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The Effect of Phorbol Diesters on Chicken Embryo Fibroblasts¹

Department of Pharmacology, Harvard Medical School, Boston, Massachusetts 02115

The tumor promoter phorbol myristate acetate (PMA) at concentrations of 1 to 30 ng/ml (1.5 to 45 x 10^–9 M) had dramatic effects on chicken embryo fibroblasts (CEF). Cell morphology, size, growth, and deoxyglucose transport were among the altered properties. Of the morphological changes, the typical parallel orientation of cells on the plate was lacking for CEF grown to confluence in the presence of PMA (30 ng/ml). In sparse as well as dense cultures, PMA caused the cells to appear thinner and more elongated, with prominent processes; initial changes were discernible within 3 hr. Cell volume was decreased by PMA over the course of 2 to 3 days. The plateau value for cell volume was 62% of that of the control cultures. The exponential growth rate of CEF cultures in PMA at 50 ng/ml was not significantly altered. However, as growth rates leveled off after 3 days, cells in PMA eventually reached a 1.8-fold higher density than did those in control cultures. Resting CEF were stimulated to divide by PMA; cell number increased by 80% within 24 hr of PMA addition. The half-maximally effective dose of PMA for mitogenic stimulation was 2.3 ng/ml. PMA showed a similar half-maximally effective dose for stimulation of [G³H]-2-deoxy-D-glucose uptake. Uptake reached a peak quickly, 4 to 6 hr after PMA addition, and attained a level 90% of that induced by addition of chicken serum (10%, final concentration). Stimulation of [G-³H]-2-deoxy-D-glucose uptake by other phorbol derivatives was investigated. The half-maximally effective doses for phorbol 12,13-dibenzoate, 4-O-methyl phorbol 12-myristate 13-acetate, phorbol 12,13-diacetate, and phorbol 12,13,20-triacetate occurred at 5.7, 240, 405, and 6400 ng/ml, respectively, whereas phorbol itself was completely inactive at concentrations up to 20,000 ng/ml. The results lend additional support to a model for the mechanism of action of tumor promoters.

¹ This work was supported by Grant CA18294 from the National Cancer Institute. A preliminary report on a portion of the work has appeared previously (5).

² Predoctoral Fellow of the National Science Foundation.

Received 3/10/77. Accepted 5/27/77.

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