Cleavage of vimentin in dense cell cultures: Inhibition upon transformation by type 5 adenovirus

Abstract

The analysis on two-dimensional isoelectric focusing and SDS polyacrylamide gels (2D gels) of the Triton X-100 and high salt-insoluble fraction of fibroblast cell lines, certain epithelial cell lines and granulosa cells revealed various amounts of a vimentin cleavage product, with a more basic pI and with a MW (1500–2000) lower than that of intact vimentin. This cleavage product of vimentin which constituted as much as 30% of the total vimentin in an established rat embryo fibroblast cell line (CREF), was detected by a monoclonal antivimentin antibody in whole cell and Triton-insoluble extracts, and it has a phosphorylated variant which can be degraded to form the “staircase pattern” on 2D gels similarly to intact vimentin. This processing of vimentin occurred mainly in dense cell cultures and it could not be induced in sparse cell cultures by inhibiting DNA synthesis with ara C, or by arresting cell growth in medium containing 0.1% serum. Transformation of CREF cells with intact wild-type (H5wt) and host-range cold-sensitive mutants (H5hr1 or H5d1101) of type 5 adenovirus (Ad5), or transformation of CREF cells by Ca 2+-mediated DNA transfection with the transforming E1a (0–4.5 map units) or E1a + E1b (0–11.5 map units) region of Ad5 inhibits the cleavage of vimentin in dense cultures only at temperatures which are permissive for expression of the transformed phenotype. The transformation of cells with bovine papilloma virus type 1, with T24 ras oncogene, or with RSV does not interfere with the cleavage of vimentin. The organization of the vimentin network in dense cultures, where the vimentin cleavage occurs, is very different from that of sparse untransformed and sparse or dense Ad5-transformed cells. The possibility that the acidic amino acid-rich C-terminus of vimentin is cleaved in dense cell cultures in conjunction with the reorganization of the vimentin network and the inhibition of this cleavage by transformation with Ad5, are discussed.