Louise Harel

IGFBP-1, AN INSULIN LIKE GROWTH FACTOR BINDING PROTEIN, IS A CELL GROWTH INHIBITOR

A novel cell growth inhibitor, IDF45 (inhibitory diffusible factor), was recently purified to apparent homogeneity. It is a bifunctional molecule: able to bind Insulin like growth factor (IGF) and to 100% inhibit DNA synthesis stimulated by serum in fibroblasts. It was of interest to verify whether other members of the IGF-binding protein (IGFBP) family show the same bifunctional growth inhibitory properties. In this paper we show that purified IGFBP-1 derived from amniotic fluid is a cell growth inhibitor. In chick embryo fibroblasts, it inhibited DNA synthesis stimulated by serum. However the stimulation was maximally 60% inhibited and half of the inhibition was observed with 100ng/ml IGFBP-1. So the specific activity of IGFBP-1 is lower than that of IDF45. IGFBP-1 also reversibly prevented the CEF growth. In the same cells IGFBP-1 inhibited DNA synthesis stimulated by IGF-I. We demonstrated that the same protein IGFBP-1 is able to inhibit DNA synthesis stimulated by serum and by IGF-I. The possibility that IGFBP-1 is a bifunctional molecule is discussed.

Persistance d'une synthèse de D-RNA dans le foie de rat traité par l'actinomycine

Abstract Actinomycin D-treated rats were injected with 32 P-labelled orthophosphate, 3 h later the animals were sacrificed and both RNA and DNA were extracted from their livers using phenol plus 0.5% or 1.0% sodium dodecylsulfate. RNA was fractionated by centrifugation in sucrose gradients. Separation of ribosomal and DNA-like RNA from s-RNA was achieved using 1.5 M NaCl or 0.2 M MgCl 2 . Complete inhibition of the synthesis of ribosomal RNA and s-RNA was found in the livers of actinomycin-treated rats, since, whether or not the RNA was treated with snake-venom phosphodiesterase (EC 3.14.1) before analysing the labelled 2′,3′-nucleotides, residual labelling of s-RNA could be attributed exclusively to the end groups pC-pC-pA. Synthesis of another fraction of RNA (8–10 S) was only partially inhibited (50–70%) by actinomycin. The (A+U)/(C+G) ratio of this fraction was identical to the (A+T)/(C+G) ratio of DNA, but generally the proportion of A was much larger (32–33%) than the proportion of U (20–22%). This DNA-like RNA could be recovered in a form closely associated with DNA itself. A similar DNA-like RNA was found in normal rat liver. These findings suggest that synthesis of all cellular RNA fractions is DNA dependent, but synthesis of DNA-like RNA is much less sensitive to actinomycin than synthesis of ribosomal and s-RNA. These facts pose certain questions about the manner in which ‘messenger’ RNA is replicated in mammalian cells.

Presence of IDF45 (mlGFBP-3) binding sites on chick embryo fibroblasts

IDF45 (inhibitory diffusible factor) a mouse insulin-like growth factor binding protein (mlGFBP-3) has been shown to 100 percent inhibit DNA synthesis stimulated by serum in chick embryo fibroblasts (CEF). Our previous results suggested that this large inhibition by IDF45 of serum stimulation was not just the result of its inhibitory activity toward IGF present in serum. The addition of Mn2+ (10(-3)M) in the incubation medium enables us to show the presence of numerous binding sites per cells (about 60,000) of mlGFBP-3. However the dissociation constant (10(-8)M) indicated that this mouse IGFBP-3 bound to the membrane with low affinity. These findings lend new support to the assumption of the bifunctional property of IGFBP-3, which would have an effect outside the cell (binding of IGF in the medium) and another effect within cells or on the surface.

IGF-I and IGF-Binding Proteins: Stimulatory and Inhibitory Factors Secreted by Human Prostatic Adenocarcinoma Cells

Deregulation of growth observed in malignant cell cultures has been assumed to be the result of increased secretion by these cells of autocrine growth factors, as well as the decreased sensitivity of these cells to inhibitory molecules which are diffused from normal or transformed cells. Our results show that PC-3 cells secreted into the medium, factors having stimulatory and inhibitory activities. We found an IGF-like molecule in medium conditioned by PC-3 cells. Its concentration was less than 1 ng/ml of conditioned medium. We demonstrated that PC-3 cells have receptors for IGF-I and are stimulated by this growth factor. However, the dose response curve shows that 1 ng/ml of IGF-I is not sufficient to indicate autocrine growth regulation by IGF of prostatic carcinoma cells. IGF-binding proteins of 90,000, 45,000, 34,000 and 28,000 molecular weight were also secreted by PC-3 cells. It is noteworthy that the secreted proteins which had the greatest inhibitory effect on chick embryo fibroblast growth also has the strongest IGF-binding activity. The probability that the IGF-binding protein secreted by PC-3 cells inhibited serum stimulation of DNA synthesis by preventing stimulation induced by IGF present in the serum is discussed. It is of interest that these IGF-binding proteins inhibited chick embryo fibroblast proliferation but did not inhibit PC-3 cells. This is in agreement with the assumption that IGF present in the medium is not an autocrine growth factor for these cells.

FRACTIONS WITH DIFFERING BASE COMPOSITION IN RNA FROM MALIGNANT CELLS OF MOUSE.

RNA labeled with 32 P was extracted from mouse ascites tumor cells, and centri-fuged in a linear sucrose gradient. After a pulse of 30 minutes most of the label was in a “heavy” (>30 s) and a “light” (4 to 10 s) fractions. The base composition of the fractions differed depending on whether or not sodium dodecyl-sulfate was included in the phenol extraction procedure. Without sodium dodecylsulfate the radioactivity of the “light” fraction predominated. This fraction contained a high proportion of cytosine which disappeared after treatment with phosphodiesterase. After the enzymic treatment, the base-ratios resembled those of transfer RNA. The “heavy” fraction differed from ribosomal RNA by a low proportion of adenine and a high proportion of uracil. More radioactivity was recovered after a short pulse when sodium dodecylsulfate and phenol were used. The “heavy” fraction differed from ribosomal RNA (28 8) only by slightly higher adenine and uracil contents. A DNA-like RNA was separated from transfer RNA contained in the light fraction (4 to 10 s). DNA-like RNA was also directly separated from DNA fibers. After labeling for 4·5 to 7 hr RNA was uniformly labeled, but there were still differences in the base ratios of the fractions. Ribosomal 26 to 28 s RNA had lower adenine and uracil contents than ribosomal 16 to 18 s RNA.

Early stimulation by EGF plus insulin of rRNA, c-fos, and actin mRNA expression: Inhibition by cytochalasin D

Stimulation of membrane ruffling is one of the first events induced by addition of growth factors to quiescent cultures. In order to assess the importance of intact cytoskeleton in induction, by EGF + insulin, of early events such as stimulation of rRNA, c-fos, and actin mRNA expression, we studied the effect of cytochalasin D (CD) on these metabolisms. We observed that CD slightly increased rRNA synthesis in nonstimulated cells; conversely, it decreased rRNA synthesis in cells stimulated by EGF + insulin. The maximal inhibition observed was 60%. The c-fos mRNA was not expressed in control cells and was accumulated in cells stimulated by the mixture of EGF + insulin; this accumulation was inhibited by CD. Actin mRNA was expressed in control cells and its expression was stimulated by EGF + insulin. Addition of CD decreased actin mRNA accumulation in stimulated cells but increased this accumulation in unstimulated cells. Our results, taken together, show that CD specifically affected the stimulation of rRNA and mRNA expression induced by growth factors and suggest that intact cytoskeleton and possibly membrane ruffling favored this stimulation.

Stimulation by TGFβ of chick embryo fibroblasts—Inhibition by an IGFBP-3

The multiple effects of TGF beta on cell proliferation are not well understood. Our results show that TGF beta was a good but transient mitogen for chick embryo fibroblasts. DNA synthesis was three- to fourfold increased, even at high concentrations of TGF beta. We did not show a bimodal effect. An inhibitor of cell growth, that inhibits 100% of stimulation induced by serum in CEF, was purified to homogeneity from medium conditioned by mouse 3T3 cells. This inhibitor has been shown to be an IGF-binding protein (mIGFBP-3). In the present work, this mIGFBP-3 inhibited the TGF beta stimulation by about 50%, while the stimulation induced by PDGF or insulin was not inhibited by mIGFBP-3. Furthermore, TGF beta stimulation, in the presence of a high concentration of insulin in conditions which would saturate IGF receptors, was not significantly inhibited by mIGFBP-3. All together these results suggest that a part of the mitogenic effect of TGF beta may be through increasing IGF secretion and eventually other growth factors such as PDGF (as suggested previously).

Early effect of growth factors (EGF+insulin) upon ATP turnover in 3T3 cells

Various early biochemical events have been observed after the addition of growth factors to quiescent cultures of 3T3 cells; however, the cascade of events which take place in the cells after growth-factor addition is not yet entirely known. Our results show that the addition of a mixture of two growth factors, i.e., Epidermal Growth Factor (EGF) and insulin, to quiescent cultures of 3T3 cells rapidly stimulated phosphate uptake and ATP turnover. Our present and previous results suggest that the increase in phosphate uptake is the consequence of the stimulation of ATP synthesis. This stimulation was not simply a consequence of an increase in oxidative phosphorylation or in glucose transport and metabolism. The change in ATP turnover was an early event observed as soon as 5 min after growth-factor addition; furthermore, it was not dependent on protein synthesis. This change may therefore be the result of post-synthetic modification of enzymes by phosphorylation. We do not know what cellular process is responsible for the increase in ATP turnover. Since growth-factor addition rapidly enhanced ATP degradation in quiescent 3T3 cell cultures, we assumed that this increase is the result of an increase in ATP degradation. We know that it was not due to a stimulation of an oligomycin-sensitive ATPase. We verified that it was not the consequence of early biochemical events like an increase in Na+/K+ ATPase or a stimulation of RNA or protein synthesis. However, it is of interest to note that the stimulation of ATP turnover due to the growth-factor addition was inhibited by quercetin.

Control by cell interaction of phosphate uptake in 3T3 cells

Abstract Phosphate uptake by monolayers of 3T3 cell decreases when the cultures enter the stationary phase, even when incubated in fresh medium containing 10% serum. However, SV 3T3 cultures retain a high rate of phosphate uptake when the cells reach saturation densities. We have observed that 3T3 cells grown to stationary phase in monolayers and then trypsinized and incubated in suspension, display an increase in phosphate uptake when the cell concentration is decreased from 10 6 cells/ml to 10 5 cells/ml. Where the cell concentration is further reduced from 10 5 cells/ml to 2.5 × 10 4 cells/ml there is no further increase in the rate of phosphate uptake. We observed, on the contrary, a small decrease. The “concentration effect” (the decrease of phosphate uptake when the cell concentration increases from 10 5 to 10 6 cells/ml) is larger when cells originate from a culture in stationary phase than when they originate from a culture in log phase. The “concentration effect” may be observed 10 min after cell incubation but is larger after a lag time of 40 min incubation. Differences in the “concentration effect” may be noted between 3T3 and SV 3T3 cells. In SV 3T3 cells no significant variations of phosphate uptake were observed when the cell concentration was changed. Thus, differences between phosphate uptake in 3T3 and SV 3T3 cells are large when cells are incubated at high concentrations or at high densities and small when they are incubated at low concentrations or at low densities. The “concentration effect” in 3T3 cells supports the assumption that interactions between cells cause the decrease of phosphate metabolism in dense culture. Diffusion of an inhibitor into the medium remains the more plausible explanation of the data.

Early stimulation of ATP turnover induced by growth factors: Synergistic effect of EGF and insulin and correlation with DNA synthesis

Addition of a mixture of EGF + insulin to quiescent cell cultures synergistically stimulates the cells to reinitiate DNA synthesis and cell division. We have previously demonstrated that this mixture rapidly increases ATP turnover in quiescent cells. The present work shows that each of the two growth factors, EGF and insulin, when added separately to quiescent cells was able to stimulate the phosphorylation of the organic acid-soluble compounds (Po) pool and ATP turnover. The stimulation of ATP turnover was closely correlated with the increase in phosphorylation of the Po pool which suggests that Po labelling reflects the ATP turnover. In many experiments, the synergy between the two growth factors on the early increase in phosphorylation of the Po pool was clearly shown. Doubling the concentration of EGF (12-24 ng/ml) or insulin (50-100 ng/ml) did not increase early stimulation of phosphorylation of the Po pool, whereas simultaneous addition of the two growth factors induced a greater stimulation than that of each growth factor separately added. The augmentation in Po labelling after addition of EGF or insulin alone was transient. The synergistic effect of the two growth factors was more significant when determined 150 or 300 min after growth-factor addition. In our experimental conditions, each of the two growth factors, EGF and insulin, was able to induce a stimulation of DNA synthesis. However, the best stimulatory effect was observed with the mixture of the two which synergistically increased DNA synthesis determined between 6 and 24 h after growth-factor addition. The comparison between DNA replication and Po labelling suggests a correlation between the increase in DNA replication and in the total ATP synthesized in the first 5 h after cell stimulation by growth factors added separately or in combination.