Hairless Mouse Epidermis Research Articles

Cell population kinetics in hairless mouse epidermis following a single topical application of 12-O-tetradecanoylphorbol-13-acetate I.

The alterations in the cell population kinetics of mouse epidermis (hr/hr. Oslo strain) following a single topical application of 17 nmol 12-O-tetradecanoylphorbol-13-acetate (TPA) were studied using serveral methods simultaneously. During a period from 2-96 h following treatment, 452 mice were used to measure incorporation of tritiated thymidine into DNA (labeling index and specific activity), mitotic index, Colcemid arrested metaphase rates, and flow cytometric DNA measurements. The time course of the kinetic response to TPA could be divided into two different periods. Period I (0-12 h) was characterized by a temporary block of cells in S-phase and mitosis. In period II (12-96 h), partly synchronized cells displayed multiple waves of DNA synthesis and cell division, with a considerable reduction of the cell cycle time from 54 h down to 10-12 h. The resulting hyperplastic epidermis is therefore composed of a relatively high proportion of young, immature cells. Hence, some of the early biochemical alterations in mouse epidermis assumed to be specific for TPA as a tumor promotor, may merely be the result of a rapid population shift.

Changes in epidermal polyamine biosynthesis and specific activity of DNA following a single application of 12-O-tetradecanoyl-phorbol-13-acetate to hairless mouse skin.

A single application of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) to hairless mouse skin induces increased activity of epidermal L-ornithine carboxy-lyase (E.C.4.1.1.17) (ODC) with a peak at 5 h, and S-adenosyl-L-methionine carboxy-lyase (E.C.4.1.1.50) (SAM-D) with a broad peak at 20-36 h. The temporal sequence of the accumulation of polyamines; i.e. putrescine, spermidine and spermine, and the rate of DNA synthesis was investigated. All four parameters were measured in the same tissue-samples and multiple peaks of DNA synthesis and of individual polyamines were demonstrated. In the period from 0-12 h, there was an initial decrease in the rate of DNA synthesis. In this period changes in the molar ratio of spermidine/spermine were negatively correlated to the rate of DNA synthesis. From 12-48 h, however, changes in the molar ratio of spermidine/spermine had an almost identical time course with rates of change of DNA synthesis. Based on corresponding cell kinetic results it is suggested that the spermidine/spermine ratio reaches a maximum peak during the S-phase of the cell cycle. The relation between the rate of DNA-synthesis and the spermidine/spermine ratio as well as the ordered time sequence for the accumulation of putrescine and the induction of ODC and SAM-D activities, suggest a strong interdependence and a strict regulation of these events in hairless mouse epidermis induced to proliferate by TPA.

The time needed for normalization of hairless mouse epidermis after treatment with twice weekly topical skin applications of 10 nmol 12-O-tetradecanoylphorbol-13-acetate in acetone, or acetone alone, for 18 weeks. A morphologic and cell kinetic study.

The time needed for normalization of hairless mouse epidermis after treatment with twice weekly topical skin applications of 10 nmol 12-O-tetradecanoylphorbol-13-acetate in acetone, or acetone alone, for 18 weeks. A morphologic and cell kinetic study.

The effect of local UVB skin irradiation on the rate of formazan deposition in the epidermis of hairless mice studied by means of a tetrazolium-reduction method

One-hundred-and-twenty hairless mice were irradiated with UVB (310 nm, exposure 60 mJ/cm2) on a limited area of the dorsal skin. At different time intervals after irradiation, the rate of endogenous dehydrogenase activity per mg dry epidermis was measured by the tetrazolium reduction method. The amount of formazan deposited remained normal for 18 h, and then increased, reaching a peak significantly higher than normal at 24 h, and thereafter returned to normal. At day 8 there was a new, probably significant peak. The reaction was followed for 14 days. It was concluded that UVB irradiation provokes a period of increased formazan deposition in the epidermis, similar to what has been observed after ionizing radiation and chemical carcinogens. The validity of the tetrazolium test for skin carcinogenic irritaments was thus also confirmed.

Inhibition by epidermal extracts of the 12-O-tetradecanoylphorbol-13-acetate induced peak of ornithine decarboxylase activity in the mouse epidermis.

The time course of induction of epidermal ornithine decarboxylase (E.C. 4.1.117) (ODC) activity following a single topical application of 17 nmoles of 12-O-tetradecanoylphorbol-13-acetate (TPA) on hairless mouse skin was established. Prior intraperitoneal (i.p.) administration of a crude epidermal extract prepared from hairless mouse epidermis led to a time-dependent, 50% inhibition of the peak level of TAP-induced ODC activity. Maximum inhibition was observed when the extract was injected 1.5 h before TPA treatment. The crude epidermal extract did not affect ODC activity in vitro. Following the administration of epidermal extracts, the inhibition of the TPA-induced ODC-response correlated positively with the presence of epidermal G2-chalone activity (determined by a stathmokinetic method) whereas myocardial, skeletal muscle, or heat-inactivated epidermal extracts with no epidermal G2-chalone activity, had no effect on TPA-induced ODC activity. These results indicate a possible relationship between ODC-activity and the control of mitotic rate by G2-chalone.

Topical spermine and putrescine stimulated DNA synthesis in the hairless mouse epidermis.

Polyamines were applied topically to the skin of the hairless mouse. Putrescine stimulated the incorporation of thymidine after a 24-h application period. The effect of polyamines upon skin pretreated with a potent topical steroid was also examined; in this model thymidine incorporation was stimulated by both spermine and putrescine. Pretreatment was performed in order to reduce endogenous polyamine biosynthesis and increase the sensitivity of the epidermis to exogenous polyamines. Depletion of the activity of ornithine decarboxylase, the rate-limiting polyamine biosynthetic enzyme, by topical steroids was confirmed in the hairless mouse following induction of the enzyme by UV-B. The results are consistent with those in vitro studies suggesting a role for polyamines in the control of DNA synthesis; the effect of corticosteroids upon proliferative skin disorders may be mediated through this mechanism.

Percutaneous absorption in experimental epidermal disease.

Little is known about the absorption of topical drugs in epidermal disease associated with epidermal proliferation and altered keratinization. An abnormal hairless mouse epidermis was produced by three different methods: ultraviolet light irradiation, topical vitamin A acid and topical acetic acid. An increased epidermal thickness resulted and the in vitro percutaneous absorption of 0.1% (4-14C) hydrocortisone was found to be increased when compared with normal hairless mouse epidermis. This is evidence for defective skin barrier function in experimental epidermal disease.

Circadian rhythms in mouse epidermal basal cell proliferation. Variations in compartment size, flux and phase duration.

Several kinetic parameters of basal cell proliferation in hairless mouse epidermis were studied, and all parameters clearly showed circadian fluctuations during two successive 24 hr periods. Mitotic indices and the mitotic rate were studied in histological sections; the proportions of cells with S and G2 phase DNA content were measured by flow cytometry of isolated basal cells, and the [3H]TdR labelling indices and grain densities were determined by autoradiography in smears from basal cell suspensions. The influx and efflux of cells from each cell cycle phase were calculated from sinusoidal curves adapted to the cell kinetic findings and the phase durations were determined. A peak of cells in S phase was observed around midnight, and a cohort of partially synchronized cells passed from the S phase to the G2 phase and traversed the G2 phase and mitosis in the early morning. The fluctuations in the influx of cells into the S phase were small compared with the variations in efflux from the S phase and the flux through the subsequent cell cycle phases. The resulting delay in cell cycle traverse through S phase before midnight could well account for the accumulation of cells in S phase and, therefore, also the subsequent partial synchrony of cell cycle traverse through the G2 phase and mitosis. Circadian variations in the duration of the S phase, the G2 phase and mitosis were clearly demonstrated.

A COMPARISON OF IN VIVO AND IN VITRO TESTING OF SUNSCREENING FORMULAS

Abstract— Seven commercially available sunscreens were compared by three different methods. Absorbance spectra were measured for each product in isopropanol solution and also on hairless mouse epidermis. In vivo tests were performed on human volunteers using a Xe arc solar simulator. Sun Protection Factors (SPF) were calculated by each method for each product tested and the results compared. By all methods used, the combination of 7% octyl dimethyl para‐aminobenzoic acid and 3% oxybenzone provided the most protection from U.V. light. While estimates of the effectiveness of all products were much too high when calculated by the isopropanol solution method, the hairless mouse epidermis technique seems to be an accurate tool for predicting product efficacy in vivo.

Specificity of epidermal chalone extracts for human epidermoid tumour cells in vitro: a preliminary report.

In order to test the mitosis-inhibiting effect and the tissue specificity of the epidermal G2 chalone for tumour cells, extracts from hairless mouse epidermis were tested in short-term tissue cultures of cells from human respiratory tract epidermoid carcinomas and adenocarcinomas. The chalone inhibited strongly the mitotic activity in two cases of histologically proven epidermoid carcinoma, and had no effect in two cases of adenocarcinoma. In one case of a supposed epidermoid carcinoma, the chalone had no effect. Revision of the histology, and the result of autopsy 11 months later, showed that in this case the lesion in the lung had been a poorly differentiated metastasis from an adenocarcinoma of the ovary. Liver extracts produced in the same way as the epidermal extracts showed no mitotic inhibition in any of the cultures. These results indicate that epidermal G2 chalone produced from mouse skin is tissue specific for human epidermoid tumour cells, and also indicate that a chalone test might be used as a diagnostic tool for poorly differentiated carcinomas to see whether they are of epidermoid origin or not.

Absence of photoreactivation of pyrimidine dimers in the epidermis of hairless mice following exposures to ultraviolet light.

Abstract—The influence of photoreactivating light on the fate of UV‐induced DNA damage has been measured in the epidermis of hairless mice using damage‐specific endonuclease from Micrococcus luteus. Groups of mice were exposed to varying fluences of UV at 297nm or from an FS40 fluorescent sun lamp to induce UV photoproducts. The same fluence‐dependent DNA damage was observed in high molecular weight epidermal DNA regardless of whether the mice were killed immediately, or maintained in the dark or under photoreactivating light for 20 h after UV. Thus, no detectable photoreactivation of UV‐induced pyrimidine dimers could be demonstrated in mouse epithelial cells in vivo.

Immunofluorescent studies of epidermal protein during UV induced carcinogenesis

Previous studies with agar diffusion technique demonstrated that antibodies produced in rabbits by injection of urea extractable proteins of rat cornfied cells cross react with proteins extracted from normal epidermis of hairless mice using the same technique. In the present study we investigated by indirect immunofluorescence microscopy the immunoreactivity of epidermal proteins in normal and ultraviolet light (UVB) induced hyperplasia and malignant transformation. Reactivity to the antibody was seen over the entire epidermis of nontreated skin and hypertrophied epidermis which occurred at 6-8 weeks after initiation of UVB irradiation. However, the reactivity diminished when malignant changes took place in the epidermal cells. Almost complete disappearance of the immunoresponse was observed in squamous cell carcinoma produced by further UVB radiation. These results suggest that the reactivity of this urea extractable protein serves as an additional immunologic marker for normal epidermal cells. Alterations in the immunoreactivity parallels UVB induced carcinogenesis.

Growth kinetics in newborn mouse epidermis: response to epidermal chalone.

Epidermal DNA synthesis, the epidermal mitotic rate, and the responsiveness to the epidermal G1 and G2 inhibitors were examined in newborn mice at different times after birth. The rate of epidermal cell renewal was in general low during the first two weeks of life. Later the two growth parameters increased and reached very high values at 32-33 days after birth. The rate of epidermal cell proliferation then decreased to a level comparable with that found in adult hairless mouse epidermis at 40-45 days. A single i.p. injection of skin extract containing the two epidermal growth inhibitors induced varying types of responses. The epidermal G2 inhibitor stimulated the mitotic rate on day 2 and day 10, but inhibited it on all other days. The epidermal G1 inhibitor brought about an increase in epidermal DNA synthesis on day 6 and possibly on the following days. No response at all seen at 2, 4, 17, and 32 days after birth. At the other examined times the inhibition was similar to that found in adult mice. These findings differed from those made in vitro on separated newborn mouse epidermal cells (our own unpublished data), and we suggest that the variability of newborn mouse epidermis could be an expression of the immaturity of the skin as a whole, and that dermis in some way modifies the response of epidermis to exogenous epidermal chalone. Our study did not support the theory that the nonresponsiveness of newborn mouse epidermal at certain times could be due to the presence of nonresponsive stem cells in epidermis.

Biosynthesis of Pyrrolidone Carboxylic Acid in Hairless Mouse Epidermis

[3H]Glutamic acid (PCA) was followed with time after a single subcutaneous injection. PCA specific activity increased slowly, reaching a peak at 3 to 4 days after injection of the labeled amino acid, after which it slowly decline. Incorporation of [3H]glutamic acid into epidermal PCA was markedly inhibited by a single topical application of cycloheximide. Topical application of cycloheximide 2 hr prior to [3H]glutamate injection caused a significantly greater reduction in PCA specific activity (determined 3 days after injection) than cycloheximide treatment 3 hr after administration of the labeled amino acid. Ninety-seven percent of the PCA content of hairless mouse epidermis was shown to reside in the stratum corneum. These observations indicate the involvement of protein synthesis in the formation of PCA from glutamic acid rather than a direct conversion of the amino acid. The high level of PCA in mammalian epidermis appears to be caused by its accumulation in the stratum corneum accompainied by a relatively slow rate of PCA turnover in comparison to other tissues.

The effect of ultraviolet light (UVL) on the lysosomes of hairless mouse epidermis.

An increased release of acid phosphatase from the lysosomes of UVL irradiated hairless mouse epidermis is demonstrated. The results indicate that lysosomal membrane stability is decreased when the hairless mouse is exposed to either acute or chronic UVL.

EFFECTS OF TOPICAL PROSTAGLANDIN E2 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.

Tachyphylaxis to Topically Applied Steroids

• Acute tolerance to the vasoconstrictive effect of topically applied glucocorticoids in humans has recently been described. Similar acute tolerance to the antiproliferative properties of topically applied steroids in proliferating and normal hairless mouse epidermis is now shown to occur. Thus, at first, topically applied steroids cause profound inhibition of DNA synthesis and mitosis in the epidermis, but with continued treatment, DNA synthesis and mitosis recover and the tissue becomes insensitive to further stimulation. This suggests that there is an optimum approach to therapy with topically applied steroids that might reduce the incidence of side effects and improve therapeutic efficacy. (<i>Arch Dermatol</i>112:1245-1248, 1976)

Tachyphylaxis to topically applied steroids.

Acute tolerance to the vasoconstrictive effect of topically applied glucocorticoids in humans has recently been described. Similar acute tolerance to the antiproliferative properties of topically applied steroids in proliferating and normal hairless mouse epidermis is now shown to occur. Thus, at first, topically applied steroids cause profound inhibition of DNA synthesis and mitosis in the epidermis, but with continued treatment, DNA synthesis and mitosis recover and the tissue becomes insensitive to further stimulation. This suggests that there is an optimum approach to therapy with topically applied steroids that might reduce the incidence of side effects and improve therapeutic efficacy.

Ultraviolet Light Carcinogenesis In Hairless Mice: Cell Kinetics During Induction And Progression Of Squamous Cell Carcinoma As Estimated By The Double-Labeling Method

The double-labeling method for studying cell cycle kinetics was applied to uninvolved epidermis of hairless mice (irradiated and unirradiated), and at various stages of ultraviolet light (UVL)-induced squamous cell carcinoma. The growth fraction (GF) was determined by continuous labeling with tritiated thymidine (3HTdR). The data indicate that the double-labeling technique is an acceptable method for studying cell kinetics in UVL-induced squamous cell carcinoma. The GF in normal epidermis (unirradiated and irradiated), hyperplastic epidermis, and lesions of squamous cell carcinoma is 1. The duration of cell generation (Tc) and DNA synthesis (Ts) periods decreased progressively with induction and progression of squamous cell carcinoma. For normal epidermis (irradiated mice) the Tc and Ts were 91 and 6.3 hr, respectively. In the hyperplastic epidermis, the Tc and Ts were reduced to 44 and 4.5 hr, respectively, while even lower values were obtained for the tumor (Tc = 16 hr, Ts = 3.1 hr). The labeling index (LI) in normal epidermis was 7% and increased progressively in hyperplastic epidermis (10%) and squamous cell carcinoma (19%). Epidermal cell differentiation in hyperplastic and tumor tissues appears to have been delayed (GF = 1), since cells above the basal layer, which in normal epidermis keratinize, retain the ability to proliferate, as evidenced by extensive incorporation of [3H]TdR in these cells. The results suggest that tumor production is associated with a progressive shortening of the cell cycle and delayed keratinization of epidermal cells.

Acute tolerance to effects of topical glucocorticosteroids.

Tachyphylaxis (acute tolerance) to two important actions of topical glucocorticosteroids is demonstrated. In humans, topically active glucocorticoids applied to the skin cause vasoconstriction initially but this response fades with successive applications. In hairless mouse epidermis, inhibition of DNA synthesis and mitosis is caused by one application of a glucocorticoid but with repeated administration of the drug this effect is lost. The degree of inhibition of cell division appears to be similar 30 and 54 h after one application of steroid to that following three or five applications during such a time period. These findings suggest that an optimum therapeutic approach to the use of these agents exists.