Theresa Ben

Phorbol ester tumor promoters induce epidermal transglutaminase activity

Abstract Epidermal basal cells in culture have low levels of epidermal transglutaminase, the enzyme responsible for the formation of the cross-linked envelope in differentiated cells. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate and other active (but not inactive) phorbol ester skin tumor promoters induce transglutaminase activity. Sloughing of differentiated cells accompanies the rise in transglutaminase activity. Phorbol esters do not affect transglutaminase activity when added directly to cell lysates. Corticosteroids have little influence on transglutaminase induction by phorbol esters. Retinoic acid induces transglutaminase activity, but activity does not further increase when basal cells are treated with both retinoic acid and 12-O-tetradecanoylphorbol-13-acetate.

Retinoids induce tissue transglutaminase in NIH-3T3 cells.

We report that all-trans and 13-cis-retinoic acid as well as the synthetic compound CH-55 enhance tissue transglutaminase activity as they increase NIH-3T3 cell adhesiveness. The 4-hydroxyphenylretinamide (4-HPR) with low activity in inducing attachment, lectin binding and growth inhibition also fails to induce transglutaminase. Thyroxine (Thy), a compound with a response element common to RA, is inactive. The tumor promoter 12-tetradecanoyl-phorbol-13-acetate (TPA), which increases adhesiveness with different kinetics than RA, failed to enhance tranglutaminase. We conclude that retinoids with biological activity in inducing adhesion, inhibition of growth and increase of lectin binding, are also active in inducing transglutaminase activity.

Effects of retinoic acid on the growth and morphology of hamster tracheal epithelial cells in primary culture

SummaryHamster tracheal epithelial cells were grown in primary culture on a collagen gel substrate in hormone-supplemented serum-free Ham’s F12 medium with 10-8 M retinoic acid (RA +), or without retinoic acid (RA -). On days 1 and 2, the colonies were composed of large (secretory) cells and lesser numbers of small (basal) cells; ciliated cells were rare. At these times, cell number, thymidine incorporation, and total labelling indices (small and large cells, combined) were similar in RA+ and RA-cultures, but the large cells became flat in RA-medium on day 2. On days 3–5, thymidine incorporation and total labelling indices were less in RA-than RA+ cultures, and on days 4–6, cell numbers were decreased in RA-cultures. On day 3, the large cells of the RA-colonies had flattened further and clusters of small basal cells had formed. On day 4, the RA+ colonies were composed of densely-packed cuboidal secretory cells, small basal cells were inconspicuous; the total labelling index was about 27%. The RA-colonies were composed of large flat secretory cells and numerous small basal cells which were clustered in groups; the total labelling index was about 7%. Since large and small cells could be discriminated by size in RA-colonies, a labelling index was generated based on cell size. On days 2, 3 and 4, the labelling index of the small basal cells in the RA-colonies was 44%, 43% and 24% respectively, whereas the labelling index of the large secretory cells fell rapidly over the same period (56%, 14% and 2%). On days 5 and 6, the cuboidal secretory cells in the RA + cultures had differentiated further and the cells were stratified focally. Some new ciliated cells had formed on day 6. In RA cultures, mucous granules were not observed in the large flat cells and ciliated cells were not seen. The total labelling indices were 11% and 0.35% in RA+ cultures, and 0.5% and 0.25% in RA-cultures on days 5 and 6, respectively.The study shows that the target cell for vitamin A in the hamster tracheal epithelium is the secretory (mucous) cell. When retinoic acid was deficient, the secretory cells flattened and their capacity to divide was greatly diminished. Since the basal cells continued to replicate when the secretory cells did not, the population density of the basal cells increased disproportionally, which could be interpreted erroneously as a “basal cell hyperplasia.” Real hyperplasia and epidermoid metaplasia were late secondary events which occurred in this study following focal disintegration of the epithelial cell sheet. Then the large secretory cells became keratinized and expressed an epidermoid phenotype.

RETINOIDS INHIBIT PROMOTER‐DEPENDENT PRENEOPLASTIC PROGRESSION IN MOUSE EPIDERMAL CELL LINES

We have recently described a cell culture system for studying promoterdependent preneoplastic progression.l.2 The JB6 cell line has been derived from primary mouse epidermal cultures and found to respond to tumor-promoting but not to nonpromoting phorbol esters with an irreversible induction of tumor-cell phenotype, as measured by colony formation in 0.33% agar at 14 days. The mechanism appears to involve induction of a new phenotype rather than selection of preexisting anchorage-independent cells.3 The JB6 precursor cells are noatumorigenic while the phorbol ester-induced anchorage-independent transformants are tumorigenic (Colburn, submitted). Thus in view of its rapidity and irreversibility this anchorage-independence response to phorbol esters by JB6 cells appears to be analogous to late-stage skin tumor promotion in vivo. Inhibition of preneoplastic progression by retinoids has been shown for a number of epithelial systems.4~5 Boutwell and coworkers have reported that retinoids applied to mouse skin one hour before TPA during the promotion phase inhibited tumor yield substantially.6,7 The experiments described in this report were undertaken (1) to determine whether retinoids are active in inhibiting late-stage promotion in cell culture; (2) to use retinoid inhibition as an approach to identifying molecular and cellular events that are required in the process of tumor promotion, and (3) to investigate the mode of action of retinoids in tumor prophylaxis.

MODULATION OF TERMINAL DIFFERENTIATION AND RESPONSES TO TUMOR PROMOTERS BY RETINOIDS IN MOUSE EPIDERMAL CELL CULTURES

For more than 25 years, retinoids have been known to alter the state of differentiation of epidermis or similar stratifying squamous The nature of this alteration often varied in experimental systems depending on species studied, dose or analog used, or method of application^.^ More recently retinoids have been shown to modify epidermal carcinogenesis by preventing the occurrence of tumors during the promotion phase,6 or by causing existing tumors to regres7,8 This laboratory has utilized cultured mouse keratinocytes for a variety of studies related to differentiation and chemical carcinogenesis. Previous data have substantiated the validity of this cell culture model for defining normal epidermal function as well as alterations associated with tumor initiation and promotion.9-12 This model has also been useful for defining the molecular events associated with retinoid-induced alterations in differentiation and carcinogenesis. The results of some of these studies are summarized in TABLE 1. Retinoid concentrations of 105 10-6M have resulted in epidermal metaplasia with morphological characteristics of a secretory epithelium.g,18,19 Under these conditions retinoids have generally been growth inhibitory.g,15 While semiconservative DNA synthesis is decreased, DNA repair synthesis is quantitatively unaffected by retinoid exposure.15 Glycoprotein synthesis and the nature of epidermal glycoproteins are rapidly altered after retinoid exposure.13.14 Both metabolism and macromolecular binding of polycyclic aromatic hydrocarbon carcinogens are changed in epidermal cells exposed to retinoids. At considerably lower levels of exposure to retinoids (10-7 10-9M), the effects of phorbol ester tumor promoters in skin are modified.6,16.17 Tumor promotion itself is inhibited in uiuo at these exposure levels.6 Recently Hennings et al. have reported that ionic calcium is a critical regulator of epidermal growth and differentiation in cell culture.20 By adjusting Ca++ to a low level (0.02 0.09 mM) in culture medium, epidermal cells proliferate rapidly and do not stratify. The addition of Ca*+ to a level of 1.0 2.0 mM induces a rapid sequence of changes resulting in terminal differentiation of most or all of the epidermal cells in culture. The capability of controlling growth and differentiation in the epidermal population by manipulating Ca** prompted us to reexamine the role of retinoids in this process. A portion of this report will present results of this study. In attempting to better understand the mechanism of tumor promotion in epidermis, we have been studying the induction of the enzyme ornithine decarboxylase (ODC) which results from exposure to the potent tumor promoter 12-0-tetradecanoyl phorbol13-acetate.z1,2* Some of these studies have utilized inhibitors of tumor promotion. Such studies are useful because they provide insight

Phorbol esters enhance attachment of NIH/3T3 cells to laminin and type IV collagen substrates

The effect of phorbol esters on the adhesive properties of NIH/3T3 mouse fibroblasts was investigated using plastic substrates precoated with the extracellular matrix proteins fibronectin, collagen, and laminin. Treatment with phorbol 12-myristate 13-acetate (PMA) enhanced NIH/3T3 cell attachment to laminin and type IV collagen substrates but had little or no effect on attachment to fibronectin and type I collagen substrates. The effect of PMA in enhancing cell attachment to laminin and type IV collagen substrates was dose dependent between 10(-9) and 10(-7) M. PMA was effective as early as 30 min; the effect reached a maximum at 2 h and decreased gradually. Phorbol 12, 13-dibenzoate and phorbol 12, 13-diacetate were effective but to a lesser extent and phorbol 12-myristate and phorbol 13-acetate showed little or no effect. These results suggest that PMA may enhance NIH/3T3 cell adhesion through effects on laminin and type IV collagen receptors. Retinoic acid, which itself requires at least 6 h to show an effect on attachment, did not have any effect on cell attachment in 2 h and, if anything, slightly inhibited PMA-enhanced cell attachment to laminin and type IV collagen substrates.

GERMICIDAL ULTRAVIOLET LIGHT INDUCES ORNITHINE DECARBOXYLASE IN MOUSE EPIDERMAL CELLS AND MODIFIES THE INDUCTION CAUSED BY PHORBOL ESTER TUMOR PROMOTERS

Germicidal ultraviolet light (UVC. 8–10 J/m2) induces ornithine decarboxylase (ODC) in mouse epidermal cells in vitro in a biphasic manner with maxima of 2–3 fold induction at 4–6 h and of 10–20 fold induction at 15–18 h after irradiation. At this dose of UVC overall protein synthesis is inhibited by 10–30% and RNA synthesis by 40–50%. Induction of both ODC peaks is prevented by actinomycin D or cycloheximide. Similar culture factors appear to influence the extent of ODC induction by UVC and by the tumor promoter, 12‐O‐tetradecanoyl phorbol‐13‐acetate (TPA), since the ratio of peak activities is approximately constant at 2, whereas absolute values vary considerably between experiments. If cells are irradiated with UVC and then exposed to TPA, the effects are additive at 10 J/m2, less than additive at higher and enhanced at lower doses of UVC.

Retinol and β‐carotene concentrations in skin, papillomas and carcinomas, liver, and serum of mice fed retinoic acid or β‐carotene to suppress skin tumor formation

Abstract Using 7,12‐dimethylbenz[a] anthracene as the initiator and 12‐O‐tetradecanoyl‐13‐acetate as the tumor promoter on the dorsal skin of Sencar mice, we previously showed that pharmacological dietary all‐trans‐retinoic acid and β‐carotene inhibit the conversion of papillomas to carcinomas in a two‐stage system of chemical carcinogenesis. The purpose of this study was to determine the influence of dietary retinoic acid and β‐carotene on retinoidand β‐carotene concentrations in skin and other tissues. We were unable to measure tissue retinoic acid because of the relatively limited amount of tissue available for analysis and the fast rate of metabolism. Different dietary levels of retinoic acid or β‐carotene did not influence total retinol of skin, papilloma, and carcinoma tissues, which all showed a concentration of approximately 1 ± 0.5 μg/g wet wt. Equally refractory to dietary retinoic acid or β‐carotene was serum retinol concentration. In contrast, dietary retinoic acid protected loss of liver reti...