PGHS-1 expression remained unchanged. PGHS-2 immunoreactivity in paraffin sections from TPA-treated skin showed a nuclear in some and a perinuclear and cytoplasmic localization in other keratinocytes. This different distribution may correlate with the proliferative state, since immunofluorescence analysis of mouse keratinocytes in culture demonstrated a predominant perinuclear and cytoplasmic PGHS-2 localization in cycling keratinocytes but a prevalent staining of the nucleus and the nuclear membrane in noncycling cells. Stimulation of proliferation of murine primary keratinocytes by serum resulted in an increased PGHS-2 expression, whereas induction of terminal differentiation by Ca2+ caused a down-regulation of PGHS-2 protein. Only minor changes in PGHS-1 expression were seen. Our data suggest that expression of PGHS-2 in mouse skin epidermis is related to epithelial regeneration.

Arachidonic acid metabolism in primary irritant dermatitis produced by patch testing of human skin with surfactants.

A clinical study was performed to determine the effects of patch testing human skin with four industrially used surfactants on erythema formation, transepidermal water loss, and the contents in suction blister fluids of primary proinflammatory mediators including arachidonic acid, eicosanoids, and IL-1 alpha, which were analyzed by quantitative gas chromatography/negative ion chemical ionization mass spectrometry and by an enzyme-immunoassay, respectively. Benzalkonium chloride (BKCI) and sodium lauryl sulfate (SLS) elicited erythema and caused increased transepidermal water loss, indicating a disturbance of the epidermal barrier. Triethanolamine (TEA) and Tween 80 did not evoke these gross symptoms of inflammation. Suction blister fluids collected after a 24-h application of BKCl, SLS, and Tween 80 contained significantly increased amounts of individual eicosanoids whereas TEA induced no response. The induced eicosanoid profile was characteristic for each compound, pointing to different cell types of skin to be involved in their production. The elevation of prostaglandin and LTB4 contents correlated with the induction of erythema and the impairment of the epidermal barrier as shown for BKCl and SLS and preceded the maximum of erythema formation. IL-1 alpha contents did not correlate with these gross symptoms of inflammation. The results of this in vivo study support those of a previous study using human keratinocytes in culture indicating the release of arachidonic acid and prostaglandins to be an early event involved in the interaction of keratinocytes with surfactants. Moreover, the in vivo data with human skin underscore the mechanistic relationship to the in vitro model and support the concept that arachidonic acid and eicosanoid release from keratinocytes can be used as a marker of primary skin irritation.

Murine epidermal lipoxygenase (Aloxe) encodes a 12-lipoxygenase isoform.

Using a combination of conventional screening procedures and polymerase chain reaction cloning, we have isolated a cDNA encoding an epidermis-type 12-lipoxygenase (e12-lipoxygenase) from mouse epidermis. The open reading frame corresponds to a protein of 662 amino acids and was found to be 99.8% identical to the ORF of an epidermal lipoxygenase gene Aloxe, described recently [Van Dijk et al. (1995) Biochim. Biophys. Acta 1259, 4-8]. When expressed in human embryonic kidney cells the recombinant protein could be shown to synthesize 12(S)-HETE from arachidonic acid. By fluorescence in situ hybridization the e12-lipoxygenase gene was localized to chromosome band 11 B1-B3.

Mechanisms in tumor promotion: guidance for risk assessment and cancer chemoprevention.

In mouse skin, tumor development promoted by 'non-genotoxic' carcinogens is closely related to the wound response. In both cases endogenous factors such as cytokines and eicosanoids released primarily from 'activated keratinocytes' play a key role as mediators of inflammation and cellular hyperproliferation. The liberation of interleukin-1 alpha and arachidonic acid from human keratinocytes has been used as an in vitro parameter of irritancy. The results (from experiments with 15 different chemicals) being validated at present in a clinical study indicate a quantitative relationship between irritancy in vivo and mediator release in vitro. In the course of experimental skin carcinogenesis an overproduction of eicosanoids due to a constitutive overexpression of the corresponding enzymes (i.e. PGH synthase-II and 8- and 12-lipoxygenase) is observed. Enzyme inhibitors, for instance nonsteroidal antiinflammatory drugs (NSAIDs), exert a strong tumoristatic effect. Thus, the approach of multistage skin carcinogenesis provides a suitable animal model for a mechanistic evaluation and further improvement of chemopreventive measures such as the inhibition of colorectal tumor development in humans by NSAIDs ('aspirin effect').