Alterations in Cholesterol Sulfate and its Biosynthetic Enzyme During Multistage Carcinogenesis in Mouse Skin

Alterations in Cholesterol Sulfate and its Biosynthetic Enzyme During Multistage Carcinogenesis in Mouse Skin

Alterations in Cholesterol Sulfate and its Biosynthetic Enzyme During Multistage Carcinogenesis in Mouse Skin Kaoru Kiguchi, Miwako Kagehara,* Ryuzabu...

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Alterations in Cholesterol Sulfate and its Biosynthetic Enzyme During Multistage Carcinogenesis in Mouse Skin Kaoru Kiguchi, Miwako Kagehara,* Ryuzaburo Higo,* Masao Iwamori,* and John DiGiovanni Department of Carcinogenesis, The University of Texas, M.D. Anderson Cancer Center, Science Park-Research Division, Smithville, Texas, U.S.A.; *Department of Biochemistry, Faculty of Medicine, The University of Tokyo, Tokyo, Japan

Recent evidence suggests that cholesterol sulfate may be an important second messenger involved in signaling epidermal differentiation in skin. The activity of cholesterol sulfotransferase (Ch-ST) is increased during squamous differentiation of keratinocytes and is believed to be a marker enzyme for terminal differentiation. The primary objective of this study was to examine changes in levels of cholesterol sulfate (CS) and activity of its biosynthetic enzyme, Ch-ST, during multistage carcinogenesis in mouse skin. Using SENCAR mice, we determined the activity of Ch-ST in normal epidermis, in tumor promoter-treated epidermis, in epidermis during wound healing, and in mouse skin tumors generated by initiation-promotion regimens. A single topical application of tumor promoters led to significantly elevated levels of Ch-ST activity and of CS. Epidermal ChST activity was also elevated during wound healing. Dramatic increases in CS levels and in the activity of Ch-

ST were found in nearly all of the papillomas and squamous cell carcinomas examined. The increased levels of CS and activity of Ch-ST in tumor promoter-treated epidermis were accompanied by increased transglutaminase-I activity. In contrast, transglutaminase I activity was not elevated in primary papillomas or squamous cell carcinomas. Finally, Ch-ST activity was significantly elevated in the epidermis of newborn HK1.ras transgenic mice, whereas transglutaminase I activity did not correlate with Ch-ST activity in these mice. These results demonstrate that diverse tumor-promoting stimuli all produce elevated CS levels and Ch-ST activity and that CS levels and Ch-ST activity were constitutively elevated in both papillomas and squamous cell carcinomas. The data also suggest a mechanism for upregulation of ChST in skin tumors involving activation/upregulation of Ha-ras. Key words: cholesterol sulfotransferase/papilloma/ squamous cell carcinoma/transglutaminase. J Invest Dermatol 111:973–981, 1998

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thickening of the stratum corneum (Webster et al, 1978; Epstein et al, 1981; Maloney et al, 1984; Rearick et al, 1987), indicating that CS is one of the factors governing the orderly cohesion-desquamation behavior of the epidermis. CS has been shown to inhibit sterologenesis directly by suppressing the activity of the rate-limiting enzyme for cholesterol synthesis, 3-hydroxy-3-methylglutaryl CoA reductase, under conditions in which serum lipoprotein is not present (Ponec and Williams, 1986; Williams et al, 1985, 1987). The enzymes responsible for the synthesis of CS are known to be strictly localized in different regions of the epidermis (Elias et al, 1984; Epstein et al, 1984; Momoeda et al, 1991; Kagehara et al, 1994). Among the different layers of mouse epidermis, CS accumulates primarily in the stratum granulosum and stratum corneum (Elias et al, 1984; Lampe et al, 1983), and Ch-ST has been shown to be localized in both the basal and the spinous layers (Epstein et al, 1984). Recently, it was reported that Ch-ST was not detected in fetal murine skin but was abruptly expressed in association with the formation of a multilayered structure (at day 16 of gestation), before the period of cornified envelope formation by TG-I and the formation of large keratin bundles (Kagehara et al, 1994). Ch-ST and TG-I expression seem to be associated with the formation of a multilayered structure of the epidermis and expansion of the stratum corneum, respectively, indicating that the two enzymes are involved in different steps of keratinocyte differentiation and thus may be useful differentiation makers not only in vitro, but also in vivo. Interestingly, the potent tumor promoter 12–0-tetradecanoylphorbol-13-acetate (TPA) increases both transglutaminase (Lichti et al, 1985; Yuspa et al, 1993; Yuspa, 1994) and Ch-ST activities (Jetten et al, 1989) in epidermal cells. Treatment of normal human epidermal keratinocytes

ecent evidence suggests that cholesterol sulfate (CS) may be an important molecule involved in epithelial differentiation. Markers of epidermal differentiation include: certain keratins, fillaggrin, involucrin, loricrin, elevated transglutaminase type I (TG-I) activity, galactose-binding 14 kDa lectin; the formation of cornified envelopes; and an increase in intermediate matrix (Yuspa, 1994). Among the lipid constituents, CS has been linked closely to squamous differentiation (Lampe et al, 1983). CS is found in high concentrations in epidermis, as well as in hair and nails. The activity of cholesterol sulfotransferase (Ch-ST) is highly inducible by inducers of squamous differentiation (Rearick et al, 1987). It has been postulated that the regulation of the ratio of CS to cholesterol, the so-called CS cycle, is important for normal desquamation in human skin (Elias et al, 1984; Epstein et al, 1984, 1988). In this regard, the diminished hydrolysis of CS in the skin of patients with recessive X-linked ichthyosis results in the

Manuscript received October 25, 1998; revised June 9, 1998; accepted for publication August 3, 1998. Reprint requests to: Dr. Kaoru Kiguchi, Department of Carcinogenesis, The University of Texas, M.D. Anderson Cancer Center, Science Park-Research Division, PO Box 389, Smithville, TX 78957. Abbreviations: CHRY, chrysarobin; Ch-ST, cholesterol sulfotransferase; CS, cholesterol sulfate; DHEA, dehydroepiandrosterone; DHEA-S, dehydroepiandrosterone sulfate; DHEA-ST, dehydroepicandrosterone sulfotransferase; FTW, full-thickness wounding; SCC, squamous cell carcinomas; TG-I, transglutaminase I; TPA, 12,0-tetradecanoylphorbol-13-acetate.

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with TPA induces terminal cell division (irreversible growth-arrest) and causes time- and dose-dependent increases in the incorporation of [35S]sodium-sulfate into CS. This stimulation of sulfate incorporation appears to be specific for cholesterol and is due to increased levels of Ch-ST activity. The increase in CS levels in TPA-treated cells appears to parallel the increase in TG-I activity. Recently, Ikuta et al (1994) reported that CS can directly activate one of the protein kinase C (PKC) isoforms, PKCη, in vitro and that this isoform can phosphorylate TG-I in vitro, which suggests a possible link for this PKC isoform in signaling differentiation in keratinocytes in vivo as follows: (i) induction of Ch-ST by differentiation signals; (ii) increased formation of CS; (iii) activation of PKCη; (iv) activation of TG-I; and (v) formation of cross-linked envelopes. This study was designed to further examine the role of CS in multistage carcinogenesis in mouse skin by examining the activity and distribution of Ch-ST and the levels of CS in vivo in normal mouse epidermis, in tumor promoter-treated epidermis, during wound healing, and in skin tumors. The studies were repeated in primary mouse keratinocytes under both low (proliferating) and high (differentiated) Ca21 conditions and following exposure to tumor promoters. In addition, we further explored the effect of Ha-ras overexpression on Ch-ST activity by examining the regulation of the CS cycle in HK1.ras transgenic mice (Greenhalgh et al, 1993). The results demonstrated that the activity of Ch-ST and the levels of CS are upregulated during multistage skin carcinogenesis. In addition, overexpression of Ha-ras in skin tumors and in epidermis of HK1.ras transgenic mice correlated with elevated Ch-ST activity, suggesting a possible relationship between Ha-ras expression and the regulation and/or expression of Ch-ST. MATERIALS AND METHODS Chemicals and biologicals TPA and okadaic acid were obtained from Alexis (Woburn, MA). Chrysarobin (CHRY) was purchased from ICN Pharmaceuticals (Plainview, NY) and was purified as previously described (DiGiovanni et al, 1987). Cholesterol, cholesterol 3-sulfate, 2 hydroxypropyl β-cyclodextrin, casein hydrolysate, leupeptin, aprotinin, phenylmethylsulfonyl fluoride, dithiothreitol, and Nonidet P-40 were purchased from Sigma (St. Louis, MO). The 7,12dimethylbenz[a]anthracene was obtained from Eastman Kodak (Rochester, NY). Animals, treatments, and tumor collection Female SENCAR mice were obtained from the National Cancer Institute (Frederick, MD). At 7–9 wk of age, the backs of the mice were carefully shaved with surgical clippers, and only those mice in the resting phase of the hair-growth cycle were used. Groups of SENCAR mice were killed at various times after single or multiple treatments with TPA (3.4 nmol), CHRY (220 nmol), okadaic acid (2.5 nmol), CS (1 µmol), dehydroepiandrosterone sulfate (DHEA-S) (1 µmol), cholesterol (1 µmol), or acetone (0.2 ml). Epidermis was scraped and stored at –70°C. For the production of skin tumors, mice were initiated with 7,12-dimethylbenz[a]anthracene (10 or 25 nmol) followed 2 wk later by twice-weekly applications of TPA (3.4 nmol) on the shaved dorsal skin. Papillomas were harvested at 10, 13, 15, or 20 wk of promotion and squamous cell carcinomas (SCC) were harvested at various times thereafter as they appeared. Tumors were quickly removed with surgical scissors, trimmed to remove any normal or necrotic tissue, and then snap-frozen in liquid N2. Squamous cell carcinomas were histologically confirmed. Primary cultures of keratinocytes were prepared from adult (7–9 wk of age) SENCAR mice by a method previously described (DiGiovanni et al, 1989). Transgenic mice that express v-Ha-ras in epidermis (i.e., HK1.ras mice) were kindly provided by Dr. Dennis Roop (Baylor College of Medicine, Houston, TX). The epidermis from both transgenic and nontransgenic mice of various ages was collected and stored at –70°C. Epidermis from neonatal mice (0.5 d and 3 d after birth) was prepared by a method previously described (Hennings et al, 1980) Wound healing For wound healing experiments mice received two different types of wounds in the dorsal skin, either a full-thickness wound (FTW) or a tape-stripping wound. For the FTW, the animals were lightly anesthetized with Methophane (Pitman-Moore, Mundelein, IL), and the dorsal skin was cut with surgical scissors to produce a 4 cm sagittal FTW. The wound was then closed with stainless steel wound clips, after which the animals were allowed to recover under a heat lamp. Mice were then sacrificed at various times after wounding and the wounded area of skin was excised, placed on a 3 3 5 index card, and snap-frozen in liquid N2, and then the epidermis was separated from the dermis by scraping the frozen skin with a scalpel blade. Epidermal scrapings were stored at –70°C until analyzed. Tape-stripping was also performed as a milder form of injury because it only

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involves removal of the stratum corneum, apart from the animals were lightly anesthetized and the shaved dorsal skin was stripped by the application of masking tape (3M, St. Paul, MN), which was firmly pressed to the skin before removal. This process was repeated µ70 times until the surface of the skin had a shiny appearance. Mice were then sacrificed at various times and, after removal of the dorsal skin, the epidermis was scraped and stored at –70°C until analysis. All mice used in these experiments were treated and housed under conditions specified by the United States National Institutes of Health, Department of Health and Human Services, and the Department of Agriculture. These experiments were also approved by the Institutional Animal Care and Use Committee of The University of Texas M.D. Anderson Cancer Center. Assay of Ch-ST activity Scraped epidermis or cultured keratinocytes were suspended in 2 volumes of ice-cold 0.25 M sucrose, transferred to a microcentrifuge tube, and sonicated (Sonifer 450/Branson, 2 s interval, 10 times at an output setting of 2). The lysate was centrifuged at 10,000 3 g for 10 min at 4°C, and the resulting supernatant was used as the enzyme source. The standard assay mixture for Ch-ST comprised 0.2 mM cholesterol, 0.5 mM 2-mercaptoethanol, 1.35 µM phosphoadenosine phospho-[35S]-sulfate (2.0 Ci per mmole; NEN), 50 mM phosphate buffer, pH 7.5, and enzyme, in a final volume of 100 µl. Cholesterol micelles were prepared by injecting 100 µl of cholesterol in ethanol (10 mg per ml) into 1.9 ml of water with a syringe (26G) at 50°C. The opalescent solution thus obtained did not form a precipitate for a longer period than that in the case of detergent-solubilized cholesterol solutions. Unless otherwise indicated, incubation was performed at 37°C for 60 min. The reaction was terminated by the addition of chloroform/methanol (2:1 vol/vol) containing 0.2 µg of CS (1 ml) and 0.5 ml of water, and the lower phase after removal of the aqueous phase was evaporated to dryness. The radioactive products were separated on a plastic-coated thin-layer chromatography plate (Polygram Sil G; Macherey-Nagel, Duren, Germany) with chloroform/methanol/acetone/acetic acid/water (8:2:4:2:1 vol/vol), and the radioactivity was determined with a Visage 60 (BioImage, Millipore) and a liquid scintillation counter (Beckman LS 1800, Beckman Instruments) after cutting out the area corresponding to the position of CS. Assay of TG-I activity Scraped epidermis or keratinocytes were suspended in 20 volumes of ice-cold phosphate-buffered saline containing 10 U leupeptin per ml, 10 U aprotinin per ml, 2 mM phenylmethylsulfonyl fluoride, and 1 mM ethylenediamine tetraacetic acid and sonicated as described in the ChST enzyme-activity assay. After determination of protein, dithiothreitol was added (final concentration of 10 mM). The lysate was centifuged at 10,000 3 g for 10 min at 4°C. The pellet containing the TG-I activity was resuspended in an equal volume of ice-cold phosphate-buffered saline and dithiothreitol buffer. Nonidet P-40 (50% of final concentration) was then added. The assay was performed by determining the incorporation of [3H]putrescine (50 Ci per mmole; NEN) dihydrochloride into casein as described previously (Jetten et al, 1990). Determination of the levels of CS Total lipids were extracted from the lyophilized epidermis, papillomas, or SCC successively with chloroform/ methanol/water (20:10:1, 10:20:1, 20:10:1, and 10:20:1 vol/vol/vol) at 40°C. Aliquots of the combined extracts were used to quantitate free cholesterol, and the remainder of the extracts were applied to a column of DEAE-Sephadex (A-25, acetate form; Pharmacia Fine Chemicals, Sweden) for separation into neutral and acidic lipid fractions (Iwamori et al, 1982). The acidic lipid fraction, which was eluted from the column with 10 volumes of 0.3 M sodium acetate in methanol, was saponified with 0.5 M sodium hydroxide in methanol at 40°C for 1 h to cleave the ester-containing lipids and was then desalted by dialysis. After purification of CS from the acidic lipid fraction by Iatrobeads (6RS-8060; Iatron, Tokyo) column chromatography, CS was identified by negative ion fast-atom-bombardment mass spectrometry (Iwamori et al, 1986; Momoeda et al, 1991) with triethanolamine as the matric solution, and by gasliquid chromatography-mass spectrometry of the products solvolyzed with 9 mM sulfuric acid in dimethylsulfoxide/methanol (9:1 vol/vol) at 80°C for 1 h. To quantitate CS, the acidic lipid fraction and a known amount of chemically synthesized CS were chromatographed on a high-performance thinlayer chromatography plate (Merck, Germany) with chloroform/methanol/ acetone/acetic acid/water (8:2:4:2:1 vol/vol), and the spots were visualized by spraying with cupric acetate/phosphoric acid reagent and heating at 110°C for 10 min. The density of the spots was determined at a sample wavelength of 420 nm and a control wavelength of 700 nm with a thin-layer chromatography densitometer (CS-9000; Shimadzu, Kyoto). Determination of DNA synthesis by [3H]dthymidine incorporation into DNA Epidermal DNA synthesis in SENCAR mouse epidermis following topical treatment with TPA or CHRY was determined by measuring the incorporation of [3H]dthymidine into epidermal DNA using a modified

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Schmidt-Thannhauser procedure as described by Slaga et al (1974). The mice, four per time point, received a single application of either 3.4 nmol of TPA, 220 nmol of CHRY, or 0.2 ml of acetone (control). Groups of mice were killed at various times after treatment, and each mouse received a single intraperitoneal injection of 30 µCi of [3H]dthymidine 30 min before being killed. The epidermis was placed in 10 ml of distilled water and homogenized with a Polytron PT-10 homogenizer (Brinkmann Instrument) for 45 s at 0–4°C. Perchloric acid (0.45 ml) was added to the homogenate, and then the homogenate was centrifuged at 5000 3 g for 15 min. The resulting pellet was washed three times with 0.2 M perchloric acid. The DNA in the pellet was hydrolyzed by adding 8 ml of 0.5 M perchloric acid and heating at 90°C for 20 min before centrifugation at 5000 3 g. The amount of radioactivity in the supernatant was determined by scintillation counting, and the amount of DNA was assayed by the diphenylamine method (Burton, 1956). The specific activity of epidermal DNA (i.e., dpm DNA per µg) is expressed as a percentage of the value obtained after topical treatment with 0.2 ml of acetone. Histologic analysis Dorsal skin samples and tumors were fixed in formalin and embedded in paraffin prior to sectioning. Sections of 4 µm were cut and stained with hematoxylin and eosin. Mice were injected i.p. with bromodeoxyuridine in phosphate-buffered saline (100 µg per gm body weight) 30 min prior to sacrifice. For the analysis of epidermal labeling index, paraffin sections were stained using anti-bromodeoxyuridine antibody as previously described (Eldridge et al, 1990).

RESULTS Elevation of CS levels and Ch-ST activity in tumor promotertreated mouse epidermis To investigate whether CS levels were altered during multistage carcinogenesis in mouse skin, we first examined the levels of CS and the activity of Ch-ST in tumor promotertreated mouse skin. When alkali-stable acidic lipids, corresponding to µ3 mg of tissue (dry weight) were examined by thin-layer chromatography, a band with mobility identical to that of chemically synthesized CS was detected in control epidermis. This band was analyzed by negative ion fast-atom-bombardment mass spectrometry, which yielded the molecular ion of CS at m/z 465 and sulfate-derived ions at m/z 80 and 97 (data not shown) (Momoeda et al, 1994). The amount of CS in control mouse epidermis was 0.21 6 0.07 nmol per mg dry weight. A single topical application of either TPA or CHRY led to significantly elevated levels of CS compared with control epidermis as shown in Fig 1(A). Maximum levels of CS were observed at 72 h or 72–120 h after treatment with TPA or CHRY and were 3.9- or 5.0fold higher than the control value, respectively. CS levels had returned to control values by 120 and 240 h after treatment with either TPA or CHRY, respectively. No significant changes in the levels of cholesterol were observed in tumor promoter treated epidermis compared with control epidermis (32 µmol per gm of dry weight) (data not shown). Figure 1(B) shows the time course for changes in Ch-ST after a single topical application of either TPA or CHRY. Following treatment, the activity of Ch-ST in TPA-treated epidermis was slightly depressed until 12 h. Subsequently, the activity followed a biphasic pattern, with a major peak at 24 h (2.6-fold) and a minor peak at 72 h. In CHRY-treated epidermis the activity was significantly depressed until 24 h, at which time there was a gradual increase in activity, with a single peak at 72 h (2.8-fold). By 120 h after TPA or CHRY application, Ch-ST activity had returned to near control levels. We also found that a single topical application of the nonphorbol ester promoter, okadaic acid (2.5 nmol), elevated the level of CS, with a single peak at 72 h (2.7-fold), the amount of CS with a single peak at 72 h (2.7-fold), and the activity of Ch-ST, with a single peak at 48 h (2.4-fold) (data not shown). Thus, these three different chemical tumor promoters all elevated the amount of CS and the activity of Ch-ST. With each type of tumor promoter, the peak of Ch-ST activity preceded the peak of CS accumulation, suggesting a relationship, although other factors (e.g., sulfatase activity) may also play a role in regulating CS levels in addition to Ch-ST levels. Figure 1 also shows the results from the examination of changes in TG-I activity and epidermal DNA synthesis following single treatments with either TPA or CHRY. A single topical application of TPA or CHRY led to elevated epidermal TG-I activity, with peaks in activity

Figure 1. A single topical application of either TPA or CHRY caused an overall increase in CS levels, cholesterol sulfotransferase activity, TG-I activity, and DNA synthesis in mouse epidermis. Effects of TPA and chrysarobin on levels of CS (A) cholesterol sulfotransferase activity (B), epidermal TG-I activity (C), and DNA synthesis (D) following a single application of TPA (3.4 nmol) (d), chrysarobin (220 nmol) (m), or acetone (j) in mouse epidermis. A single topical application of either TPA or CHRY caused an initial depression in Ch-ST activity followed by an overall increase that led to significantly elevated values of CS. Following TPA treatment, ChST activity was biphasic, whereas CHRY treatment resulted in a single peak at 72 h. DNA synthesis in the epidermis closely paralleled the time course of ChST activity. An elevation in TG-I activity was seen following treatment with either tumor promoter. Although the peaks of TG-I activity preceded CS accumulation, the time course of TG-I activation corresponded to the pattern of changes in CS level. Data represent the mean 6 SD in 3–4 mice per group. Results were obtained in three repeat experiments.

at 24 h (3.6-fold) and 48–72 h (µ3.0-fold), respectively (Fig 1C). Thus, an elevation in TG-I activity was seen following treatment with either tumor promoter. Although the peaks of TG-I activity somewhat preceded the peaks for accumulation of CS, levels of CS were significantly elevated above controls at all times when TG-I activity was elevated. Following treatment with either TPA or CHRY, the time course of epidermal DNA synthesis also paralleled the time course for changes of Ch-ST activity as shown in Fig 1(D). Following TPA treatment, an initial depression in DNA synthesis was followed by an increase to above control levels by 12 h with biphasic peaks at 18 h and 48 h (Fig 1D). This pattern of biphasic DNA synthesis was very similar to the pattern of Ch-ST activity induced in epidermis by TPA treatment, although peak Ch-ST activity occurred slightly later at 24 h and 72 h (Fig 1D). CHRY treatment also stimulated an increase in DNA synthesis, which peaked at 72 h following an initial depression in a pattern that was very similar to that of Ch-ST activity in epidermis (Fig 1D). Elevated epidermal Ch-ST and TG-I activity during wound healing To further understand the mechanisms and role for altered Ch-ST activity and CS levels, we also examined changes in the activity of Ch-ST in response to two types of wounding. For these experiments we used either a FTW or tape-stripping as forms of severe or mild skin injury, respectively. For experiments using FTW, SENCAR mice received a 4 cm-long FTW in their dorsal skin as described in Materials and Methods. For tape-stripping, mice received µ70 repeated strippings as described in Materials and Methods. The histologic features of these two forms of wounding are shown at selected time points in Fig 2. As indicated in Fig 2, unlike FTW, the basement membrane remained intact after tape-stripping. Both FTW and tape-stripping induce epidermal hyperplasia with peaks at 120–160 h and 72 h, respectively (Fig 2). As shown in Fig 3(A), Ch-ST activity was suppressed until 24 h after a FTW, induced above control levels after 48 h, and peaked

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Figure 2. Both FTW and tape-stripping induced epidermal hyperplasia. Response of the skin at various time points after tape-stripping (A) and FTW (B). Periodic acid Schiff–hematoxylin staining for tape-stripping. Arrows indicate the basement membranes. Hematoxylin and eosin staining for FTW. Scale bar: 10 µm. Both FTW and tape-stripping induced epidermal hyperplasia with peaks at 120–160 h and 72 h, respectively. Unlike FTW, the basement membrane remained intact after tape-stripping.

at 96 h (µ1.7-fold). Ch-ST activity was suppressed until 24 h after tape-stripping, induced above control levels after 48 h, and exhibited a broad peak from 72 to 120 h (2.5–2.7-fold) (Fig 3A). Following a FTW, TG-I activity was initially suppressed but returned to near control levels by 48 h (Fig 3B). After tape-stripping, TG-I activity was induced and remained at a high level (µ5.0-fold) for at least 8 d after wounding (Fig 3B). Tape-stripping induced higher levels of activity for both Ch-ST and TG-I than for FTW. CS levels were induced after 48 h and 72 h following tape-stripping and a FTW, respectively (Fig 3C), and exhibited a broad peak (1.8- and 1.5-fold, respectively) for at least 8 d. The activities of Ch-ST in primary mouse keratinocytes in both low (proliferating) and high (differentiated) Ca21 conditions and following exposure to either TPA, CHRY, or okadaic acid The activity of Ch-ST in vitro, i.e., in primary keratinocytes under both low (proliferating) and high (differentiated) Ca21 conditions and following exposure to tumor promoters, was also investigated. Adult keratinocytes grown in medium containing a low amount of Ca21 (0.05–0.10 mM) maintain basal cell morphology and proliferate; increasing the Ca21 concentration in the medium to between 1.2 and 1.4 mM signals the cell to undergo terminal differentiation (Hennings et al, 1980; Jetten et al, 1989). Primary keratinocytes were prepared from adult SENCAR mice (as described in Materials and Methods) and were switched from medium containing a low amount of Ca21 to medium containing a high amount of Ca21 or treated with 1.6– 160 nM TPA, 0.1–1.0 µM CHRY, or 0.5–5 nM okadaic acid. Cells were harvested at various times thereafter to determine Ch-ST activity and TG-I. Cells treated with okadaic acid were harvested only at the 24 h time point due to the toxicity of this compound. In murine adult keratinocytes, treatment with TPA (1.6 nM) or high Ca21 (1.4 mM) led to an elevation in Ch-ST activity (Fig 4A). Under both conditions the peak in Ch-ST activity occurred at 12 h, although the effect of TPA (14.2-fold increase) was far more dramatic compared with the moderate (2.4-fold) increase seen in high Ca21 medium. In the control (low Ca21 medium), the activity was gradually elevated until 48 or 72 h, at which time the cells reached confluency. In contrast to the results with TPA, there was no change in Ch-ST activity following exposure of cultured keratinocytes to CHRY or okadaic acid. TG-I activity was induced by both TPA (1.6 nM) and high Ca21

(1.4 mM) in cultured keratinocytes (Fig 4B). The activity was highest at 12 h (5.5-fold) and 24 h (19.4-fold), respectively. Thus, TPA and high Ca21 induced an elevation in both Ch-ST and TG-I activity. In contrast, neither CHRY nor okadaic acid (not shown) treatment increased TG-I activity in cultured keratinocytes. The observation that of three chemical tumor promoters, only TPA induced Ch-ST activity in vitro, suggest that the mechanism(s) for induction of Ch-ST activity in vivo by TPA versus CHRY or okadaic acid may be different. In addition, we examined the dynamics of the CS cycle in primary keratinocytes in response to exogenous growth factors, including epidermal growth factor, keratinocyte growth factor, and insulin-like growth factor-1. All three growth factors induced Ch-ST activity and this increase was associated with a concomitant increase in cell proliferation as measured by DNA synthesis (Fig 5). There was no measurable increase in TG-I activity following exposure to any of the above growth factors (Fig 5). Changes in activities of Ch-ST and TG-I in mouse epidermis following the topical application of CS, DHEA-S, or cholesterol We examined the time course of Ch-ST activity and TG-I activity after a single topical treatment or multiple topical treatments of CS. The effects of CS were compared with the effects of equimolar doses of cholesterol and the sulfate conjugate DHEA-S. Female SENCAR mice (at 7–9 wk of age) received a single treatment of either CS (1 µmol), DHEA-S (1 µmol), cholesterol (1 µmol), or the vehicle control methanol:acetone (1:4 vol/vol, 0.2 ml) and TG-I. Following a single application of CS or DHEA-S, the activity of ChST in epidermis was depressed but returned to near control levels by 72 h after treatment (Fig 6A, B). In CS-treated epidermis, there was a gradual increase in TG-I activity, with a peak at 48 h (2.0-fold) (Fig 6A); however, there was no change in TG-I activity in DHEAS-treated epidermis (Fig 6B). There was no change in activity of either Ch-ST or TG-I following cholesterol treatment (Fig 6C). The activities of Ch-ST and TG-I were also examined 24 h after the last treatment of the multiple applications of either CS (1 µmol), DHEA-S (1 µmol), or cholesterol (1 µmol) (six treatments total, every other day). Ch-ST activity was depressed in CS- and DHEA-S-treated epidermis (72% 6 6% and 67% 6 5% of the vehicle control, respectively) (Table I). TG-I activity in CS-treated epidermis was induced (161% 6 10%); however, there was no significant induction of TG-I activity compared with control in DHEA-S-treated epidermis

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Figure 4. TPA and high Ca2F induced Ch-ST and TG-I activities in primary mouse keratinocytes. Changes in activities of Ch-ST (A) and TGI (B) in primary mouse keratinocytes following a single treatment of TPA (1.6 nM) (d), chrysarobin (1 µm) (m), or acetone (j), and in keratinocytes switched to a medium with a high concentration of Ca21 (1.4 mM) (r). Treatment with TPA and high Ca21 led to an elevation in Ch-ST and TG-I activity. There was no change in Ch-ST or TG-I activity in keratinocytes treated with either CHRY or okadaic acid. Data represent the mean 6 SD of three cultures for each group from three separate experiments.

Figure 3. Tape-stripping caused an overall increase in Ch-ST activity, TG-I activity, and CS levels. FTW caused an increase in Ch-ST activity and CS levels. Activities of Ch-ST (A), TG-I (B), and CS levels (C) during healing after a FTW or tape-stripping. Mice received a 4 cm full-thickness sagittal wound or were wounded by repeat (70 times) application of masking tape to strip already shaved dorsal skin (see Materials and Methods for further details). Activities of Ch-ST and TG-I were measured at various time points during wound healing. s, FTW; r, tape-stripping. Ch-ST activity was suppressed until 24 h after a FTW, was induced above control levels after 48 h, and peaked at 96 h. Ch-ST activity was suppressed until 24 h after tapestripping, was induced above control levels after 48 h, and exhibited a broad peak from 72 to 120 h. After tape-stripping TG-I activity was induced and remained at a high level. Tape-stripping induced higher levels of activity than FTW for both Ch-ST and TG-I. CS levels were also induced after 48 h and 72 h following a tape-stripping and FTW, respectively, and exhibited a broad peak for at least 8 d after wounding. Data represent the mean 6 SD in 3–5 mice per group from two separate experiments. Values for TG-I activities during the healing of tape-stripping wounds had a maximum variation of 10%.

(107% 6 9%). Cholesterol-treated epidermis did not show significant changes in the activity of either Ch-ST (104% 6 7%) or TG-I (97% 6 5%). There were no significant differences in epidermal

labeling index of skin treated with CS versus DHEA-S or cholesterol. Although a single treatment of CS, DHEA-S, or cholesterol did not cause any significant histologic alterations in the epidermis, multiple treatment with CS resulted in an overall increase in epidermal thickness and hyperkeratosis was also evident (Fig 7). The granular layer with keratohyalin granules was clearly observed in CS-treated epidermis (Fig 7B). These histologic observations of CS-treated epidermis are consistent with results that have been previously reported (Maloney et al, 1984; Chida et al, 1995). There was no increase in cell proliferation or evidence of inflammation in CS-treated epidermis. Analysis of CS levels and Ch-ST and TG-I activity in primary skin tumors To further explore the changes in CS levels and the activities of Ch-ST and TG-I during multistage skin carcinogenesis, we examined these parameters in primary skin tumors generated by an initiation-promotion regimen (i.e., 7,12-dimethylbenz[a]anthracene initiation-TPA promotion). For these experiments, tumors were harvested at least 1 wk after the final TPA treatment. As shown in Fig 8(A), the level of CS in papillomas and SCC was significantly elevated (4.0-fold in both tumor types), whereas total cholesterol levels were unchanged (the latter data not shown). Notably, Ch-ST activity was dramatically elevated in all 10 of the papillomas and in 11 of 12 SCC analyzed (average 4.2-fold and 3.9-fold, respectively) (Fig 8B). Surprisingly, however, TG-I activity in papillomas and SCC was essentially the same as in control epidermis (Fig 8C).

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Figure 5. EGF, KGF, and IGF-1 induced Ch-ST activity and DNA synthesis but not TG-I activity. Changes in activities of Ch-ST ( ), TG-I (u) and DNA synthesis (j), determined by thymidine incorporation in DNA into primary mouse keratinocytes 20 h after a single treatment of either epidermal growth factor, keratinocyte growth factor, or insulin-like growth factor-1 (20 ng per ml). All three growth factors induced Ch-ST activity and this increase was associated with a concomitant increase in DNA synthesis. There was no measurable increase in TG-I activity. Data represent the mean 6 SD of three separate cultures. *Significantly higher (Student t test. p , 0.002) than control; **not significantly (p . 0.4) different from control.

Analysis of Ch-ST and TG-I activity in epidermis of HK1.ras transgenic mice A common feature of papillomas and SCC induced by initiation-promotion regimens in SENCAR mice is that the Haras gene (both mutated and wild-type) is overexpressed (Pelling et al, 1986, 1987). To investigate the possible relationship between altered regulation of CS levels and expression of Ha-ras in skin tumors, we examined the activity of Ch-ST in Ha-ras transgenic mice. Transgenic mice that express v-Ha-ras in epidermis have been established by means of a targeting vector based on the human keratin 1 gene (HK1) (Greenhalgh et al, 1993). Epidermal expression of v-Ha-ras in the HK1.ras mice results in epidermal hyperplasia in neonates and hyperkeratosis in juveniles (Greenhalgh et al, 1993). This skin phenotype diminishes somewhat with age but eventually adult animals do develop papillomas (Greenhalgh et al, 1993). As shown in Fig 9, Ch-ST activity was significantly elevated in the epidermis of transgenic neonates [ø3 d after birth (5.8-fold)] compared with Ch-ST levels in the epidermis of nontransgenic littermates of the same age. Interestingly, Ch-ST activity returned to near control levels by 7 d after birth. Although TG-I activity was a little higher than control in neonates, it was not significant. In papillomas that developed in the HK1.ras transgenic mice, the Ch-ST and TG-I activities were 4.5-fold and 0.9-fold, respectively, compared with that found in the epidermis of age-matched nontransgenic littermates (data not shown). DISCUSSION CS is formed via the conversion of cholesterol by Ch-ST, which is believed to be a marker enzyme for the terminal differentiation of epidermal cells (Rearick et al, 1987; Jetten et al, 1989). It is the increase in Ch-ST that appears to be responsible for the increase in CS in the upper epidermis, i.e., the stratum granulosum and stratum corneum (Elias et al, 1984). In the study reported here, we explored the changes in CS and its biosynthetic enzyme during multistage skin carcinogenesis in SENCAR mice. The major findings of this study are several-fold. First, the levels of CS and the activity of Ch-ST were elevated in the epidermis following chemical or physical tumor-promoting stimuli. In tumor promoter-treated epidermis, the elevation of Ch-ST activity preceded CS accumulation and was correlated with an elevation in DNA synthesis. TG-I activity was also elevated in tumor promotertreated epidermis and the time course of TG-I activity paralleled that of CS levels, although TG-I activity generally peaked before the peak in CS levels. Second, TPA and high Ca21, which are known for their

Figure 6. A single topical application of CS caused an increase in TGI activity of mouse epidermis. Effects of CS (A), DHEA-S (B), and cholesterol (C) on Ch-ST activity (d) and TG-I activity (u) following a single topical application of 1 µmol of CS, DHEA-S, or cholesterol in mouse epidermis. A single topical application of either CS or DHEA-S caused a depression in Ch-ST activity. The activity had returned to near control levels by 72 h after treatment. Following CS treatment, TG-I activity was induced with a peak at 48 h after the treatment. There was no change in TG-I activity following DHEA-S treatment. There was no change in activity of either ChST or TG-I following cholesterol treatment. Data represent the mean 6 SD in four mice per group.

ability to induce terminal differentiation in keratinocytes, led to elevations in activity of both Ch-ST and TG-I in primary cultures of adult keratinocytes. In contrast, CHRY and okadaic acid did not increase the activity of Ch-ST or TG-I in cultured keratinocytes. An elevation in Ch-ST activity but not TG-I activity was also seen in

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Table I. Effects of CS, DHEA-S, and cholesterol on Ch-ST and TG-I activities following multiple application in mouse epidermisa

CS DHEA-S Cholesterol (% of control)

Ch-ST

TG-I

Epidermal labeling index

72 6 6b 67 6 5b 104 6 7c

161 6 10b 107 6 9c 97 6 5c

2.8 6 0.2c 3.6 6 0.2c 3.3 6 0.4c

aFive mice (female SENCAR at 7–9 wk of age) were used for each group. One micromole of CS, DHEA-S, or cholesterol was applied repeatedly (six times total, every other day) and 24 h after the last treatment. Epidermis were scraped for measurement of Ch-ST and TG-I activities. Data represent the mean 6 SD of percentage of control. Controls were treated with 0.2 ml acetone:methanol (4:1 vol/vol). bSignificantly different (p , 0.002) from control group by t test. cNot significantly different (p . 0.4) from control group by t test.

cultured keratinocytes in response to exogenous epidermal growth factor, keratinocyte growth factor, and insulin-like growth factor-1, and it was associated with a concomitant increase in DNA synthesis. Third, both CS levels and the activity of Ch-ST were significantly elevated, whereas TG-I activity remained similar to control values in both papillomas and SCC generated by an initiation-promotion regimen. Fourth, in neonatal mouse epidermis of HK1.ras transgenic mice, the activity of Ch-ST was significantly elevated. Finally, topical treatment of SENCAR mice with CS resulted in an increase in TG-I activity, increased epidermal thickness, and epidermal hyperkeratosis. Taken together, these results indicate that the regulation of CS levels is altered during multistage skin carcinogenesis. In this study, we found that diverse tumor-promoting stimuli all produced elevated levels of epidermal Ch-ST activity and this was accompanied by significantly elevated CS levels (CS levels were only analyzed in TPA- and CHRY-treated epidermis); however, only TPA induced elevated Ch-ST activity in cultured keratinocytes. CHRY is a relatively potent anthrone type tumor promoter (DiGiovanni et al, 1987), and okadaic acid is also a potent tumor promoter that inhibits protein phosphatases 1 and 2A (Suganuma et al, 1988; Fujiki et al, 1992), thereby causing an increase in phosphorylated proteins. Unlike TPA, these compounds do not directly activate PKC isozymes in vitro, and appear to work by initial mechanisms distinct from that of TPA. These observations suggest that the mechanism of induction of ChST activity by both CHRY and okadaic acid is indirect and may be a result of the hyperplasia induced by these tumor promoters. The fact that wounding (both tape-stripping and FTW) induced Ch-ST activity supports this hypothesis. In contrast, the increased activity of Ch-ST following TPA treatment in vivo may result from a combination of both direct and indirect effects. With respect to the direct effects of TPA, the fact that the Ca21 switch also led to increased Ch-ST activity suggests the possibility that Ca21-dependent PKC isozymes may mediate the direct effects of TPA on Ch-ST activity. Furthermore, Denning et al (1995) reported that CS induced the expression of the granular layer proteins, filaggrin and loricrin, in primary cultures of mouse keratinocytes and also induced the activity of PKCε, η, and ζ as well as PKCα and g in vitro, although activity of the latter PKC isozymes is less than the activation induced by TPA. Figures 6 and 7 and Table I show the direct effects of topical application of CS on epidermis. Both single and multiple treatment with CS resulted in an increase in TG-I activity and inhibition of ChST activity in mouse epidermis. TG-I activity was highest at 48 h (µ2.0-fold) and Ch-ST activity was lowest at 24 h (52% of control) after a single application of CS. Histologic analysis of the epidermis following multiple treatment with CS demonstrated that CS increased epidermal thickness (primarily the stratum corneum and granular layer) and induced hyperkeratosis indicative of an increase in the rate of epidermal differentiation, which further supports the hypothesis that CS may play a role in terminal differentiation. TG-I activity was induced by CS but not by cholesterol or another sulfate conjugate, DHEA-ST. DHEA-S is formed via the conversion of DHEA by one of the sulfotransferase enzymes, DHEA-ST, which catalyzes the sulfate

Figure 7. Increased thickness of epidermis and induction of scaling following multiple topical application of CS (hematoxylin and eosin staining). One micromole of CS (B), DHEA-S (C), or cholesterol (D) were applied six times (every other day). Twenty-four hours after the last treatment skins were removed for histologic examination. (A) Vehicle-treated (acetone:methanol 4:1 vol/vol).

conjugation of bile acids and steroid hormones such as estrogens (Falany et al, 1989; Radominska et al, 1990; Otterness et al, 1992). Although DHEA-S is not present in mouse epidermis, sulfation of DHEA does occur in the cytosol fraction of a epidermal homogenate (Higo et al, 1997). DHEA-S inhibits epidermal Ch-ST activity within a dosedependent manner in vitro (Higo et al, 1997). This study demonstrates that DHEA-S treatment also inhibits Ch-ST activity in epidermis in vivo. Taken together, the results suggest that the induction of differentiation and the elevation of TG-I activity may be directly attributable to CS. The inhibition of epidermal Ch-ST activity following CS treatment may be due to product inhibition, and the inhibition following DHEA-S treatment may be due to the inhibitory effect of DHEA-S on Ch-ST as shown in the in vitro experiment (Higo et al, 1997). These results suggest that the ability to induce TG-I activity in mouse epidermis is unique to CS, and that elevated CS levels in tumor

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Figure 9. Ch-ST activity was significantly elevated in epidermis of Haras transgenic neonates. Activities of Ch-ST (j) and TG-I (u) in epidermis of Ha-ras transgenics. Ch-ST activity was significantly elevated in the epidermis of transgenic neonates (the age when transgene expression is highest) compared with Ch-ST levels in the epidermis of nontransgenic littermates of the same age. Ch-ST activity returned to near control levels by 7 d after birth. TG-I activity was essentially the same level or slightly higher then control in neonates and adult mice and did not correlate with Ch-ST activity. Data represent the mean 6 SD of five mice per group from two separate experiments. Controls were nontransgenic age-matched littermates. **Not significantly (p . 0.4) different from control.

Figure 8. CS levels and Ch-ST activity were dramatically elevated in papillomas and SCC. Level of CS (A) and activities of Ch-ST (B) and TGI (C) papillomas and SCC generated by initiation-promotion regimens. Ch-ST activity was dramatically elevated in all 10 of the papillomas and in 11 of 12 SCC analyzed; however, TG-I activity in papillomas and SCC was essentially the same as in control epidermis. Data represent the mean 6 SD of 10 papillomas and 12 SCC. Results were obtained in four repeat experiments. Tumors were harvested at least 1 wk after the last TPA treatment. CTR, control epidermis. **Not significantly (p . 0.4) different from control.

promoter-treated epidermis contributes, at least in part, to increased TG-I activity during tumor promotion and wound healing. The fact that the peak in TG-I activity occurred prior to the peak in CS levels in tumor promoter-treated or wounded skin suggests that other factors, in addition to CS, may regulate TG-I activity. In wound healing, CS may be particularly important for sustaining elevated TG-I activity (Fig 3). Recently, Ikuta et al reported that CS is a specific activator of PKCη in vitro and suggested that this neutral lipid acts as a second messenger, mediating squamous differentiation by activation of TG-I through PKCη. PKCη is reportedly expressed only in the granular layer of epidermis, supporting this hypothesis (Osada et al, 1993). Furthermore, this research group reported that CS inhibited the effects of tumor promotion by delaying the onset of skin tumors when applied at pharmacologic doses (Chida et al, 1995). The mechanism of inhibition by CS on tumor promotion was postulated to occur via activation of the PKCη-mediated pathway, the end result of which is squamous differentiation; however, our data indicate that CS levels and TG-I activity are already elevated in tumor promoter-treated epidermis.

Thus, the mechanism whereby topical application of pharmacologic doses of CS inhibit TPA tumor promotion may involve additional mechanisms and this requires further study. In this study, we also examined the levels of CS and the activities of both Ch-ST and TG-I in primary skin tumors induced by an initiation-promotion regimen in SENCAR mice. Notably, our results showed that, in both papillomas and SCC, CS levels were significantly higher than in control epidermis and the activity of Ch-ST was constitutively higher than the activity in control epidermis. The ras oncogene family plays an important role in mouse epidermal carcinogenesis (DiGiovanni, 1992). Depending on the tumor initiator, a large proportion of the papillomas and carcinomas induced in mouse skin by the initiation-promotion regimen may contain an activated Ha-ras oncogene with a point mutation at either codon 12, 13, or 61 (Barbacid, 1986; Lacal and Tronik, 1988). In addition, regardless of the initiator, Ha-ras mRNA expression is significantly elevated early in tumor development in mouse skin (Pelling et al, 1986, 1987). We hypothesized that the observed elevation in Ch-ST activity in skin tumors might be due to the presence of an activated or overexpressed Ha-ras. To further explore this hypothesis we examined the activity of Ch-ST in the epidermis of HK1.ras transgenic mice and found that the activity of Ch-ST was elevated significantly in the epidermis of these neonatal mice; the age when transgene expression is highest (Greenhalgh et al, 1993). These data suggest that the constitutive elevation of Ch-ST activity in skin tumors may be due, in part, to Ha-ras activation and/or overexpression. Finally, another interesting observation in this study was the finding that there was a dissociation between elevated Ch-ST activity (and CS levels) and elevated TG-I activity in skin tumors and in epidermis and papillomas from HK1.ras transgenic mice. In contrast, these parameters were correlated in tumor promoter-treated and wounded skin. Yuspa and colleagues (Dlugosz et al, 1994) have suggested, based on a number of observations in v-Ha-ras keratinocytes, that oncogenic Ha-ras alters keratinocyte differentiation by altering signaling of the PKC pathway mediated mainly by PKCα. In this regard, it was proposed that PKCα activation in v-Ha-ras keratinocytes led to the blockade of Ca21induced expression of keratins K1 and K10 and to accelerated expression of late differentation markers (loricrin, filaggrin, and TG-I) in these cells. That cells comprising skin tumors possess a defect in the ability to undergo terminal differentiation has been suggested by several investigators (Parkinson, 1985; Parkinson and Balmain, 1990; Yuspa,

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1994). The observation that skin tumor cells produce significant amounts of CS without elevated TG-I activity suggests that a CS effector molecule is defective. Our data suggest that skin tumor cells and epidermal cells of HK1.ras mice possess a defect in the differentiation pathway that occurs somewhere between the activation of Ch-ST and the activation of TG-I. Further study of the effects of CS on normal and initiated keratinocytes may help to delineate the nature of the defect in initiated keratinocytes. In conclusion, we have shown that an elevation in the level of CS and the activity of its biosynthetic enzyme Ch-ST are common features of tumor promoter-treated skin, wounded skin, and skin tumors. In addition, a possible relationship between Ha-ras activation and/or expression and altered levels of CS, through Ha-ras induced alterations in Ch-ST activity or levels in skin tumors, was observed. Future studies will be aimed at understanding the functional significance of elevated CS and Ch-ST activity in multistage carcinogenesis and tumor promotion. In addition, future studies will examine the mechanism(s) by which Ch-ST activity is altered in epidermis by diverse promoting stimuli and the mechanism(s) for constitutive elevations in Ch-ST activity in skin tumors.

We thank Carrie McKinley for her excellent secretarial skills in the preparation of this manuscript and Linda M. Beltra´n for excellent technical support. This research was supported by U.S. Public Health Service Grants CA 37111 (J.D.) and CA 57596 (J.D.), and Core grants CA 16672 and ES 07784 and Institutional Research Support Grants of the University of Texas M.D. Anderson Cancer Center (K.K.). Portions of this work were presented at the 86th and 87th annual meetings of the American Association for Cancer Research (Abstracts #1080 and 693, respectively).

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