Actin, a highly conserved protein comprising cell stress fibers and other cellular structures, is found in both the cytoplasm and nucleus of cells and responds to both epigenetic signals and altered gene expression occurring during tumorigenesis. We have previously shown that changes in the cytoplasmic F-and G-actin ratios reflect bladder cancer risk. To determine whether nuclear actin is also altered and how nuclear and cytoplasmic actin alterations are interrelated in transformation, an in vitro model of carcinogen-induced transformation consisting of 2 human uroepithelial cell lines immortalized by infection with SV-40 was studied. One line, HUC-PC, is tumorigenic in nude mice after incubation with the carcinogen 4-ABP, the other, HUC-BC, is not. Cytoplasmic and nuclear F-and G-actin were determined by QFIA on individual cells 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 nuclear G-and Carcinogenesis is a multi-stage process resulting in the eventual emergence of a malignant phenotype that often continues to progress to an eventually fatal metastatic phenotype. Although the genetic elements of carcinogenesis, the oncogenes and tumor suppressor genes, have been considered the central theme and are relatively well understood (Bishop, 1987;Weinberg, 1991), it is clear that cancer is a more complex process also involving epigenetic interactions among cells and their environment. Rubin (1992) described reversible transformation, including stable genetic heterogeneity of the cell population, simply by manipulating cell culture growth conditions. Pienta et al. (1989) have proposed that DNA organization in nuclei is an important epigenetic factor in cancer development, whereas others have observed that cell-cell interactions such as gap junctions and adherence are also involved (Trosko et al., 1990). Other experiments studying the relationship between basement membrane and type IV procollagen mRNA expression support a direct interaction among extracellular matrix/ cytoskeleton/nuclear matrix (Schlussel et al., 1991). Other studies show a connection between cytoskeletal actin, the nucleus, and the cell periphery that is altered after transformation (Pienta and Coffey, 1992). Mutation of the b-actin gene has been shown to alter the metastatic phenotype (Leavitt, 1994). A number of genes are also known to regulate the multimolecular focal complexes associated with actin stress fibers and cell cycle progression through G 1 and DNA synthesis (Nobes and Hall, 1995;Olson et al., 1995). One candidate for being the crucial integrative factor is the actincontaining nuclear matrix.Qualitative alterations of cytoplasmic actin, the major cellular protein involved in cell-cell interaction, morphology, motility, differentiation and adhesion, have been widely investigated in cancer cells in response to interactions between cells and their environment (Rao and Cohen, 1991;Pollard...