Density‐dependent inhibition of growth: fractionation of inhibitory diffusible factor(s) released by dense cultures of 3T3 cells

The growth rate of normal cells multiplied in vitro decreases as the cell density of the culture increases. Previous results suggested that this density-dependent inhibition of growth in nontransformed cells was due to the diffusion of growth inhibitory substances in the medium of dense cultures. In this paper, we demonstrate that dense cultures of 3T3 cells secrete inhibitory and stimulatory factors. Macromolecules of conditioned medium were fractionated on Biogel P150 and the different fractions were tested on quiescent cultures of 3T3 cells stimulated or not to proliferate by addition of alpha globulin. When target cells were not stimulated to proliferate by addition of exocrine growth factors, we observed the inhibitory activity of a large molecular weight inhibitor (IDF45) and the stimulatory activity of autocrine growth factors (fraction about 35 and 10 K molecular weight), on the incorporation of 14C inosine into nucleotide pool and RNA. However, DNA synthesis was significantly stimulated with fraction 10 K only. This discrepancy between the stimulation of RNA and DNA synthesis may be explained by the presence, simultaneously, of inhibitory and stimulatory factors in fraction 35 and 10 K molecular weight. The presence of inhibitory factor was demonstrated when the fractions were tested on target cells stimulated to proliferate by alpha globulin addition and labeled with 14C thymidine. In these conditions, the stimulatory activity of autocrine growth factors was not observable, and only the inhibitory activity on DNA synthesis of fractions 35 and 10 K appeared. It is tempting to assume that the regulation of in vitro cell proliferation is determined by the balance between these antagonist stimulatory and inhibitory autocrine growth factors.

Section: Secretion B 3t3 Cells Of Stimulatory and Inhibitory Factors supporting

confidence: 94%

“…The presence of stimulating factors may mask the inhibitory ones. These results help to explain our previous dificulties in finding low molecular weight inhibitory factors (Harel et al, 1983) and they now agree with those of Steck and co-workers (1982).…”

supporting

confidence: 92%

Regulation of cell proliferation inhibitory and stimulatory factors diffused by 3T3 cultured cells

Harel

Blat

Chatelain

1985

“…On the other hand, different results have suggested that DDI of growth in 3T3 cells is under the control of growth inhibitory molecules diffusing from dense cultures (Harel et al, 1978). Growth inhibitory factors have been fractionated or purified from medium conditioned by dense cultures of 3T3 cells (Harel et al, 1983Blat et al, 1986;Hsu and Wang, 1986) and other cell types (Holley et al, 1980;Wells and Malluci, 1983;Iwata et al, 1985). However, a molecule of 25 Kd, inhibitory for BSCl cells (Holley et al, 1980), was shown to be similar to PTGF secreted by transformed and normal cells (Roberts et al, 19811, and could be a bifunctional regulator of cell growth (Tucker et al, 1984;Roberts et al, 1985;Shipley et al, 1985).…”

mentioning

confidence: 96%

Modulation by the src oncogene of the effect of inhibitory diffusible factor IDF45

Blat

Villaudy

Rouillard

et al. 1987

Self Cite

Density-dependent inhibition of growth has been assumed to be under the control of inhibitory molecules diffusing from dense cell cultures. Growth inhibitory factors have been fractionated or purified from medium conditioned by different cell types. In the present work, it was shown that IDF45 (inhibitory factor diffusing from 3T3 cells) decreased DNA synthesis in chick embryo fibroblasts (CEF) and was an inhibitor of CEF growth; this inhibition was reversible. Since similitudes between oncogene products and growth factors have been observed, it was of interest to compare the inhibitory effect of IDF45 upon the stimulation of DNA synthesis induced either by serum or by pp60-src. CEF infected by Ny68 virus (a mutant of Rous sarcoma virus ts for the expression of transformation) were density-inhibited at 41 degrees C, but were stimulated at this temperature by addition of 1% serum. This stimulation was 94% inhibited by IDF45. The same Ny68-infected cells could also be stimulated by transfer to 37 degrees C, the permissive temperature (in the absence of serum). The stimulation of DNA synthesis by src expression was poorly inhibited by IDF45. From our results, it appears that oncogene expression in CEF induces a loss in their sensitivity to IDF45. This would explain why transformed cells escape DDI of growth.

“…Steck and coworkers reported partial purification from conditioned medium of 3T3 cell cultures of growth inhibitory factors (Steck et al, 1979;1982). We recently fractionated, from the culture medium of 3T3 cells, a thermolabile inhibitory diffusible factor (IDF) with a molecular weight of about 40,000 daltons (Harel et al, 1983) This factor decreased the 14C-inosine incorporation into purine nu-cleotides and nucleic acids of stimulated target 3T3 cells.In the presence of IDF, DNA synthesis and protein accumulation also decreased.To provide new evidence for the assumption that IDF is involved in the DDI of growth and to obtain further insight into the mechanism of the release of DDI of growth in transformed cells, we compared the inhibitory activity of IDFN produced by 3T3 cells and of IDFT, (produced by Rous sarcoma virus-transformed [3T3 SRAJ H] cells), on 3T3 and 3T3 SRA/H cell metabolism. …”

mentioning

confidence: 99%

mentioning

confidence: 99%

Density‐dependent inhibition of growth: Inhibitorydiffusible factors from 3T3‐ and rous sarcoma virus (RSV)‐transformed 3T3 cells

Harel

Chatelain

Golde

1984

We recently fractionated, from the culture medium of 3T3 cells, a thermolabile inhibitory diffusible factor (IDFN) with a molecular weight of about 40,000 daltons, which decreased nucleic acids synthesis of stimulated target 3T3 cells. In the present publication the inhibitory activities of IDFN (produced by 3T3 cells) and IDFT (produced by RSV-transformed 3T3 [3T3 SRA/H] cells) on 3T3 and 3T3 SRA/H cells have been compared. The inhibitory activity of IDFN decreased (by a mean of 57%) when it was tested on transformed instead of 3T3 cells. On the other hand, IDFT was able to decrease 14C-inosine incorporation in target 3T3 cells. However, the inhibitory activity of IDFT decreased (by mean 50%) when tested on 3T3 SRA/H instead of 3T3 cells. Therefore, transformed cells produced an inhibitory factor but were less sensitive than 3T3 cells to its inhibitory activity. The inhibitory activity of IDFT on 3T3 SRA/H cells was only 20% of the inhibitory activity of IDFN on 3T3 cells. This appreciable difference is of particular interest, since it could explain the release of density-dependent inhibition of growth (DDI) in transformed 3T3 SRA/H cells. Furthermore, it provides more evidence for the hypothesis that, in 3T3 cells, DDI of growth is due to the release of an inhibitory molecule into the medium, and that IDFN is in fact, the inhibitory molecule involved in this phenomenon.