Cytochalasin D induces increased actin synthesis in HEp-2 cells.

Abstract

In HEp-2 cells treated with 0.2 to 2.0 ,uM cytochalasin D (CD) for 7.5 to 24 h there was a 20 to 50% relative increase in actin content (units of actin per microgram of total cell protein). This augmentation, which was concentration and time dependent, was prevented by treatment with cycloheximide during exposure to CD. A 15 to 20% increase in the relative rate of actin synthesis in CD-treated HEp-2 cells (0.2 to 2.0 ,uM CD) was detectable after 1 h of treatment and increased to 30 to 50% by 24 h. This increased rate of actin synthesis was apparently responsible for the higher actin content of CD-treated HEp-2 cells. The concentration dependence of these effects of CD on actin metabolism correlated with the pattern seen for CD-triggered changes in cellular morphology and the underlying rearrangements of the actin-containing cytoskeletal structures, suggesting that the effects on metabolism and morphology were interrelated. Since the rapidly occurring cytoskeletal reorganization preceded the effects of CD on actin metabolism, it is proposed that actin synthesis is induced by the cytoskeletal rearrangement resulting from exposure to CD.Cytochalasins inhibit cell motility and contractile system-dependent functions (review in reference 48). These drugs bind to actin, can reportedly inhibit in vitro polymerization (7,8,15,29) and treadmilling (46) by associating with the rapidly growing end of the actin filament (30), cause scission of filaments (22,31,44), and dissolve gels of actin formed either by direct filament-filament interactions or associations mediated by cross-linking molecules (22,30). Presumably, all of these effects also occur in the living cell. One of the effects of cytochalasins on living cells which is not observed with pure actin in vitro is a rearrangement of the actin-containing components of their cytoskeletons into aggregated masses (references 20, 21, 35, and 52, among others; review in reference 18). This rearrangement is dependent on metabolic energy (20,21,34,43) and is accompanied by little or no depolymerization of cellular actin (3, 36). We have previously reported that treatment of HEp-2 cells with cytochalasin D (CD) induced an increase in the relative content of total cellular actin (36). We have now extended these observations and report here that this rise in actin content is accompanied by an elevation in the relative rate of actin synthesis. Both of these effects occur only at concentrations of CD capable of eliciting rearrangements of actin in these cells, suggesting that these effects may be inter-

Cytoskeletal protein synthesis and organization in cultured mouse osteoblastic cells Effects of cell density

“…A correlation between the polymerization state and the gene expression of cytoskeletal elements has been described for microtubules [25,26] but it is not known whether this mechanism of regulation also applies to microfilaments. Changes in actin at-rangement may trigger actin biosynthesis in osteoblasts as in other cell types [27].…”

Cytochalasin D alters the rate of synthesis of some HEp‐2 cytoskeletal proteins

“…HEp-2 cells grown in monolayer culture [4] were, for experiments, planted at a density of 5 x lo5 cells per 60-mm plastic petri dish (Falcon Plastics), incubated for 1-2 days (to subconfluence), then treated with 0.2 pM or 2.0 pM CytD (Sigma Chemical Company) in 0.1 % dimethylsulfoxide (Me,SO) in growth medium [4] for 20 h; control dishes received 0.1 YO Me2S0. After drug treatment the cells were labeled for 1 h with ~-[~~S]methionine (approx.…”

“…Two-dimensional gel electrophoresis was performed by the procedure of O'Farrell [6] with the modifications previously described [4]. Isoelectric focusing was in cylindrical gels (2.4 x 125 mm) containing 1.6% (w/v) ampholytes of pH range 5 -7 and 0.4% (w/v) of pH range 3 -10 (Bio-Lyte 5/7 and Bio-Lyte 3/10 from Bio-Rad).…”

“…1340 Ci/mmol: Amersham Corporation) at a concentration of 20 pCi/ml in methionine-free growth medium containing either 0.1% (v/v) MezSO or CytD, as appropriate. Cells were then harvested into 0.3 ml lysis buffer A [6] plus 1 mM phenylmethylsulfonylfluoride (PheMeS02F) and 1 mM tosylphenylalanylchloromethane (TosPheCH,C1), processed and stored as previously described [4]. Cells labeled for longer time periods (approx.…”

“…During treatment of HEp-2 cells (among others) with cytochalasin D (CytD), cytoskeletal structures containing filamentous actin and associated proteins become rearranged into massive aggregates [3, 41. This morphological alteration is accompanied by an elevation in the relative rate of synthesis and the content of actin in HEp-2 cells [4], as well as in a variety of other cell types [5]. It was of interest to determine if this effect of CytD on actin synthesis extended to any other cytoskeletal proteins.…”