GROWTH-INHIBITORY FUNCTIONS OF A MUTATED GELSOLIN (HIS321) IN NIH 3T3MOUSE FIBROBLASTS/

We have previously established a murine flat revertant cell line R1 fr om an activated H-ras transformant EJ-NIH/3T3 by subjecting it to ethy l methanesulfonate. From the R1 cells, we cloned a mutated gelsolin ge ne His321 and have shown the inhibitory activity of His321 against EJ- NIH/3T3 tumors. Our present experiments were conducted to find out whe ther the His321 gene has any effects on untransformed NIH/3T3 fibrobla sts. Rhodamine-phalloidin staining revealed that two NIH/3T3 clones ex pressing His321 (NIH/lambda 2S-3 and NIH/lambda 2S-6) form organized a ctin stress fibers as two clones transfected with the vector alone (NI H/neo-3 and NIH/neo-5). We also found that in a liquid medium, NIH/lam bda 2S-3 and NIH/lambda 2S-6 grew more slowly than NIH/neo-3 and NIH/n eo-5 and that the doubling times of the former were about 10 h slower than those of the latter. To investigate the effects of His321 on the signal transduction pathway necessary for cell growth, we stimulated t he cell lines by prostaglandin E1 (PGE1), a platelet-derived growth fa ctor (PDGF), or the epidermal growth factor (EGF). Although stimulatio n by PGE1 increased intercellular cyclic AMP in R1 cells, it did not d o so in NIH/lambda 2S-3 and NIH/lambda 2S-6 cells. On the other hand, stimulation by PDGF or EGF induced far less DNA synthesis in NIH/lambd a 2S-3 and NIH/lambda 2S-6 than in NIH/neo-3 and NIH/neo5. These resul ts suggest that through the effects on the signal transduction pathway of PDGF and/or EGF His321-mutated gelsolin inhibits the growth of NIH /3T3. (C) 1996 Academic Press, Inc.