Cutaneous changes during prolonged application of 12-O-tetradecanoylphorbol-13-acetate on mouse skin and residual effects after cessation of treatment.

The epidermal and dermal effects of protracted 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment (2 micrograms TPA twice weekly) of Sencar mouse skin were studied using cell kinetics and morphometric techniques. In addition, regression of TPA-induced changes was evaluated after cessation of 56 topical applications. During the first week of treatment a reactional hyperplasia, characterized by cell damage, edema, and acute inflammation in both epidermis and dermis, occurred. This picture changed gradually during the following 3 weeks: an epidermal hyperplasia devoid of involutional or inflammatory features was accompanied by a moderate to mild chronic inflammation of the dermis and a hyperplasia of the hair follicles. This remained throughout the experimental period until the topical TPA treatment ceased. Although TPA induced papillomas in only 5% of the animals (maximum = 2 papillomas/animal and no carcinomas), all sustained marked epidermal hyperplasia of approximately 4 to 5 times the normal thickness, and increased the number and volume of hair follicles. The [3H]thymidine pulse-labeling index of the basal layer was approximately 32% (normal congruent to 6%). The level of dark keratinocytes remained constant; i.e., 8% of the basal cells were identified as dark cells during the entire experiment. At the subepidermal level the dermal thickness and total cellularity increased, although the proportion of the different cell types changed during the treatment. The mast cell population increased remarkably. After TPA treatment ceased, most of these parameters regressed abruptly during the first 2 weeks. Two to 4 months later, the epidermis was slightly thinner, and the labeling index was 50% lower than normal (2.8%). This study shows that prolonged repetitive TPA applications induced a steady-state hyperplasia without tachyphylaxis, and that this alteration regressed rapidly after treatment ceased. In addition, labeling-index values lower than normal were reached soon after normalization, suggesting that a possible selection of keratinocytes, dependent on TPA for proliferation, took place during the chronic administration of topical TPA. The number of hair follicle, capillary vessels, mast cells, and the dermal thickness never reached normal values after treatment. These important changes in the dermis and hair follicles indicate that the target cells for tumor promoters are not confined to the epidermis alone, and that these tissues could participate actively in carcinogenesis directly, either as tumor-originating tissues (hair follicles), or as inducers or helpers of neoplastic growth (connective tissue cells).

Expression of epidermal growth factor receptor, polyamine levels, ornithine decarboxylase activity, micronuclei, and transglutaminase I in a 7,12-dimethylbenz(a)anthracene-induced hamster buccal pouch carcinogenesis model.

The expression of epidermal growth factor receptor and transglutaminase type I, polyamine (putrescine, spermidine, and spermine) levels, ornithine decarboxylase activity, and micronuclei occurrence were assessed in the 7,12-dimethylbenz(a)anthracene (DMBA)-induced hamster buccal pouch model to elucidate the role and timing of changes in different growth and differentiation markers during carcinogenesis. DMBA (0.5%) in heavy mineral oil was applied to the right buccal pouch 3 times per wk for up to 16 wk; controls received heavy mineral oil alone. Hamsters were killed after 0, 4, 8, and 16 wk. Frozen tissue was chemically analyzed for polyamine levels and ornithine decarboxylase activity and was also used for immunohistochemical analysis of transglutaminase I. Paraffin-embedded sections were used for epidermal growth factor receptor immunohistochemical determinations and for micronucleated cell assays. Hyperplasia was detected by histological analysis at 4 wk, dysplasia with or without papillomatous changes at 8 wk, and squamous cell carcinoma at 16 wk. Epidermal growth factor receptor was not expressed in the normal buccal epithelial layer, at a moderate level in both the superficial keratin and basal cell layers in hyperplastic epithelium, and at very high levels in both dysplasia and squamous cell carcinoma. Transglutaminase I was expressed at a limited level in normal buccal mucosa, was expressed at a low level in the basal layer of hyperplastic lesions, was somewhat elevated in dysplasia, and was markedly enhanced in squamous cell carcinoma. Putrescine and spermidine levels and ornithine decarboxylase activity increased dramatically after 8 and 16 wk of DMBA. Micronucleated cells increased after 4 wk of DMBA treatment, that high level sustained during all stages of carcinogenesis. We suggest that these biological markers could be excellent intermediate end points in assessing the effects of various chemopreventive agents to be tested in the hamster buccal pouch model and in human clinical trials.

Microspectrophotometric determination of DNA in oral lesions.

The DNA content of the epithelial basal cells of oral lesions was determined by Feulgen microspectrophotometry. The DNA histograms of the radicular cysts showed a definite peak in the diploid range and a slight deviation to the right. The keratocysts also showed a well-defined modal peak in the diploid range; however, another cell population defined a peak in the tetraploid range. In leukoplakias of the oral cavity, two different profiles of DNA distribution were found. Where both the clinical and microscopic traits defined a nondysplastic lesion, the histogram showed a diploid peak and a percentage of cells in diploid-tetraploid range. For the more active lesion, a shift of the mode towards higher values was evident. The microspectrophotometric studies of oral epidermoid carcinomas showed a broad distribution towards the right and ill-defined modal values.

Microspectrophotometric quantitation of nucleic acid and protein in irradiated epidermis.

Nucleic acid and proteins of newborn rat tail subjected to local X-irradiation were microspectrophotometrically studied. Feulgen, gallocyanine chrom-alum and naphthol yellow S methods were performed for demonstration of DNA, total nucleic acid and proteins respectively. The amount of proteins and total nucleic acid increases concomitantly with reactional acanthosis. However, the proteins and nucleic acid decrease as from day 3 post-irradiation. A tentative interpretation of the results would point to a giantization of the epidermic cells not only caused by aqueous imbition but also by an actual increase of the cellular protoplasm.

Immunodetection of involucrin in lesions of the oral mucosa.

The immunoperoxidase method for involucrin detection was applied to the study of the maturation of epithelial lesions of the oral mucosa that included specimens of leukoplakia, lichen planus, verrucous carcinoma, carcinoma in situ and invasive carcinoma. Areas of orthokeratinized, parakeratinized, and non-keratinized normal mucosa were also studied. Normal orthokeratinized epithelia showed intracytoplasmic or pericellular staining in the suprabasal epithelial layers in a pattern similar to that of the normal epidermis. Parakeratinized and non-keratinized epithelia were less stained. Intense staining was observed in leukoplakia, whereas the staining of lichen planus was less intense but exhibited a more homogeneous pericellular staining pattern than leukoplakia. Verrucous carcinoma was markedly and very irregularly stained. Carcinomas in situ and invasive carcinoma exhibited a slightly positive and patchy reaction. The distribution patterns of involucrin in the lesions correlated very well with the degree of epithelial differentiation. In addition, irregular patchy distribution correlated with the degree of atypia, and was especially evident in carcinomas.