Effect of a Fibroblast Growth Factor, Insulin, Dexamethasone, and Serum on the Morphology of BALB/c 3T3 Cells

Abstract

The effects of serum, fibroblast growth factor, dexamethasone, and insulin on the morphology of two lines of BALB/c 3T3 cells are described and illustrated. Fibroblast growth factor, a polypeptide purified from bovine brain and pituitary glands, stimulates DNA synthesis and cell division in both sparse and confluent cultures of quiescent 3T3 cells. Cell Culture. Two lines of BALB/c 3T3 cells maintained in this laboratory were used. They have been described elsewhere (2). Cells were cultured at 370 in a CO2 incubator in Dulbecco's modified Eagle's medium.For the study of growing cultures, confluent cultures grown in 10% calf serum were trypsinized with 0.25% (w/v) trypsin. Trypsinization was stopped with 30% horse serum, and the cell suspension was centrifuged. The pellet was resuspended in medium, and 8 X 104 cells were plated per 6-cm dish in 5 ml of medium containing 5%0 calf serum. One day later the medium was removed, the cells were washed once with Eagle's medium, and 5 ml of medium with 0.6% calf serum was added. Two to 4 days later, the materials to be investigated were added to the dishes. Fibroblast growth factor was added in 251AI of a 0.5% solution of crystalline bovine serum albumin to make a final concentration of 25 ng/ml. Dexamethasone was added in 25 M1 of absolute ethanol to a final concentration of 1 /Ag/ml. Insulin was added in 25 1A of Eagle's medium adjusted to pH 2.0 with HCl to a final concentration of 1 /Ag/ml. All materials were added daily except serum, which was added only once.For (Fig. 1A) were supplemented with 10% serum, they grew to confluency and appeared as typical contact-inhibited cells (Fig. 1B). In contrast, cells grown in the presence of growth factor (Fig. 1G) grew to a lower density (Table 1); they ceased dividing after one or two cycles (3). These cultures were characterized by two morphologically distinct cell populations; the majority were rounded and attached to the plates by a few long processes ("spidery" cells); the remainder, 10-20%, were flat and looked somewhat like the controls (Fig. 1A). When cells were maintained in the presence of growth factor plus dexamethasone (Fig. ID), the two distinct populations were found again, but in approximately equal numbers. No contact inhibition was noticed; the "spidery" cells had grown on top of the flat 4648 Abbreviation: Bt2cAMP; dibutyryl cyclic AMP.

Transformation of mouse BALB/c 3T3 cells with human basic fibroblast growth factor cDNA.

“…In contrast, the parental A31 cells showed contact inhibition, with a regular cobblestone pattern in the monolayer culture. A morphological change similar to that of the transformed cells was observed when A31 cells were cultured in the presence of bFGF, as reported by Gospodarowicz and Moran (15).…”

Dexamethasone modulates ErbB tyrosine kinase expression and signaling through multiple and redundant mechanisms in cultured rat hepatocytes

“…In a given cell type, the actions of dexamethasone can vary between growth factors. In 3T3 cells, dexamethasone inhibits the growth promoting action of EGF but potentiates that of fibroblast growth factor (18,19). This suggests that the action of dexamethasone does not target a single cytoplasmic or nuclear mechanism distal to the interaction between growth factors and their cell surface receptors.…”

Stimulation of Division of Sparse and Confluent 3T3 Cell Populations by a Fibroblast Growth Factor, Dexamethasone, and Insulin

“…Dexamethasone is necessary to condition the cells in order that they might divide to reach a final density comparable to that seen with serum. When dexamethasone is present, insulin has little effect on cell growth; however, its effect on morphology is marked (10). We conclude that dexamethasone either prevents the degeneration that takes place in cells maintained in medium containing little serum, or it permits the cells to complete a division cycle initiated by FGF.…”

“…Final cell densities observed after the addition of 10% serum were: 3.2 X 106 for cultures grown in FGF plus dexamethazone plus insulin, 2.2 X 106 for cultures grown in FGF plus dexamethasone, and 1.4 X 106 for cultures grown in 10% serum. Morphological studies (10) indicated that the addition of 10% serum to cells grown to confluency in the presence of 10% serum did not result in overcoming "contact inhibition." However, when 10% serum was added to cells grown in the presence of FGF plus dexamethasone or of FGF plus dexamethasone plus insulin, "contact inhibition" was not observed; the cells grew in double or triple layers and had a morphology similar to that of transformed cells (10).…”

“…The morphological transformation brought about by the addition of FGF or of FGF plus dexametlhasone to a confluent, "'contact-inhibited" culture is described in ref. 10 (which is a protein concentration of 150 ,ug/ml), a lack of serum factors may result in a diminution of responsiveness to growth factor such that the response may, eventually, become negligible.…”