The effects on epidermal DNA synthesis of topically applied cycloheximide

An animal model for investigating topically applied epidermal cytotoxic drugs to determine optimum therapeutic doses and schedules is described. It is used here to examine the effects of cycloheximide on epidermal DNA synthesis, as measured by 3H-thymidine incorporation in vivo, in terms of dose/response and activity/time profiles and potential systemic toxicity.     

Differential effects of retinoids on DNA synthesis in calcium-regulated murine epidermal keratinocyte cultures

To study the possibility that the state of proliferation of epidermal keratinocytes can influence the action of retinoids, the rate of proliferation of murine epidermal keratinocytes was manipulated by growing the cells in media containing high or low concentrations of Ca++. In contrast to what other investigators have reported, keratinocytes cultured in medium containing 1.4 mM Ca++ proliferate faster, instead of slower, than cells cultured in medium with 0.09 mM Ca++. Other experiments showed that Ca++ was stimulatory to keratinocytes in medium containing a low level of growth factors, and inhibitory in medium containing a high level of growth factors, suggesting that the discrepancy could be due to a difference in the sera used. The high Ca++ cells prominently expressed the 48kD/56kD pair of keratin, showing that they were in a hyperproliferative state. Exposure of the faster growing high Ca++ cells to all-trans retinoic acid, 13-cis retinoic acid, etretinate, etretin, and arotinoid ethyl ester caused dose-dependent inhibition of DNA synthesis. In contrast, exposure of the slower growing low Ca++ cells to these retinoids resulted in dose-dependent stimulation of DNA synthesis. In addition, all-trans retinoic acid caused dose-related increases in cell number in the low Ca++ cultures. These findings correlate with the reported differential effects of retinoids on normal and hyperproliferative epidermis, and suggest that Ca++ and low growth factor-regulated keratinocyte cultures are useful for studying the mechanism of hyperproliferation and retinoid actions.     

Barrier function regulates epidermal lipid and DNA synthesis

The stratum corneum, the permeability barrier between the internal milieu and the environment, is composed of fibrous protein-enriched corneocytes and a lipid-enriched intercellular matrix. The lipids are a mixture of sphingolipids, cholesterol and free fatty acids, which form intercellular membrane bilayers. Lipid synthesis occurs in the keratinocytes in all nucleated layers of the epidermis, and the newly synthesized lipids are delivered by lamellar bodies to the interstices of the stratum corneum during epidermal differentiation. Disruption of barrier function by topical acetone treatment results in an increase in the synthesis of free fatty acids, sphingolipids and cholesterol in the living layers of the epidermis, leading to barrier repair. Cholesterol and sphingolipid synthesis are regulated by the rate-limiting enzymes HMG CoA reductase and serine palmitoyi transferase (SPT). respectively. Acute barrier disruption leads to an increase in both enzymes, but with a different time curve: increase in HMG CoA reductase activity begins at 1.5 h, whereas the increase in SPT activity occurs 6 h after barrier impairment. Topical application of HMG CoA reductase or SPT inhibitors after acetone treatment delays barrier repair, providing further evidence for a role of cholesterol and sphingolipids in epidermal barrier function. Repeated application of lovastatin to untreated skin results in disturbed barrier function accompanied by increased DNA synthesis and epidermal hyperplasia. Therefore, we have examined the specific relationship between barrier function and epidermal DNA synthesis. After acute and chronic disturbances not only lipid, but also DNA synthesis, is stimulated. Thus, stimulation of DNA synthesis leading to epidermal hyperplasia may be a second mechanism by which the epidermis repairs defects in barrier function. The link between barrier function and both lipid and DNA synthesis is supported further by occlusion studies. Artificial barrier repair by latex occlusion prevents an increase in both lipid and DNA synthesis. In addition, increased epidermal lipid and DNA synthesis in essential fatty-acid deficiency can be reversed by topical applications of the n-6 unsaturated fatty acids, linoleic or columbinic acid. These studies may be of relevance in understanding the pathogenesis of hyperproliferative skin diseases, such as ichthyosis, psoriasis, atopic dermatitis, and irritant contact dermatitis.     

Linoleic acid effects on epidermal DNA synthesis and cutaneous prostaglandin levels in essential fatty acid deficiency

An essential fatty acid (EFA) deficient state has been induced in hairless mice. The epidermal changes included hyperkeratosis, hypergranulosis and acanthosis. Epidermal DNA synthesis was increased 3-fold compared with normal diet mice. Prostaglandin E (PGE) and prostaglandin F (PGF) levels, measured by radioimmunoassay, were much reduced in the EFA deficient mice skin. 10% Linoleic acid applied topically for 2 weeks corrected the gross and histological skin abnormalities and reduced epidermal DNA synthesis to normal values. The levels of PGE and PGF were only partially corrected. Linoleic acid applied to normal diet mice increased skin levels of PGE and PGF compared with the control vehicle treated normal diet mice. These results provide further evidence for the importance of essential fatty acids in the control of epidermal proliferation and differentiation. The importance of PGE and PGF in controlling epidermal DNA synthesis in EFA deficiency is less clear.     

Effects of topical prostaglandin E analogue on normal hairless mouse epidermal DNA synthesis.

The in vivo topical effects of a synthetic analogue of prostaglandin E2 (15(S)-15-methyl PGE2 methyl ester [PGE2 analogue]), have been studied on the epidermis of hairless mice. One microgram of the PGE2 analogue increased DNA synthesis significantly by 5 hr, a maximum increase of 390% was reached by 12 hr, and DNA synthesis returned to control levels by 24 hr. Twenty micrograms of the PGE2 analogue reduced epidermal DNA synthesis for 12 hr after application, DNA synthesis was increased at 24 hr, returning to control levels by 48 hr. One hundred micrograms of topical PGE2 had no significant effect on epidermal DNA synthesis over 48 hr, but 1 mug of intradermal PGE2 increased DNA synthesis by 160% at 24 hr. These results suggest that topical 15(S)-15-methyl PGE2 methyl ester is biologically active compared with PGE2.     

Products of epidermal protein synthesis.

The products of epidermal protein synthesis in tissue slice and cell-free systems have been characterized by chromatographic, electrophoretic, and immunologic methods. Radiolabeled proteins of heterogeneous size are found in the supernatant and particulate fractions from both systems. The major labeled proteins in the supernatant fraction have molecular weights of approximately 43 000 and 65 000. The natively-insoluble proteins of the particulate fraction were solubilized either with desoxycholate or alkaline urea. Subsequent analysis of theses fractions yielded a labeled, high molecular weight, urea-soluble protein whose electrophoretic mobility was slower than that of the heavy chain of myosin. The immunologic reaction between the radiolabeled fraction and antibody raised against filamentous protein suggests that synthesis of proteins identical to epidermal filamentous protein occurs in the in vitro systems.     

DNA synthesis among uninvolved and involved psoriatic epidermal cells and normal epidermal cells in vitro

Epidermal cells from 8 patients with psoriasis (involved and uninvolved areas) and from 7 normal individuals, were propagated in vitro. DNA synthesis was determined autoradiographically by measuring the labeling index (1 hr) and a continuous labeling index (72 hr) on 8-10 day old cell cultures. No differences in these 2 kinetic parameters were obtained with respect to involved and uninvolved psoriatic cells. However, when psoriatic cells (involved and uninvolved) were compared to normal cells, the psoriatic cells exhibited a much higher labeling index and continuous labeling index, 200-300% and 60-70% respectively. These data clearly show that in vitro (1) epidermal cells from clinically uninvolved skin of psoriatics proliferate at a rate equal to that of involved psoriatic cells and (2) psoriatic epidermal cells proliferate at a rate greater than normal cells.     

An animal model for evaluating the local and systemic effects of topically applied corticosteroids on epidermal DNA synthesis

A simple animal model for studying topically applied glucocorticosteroids for their effect on epidermal DNA synthesis is described. It is designed so that their potential for systemic side effects may be predicted and its usefulness in this respect is illustrated by the results obtained with two of the steroids investigated. One, a new formulation Temetex, is predicted to have considerable systemic potential. The other, Molivate, is remarkable for having moderate local, without demonstrable systemic activity.     

Pig-skin epidermal calmodulin: Effects of antagonists of calmodulin on DNA synthesis of pig-skin epidermis

Summary Although epidermal keratinocytes contain significant amounts of calmodulin, the exact role of calmodulin in epidermal biological activity remains to be determined. Pig-skin (epidermal) calmodulin was purified to homogeneity by DEAE/Sepharose- and phenothiazine-affinity-column chromatography. The characteristics of the purified calmodulin proved to be in good agreement with those of calmodulin obtained from other sources. Phenothiazines (trifluoperazine and chlorpromazine), mepacrine, propranolol, and colchicine inhibited the effect of the purified epidermal calmodulin on the calmodulin-deficient phosphodiesterase of bovine heart. These calmodulin antagonists all had inhibitory effects on the thymidine incorporation of pig-skin epidermal keratinocytes. These observations support the assumption that calmodulin might play an important role in epidermal keratinocyte proliferation.Abbreviations EGTA Ethylene glycol bis (-aminoethyl)-N,N,N,N-tetraacetic acid - 2ME 2-mercaptoethanol - SDS sodium dodecylsulfate     

Inhibitors of epidermal cell DNA synthesis in surviving pig skin in vitro

Keratome slices of domestic pig skin were used to study the DNA synthesis phase of epidermal cell DNA synthesis. Cyclic AMP and agents which elevate intracellular concentrations of cyclic AMP have no direct effect on the "S" phase of DNA synthesis. Theophylline, isobutylmethylxanthine, and adenosine inhibit DNA synthesis immediately by a mechanism which is reversible and is not dependent on cyclic AMP. This inhibition is not associated with an increase in intracellular thymidine phosphates. Hydroxyurea, however, inhibits DNA synthesis immediately and does produce an elevated pool of thymidine phosphates.     

Effects of gas tension on epidermal keratinocyte DNA synthesis and prostaglandin production

The gas phase partial pressure of O2 (PO2) overlying mouse keratinocyte cultures controls the rate of DNA synthesis of these cells by an undefined mechanism. In these studies, both PO2 and PCO2 tensions overlying primary cell cultures were varied within the physiologic range. The prostaglandin (PG) production of cells grown under several varying gas tensions was then determined using radioimmune assay. The cultures were grown under a PO2 of either 7.5% (physiologic for in vivo epidermis) or 21% (atmospheric; culture conditions and wound healing) for 5 days. The PCO2 was either 5 or 10%, 2 CO2 tensions routinely used in tissue culture studies. DNA synthesis was quantitated using [3H]dThd uptake into DNA and autoradiography. The PGE2 and PGF2 alpha syntheses by the cultures over specific time periods were determined. Changing the PO2 from 21 to 7.5% decreased the rate of DNA synthesis, while PG production remained constant. When the PCO2 was varied from 5 to 10%, keratinocyte DNA synthesis remained unchanged but PG production was markedly stimulated. The PCO2 effect on PG production was greatest at the highest oxygen tension. The data indicated that under this set of variables, PG production by keratinocytes is not directly related to the rate of DNA synthesis of the cells. Apparently the amount of oxygen in the gas phase can have a permissive effect on epidermal keratinocyte PG production.     

Prostaglandin and DNA synthesis in human skin: possible relationship to ultraviolet light effects.

The effect of prostaglandin E2 (PGE2) on DNA synthesis in human skin was evaluated. PGE2 (1 mug) was infected intradermally into normal buttock skin of 15 volunteers followed by tritiated thymidine for autoradiographic quantitation of DNA synthesizing cells. Controls of normal saline, histamine (50 mug), and lower doses of PGE2 were also injected into 8 of the volunteers. Forty-eight hours after injection of 1 mug and 0.1 mug PGE2 there was a 264% and 62% increase, respectively, in the number of DNA synthesizing epidermal cells/high-power field as compared to saline controls. These differences were statistically significant (p smaller than 0.01). Histamine (50 mug) produced a statistically significant 36% higher labeling index compared to its saline controls (p smaller than 0.05). Many types of skin injury, including ultraviolet light (UVL) irradiation, produce an increase in the number of DNA synthesizing cells about 48 hr after the stimulus. Our findings suggest that PGE, a putative mediator of UVL-induced inflammation, may be one of the chemical mediators for the UVL-induced increase in DNA synthesizing cells. Histamine may also contribute to the increase in DNA synthesizing cells following UVL-induced inflammation.     

The effects on epidermal DNA synthesis of the butyrate esters of clobetasone and clobetasol, and the propionate ester of clobetasol

An animal model for evaluating the effects of drugs on epidermal DNA synthesis has been used to determine the time of onset, period of maximum activity and duration of effect of two topically applied proprietary glucocorticosteroids, clobetasone butyrate and clobetasol propionate. Both drugs produced maximum inhibition of epidermal DNA synthesis by 24 h after application. Clobetasol propionate exhibited a significantly earlier onset (4 1/2 h) and longer duration (36 h) of activity than clobetasone butyrate (6 and 24 h respectively). A systemic effect was not observed at any time after the application of clobetasone butyrate but was observed for up to 24 h after the application of clobetasol propionate. Clobetasol butyrate was also assayed in this system and was found to be as potent as clobetasol propionate. It is concluded that the alcoholic, as opposed to the ketonic, grouping in the 11 positions of the steroid ring profoundly alters the potentials of this molecule for systemic activity.     

Effects of retinoids on DNA synthesis of pig epidermis: its relation to epidermal beta-adrenergic adenylate cyclase response and to epidermal superoxide dismutase activity

It has been suggested that the epidermal beta-adrenergic adenylate cyclase response and the epidermal superoxide dismutase (SOD) activity is inversely associated with keratinocyte cell proliferation. Effects of various retinoids on the thymidine incorporation of pig epidermis were compared with their effects on the beta-adrenergic response and the SOD activity. Following 24 h incubation with synthetic retinoids (etretin and E-5166 (3,7,11,15-tetramethyl-2,4,6,10,14-hexadecapentaenoic acid)), thymidine incorporation of epidermis was significantly decreased. The effect of etretin was more potent than that of E-5166; the former revealed the inhibitory effect at lower concentrations than the latter. The effect of etretinate was not statistically significant. Following the 24 h incubation with the synthetic retinoids, the epidermal beta-adrenergic adenylate cyclase responses were increased. Etretin was again more potent than E-5166, while etretinate showed also little effect on the beta-adrenergic response of epidermis. Thus the inhibitory effect on the thymidine incorporation was inversely correlated with the beta-adrenergic augmentation effect among these synthetic retinoids. On the other hand, physiologic retinoids (retinol and retinoic acid) revealed no correlation between these two parameters; whereas both compounds decreased the thymidine incorporation to a similar extent, only retinoic acid revealed a marked beta-adrenergic augmentation effect. Decreased SOD activity has been observed in various hyperproliferative epidermis. The SOD activity, however, was totally unaffected by the retinoid-treatments.     

Regional variation in the deposition of subepidermal immunoglobulin in NZB/W F1 mice: association with epidermal DNA synthesis.

We have previously reported that aging NZB/W F1 mice develop irregular immunoglobulin deposits along the dermalepidermal junction similar in appearance to those seen in the visibly normal skin of systemic lupus erythematosus patients. In this report we have extended this observation by showing that the frequency of these deposits varies from site to site in the skin of these animals. This regional variation in frequency of deposits was found to correlate positively with regional differences in rates of epidermal basal cell DNA synthesis as determined by autoradiography. These findings provide an explanation for the observed regional variation in frequency of subepidermal immunoglobulin deposition that occurs in human systemic lupus erythematosus.     

Effects of retinoids on phorbol ester-stimulated epidermal DNA synthesis and hyperplasia in hairless mice

We investigated the effects of the retinoids, all-trans retinoic acid (t-RA), 13-cis retinoic acid, etretinate, and arotinoid ethyl ester, on 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced DNA synthesis, and epidermal hyperplasia in hairless mouse skin. Topical application of these retinoids produced dose-dependent inhibition of the TPA-induced epidermal DNA synthesis as measured by [3H]thymidine incorporation at 15 h after TPA application. However, this inhibition was only transient and did not affect the corresponding increase in epidermal cell layers measured at 40 or 70 h after TPA application. Fluocinonide also inhibited the epidermal DNA synthesis and failed to block TPA-induced epidermal hyperplasia. However, fluocinonide did effectively suppress the inflammation caused by TPA. In this paper we have shown that the suppression of TPA-stimulated DNA synthesis is a general property of topically applied retinoids. The biologic significance of a temporary suppression of TPA-stimulated epidermal DNA synthesis by the retinoids and fluocinonide is not understood at this time.     

Loss of water from the stratum corneum induces epidermal DNA synthesis in hairless mice

Abstract Many clinical studies have shown that low humidity has a deleterious effect on skin, but the mechanisms involved are poorly understood. To clarify the changes that occur in skin, we examined epidermal cell proliferation in mice kept in a dry (relative humidity < 10%) or a moist (relative humidity > 90%) environment. In animals exposed to low humidity, epidermal DNA synthesis started to increase within 12 h, reaching twice the original level, and the increased level was maintained for up to 5 days. The transepidermal water loss (TEWL) of mice kept for 12 h in the dry environment was the same as that of mice kept in the moist enviroment, but the skin conductance was lower. The increase in epidermal DNA synthesis following exposure to the dry environment was inhibited by topical application of petrolatum. It is concluded that loss of water from the stratum corneum induces epidermal cell proliferation within 12 h, and this change occurs in the absence of apparent cutaneous barrier dysfunction. Received: 20 April 1998 / Received after revision: 2 July 1998 / Accepted: 31 July 1998