QUANTITATIVE CHANGES IN CYTOSKELETAL AND NUCLEAR ACTINS DURING CELLULAR-TRANSFORMATION

Actin, a highly conserved protein comprising cell stress fibers and ot her cellular structures, is found in both the cytoplasm and nucleus of cells and responds to both epigenetic signals and altered gene expres sion occurring during tumorigenesis. We have previously shown that cha nges in the cytoplasmic F- and G-actin ratios reflect bladder cancer r isk. To determine whether nuclear actin is also altered and how nuclea r and cytoplasmic actin alterations are interrelated in transformation , an in vitro model of carcinogen-induced transformation consisting of 2 human uroepithetial cell lines immortalized by infection with SV-40 was studied. One line, HUG-PC, is tumorigenic in nude mice after incu bation with the carcinogen 4-ABP, the other, HUC-BC, is not. Cytoplasm ic and nuclear F- and G-actin were determined by QFIA on individual ce lls using fluorochrome-labeled phallicidin and DNase, I, respectively. Before exposure to 4-ABP, the PC cells had lower cytoplasmic F-actin content, higher cytoplasmic G-actin content, but similar levels of nuc lear G- and F-actin in comparison to the BC cells. After incubation wi th 4-ABP, F-actin decreased and G-actin increased in both cytoplasm an d nuclei of PC cells and cytoplasmic F-actin fibers were lost, but onl y cytoplasmic actin was altered in the BC cells. Northern blot analysi s showed the expression of the B-actin gene was only approximately 20% lower in 4-ABP-treated PC cells than in untreated controls, indicatin g the cellular change in actin was attributed to a shift between F- an d G-actin proteins rather than to net actin synthesis. (C) 1997 Wiley- Liss, Inc.