A single application of 12-O-tetradecanoyl-phorbol-13-acetate to mouse skin induces an ordered sequence of ultrastructural changes in the cells of all layers of the epidermis, which correlate well with the changes in the rate of precursor incorporation into protein, RNA, and DNA in the whole skin and also with changes in the thickness, number of nucleated cell layers, and mitotic index of the interfollicular epidermis.

The incorporation of orotic acid-5-³H into RNA is increased within 1 hr and reaches a peak by 3 hr. An increase in leucine-¹⁴C incorporation into protein is observed at 2 hr, but it is marked only after 6 to 7 hr and reaches a high plateau by 12 hr. The incorporation of thymidine-³H into DNA is markedly depressed for up to 9 hr and then sharply increases, to reach a first peak by 18 hr.

The mitotic index is depressed at 8 hr, reverting to control levels at 12 to 15 hr followed by an increase in mitotic activity, and reaching a peak by 24 hr. At 16 hr, there is a marked increase in the thickness of the interfollicular epidermis and in the number of its nucleated cell layers. At this time, the mitotic index and DNA synthesis are depressed or are at control level, indicating that the increase in nucleated cell layers is due to hypertrophy, not to proliferation.

Two hr after administration of the 12-O-tetradecanoyl-phorbol-13-acetate, the nucleoli of the basal cells are markedly enlarged, with an increase of the granular and fibrillar components. By 4 hr, the granular component is further increased, polysomes appear, and the intercellular spaces between basal cells are dilated. By 6 hr, the cytoplasm of the basal cells is enlarged and filled with polysomes. The mitochondria are enlarged and have more cristae and a less dense matrix. By 12 hr, the number of polysomes is further increased, and profiles of the granular reticulum and Golgi complexes are more numerous so that the stimulated basal cells resemble those of embryonic epidermis. By 24 hr, markedly hypertrophic cells and granular mitochondrial inclusions are frequently seen. By 48 hr, flattening, involution of the organelles, and accumulation of numerous keratohyalin granules occur in the cells of the upper layers.

Granular electron-dense material appears in the dilated intercellular spaces at 6 hr and increases until 36 hr, when it is also in the cisternae of the endoplasmic reticulum.

The superficial cells, normally flattened, are swollen by 2 hr, and they progressively regain their nuclear and cytoplasmic organization so that, by 12 hr, they resemble the stimulated basal cells.

Dark cells with ultrastructural features distinct from the other epidermal cells are observed after administration of 12-O-tetradecanoyl-phorbol-13-acetate, but are not found in normal or acetone-treated controls.

The relevance of the cellular events to tumor promotion is analyzed, and we propose that tumor promoters act by altering the normal differentiation pathway, thus enabling expression of the neoplastic phenotype.