Portal de Periódicos da CAPES

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

1998; Elsevier BV; Volume: 111; Issue: 6 Linguagem: Inglês

10.1046/j.1523-1747.1998.00404.x

1523-1747

Kaoru Kiguchi, John DiGiovanni, Miwako Kagehara, Ryuzaburo Higo, Masao Iwamori,

Monoclonal and Polyclonal Antibodies Research

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 Ch-ST 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 Ch-ST in skin tumors involving activation/upregulation of Ha-ras. 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 Ch-ST 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 Ch-ST in skin tumors involving activation/upregulation of Ha-ras. chrysarobin cholesterol sulfotransferase cholesterol sulfate dehydroepiandrosterone dehydroepiandrosterone sulfate dehydroepicandrosterone sulfotransferase full-thickness wounding squamous cell carcinomas transglutaminase I 12,0-tetradecanoylphorbol-13-acetate Recent 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, 1994Yuspa S.H. The pathogenesis of squamous cell cancer: lessons learned from studies of skin carcinogenesis.Cancer Res. 1994; 54: 1178-1189PubMed Google Scholar). Among the lipid constituents, CS has been linked closely to squamous differentiation (Lampe et al., 1983Lampe M.A. Williams M.L. Elias P.M. Human epidermal lipids: characerization and modulations during differentiation.J Lipid Res. 1983; 24: 131-140Abstract Full Text PDF PubMed Google Scholar). 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., 1987Rearick J.I. Albro P.W. Jetten A.M. Increase in cholesterol sulfotransferase activity during in vitro squamous differentiation of rabbit tracheal epithelial cells and its inhibition by retinoic acid.J Biol Chem. 1987; 262: 13069-13074Abstract Full Text PDF PubMed Google Scholar). 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., 1984Elias P.M. Williams M.L. Maloney M.E. Bonifas J.A. Brown B.E. Grayson S. Epstein E.H. Startum corneum lipids in disorders of cornification: steroid sulfatase and cholesterol sulfate in normal desquamation and the pathogenesis of recessive X-linked ichthyosis.J Clin Invest. 1984; 74: 1414-1421Crossref PubMed Scopus (177) Google Scholar;Epstein et al., 1984Epstein Jr, E.h. Bonifas J.M. Barber T.C. Haynes M. Cholesterol sulfotransferase of newborn mouse epidermis.J Invest Dermatol. 1984; 83: 332-335Abstract Full Text PDF PubMed Scopus (25) Google Scholar,Epstein et al., 1988Epstein E.H. Langston A.W. Leung J. Sulfation reactions of the epidermis.Ann NY Acad Sci. 1988; 548: 97-101Crossref PubMed Scopus (6) Google Scholar). In this regard, the diminished hydrolysis of CS in the skin of patients with recessive X-linked ichthyosis results in the thickening of the stratum corneum (Webster et al., 1978Webster D. France J.T. Shapiro L.J. Weiss R. X-linked ichthyosis due to steroid-sulphatase deficiency.Lancet. 1978; 1: 70-72PubMed Google Scholar;Epstein et al., 1981Epstein Jr, E.h. Williams M.L. Elias P.M. Steroid sulfatase, X-linked ichthyosis, and stratum corneum cell cohesion.Arch Dermatol. 1981; 117: 761-763Crossref PubMed Scopus (86) Google Scholar;Maloney et al., 1984Maloney M.E. Williams M.L. Epstein E.H. Law Myl Fritsh P.O. Elias P.M. Lipids in the pathogenesis of ichthyosis: topical cholesterol sulfate-induced scaling in hairless mice.J Invest Dermatol. 1984; 83: 252-256Abstract Full Text PDF PubMed Scopus (48) Google Scholar;Rearick et al., 1987Rearick J.I. Albro P.W. Jetten A.M. Increase in cholesterol sulfotransferase activity during in vitro squamous differentiation of rabbit tracheal epithelial cells and its inhibition by retinoic acid.J Biol Chem. 1987; 262: 13069-13074Abstract Full Text PDF PubMed Google Scholar), 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, 1986Ponec M. Williams M.L. Cholesterol sulfate uptake and outflux in cultured human keratinocytes.Arch Dermatol Res. 1986; 279: 32-36Crossref PubMed Scopus (23) Google Scholar;Williams et al., 1985Williams M.L. Hughes-Fulford M. Elias P.M. Inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and sterol synthesis by cholesterol sulfate in cultured fibroblasts.Biochimica et Biophysica Acta. 1985; 845: 349-357Crossref PubMed Scopus (45) Google Scholar,Williams et al., 1987Williams M.L. Rutherford S.L. Feingold K.R. Effects of cholesterol sulfate on lipid metabolism in cultured human keratinocytes and fibroblasts.J Lipid Res. 1987; 28: 955-967Abstract Full Text PDF PubMed Google Scholar). The enzymes responsible for the synthesis of CS are known to be strictly localized in different regions of the epidermis (Elias et al., 1984Elias P.M. Williams M.L. Maloney M.E. Bonifas J.A. Brown B.E. Grayson S. Epstein E.H. Startum corneum lipids in disorders of cornification: steroid sulfatase and cholesterol sulfate in normal desquamation and the pathogenesis of recessive X-linked ichthyosis.J Clin Invest. 1984; 74: 1414-1421Crossref PubMed Scopus (177) Google Scholar;Epstein et al., 1984Epstein Jr, E.h. Bonifas J.M. Barber T.C. Haynes M. Cholesterol sulfotransferase of newborn mouse epidermis.J Invest Dermatol. 1984; 83: 332-335Abstract Full Text PDF PubMed Scopus (25) Google Scholar;Momoeda et al., 1991Momoeda M. Taketani Y. Mizuno M. Iwamori M. Nagai Y. Characteristic expression of cholesterol sulfate in rabbit endometrium during the implantation period.Biochem Biophys Res Comm. 1991; 178: 145-150Crossref PubMed Scopus (24) Google Scholar;Kagehara et al., 1994Kagehara M. Tachi M. Harii K. Iwamori M. Programmed expression of cholesterol sulfotransferase and transglutaminase during epidermal differentiation of murine skin development.Biochimica et Biophysica Acta. 1994; 1215: 183-189Crossref PubMed Scopus (25) Google Scholar). Among the different layers of mouse epidermis, CS accumulates primarily in the stratum granulosum and stratum corneum (Elias et al., 1984Elias P.M. Williams M.L. Maloney M.E. Bonifas J.A. Brown B.E. Grayson S. Epstein E.H. Startum corneum lipids in disorders of cornification: steroid sulfatase and cholesterol sulfate in normal desquamation and the pathogenesis of recessive X-linked ichthyosis.J Clin Invest. 1984; 74: 1414-1421Crossref PubMed Scopus (177) Google Scholar;Lampe et al., 1983Lampe M.A. Williams M.L. Elias P.M. Human epidermal lipids: characerization and modulations during differentiation.J Lipid Res. 1983; 24: 131-140Abstract Full Text PDF PubMed Google Scholar), and Ch-ST has been shown to be localized in both the basal and the spinous layers (Epstein et al., 1984Epstein Jr, E.h. Bonifas J.M. Barber T.C. Haynes M. Cholesterol sulfotransferase of newborn mouse epidermis.J Invest Dermatol. 1984; 83: 332-335Abstract Full Text PDF PubMed Scopus (25) Google Scholar). 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., 1994Kagehara M. Tachi M. Harii K. Iwamori M. Programmed expression of cholesterol sulfotransferase and transglutaminase during epidermal differentiation of murine skin development.Biochimica et Biophysica Acta. 1994; 1215: 183-189Crossref PubMed Scopus (25) Google Scholar). 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 onlyin vitro, but alsoin vivo. Interestingly, the potent tumor promoter 12–0-tetradecanoylphorbol-13-acetate (TPA) increases both transglutaminase (Lichti et al., 1985Lichti U. Ben T. Yuspa S.H. Retinoic acid-induced transglutaminase in mouse epidermal cells is distinct from epidermal transglutaminase.J Biol Chem. 1985; 260: 1422-1426Abstract Full Text PDF PubMed Google Scholar;Yuspa et al., 1993Yuspa S.H. Hennings H. Lichti U. Organ specificity and tumor promotion.Basic Life Sci. 1993; 24: 157-171Google Scholar;Yuspa, 1994Yuspa S.H. The pathogenesis of squamous cell cancer: lessons learned from studies of skin carcinogenesis.Cancer Res. 1994; 54: 1178-1189PubMed Google Scholar) and Ch-ST activities (Jetten et al., 1989Jetten A.M. George M.A. Nervi C. Boone L.R. Rearick J.I. Increased cholesterol sulfate and cholesterol sulfotransferase activity in relation to the multi-step process of differentiation in human epidermal keratinocytes.J Invest Dermatol. 1989; 92: 203-209Abstract Full Text PDF PubMed Google Scholar) in epidermal cells. Treatment of normal human epidermal keratinocytes 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., 1994Ikuta T. Chida K. Tajima O. et al.Cholesterol sulfate, a novel activator for the eta isoform of protein kinase C.Cell Grow Different. 1994; 5: 943-947PubMed Google Scholar reported that CS can directly activate one of the protein kinase C (PKC) isoforms, PKCη,in vitro and that this isoform can phosphorylate TG-Iin vitro, which suggests a possible link for this PKC isoform in signaling differentiation in keratinocytesin 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 CSin 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) Ca2+ 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., 1993bGreenhalgh D.A. Rothnagel J.A. Quintanilla M.I. et al.Induction of epidermal hyperplasia, hyperkeratosis, and papillomas in transgenic mice by a targeted v-Ha-ras oncogene.Molec Cacinog. 1993 b; 7: 99-110Crossref PubMed Scopus (113) Google Scholar). 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. 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., 1987DiGiovanni J. Kruszewski F.H. Chenicek K.J. Studies on the skin tumor promoting actions of chrysorobin (1,8-dihydroxy-3-methyl-9-anthrone).in: Butterworth B. Slaga T.J. Nongenotoxic Mechanisms of Carcinogenesis. Cold Spring Harbor, Cold Spring Harbor, New York1987: 25-39Google Scholar). 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 7,12-dimethylbenz[a]anthracene was obtained from Eastman Kodak (Rochester, NY). 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., 1989DiGiovanni J. Gill R.D. Nettikumara A.N. Colby A.B. Reiners Jr, J.j. Effect of extracellular calcium concentration on the metabolism of polycyclic aromatic hydrocarbons by cultured mouse keratinocytes.Cancer Res. 1989; 49: 5567-5574PubMed Google Scholar). 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., 1980Hennings H. Michael D. Cheng C. Steinert P. Holbrook K. Yuspa S.H. Calcium regulation of growth and differentiation of mouse epidermal cells in culture.Cell. 1980; 19: 245-254Abstract Full Text PDF PubMed Scopus (1505) Google Scholar) 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 × 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 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 ≈70 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. 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 ×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. 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 Ch-ST enzyme-activity assay. After determination of protein, dithiothreitol was added (final concentration of 10 mM). The lysate was centifuged at 10,000 ×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., 1990Jetten A.M. George M.A. Rearick J.I. Down-regulation of squamous cell-specific markers by retinoids: transglutaminase type I and cholesterol sulfotransferase.Meth Enzymology. 1990; 190: 42-49Crossref PubMed Scopus (6) Google Scholar). 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., 1982Iwamori M. Sawada K. Hara Y. Nishio M. Fujisawa T. Imura H. Nagai Y. Neutral glycosphingolipids and gangliosides of bovine thyroid.J Biochem. 1982; 91: 1875-1887PubMed Google Scholar). 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., 1986Iwamori M. Kiguchi K. Kanno K. Kitagawa M. Nagai Y. Gangliosides as markers of cortisone-sensitive and cortisone-resistant rabbit thymocytes: characterization of thymus-specific gangliosides and preferential changes of particular gangliosides in the thymus of cortisone-treated rabbits.Biochem. 1986; 25: 889-896Crossref PubMed Scopus (23) Google Scholar;Momoeda et al., 1991Momoeda M. Taketani Y. Mizuno M. Iwamori M. Nagai Y. Characteristic expression of cholesterol sulfate in rabbit endometrium during the implantation period.Biochem Biophys Res Comm. 1991; 178: 145-150Crossref PubMed Scopus (24) Google Scholar) with triethanolamine as the matric solution, and by gas-liquid 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 thin-layer 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). 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 Schmidt-Thannhauser procedure as described bySlaga et al., 1974Slaga T.J. Bowden G.T. Shapas B.G. Boutwell R.K. Macromolecular synthesis following a single application of polycyclic hydrocarbons used as initiators of mouse skin tumorigenesis.Cancer Res. 1974; 34: 771-777PubMed Google Scholar. 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 ×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 ×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, 1956Burton K. A study of the conditions and mechanisms of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid.Biochem J. 1956; 62: 312-322Google Scholar). 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. 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., 1990Eldridge S.R. Tilbury L.F. Godsworty T.L. Butterworth B.E. Measurement of chemically induced cell proliferation in rodent liver and kidney: a comparison of 5-bromo-2′-deoxyuridine and [3H]thymide aministered by injection or osmotic pump.Carcinogenesis. 1990; 11: 2245-2251Crossref PubMed Scopus (217) Google Scholar). 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 promoter-treated mouse skin. When alkali-stable acidic lipids, corresponding to ≈3 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 atm/z 465 and sulfate-derived ions atm/z 80 and 97 (data not shown) (Momoeda et al., 1994Momoeda M. Cui Y. Sawada Y. Taketani Y. Mizuno M. Iwamori M. Pseudopregnancy-dependent accumulation of cholesterol sulfate due to up-regulation of cholesterol sulfotransferase and concurrent down-regulation of cholesterol sulfate sulfatase in the uterine endometria of rabbits.J Biochem. 1994; 116: 657-662PubMed Google Scholar). The amount of CS in control mouse epidermis was 0.21 ± 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 inFigure 1a. Maximum levels of CS were observed at 72 h or 72–120 h after treatment with TPA or CHRY and were 3.9- or 5.0-fold 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 1b 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