Retinoic acid (RA) exerts its pleiotropic effects on cell growth and differentiation through the activation of a family of transcription factors-the RA receptors (RARs). Three subtypes of these receptors exist, RARa, RAR(3, and RARly. The receptors are differentially expressed in different cell types and stages of development, suggesting that they may regulate different sets of genes. We have identified a synthetic retinoid with the characteristics of a selective RARa antagonist. This antagonist counteracts RA effects on HL-60 cell differentiation and on B-lymphocyte polyconal activation.Beyond its potential practical relevance, this and other specific antagonists will be useful to dissect the RAR system and to assign to one given receptor each of the many RA-regulated functions.The natural retinol (vitamin A) derivative retinoic acid (RA) is known to have profound effects on cell growth and differentiation (1) and to be essential for normal embryonic development (2). While RA and some synthetic analogs (retinoids) are useful in the control of some tumors (3) as well as of nonmalignant hyperproliferative conditions of the skin (4), they are, at high concentrations, teratogenic (5).The pleiotropic effects of retinoids are mediated by two known families of nuclear receptors, both belonging to the steroid-thyroid hormone receptor superfamily of ligandinducible transcriptional regulators (6, 7). The RA receptor (RAR) gene family comprises three subtypes-RARa (8, 9), RAR,[8][9][10][11][12], and RAR'y (13, 14)-with each gene encoding a variable number of isoforms arising by differential splicing of two primary . All receptors of the RAR family bind RA with comparable affinity (18). The retinoid receptors of the second family (RXR) do not bind the major form of RA (all-trans-RA) (19). They bind instead the 9-cis stereoisomer of RA (20, 21).Transcription of some RAR genes themselves is RA sensitive (22-25). Also, the expression of some of the cellular retinol-or RA-binding proteins (CRBP and CRABP), putatively involved in the storage, transport, and/or metabolism of retinol and RA, is differentially regulated by RA in a receptor-specific manner (26-28). The RA-related molecules represent, therefore, an autoregulated system. RAR types and isoforms, as well as RXRa and RXRB, are differentially expressed both spatially and temporally (15-18, 29-32). They might therefore regulate different target genes during embryonic and adult life, as well as in specific cell types at different stages of differentiation. RARa is the most ubiquitously expressed, while RAR8 and RARy display a more restricted pattern of distribution, with RARy being predominantly expressed in the skin (31).It seems reasonable to assume that the multiple effects of RA could be dissociated by specific ligands for each of the known receptors, and/or by receptor-specific antagonists, so as to obtain the desired beneficial effects while limiting the unwanted side effects. Retinoids with a good degree of selectivity have been described (33), and we have o...

In order to shed further light on the potential role of mast cells during tissue turnover, we have investigated the number of mast cells containing only tryptase and those storing both tryptase and chymase by enzyme histochemistry in normal versus healing skin. Furthermore, we have studied the in vitro effect of these enzymes on the mitogenesis of subconfluent quiescent fibroblast and HaCaT keratinocyte cultures, using flowcytometric DNA analysis. Chymase-containing mast cell numbers were markedly decreased in scars (P<0.001), whereas the overall number of tryptase-containing mast cells was not decreased, although these cells were smaller and stained more faintly in scars. Chymase (5 to 300 mU/ml) induced a marked, dose-dependent in vitro mitogenic response in 3T3 fibroblasts, whereas the effects of tryptase, at up to 60 nM, were only moderate, compared to the known fibroblast mitogens EGF, TGF-alpha, alpha-thrombin and trypsin at optimal concentrations. Coincubation of either protease with EGF or alpha-thrombin had additive effects. In contrast to fibroblasts, keratinocytes showed only minor mitogenic responses to tryptase and chymase, also in comparison to other known mitogenic stimuli, and responses to EGF and alpha-thrombin were inhibited on costimulation of cells with the proteases. These findings document for the first time a potential role of mast cell chymase in connective tissue repair, with tryptase being less active on fibroblasts, and with inhibitory effects of both mast cell proteases on keratinocytes.

Flow cytometric measurements of the DNA content were performed on a large number of skin biopsies by an automated technique. Expressed as a percentage of all viable cells in the epidermis, the figures for cells in S-phase averaged 1.8% and for G2M 0.9%. No significant differences due to sex were found. Concomitantly with age the ratio S/G2M (representing the duration of S to the duration of G2M) increased. Also seasonal effects were clear, showing higher values for S and G2M in June compared to November and December. Lastly we found small differences dependent on body-site, the ratio S/G2M being greater in legs than in arms. The present status is discussed together with future lines of development.

This report describes an immunocytochemical procedure for the simultaneous quantification of bromodeoxyuridine (BrdUrd) incorporated into cellular DNA and total DNA content in individual cells in suspension. Improvement of existing methods was achieved by combining acid denaturation and proteolytic enzyme digestion (0.2 mg/ml pepsin in 2N HCl for 30 min at room temperature). Acid denaturation preceded by enzyme digestion resulted in a large amount of debris and the occurrence of naked nuclei. In contrast, the Simultaneous measurement of incorporated bromodeoxyuridine (BrdUrd) and relative DNA content by flow cytometry allows detailed analysis of cell cycle kinetics (4,8,11,14,15). Several immunochemical procedures for cell staining have been described using anti-BrdUrd and propidium iodide, measuring simultaneously DNA synthesis and relative DNA content (5,7,8,13). Binding of the monoclonal anti-BrdUrd antibody requires regions of single-stranded DNA, while propidium iodide, an intercalating dye, requires double-stranded DNA, Acid treatment (7) or thermal denaturation (81, the best compromises, have most often been used. Improvements on these techniques have recently been reported using additional cell treatments, such as extraction of chromatin proteins by detergent, reduction of autofluorescence by sodium borohydride (51, or an extra protein degradation step using a proteolytic enzyme (13). However, these protocols are either time-consuming or, at least in our hands, cause substantial cell loss, especially when applied to hematopoietic cells.Here we describe a n improvement of the method of Schutte et al. (13), combining the protein digestion and acid hydrolysis steps and using FITC-conjugated antiBrdUrd. MATERIALS AND METHODSCell Culture and Tissue Preparation Human neonatal foreskin keratinocytes were cultured on 3T3 feeder cells (12). Epidermal cells were seeded on simultaneous denaturationlprotein digestion procedure did not damage the cellular structure, is rapid and reproducible, and has cell recoveries of more than 85%. Although experimental conditions were tested on human cultured keratinocytes, this method also appeared applicable to bone marrow cells and cells obtained from solid tissues.

A sequential double immunoenzymic staining procedure was developed using the monoclonal antibody anti-BrdUrd and Ki67 in order to determine whether hyperproliferative skin disorders, such as psoriasis, are characterized by an increased growth fraction rather than a much shorter cell cycle time of all germinative cells. Ki67 binds to a proliferation-associated nuclear antigen in a variety of human cell types, and anti-BrdUrd can be used to identify DNA-synthesizing cells. Although in hyperproliferative epidermis the absolute numbers of BrdUrd-positive cells as well as Ki67-positive cells were grossly increased, the ratio of these values was not changed compared to the ratio found in the epidermis of the clinically uninvolved skin of psoriatic patients and in normal epidermis. This suggests an increased growth fraction in hyperproliferative epidermis. Our data show that immunohistochemical double-staining techniques can be a valuable tool in the study of cell cycle kinetics in epithelial tissues.