Studies on mechanism of action of anti-tumor-promoting agents: their specificity in two-stage promotion.

Abstract

The effects of fluocinolone acetonide (FA), retinoic acid (RA), and tosylphenylalanine chloromethyl ketone (TPCK) on two-stage promotion after 7,12-dimethylbenzfaJ anthracene (DMBA) initiation in female Sencar mice were investigated. The two-stage promotion protocol was achieved by twice weekly applications of 2 sg of 12Otetradecanoylphorbol 13-acetate (TPA) for 2 weeks (stage I) followed by twice Carcinogenesis in the skin can be induced by the sequential application of a subthreshold dose of a carcinogen (initiation phase) followed by repetitive treatment with a noncarcinogenic tumor promoter. The initiation phase requires only a single application of either a direct-acting carcinogen or a procarcinogen (which has to be metabolized before being active) and is essentially irreversible; the promotion phase is initially reversible but later becomes irreversible. This system not only has provided an important model for studying carcinogenesis and for bioassaying carcinogenic agents, but it is also one of the best systems for investigating the effects of inhibitors of chemical carcinogenesis (1). Recent data from our laboratory suggest that the tumor promotion stage can be divided into two stages (2, 3). The first stage can be effectively brought about by limited applications of 12-0-tetradecanoylphorbol 13-acetate (TPA), or, to a lesser degree, of 4-0-methyl-TPA. Mezerein, a diterpene similar to TPA in its biochemical and morphological effects but a weak promoter, is a-potent stage II promoter when given repetitively after stage I promotion (2-4). The combined treatment after tumor initiation will give a tumor response similar to single-stage or complete promotion by TPA (3, 4).We have recently reported (3) that the only major biochemical and morphological difference between the effects of TPA and mezerein on the skin is the ability of TPA to induce a large number of dark basal keratinocytes, which suggests that these dark cells are important in stage I of promotion. The importance of these dark basal keratinocytes in tumor promotion was originally reported by Raick and coworkers (5-8). They found that TPA induced the appearance of these cells in the epidermis whereas ethylphenylpropiolate did not (5-8).The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 2251Inhibitors of tumor promotion have been useful in the identification of important events in tumor promotion. This has been especially true for anti-inflammatory steroids, retinoids, and protease inhibitors (1,(9)(10)(11)(12)(13)(14). These agents all are potent inhibitors of tumor promotion and chemical carcinogenesis in general (9-18). The purpose of this study was to determine the effects of some inhibitors of tumor promotion on the first and second stages of promotion and their effect on TPA-induced dark, basal keratinocytes. Tosylphenylalanyl chloromethyl ketone (TPCK) was found to inhibi...

Defective responses of transformed keratinocytes to terminal differentiation stimuli. Their role in epidermal tumour promotion by phorbol esters and by deep skin wounding

“…Stage I promotion has also been directly shown to require DNA synthesis (Kinzel et al, 1984a). Taken together these studies indicate that epidermal hyperplasia or an event closely associated with it, is important for the completion of both stage I and stage II promotion, and this is supported by the work of Slaga et al (1980a) who reported that steroids [potent inhibitors of epidermal hyperplasia (Belman & Troll, 1972;Scribner & Slaga, 1973;Schwarz et al, 1977)] were potent inhibitors of both stage I and stage II promotion in SENCAR mice.…”

“…Additional evidence that there are indeed two qualitatively different stages in promotion comes from the observations that the two stages are inhibited by different antagonists (Slaga et al, 1980a) and are dependent to different extents on the synthesis of prostaglandins F2a and E2 (Fustenberger & Marks, personal communication). This suggests that the two stages have a different molecular basis.…”

“…Similarly, preliminary reports suggest that the labelretaining putative stem cells (Mackenzie & Bickenbach, 1982) are not stimulated to divide by either mezerein (McCutcheon et al, 1984) or RPA (McCutcheon et al, 1985) in contrast to their stimulation by TPA (Morris et al, 1985). Dark cells thought by some to primitive keratinocytes (Raick, 1973) are also observed in relatively fewer numbers in mezerein-treated, as opposed to TPA-treated Sencar mouse skin (Klein-Szanto et al, 1980; and there is evidence that dark cell induction is a good indicator of stage I promoting power in this system (Klein-Szanto et al, 1982;Slaga et al, 1980a). The apparent toxic action of second stage promoters may result from their ability to induce terminal differentiation in all the basal cells equally whereas TPA appears to preferentially affect the more mature population (Parkinson et al, 1983;Morris et al, 1985).…”

“…One could speculate that a reduced requirement for certain growth factors might also allow keratinocytes initiated by rasH activation to grow autonomously in zones of the epidermis where previously they could not (see Yuspa et al, 1985). Whether the irreversible change from a promoterdependent to a promoter-independent lesion is spontaneous or whether it involves TPA-induced chromosome changes (Kinsella & Radman, 1978;Emeritt & Cerutti, 1981;Parry et al, 1981) or gene amplications (Varshavsky, 1981) as a possible consequence of the ability of TPA to generate free radicals (Troll et al, 1982) (Schwarz et al, 1984) and the effect of TPA on anchorage independence is blocked by both radical scavengers (Nakamura et al, 1985) and retinoids (Colburn, 1979) the last of which specifically inhibits stage II promotion (Slaga et al, 1980a).…”

Initiation and promotion at different ages and doses in 2200 mice. III. Linear extrapolation from high doses may underestimate low-dose tumour risks

“…If there is indeed an irreversible cellular effect which can be produced by brief promotion, the simple dichotomy between normal cells and initiated cells will need to be extended to allow various categories of initiated cells (or microscopic clones of cells), and promotion itself may prove to be a multi-step process, as was suggested by Boutwell (1964) and Slaga et al (1980). Initiation w)ith promotion…”