Cytoarchitecture of kirsten sarcoma virus‐transformed rat kidney fibroblasts: Butyrate‐induced reorganization within the actin microfilament network

Abstract

Murine sarcoma virus-transformed rat fibroblasts (KNRK cells) undergo marked cytoarchitectural reorganization during in vitro exposure to sodium-n-butyrate (NaB) resulting in restoration of (1) a more typical fibroblastoid morphology, (2) proper cell-to-cell orientation, and (3) substratum adherence. Augmented cell spreading, involving greater than 90% of the population, was a function of culture density and time of exposure to NaB (2 mM final concentration). Induced cell spreading reflected a 2.5- to 3.0-fold increase in both total cellular actin content and deposition of actin into the detergent-resistant cytoskeleton. Cytoskeletal actin deposition in response to NaB was accompanied by the formation of occasionally dense, parallel alignments of F-actin-containing microfilaments and by a dramatic increase in the size and incidence of actin-enriched membrane ruffles. Long-term NaB-treated cells exhibited parallel orientations of microfilaments similar to those found in untransformed fibroblasts. Increased cytoskeletal actin occurred within 24 hr of NaB exposure, correlating with the initial reorganization of actin-containing microfilaments detected microscopically, and reflected concomitant 3-fold increases in cellular alpha-actinin and fibronectin content. In contrast, the amount of vimentin, tropomyosin, and tubulin in NaB-treated cells was significantly decreased. NaB-induced morphologic restructuring of sarcoma virus-transformed fibroblasts, thus, impacts on all three basic cytoskeletal systems. Selective increases, however, were evident in particular cytoskeletal proteins (actin, alpha-actinin, fibronectin) implicated in microfilament networking and cell spreading.

PAI-1 gene expression is regionally induced in wounded epithelial cell monolayers and required for injury repair

“…Saponin-resistant (SAP fraction) residues were scraped into sample buffer (50 mM Tris/HCl, pH 6.8, 10% glycerol, 1% SDS, 1% 2-mercaptoethanol) and boiled. For electrophoresis, 25,000 cpm trichloroacetic acid-insoluble SAP-fraction protein were separated on SDS/9% acrylamide slab gels (Ryan and Higgins, 1988). Labeled protein bands were visualized by fluorography and quantified by computerized densitometry (Smith et al, 1992).…”

Growth state-dependent regulation of plasminogen activator inhibitor type-1 gene expression during epithelial cell stimulation by serum and transforming growth factor-?1

“…and gel electrophoresis Quiescent cells were labeled with 50 Ci 35 S-methionine/ml (Ryan and Higgins, 1988) upon stimulation with 20% dialyzed FBS (Kutz et al, 1997). Conditioned labeling media were aspirated, clarified at 13,000 ϫ g, and IP of PAI-1 and SPARC was done simultaneously utilizing procedures and antibodies developed previously (Higgins et al, 1990).…”

Differential growth state‐dependent regulation of plasminogen activator inhibitor type‐1 expression in senescent IMR‐90 human diploid fibroblasts

“…Low-density early-and late-passage IMR-90 cells were washed once with PBS, fixed in situ with 10% (v/v) formaldehydePBS for 20 min, then permeabilized with 1% (v/v) Nonidet P-40 in PBS for 20 min (Wahrman et al, 1985;Ryan and Higgins, 1988). After a brief PBS rinse, cultures were incubated in Rh-phalloidin for 2 hr at 37"C, rinsed with PBS, and coverslips mounted with 50% (vh) glyceroVPBS.…”