Synthesis of proto-oncogene proteins and cyclins depends on intact microfilaments

Abstract

It is well established that microfilament disintegration by cytochalasin D (CD) as well as latrunculin (LAT)-A and LAT-B causes an inhibition of S phase entry of various nontransformed cell lines. Our experiments extended these observations to human embryonal diploid fibroblasts (Wi-38). To investigate the question whether this stop of DNA synthesis is due to a decline of the synthesis of proteins that are necessary for G1 progression and S phase entry, we examined the expression of two proto-oncogenes (c-fos, c-jun) and three cyclins (D1, E, A) after altering the microfilament system. Disintegration of microfilaments by CD, LAT-A, or LAT-B of asynchronously growing fibroblasts caused a strong dose-dependent and time-dependent inhibition of total protein synthesis. Expression of c-jun, cyclins D1, E, and A decreased by about the same percentage as total protein synthesis. The strong induction of total protein synthesis after reactivating serum-starved fibroblasts by adding fetal calf serum was suppressed, when CD or LAT-A were added to the culture medium during this reactivation process. While expression of cyclin E as well as cyclin A decreased by about the same percentage as total protein synthesis, cyclin D1 was more suppressed after microfilament disintegration. After reactivating growth-arrested Wi-38 fibroblasts, cultured in suspension for 12 h, by transferring them to a rigid substratum they could adhere to, total protein synthesis was strongly induced. Again alteration of microfilaments by CD suppressed that increase. The expression of cyclin D1 was slightly more suppressed than total protein synthesis after addition of CD during that reactivation process. Our results suggest that alteration of microfilaments causes a strong decline of total protein synthesis accompanied by a decrease of the expression of proteins that are required for G1 progression and S phase entry. The diminished presence of proteins that are important for cell cycle progression could explain the inhibition of DNA synthesis after microfilament disintegration by various drugs.