Multistage carcinogenesis in mouse skin

Multistage carcinogenesis in mouse skin

Pharmac. Ther. Vol. 54, pp. 63-128, 1992 Printed in Great Britain. All rights reserved 0163-7258/92 $15.00 © 1992 Pergamon Press Ltd Associate Edito...

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Pharmac. Ther. Vol. 54, pp. 63-128, 1992 Printed in Great Britain. All rights reserved

0163-7258/92 $15.00 © 1992 Pergamon Press Ltd

Associate Editor: D. GRUNBERGER

MULTISTAGE CARCINOGENESIS IN MOUSE SKIN JOHN DIGIOVANNI Department of Carcinogenesis, University of Texas M.D. Anderson Cancer Center, Science Park--Research Division, P.O. Box 389, Smithville, TX 78957, U.S.A. Abstract--The mouse skin model of multistage carcinogenesis has for many years provided a conceptual framework for studying carcinogenesis mechanisms and potential means for inhibiting specific stages of carcinogenesis. The process of skin carcinogenesis involves the stepwise accumulation of genetic change ultimately leading to malignancy. Initiation, the first step in multistage skin carcinogenesis involves carcinogen-induced genetic changes. A target gene identified for some skin tumor initiators is c-Ha-ras. The second step, the promotion stage, involves processes whereby initiated cells undergo selective clonal expansion to form visible premalignant lesions termed papillomas. The process of tumor promotion involves the production and maintenance of a specific and chronic hyperplasia characterized by a sustained cellular proliferation of epidermal cells. These changes are believed to result from epigenetic mechanisms such as activation of the cellular receptor, protein kinase C, by some classes of tumor promoters. The progression stage involves the conversion of papillomas to malignant tumors, squamous cell carcinomas. The accumulation of additional genetic changes in cells comprising papillomas has been correlated with tumor progression, including trisomies of chromosomes 6 and 7 and loss of heterozygosity. The current review focuses on the mechanisms involved in multistage skin carcinogenesis, a summary of known inhibitors of specific stages and their proposed mechanisms of action, and the relevance of this model system to human cancer. CONTENTS 1. Introduction and Historical Perspective 2. Overview of Multistage Carcinogenesis in Mouse Skin 3. Tumor Initiation 3.1. Nature of skin tumor initiators 3.2. Role of DNA interactions in skin tumor initiation 3.3. Target cells for tumor initiation 3.4. Genes and genetic changes associated with skin tumor initiation 3.5. Modifying factors affecting tumor initiation in mouse skin

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Abbreviations--AHH, aryl hydrocarbon hydroxylase; B[a]P, benzo[a]pyrene; B[e]P, benzo[e]pyrene; BCG, bacillus Calmette-Gu6rin vaccine; BHA, butylated hydroxyanisole; BHT, butylated hydroxytoluene; BHTOOH, butylated hydroxytoluene hydroperoxide; BPDE, benzo[a]pyrene diol-epoxide; 7-BrMe-BA, 7-bromomethylbenz[a]anthracene; Cu 2÷ DIPS, copper (II) bis (diisopropylsalicylate); dAdo, deoxyadenosine; DAG, diacylglycerol; DB[a,j]A, dibenz[a,j]anthracene; dCyto, deoxycytosine; DC, dark basal cells; DDTC, diethyl dithiocarbamate; c~-DFMO, a-difluoromethyl ornithine; DMBA, 7,12-dimethylbenz[a]anthracene; EA, ellagic acid; EGFr, epidermal growth factor receptor; ENU, ethyl nitrosourea; EPP, ethyl phenylpropiolate; ETYA, 5,8,11,14-eicosatetraynoic acid; FA, fluocinolone acetonide; GSH, reduced glutathione; GSSG, oxidized glutathione; G6PDH, glucose-6-phosphate dehydrogenase; dGuo, deoxyguanosine; HA, hydroxyapatite; hTGF~, human transforming growth factor alpha; IP3, inositol-l,4,5-trisphosphate; MCA, 3-methylcholanthrene; MGBB, methyl-glyoxal bis (butyl-amidinohydrazone); MNNG, N-methyl-N'nitro-N-nitrosoguanidine; MNU, methyl nitrosourea; ODC, ornithine decarboxylase; PAH, polycyclic aromatic hydrocarbon; 4~PDD, 4-a-phorbol-12,13,didecanoate; PDGF, platelet-derived growth factor; PIP 2 phosphatidylinositol-4,5-bisphosphate; PKC, protein kinase C; PLC-7, phospholipase-c-gamma; PMNs, polymorphonuclear leukocytes; RA, retinoic acid; RPA, 12-O-retinoylphorbol-13-acetate; SCC, squamous cell carcinoma; sn-I,2-DDG, sn-l,2-didecanoylglycerol; SOD, superoxide dismutase; TAME, tosylalanine methyl ester; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TGF~, transforming growth factor alpha; TGFfl, transforming growth factor beta; TLCK, tosylysine chloromethyl ketone; TPA, 12-O-tetradecanoylphorbol-13-acetate; TPCK, tosylphenylalanine chloromethylketone; UVB, ultraviolet B radiation. 63

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4. Tumor Promotion 4.1. Nature of skin tumor promoters 4.2. Cellular, biochemical and molecular changes associated with skin tumor promotion 4.2.1. Cellular changes 4.2.2. Biochemical and molecular changes and the role of protein kinase C 4.2.3. Prooxidant related changes 4.2.4. Other changes associated with non-phorbol-ester promoters 4.3. Role of cell selection during tumor promotion 4.4. Multistage promotion 4.5. Modifying factors affecting tumor promotion in mouse skin 5. Tumor Progression 5.1. Biology of skin tumor growth and progression 5.2. Genetic alterations accompanying tumor progression in mouse skin 6. Relevance of the Mouse Skin Model to Human Cancer 7. Conclusions and Future Directions Acknowledgements References

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1. I N T R O D U C T I O N A N D H I S T O R I C A L PERSPECTIVE Skin carcinogenesis in mice can be accomplished using either multistage or complete protocols (Boutwell, 1964, 1974). Complete carcinogenesis experimental protocols involve the administration of a single dose or repeated applications of smaller doses of a carcinogen to an experimental animal. In the mouse skin tumorigenesis system, multiple papillomas and carcinomas can be produced on the backs of mice following a single application of as little as 600-800 nmol of a pure polycylic aromatic hydrocarbon (PAH) such as 7,12-dimethylbenz[a]anthracene (DMBA) (Terracini et al., 1960; Turusov et al., 1971). The induction of mouse skin tumors can also be accomplished by using a multistage model that involves the processes defined operationally and mechanistically as initiation and promotion (Boutwell, 1964, 1974; Slaga, 1989). Initiation is generally accomplished by topical application of a single subcarcinogenic dose of a skin carcinogen, such as DMBA. An initiating dose of a carcinogen, per se, will not lead to the development of visible tumors. Visible tumors will result only following prolonged and repeated topical applications of a tumor promoter, such as croton oil or its most active constituent, 12-O-tetradecanoylphorbol-13-acetate (TPA), to the initiated skin (Boutwell, 1964, 1974). It is generally assumed that both the initiating and promoting components are present during complete carcinogenesis experimental protocols. The multistage model of mouse skin carcinogenesis has its origins dating back over 60 years to studies by Deelman (1924, 1927) who found that wounding led to the appearance of skin tumors in mice that had been first treated with a carcinogenic tar. These, and other early studies, suggested a role for cell proliferation and hyperplasia in tumor development and skin carcinogenesis (Twort and Twort, 1939; Rous and Kidd, 1941; Friedewald and Rous, 1944). In their hallmark paper in 1944, Friedewald and Rous first defined the terms initiation and promotion after applying a two-stage technique for the development of skin tumors in rabbits. They demonstrated that 'latent' tumor cells initiated in rabbit skin by one treatment with 3-methylcholanthrene (MCA) could be 'forced' or 'promoted' to reveal themselves by subsequent treatment of the skin with agents which did not themselves initiate neoplastic change (e.g. turpentine, chloroform, wounding). The discovery of croton oil (Berenblum, 1941; Berenblum and Shubik, 1947) as a potent promoting agent led Mottram (1944) and others (Berenblum and Shubik, 1949; Berenblum, 1954; Boutwell, 1964) to ultimately develop the two-stage protocol of mouse skin tumorigenesis as it is generally used today. In his early experiments, Mottram (1944) elicited skin tumors by treating the backs of mice with a single, subcarcinogenic dose of benzo[a]pyrene (B[a]P) and followed this with repeated applications of croton oil, obtained from the seeds of Croton tiglium. Boutwell (1964) later reported that the process of mouse tumor promotion could be subdivided into operational stages he termed 'conversion' and 'propagation' using limited doses of croton oil followed by turpentine. In addition, Boutwell (1964) developed a selective breeding protocol that led to the production of a mouse line highly sensitive to multistage carcinogenesis using a variety of agents (DiGiovanni et al., 1980, 1991b). This mouse line, called SENCAR, has become a standard for studies of

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multistage carcinogenesis in many laboratories (Slaga and Nesnow, 1985; Eastin, 1989). The ultimate discovery and isolation of phorbol diesters and, in particular TPA, the most active phorbol diester from croton oil (Van Duuren, 1969; Van Duuren and Orris, 1965; Hecker, 1968, 1971) ultimately paved the way for the modern era of studies in the field of multi-stage skin carcinogenesis in mice and to a greater understanding of biochemical and molecular mechanisms associated with the tumor promotion process.

2. OVERVIEW OF MULTISTAGE CARCINOGENESIS IN MOUSE SKIN A schematic representation of multistage carcinogenesis in mouse skin is depicted in Fig. 1. As noted above, the initiation stage of mouse skin tumorigenesis is effected by a single application or exposure to a subcarcinogenic dose of a skin carcinogen. Initiation is a rapid process that produces no apparent morphological alterations in the epidermis (reviewed in Boutwell, 1964, 1974, 1976; Scribner and Suss, 1978). The initiation event occurs as a result of interaction of a reactive form of a skin carcinogen with the DNA of an epidermal target cell (Brookes and Lawley, 1964; Yuspa and Poirer, 1988; Slaga, 1989). As a result of interaction of a reactive carcinogenic intermediate with DNA one can measure a transient inhibition of epidermal DNA synthesis (Slaga et al., 1973, 1974). One of the hallmarks of tumor initiation in mouse skin is that it persists for the lifetime of the animal (Berenblum and Shubik, 1949; Roe et al., 1972; Van Duuren et al., 1975; Loehrke et al., 1983) despite the fact that the epidermis renews itself approximately once every 6-8 days (Patten, 1983). Thus, some important characteristics of skin tumor initiators can be summarized as follows (reviewed in Slaga and Fischer, 1983; Yuspa and Poirer, 1988; Slaga, 1989): (i) react covalently with or indirectly modify cellular macromolecules such as DNA, RNA, and protein; (ii) produce an essentially irreversible event after a single application or exposure; and (iii) are mutagenic in bacterial and mammalian cells.

Multistage Carcinogenesis in Mouse Skin Initiation

Promotion 1 week tO 1 year

...~~==.

I

Progression I

1. Metabolic activation of procarclnogens and covalent binding to DNA

1. Incmaesd DNA synthesis 2. increased production of

1. Production and maintenance of chronic cell proliferation

2. DNA repair / cell replication and fixation of mutation

proataglendlns and other growth regulatory molecules 3, Eleval~l ODC activity and elevated polyamlnes

2. Development of ¢lonal outgrowths; benign papillomas

3. Mutation Induction In critical target genes of stem cell e.g. Ha.ros 4. Phenotyplcelly "normal" epldermls

4. Expansion of Inltlated stem cells through eplgsnetlc mechsnlsms

3. Altered patterns of differentiation 4. Dlplold leslohs

I

1. Addltionel genatlc events occurlng stochastically 2. Anu~loldy e.g. nonrendom trlsomlu of chromesomes 6 and 7 3. Loss of heterozygoslty 4. Further eltaratlons In dlfferentiatlon pattems 5. Presence of dysplaela

I

1. Invasion 2. Metestlsls 3. LOllS of tumor supprmmor ectlvlty e.g. p~l mutation 4. Geno amplification e,g. mut~ed Ha-ros allele

6. Conversion of papllloma to squamous cell carcinoma

FIG. 1. Overview of multistage carcinogenesis in mouse skin. Carcinogenesis in mouse skin can be divided into 3 main stages: initiation, promotion and progression. The promotion and progression stages are further clarified by indicating early vs later events associated with these stages. Major events and/or changes associated with each stage are summarized below the cartoon representing the physical appearance of the skin or major lesion at that particular stage.

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In contrast to the initiation stage, treatment of mouse skin with a tumor promoting agent produces dramatic morphological and biochemical effects which are reversible in the absence of continued treatment (Boutwell, 1974, 1976; Argyris, 1981; Klein-Szanto, 1984; Aldaz et al., 1985), although some effects may persist for longer periods of time after discontinuation of promoter treatment (Fiirstenberger et al., 1983, 1985; Klein-Szanto, 1984; Aldaz et al., 1985). Thus, tumor promoters must be given at an optimal frequency and duration in order to effect tumor development. The process of tumor promotion in mouse skin is believed to involve the selective clonal expansion of initiated cells into visible clonal outgrowths (papillomas) by one or a combination of several mechanisms (reviewed in Parkinson, 1985; Yuspa and Poirer, 1988: Slaga, 1989; and see Section 4.3). While genetic mechanisms have been postulated to play a role in the tumor promotion stage (Petrusevska et al., 1988; Ffirstenberger et al., 1989b; and reviewed in Cerutti, 1985; Kensler and Taffe, 1986; Perchellet and Perchellet, 1988), the eventual reversibility of promoter-induced effects (Ffirstenberger et al., 1985; Aldaz et al., 1985; Ewing et al., 1988b) argues that this stage is accomplished primarily through epigenetic mechanisms as originally postulated by Boutwell and others (Boutwell, 1974, 1976). Since the early studies of Boutwell (1964), several laboratories have extended the observation that the tumor promotion stage of mouse skin tumorigenesis can be subdivided operationally and into two distinct stages (Slaga et al., 1980a,b; Ffirstenberger et al., 1981). However, this concept has been challenged by recent studies demonstrating that the first or 'conversion' stage can, in fact, be effected for a limited time, prior to application of the initiator (F/irstenberger et al., 1985; Ordman et al., 1985; Ewing et al., 1988b; Marks and Fiirstenberger, 1990). The first tumors that appear during a two-stage mouse skin tumorigenesis protocol using DMBA and TPA as the initiator and promoter, respectively, are premalignant lesions called papillomas (reviewed in Klein-Szanto, 1989). The papillomas that initially develop during mouse skin initiation-promotion procotols have long been considered heterogeneous in that some will persist, some will disappear or regress, and only a small proportion will 'progress' to an invasive squamous cell carcinoma (SCC) during the time frame of most experiments (Shubik et al., 1953a; Burns et al., 1976, 1978; Verma and Boutwell, 1980; Scribner et al., 1983; Hennings et al., 1985). This information has led to the hypothesis that different subclasses of papillomas exist with different probabilities of malignant progression and, indeed, to the hypothesis that some papillomas (referred to as 'terminally benign') (Burns et al., 1976, 1978; Scribner et al., 1983; Reddy et al., 1987) are not at risk for progression to SCC. On the other hand, other studies (Aldaz et al., 1987) have suggested, based on a sequential cytogenetic and histopathological study, that skin papillomas generated during an initiation-promotion regimen progress to SCC at different rates and that many of the papillomas generated during initiation-promotion have the potential to become SCC. Furthermore, recent studies (Ewing et al., 1988a, 1989; Aldaz et al., 1991) have demonstrated that many factors (e.g. tumor burden) can dramatically influence the apparent growth potential of papillomas and hence influence final carcinoma yields. Studies such as these have provided additional support for the hypothesis that a significant proportion of papillomas generated by initiation-promotion protocols in sensitive mouse strains should be considered premalignant lesions (Aldaz and Conti, 1989). Tumor progression in the mouse skin initiation-promotion model invo!ves the conversion of papillomas to SCC. Based on the available evidence (reviewed in Burns et al., 1984; Aldaz and Conti, 1989; Klein-Szanto, 1989) most, if not all, SCCs arise from preexisting papillomas. The process of tumor progression appears to involve the accumulation of additional genetic changes in cells comprising skin papillomas (Hennings et al., 1983; O'Connell et al., 1986a; Aldaz et al., 1987, 1989; Bremner and Balmain, 1990; Bianchi et al., 1990, 1991). These genetic changes appear to occur stochastically in that once a maximum papilloma yield is obtained on a given mouse, further promoter treatment is not necessary to produce a maximum SCC yield (Burns et al., 1978; Verma and Boutwell, 1980; Hennings et al., 1983, 1985; Ewing et al., 1988a). As noted above, the major types of tumors produced in mouse skin using initiation-promotion regimens are squamous papillomas and SCCs. However, other tumor types have been noted to various extents such as keratoacanthomas, melanomas, and adnexal tumors (Berkelhammer and Oxenhandler, 1987; Husain et al., 1991; Jaffe and Bowden, 1987; Monks et al., 1990;

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O'Connell et al., 1986b and reviewed in Klein-Szanto, 1989). The appearance of these tumor types may be highly dependent on the type of initiator and/or promoter used and on the genetic background of the mouse stock or strain utilized.

3. TUMOR INITIATION 3.1. NATUREOF SKIN TUMORINITIATORS The initiation stage of mouse skin carcinogenesis can be effected by a variety of both direct and indirect acting chemical carcinogens (Pereira, 1982; Slaga and Nesnow, 1985). In general, where appropriately tested, complete carcinogens for mouse skin possess at least some tumor-initiating activity (Slaga and Fischer, 1983; Slaga and Nesnow, 1985). However, several compounds, including urethane, diol-epoxide derivatives of B[a]P and benz[a]anthracene, and possibly several other compounds, have been suggested to possess only skin tumor initiating activity (Slaga and Fischer, 1983; Slaga and Nesnow, 1985). While these observations require further substantiation, the significance of the existence of so called 'pure' tumor initiators is two-fold: (i) carcinogens lacking promoting ability cannot be detected using a complete carcinogenesis protocol in the mouse skin model; and (ii) such compounds would provide unique mechanistic tools to study which cellular, biochemical, and molecular effects are lacking and hence necessary for the tumor promotion process that occurs during complete carcinogenesis in this tissue. PAH are one of the major chemical classes of carcinogens that possess both tumor initiating as well as complete carcinogenic activity on mouse skin (Gelboin and Ts'o, 1978; Slaga and Fischer, 1983; Slaga and Nesnow, 1985). These compounds are widely utilized and studied for their mechanism(s) of skin tumor initiation. In addition to PAH, many other chemical classes of compounds possess, albeit in some cases low activity, the ability to initiate skin tumors in mice. Comprehensive listings of known chemical carcinogens and tumor initiators on mouse skin can be found elsewhere (Nesnow et al., 1981; Pereira, 1982; Slaga et al., 1982a; Slaga and Fischer, 1983; Slaga and Nesnow, 1985) but examples of some of the major types are listed in Table 1. Note that chemical skin tumor initiators may be effective when administered topically (Boutwell, 1964), intragastrically (Ritchie and Saffiotti, 1955; Goerttler et al., 1980), or intraperitoneally (Hennings et al., 1969, 1981) and, can also act transplacentally (Goerttler and Loehrke, 1976, 1977; Goerttler et al., 1980). Some chemicals that are not tumor initiators when applied topically (e.g. 2-acetylaminofluorene) can initiate skin tumorigenesis when administered orally (Ritchie and Sattiotti, 1955). This observation suggests that metabolic transformation in the liver, to an intermediate that is then transported to the skin for further metabolism, can be a process whereby some chemicals are able to initiate skin carcinogenesis. Further study of this phenomenon may reveal additional chemicals not previously considered capable of initiating skin carcinogenesis that fit in this category. In addition to the wide variety of chemical initiators, both UV light and ionizing radiation TABLE 1. Chemicals that Possess Tumor Initiating Activity in Mouse Skin Chemical class . Examples Arylamines 2-acetylaminofluorene* N-Arylacetohydroxamic N-acetoxy-4-acetamidobiphenyl,N-acetoxy-2-acetamidofluorene acids PAH DMBA, B[a]P, MCA DB[a,j]A, 5-MeC, B[c]Ph, DB[a,h]A, 11-Me-CPP-17-one Carbamates urethane, vinyl carbamate Haloalkylethers bis(chloromethyl)ether Haloaromatics 2,3,4,5-trichloronitrobenzene Lactones -propiolactone Multifunctionals 4-nitroquinoline-N-oxide Nitro-aromatics 6-nitro-chrysene Nitrosamides MNNG Ureas NMU Abbreviations--5-Mec, 5-methylchrysene; B[c]Ph, benzo[c]phenanthrene; DB[a,h]A, dibenz[a,h]anthracene; ll-Me-CPP-17-one, ll-methylcyclopentaphenanthrene-17-one. *Only active as a skin tumor initiator when administered orally (Ritchie and Saffiotti, 1955).

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have been shown to initiate skin tumors in mice (Shubik et al., 1953b; Epstein and Roth, 1968; Pound, 1970; Stenback, 1975; Jaffe and Bowden, 1987). Some chemicals, such as certain psoralens, require interaction with u.v.-light to effect tumor initiation on mouse skin (Stern, 1989). Furthermore, infection of mouse epidermal cells with v-Ha-ras leads to an initiating event (Brown et al., 1986). This latter observation, in addition to other data discussed below (Section 3.4), has provided evidence for the involvement of the c-Ha-ras gene in the process of skin tumor initiation in mice. Many chemical carcinogens and skin tumor initiators, including the widely studied PAH, require metabolic activation in order to express their carcinogenic and tumor-initiating properties in mouse skin (reviewed in Miller, 1978; Gelboin and Ts'o, 1978; Beland and Poirer, 1989; Cooper and Grover, 1990a). PAHs are metabolized by cytochrome P-450-dependent monooxygenase enzymes to a wide variety of primary metabolites, including epoxides, dihydrodiols, quinones, and phenols (reviewed in Gelboin, 1980; Pelkonen and Nebert, 1982; Dipple et al., 1984; Hall and Grover, 1990). Some of these primary oxidation products can serve as substrates for conversion to water-soluble glutathione, glucuronide and sulfate conjugates (Gelboin, 1980; Pelkonen and Nebert, 1982; Hall and Grover, 1990). A number of the products of initial oxidation (i.e. phenols and diols) can be reoxidized and recycled through these pathways (Gelboin, 1980; Pelkonen and Nebert, 1982; Dipple et al., 1984; Hall and Grover, 1990). Of particular interest is the formation of the highly reactive diol-epoxides. Studies in a variety of tissues and species of the ubiquitous PAH, B[a]P, for example, have shown that it is activated to mutagenic and carcinogenic derivatives by a two-step mechanism leading to the formation of a 'bay-region' diol-epoxide (Dipple et al., 1984; Hall and

12

1

11 10

5

~

BENZO (a) PYRENE

OH

EH

OH

,~..A

(+) anti BPDE

"

(') BP-7,8-dlol

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H

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HOlt,, (MFO=-mlxedfunction oxldase~H \ EH=epoxldehydrltue /

O

'

'

~

OH(+) antl-BPDE-dGuo

FIG. 2. Two-step oxidation pathway leading to the metabolic activation and DNA binding of B[a]P. The major B[a]P-DNA adduct in most target tissues including mouse epidermis is shown, (+)-anti-BPDE-trans-dGuo. Abbreviations--MFO, cytochrome P-450-mediated mixed-function oxidase; EH, epoxide hydratase; BPDE, benzo[a]pyrene-diol-epoxide.

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Grover, 1990) and that the various isomers of this diol-epoxide bind covalently to different extents at various positions on the DNA bases, particularly on deoxyguanosine (dGuo) residues (Dipple et al., 1984; Baird and Pruess-Schwartz, 1988; Hall and Grover, 1990). Growing evidence indicates that other PAHs are activated in a similar way (reviewed in Dipple et al., 1984; Lehr et al., 1985; Baird and Pruess-Schwartz, 1988; Hall and Grover, 1990). Figure 2 highlights this two step oxidation pathway, showing the chemical structure of the major B[a]P-DNA adduct formed in a variety of tissues that are targets for PAH carcinogens. This two-step oxidation mechanism appears to be the major pathway of metabolic activation of B[a]P and other PAH for tumor initiation in mouse skin (reviewed in Baird and Pruess-Schwartz, 1988; DiGiovanni, 1989a). The epidermal target cells of mouse skin contain a variety of enzymes capable of metabolizing PAHs and other carcinogens to both active and inactive derivatives. The major enzymes catalyzing phase I (i.e. oxidative) reactions are the monooxygenases (cytochrome P-450-dependent), peroxidases (cyclooxygenase), and hydrolases (epoxide hydratase); those catalyzing phase II (i.e. conjugative) reactions are glucuronyltransferases, sulfotransferases, and glutahione-S-transferases. The mouse epidermal aryl hydrocarbon hydroxylase enzyme system (AHH, EC 1.14.14.2), the cytochrome P-450-dependent enzyme complex responsible for oxidizing PAH, is highly active and inducible by a variety of PAH substrates (Weibel et al., 1975; Akin and Norred, 1976; Thompson and Slaga, 1976a; Pohl et al., 1976; Manil et al., 1981). In early studies (Weibel et al., 1975), the highest AHH enzyme activity was found in the superficial layer of the dermis; more detailed studies (Akin and Norred, 1976; Thompson and Slaga, 1976a) revealed, however, that the epidermal layer contains the highest AHH activity in mouse skin. Although the cytochrome P-450-dependent monooxygenase system (i.e. AHH) is responsible for the metabolic activation of PAHs in a variety of target tissues, some studies have implicated enzymes involved in prostaglandin biosynthesis in carcinogen metabolism (Marnett et al., 1978; Guthrie et al., 1982; Robertson et al., 1983; Battista and Marnett, 1985; Eling and Krauss, 1985). Mouse epidermis contains significant amounts of the enzymes involved in arachidonic acid metabolism (e.g. cyclooxygenase and iipoxygenase) (Ftirstenberger and Marks, 1980; Fischer et al., 1982a; Kondoh et al., 1985; Nakadate et al., 1985; Eling et al., 1986; Nakadate et al., 1986a; Wheeler and Berry, 1986; Fischer et al., 1987, 1988). However, several lines of evidence suggest that these enzymatic pathways play only a lesser role in the production of DNA-binding intermediates from parent PAHs such as B[a]P (DiGiovanni et al., 1980, 1989; and reviewed in DiGiovanni, 1989a). Finally, evidence is emerging (DiGiovanni et al., 1980; Rettie et al., 1986; Ichikawa et al., 1989; reviewed in DiGiovanni, 1989a) to suggest that in epidermal cells the relative content of MCA-P-450 (or P450IA1, the form that is induced by MCA (Ioannides and Parke, 1990)) may be high in the skin of uninduced mice as compared with tissues such as the liver (Kahl et al., 1976). These observations may help explain why mouse epidermis is relatively sensitive to PAH carcinogens because it constitutively expresses a form of cytochrome P-450 that is highly active in converting B[a]P to the ( - )-7,8-diol and ultimately to the ( + ) - a n t i - B P D E (reviewed in Ioannides and Parke, 1990). 3.2. ROLE OF DNA INTERACTIONSIN SKIN TUMOR INITIATION Skin tumor initiators have been shown to bind covalently to a wide variety of cellular macromolecules including DNA, RNA and protein (Brookes and Lawley, 1964; Heidelberger, 1975; Miller, 1978). However, it is the interaction with DNA that correlates most closely with skin tumor initiating activity (Brookes and Lawley, 1964; Slaga et al., 1982b; Slaga and Fischer, 1983). Considerable effort has been expended in characterizing covalent interactions between activated carcinogen metabolites (primarily PAH) and the DNA of epidermal cells, the target cells for tumor initiation in mouse skin. Following topical application of the prototypic PAH, B[a]P, to mouse skin in vivo, the major DNA adduct formed is ( + ) - a n t i - B P D E bound through trans addition of the exocyclic amino (N 2) group of dGuo (( + ) - a n t i - B P D E - N 2 - d G u o ) (Fig. 2, reviewed in Dipple et al., 1984; DiGiovanni, 1989a). Although much attention has focused on ( + ) - a n t i - B P D E - N ~ - d G u o , a number of studies in various tissues including mouse epidermis (Ivanovic et al., 1978; Ashurst and Cohen, 1981a,b; Ashurst et al., 1983; DiGiovanni et al., 1985b; reviewed in Sims, 1980; Dipple et al., 1984; Baird and Pruess-Schwartz, 1988; Hall and Grover, 1990) have found DNA-adducts JPT

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arising from reaction of the anti B[a]P-diol-epoxides with deoxyadenosine (dAdo). In addition, at least one dGuo adduct with a syn-B[a]P-diol epoxide has been identified in mouse epidermal DNA following topical application of B[a]P to mouse skin (Ashurst et al., 1983; DiGiovanni et al., 1985b). Furthermore, studies of B[a]P diol-epoxide DNA binding in titro have indicated the formation of 06 and N 7 dGuo and N 4 deoxycytidine (dCyto) derivatives (Straub et al.. 1977; Osborne et al., 1978; Meehan et al., 1977; Meehan and Straub, 1979; Osborne et al., 1981) however, less is known about their formation in mouse skin or other target tissues in vitro. Less is known about the total spectrum of DNA-adducts formed in mouse epidermis from other PAH skin tumor initiators. DMBA, another widely studied PAH, is metabolized to a variety of reactive intermediates that bind extensively to epidermal DNA, including both syn and anti diol-epoxides (Dipple et al., 1984). Interestingly, these diol-epoxide metabolites of DMBA bind much more extensively to dAdo residues in epidermal DNA than B(a)P and it has been hypothesized that this preference for dAdo residues could account, ill part, for the difference in biological potency between B[a]P and DMBA (Dipple et al., 1984; DiGiovanni et al., 1986). The reader is referred to several extensive reviews on the subject of PAH DNA adduct formation in various cellular systems including mouse skin (Dipple et al., 1984; Baird and Pruess-Schwartz, 1988). Each PAH skin tumor initiator so far studied in sufficient detail appears to produce a somewhat unique spectrum of DNA adducts in vivo. This spectrum of DNA adducts appears to be reflected in the types of mutations observed in in vitro mutagenesis systems (Eisenstadt et al., 1982; Marshall et al., 1984; Vousden et al., 1986; Basu and Essigmann, 1988: Singer and Essigmann, 1991) and to a certain extent in a target gene in vivo (i.e. c - H a - r a s ) believed to be responsible for an initiating event in mouse epidermis (Yuspa and Poirer, 1988; Beland and Poirer, 1989; Cooper, 1990; see Section 3.4). Despite our extensive knowledge of the reactions of PAH with DNA, it remains to be precisely determined which PAH DNA adducts are most critical for the process of tumor-initiation. Several investigators have suggested that the major DNA lesions responsible for mutation induction and tumor initiation by bulky carcinogens such as the PAH could be carcinogen-induced apurinic/apyrimidinic sites (Drinkwater et al., 1980; Schaaper et al., 1982; Foster et al., 1983: Kunkel, 1984; Loeb and Preston, 1986; Takeshita et al., 1987). With regard to other types of skin tumor initiators such as the direct acting alkylating agents, u.v.-light, ionizing radiation, psoralens and other chemicals, much less is known about their interaction with mouse epidermal DNA in vivo. Nevertheless, some of these agents have been studied in detail in other model systems and specific DNA modifications (e.g. O6-alkyl-dGuo and O4-alkyl-deoxy-thymidine for alkylating agents; u.v.-induced cyclobutane dimers) have been correlated with their carcinogenic properties. The reader is referred to several recent articles and reviews dealing with the nature of DNA modifications by various classes of carcinogens and their relationship to mutagenesis, tumor initiation and carcinogenesis in various systems (Eisenstadt et al., 1982; Huttermann et al., 1983; Pegg, 1984; Singer, 1984; Lawley, 1984; Cimino et al., 1985: Hutchinson, 1985; Franklin and Haseltine, 1986; Singer, 1986; Vousden et al., 1986: Teoule, 1987~ Basu and Essigmann, 1988; Singer and Essigmann, 1991). 3.3. TARGET CELLS FOR TUMOR INITIATION

Because mouse epidermis is a differentiating tissue, this raises the question as to what are the exact target cells for tumor initiation. Mouse epidermis consists of a heterogeneous population of cells at different stages of terminal differentiation (Fischer et al., 1982b; Potten, 1983; Parkinson, 1985; Klein-Szanto et al., 1987). The fact that tumor initiation is essentially an irreversible event strongly supports the hypothesis that the target cells for the initiation stage of chemical carcinogenesis in mouse skin are epidermal stem cells (reviewed in Parkinson, 1985; Yuspa and Poirer, 1988). Evidence suggests that epidermal stem cells may reside both in the interfollicular as well as follicular epidermis (Potten, 1983) and it remains a controversial issue as to which of these stem cell populations are the major target for tumor-initiation and epidermal carcinogenesis (Morris et al., 1986; Klein-Szanto et al., 1987; Morris et al., 1990; Cotsarelis et al., 1990). Slaga and coworkers (Klein-Szanto and Slaga, 1981; Slaga and Klein-Szanto, 1983; Slaga, 1985) have suggested that the dark basal keratinocytes or 'dark cells' (DCs) found in mouse epidermis may be targets for skin tumor initiators based on a number of observations, including: (i) DCs are

Multistage carcinogenesis in mouse skin

71

present in the skin in large numbers during embryogenesis, in moderate numbers in newborns, in low numbers in young adults, and in very low numbers in old adults (Klein-Szanto and Slaga, 1981; Slaga and Klein-Szanto, 1983). The initiating potential of mouse skin decreases with the age of the mouse to the point that it is very difficult to initiate skin from mice older than one year; a time when few dark basal keratinocytes are present (Klein-Szanto and Slaga, 1981; Slaga and Klein-Szanto, 1983); and (ii) the sensitivity to transplacental initiation with DMBA, which is demonstrated postnatally by topical treatments with TPA, varies markedly according to the prenatal day of initiation, similar to the numerical distribution of DCs as a function of gestational age (Goerttler and L6ehrke, 1976; Goerttler et al., 1980; Slaga and Klein-Szanto, 1983). Some investigators have suggested that DCs are simply dead or dying cells (Glaso et al., 1986, 1989; Glaso and Hovig, 1987; Glaso and Haskjold, 1989; Glaso and Wetteland, 1990). However, Klein-Szanto and coworkers (Chiba et al., 1984; Klein-Szanto, 1984) have further characterized several types of DCs, including both viable and nonviable types. While these data are interesting, DCs remain essentially an enigma at the present time. Furthermore, the correlations noted above for DC number and susceptibility to initiation could be coincidental since elevated numbers of DCs are closely linked to the extent of cell proliferation in most mouse strains (Naito et al., 1988). As will be discussed in Section 3.5, one of the major factors affecting the extent of tumor initiation is the rate of cell proliferation at the time of initiation. Further work will be necessary to establish whether certain types of DCs are indeed targets for tumor initiators in mouse skin. In recent studies using mouse epidermis, density gradient centrifugation (i.e. continuous Percoll ~": gradients) has been used to separate keratinocytes into as many as 5 different subfractions based on their bouyant densities (Fischer et al., 1982; Klein-Szanto et al., 1987; Morris et al., 1990). Morphological, immunological, and autoradiographic studies have identified the 3 most dense fractions as consisting primarily of basal cells, while the uppermost fractions consist primarily of maturing keratinocytes (Fischer et al., 1982b; Baer-Dubowska et al., 1990; Morris et al., 1990 and reviewed in Klein-Szanto et al., 1987). Further analyses have revealed that those cells making up the most dense Percoll fraction (i.e. Fraction 5) are enriched with clonogenic cells, DNA synthetic activity in vivo, and [3H]thymidine 'label-retaining' cells. While the significance of these findings is not fully understood at the present time, such properties are believed to be characteristic of epidermal stem cells (Potten, 1983; Parkinson, 1985). Interestingly, of the 3 basal cell fractions obtained from continuous Percoil gradients, Fraction 5 cells (which contain both follicular as well as interfollicular 'label retaining' cells (Morris et al., 1990)) become enriched with B[a]P, DMBA, and ( + ) - a n t i - B P D E DNA adducts within a few hours after topical application of each compound (Baer-Dubowska et al., 1990). These results, using biochemical techniques, have confirmed other observations (Morris et al., 1986, 1990; Klein-Szanto et al., 1987) using autoradiographic techniques, demonstrating that the radioactive carcinogen 'label' was higher in the slowly cycling population of epidermal cells (i.e. [3H]thymidine 'label retaining' cells) one month after topical application of radiolabeled B[a]P. Subsequent experiments have identified these slowly cycling epidermal cells as part of Fraction 5 cells from the same type of continuous Percoll gradients (Morris et al., 1986, 1990). Thus, it appears that cells with the characteristics of epidermal stem cells could be at higher risk for an initiating event due to the rapid accumulation and persistence of carcinogen-induced DNA damage. The exact mechanism(s) for the observed differences in adduct levels among mouse epidermal subpopulations remain(s) unknown at present; however, several possibilities exist. The two most obvious are: (i) differential repair; and (ii) differential metabolism. With regard to the first possibility, several studies have suggested that the differentiation state of epidermal cells may play a role in their ability to undergo a DNA repair response (Yuspa and Harris, 1974; Bowden et al., 1977; Liu et al., 1983; Nakayama et al., 1984; Sawyer et al., 1988). Nakayama et al. (1984) reported that differentiating populations of newborn epidermal keratinocytes in culture efficiently removed DNA adducts formed from B[a]P. Furthermore, it has been recently reported (Sawyer et al., 1988; Gill et al., 1991) that cultures of differentiating mouse epidermal cells displayed significant DNA repair capacity (measured as unscheduled DNA synthesis) when exposed to a variety of chemical carcinogens including B[a]P, DMBA, and (+)-anti-BPDE. In contrast, Bowden et al. (1977) suggested that epidermal basal cells, but not epidermal cells that have been committed to the differentiation pathway, were capable of repairing u.v.-induced DNA damage. Liu et al. (1983)

72

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reported that the repair of u.v-light-induced DNA damage in human epidermal basal cells was much greater than in differentiated cells from the same skin preparation. Further experiments, examining the DNA repair capacity of epidermal subpopulations, will be necessary to determine the role this process plays in determining different DNA adduct levels among epidermal subpopulations and ultimately in the process of tumor initiation. An alternate hypothesis for the observed differences in DNA adduct levels in epidermal subpopulations is that metabolic differences may lead to higher initial adduct levels in some epidermal subpopulations. Differences in metabolic capability among epidermal subpopulations as a function of differentiation have been reported (Pohl et al., 1983; Coomes et al., 1983, 1984; DiGiovanni et al., 1989; Guo et al., 1990; Reiners et al., 1990, 1991a). In this regard, differentiating keratinocytes have been shown to have higher 7-ethoxycoumarin-O-deethylase activities (Pohl et al., 1983; Reiners et al., 1991a), as well as greater ability to metabolize both B[a]P and DMBA to DNA-binding metabolites (DiGiovanni et al., 1989). However, these differences may not be responsible for the differences in DNA adduct levels observed in epidermal subpopulations since higher DNA adduct levels were also observed following application of (_+)-anti-BPDE (Baer-Dubowska et al., 1990). Nevertheless, these observations raise the interesting possibility that some carcinogen metabolites may be produced in the more differentiated epidermal cell populations and transported to the less differentiated populations within the epidermis. Further work studying the role of various epidermal subpopulations in tumor initiation should provide greater insight into the carcinogenic process in this tissue. 3.4. GENES AND GENETIC CHANGES ASSOCIATEDWITH SKIN TUMOR INITIATION The capacity of many carcinogens to cause point mutations in DNA (Huberman and Barr, 1985; Cooper and Grover, 1990b), together with the irreversible nature of skin tumor initiation (reviewed in Boutwell, 1964; Slaga et al., 1982b; Huberman and Barr, 1985; Yuspa and Poirer, 1988) led to the hypothesis that initiation involves the induction of point mutations in a gene(s) that confers some selective growth advantage to the target epidermal cell(s). A number of genes have been found to be frequently altered by translocation or mutation in human or animal tumors (reviewed in Klein and Klein, 1984; Balmain, 1985; Weinberg, 1985; Barbacid, 1987; Guerrero and Pellicer, 1987; Balmain and Brown, 1988). Most genes in this category are members of the ras family, comprising Harvey-(Ha-), Kirsten-(Ki-), and N-ras, although several other genes have also been identified (Weinberg, 1985; Barbacid, 1987). Members of this gene family all encode a 21,000 Da membranebound protein (p21) which binds GTP and has intrinsic GTPase activity, although the exact cellular function of this protein has not been determined (Barbacid, 1987; Wakelman, 1990). Members in this gene family are almost invariably activated by point mutations in a select set of codons (primarily codons 12, 13 and 61) (Barbacid, 1987; Cooper, 1990). Activating mutations lead to single amino acid changes that alter the properties of the ras p21 and these altered proteins are characterized by greatly reduced GTPase activity (Trahey and McCormick, 1987; Wakelman, 1990). It has been postulated that ras p21 proteins may be similar to G-proteins and that the reduced GTPase activity could lead to the protein being in a permanent, GTP-bound state and hence locked in the 'on' position (reviewed in Balmain, 1985; Barbacid, 1987; Wakelam, 1990). This would have dramatic consequences for specific signal transduction pathways within the cell. Balmain et al. (1984) demonstrated that a high percentage of DMBA-induced mouse skin carcinomas and papillomas had an activated c-Ha-ras. In addition, the fact that this gene was activated in papillomas led to the conclusion that this alteration must have occured at a relatively early stage in the carcinogenic process. Further support for a role of H a - r a s activation in the process of skin tumor initiation comes from studies where an activated H a - r a s gene (i.e. v - H a - r a s ) has been introduced directly into epidermal cells in culture, allowing them to form papillomas in growth chambers on nude mice (Roop et al., 1986) or in vivo followed by treatment with TPA (Brown et al., 1986). In the latter study, although introduction of v - H a - r a s alone did not lead to the development of skin tumors, subsequent promoter treatment led to the formation of both papillomas and SCC. Finally, the importance of activated ras in mouse skin tumor initiation has been demonstrated by the development of skin tumors, primarily at sites of wounding in transgenic mice harboring an activated ras under the control of an epidermal keratin promoter

Ha-ras Ha-ras Ha-ras Ha-ras

G35+T Ala2-+T A18*+T G3’+A

N*-dGuo

not (1, not (1, not

not established (06-Me-dGuo) cyclobutane dimer, (6-4)photoproducts

WalP

/?-Propiolactone

NMU u.v.-light

et al., 1989

et al., 1988

et al., 1989

Brown et al., 1990 Pierceall et al., 1992

Brown et al., 1990

Bonham

Hochwalt

Akhurst

Bizub et al., 1986

Gill et al., 1992 Brown et al., 1990

Quintanilla et al., 1986; Bizub et al., 1986; Brown et a[., 1990 Husain et al., 1991

References

*Where limited data is available regarding the nature and spectrum of DNA adducts formed in mouse epidermal cells for a particular compound the words ‘not established’ have been entered. For those compounds where potential mutagenic lesions have been established in other tissues or from in vitro reactions, these are indicated in parentheses. References for these latter studies are as follows: 3-MCA (Osborne et al., 1986); fi-propiolactone (Sega et al., 1981; Chen et al., 1981; Mate et al., 1977); urethane (Miller and Miller, 1983); MNNG and MNU (Singer, 1986; Basu and Essigmann, 1988). fin this study, DMBA was used as the initiator followed by UVA-radiation as the promoter.

MNNG

established N6-etheno-dAdo) established N6-etheno-dAdo) established (06-Me-dGuo) Ha-ras N-ras N-ras N-ras

Ha-ras Ha-ras

Als2+T A”‘*+T

not established

DB(c,h)ACR

Papillomas; carcinomas Papillomas; carcinomas Papillomas Carcinomas Carcinomas Melanoma

Papillomas Papillomas; carcinomas Papillomas Papillomas; carcinomas Papillomas; carcinomas Carcinoma

Ha-ras Ha-ras

N6-dAdo not established N6-dAdo)

DB[aj]A MCA

Urethane

Melanotic tumors inc. malignant melanomas

N-ras

A’83+T, AJ8*+T, C”” +A Als2+T G’*+T

N6-dAdo

DMBAt

(N2-dGuo;

Papillomas

Ha-ras

Tumour type

A’82-+T

ras gene

Observed mutations

in ras Genes of Mouse Skin Tumors

N”-dAdo

initiator

and Point Mutations

DMBA

Tumor

between Carcinogen DNA-adducts

Potential mutagenic DNA adduct found in mouse epidermis*

TABLE 2. Relationship

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J. DIGIOVANNI

(Bailleul et al., 1990). These studies as well as others (reviewed in Balmain, 1986) strongly implicate ras gene involvement in mouse skin tumor initiation. Evidence also has been emerging that point mutations in the mouse c - H a - r a s gene obtained from skin papillomas and carcinomas can be highly dependent on the initiator (Quintanilla et al.. 1986; Bizub et al., 1986; Barbacid, 1987; Guerrero and Pellicer, 1987; Balmain and Brown, 1988; Cooper, 1990). Whereas tumors from mice initiated with DMBA, dibenz[c,h]acridine, and urethane produced almost exclusively A~S2~T transversions at codon 61 of the second exon of c-Ha-ras, methylnitrosourea (MNU)-, or N - m e t h y l - N ' - n i t r o - N - n i t r o s o g u a n i d i n e (MNNG)-induced papillomas and carcinomas did not have this point mutation in their DNA (Quintanilla et al., 1986; Bizub et al., 1986; Bonham et al., 1989; Brown et al., 1990). In fact, M N U or MNNG-induced papillomas contained primarily G35--*A transition mutations in codon 12 of c-Ha-ras (Brown et al., 1990). These observations are similar to those of Barbacid and coworkers (Zarbl et al., 1985; Barbacid, 1986, 1987) where DNA from rat mammary carcinomas induced by M N U contained an activated c - H a - r a s gene with a point mutation in codon 12, whereas, with DMBA the same A~-~-,T transversion in codon 61 was observed. Recent studies have characterized the DNA adducts found in mouse epidermal D N A (Nair et al., 1991; DiGiovanni et al., 1991a) and the c-Ha-ras mutations in skin tumors resulting from topical application of dibenz[a,j]anthracene (DB[a,j]A) (DiGiovanni et al., 1991a; Gill et al., 1992). A high percentage (80%) of skin papillomas initiated by this compound possessed A~s2---}T transversion mutations in codon 61 of c-Ha-ras. In addition, papillomas induced by initiation with the putative ultimate tumor initiating diol-epoxide of DB[a,j]A (anti-DB[a,j]A-3,4-diol-l,2-epoxide) possessed the same mutation as those found in tumors initiated with the parent hydrocarbon, i.e. AtS2--}T transversions in codon 61. These mutations in c - H a - r a s are consistent with the formation of specific dAdo adducts from the anti-diol-epoxide formed in mouse epidermal cells following exposure to the parent hydrocarbon, DB[a,j]A (Nair et al., 1991; DiGiovanni et al., 1991a). Table 2 summarizes what is currently known concerning the relationship between carcinogen-induced DNA modification in mouse skin and point mutations in ras genes of mouse skin tumors. The reader is referred to several reviews which deal with this relationship in various tissues in addition to mouse skin (Barbacid, 1987; Guerrero and Pellicer, 1987; Cooper, 1990). It should be noted that skin papillomas and carcinomas induced by promoter treatment alone have been reported to contain an activated c-Ha-ras (61 ~ codon mutation) (Pelling et al., 1988). While it was hypothesized that the observed mutations arose 'spontaneously' the possibility that these mice, as well as other mice reported to develop skin tumors in the absence of initiator treatment, were exposed to some endogenous or exogenous initiator cannot be ruled out at the present time. Although there are still many questions that remain to be answered about proto-oncogene activation and chemical carcinogenesis, the collective data using the mouse skin model suggest that: (i) activation o f r a s is an initiating event in this tissue; and (ii) where sufficient information is available there is a good correlation between carcinogenspecific DNA damage and mutation induction in this target gene.

3.5. MODIFYING FACTORS AFFECTINGTUMOR INITIATIONIN MOUSE SKIN Table 3 lists some of the modifying factors that can affect the process of tumor-initiation in mouse skin. Potential modifiers or modifying factors of tumor initiation are diverse and may affect various aspects of this process. The study of modifying factors has in many instances greatly aided our understanding of the tumor initiation stage of carcinogenesis in mouse skin as well as other tissues. As discussed above, PAHs, the most widely studied mouse skin tumor initiators, are metabolized by a variety of enzyme systems to many metabolites in addition to the ultimate tumor initiating diol-epoxide metabolites. A majority of the exogenous and endogenous modifying factors affecting skin tumor initiation, that have been studied to date, primarily affect the metabolic activation and/or detoxication of skin tumor initiators such as PAHs. Table 3 lists some of the major classes or types of exogenous chemicals that have been shown to inhibit tumor initiation in mouse skin. A number of antioxidants have been shown to inhibit mouse skin tumor initiation by DMBA including: BHA, BHT, vitamin E, and vitamin C (Slaga and DiGiovanni, 1984; DiGiovanni, 1990). Antioxidants may protect against the initiation of chemically induced tumors in a variety of tissues,

Multistage carcinogenesis in mouse skin

75

TABLE3. General Factors Modifying Skin Tumor Inith~tion in Mice Factor Exogenous chemicals Antiinitiators Coinitiators Age Sex Hormonal balance Diurnal rhythms Dietary factors Viral Radiation exposure Genetic factors

Specific examples Antioxidants, flavones and polyphenols, chlorinated hydrocarbons and other enzyme inducers, weakly or non carcinogenic PAH, antiinflammatory steroids, prostaglandin synthesis inhibitors, etc. Weakly or noncarcinogenic PAH, catechols, tumor promoters and other agents stimulating epidermal DNA synthesis, prostaglandin precursors, etc.

Dietary fat, caloric restriction Constitutive expression and inducibility of metabolizing enzymes e.g. cytochromes P-450; genetic differences in DNA repair

including mouse skin, primarily by altering the enzymes responsible for both the activation and detoxification of carcinogens (Slaga and DiGiovanni, 1984; DiGiovanni, 1990). The mechanism of inhibition of PAH skin tumor initiation by flavones (e.g. 7,8-BF, 5,6-BF, and quercetin) when applied simultaneously with several PAHs, results, at least in part, from inhibition of the epidermal enzyme system(s) (i.e. cytochrome(s) P-450) responsible for generating the electrophilic diol-epoxide intermediates that bind to epidermal DNA. On the other hand, prior exposure to some flavones that also possess enzyme-inducing activity (e.g. 5,6-BF) may reduce PAH skin tumor initiation by enhancing detoxification pathways (Slaga and DiGiovanni, 1984; DiGiovanni, 1990). Ellagic acid (EA), a widely distributed plant phenol has been shown to inhibit PAH-induced skin tumorigenesis (Lesca, 1983; Mukhtar et al., 1984, 1986). The exact mechanism(s) of inhibition by EA is not known, but several have been postulated (DiGiovanni, 1990) including: (i) inhibition of the metabolism and metabolic activation of PAH; (ii) promoting the detoxification of PAH, and/or (iii) serving as a scavenger of the reactive diol-epoxides of the PAH. Enzyme induction has been postulated as a mechanism for the anti-initiating activity of a number of compounds including certain flavones as noted above (see again Table 3). The most potent member and prototype of this group of compounds is 2,3,7,8-tetrachlorodibenzo-p-doxin (TCDD). The mechanistic studies that have been conducted in mouse skin, indicate that inhibition of tumorigenesis correlates with increased enzyme activities, both oxidative and conjugative, and reduced formation of DNA adducts in target epidermal cells (DiGiovanni and Slaga, 1981; Slaga and DiGiovanni, 1984; DiGiovanni, 1990). A number of other classes or types of chemicals have been shown to consistently inhibit tumor initiation by PAH, including: (i) weakly carcinogenic or noncarcinogenic PAHs (reviewed in DiGiovanni, 1990); (ii) anti-inflammatory steroids (Ghadially and Green, 1954; Baserga and Shubik, 1954; Slaga and Scribner, 1973; Thompson and Slaga, 1976b; Viaje et al., 1978; and reviewed in DiGiovanni, 1990); (iii) inhibitors of arachidonic acid metabolism (Fischer et al., 1979, 1989; and reviewed in DiGiovanni, 1990); and (iv) suicide inhibitors of cytochrome P-450 (Viaje et al., 1990; Alworth et al., 1991). In addition to these major classes or types of exogenous or chemical modifiers of tumor initiation listed in Table 3, other miscellaneous agents have been identified. The reader is referred here to several recent, more detailed reviews on exogenous inhibitors of tumor initiation and chemical carcinogenesis and their proposed mechanism(s) of action (Slaga and DiGiovanni, 1984; DiGiovanni, 1990). A number of exogenous chemical agents are capable of acting as coinitiators. A coinitiator is defined as a chemical devoid of tumor initiating activity at the dose utilized but capable of enhancing the initiating activity of another skin tumor initiator. Some chemical agents that fit this definition are also listed in Table 3. Van Duuren and co-workers (Van Duuren and Goldschmidt, 1976; Van Duuren et al., 1978b) found that several weakly or noncarcinogenic PAH including pyrene, fluoranthene, and benzo[e]pyrene (B[e]P) were co-carcinogenic when applied repeatedly with B[a]P to the skins of mice. Subsequent studies using initiation-promotion regimens

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J. DIGIOVANNI

demonstrated that this action could be attributed, in part, to a co-initiating effect (reviewed in DiGiovanni, 1990). Catechol, a major constituent in cigarette smoke is also an effective cocarcinogen when applied simultaneously with B[a]P to mouse skin (Van Duuren and Goldschimdt, 1976; Van Duuren et al., 1978b). Recent studies using the mouse skin system have demonstrated only a weak coinitiating activity of catechol (Melikian et al., 1989b). The mechanism for this weak coinitiating effect of catechol, like that of B[e]P, may be related to alterations in the metabolic activation of B[a]P leading to slight changes in the relative proportions of B[a]P-diol-epoxide DNA adducts formed (Melikian et al., 1986, 1989a; Smolarek et al., 1987). It is interesting to point out that B[e]P, when applied simultaneously with DMBA, inhibited tumor initiation (DiGiovanni, 1990). These differences are believed to be the result of differences in the pathway(s) of metabolic activation for B[a]P vs DMBA in mouse epidermis. These observations serve to illustrate some of the complexities of studying exogenous modifiers of tumor initiation and carcinogenesis. It has been known for some time that the rate of epidermal DNA synthesis at the time of initiator application can affect the initiating response. In this regard, agents or manipulations that increase epidermal DNA synthesis rates at the time of carcinogen application can act as coinitiators. For example, the diurnal variation of susceptibility to initiation directly follows the diurnal variation in epidermal DNA synthesis (Frei and Ritchie, 1964). Treatment of mouse epidermis with colchicine followed 9 hr later (peak enhancement in epidermal DNA synthesis) by application of the initiator, urethan, leads to an enhanced initiating response to this agent (Berenblum and Armuth, 1977). Finally, chemical agents such as croton oil which lead to increased epidermal DNA synthesis, when given at appropriate times prior to initiation enhance initiation with urethan, DMBA and MNNG (Hennings et al., 1969, 1973, 1978). Thus, it would appear from these studies that the fraction of ceils that are cycling through a critical phase of the cell cycle (S phase) at the time of carcinogen application is a very important determinant in the magnitude of the tumor initiation response. Only recently have studies begun to re-address questions regarding the role of normal dietary constituents and parameters such as dietary fat, protein, fiber and total calories in modulating skin carcinogenesis using the mouse skin model (Black et al., 1983, 1985; Reeve et al., 1988; DiGiovanni, 1991). Boutwell (1964) reported some 40 years ago that caloric restriction was without effect on the initiation stage of mouse skin carcinogenesis (Boutwell and Rusch, 1951; Boutwell, 1964). More recent studies have reported that feeding a high corn oil (20% corn oil) diet during the initiation phase (using DMBA as initiator) reduced the number of papillomas per mouse (Birt et al., 1989). The mechanism for this effect of corn oil (i.e. increased dietary saturated fat) is not known but could involve effects on DMBA metabolism or possibly on epidermal DNA synthesis rates. On the other hand, Locniskar et al. (1991) found little effect of different types of dietary fat up to a maximum of 10% total dietary fat on skin tumor initiation by DMBA. While these studies differ somewhat, the mouse skin model would appear to be ideal for studying the effects of dietary parameters on both the initiation and promotion stages of carcinogenesis (also see Section 4.5). Genetic differences can play an important role in modulating susceptibility to multistage mouse skin carcinogenesis. A number of studies have demonstrated that a major determinant of susceptibility to multistage skin carcinogenesis among mouse strains and between species is their susceptibility to the tumor promotion stage (DiGiovanni, 1989b,c; see Section 4.5). Nevertheless, numerous studies have suggested a positive correlation between the level of AHH (both basal and induced) and susceptibility to tumorigenesis by PAH in several tissues and within the same tissue when comparing different mouse strains (Kouri et al., 1973, 1974, 1978; Kouri, 1976; Nebert et al., 1977, 1978; Nebert, 1980). Nebert and Gelboin (1969) found that certain strains of mice could be classified as being 'responsive' to induction of AHH activity in liver by PAH, whereas others were 'non-responsive'. This variation in enzyme induction has been extensively studied in the mouse and has been linked to the expression of a cytosolic protein, MW 95 kDa (i.e. the Ah-receptor), which is capable of binding PAH within the cell (Poland and Glover, 1988; Fernandez et al., 1988), an event postulated to result in its translocation to the nucleus ultimately leading to the expression of a number of genes encoding cytochrome P-450 (primarily the P-450I family) and other enzyme activities (Nebert and Jensen, 1979; Nebert and Gonzalez, 1987). It was hypothesized that 'responsive' strains would demonstrate increased susceptibility to PAHinduced tumorigenesis compared with equivalent 'non-responsive' animals. In general this has been

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77

found to be the case when using complete carcinogenic regimens, but the correlation is not necessarily a strict one (Kinoshita and Gelboin, 1972). For instance, which PAH is used as the carcinogen (Kouri et al., 1973; Nebert and Jensen, 1979), the route of its administration (Legraverend et al., 1980) or the tissue being examined (Nebert and Gelboin, 1969; Seifried et al., 1977; Okey et al., 1979) may all be important determining factors. Again, there are particular strains (Seifried et al., 1977) and species (Bickers et al., 1983) that may be defined as being ‘responsive’ and yet they are resistant to hydrocarbon-induced tumorigenesis. In theory, such differences could have a dramatic influence on susceptibility to skin tumor initiation in mice. Legraverend et al. (1980) have reported a correlation between the relative amounts of induced B[a]P metabolism in mouse skin microsomes and B[a]P-induced complete carcinogenesis in mouse skin using three inbred mouse strains. However, to date it has not been possible to clearly establish a correlation between levels of tissue monooxygenase activity (basal or induced) and tumor initiation by PAH in mouse skin (Nebert et al., 1972; Benedict et al., 1972; Burki et al., 1973; DiGiovanni et al., 1979, 1980; Reiners et al., 1984). This could be due to the fact that mouse epidermis constitutively expresses the major cytochrome P-450 specie(s) (e.g. P4501Al) responsible for metabolic activation of PAH carcinogens to reactive diol-epoxides at levels sufficient for tumor initiation under most experimental conditions (see DiGiovanni, 1989a). Alternatively, as discussed above and in more detail in Section 4.5, this may be due to the dramatic differences in susceptibility to tumor promoting agents that may overshadow more subtle differences related to initiation events (DiGiovanni, 1989b,c). It has been postulated that the initiation phase of mouse skin tumorigenesis is the result of persistent lesions which are not adequately removed by DNA repair systems prior to DNA replication (Trosko and Chu, 1975; Roberts, 1980). Alternatively, such lesions may be repaired in an error-prone fashion thus introducing mutagenic lesions in the DNA (Trosko and Chu, 1975; Roberts, 1980). Several studies have investigated the ability of genotoxic agents to induce a DNA repair response in skin cells (Taichman and Setlow, 1975; Sutherland et al., 1980; Hanawalt et al., 1981; Gibson-D’Ambrosio et al., 1983; Eggset et al., 1983; Hosomi and Kuroki, 1985; Sawyer et al., 1988; Gill et al., 1991). u.v.-Light has been shown to induce DNA repair in normal human skin, although irradiation of skin from patients suffering from xeroderma pigmentosum induces little, if any, repair synthesis (Epstein et al., 1970; Cleaver and Gruenert, 1984). This disease, which is a genetic disorder characterized by a deficiency in DNA excision repair and a heightened sensitivity to u.v.-irradiation, results in multiple skin cancers during the first few years of life (Lynch et al., 1980). u.v.-Induced DNA repair has also been demonstrated in primary cultures of neonatal mouse epidermal cells (Bowden et al., 1975, 1977, 1978) and in mouse skin in vivo (Kodama et al., 1984; Ishikawa and Sakurai, 1986). Hennings and coworkers (Hennings et al., 1974; Hennings and Michael, 1976) measured guanine-specific DNA repair in the DNA of non-replicating cells in mixed epidermal-fibroblast cultures treated with either b-propiolactone or MNNG and adult mouse epidermal cells have been used to examine unscheduled DNA synthesis in response to a variety of genotoxic agents (Sawyer et al., 1988; Gill et al., 1991). However, little additional work has been done on chemically induced DNA repair in skin of mice, especially with regard to PAH-induced DNA damage and tumor initiation. It is generally believed that rodent cells are less efficient at DNA repair than human cells (Sutherland et al., 1974, 1980; Ley et al., 1977; Hewitt and Meyn, 1978; Yagi et al., 1984). This could help explain why rodents (i.e. mice) are, in general, quite susceptible to chemical carcinogenesis. At present, little is known about genetic differences in DNA repair between mouse strains and their role in modifying tumor initiation and multistage carcinogenesis in mouse skin (Ley et al., 1977; Strickland and Strickland, 1984; Sanford et al., 1989). Other factors have been shown to modify or have the potential to modify tumor initiation and the reader is referred to specific articles for additional information; age (Roe et al., 1972; Ebbesen, 1974; Van Duuren et al., 1975, 1978a; Peto et al., 1975; Loehrke et al., 1983); sex (Naito et al., 1988; Eastin, 1989; Bangrazi et al., 1990); hormonal balance (Boutwell, 1964; Slaga, 1980); immune status (Curtis et al., 1975; Stenback et al., 1979; Slaga, 1980; Inoue et al., 1981); viral infection (Tennant and Rascati, 1980; Burns and Murray, 1981; Peck et al., 1983); and radiation exposure (Ullrich, 1980; Slaga, 1980).

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J. DIGIOVANNI 4. T U M O R PROMOTION 4.1. NATUREOF SKIN TUMOR PROMOTERS

As noted earlier in this review, the phorbol esters are the most widely studied skin tumor promoters. These compounds are derivatives of the tetracyclic diterpene phorbol, esterified in the 12 and 13 positions (see Fig. 3). The tumor-promoting activity of the phorbol esters appears to be related to a delicate hydrophobic-hydrophilic balance determined by positions 12-O, 13-O and 20. TPA is the most potent of the phorbol ester series (Hecker, 1971, 1978). The structure-activity relationships of phorbol esters, as well as other diterpene esters, have been reported in detail by Hecker (1971, 1978). The parent alcohol, phorbol, the monoesters, the very short chain diesters, 4~-phorbol-12,13-didecanoate (4~PDD) and 4-O-methyl TPA are inactive as skin tumor promoters (Hecker, 1978). It is of interest to point out that the overall configuration of the phorbol molecule is critical, since changing the C-4 hydroxyl to the a position as in 4~PDD dramatically changes the three-dimensional shape of the molecule and destroys its skin tumor promoting activity (Hecker, 1978). Although 4-O-methyl TPA is non-promoting, its three-dimensional structure is very close to that of TPA (Hecker, 1978). An explanation for its lack of promoting activity is presently not known. Although the phorbol esters have been the most widely studied skin tumor promoter to date, many other chemical compounds have been shown to possess skin tumor promoting properties.

OCO(CH2)12C3H 19~1~""16

HI,~ O ' ~O~_ R~I~..,.,. 0

CH~O R

31 0 OH

OH

0CH7 OH Debromoaplyslatoxin,R=H Aplyslatoxin,R=Br

TPA

"•

H

HO

0

OH

~ C H 3 1,8-dlhydroxy-3-methyl-9-anthrone (Chrysarobln)

,~

~

TeleocldlnA

o

o

II

II

O'o-oJ'O " ~

CH2B r 7-bromomethylbenz[a]anthracene

Benzoylperoxide FIG. 3. Chemical structures of representative skin tumor promoters, including TPA (upper left).

Multistage carcinogenesis in mouse skin

79

Teleocidin, isolated from Streptomyces mediocidicus, is an indole alkaloid composed of telocidin A and telocidin B and their isomers. Both teleocidin and dihydroteleocidin B, a catalytically hydrogenated derivative of telocidin B, induce many of the same biologic and biochemical effects and have promoting activity comparable to that of TPA (Fujiki et al., 1981, 1982a; Suganuma et al., 1982). Lyngbyatoxin was first isolated from the Hawaiian seaweed L y n g b y a majuscula by Cardellina and co-workers (1979). It was subsequently shown to be structurally identical to telocidin A (Sugimura, 1982). Two polyacetates that differ in chemical structure only by the presence or absence of a bromine residue in the hydrophilic region of the molecule were isolated from the seaweed L y n g b y a gracilis (Sugimura, 1982). Both induce many of the same effects as TPA, but aplysiatoxin is a good promoter, whereas debromoaplysiatoxin, which lacks the bromine residue, is a very weak promoter (Sugimura, 1982; Fujiki et al., 1982b; Shimomura et al., 1983). A number of more recently discovered skin tumor promoting agents include: palytoxin (Fujiki et al., 1986, 1989); thapsigargin (Haikii et al., 1986; Fujiki et al., 1989); calyculin A (Suganuma et al., 1990; Fujiki et al., 1989); and okadaic acid (Fujiki et al., 1987, 1989; Suganuma et al., 1988). These agents appear to be distinct from the phorbol esters and similar acting compounds in many of their initial biochemical actions and as discussed below also in their proposed initial mechanism(s) of action (Fujiki et al., 1989). Other examples of chemical mouse skin tumor promoters include: fatty acid methylesters (Arffman and Glavind, 1971), anthrones such as anthralin (Bock and Burns, 1963; Van Duuren et al., 1978b) and chrysarobin (DiGiovanni and Boutwell, 1983), iodoacetic acid (Gwynn and Salaman, 1953), the weakly acidic fraction of cigarette smoke condensate (Hecht et al., 1975; Wynder and Hoffman, 1961; Bock et al., 1971), hydrocarbons such as 7-bromomethyibenz[a]anthracene and B[e]P (Scribner and Scribner, 1980; Slaga et al., 1979), benzoyl peroxide (BzP) (Slaga et al., 1981), retinoic acid (RA) (Hennings et al., 1982), and 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD) (Poland et al., 1982). Table 4 lists many of these chemical skin tumor promoting agents and their relative potency compared with the phorbol esters. The chemical structures of some selected skin tumor promoters are also shown in Fig. 3. In addition to chemical promoting agents, a number of other types of stimuli can act as promoters of skin tumors in this model system, u.v.-Light has a strong promoting action in mouse skin (Verma et al., 1979a; Lowe et al., 1978; Husain et al., 1991). Physical trauma of sufficient magnitude has long been known to promote skin tumor formation in previously initiated mice (Pullinger, 1945; Hennings and Boutwell, 1970; Clark-Lewis and Murray, 1978; Argyris, 1980,

TABLE4. Diversity of Chemical Skin Tumor Promoters Promoters

Activity

Croton oil Certain phorbol esters found in croton oil Some synthetic phorbol esters Certain euphorbia lattices Teleocidins Polyacetates (e.g. aplysiatoxin) Okadaic acid Calyculin A Palytoxin Thapsigargin Anthrones (e.g. anthralin, chrysarobin) Extracts of unburned tobacco Tobacco smoke condensate l-Fluoro-2,4-dinitrobenzene 7-Bromomethylbenz[a]anthracene Benzo(e)pyrene Benzoyl peroxide Certain fatty acids and fatty acid methyl esters Certain long chain alkanes A number of phenolic compounds Surface-active agents (sodium lauryl sulfate, Tween 60) Citrus oils Iodoacetic acid

Strong Strong Strong Strong Strong Strong Strong Strong Strong Strong Moderate Moderate Moderate Moderate Moderate Moderate Moderate Weak Weak Weak Weak Weak Weak

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J. DIGIOVANNI

1985, 1989). In this regard, repeated physical abrasion can promote skin tumors (Argyris, 1980, 1985, 1989). Finally, full thickness skin wounding is a very strong promoting stimulus for epidermal tumorigenesis (Hennings and Boutwell, 1970; Clark-Lewis and Murray, 1978). Recently, another type of physical skin tumor promoting agent has been identified; biogenic silica fibers (Bhatt et al., 1984). These fibers, obtained from the grain of Phalaris canariensis, are effective skin tumor promoters when rubbed on the backs of previously-initiated mice (Bhatt et al., 1984). Interestingly, recent evidence suggests that these biogenic silica fibers work by a mechanism distinctly different from physical abrasion (Bhatt et al., 1992). One has to marvel at the diversity of stimuli that possess skin tumor promoting activity and whether there are some common effects at the biochemical and molecular level responsible for tumor promotion by all these different agents. Unlike the vast majority of skin tumor initiators that are chemically reactive, per se, or require 'activation' (spontaneously or metabolically) to be effective, many chemical skin tumor promoters do not appear to require metabolic activation for their effects. Investigators in several laboratories have clearly shown that TPA does not require metabolic activation for its promoting action in mouse skin (Kreibich et al., 1974; Berry et al., 1977, 1978). The major metabolic pathways for TPA in mouse skin involve formation of monoesters and the parent alcohol, phorbol. All of these metabolites are significantly less active than TPA and the parent alcohol, phorbol, is devoid of promoting activity as noted above. Little information is available on potential metabolic pathways for other classes of skin tumor promoters. Hagiwara et al. (1987) recently reported that the indole alkaloid promoter ( - )-indolactam V (chemically related to the teleocidins) is metabolized by the microsomal cytochrome P-450 system to several less biologically active derivatives. These data suggest that other indole alkaloids such as the teleocidins may follow similar metabolic pathways leading primarily to detoxification products. Interestingly, some classes of tumor promoters such as the anthrones, organic peroxides, hydrocarbons, and quinones, do appear to require conversion, either spontaneously or enzymatically, to reactive intermediates for their promoting action. Studies of the skin tumor promoter, butylated hydroxytoluene hydroperoxide (BHTOOH), suggest that one or more reactive intermediate including: phenoxyl, peroxyl, quinoxyl or quinone methide, may be involved in its promoting action (Taffe et al., 1987, 1989; Taffe and Kensler, 1988). Anthrone derivatives such as anthralin and chrysarobin autoxidize to a variety of reactive intermediates including: anthranyl, peroxyl, and semiquinone (Martinmaa et al., 1978, 1981; Mustakallio, 1981; Whitefield, 1981; Krebs et al., 1981; DiGiovanni et al., 1988a). In addition, anthrones generate superoxide anion (02) during their autoxidation (Mustakallio, 1981). The available evidence indicates that one or more of these free radical intermediates are responsible for the promoting action of anthrones (reviewed in DiGiovanni et al., 1988a). The hydrocarbons and quinones have not been studied in detail in terms of their potential metabolic activation for tumor promotion. However, redox-cycling (Cerutti, 1985) does not appear to be responsible for the tumor promoting actions of some quinones (Monks et al., 1990). For the other compounds listed in Table 4, there are no compelling data at present to suggest that spontaneous or metabolic activation plays an important role in their tumor promoting actions.

4.2. CELLULAR, BIOCHEMICALAND MOLECULAR CHANGESASSOCIATEDWITH SKIN TUMOR PROMOTION 4.2.1. Cellular Changes Tumor promoting agents produce substantial cellular changes when topically applied to mouse epidermis. Within a few hours after application of a single effective dose of the phorbol ester, TPA, to mouse skin, localized edema and erythema characteristic of inflammation and irritation are evident and, by 24 hr, there is leukocytic infiltration of the dermis (Stenback et al., 1974; Scribner and Suss, 1978). At that time there is also a 5- to 10-fold increase in the percentage of DCs in the interfollicular epidermis (Raick, 1973a; Klein-Szanto et al., 1980; Klein-Szanto and Slaga, 1981). These DCs are characterized by their strong basophilia, dense chromatin, and large numbers of free ribosomes. They increase in number in TPA-induced hyperplasia to a greater extent than in hyperplasia induced by mezerein or more weakly promoting hyperplastic agents (Raick and

Multistage carcinogenesis in mouse skin

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Burdzy, 1973; Klein-Szanto et al., 1980). These observations have led to the hypothesis that an increase in their number may be an important component of the promotion stage of skin carcinogenesis (Klein-Szanto and Slaga, 1981; Slaga and Klein-Szanto, 1983; Slaga, 1985). Again, various investigators have reported the presence of both viable and nonviable DCs in promotertreated epidermis (Parsons et aL, 1983; Glaso et aL, 1986, 1989; Glaso and Hovig, 1987; Glaso and Haskjold, 1989; Glaso and Wetteland, 1990; Murakami et al., 1985; Naito et al., 1988; Chiba et al., 1984; Klein-Szanto, 1984). The importance of any or all of these types of DCs in the process of tumor promotion remains an open question at present. Within 1-2 days after a single promoter treatment, stimulation of mitotic activity in the basal cell layer of the epidermis continues for several days and results in an increased number of nucleated cell layers (Raick, 1973a). This is followed by a phase of increased keratinization of the upper layers of the epidermis (Bach and Goerttler, 1971; Raick, 1973a; Balmain, 1976). Without additional promoter treatments, all these responses to the promoter gradually subside and the epidermis regains its normal appearance within approximately 2-3 weeks of treatment (Raick, 1973b). Repeated promoter treatment, however, prevents this decrease in response, and the skin appears to be in a chronic state of irritation and regenerative hyperplasia (Aldaz et al., 1985). In fact, repeated treatment with TPA leads to a potentiation of the hyperplasia response in species and mouse strains that are susceptible to skin tumor promotion by phorbol esters (Sisskin et al., 1982; Naito and DiGiovanni, 1989). Table 5 illustrates the potentiated hyperplasia that occurs after 4 or more topical applications of TPA or chrysarobin to a promotion responsive mouse stock (i.e. SENCAR) compared to a single application. Note that where adequately tested all tumor promoters produce such a potentiated hyperplasia although the magnitude and kinetics of this response can differ with each type of promoting agent (Argyris, 1985, 1989; Kruszewski et al., 1989). The ability to produce a potentiated hyperplasia after multiple treatments and the magnitude of this response appear to correlate most closely with the tumor promoting ability of various compounds (Argyris, 1985, 1989; Naito et al., 1987; Kruszewski et aL, 1989). It has been argued for many years that the induction of cell proliferation and hyperplasia was a necessary but not sufficient condition for tumor promotion in the mouse skin model (Boutwell, 1974, 1976; Slaga et al., 1975; Scribner and Suss, 1978). This argument was based on the observations that certain chemicals could produce dramatic epidermal hyperplasia after a single application (e.g. acetic acid, mezerein, ethyl phenyl propiolate (EPP)) and yet these compounds exhibited only poor papilloma promoting ability (Raick and Burdzy, 1973; Slaga et al., 1975; Slaga, 1984b; Mufson et al., 1979). However, careful examination of these compounds revealed that they are unable to maintain a potentiated epidermal hyperplasia and cell proliferation when given repeatedly due, in part, to severe epidermal toxicity (Argyris, 1983a,b, 1985, 1989; Naito et al., 1987; Baxter et al., 1989). Epidermal toxicity appears to be an important limiting factor in the promoting activity of anthrones (Kruszewski et al., 1987) and EPP (Cameron et al., 1991). These TABLE5. Comparison of Epidermal Hyperplasia Induced by TPA and Chrysarobin Following Single and Multiple Treatments* Treatment Epidermal protocol thickness(#m) Single 15.8 + 1.0 Multiple 15.0 _ 1.0 TPA (3.4 nmol) Single 42.8 + 2 Multiple 71.5 _ 4.9t Chrysarobin (220 nmol) Single 20.8 + 1.2 Multiple 54.2 _ 3.2t *Three female SENCAR mice were used for each experimental group. TPA (3.4 nmol) was applied either as a single application or as 5 applications given twice-weekly over a 2½ week period. Chrysarobin (220 nmol) was applied either as a single application or as 5 applications given once-weekly over 5 weeks. Animals were sacrificed 48 hr after the last application. Data are from Kruszewski et al., 1989. tSignificantly greater (p < 0.05) than the value single application. Compound (dose) Acetone (0.2 ml)

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J. DIGIOVANNI

observations as well as the fact that regenerative hyperplasia alone can promote skin tumors (Argyris 1980, 1985) supports the hypothesis that epidermal hyperplasia and cell proliferation of a specific type, magnitude, and duration is sufficient for skin tumor promotion in susceptible mouse strains and stocks (Argyris, 1985, 1989). With repeated TPA treatment of initiated skin, benign tumors begin to appear in about 6 weeks (Boutwell, 1964) and, with some mouse stocks such as the selectively bred SENCAR, there can be as many as 20-30 papillomas per mouse after 15 weeks of promoter treatment (DiGiovanni et al., 1980).

4.2.2. Biochemical and Molecular Changes and the Role o f Protein Kinase C Phorbol diesters with promoting activity produce an initial inhibition of tritiated thymidine incorporation into epidermal DNA (Paul and Hecker, 1969; Baird et al., 1971; Raick, 1973a). This is soon followed by greatly increased rates of nucleic acid and protein synthesis (Paul and Hecker, 1969; Baird et al., 1971; Hennings and Boutwell, 1970). Promoter-treated skin shows an increase in phospholipid turnover (Suss et al., 1971; Rohrschneider et al., 1972; Balmain and Hecker, 1974) and prostaglandin accumulation (Verma et al., 1980a; Fiirstenberger and Marks, 1980), a decreased responsiveness to epidermal chalones (Marks et al., 1978; Marks, 1976) and/3-adrenergic agonists (Marks and Grimm, 1972; Grimm and Marks, 1974; Verma and Murray, 1974; Mufson et al., 1977), a decrease in the basal activities of epidermal superoxide dismutase (SOD) and catalase (Solanki et al., 1981a; Reiners et al., 1991b), a decrease in the glucocorticoid receptor (Davidson and Slaga, 1982, 1983; Warren et al., 1991), and an increase in the activity of xanthine oxidase (Reiners et al., 1991b). Tumor promoter treatments also lead to a decrease in epidermal histidase (Colburn et al., 1975) and histidine decarboxylase (Watanabe et, al., 1981), modification of epidermal keratins and keratin expression (Balmain, 1976; Schweizer and Winter, 1982; Nelson and Slaga, 1982; Roop et al., 1983), increased synthesis and phosphorylation of histones (Raineri et al., 1973, 1978; Link and Marks, 1981), and a large induction of ornithine decarboxylase (ODC) (O'Brien et al., 1975a,b), the rate-limiting enzyme in polyamine biosynthesis. As a result of the induction of epidermal ODC, the levels of putrescine and especially spermidine in the epidermis become elevated (O'Brien, 1976; Weeks and Slaga, 1979; Astrup and Paulsen, 1981; Kruszewski and DiGiovanni, 1988). The elevated ratio of spermidine:spermine that occurs after TPA treatment is tightly linked to the DNA synthesis response induced by this promoter (Astrup and Paulsen, 1981 ; Kruszewski and DiGiovanni, 1988). Specific high-affinity phorbol ester membrane-binding sites were initially demonstrated in a variety of tissues, including mouse epidermis (using either particulate fractions or whole cells) (Delclos et al., 1980; Shoyab and Todaro, 1980; Driedger and Blumberg, 1980; Dunphy et al., 1980; Nagle et al., 1981; Ashendel and Boutwell, 1981; Solanki and Slaga, 1981; Solanki et al., 1981b~ Sando et al., 1982; Biumberg et al,, 1984). Castagna et al. (1982) also demonstrated that phorbol esters capable of promoting skin tumors directly activated a partially purified protein kinase from rat brain, termed protein kinase C (PKC). This widely distributed protein kinase in mammalian tissues, especially brain, is cyclic nucleotide- and calmodulin-insensitive but Ca z+- and phospholipid-dependent (Nishizuka, 1983, 1986). Castagna et al. (1982) also showed that low concentrations of TPA could substitute for 1,2-diacylglycerol in the activation of PKC in ~itro. Subsequently, it was found that saturable phorbol ester receptors in particulate or cytosolic preparations from several cells and tissues copurified with PKC (Niedel et al., 1983; Leach et al., 1983; Ashendel et al., 1983; Vandenbark et al., 1984). Sharkey et al. (1984) showed that diolein and other diacylglycerols competitively inhibited specific phorbol diester binding and suggested that diacylglycerol may be the endogenous ligand for the phorbol ester receptor. TPA also has been found to increase diacylglycerol levels in several cell systems, suggesting possible activation of phospholipase C (Mufson, 1984). Thus, phorbol esters may exert some of their effects by initially binding to high-affinity sites on PKC (and/or increasing membrane levels of 1,2-diacylglycerol) with a resultant increase in membrane-associated kinase activity and subsequent changes in the phosphorylation of cellular proteins (Weinstein, 1983). Evidence has accumulated recently indicating that activation of PKC by TPA leads to the induction of epidermal ODC (Jetten et al.,

Multistage carcinogenesis in mouse skin

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1985; Smart et al., 1986; Verma et al., 1986c) as well as other genes (Denhardt et al., 1989; Karin and Herrlich, 1989 and see below). PKC exists as a family of nine related kinases (reviewed in Nishizuka, 1988, 1989; Bell and Burns, 1991). The members of this family have been divided into two distinct subclasses. The first subclass comprises four PKC isozymes (ct, ill,/311, and 7) which can be isolated through hydroxylapatite (HA) chromatography, and appear to be dependent upon Ca 2÷ for maximal activity (Huang et al., 1986, 1988; Jaken and Kiley, 1987; Marais and Parker, 1989). These isozymes are all activated by TPA. The second subclass consists of Ca 2÷ independent but phosholipid-responsive isozymes (also referred to as nPKC), including PKC6, E, ~, r/and L (Ono et al., 1988; Schaap and Parker, 1990; Osada et al., 1990; Bacher et al., 1991). Some of these latter PKC isozymes are TPA-responsive (& E), at least one is not TPA-responsive (~), and little is known about the phorbol ester responsiveness o f P K C ~/and L at present (Ono et al., 1988, 1989; Nishizuka, 1989; Hagiwara et al., 1990; Osada et al., 1990; Bacher et al., 1991). Data emerging in the literature for individual PKC isozymes regarding their different structures, tissue distribution, and functional properties strongly suggest that they have different functions in signal transduction. Most information regarding the biochemical characteristics of the first group of PKC isozymes (i.e. ~t, ill, fill, and 7) has come from studies using brain tissue (Huang et al., 1986, 1988; Jaken and Kiley, 1987; Marais and Parker, 1989). Much less is known about these isozymes in peripheral tissues such as epidermis (Hirabayashi et al., 1990). Information regarding biochemical characteristics of the second group of PKC isozymes (i.e. PKC6, c, ~, r/and L) has been primarily obtained from cloned expression systems (Ono et al., 1988; Schaap and Parker, 1990; Osada et al., 1990; Bacher et al., 1991) and there is only limited data concerning nPKC isozymes isolated directly from tissue (Gschwendt et al., 1989; Leibersperger et al., 1990; Hagiwara et al., 1990). An important question regarding the role of PKC in tumor promotion by phorbol esters and other tumor promoters that interact with this kinase is which PKC isozyme(s) is most critical for mediating specific effects associated with this process. In addition, phorbol ester treatment of cells and tissues invariably leads to the loss of PKC activity and protein due to proteolysis by Ca2+-dependent proteases (Nishizuka, 1989). It remains to be determined what role phorbol ester-induced proteolytic degradation of PKC isozymes (i.e. down-regulation) plays in the process of tumor promotion (Nishizuka, 1989; Droms and Malkinson, 1991 and see below). In addition to phorbol esters, several classes of compounds which differ in chemical structure but induce many of the same cellular and biochemical responses as the phorbol esters, including: teleocidins and aplysiatoxins (Fujiki and Sugimura, 1987; Fujiki et al., 1989); directly interact with PKC and appear to exhibit a similar initial mechanism(s) of action (Fujiki and Sugimura, 1987; Fujiki et al., 1989). Other skin tumor promoters such as anthralin (or chrysarobin), iodoacetic acid, palytoxin, thapsigargin, okadaic acid, calyculin A, benzoyl peroxide, and cantharidin (and many others), do not interact directly with the phorbol ester receptor (Fujiki and Sugimura, 1987; DiGiovanni et al., 1987; Suganuma et al., 1988; Fujiki et al., 1989; Imamoto et al., 1990). As noted above, many of the biochemical effects of phorbol esters and related tumor promoters, including those associated with epidermal hyperplasia, are believed to result from the initial interaction with PKC (Blumberg, 1988; Weinstein, 1988; Nishizuka, 1989). Phorbol esters such as TPA, by activating PKC, are believed to bypass a normal cellular mechanism(s) for regulating cell proliferation (Berridge and Irvine, 1984; Nishizuka, 1986, 1989; Pandiella et al., 1989). In keratinocytes of mouse skin, an important mechanism presumably involves the interaction of growth factors with their receptors, e.g. the epidermal growth factor receptor (EGFr) (King et al., 1990). The binding of a ligand such as EGF or transforming growth factor alpha (TGFct) to the EGFr leads to a cascade of events that result from activation of the EGFr tyrosine kinase (Pandieila et al., 1989; King et al., 1990; Carpenter and Cohen, 1990). EGFr activation rapidly triggers phosphatidylinositol-4,5-biphosphate (PIP2) hydrolysis, through phosphorylation ofphospholipase C-y, (PLC-?) which generates at least two second messengers: (i) diacylglycerol (DAG), which activates PKC as noted above; and (ii) inositol 1,4,5-trisphosphate (IP3), which binds to its receptor at the surface of an intracellular Ca 2+ storage organelle and induces release of stored Ca 2÷ to the cytoplasm. In addition, EGFr activation triggers phosphorylation of other substrates mediating multiple signalling pathways (Carpenter and Cohen, 1990). While much of the signal transduction pathway(s) beyond this and many of the nuclear gene products associated with the initiation of

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DNA synthesis resulting from growth factor stimulation are unknown, it is clear that a temporally ordered expression of a set of competence genes and progression genes occur. Among these competence genes are c-myc, c-los, and c-jun. Growth factor stimulation rapidly leads to the transient, elevated expression of these genes (reviewed in Pardee, 1989; Brenner et al., 1989). Tumor promoting phorbol esters are also able to modulate the transcription of a number of cellular and viral genes (Fisher et al., 1978; zur-Hausen et al., 1978; Monier et al., 1980; Amtmann and Sauer, 1982; Cochran et al., 1984; Greenberg and Ziff, 1984; Lau and Nathans, 1985; Angel et al., 1986; Verma et al., 1986b; Akhurst et al., 1988; Kreig et al., 1988; Rose-John et al., 1988; Pittelkow et al., 1989; Denhardt et al., 1989; Karin and Herrlich, 1989). Some of these genes, including c - m y c and c q b s , whose transcription is induced by phorbol esters in cultured cells (Cochran et al., 1984; Greenberg and Ziff, 1984) and mouse skin in vivo (Rose-John et al., 1988) are the same competence genes induced by activation of the EGFr as noted above. In many of these cases again, activation of PKC by TPA has been implicated in the mechanism of altered transcription and presumably mitogenesis. However, several recent studies using cultured fibroblasts have provided evidence that PKCindependent pathways may also play a role in growth factor-mediated gene expression and mitogenesis (Coughlin et al., 1985; McCaffrey et al., 1987; Hill et al., 1990). An important question regarding the mechanism of skin tumor promotion is whether the activation of PKC, per se, by phorbol esters and related compounds is sufficient for tumor promotion. In particular, Coughlin et al. (1985) reported that depletion of PKC in human foreskin fibroblasts abolished the induction of c - m y c by a second exposure to TPA but did not affect the mitogenic response to platelet-derived growth factor (PDGF). Furthermore, in this study TPA produced only ~ 1/10th the mitogenic response elicited with PDGF. Boynton and coworkers (Hill et al., 1990) have also provided evidence that activation of PKC, per se, through PLC7 activation and subsequent production of DAG is not required for induction of DNA synthesis in C3H10TI/2 mouse fibroblasts exposed to PDGF. In addition, McCaffrey et al. (1987) have shown that the induction of c - m y c and c-los mRNA by EGF in Swiss 3T3 cells is completely independent of PKC activation. Thus, these studies raise some important questions as to the role of PKC activation in mitogenic responses to external stimuli and indicate that cells, including mouse epidermal cells, may have multiple signalling pathways that mediate mitogenic responses (Ullrich and Schlessinger, 1990). In further support of this idea is the fact that many skin tumor promoters listed in Table 4 do not have the ability to directly activate PKC and yet they bring about dramatic proliferative responses as well as some of the same changes in gene expression in mouse epidermis after topical treatment. In mouse epidermis, a single application of TPA induces two major peaks of epidermal DNA synthesis at ~ 18 hr and ~ 4 4 4 8 hr (Baird et al., 1971; Raick, 1973a; Astrup and Paulson, 1981; Kruszewski and DiGiovanni, 1988). Interestingly, sn-l,2-didecanolyglycerol (sn-I,2-DDG), a membrane permeable DAG analog, when applied topically as a single application to CD-l mice also leads to increased epidermal DNA syntheses with a peak at ~ 18 hr implicating a role for PKC in the early transient DNA synthesis response to TPA (Smart et al., 1986). However, it is not known whether sn-I,2-DDG induces the later wave(s) of epidermal DNA synthesis (Smart et al., 1986). In addition, whereas both single and multiple (twice-weekly) applications of TPA induce significant hyperplasia, similar treatments with sn-I,2-DDG even up to 20 #mol per mouse fail to induce a dramatic hyperplasia in either CD-I (Smart et al., 1989; Hansen et al., 1990) or SENCAR mice (Hirabayashi et al., 1988). Thus, these data raise the interesting question of whether PKC activation, per se, can lead to sustained, potentiated epidermal cell proliferation and hyperplasia necessary for skin tumor promotion, even by the phorbol esters. Interestingly, recent studies have shown that if sn°I,2-DDG is given quite frequently (i.e. twice-daily) it will produce a significant hyperplasia response in CD-1 mice and promote skin tumors (Smart et al., 1989). This hyperplasia produced by the frequent application of sn-I,2-DDG actually correlated with the downregulation of epidermal PKC. These latter data provide support for the hypothesis that the downregulation of PKC may also play an important role in the production of hyperplasia and tumor promotion in vivo. In this regard, Nishizuka (1989) has suggested that under physiological conditions the activation of PKC is transient, since DAG once produced in membranes disappears within a few seconds or at most several minutes after its formation. On the other hand, treatment of cells with TPA causes sustained activation of PKC, ultimately resulting in depletion of the enzyme

Multistage carcinogenesis in mouse skin

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(Nishizuka, 1989). As a consequence, the cell may be relieved from the negative feedback control of a growth factor receptor, such as the EGFr, so that uncontrolled cell proliferation may occur in the presence of a mitogenic stimulus. Thus, TPA may provide a dual effect, furnishing a positive short-term activation of PKC, and then a more long-term effect due to degradation of the enzyme (Nishizuka, 1989). Phorbol ester tumor promoters have been shown in a variety of studies to alter the binding of EGF to its cellular receptor (Shoyab et al., 1979; Lee and Weinstein, 1979; Magun and Bowden, 1984; Imamoto et al., 1990). This change is apparently due to the loss of high affinity binding of EGF; however the exact mechanism for this alteration is currently unknown. TPA activation of PKC induces phosphorylation of Thr-654 which may lead to reduced affinity of the receptor for its ligand (King and Cooper, 1986; Davis, 1988; Downward et al., 1985). However, TPA also alters receptor affinity in cells transfected with an Ala-654-containing EGFr, in addition to altering affinity of the wild type EGFr (King and Cooper, 1986), suggesting that alternate pathway(s) (other than PKC activation) may also mediate alterations in EGFr affinity (Countaway et al., 1989). Recently, several non-phorbol ester type skin tumor promoters, such as thapsigargin (Friedman et al., 1989), okadaic acid (Hernandez-Sotomayor et al., 1991), palytoxin (Wattenberg et al., 1989), and chrysarobin (Imamoto et al., 1990), have been shown to inhibit the binding of EGF to its receptor through a protein kinase C-independent pathway(s). In addition, ultraviolet-B (UVB) irradiation (Matsui et al., 1989) as well as phenobarbital (Meyer et al., 1989) have been shown to inhibit EGF binding to its receptor on specific cell types. Thus, many known skin tumor promoters appear to initially inhibit EGF binding, especially using cultured cells. These relatively short-term effects of various tumor promoters on the EGFr appear, on the surface, unrelated to their ability to induce rather marked proliferative responses in mouse skin in vivo and various cells in culture, including keratinocytes (Boutwell, 1974, 1976; Yuspa et al., 1976; Diamond et al., 1978; Diamond, 1982; Parkinson, 1985). However, Imamoto et al. (1991) have recently provided evidence that topical application of both TPA and chrysarobin lead to the loss of epidermal PKC and ultimately elevation of ~25I-EGF binding to its membrane receptor. In addition, these authors have shown that both tumor promoters increase the levels of TGF~ mRNA and protein suggesting a possible role for the EGFr in tumor promoter induced cell proliferation and a possible common mechanism among some tumor promoters for induction of cell proliferation in mouse epidermis (Imamoto et al., 1991). Both TPA and 2,3,7,8-TCDD have been shown to induce TGF~ mRNA and protein synthesis in human keratinocytes (Pittelkow et al., 1989; Choi et al., 1991). In addition, UVB-radiation leads to increased expression of TGFct protein in cultured human melanocytes (Ellem et al., 1988). All of these studies support a possible role of both the EGFr and TGFct in the process of skin tumor promotion. The ability of tumor promoters to modulate the expression of growth factors (both positive and negative) in mouse epidermis is becoming increasingly apparent (Parkinson and Balmain, 1990). In addition to TGF~ (lmamoto et al., 1991), several laboratories (Akhurst et al., 1988; Krieg et al., 1991) have demonstrated that promoter treatment of mouse skin leads to increased production of TGFfl mRNA and presumably TGFfl protein. TGFfl has been shown to inhibit DNA synthesis in human and mouse keratinocyte (Shipley et al., 1986; Coffey et al., 1988; Mansbridge and Hanawalt, 1988; Ffirstenberger et al., 1989a; Partridge et al., 1989). However, many transformed epithelial cell lines are resistant to this effect (Sporn and Roberts, 1985; Roberts et al., 1988). It has also been postulated that initiated cells respond differently to growth inhibitory signals (Parkinson, 1985; Yuspa and Poirer, 1988; Parkinson and Balmain, 1990) such as those mediated by TGFfl or other growth regulatory molecules and that this may play a role in the selection process that takes place during tumor promotion (see Section 4.3). Recently, combined intradermal injections of both TGF~ and TGFfl were reported to substitute for TPA in the first stage of a two-stage promotion protocol, supporting this hypothesis (Ffirstenberger et al., 1989a). 4.2.3. Prooxidant Related Changes Evidence is emerging that the generation of free radicals may be involved in the skin tumor promoting actions of several classes of promoting agents as noted above under Section 4.1 (Cerutti, IPT 54/I--G

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1985; Kensler and Taffe, 1986), including the phorbol esters. For example, TPA stimulates production of O~ and possibly other free radicals by polymorphonuclear leukocytes (PMNs) (Repine et al., 1974; Kensler and Trush, 1981; Troll et al., 1982), probably by activating the ubiquitous membrane bound NADPH oxidase system (Cox et al., 1985: Papini et al., 1985). Fischer et al. (1986) demonstrated the production of 02 in isolated epidermal cells by active, but not inactive, phorbol ester analogs using a chemiluminescence assay and its suppression by antiprorooters (Fischer and Adams, 1985). More direct evidence for the involvement of free radicals in tumor promotion comes from studies with free radical generating compounds such as the organic peroxides and anthrones. Benzoyl peroxide and other organic peroxides are effective skin tumor promoters in sensitive mouse stocks and strains (Slaga et al., 1981, 1983). In addition, structure-activity studies for tumor promoting activity with anthrone derivatives strongly suggest that oxidation at C~0 of the molecule with subsequent generation of free radical intermediates is crucial for their tumor promoting actions (DiGiovanni et al., 1987, 1988a). A number of additional studies have also demonstrated that free radical generating systems such as xanthine/xanthine oxidase can mimic the effects of phorbol esters on enhancing cell transformation in cultured C3HIOTI/2 (Zimmerman and Cerutti, 1984) and mouse epidermal JB6 cells (Cerutti, 1987). Antioxidants are effective inhibitors of chemical carcinogenesis and skin tumor promotion which further supports a role for free radicals in tumor promotion (reviewed in Kensler and Taffe, 1986; Slaga and DiGiovanni, 1984 and see Section 4.5). TPA, benzoyl peroxide and anthralin have also been shown to decrease the activities of SOD and catalase in mouse epidermis, shortly after their application (Solanki et al., 1981a; Slaga et al., 1983; Reiners et al., 1991b). Perchellet et al. (1986) have demonstrated that a wide variety of tumor promoters decrease the ratio of reduced (GSH)/oxidized (GSSG) glutathione in mouse epidermal cells treated with a variety of promoting agents. TPA was also found to stimulate a rapid, transient increase in GSH-peroxidase followed by a prolonged depression in the activity of this enzyme (Perchellet et al., 1986). These changes presumably reflect the induction of a prooxidant state in the epidermal cells by TPA and other types of tumor promoters. More recent studies by Perchellet and coworkers (Perchellet et al., 1988; Perehellet and Perchellet, 1989) have demonstrated that tumor promoters can induce the production of hydroperoxides in mouse epidermal homogenates. Taken together, all these studies support a role for free radicals in tumor promotion by certain types of compounds. The exact biochemical and molecular mechanism(s) whereby certain free radical intermediates might lead to the process of tumor promotion remain unknown. Both genetic as well as epigenetic mechanisms have been postulated (reviewed in Cerutti, 1985, 1991; Kensler and Taffe, 1986; Perchellet and Perchellet, 1988). Cerutti (1985, 1991) has proposed that the induction of a prooxidant state leads to altered gene expression through activation of poly (ADP-ribose) synthetase and subsequent ADP-ribosylation of chromosomal proteins. The activation of poly (ADP-ribose) synthetase is proposed to result from oxidant induced DNA strand breaks and increased levels of oxidized pyridine nucleotides. Because of the reactivity of unsaturated and sulfur-containing molecules with free radicals, proteins containing such functional groups will be susceptible to free radical mediated amino acid modification (Pryor, 1976; Freeman and Crapo, 1982). A variety of cellular proteins and/or enzymatic pathways could thus be changed leading to altered phenotypic characteristics of a cell (reviewed in Freeman and Crapo, 1982; Kensler and Taffe, 1986; Cerutti, 1991). In this regard, anthralin is known to inhibit glucose-6-phosphate dehydrogenase (G6PDH) in vitro as well as in epidermal preparations of human skin (Hammar, 1970; Cavey et al., 1982). The ability of anthralin to inhibit G6PDH in vitro correlates with its ability to undergo oxidation to 1,8-dihydroxyanthraquinone and the anthralin dimer (Cavey et al.~ 1982). PKC may be regulated to a certain extent by direct oxidation. In this regard, mild oxidation of the regulatory domain of PKC may eliminate the requirement for Ca ~+ and phospholipid for its activation (Gopalakrishna and Anderson, 1989). Furthermore, H202 has been reported to alter the distribution of PKC in JB6 cells (Larsson and Cerutti, 1989) and benzoyl peroxide to alter PKC distribution in mouse epidermis (Donnelly et al., 1987). The activities of other proteins may also be regulated to a certain extent directly by redox reactions including: c-fos (Abate et al., 1990); c-jun (Abate et al., 1990); a tyrosine kinase located in the endoplasmic reticulum (Bauskin et al., 1991); GSSG reductase and Mg2+-dependent, Na+/K + stimulated ATPase (Thor and Orrenius, 1980), and possibly many others.

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During oxidative stress, most cells suffer from compromised energy homeostasis due to uncoupling of oxidative phosphorylation, decreased levels of GSH, and decreased levels of NADPH as a result of its utilization by the GSH-peroxidase redox cycle leading to the subsequent release of intracellular Ca 2+ stores (Trump and Berezesky, 1987; Richter and Frei, 1988; Reed, 1990). The resulting increase in intracellular Ca 2+ concentrations could lead to the activation of a cascade of biochemical pathways (reviewed in Trump and Berezesky, 1987; Reed, 1990). It is interesting to note that PKC and Ca 2+ are believed to act synergistically in stimulating various cellular responses (Nishizuka, 1983; Berridge and Irvine, 1984). In addition, release of intracellular Ca 2÷ as been postulated to account for the phosphorylation of the ribosomal protein, $6, in cells treated with H202 (Larsson and Cerutti, 1988) through an intermediate Ca2+/-calmodulin sensitive kinase (Larsson and Cerutti, 1988; Cerutti, 1991). The reported down-regulation of epidermal PKC by anthrone tumor promoters (Imamoto et al., 1991) could result from the activation of Ca2+-dependent proteases (Mellgren, 1987) through a similar mechanism. Despite the fact that tumor promoters are not mutagenic in different test systems (Lankas et al., 1977; Thomson et al., 1980) and do not bind covalently to DNA, there is cumulative evidence that TPA induces alterations at the genetic level which could result in toxicity and/or alterations in gene expression. It has been shown that TPA induces replication of endogeneous and integrated viral genomes (Imbra and Karin, 1986; Fisher et al., 1978; zur-Hausen et al., 1978, 1979), enhances sister chromatid exchanges in hamster fibroblasts (Kinsella and Radman, 1978) and induces DNA single-strand breaks in human leukocytes (Birnboim, 1982) and mouse keratinocytes (Dutton and Bowden, 1985; Hartley et al., 1985). Moreover, TPA treatment induces and/or enhances numerical and structural chromosomal aberrations in different systems, such as yeast (Parry et al., 1981), human leukocytes (Callen and Ford, 1983; Emerit and Cerutti, 1981) and mouse keratinocytes (Dzarlieva and Fusenig, 1982; Fusenig and Dzarlieva, 1982; Dzarlieva-Petrusevska and Fusenig, 1985). Recently it has been reported that TPA also induced cytogenetic changes in cultures of primary mouse keratinocytes (Petrusevska et al., 1988).

4.2.4. Other Changes Associated with N o n - P h o r b o l - E s t e r

Promoters

Chemicals such as okadaic acid, calyculin A, and similar compounds appear to be relatively specific inhibitors of cellular serine/threonine protein phosphatases (primarily PP-I and PP-2A (Cohen, 1989)) which may mediate, in part, their skin tumor promoting activities (Hesheler et al., 1988; Haystead et al., 1989; Suganuma et al., 1989, 1990). The sesquiterpene lactone, thapsigargin, promotes tumorigenesis in mouse skin but does not activate PKC and is classified as a non-phorbol ester-type tumor promoter (Haikaii et al., 1986; Fujiki et al., 1989). Thapsigargin has been demonstrated to specifically release Ca 2+ from the inositol trisphosphate-sensitive intracellular pool in parotid acinar cells (Takemura et al., 1989). Studies in rat hepatocytes suggest that the mechanism of action of thapsigargin involves inhibition of the Ca2+-activated endoplasmic reticulum ATPase, the presumptive intracellular Ca z+ pump (Thastrup et al., 1990). Thapsigargin elevates intracellular Ca z+ in a dose-dependent manner, providing a unique tool to study the effect of intracellular Ca 2+ concentration on gene transcription and tumor promotion. The biochemical and molecular mechanism(s) by which the many other nonphorbol ester skin tumor promoters work remain(s) to be determined. However, it should be stressed again that all known skin tumor promoters, that have been adequately studied to date, induce a sustained and potentiated hyperplasia and cell proliferation response following multiple treatments (Argyris, 1985, 1989; Naito et al., 1987) and it is this latter response that appears to be the most universal among the different types of promoting stimuli.

4.3. ROLEOF CELL SELECTIONDURINGTUMORPROMOTION The process of tumor promotion in mouse skin is believed to involve the selective clonal expansion of initiated cells into visible clonal outgrowths (papillomas) by one or a combination of several postulated mechanisms. The clonal origin of skin papillomas and carcinomas generated

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using both initiation promotion and complete carcinogenesis regimens (reviewed in Yuspa and Poirer, 1988) argues strongly in favor of such a mechanism. Several mechanisms have been proposed to explain such a cell selection process during tumor promotion and all involve differential responsiveness of the initiated cell to the tumor promoter induced effects (directly or indirectly). A first possible mechanism for cell selection involves a stimulatory effect of the tumor promoter on initiated cells causing them to divide and expand in number. With this mechanism it is necessary to invoke a differential responsiveness between initiated vs normal mouse keratinocytes with the former being more sensitive. The data supporting such a mechanism are generally lacking, however some recent studies could theoretically, provide a basis for such a differential response. In this regard, a close correlation between activation of Ha-ras and TGF~ expression in mammary epithelial cells has been reported (Ciardiello et al., 1988, 1990). Treatment of activated c-Ha-ras-transfected mammary epithelial cells with anti-TGF~ neutralizing or anti-EGFr blocking monoclonal antibody leads to > 50% inhibition of colony formation of these cells. In addition, the growth of TGF~-transfected mammary epithelial cells in soft agar was completely inhibited by treatment with the same antibodies (Ciardiello et al., 1990). These observations suggest that Ha-ras transfected mammary epithelial cells may contain at least two growth advantages: (i) elevated synthesis of TGF~; and (ii) altered signal transduction due to activated Ha-ras protein. TGF~ is a potent mitogen for cultured mouse keratinocytes (Wille et al., 1984; Coffey et al., 1988). Introduction of a human TGF~ gene (hTGF~) into cultured primary mouse keratinocytes, using a defective retroviral vector, led to normal epidermis upon grafting these cells to nude mice (Finzi et al., 1988). In contrast, grafts containing primary keratinocytes expressing hTGF~ and mixed with non hTGF~-expressing papilloma cells (or the converse) led to growth of larger tumors than obtained with papilloma cells alone (Finzi et al., 1988). Thus, increased expression of this growth factor alone did not lead to a transforming event nor did it affect the progression of the tumors in this system (Finzi et al., 1988). However, while the low levels of hTGF~ secreted by these cells did not appear to alter the growth status of normal keratinocytes, the growth of the SP-1 papilloma cells was apparently enhanced as judged by tumor size. While these studies are far from conclusive and more detailed analyses of the proliferative response induced by TGF~ in normal vs initiated keratinocytes are necessary, they suggest that initiated cells may have a higher sensitivity to the mitogenic action of TGF~. Since several tumor promoters, including the phorbol esters and anthrones as noted above are known to induce TGF~ synthesis in mouse and human keratinocytes (Pittelkow et al., 1989; Imamoto et al., 1991), this type of mechanism could be feasible. A second possible mechanism for selective clonal expansion of initiated cells during promotion has been postulated to involve an altered response to the terminal differentiation inducing effects of phorbol esters and similarly acting compounds. Reiners and Slaga (1983) found that tumor promoters induce a subpopulation of basal cells to commit to terminal differentiation and accelerate the rate of differentiation of committed cells. Yuspa et al. (1982) also found that tumor promoters can induce subpopulations of basal cells in culture to differentiate. This could be an important mechanism in the expansion of the initiated cell population if initiated cells respond differentially to this stimuli. Yuspa and Morgan (1981) found that putative 'initiated' epidermal cells in culture do not differentiate under a physiological stimulus to differentiation (i.e. elevated extracellular Ca2+). In this regard, cells with a putative 'initiated' phenotype could be selected by their ability to grow in high Ca 2+ medium, which induced normal cells to differentiate (Hennings et al., 1980). These putative 'initiated' cells were non-tumorigenic, but became tumorigenic after repeated passage in vitro. The observation that similar cells could be isolated after initiation of mouse skin in vivo (Yuspa and Morgan, 1981; Morris et al., 1988) has led to the suggestion that the processes of initiation in vivo and in vitro and hence cell selection may be very similar. However, recent evidence indicates that significant differences may exist at the molecular level between 'initiated' cells isolated in vitro by this Ca 2+ selection method and those present in mouse skin in vivo that give rise to skin papillomas (Quintanilla et al., 1991). Nevertheless, the following lines of evidence suggest that differential response to a negative growth regulatory molecule could play a role in tumor promotion: (i) several laboratories (Akhurst et al., 1988; Krieg et al., 1991) have demonstrated that promoter treatment of mouse skin leads to increased production of TGFfl m R N A and presumably TGF~ protein; (ii) TGFfl has been shown to inhibit DNA synthesis in

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human and mouse keratinocyte (Shipley et al., 1986; Coffey et al., 1988; Mansbridge and Hanawalt, 1988; Fiirstenberger et al., 1989a; Partridge et al., 1989); (iii) many transformed epithelial cell lines are resistant to this effect (Sporn and Roberts, 1985; Roberts et al., 1988). Note that with this mechanism there must also be a stimulus for cell division (directly or indirectly) supplied by the tumor promoter. In this regard, studies have been reported showing that simultaneous intradermal injection of both TGF~ and TGFfl mimicked the first stage of a two-stage promotion protocol in NMRI (Fiirstenberger et al., 1989a). Tumor promoter induced production of TGFct or other direct or indirect effects of the tumor promoter may provide the necessary stimulus for cell proliferation under these conditions. A third possible mechanism for cell selection during the process of tumor promotion involves selective cytotoxicity toward normal epidermal cells vs initiated cells allowing the latter a selective growth advantage (reviewed in Parkinson, 1985; Yuspa and Poirer, 1988; Slaga, 1989). This could be an important mechanism for some classes of tumor promoters such as the anthrones and organic peroxides as well as others. In support of this hypothesis are studies demonstrating that 'initiated' keratinocytes in culture are resistant to the cytotoxic effects of benzoyl peroxide (Hartley et al., 1987). Furthermore, histologic studies of mouse skins treated with the anthrone derivative chrysarobin, show that a certain degree of epidermal toxicity is an important component of the promoting action of this compound (Kruszewski et al., 1989). Finally, skin tumors generated by standard initiation-promotion protocols have elevated levels of reduced glutathione (GSH) (Reiners et al., 1991c). These results suggest the possibility that initiated cells may have a phenotype conducive to resistance to oxidative stress and thus more resistant to oxidant-induced toxicity. Again, with this mechanism, there must also be a stimulus (directly or indirectly) for cell division supplied by the tumor promoter. Regardless of the exact mechanism, the critical aspect of skin tumor promotion and possibly tumor promotion in other tissues is the selective expansion of the initiated cells by the continued exposure to the tumor promoter. It should also be stressed that none of the above mechanisms of cell selection should be viewed as necessarily independent mechanisms. It is entirely possible that with many compounds, more than one mechanism may be operating simultaneously.

4.4. MULTISTAGEPROMOTION Studies by Boutwell (1964) first suggested that the tumor promotion phase of mouse skin carcinogenesis had two operationally distinct stages. These early studies used limited treatments with croton oil, insufficient to produce skin papillomas by itself, followed by repeated treatments with turpentine (Boutwell, 1964). These combined treatments led to a significant tumor response. More recent studies by Slaga et al. (1980a) and F/irstenberger et al. (1981) led to further development of this two-stage promotion concept. Currently, the standard twostage promotion protocol involves initiation followed by one to four applications of TPA (stage I) and then by multiple applications of a weak papilloma-promoting agent, such as mezerein or 12-O-retinolphorbol-13-acetate (RPA) (stage II) (Slaga, 1983). Several cellular and biochemical changes have been suggested to be associated with these operational stages of skin tumor promotion. The induction of DCs and the synthesis of prostaglandins and possibly other metabolites of arachidonic acid as well as the growth factors TGFct and TGFfl are reported to be associated with stage I (Klein-Szanto et al., 1982; Slaga et al., 1982b,c; Fiirstenberger et al., 1989a). Stage I promotion also has some persistent effects that last 5-8 weeks, while stage II promotion is apparently immediately reversible (Slaga, 1983; Fiirstenberger et al., 1983; Ewing et al., 1988b). The induction of epidermal ODC, elevated levels of polyamines, and the maintenance of chronic cellular proliferation have been reported to be associated more specifically with stage II promotion (Slaga et al., 1982b,c; Slaga, 1983). The protease inhibitor tosyl phenylalanine chloromethyl ketone (TPCK) has been reported to specifically block stage I, and retinoic acid (RA) inhibits stage II promotion (Slaga et al., 1982b,c; Slaga, 1983). These observations, especially with various inhibitors (see Section 4.5 for further promotion stage inhibitors), provide the strongest support for the hypothesis that these operational stages of tumor promotion may have distinct mechanistic bases.

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The mouse stocks most commonly used in two-stage promotion protocols are SENCAR and N M R I (reviewed in Hennings and Yuspa, 1985). To date, several important differences between the two stocks have been found in response to the various first and second stage promoters. TPA is the most effective stage I promoter in both mouse stocks (Slaga et al., 1980a; F/irstenberger et al., 1981), however, in SENCAR mice 4-O-methyl-TPA and the calcium ionophore A23187 are also reported to be effective as stage I promoters (Slaga et al., 1980a, 1983). In contrast, these latter two compounds do not appear to serve as stage I promoters in N M R I mice (F/irstenberger et al., 1981). Mezerein is a good stage II promoter in SENCAR as noted above (Slaga et al., 1980a,b) but appears to be inactive as a complete or second stage promoter in N M R I mice (F/irstenberger et al., 1983). On the other hand, RPA is a good stage II promoter in N M R I mice (F~rstenberger et al., 1981) while this compound has been reported to be a complete promoter in SENCAR mice (Fischer et al., 1985). Despite these differences in response to the various compounds, it is clear that both of these mouse stocks respond to an operational two-stage promotion protocol (with the respective compounds) giving rise to a significant tumor yield. In addition, treatment with stage I or stage II promoters alone in either mouse stock results in significantly fewer papillomas. Thus, considerable evidence exists in the literature supporting the concept that the process of skin tumor promotion can be operationally divided into several stages. On the other hand, such differences in mouse stocks have raised questions about the generality and mechanistic basis of this phenomenon (Hennings and Yuspa, 1985). Despite the evidence supporting a two-stage promotion concept, recent studies in N M R I and SENCAR mice showing a partial inversion of the multistage promotion phenomenon have further complicated its interpretation and relevance. In this regard, F/irstenberger et al. (1985) reported that when TPA is given prior to initiation in N M R I mice it enhanced the subsequent promoting activity of RPA. In addition, this effect was abolished by inhibition of DNA synthesis with hydroxyurea (Kinzel et al., 1986). The implications of these data are that the first or "conversion' stage of promotion can occur prior to initiation. In SENCAR mice, pretreatment with TPA prior to initiation also enhances the subsequent promoting response to mezerein treatments (Ordman et al., 1985; Ewing et al., 1988b). Further complicating the interpretation of multistage promotion is the observation that limited applications of mezerein (i.e. two applications) given before initiation are capable of enhancing its own papilloma-promoting activity (Ewing et al., 1988b). Based on these data, it has been suggested that mezerein may possess additional properties that prevent the full expression of its complete promoting activity (Ewing et al., 1988b). One property of this compound (discussed in Section 4.2.1) that could modulate its promoting activity may be the ability to produce significant epidermal toxicity especially after repetitive treatments, and Argyris (1983a, 1985, 1989) has presented data that support this concept. Whether a similar explanation applies to RPA remains to be determined. Another important question about multistage promotion is whether it generally applies to other classes of skin tumor promoters. Although limited, the available data suggest that this is not a general phenomenon among all classes of tumor promoters. Neither benzoyl peroxide nor chrysarobin were effective as stage I promoters in SENCAR mice nor were their promoting activities enhanced by stage I treatments with TPA (DiGiovanni et al., 1985a; Ewing et al., 1988b). Therefore, the concept of multistage promotion appears to remain an open question at present and may apply only to phorbol esters and select compounds that produce similar short-term effects in skin. Further work is clearly warranted to resolve the generality of this phenomenon and the various inconsistencies noted above.

4.5. MODIFYING FACTORS AFFECTING TUMOR PROMOTION IN MOUSE SKIN Table 6 lists some of the modifying factors that can affect the process of tumor promotion in mouse skin. It should be apparent that many of the same general factors that can modify the process of tumor initiation have the potential to modify the tumor promotion process in mouse skin, although the mechanisms involved are likely to be different. Again, as with the modifiers of initiation, many studies on potential mechanism(s) of skin tumor promotion have been aided through the use of specific chemical inhibitors. The mechanism(s) whereby various agents inhibit

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TABLE6. Modifying Factors Affecting Tumor Promotion in Mouse Skin Factor Exogenous chemicals Antipromoters Copromoters Dietary factors Immune status Viral Radiation exposure Genetic factors Age Sex Hormonal balance

Specific examples Antiinflammatory steriods, retinoids, protease inhibitors, inhibitors of arachidonic acid metabolism, antioxidants, polyamine synthesis inhibitors, PKC inhibitors, etc. Prostaglandins F2~ and E2, chrysarobin, 7-interferon Calories, fat u.v.-Light

Glucocorticoids

skin tumor promotion are quite diverse owing to the many effects produced by promoting agents such as the widely studied phorbol esters as detailed in Section 4.2. The discovery that PKC is a phorbol ester receptor (Nishizuka, 1989) has led to the identification of several inhibitors of PKC. Nevertheless, the majority of chemical inhibitors of tumor promotion by phorbol esters do so through mechanisms other than inhibition of PKC activity. In addition, much less work has been done to determine whether inhibitors of phorbol ester promotion also inhibit promotion by other classes of chemical promoters, especially those that do not directly activate PKC, or by physical promotion mechanisms. The major classes of chemicals that inhibit skin tumor promotion include (see also Table 6): antiinflammatory steroids; retinoids; protease inhibitors; prostaglandin synthesis inhibitors; antioxidants; polyamine synthesis inhibitors; PKC inhibitors; as well as numerous miscellaneous inhibitors. The reader is again referred to several more extensive listings ofinhibitors of tumor promotion and multistage skin carcinogenesis (Slaga et al., 1982b,c; Slaga, 1984a,b; Slaga and DiGiovanni, 1984; DiGiovanni, 1990, 1991). Antiinflammatory steroids are potent inhibitors of mouse skin tumor promotion by phorbol esters, as well as chrysarobin and 7-bromomethyl-benz[a]anthracene (7-BrMe-BA) (reviewed in DiGiovanni, 1990). Fluocinolone acetonide (FA) is a potent inhibitor of epidermal DNA synthesis (Schwarz et al., 1977), and the ability of the various steroid analogs to inhibit epidermal DNA synthesis correlates with their antiinflammatory abilities as well as with their abilities to inhibit skin tumor promotion (Schwarz et al., 1977). A number of retinoids are specific inhibitors of mouse skin tumor promotion (Slaga and DiGiovanni, 1984; DiGiovanni, 1990). All trans-RA is one of the most potent retinoids in counteracting skin tumor promotion by TPA (Slaga and DiGiovanni, 1984; Slaga, 1984b; DiGiovanni, 1990). Verma et al. (1979b) reported a strong correlation between the ability of retinoids to inhibit TPA-induced mouse epidermal ODC and their ability to inhibit TPA promotion. Interestingly, RA does not inhibit epidermal ODC induced by a complete carcinogenic dose of DMBA, nor does it inhibit complete carcinogenesis by DMBA (Verma et al., 1980b). Recent studies by Dawson et al. (1987) and DiGiovanni et al. (1988a), however, indicate that retinoids can inhibit skin tumor promotion by the anthrone class of skin tumor promoters (e.g. anthralin and chrysarobin). In addition, RA inhibited skin tumor promotion and ODC induction by 7-BrMe-BA after DMBA initiation (Verma et al., 1983). Recent evidence also indicates that retinoids may bring about many of these effects by interacting with nuclear receptors (Giguere et al., 1987; Petkovich et al., 1987). These nuclear receptors appear to be trans-acting factors which can regulate the expression of specific genes (Giguere et al., 1987; Petkovich et al., 1987) and this may explain the ability of retinoids to inhibit transcription of ODC mRNA that is induced following treatment with TPA (Verma, 1988). The use of protease inhibitors as possible inhibitors of promotion and carcinogenesis was started by the observation that TPA and croton oil caused the appearance of a trypsin-like protease in mouse skin (Troll et al., 1970). The protease inhibitors tosyllysine chloromethyl ketone (TLCK), TPCK, and tosylalanine methyl ester (TAME) are all effective inhibitors of croton oil and TPA promotion after DMBA initiation (Troll et al., 1970). Another protease inhibitor, leupeptin, which

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does not alkylate sulfhydryl groups is also an inhibitor of croton oil promotion in mouse skin (Hozumi et al., 1972). Interestingly, protease inhibitors reportedly have little effect on TPA-induced epidermal hyperplasia or on TPA-induced ODC activity (Slaga et al., 1982b,c; Slaga, 1984b). However, TPCK reportedly counteracts the induction of DCs by TPA (Slaga, 1984b). It has been postulated that protease inhibitors may also act through suppression of the formation of reactive oxygen species (Troll et al., 1987). Protease inhibitors are known to prevent the formation of superoxide anion (O£) and hydrogen peroxide (H202) by polymorphonuclear leukocytes (PMNs) that are activated following exposure to TPA (reviewed in Troll et al., 1987). Extensive evidence suggests that prostaglandins or, more correctly, the end products of arachidonic acid metabolism may play an important role in tumor promotion and in carcinogenesis in general (reviewed in Ffirstenberger and Marks, 1983; Slaga, 1984a, 1989; Fischer et al., 1989). These studies also support the hypothesis that promoter-induced inflammatory reactions are an important component of the tumor promotion process in mouse skin. A number of prostaglandin synthesis inhibitors are effective in counteracting tumor promotion and carcinogenesis (Fischer et al., 1979, 1982a, 1989). Inhibitors of phospholipase A 2 activity such as antiinflammatory steroids (Blackwell et al., 1978) and dibromoacetophenone (Fischer et a l , 1982a), are effective inhibitors of skin tumor promotion. The next most consistent inhibitors of tumor promotion in this general category are phenidone and 5,8,11,14-eicosatetraynoic acid (ETYA); these compounds inhibit both the cyclooxygenase and lipoxygenase pathways (Fischer et al., 1982b). This mode of inhibition as with the phospholipase A 2 inhibitors leads to a decrease in all the important end products of arachidonic acid metabolism (Fischer et al., 1982a, 19~9). Cyclooxygenase inhibitors such as indomethacin and flurbiprofen have been shown to inhibit skin tumor promotion (F/irstenberger and Marks, 1978; Fischer et al., 1987; Verma et al., 1980a). In addition, a number of lipoxygenase inhibitors are known to be effective inhibitors of TPA-induced ODC and skin tumor promotion including: quercetin (Kato et aL, 1983), nordihydroguaiaretic acid (NDGA) (Nakadate et al., 1982), 2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)- 1,4-benzoquinone (AA861) (Aizu et al., 1986), and 3,4,2',4'-tetrahydrochalcone (Aizu et al., 1986). Finally, antihistamines, such as diphenyldramine, also inhibit phorbol ester tumor promotion and vascular permeability changes in SENCAR mouse skin (Fischer et aL, 1990). Various antioxidants that have been shown to effectively inhibit skin tumor promotion by both TPA and benzoyl peroxide include: BHA, BHT, disulfiram, and 4-p-hydroxyanisole (reviewed in Slaga and DiGiovanni, 1984; DiGiovanni, 1990, 1991). Kensler and colleagues (reviewed in Kensler and Taffe, 1986) reported that BHA and other antioxidants inhibited TPA-induced epidermal ODC activity over the same dose range as that used for inhibition of tumor promotion (Slaga et al., 1983). The mechanism by which the antioxidants inhibit tumor promotion in mouse skin is not presently known, however, they may be scavenging radicals generated directly, in the case of benzoyl peroxide, or indirectly by TPA. Copper (II) bis(diisopropylsalicylate) (Cu2+DIPS), an SOD mimetic, inhibits skin tumor promotion (Kensler et al., 1983) and ODC induction (Egner and Kensler, 1985) by TPA. Cu2+DIPS also moderately inhibited skin tumor promotion by the anthrone derivative chrysarobin (DiGiovanni et al., 1988a). These results suggest the involvement of O2 in skin tumor promotion by both phorbol esters and anthrones. Shamberger (I 970) reported that Se was an effective inhibitor of skin tumor promotion by croton oil. The antiproliferative actions of Se may be involved in its antipromoting effects (reviewed in Slaga and DiGiovanni, 1984; DiGiovanni, 1990). Shamberger and Rudolph (1966) have also shown that ~-tocopherol and ascorbic acid significantly reduced tumor formation induced by DMBA initiation and croton oil promotion. Smart et al. (1987) have more recently demonstrated that topical application of ascorbic acid or ascorbyl palmitate effectively inhibited TPA-induced ODC, epidermal DNA synthesis and tumor promotion in CD-1 mice. Perchellet et al. (1987a) have further shown that combinations of Se and ~-tocopherol were highly effective at inhibiting TPA promotion. Finally, the antioxidant diethyldithiocarbamate (DDTC) has been shown to inhibit skin tumor promotion by phorbol ester (Perchellet et al., 1987b) although only at very high doses. Certain other agents that raise glutathione levels (including DDTC) appear to be capable of inhibiting TPA promotion, including glutathione itself (Perchellet et al., 1985a,b). These agents may be effective inhibitors due to the fact that a wide variety of tumor promoters decrease the ratio of reduced (GSH)/oxidized (GSSG) glutathione in mouse epidermis (Perchellet et aL, 1986)

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although a recent study failed to demonstrate significant changes in epidermal GSH levels in TPA treated skin (Reiners et al., 1991b). Weeks et al. (1982) and Takigawa et al. (1982) reported that ~t-difluoromethylornithine (~t-DFMO), a specific and irreversible inhibitor of ODC was able to inhibit significantly skin tumor promotion by TPA. Verma et al. (1986a) more recently demonstrated that small amounts of ~t-DFMO given in the drinking water could effectively inhibit epidermal ODC induction and skin tumor promotion by TPA. Methylglyoxal bis(butylamidinohydrazone) (MGBB), a reversible inhibitor of both ODC and S-adenosylmethionine decarboxylase inhibited TPA-induced ODC, accumulation of putrescine and spermidine, and tumor promotion by TPA in mouse skin (Hibasami et al., 1988). DiGiovanni and coworkers (1988b) have also shown that ~-DFMO given as a drinking-water supplement was a very effective inhibitor of skin tumor promotion by chrysarobin in SENCAR mice. A number of PKC modulators have been discovered. Several of these compounds have been shown to inhibit skin tumor promotion by TPA including: quercetin (Kato et al., 1983), bryostatin 1 (Hennings et al., 1987) and palmitoylcarnitine (Nakadate et al., 1986b), staurosporine (Yamamoto et al., 1989). However, with the exception of bryostatin 1, it is questionable whether interaction with PKC is the major mechanism of antipromoting activity for these compounds. Quercetin is an effective inhibitor of lipoxygenase (Kato et al., 1983), and palmitolycarnitine appears to affect other kinases such as the calmodulin-sensitive, Ca2÷-dependent protein kinases (Katoh et al., 1981). Staurosporine, a potent inhibitor of PKC activity in vitro, was found to inhibit skin tumor promotion by TPA (Yamamoto et al., 1989). However, this compound failed to inhibit TPA-induced epidermal ODC, hyperplasia, inflammation and DNA synthesis. The role of inhibition of PKC in the antipromoting action of this compound remains open at present. Finally, a number of miscellaneous inhibitors of skin tumor promotion have been identified including: IBMX; glycyrrhetic acid; o-limonine; calcium glucarate; DMSO; butyrate; BCG; Poly I-C; cAMP; acetic acid; and hyperthermia (Mitchel et al., 1986, 1987 and reviewed in Slaga and DiGiovanni, 1984; DiGiovanni, 1990, 1991). The promotion stage specificity of a number of antipromoters has been reported. These types of studies have led to hypotheses concerning cellular, biochemical, and molecular events critical for stage I and stage II promotion (see Section 4.4 above). For example, FA has been examined for its ability to affect specific stages of tumor promotion in mouse skin. In this regard, FA was an effective inhibitor of both stage I and stage II promotion in SENCAR mice (Slaga et al., 1982c; Slaga, 1984a,b, 1989). RA has also been examined for its ability to inhibit specific stages of tumor promotion in mouse skin. Slaga and coworkers (Slaga et al., 1982c; Slaga, 1984a,b, 1989) reported that RA was a more effective inhibitor of stage II than stage I promotion. More recently, however, Verma (1987) reported that RA effectively inhibited both stage I and stage II promotion when given at doses similar to those used in studies cited above by Slaga and coworkers. In light of these conflicting results, further work is necessary to establish the existence of stages and/or the promotion-stage specificity of retinoids. Protease inhibitors have been examined for their effects on specific stages of promotion in mouse skin. TPCK was reported to inhibit specifically stage I of promotion in SENCAR mice (Slaga et al., 1982b,c; Slaga, 1984a,b, 1989). Studies to date suggest that polyamine synthesis inhibitors inhibit primarily stage II of promotion in SENCAR mouse skin (reviewed in Weeks et al., 1984) and support the view that elevated ODC activity and polyamine levels are important for later stages of skin tumor promotion (Slaga et al., 1982c; Slaga, 1984a,b). In addition, the promotion stage specificity of several antioxidants has been examined. In particular, BHA was reported to be a more effective inhibitor of stage II promotion as was vitamin E (Slaga, 1984b). Furthermore, Perchellet and coworkers (Perchellet et al., 1987a,b) have reported that DDTC inhibited both stage I and stage II while combinations of Se and vitamin E were very effective at inhibiting stage II of promotion. Only a limited number of studies have identified co-promoting agents. Fischer et al. (1980b) reported that, although prostaglandin F:~ and E2 were not skin tumor promoters they effectively enhanced promotion by TPA. Likewise, Lupulescu (1978) found that prostaglandin F2~ and E2 enhanced complete skin carcinogenesis in mice by MCA. Certain doses of indomethacin and flurbiprofen when given with TPA can enhance skin tumor promotion with this compound (Fischer et al., 1979, 1980a,b). Chrysarobin, when applied at a dose of 0.5 #g (2.2 nmol) 30min prior

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to each application of TPA, produced a marked potentiation (100%) in the tumor promoting response to TPA (DiGiovanni and Boutwell, 1983). A 5 ~tg dose of chrysarobin also gave rise to a slight potentiating effect on TPA promotion (29%). Neither the 0.5/~g or 5/~g doses of chrysarobin alone (given twice weekly) gave rise to papilloma development in previously initiated mice. The observed response, therefore, represented a true synergism or potentiation and not simply an additive effect. Finally, a recent report demonstrated that systemic administration of 7-interferon could enhance the tumor promoting activity of TPA (Reiners et al., 1989). The mechanism(s) for the co-promoting effects in all of these studies remain to be determined. The effect of restricted dietary and caloric intake on the development of spontaneous and induced tumors was first reported by Tannenbaum (1940). Early initiation-promotion studies in mouse skin suggested that dietary and caloric restriction were most effective at modifying the promotional stage of tumor formation (Boutwell, 1964). One possible mechanism by which dietary and caloric restrictions inhibit tumor promotion might be related to the increase in endogenous glucocorticoids that follows such caloric restriction. As previously discussed, antiinflammatory steroids (glucocorticoids) are potent inhibitors of skin tumor promotion. Several reports have indicated that high-fat diets act as tumor promoters in mammary gland and colon carcinogenesis (Wynder et al., 1978; Hopkins and Caroll, 1979; Ip et al., 1986). Low-fat diets, especially those containing polyunsaturated fats, decrease the level of tumors in both the mammary gland and the colon induced by chemical carcinogenesis protocols (Wynder et al., 1978; Hopkins and Carroll, 1979; Ip et al., 1986). In the mouse skin model, increasing the levels of unsaturated fat (as corn oil) leads to enhancement of u.v.-light induced skin carcinogenesis (Black et al., 1985). Further studies using the initiation promotion regimen have demonstrated that feeding a high corn oil diet during the promotion phase (using TPA) led to an enhancement in both the rate of tumor development as well as in the total number of skin papillomas and carcinomas (Birt et al., 1989). It should be noted that in this latter study mice apparently consumed equal amounts of calories in the control and high-fat diets, thereby excluding calorie-related effects. In contrast to these studies in the skin model, which are consistent with dietary fat effects on carcinogenesis in other model systems, Leyton et al. (1991) found an inverse relationship between dietary corn oil and tumor promotion response. In these studies, the highest dietary corn oil groups had the lowest papillomas per mouse. In addition, dietary fish oil (i.e. Menhaden oil) rich in omega-3 fatty acids while protective against mammary carcinogenesis in rats (Jurkowski and Cave, 1985; Abou-E1-Ela et al., 1988), failed to protect against TPA tumor promotion in mouse skin (Locniskar et al., 1990). Many factors may contribute to such conflicting results and will require further detailed investigations. The mouse skin model would again appear to be ideal for further studies of dietary modulation of skin carcinogenesis and carcinogenesis in general. Modulation of the immune system is known to modify epidermal carcinogenesis and this is one mechanism postulated for how dietary fat can modulate skin carcinogenesis and tumor promotion (Hwang, 1989). de Gruijl and van der Leun (1982) reported that repeated exposure of hairless mice to u.v. irradiation on one side accelerated the subsequent induction of epidermal skin cancers by chronic u.v.-irradiation on the opposite side, which had been shielded during the first irradiation period. Their study demonstrated that some systemic effect of u.v. radiation markedly enhanced the formation of primary u.v.-induced skin tumors. Experiments by Fisher and Kripke (1982) showed, in addition, that u.v. radiation-induced suppressor lymphocytes could accelerate the development of primary skin cancers induced by chronic u.v. irradiation. This finding indicated that at least some of the systemic effects of u.v. radiation on u.v.-induced carcinogenesis were immunologically mediated and that the immune system played an important role in skin carcinogenesis from chronic u.v. irradiation. More recent studies by Strickland et al. (1985) have demonstrated that the systemic effect of u.v. radiation primarily modulates the promotion stage using a two-stage protocol with UVB-TPA. Gensler (1988a,b) has also recently reported that distal irradiation with u.v.-light enhanced complete carcinogenesis by B(a)P, however, additional studies revealed that u.v. radiation inhibited two-stage carcinogenesis with DMBA-TPA. That tumor promoters such as the phorbol esters, interact with immunofunctional cells in vitro has been known for some time (Baxter, 1984). Baxter et al. (1988, 1989) reported that multiple treatments with several tumor promoters, including TPA, led to reduction in the number of epidermal Langerhans cells. In addition, tumor promoters such

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95

as TPA, benzoyl peroxide, anthralin, n-dodecane, and BHTOOH have been shown to suppress contact hypersensitivity (Kodari et al., 1991) but whether this plays an important role in tumor promotion remains to be determined. Clearly more work is necessary in this relatively understudied area to understand the role of the immune system in multistage skin carcinogenesis using both chemical and physical agents. Mouse skin is generally considered most sensitive to epidermal carcinogenesis by application of either the complete carcinogenesis protocol or the initiation-promotion protocol (reviewed in Slaga and Fischer, 1983; Naito and DiGiovanni, 1989). Other species such as the rat, hamster, and rabbit are less sensitive, and the guinea pig is very resistant (reviewed in Slaga and Fischer, 1983; Naito and DiGiovanni, 1989). In addition to these species differences, there are marked strain differences with respect to epidermal two-stage carcinogenesis. Table 7 shows the relative rankings of several species and a variety of mouse strains used in epidermal carcinogenesis studies for their sensitivity to complete (using PAH as carcinogens) and initiation-promotion (using phorbol esters as promoters) carcinogenesis protocols (reviewed in Naito and DiGiovanni, 1989). Several laboratories have atter.~pted to determine the basis for altered susceptibility to epidermal carcinogenesis among mouse strains. Although it is known that genetic differences exist in the enzyme systems responsible for metabolism of PAH and other skin carcinogens (see Section 3.4), the available data suggest that certain aspects of initiation with PAH are qualitatively and quantitatively similar in mouse strains that differ in their sensitivity to epidermal carcinogenesis (reviewed in DiGiovanni, 1989b,c; Naito and DiGiovanni, 1989). Additional data have led to the conclusion that the primary determinant in strain differences to epidermal chemical carcinogenesis is at the level of response to the tumor promoter (reviewed in DiGiovanni, 1989b,c; Naito and DiGiovanni, 1989). A tentative genetic model has been developed using inbred mouse strains that are relatively sensitive (DBA/2, C3H/He) and relatively resistant (C57BL/6) to phorbol ester skin tumor promotion (Reiners et al., 1984; DiGiovanni et al., 1984, 1992a; Naito et al., 1988). This model has the following characteristics: (i) susceptibility to TPA promotion in B6D2F~ and B6C3F1 hybrid mice is inherited as an incomplete dominant trait; (ii) neither cytoplasmic genetic determinants nor the X chromosome appear to play a major role in susceptibility of mice to phorbol ester promotion; (iii) the degree of sustained epidermal hyperplasia and DC induction after multiple TPA treatments show an excellent correlation with inherited susceptibility to promotion; (iv) the incidence of tumors in backcrosses between F~ mice and TPA-resistant C57BL/6 mice, in B6D2F2 and B6C3F 2 mice, and in BXD and BXH recombinant inbred mice can be explained by a model with a minimum of three loci (two dominant and one recessive locus) controlling TPA promotion sensitivity. It is interesting to note that C57BL/6 mice, although relatively refractory to two-stage carcinogenesis and skin tumor promotion by TPA, are quite sensitive to complete carcinogenesis protocols with both B(a)P and DMBA (Table 7). Bock and Burns (1963) presented earlier data suggesting that C57/st mice were less sensitive to anthralin than Swiss mice initiated with the same dose of DMBA, although small numbers of animals were used in the experimental groups of this

TABLE 7. Species and Strain Comparison of Response to Complete and Two-stage Epidermal Carcinogenesis and Epidermal Hyperplasia using TPA Species/ strain Hamster

Rat Mouse SENCAR DBA/2 CD-I C3H BALB/c C57BL/6

Complete carcinogenesis

Responsiveness to two-stage epidermalcarcinogenesis

Hyperplasiaafter multiple treatments

+ + + +

+ / +

+ + +

+ + + + + + + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + + + + +/-

+ + + + + + + + + ++ + + + + + + +

+ = responsive, the number of +s indicates the relative degree of responsiveness. + / - = marginal or nonresponsiveness.

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study. In further studies, CD-1 mice appeared to be less sensitive than SENCAR mice to the promoting effects of 7-bromomethylbenz(a)anthracene (Scribner et al., 1983) and u.v.-light (Strickland, 1982). More detailed studies (DiGiovanni et al., 1991b) have clearly shown that mouse stocks and strains, when tested under appropriate conditions, generally show similar strain distribution patterns for sensitivity to several different classes of promoting agents. In addition, C57BL/6 mice are highly refractory to wounding as a promoting stimulus (J. DiGiovanni, unpublished). In contrast to these studies, Reiners et al. (1984) reported that C57BL/6 mice were highly sensitive to benzoyl peroxide tumor promotion. Despite the latter inconsistency, the current body of data suggest that there may be some common genetic factors controlling responsiveness to tumor promoting agents and by inference some common biochemical and molecular events in tumor promotion by diverse chemical and physical stimuli.

5. TUMOR PROGRESSION 5.1. BIOLOGYOF SKIN TUMOR GROWTH AND PROGRESSION

The papillomas that initially develop during mouse skin initiation-promotion protocols are considered by many investigators to be heterogeneous in that some will persist, some will disappear or regress, and only relatively few (5-7%) are observed to 'progress' to an invasive SCC during the time frame of most reported initiation-promotion experiments using phorbol esters as the promoter (Shubik el aL, 1953a; Burns el aL, 1976, 1978; Verma and Boutwell, 1980; Scribner el al., 1983; Hennings el al., 1985; Kruszewski el aL, 1987). In contrast, a complete carcinogenic regimen with DMBA, for example, produces a relatively low yield of papillomas many or all of which progress to SCC (Burns el aL, 1978; Scribner el al., 1983; Slaga, 1989). In early experiments with this model, Shubik (1950) reported a high regression rate among papillomas produced by protocols using high concentrations of DMBA and croton oil. Burns el al. (1976, 1978) reported that about 95% of the papillomas regressed in HA/ICR mice initiated with relatively low doses of DMBA followed by limited promotion with TPA. On the basis of these earlier studies, as well as their own work, several other investigators have suggested the existence of papilloma subpopulations with a majority of the tumors produced during initiation-promotion having little or no chance to progress (Scribner el aL, 1983; Hennings el al., 1985). These tumors have been referred to as 'terminally benign tumors'. Reddy el aL (1987) reported that 80% of the DMBA-TPA induced papillomas were promoter dependent and regressed after cessation of promoter treatment in BALB/cPGK mice initiated with high doses of DMBA. In studies with low doses of DMBA using BALB/cPGK mice, these same investigators (Reddy and Fialkow, 1988) concluded that essentially all tumors regressed following cessation of limited TPA treatment. Hennings el aL (1985) reported that approximately 30-40% of the papillomas produced during promotion with TPA in SENCAR mice initiated with relatively high doses of DMBA regressed whether TPA was given for 10, 20, or 40 weeks, whereas in CD-1 mice, only in mice where TPA treatment was terminated after 12 weeks did papillomas regress. On the other hand, Ewing el aL (1988a) reported that regardless of the length of promoter treatment (3, 5, 7, 10, or 60 weeks), in mice initiated with l0 nmol DMBA the average number of papillomas/mouse remained essentially at a plateau level during the remaining experimental period, suggesting only minimal papilloma regression in SENCAR mice. However, loss or disappearance of tumors was observed in those animals treated continuously with TPA that received higher initiating doses and with high tumor burdens (Ewing et aL, 1988a, 1989). Therefore, discrepancies exist in the literature regarding the actual proportion of papillomas that are so-called 'promoter dependent' and that regress after termination of TPA treatment. It is important to note that several factors may contribute to apparent tumor loss during the course of a typical initiation-promotion experiment. These factors include papilloma-papilloma and papilloma--carcinoma coalescence, ischemic necrosis, tumor infections, dermatitis, biting, tumor regression, and, of course, animal death. The importance of some of these factors appears to be directly related to tumor load per animal (e.g. coalescence) which is a function of the initiator and/or promoter dose. In addition, the available data suggest that genetic differences in response to TPA can affect the latency of tumor development (and possibly tumor regression kinetics) among

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specific stocks and strains of mice during promotion with phorbol esters (Naito and DiGiovanni, 1989; DiGiovanni, 1989b,c). Finally, the growth properties of nearby tumors may be dramatically affected by a developing SCC as a result of angiogenesis, overproduction of growth regulatory molecules and possibly other factors. Taking some of these factors into consideration, recent studies (Aldaz et al., 1991) have shown that the number of promoter-dependent papillomas that regressed over a 21-week period after discontinuation of a 10 week TPA treatment regimen in SENCAR mice represented a relatively small percentage of the total papillomas produced using a standard initiation-promotion protocol. Furthermore, different initiator doses (in the range of 0.25-2 pg/mouse) did not appear to have a major influence on the growth characteristics of the papillomas produced in this mouse stock. These latter observations may be consistent with a model (Aldaz et al., 1987), based on sequential cytogenetic and histopathological studies, suggesting that skin papillomas progress to SCC at different rates and that many of the papillomas generated during initiation-promotion do have the potential to become SCC. Clearly, more work is necessary to understand the biological aspects of tumor progression in mouse skin and the various factors influencing this process. As noted earlier in this review and discussed in more detail below in Section 5.2, the process of tumor progression appears to involve the accumulation of additional genetic changes in cells comprising skin papillomas. Hennings and coworkers (1983) reported a significant increase in the rate of conversion of papillomas to carcinomas when mice with papillomas generated during promotion with TPA were treated repetitively with MNNG. Similar results have been reported with limited treatment of ethylnitrosourea (ENU) (O'Connell et al., 1986a). This type of treatment (initiation-promotion-initiation) produces a carcinoma response more similar to a complete carcinogenesis protocol. Interestingly, benzoyl peroxide and several other free radical generating compounds have been reported capable of enhancing the conversion of papillomas to carcinomas (O'Connell et al., 1986b; Athar et al., 1989). The mechanism whereby a different type of promoter, like benzoyl peroxide, can increase the conversion of papillomas generated by TPA to SCCs is presently unknown although Rotstein et al. (1987) have suggested a role for free radicals in this process. In addition, these investigators reported that glutathione and disulfiram inhibited malignant progression in this model (Rotstein and Slaga, 1988). Conversely, the glutathione depleting agent, diethylmaleate, reportedly enhanced tumor progression (Rotstein and Slaga, 1988). Although much more work is needed, these studies suggest that certain types of free radical generating tumor promoters may enhance the conversion of papillomas to carcinomas and that it may be possible to identify an effective inhibitor of malignant progression in this model system. In contrast to these studies with benzoyl peroxide, studies with the free radical generating anthrone derivative, chrysarobin, failed to demonstrate a malignant conversion enhancing property of this compound (Ewing et al., 1988a). Furthermore, recent studies (DiGiovanni et al., 1992b) have demonstrated that the rate of malignant conversion of papillomas with a standard initiationpromotion protocol using benzoyl peroxide as the promoter is no greater than other promoters lacking the reported malignant conversion enhancing properties (i.e. TPA, chrysarobin). Further studies to determine the nature and relevance of this so-called 'progressor' property of certain free radical generating compounds remains an important area of research using the skin model system. 5.2. GENETICALTERATIONSACCOMPANYINGTUMOR PROGRESSIONIN MOUSE SKIN As noted above, tumor progression in mouse skin is the process whereby papillomas and possibly other premalignant skin lesions progress to SCCs. It is generally accepted that additional genetic changes within cells that comprise papillomas are necessary for tumor progression in mouse skin (Yuspa, 1985; Balmain, 1986; Aldaz and Conti, 1989; Slaga, 1989). Chromosomal studies have shown that SCCs induced during promotion with TPA are highly aneuploid lesions often exhibiting hyperdiploid stem lines (Conti et al., 1986; Aldaz et al., 1987). Although initial papillomas (10 weeks of promotion) are diploid, they progressively show chromosomal changes and eventually become aneuploid after 30 to 40 weeks of promotion (Aldaz et al., 1987; Aldaz and Conti, 1989). Additional evidence indicates that specific chromosome alterations such as the sequential trisomy of chromosome 6 followed later by trisomy of chromosome 7 may play an important role in

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malignant progression in mouse skin (Aldaz et al., 1989). These latter genetic alterations are nonrandom chromosomal changes present in most aneuploid papillomas and a high percentage of the SCC generated during a DMBA initiation-TPA promotion regimen (Aldaz et al., 1989). Recent studies (C. M. Aldaz, C. J. Conti and J. DiGiovanni, unpublished) have also demonstrated similar nonrandom chromosomal changes in tumors generated during tumor promotion with the anthrone tumor promoter, chrysarobin. These latter studies support the hypothesis that such chromosomal changes may be critical events in the progression of papillomas to SCC, and may occur independent of the type of tumor promoter. Recent studies have also implicated the Ha-ras gene in the process of tumor progression (Quintanilla et al., 1986; Dotto et al., 1988; Bianchi et al., 1990). Quintanilla et al. (1986) observed that some SCCs showed either homozygosity or amplification of the mutated Ha-ras allele. Furthermore, it has recently been reported (Bianchi et al., 1990) that the trisomy of chromosome 7 occurs as a result of duplication of the chromosome containing the mutated Ha-ras allele. The result is that most SCCs induced by initiation promotion regimens in mouse skin present a 1:2 or 0:3 (normal:mutated) Ha-ras ratio. These data implicate a possible role of mutated Ha-ras gene dosage in the process of malignant progression in mouse skin. Transfection studies with papilloma-derived keratinocytes in culture have demonstrated that introduction of activated Ha-ras (i.e. v-Ha-ras in a retroviral vector) induced aggressive carcinomas when these cells were subsequently introduced into growth chambers implanted on the backs of syngeneic hosts (Dotto et al., 1988). Yuspa and coworkers (Greenhalgh and Yuspa, 1988; Greenhalgh et al., 1990) have also reported that changes in the f o s gene may be involved in skin tumor progression and that Ha-ras a n d f o s may cooperate in converting papilloma-derived keratinocytes to malignant tumors. The expression of recessive mutations (i.e. tumor suppressor genes) has been shown to be involved in carcinogenesis in both humans and animals. These genes probably play a role in carcinogenesis when their function is inactivated by point mutations, deletions or chromosomal loss (monosomy). In the last few years, evidence supporting the existence of suppressor genes has accumulated and several putative tumor suppressor genes have been identified and cloned (Knudson, 1985; Sager, 1989; Weinberg, 1989; Stanbridge, 1990; Bishop, 1991; Marshall, 1991). In the mouse skin model, Bianchi et al. (1991), have recently reported that mitotic recombination occurs resulting in the loss of parental alleles in the distal part of chromosome 7. These data suggest that a tumor suppressor gene may be located in this region of chromosome 7 and could be important in malignant progression in this mouse model system. Finally, Ruggeri et al. (1991) have reported that alterations in the putative tumor suppressor gene, p53, occur in 25 50% of murine SCCs induced by the two stage initiation-promotion protocol. These changes in p53 may be relatively late events in the progression of SCCs to more malignant and/or poorly differentiated phenotypes (Ruggeri et al., 1991) although another study found similar alterations in p53 in well differentiated SCCs (Burns et al., 1991). The mouse skin model of multistage carcinogenesis is ideally suited for studying the stepwise evolution of genetic events associated with tumor progression due to the ease of generating large numbers of tumors at defined time points during the overall carcinogenesis process.

6. RELEVANCE OF THE MOUSE SKIN MODEL TO HUMAN CANCER The multistage model of carcinogenesis in mouse skin has, for more than 50 years, provided a conceptual framework from which to study the carcinogenesis process. Many concepts now currently applied to other tissues and model systems, including cell culture models for multistage carcinogenesis and transformation, were originally derived from the mouse skin model (Diamond, 1982; Slaga et al., 1989; Boyd and Barrett, 1990; Drinkwater, 1990). That these concepts also apply to human cancer has been confirmed by epidemiological studies indicating that human carcinogenesis also occurs via a multistep process involving initiation and promotion mechanisms (Wynder et al., 1978; Hayes and Campbell, 1980; Slaga, 1980; Borzonyi et aL, 1984; Cartwright, 1984). In addition, Vogelstein and colleagues have used human colorectal carcinoma as a model system to provide convincing evidence of multistep tumorigenesis at the molecular level in a human cancer.

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In this regard, all colorectal tumors examined, including the smallest adenomas, were found to be clonal in origin (Fearon et al., 1987). These adenomas also exhibited DNA hypomethylation, which is hypothesized to lead to chromosomal defects resulting in loss of tumor suppressor genes (Goelz et al., 1985). Among the chromosomal deletions known to occur in human colorectal carcinoma, those at 5q, 17p and 18q occur most commonly (Fearon et al., 1987; Vogelstein et al., 1988; Law et al., 1988). These genetic alterations appear to occur sequentially during tumor progression; deletions at 5q and activation of the K - r a s gene (Bos et al., 1987; Forrester et al., 1987; Vogelstein et al., 1988) often precede allelic deletion at chromosomes 17p and 18q. These results suggest a model, in which the acquisition of a ras gene mutation coupled with the loss of several tumor suppressor genes (including the p53 gene on chromosome 17p) leads to human colorectal tumorigenesis (Vogelstein, 1989). Thus, the multistep nature of human tumorigenesis may involve the activation of proto-oncogenes as well as the inactivation of one or more tumor suppressor genes. As stressed in this review, the multistage model of mouse skin carcinogenesis also occurs by a number of sequential cellular, biochemical, and molecular-genetic events. Although the exact sequence of events may differ between tissues and species, the overall concept appears to be directly applicable to human cancer. Thus it is likely that the further study of the mouse skin model of multistage carcinogenesis will continue to provide important insight into mechanisms of epithelial carcinogenesis in humans. In terms of more direct relevance to human skin cancer, comparison of skin tumors in man and mouse has shown both similarities and differences in the incidence and biological behavior of certain tumor types. Certain tumors in humans have not been described or occur less frequently during epidermal carcinogenesis in mice (reviewed in Klein-Szanto, 1989). Most notably are the lack of basal cell carcinoma and melanoma using standard initiation-promotion regimens with PAH as initiators and phorbol esters as promoters (Elwood et al., 1989; Klein-Szanto, 1989). Although carcinogen-induced mouse skin papillomas do not have an exact human equivalent, they appear to represent an immediate precursor to SCCs (Klein-Szanto, 1989; Aldaz and Conti, 1989; Aldaz et al., 1991). Keratoacanthomas are relatively less common in mouse skin than in human skin. Although their histopathological features are identical, in mice they appear also to be precursors of SCCs, while in humans this is not the case (Klein-Szanto, 1989). On the other hand, SCCs are similar in histological features and, to a certain extent, in their invasive and metastasizing potential when comparing tumors from mouse and man. Since SCCs are the final end point for initiation-promotion protocols, it is this human skin tumor type where direct relevance can be most readily studied at present by the mouse skin model of multistage carcinogenesis. However, this limitation of the mouse skin model in terms of relevance to other types of human skin cancers may be due, in part, to our limits of knowledge about the model system. To date the vast majority of studies with this model system have employed PAH and phorbol esters (i.e. croton oil or TPA) as the initiating and promoting agents, respectively. It should be noted that experimental protocols using different agents may lead to the development of higher proportions of certain types of tumors allowing a more detailed study of their development using the mouse skin model. Jaffe and Bowden (1987) reported the appearance of basal cell carcinoma in CD-1 mice exposed to ionizing radiation. Monks et al. (1990) reported a higher incidence of both keratoacanthomas and sebaceous squamous cell carcinomas in mice initiated with DMBA and promoted with the quinone, juglone. O'Connell et al. (1986a) also reported a relatively high incidence of keratoacanthomas in an initiation-promotion-progression protocol where benzoyl peroxide was administered following 20 weeks of promotion with TPA. Berkelhammer and Oxenhandler (1987) reported the induction malignant melanoma (16% incidence) in C57BL/6 mice exposed to a DMBA-initiation--croton oil-promotion protocol. Finally, Husain et al. (1991) have reported the induction of melanotic tumors (including malignant melanomas) in hairless mice exposed to DMBA followed by UVA-irradiation as the promoter. As we learn more about the mouse skin model and identify and define other initiators and promoters besides the most commonly (and historically) used PAH and phorbol esters, it may be possible to study in greater detail other tumor types that have relevance to human skin cancers. In addition, further understanding the tumor response in different mouse strains with different genetic backgrounds to specific initiating and promoting agents may also facilitate detailed study of other tumor types. A major advantage of using an animal model system, such as mouse skin, is that it is ideally suited

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to study early stages of carcinogenesis, especially the transition between premalignant and malignant stages. With regard to molecular mechanisms, it has already been pointed out earlier in this review that activation of the H a - r a s gene occurs with a high frequency in mouse skin tumors initiated with certain chemical carcinogens, especially PAH. Recent studies have shown that ras gene activation can be detected in a significant proportion of both melanoma (20%, primarily N - r a s ) (Albino et al., 1989; van't Veer et al., 1989) and nonmelanoma (46% SCCs, 31% basal cell carcinomas, primarily H a - r a s ) (Pierceall et al., 1991) human skin cancers. Furthermore, recent studies (Husain et al., 1991) have shown that chemical carcinogen exposure (i.e. DMBA) followed by UVA treatments as the promoter to hairless mice using an initiation promotion type regimen leads to activating mutations in the N - r a s gene and that these mutations are present in early pigmented lesions as well as malignant melanomas. These results constitute the first report of the presence of ras mutations in melanomas and pigmented nevi induced in a mouse skin model. They suggest that ras mutations may have some role in the induction of melanomas in this species and demonstrate the potential utility and relevance of this murine model to the study of the development and biology of human melanoma. Finally, ras mutations have been detected in mouse skin tumors induced by u.v.-light ( ~ 2 0 % , N - r a s ) (H. N. Ananthaswamy, personal communication). All of these data suggest that the mouse skin model under appropriate conditions, is relevant for studying molecular mechanisms of skin carcinogenesis in humans.

7. C O N C L U S I O N S A N D F U T U R E D I R E C T I O N S The initiation stage of mouse skin carcinogenesis involves genetic damage in the form of DNA adducts or initiator-induced base changes. These changes ultimately lead to mutations in critical target genes of epidermal stem cells. The c-Ha-ras, and to a limited extent, N - r a s genes have been identified as target genes for certain tumor-initiators in this model system. Despite our knowledge, important questions about the process of tumor initiation in mouse skin still remain. First, while some data exists showing the correlation between specific DNA adduct formation and mutations in the H a - r a s gene, sufficient data exists for only a few compounds (see Table 2). Further work establishing the relationship between the type of D N A damage and the point mutations observed in ras genes should help further substantiate this relationship. The report that tumors induced by TPA treatment alone possessed 61 s~codon H a - r a s mutations (Pelling et al., 1988) raises questions about the mechanism(s) of mutation induction in this gene in vivo which must be addressed. Second, although ras gene(s) are activated in a high percentage of skin papillomas induced by certain chemicals, in some instances the frequency of observed ras mutations is less than 50% (Hochwalt et al., 1988; Brown et al., 1990; Husain et al., 1991; Pierceall et al., 1992) or in the case of ionizing radiation activated ras genes are absent (Jaffe and Bowden, 1989). These observations suggest the involvement of other genetic loci as targets for tumor-initiation and their identification will be an important goal using the mouse skin model. Third, while we have learned much about the types of mutations and even the amino acid changes that are found in the mutant ras p21 proteins (Barbacid, 1987) little is known about these altered proteins in terms of the 'initiated' phenotype. To date, it has not been possible to identify initiated cells in vivo and study their properties. Cell culture models may continue to offer an important opportunity to study potential initiated cells (Yuspa and Poirer, 1988) although as already noted in Section 4.3 the selection pressure for putative initiated cells in vitro may not be the same as that in vivo (Quintanilla et al., 1991). Thus further studies evaluating possible alternate modes of cell selection in these cell culture models are warranted. The development of transgenic mouse models where the transgene expression is directed to the skin may ultimately allow study of initiated cells in the skin in vivo. Transgenic mice have been developed that have an activated H a - r a s whose expression is driven by a specific keratin promoter (Bailleul et al., 1990). However, in this case, the transgene is expressed at levels much higher than observed under actual initiation conditions. In addition, the skin of these animals displayed considerable morphological changes not observed in initiated mouse skin. As techniques improve, it will likely be possible to construct transgenes that behave more closely to the actual

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gene in an initiated cell. Such studies will likely yield important clues about the growth properties of initiated cells. The promotion stage of mouse skin carcinogenesis involves the production and maintenance of a chronic state of hyperplasia and cell proliferation and ultimately the selective clonal expansion of initiated cells. The hallmark of all tumor promoters, that have been adequately tested, is their ability to induce a potentiated hyperplasia after several treatments that is greater than that observed after a single application. Tumor promoters produce many effects when applied topically to mouse skin. Many of the effects that occur after a single application of phorbol esters such as TPA appear to be mediated by its interaction with PKC (Nishizuka, 1989). An important question is whether the activation of PKC, per se, is responsible for tumor promotion by TPA. Since repetitive treatments with TPA lead to a sustained loss of PKC it is possible that other effects, not mediated by PKC, but produced by phorbol esters and related compounds may play an important role in the production and maintenance of chronic hyperplasia and cell proliferation in the skin and for skin tumor promotion, In addition, we must learn more about the promoting actions of other compounds outside of the most commonly studied phorbol esters. Studies of some of these compounds already have and will continue to provide important clues regarding possible common pathways shared by diverse promoting agents. One such pathway may involve the EGFr and its ligand TGFa. As discussed in Section 4.2.2 it is now evident that many different types of promoting agents increase production ofTGF~t (Ellem et al., 1988; Pittelkow et al., 1989; Imamoto et al., 1991; Choi et al., 1991). Although many tumor promoters initially decrease the binding of 125I-EGF to the EGFr in specific cell types, including mouse epidermal cells, the long term effects of tumor promoters especially after repetitive treatments, may be considerably different. Tumor promoters may ultimately lead to a sensitization of this receptor pathway (Nishizuka, 1989; Imamoto et al., 1991) as well as other pathways regulated by PKC. Further studies to determine the role of the EGFr and possibly other growth factor mediated pathways in tumor promoter-induced hyperplasia and cell proliferation will likely yield important insights into the overall promotion process. Other evidence supporting some common mechanism(s) shared by diverse tumor promoting agents comes from studies of genetic differences in response to diverse promoting stimuli. The available evidence suggests that, in general, mice less sensitive to phorbol esters are also less sensitive to other chemical classes of promoters (DiGiovanni, 1991; DiGiovanni et aL, 1991b). Furthermore, C57BL/6 mice, which are very resistant to phorbol ester promotion are also very resistant to tumor promotion by full thickness skin wounding (J. DiGiovanni, unpublished observations). Collectively, these studies support the hypothesis that tumor promotion by diverse chemical agents may mimic events occurring during the process of wound healing (Argyris, 198 l, 1985, 1989; Parkinson, 1985; Parkinson and Balmain, 1990). Thus, a major emphasis should be placed on studying the nature and mechanism(s) of wounding-induced tumor promotion in mouse skin and its similarities and differences to the mechanisms of chemical and physical promoting stimuli. The study of the process of tumor progression in the mouse skin model should be considered in its infancy. Few, if any, studies have adequately considered physiologic factors and their influence on the growth potential of papillomas. In addition, the existence of dramatic genetic differences in response to promoting agents needs to be more adequately addressed when comparing tumor progression in different mouse stocks and strains. The further study of optimum design for analyzing tumor progression will likely improve our understanding of the biology of this process and the premalignant nature of papillomas. The genetic changes that occur during the progression of mouse skin papillomas are only beginning to be unravelled. It is likely that multiple genetic changes will be identified as necessary for the conversion of premalignant papilloma cells to malignant SCCs. The further study and identification of these changes will undoubtedly yield important gains in our knowledge of multistage carcinogenesis in the mouse skin model. The observed trisomies of both chromosomes 6 and 7 in mouse skin SCCs (Aldaz et al., 1989) and the identification of a putative tumor suppressor gene on mouse chromosome 7 (Bianchi et al., 1991) provide support for such a model, In addition, it is likely that further identification of genetic changes during the tumor progression stage will demonstrate the utility of mouse skin model for studying the ontogeny of the progression process and its relevance to human cancer. JPT 54/I--H

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With regard to using the mouse skin model as a model for h u m a n skin cancer, more emphasis should be placed on other etiologic factors outside o f the c o m m o n l y used P A H initiators and p h o r b o l ester promoters. The available evidence, although limited, suggests that mouse skin models for both basal cell carcinoma as well as malignant m e l a n o m a (Jaffe and Bowden, 1987; Husain et al., 1991) might be possible u p o n further development. In conclusion, the mouse skin model o f multistage carcinogenesis continues to serve as a major in vivo model for studying the sequential and stepwise evolution o f the cancer process by chemical and physical carcinogens. While no rodent carcinogenesis model system can define mechanisms for all types o f cancer and cancer causing agents, further understanding o f the process in this specific epithelial model system will likely continue making significant contributions to our understanding o f multistage carcinogenesis in humans. Acknowledgements The author wishes to acknowledge Carrie McKinley for her time, eflbrt and secretarial skills in the preparation of this manuscript. The author also wishes to thank and acknowledge Drs Thomas J. Slaga, Susan M. Fischer and Claudio J. Conti for their critiques and helpful discussions of this review. Finally, original research was supported by DHHS grants CA 3711 I, CA 36979, CA 38871, an American Cancer Society Faculty Research Award FRA-375 and Core grant CA 16672 to the University of Texas M. D. Anderson Cancer Center.

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