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A method is described which allows the isolation undergraded total cellular RNA from living epidermis in a one-strep procedure. Epidermal tissue is homogenized in 4 M guanidinium thiocyanate/2% sarkosyl buffer and centrifuged through a 5.7 M CsCl cushion. Under appropriate density conditions in the homogenate, large amounts of RNA are sedimented free of DNA and protein to the bottom of the tubes. A prerequisite for the applicability of this method is the preparation of pure epidermis under conditions which efficiently inactivate endogenous R Nases. Oligo (dT) affinity chromatography of total RNA yields 3–4% of polyadenylated RNA. Translation of poly(A)-rich fractions from both newborn and adult mouse epidermis in a cell-free reticulocyte system leads to the synthesis of proteins with electrophoretic mobilities identical to those of native keratin polypeptides of the corresponding tissue. Similar to native keratin polypeptides, these proteins are highly insoluble and can selectively be enriched by high-salt extraction of the translation assays. The CNBr cleavage pattern of these purified proteins is identical to that of native keratins. There is no evidence for the synthesis of precursors of these proteins. [ABSTRACT FROM AUTHOR]

A two-stage carcinogenesis experiment was performed in Syrian golden hamsters using a single intragastric initiation with 10 mg/kg body weight of 7,12-dimethylbenz[a]anthracene (DMBA) and repetitive topical promotion on the back skin with two different doses (80 and 160 mg, respectively) of benzoyl peroxide in 1 ml acetone. Benzoyl peroxide was administered three times per week over a period of 16 months. The treatment with benzoyl peroxide alone leads to both a generalized hyperpigmentation and skin scaling without formation of any tumors. DMBA initiation alone induces a moderate number of melanotic foci and a small number of palpable melanotic tumors. Both lesions are located in the dermis. Papillomas develop in the epithelia of the tongue, esophagus and especially of the forestomach but not, however, on the back skin. The combined treatment with DMBA and both doses of benzoyl peroxide drastically increases the incidence of dermal melanotic foci and at late stages also that of melanotic tumors. Therefore, in addition to its known ability to promote papilloma and carcinoma formation in the back skin of mice, benzoyl peroxide is also able to promote the formation of melanotic tumors in the dermis of hamsters. [ABSTRACT FROM PUBLISHER]

Initiation with the carcinogen 7,12-dimethylbenz[a]anthracene followed by promotion with the hyperplasiogenic phorbol ester 12-O-tetradecanoylphorbol-13-acetate carried out on the dorsal skin of European hamsters ( L.) led to the formation of a broad spectrum of skin tumors in terms of a two-stage principle. Tumor formation involved the epidermis and its appendages, the connective tissue, and the vascular and pigmentary system. With regard to the sensitivity to initiation with DMBA alone and initiation with DMBA and promotion with TPA the European hamster occupies a special position since all of the DMBA-TPA sensitive two-stage target organs known from the mouse, rabbit, Syrian golden hamster and rat are combined in this animal. [ABSTRACT FROM PUBLISHER]

The persistence of the initiated state during two-step car-cinogenesis in mouse epidermis is a generally accepted phenomenon, however, conflicting results exist with regard to the degree of irreversibilty relative to the age of the animals. Several factors such as age-dependent alterations in the response of the epidermis to the promoter and skin damage following the initiation step have been proposed to account for the observed discrepancies. In the present investigation we have tried to circumvent skin-damaging effects of topically applied 7,12-dimethylbenz[a]anthracene by intragastric administration of the drug. Tumor production by topical promotion with 12-O-tetradecanoylphorbol-13-acetate was subsequently determined in 600 female NMRI mice using intervals of 4, 8, 16, 24, 32 and 40 weeks between initiation and promotion. Independent of the delay between the initiating and promoting step, we observed a similar time course and extent of tumor production in the different experimental groups. This indirectly proves that the promoting capacity of 12-O-tetradecanoylphorbol-13-acetate is age-independent and that during aging no substantial loss of initiated cells occurs in mouse epidermis. [ABSTRACT FROM PUBLISHER]

In contrast to a previous report by Shubik and in accordance with a pilot study from our laboratory with female rats, the 2-stage skin carcinogenesis experiment could be successfully accomplished in male Sprague-Dawley rats. Male animals of this species are better suited for this long-term experiment since, under the conditions used, females are very sensitive to mammary gland tumor formation and show a drastically reduced survival time. A broad spectrum of tumors of epidermal origin could be induced by topical initiation with 7,12-dimethylbenz[a]anthracene (DMBA) and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) as promoter. The tumors were not only localized in the interfollicular epidermis but to a large extent also in the epidermal appendages. More importantly, the DMBA — TPA treatment led to the development of a variety of tumors of the connective tissue and the angiofibrous matrix. The incidence of these tumors was almost comparable to that found for epidermal tumors. In some animals we observed up to 12 histologically different tumors. Compared to the mouse, the tumor incidence in organs other than the skin was definitely lower. Ethyldiazoacetate (EDA), a substance which has been shown to induce skin tumors when administered i.v. to rats, was tested with regard to its initiating capacity. The combination EDA — TPA led to a skin tumor spectrum comparable to that produced by DMBA — TPA. However, the survival time of the EDA — TPA treated animals was significantly higher than those exposed to DMBA — TPA. EDA, therefore, seems to be a suitable alternative to DMBA for systemic initiation of skin tumors. [ABSTRACT FROM PUBLISHER]

In this study a new modification of the systemic 2-stage carcinogenesis experiment is described. Tumors were induced in NMRI-mice using a single intravaginal application of the carcinogen 7, 12-dimethylbenz[a]anthracene (DMBA) (initiator) followed by intravaginal treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) (promotor) as a cocarcinogen over a period of 52 weeks. The substances were applied using small tampons. Development of preneoplastic lesions or neoplasias was not found in the group of untreated control animals (n = 50) and in TPA treated animals (n = 50). However, mice which had been treated with TPA alone showed pronounced hyperplasia. When DMBA was applied without subsequent treatment with TPA (n = 100), formation of preneoplastic lesions was frequently observed. Fibroepitheliomas (papillomas), carcinomas and sarcomas, typically located intravaginally and at the external region of the vagina, were observed after the combined treatment with DMBA and TPA (n = 100). In addition, benign as well as malignant tumors were found, mainly in the forestomach, in DMBA treated animals and more frequently after application of DMBA and TPA. Tumors of the lung and leukemias were identified more frequently in comparison to the normal spontaneous tumor rate in NMRI mice, and were probably in part caused by the promoting substance TPA. On the other hand, no significant tumor formation in the mammary gland and in the ovaries was observed after DMBA/TPA application. The tumors observed in these organs were, however, caused by treatment with the carcinogen DMBA only. This new modification of the 2-stage experiment, i.e. the direct intravaginal application of initiating and promoting substances, has special relevance for the elucidation of the mechanisms involved in the development of carcinomas located at the portio vaginalis uteri in humans. [ABSTRACT FROM PUBLISHER]

A comparative study of the keratin composition of adult and neonatal mouse epidermis, benign and malignant tumors of mouse skin and murine epidermal cells grown in culture revealed striking differences in the keratin polypeptide patterns when analyzed by one-dimensional gel electrophoresis. Not only did the study confirm body-site specific alterations in the keratin patterns within one species, but it also demonstrated that similar to cultured epidermal cells, three malignant skin tumors investigated specifically lacked a group of keratin components with molecular weights larger than 61,000 daltons, which were invariably present in all normal and also in benign tissues. These findings offer the possibility of using keratins as molecular markers of the malignant state of epidermal cells. Two-dimensional gels of the keratin polypeptides of normal epidermis and of benign tumors exhibited spot patterns which could be divided at the 61,000 dalton level into an essentially basic subgroup, comprising the high molecular weight keratins, and an acidic subgroup including the low molecular weight components. The two uppermost proteins of cultured epidermal cells and carcinomas (molecular weights 61,000 and 58,000 daltons) belong to the acidic subgroup in normal tissues as well as in papillomas. However, in the case of malignant tumors and transformed epidermal cells they showed distinct alterations in charge in that they migrated into the more alkaline part of the gel. The number of spots appearing in the more basic region of the gel could be inversely related to the degree of differentiation exhibited by tumors or in vitro cells. [ABSTRACT FROM PUBLISHER]

Diaplacental initiation with the carcinogens DMBA or urethane followed by repeated topical treatment of mice of the F generation with the tumor promoter TPA leads to the formation of benign and malignant tumors on the skin of the back as well as in other tissues and organs. The tumor yield in this modified 2-stage Berenblum/Mottram experiment considerably exceeds the number of spontaneously formed tumors and of tumors produced by initiation alone. Further differences can be demonstrated in the malignancy rate, the formation of multiple tumors in various organs, additional non-neoplastic alterations and in a reduction of the lifetime of the animals. The effect of the tumor promoter TPA is not restricted to carcinogenesis in the back skin. Obviously, TPA is able to activate inititated tumor cells in internal organs to form tumors. This, in turn, implies the absorption of the substance via the blood vessels and its distribution throughout the body. The preferential occurrence of tumors in the genital tract of female mice (carcinomas and sarcomas of the vaginal wall, granulosa cell tumors of the ovaries) points to a possible hormonal involvement; in this context, relevance to prenatally induced tumors in human pathology is discussed. The results emphasize the important role of prenatal carcinogenesis and indicate the increased risk to man by either prenatal initiation or postnatal promotion. [ABSTRACT FROM AUTHOR]

7, 8-Benzoflavone (BF) was applied orally via stomach tube in doses of 50, 100, 200 and 400 mg/kg body weight to pregnant NMRI mice on the 18th day of gestation. BF application was followed 1 h later by oral administration of 60 mg/kg body weight dimethylbenz[a]anthracene (DMBA). As a rule this dose of DMBA does not lead to prenatal secondary effects and is not carcinogenic to either the mother animals or the Fl generation. Subsequent promotion of the Fl generation with the tumour promoter 12-O-tetradecanoylphorbo13-acetate over a period of 12 weeks resulted in a high yield of skin papillomas and lung adenomas when no BF had been applied. With prior application of BF, the tumour yield could be significantly reduced. [ABSTRACT FROM PUBLISHER]