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A single topical application of 17 nmol 12-O-tetradecanoyl-phorbol-13-acetate (TPA) to the skin of hairless mice induces characteristic transient alterations in the epidermal cells turnover and maturation (0.96 h), associated in time with characteristic changes in the activities of L-ornithine carboxy-lyase (E.C. 4.1.1.17) (ODC) and S-adenosyl-L-methionine carboxy-lyase (E.C.4.1.1.50) (SAM-D) and in the accumulation of polyamines. The effects on these responses of local pretreatment of the skin with retinoic acid 1 h prior to TPA were investigated at selected time points. Retinoic acid inhibited the TPA-induced ODC activity and the ensuing accumulation of putrescine, but did not alter the TPA-induced SAM-D activity or the molar ratio of spermidine/spermine. This pretreatment also decreased in number of dividing basal cells in the first TPA-induced synchronized wave of proliferating cells. However, during the subsequent period of proliferation, the number of dividing cells in the retinoic acid pretreated group was comparatively increased. Hence, at four levels of retinoic acid (0.17, 1.70, 17.0 and 170 nmol), which all inhibited the TPA-induced ODC effectively, there was no change in the total number of basal cells that divided during 16-48 h after TPA-application. Theory is put forward the retinoic acid might exert its antitumorigenic effect during tumor promotion with TPA by interfering with the rate and/or quality of epidermal cell maturation, rather than by inhibiting cell proliferation.

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.

A single application of 17 nmol of 12-O-tetradecanoyl phorbol-13-acetate (TPA) to mouse skin caused a marked (200- to 400-fold) induction of ornithine decarboxylase (EC 4.1.1.17, L-ornithine carboxy-lyase) activity in mouse epidermal and epidermal-dermal preparations. No change in the basal level of 3':5'-cyclic AMP occurred in epidermal-dermal preparations within 30 min of TPA application. Intraperitoneal injection of the beta-agonist isoproterenol resulted in a dose-dependent accumulation of 3':5'-cyclic AMP occurred in epidermal-dermal preparations within 30 min of TPA application. Intraperitoneal injection of the beta-agonist isoproterenol resulted in a dose-dependent accumulation of 3':5'-cyclic AMP 10 min after injection, but caused no induction of ornithine decarboxylase. When isoproterenol was injected 10 min prior to an application of either 1.7 or 17 nmol of TPA, the magnitude of the ornithine decarboxylase induction was the same as induction with TPA alone. Topical application of 17 nmol of TPA caused no increase in the level of 3':5'-cyclic GMP present in the mouse epidermal-dermal preparations 2-20 min after application. Intraperitoneal injection of 1.75 mumol of dibutyryl 3':5'-cyclic GMP and/or butyryl derivatives of cyclic GMP caused a 6-fold increase in the level of cyclic GMP and/or butyryl derivatives of cyclic GMP in epidermal-dermal preparations within 5 min of injection, and the level remained elevated for at least 20-30 min. This dose of dibutyryl 3':5'-cyclic GMP was incapable of inducing ornithine decarboxylase. Injection of dibutyryl 3':5'-cyclic GMP 5 min before application of 1.7 nmol of TPA or 30 min before application of 17 nmol of TPA did not alter the magnitude of the ornithine decarboxylase induction produced by TPA alone. These results suggest that early increases in the total intracellular levels of either 3':5'-cyclic AMP or 3':5'-cyclic GMP are not part of the mechanism by which TPA induces ornithine decarboxylase in the epidermis.

Quantitative and qualitative changes in epidermal polyamine levels and DNA synthesis (specific activity and labeling index) after a single topical application of 12-O-tetradecanoylphorbol-13-acetate (TPA), mezerein (MEZ), or ethylphenylpropiolate (EPP) in acetone were studied concomitantly in the same epidermal cell population from each treated mouse. The doses tested were 17 nmol of TPA; 1.7, 8.5, and 17 nmol of MEZ; and 20 mumol of EPP. With relatively small variations in time patterns, both the tumor promoter TPA and the mitogens MEZ and EPP caused similar sequential changes: initial inhibition of DNA synthesis; induction of L-ornithine decarboxylase activity; and subsequent peaks of putrescine levels preceding peaks in the rate of DNA synthesis. A remarkably good correlation between the molar ratio of spermidine/spermine and the increase in DNA synthesis was seen after all three of the compounds. However, in the initial period with inhibited DNA synthesis, a negative correlation between spermidine/spermine and DNA synthesis was observed after all of the treatments. TPA and MEZ induced pronounced biphasic increases in DNA synthesis, accumulation of putrescine, and the spermidine/spermine ratio, whereas EPP induced single-peaked increases in the same variables. The fluctuations in polyamine levels and DNA synthesis were associated with cohorts of partly synchronized cells passing the cell cycle multiple turns. Thus, the induction of L-ornithine decarboxylase and the polyamines does not seem to be specific for tumor promotion but merely seems to be associated with the cell kinetic events during stimulated cell proliferation. It is suggested that quantitative aspects of hyperproliferation may be essential for tumor promotion.

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.