tretinoin and cholesteryl-sulfate

Vitamin A and some of its metabolites such as beta-all-trans retinoic acid (RA) have been implicated in the regulation of differentiation of normal and malignant epithelial cells in vivo and in vitro. In the present study the effects of RA on the growth and differentiation of 7 cell lines derived from human head and neck squamous-cell carcinomas (HNSCCs) were examined. RA (greater than 0.01 microM) inhibited the proliferation in monolayer culture of 6 of 7 HNSCC cell lines. One cell line (UMSCC-35) was very sensitive, 5 (UMSCC-10A, -19, -30, -22B and HNSCC 1483) were moderately sensitive, and 1 (HNSCC 183) was insensitive. Three of the cell lines (UMSCC-22B, -30, and HNSCC 1483) were capable of forming colonies in semisolid medium--a capability that was suppressed by RA. The HNSCC cell lines expressed various levels of the squamous-cell differentiation markers type I (particulate, epidermal) transglutaminase (TGase) and cholesterol sulfate (CS). RA treatment (I microM, 6 days) decreased TGase activity by more than 50% in 3 (UMSCC-10A, -22B and 1483) of the 7 cell lines, and the effect on UMSCC-22B was dose-dependent. Type II TGase (soluble, tissue type) activity was detected in 3 cell lines, and after RA treatment its activity increased in HNSCC 1483 and 183 cells and decreased in UMSCC-19. Following RA treatment, CS levels decreased by 20, 25, 70, 76, 89 and 91% in cell lines UMSCC-30, -10A, 183, UMSCC-35, -22B, and HNSCC 1483, respectively. The suppression by RA of CS accumulation in the 1483 cells was dose-dependent. Cholesterol sulfotransferase activity, which is responsible for CS synthesis, was suppressed by 40-97% after RA treatment of UMSCC-19, -22B, and HNSCC 1483. Our results demonstrate that RA inhibits the growth and decreases the level of 2 squamous differentiation markers in HNSCC cells.

Topics: Carcinoma, Squamous Cell; Cell Line; Cell Transformation, Neoplastic; Cholesterol Esters; Depression, Chemical; Dose-Response Relationship, Drug; Head and Neck Neoplasms; Humans; Mouth Neoplasms; Sulfotransferases; Transglutaminases; Tretinoin; Tumor Cells, Cultured

In this study the synthesis of cholesterol sulfate is examined in relation to the process of squamous differentiation in normal human epidermal keratinocytes (NHEK) in culture. During the exponential growth phase, NHEK cells exhibit a relatively high colony-forming efficiency and appear undifferentiated on the basis of their morphology and expression of biochemical characteristics. At confluence, the cells undergo terminal differentiation that is characterized by the commitment to terminal cell division (reduction in colony-forming ability) and expression of the differentiated phenotype. An accumulation of cholesterol sulfate accompanies this program of differentiation. This accumulation of cholesterol sulfate parallels the increase in transglutaminase type I activity and the competence to form cross-linked envelopes, whereas it precedes the "spontaneous" formation of cross-linked envelopes. Increased cholesterol sulfotransferase activity appears to account for the increase in cholesterol sulfate. The cholesterol sulfate accumulation, as well as the increase in cholesterol sulfotransferase and transglutaminase activity, are inhibited by retinoids. However, the presence of retinoids does not prevent NHEK cells from undergoing terminal cell division at confluence. Two NHEK cell lines expressing SV40-large T antigen also undergo terminal differentiation at confluence and start to accumulate cholesterol sulfate. Two other, differentiation-defective cell lines do not exhibit an increase in cholesterol sulfate at confluence. These results show that epidermal keratinocytes in culture, like cells in the epidermis, accumulate cholesterol sulfate when undergoing squamous differentiation. This program appears to consist of a retinoid-insensitive step (commitment to terminal cell division) and a retinoid-sensitive step (expression of the squamous differentiated phenotype).

Topics: Calcium; Cell Differentiation; Cell Line, Transformed; Cells, Cultured; Cholesterol Esters; Epidermal Cells; Epidermis; Humans; Keratins; Sulfotransferases; Tretinoin

This study examines the action of phorbol 12-myristate 13-acetate (PMA) on the synthesis of cholesterol sulfate in cultured normal and transformed human epidermal keratinocytes and assesses the antagonistic effects by retinoids and bryostatins on PMA action in relation to the multistep program of squamous differentiation. Treatment of normal human epidermal keratinocytes (NHEK) with PMA induces terminal cell division (irreversible growth-arrest) and causes a time- and dose-dependent increase in the incorporation of Na2(35)SO4 into cholesterol sulfate, a marker for squamous cell differentiation. This stimulation in sulfate incorporation appears specific for cholesterol sulfate and is due to increased levels of cholesterol sulfotransferase activity. The increase in cholesterol sulfate accumulation parallels the increase in transglutaminase type I, another marker for squamous differentiation. Several transformed NHEK cell lines do not exhibit increased levels of cholesterol sulfate and transglutaminase type I activity after PMA treatment, indicating that they acquired defects in the regulation of squamous differentiation. Bryostatins 1 and 2, and several diacylglycerol analogues neither inhibit cell proliferation nor increase cholesterol sulfate synthesis or transglutaminase activity, indicating that these agents do not induce terminal differentiation. In contrast, the bryostatins block the increase in cholesterol sulfate and transglutaminase activity as well as the commitment to terminal cell division by PMA. Bryostatin 1 inhibits the commitment to terminal cell division and the accumulation of cholesterol sulfate significantly even when added 8 h after PMA administration. Retinoids inhibit cholesterol sulfate accumulation and the increase in transglutaminase activity by PMA but do not affect the commitment to terminal cell division. In summary, phorbol esters induce in NHEK cells a program of squamous differentiation. This process of differentiation consists of the commitment to terminal cell division and expression of a squamous phenotype. Expression of this phenotype is accompanied by an accumulation of cholesterol sulfate and increased cholesterol sulfotransferase activity. Bryostatins 1 and 2 and retinoic acid affect this differentiation process at different stages.

Topics: Bryostatins; Cell Differentiation; Cell Line, Transformed; Cholesterol Esters; Diglycerides; Epidermal Cells; Humans; Keratins; Lactones; Macrolides; Tetradecanoylphorbol Acetate; Tretinoin

One of the primary events in the pathogenesis of acne vulgaris is abnormal follicular keratinization. Since oral isotretinoin therapy reduces follicular hyperkeratinization in acne, our study has been designed to determine whether epidermal lipid composition of the epithelium of sebaceous follicles is affected by isotretinoin treatment. Noninflamed early comedones obtained from ten patients with nodulocystic acne before and after the 6th week of isotretinoin therapy (mean daily dose 0.7 mg/kg b. wt.) were used as probes of the hyperkeratinizing follicular epithelium. Comedonal lipids were analyzed by high-performance thin-layer chromatography. Oral isotretinoin caused a decrease of the comedonal glyceride fraction by 36% (P less than 0.01), whereas free sterols and total ceramides increased by 34% (P less than 0.10) and 19%, respectively. The changes of comedonal lipids were associated with a significant elevation of the free sterols/cholesterol sulfate ratio of 86% from pretreatment levels (P less than 0.05). The isotretinoin-induced changes of the comedonal lipid composition in direction to a pattern of epidermal lipids of normal desquamating stratum corneum are discussed as a possible comedolytic mechanism of oral isotretinoin treatment.

Topics: Acne Vulgaris; Adult; Ceramides; Cholesterol Esters; Dermatologic Agents; Female; Humans; Isotretinoin; Lipid Metabolism; Male; Sebaceous Glands; Sterols; Tretinoin; Triglycerides

In this paper we show that the expression of the squamous differentiated phenotype and mucosecretory phenotype by cultured rabbit tracheal epithelial cells can be regulated by substratum and the presence of retinoic acid. Cells grown on a type I collagen gel matrix in the absence of retinoic acid stratify and undergo squamous differentiation as indicated by the appearance of squamous, cornified cells. Under these conditions cells are rich in desmosomes and heavy tonofilament bundles. These cells also express several biochemical markers for squamous differentiation such as high levels of type I transglutaminase and cholesterol sulfate. High levels of transglutaminase were also observed in areas of squamous metaplasia in tracheas of vitamin A-deficient hamsters. Treatment with retinoic acid not only blocked squamous differentiation as evidenced by the inhibition of the biochemical markers for squamous differentiation but induced the appearance of columnar, polarized cells many of which contained secretory granules. These granules stained positively with periodic acid thiocarbohydrazide and certain lectins indicating the presence of glycoconjugates. Analysis of radiolabeled glycoconjugates released into the medium indicated the synthesis of mucous glycoproteins. It appears that retinoic acid determines the pathway of differentiation whereas the collagen gel matrix is permissive for the expression of both phenotypes. The morphological and biochemical similarities between this in vitro cell system and the normal and metaplastic tracheal epithelium suggest that this rabbit tracheal epithelial cell system is a useful and relevant model to study the regulation of differentiation of the tracheobronchial epithelium.

Topics: Animals; Cell Differentiation; Cell Division; Cells, Cultured; Cholesterol Esters; Collagen; Cytoplasmic Granules; Epithelial Cells; Epithelium; Extracellular Matrix; Keratins; Male; Mucins; Phenotype; Rabbits; Trachea; Transglutaminases; Tretinoin

Rabbit tracheal epithelial (RbTE) cells in culture undergo terminal squamous differentiation characterized by enhanced transglutaminase activity, synthesis of specific keratins, and the formation of cross-linked envelopes. The expression of each of these markers of differentiation occurs spontaneously after the cells reach confluency, but this expression can be inhibited by the inclusion of retinoids in the extracellular medium. In the current work, we demonstrate that radioactive sulfate incorporation into the organic phase of a CHCl3/CH3OH (2:1) extract of RbTE cells increases 50- to 100-fold upon differentiation and that this accumulation can be completely blocked by the inclusion of retinoic acid in the culture medium. By the techniques of specific metabolic radiolabeling, thin layer chromatography, gas chromatography-mass spectrometry, and fast atom bombardment-mass spectrometry, the sulfated amphiphile was shown to be cholesterol 3-sulfate. Cholesterol sulfate accumulation begins 1 to 2 days after the RbTE cells reach the stationary phase of growth which is the same time that other differentiated functions begin to be expressed. The inhibition of accumulation by retinoic acid is concentration-dependent and half-maximal at 5 X 10(-11) M. The relative efficacy of a series of synthetic retinoids in inhibiting cholesterol sulfate accumulation correlated with their binding to the cellular retinoic acid-binding protein. These data taken together indicate that cholesterol sulfate is a marker of squamous differentiation in RbTE cells in culture. Possible biochemical mechanisms of the regulation of cholesterol sulfate levels during differentiation are discussed.

Topics: Animals; Cell Differentiation; Cholesterol Esters; Chromatography, Ion Exchange; Chromatography, Thin Layer; Dose-Response Relationship, Drug; Epithelial Cells; In Vitro Techniques; Mass Spectrometry; Rabbits; Retinoids; Structure-Activity Relationship; Trachea; Tretinoin