"Contact inhibition" of cell division in 3T3 cells.

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.

Section: Discussionmentioning

confidence: 78%

“…cessation of growth at confluency may reflect a transition from a proliferative stage to a resting stage, under very strict control of serum growth factors (7). It has been observed that when 3T3 cells are maintained in 30% serum with a change of fluid daily, contact inhibition is not seen; the cells grow to a very high density (7).…”

Section: Discussionmentioning

confidence: 97%

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

Gospodarowicz

Moran

1974

“…Without rinsing, the cells were fixed with 2 ml per plate of cold 5% trichloroacetic acid (TCA) and incubated at 40 for an hour. Then the TCA suspensions were transferred to a cold centrifuge tube, pooled with a 5% TCA wash of the plates, and pelleted at 10,000 rpm for 5 (Table 2). However, the lines A-ySV4 and AySV5, which were selected for their inability to grow in 10% agamma-depleted calf serum (7), retained the low cAMP level of SV3T3101 ( (Table 3).…”

Section: Methodsmentioning

confidence: 99%

Intracellular Cyclic AMP Concentration Responds Specifically to Growth Regulation by Serum

Oey

Vogel

Pollack

1974

AB STRACTIntracellular concentrations of cyclic AMP increase when cells are deprived of serum. Studies with the mouse fibroblast line 3T3, an SV40-transformed subline of 3T3, and six different revertant lines derived from this clone show that a marked increase in cyclic AMP occurs only when the serum concentration is reduced below the minimum necessary for growth of a given line. Conversely, density-dependent inhibition of growth is not accompanied by an increase in cyclic AMP concentration in any line.The regulation of proliferation of fibroblast cultures is complex. Well regulated cell lines, such as the mouse line 3T3, are sensitive to inhibition by at least three different environmental signals: contact with other cells (1-3), deprivation of serum (4, 5), and absence of a solid substrate (6). SV40 virus transformed 3T3 clones may lose sensitivity to all three signals, and thus grow despite cell-cell contact (2), grow even when serum is reduced by 10-fold (7), and form spherical colonies when suspended in methyl cellulose gel (8, 9) ( Table 1). This loss of growth control can be reversed. By applying negative selection pressures to transformants, we have obtained cell lines, called revertants, which exhibit growth control comparable to that of normal cells (7, 9-11). Some revertants have regained sensitivity to contact-regulations and anchorage-regulations without regaining a high serum requirement for growth. We term these "density-revertants" (10, 11) ( Table 1). Other revertants have regained a high serum requirement for growth and, in addition, sensitivity to either contact alone, or to both contact and anchorage. We term these "serum-revertants" (7) ( Table 1).Many studies have suggested a possible inhibitory role for 3': 5'-cyclic AMP (cAMP) in the regulation of proliferation of fibroblast cultures (12-19). When cAMP is measured directly in cultures of normal and transformed cell lines, the concentration of cAMP is consistently found to be about half as high in growing cultures of transformed lines (13)(14)(15)(16)(17)

“…It has been suggested that one factor responsible for the cessation of growth of contact-inhibited cells is the depletion of growth factors in the medium, especially serum growth factors (5,6). If contact inhibition in the BrdU-dependent cells in the absence of BrdU is due to such depletion of growth factors, then BrdU seems to alter the cells in such a way that these factors are no longer required.…”

Section: Discussionmentioning

confidence: 99%

Reversible “Transformation” of Bromodeoxyuridine-Dependent Cells by Bromodeoxyuridine

Davidson

Horn

1974

The growth characteristics of a bromodeoxyuridine-dependent cell line that was derived from a non-contact-inhibited Syrian hamster melanoma line were studied. The dependent cells require high concentrations of bromodeoxyuridine not only for optimal growth but also for the maintenance of the non-contact-inhibited state. When grown in the absence of bromodeoxyuridine, the dependent cells become contact-inhibited. The transition to the contact-inhibited state is reversed when bromodeoxyuridine is added back to the medium, if the bromodeoxyuridine is incorporated into dividing cells. The effects of bromodeoxyuridine on growth rate and on contact inhibition are separable.We recently described a new mutation in mammalian cellsbromodeoxyuridine dependence (1). The mutant cells require the drug 5-bromodeoxyuridine (BrdU) for optimal growth, and the concentration of BrdU at which these cells grow most rapidly is toxic to most other cells. In the absence of BrdU, the mutant cells continue to multiply but at a significantly reduced rate. The requirement for BrdU in these cells is specific, not being satisfied by thymidine or related analogues such as iododeoxyuridine. The mutant cells incorporate BrdU into nuclear DNA, replacing approximately 50% of the thymidine residues with BrdU. In the presence of BrdU plus inhibitors of thymidine biosynthesis, the cells grow with essentially 100% of the thymidine replaced by BrdU (2).The BrdU-dependent cells were derived from a highly malignant Syrian hamster melanoma cell line. Like other malignant cells in culture, the melanoma cells are not contactinhibited, i.e., they do not cease growing when they have formed a confluent monolayer. In the presence of BrdU, the BrdU-dependent cells are similarly not contact-inhibited. However, in the absence of BrdU, the dependent cells become contact-inhibited. This change in the pattern of growth is of special interest because contact inhibition is a characteristic generally associated with nonmalignant cells. (The mechanisms of contact inhibition are not understood, and the role of cell contact per se remains to be demonstrated.) This paper describes the requirement of BrdU for the maintenance of the non-contact-inhibited state in the BrdU-dependent cells. MATERIALS AND METHODSCell Lines and Growth Media. The isolation of the BrdUdependent cell line B4 from the Syrian hamster melanoma line RPMI 3460 was described previously (1). The 3460 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal-calf serum (E medium). The B4 cells were maintained in E medium containing 0.1 mM BrdU (E-B medium). A revertant cell line, able to grow well in BrdU but no longer requiring it, was isolated from B4. The B4 cells were first cloned in E medium, and a clone of rapidly growing cells, called B4-E18, was isolated. The B4-E18 cells were then cloned in E-B medium, and a subclone, B4-E18-B2, which grew well in the presence of BrdU was isolated. All of the cell lines were protected from exposure to light of wavelengths below 550 n...