Full-thickness skin plugs from immature and adult rats have been shown to incorpDrate 3H·thymidine in vitro in a semisynchronous fashion for up to 54 hr. DNA synthesis is minimal at 16 hr post sacrifice but increases again at 24 and 48 hr, and cells in S phase at 48 hr have passed through at least one mitosis in vitro (between 32 and 39 hr). Advantage can be taken of this semisynchronous burst of DNA synthetic activity to test the effects of potential inhibitors of skin cell proliferation, and

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... o determine at which phase of the cell cycle these inhibitors act. High concentrations of epinephrine or isoproterenol (-10 -5 M) are required to cause inhibition but the effect is specific for the G, phase of the cell cycle. Propranolol does not demonstrate /3-antagonism in this system, since it is a very potent inhibitor acting in G 1 phase. Dibutyryl cyclic AMP and theophylline have two effects: one is a specific inhibitory action on cells in the G. phase and the second is a short-term action limiting thymidine uptake by cells in S phase without affecting the transition of cells from G, to S. With the increasing interest in tissue-specific growth factors [1J and chalones [2J in skin, and the search for specific agents for the control of hyperproliferative diseases such as psoriasis [3], it has become necessary to develop adequate model systems for the assay of potential stimulators or inhibitors of skin cell proliferation. Time-honored techniques [4-6J dependent upon counting of mitoses or labeled cells are laborious and do not lend themselves to the assay of large numbers of compounds. Recently more attention has been paid to direct scintillation counting of the thymidine incorporated either in vivo [7J or in vitro [8,9J into the DNA of proliferating epidermal cells, and in this paper we describe the applications of these latter techniques to the investigation of an in vitro wound response model [10,11 J. MATERIALS AND METHODS Tissue preparation. Male Sprague-Dawley rats were received at 17 days and maintained at least 4 days on a constant (l2-hr) light-dark cycle prior to use. Animals were sacrificed at the same time each day (7:00-8:00 AM) in order to reduce any diurnal variation effects. The rats (21-27 days old) were killed in a CO, atmosphere, the backs were shaved with electric clippers, swabbed with a Nystatin suspension in 70% ethanol (left to evaporate over 15 min), and the dorsa! skin was excised and immersed in medium. During all subsequent procedures, precautions were taken to keep the skin moistened with medium. Subcutaneous fat and muscle were removed from the underside of the dermis by vigorous scraping with a dull scalpel. After rinsing, the skin was uniformly flattened (dermis down) on a Teflon sheet, and 6-mm circles were cut with Ii biopsy punch; up to 50 pieces could be obtained from each rat. Incubations. Two circles of skin (10-20 mg total) from Manuscript