C. Blat

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.

Phosphoprotéines des membranes plasmiques isolées du foie de rat. Incorporation du 32P dans ces phosphoprotéines

Abstract Three different sorts of phosphoproteins have been characterized in plasma membranes from rat liver cells: alkali unstable phosphoproteins, containing phosphoseryl and phosphothreonyl groups: acid-unstablephosphoproteins containing phosphohistidine; and acyl phosphate-bound phosphoproteins. Alkali-unstable phosphoproteins from plasma membranes are more abundant than in any other structure (7 μg P/mg N protein as compared with 1.3 μg in microsomes). They are also more rapidly labelled. About 30 min after injecting 32P into animals, they attain a maximum specific radioactivity, which is 4 times greater than that of the total cell phosphoproteins. The maximum specific radioactivity is reached within the same time in acid-unstable phosphoproteins as well as in Pi and ATP, which suggests that a rapid exchange of 32P is occurring between these different compounds. In contrast, after labelling in vivo, acyl phosphate-bound phosphoproteins from plasma membranes are found to be 10–20 times less radioactive than alkali-unstable phosphoproteins, but they are very rapidly labelled in vitro when isolated membranes are incubated with [γ-32P]ATP. The radioactivity is K+ and Na+ dependent.

Effect of transformation of chicken cells by Rous sarcoma virus on in vitro phosphorylation of nuclear non-histone proteins☆

Abstract The use of virus temperature-sensitive (ts) mutants for transformation allows us to determine the effect of transformation on different alterations in cell morphology and cell functions. We studied the kinase activity of nuclear non-histone proteins (NHP) extracted from chicken embryo fibroblasts (CEF), both infected and non-infected with a ts mutant of Rous sarcoma virus (Schmidt Ruppin strain, subgroup A, T class) and cultivated at the restrictive (41°C) and permissive (37 °C) temperature for transformation. The endogenous in vitro phosphorylation of NHP extracted from stationary ts-infected cultures increased by 300% when the cells were cultivated at 37 instead of 41°C. This increase is probably a consequence of the expression of transformation, since it was not observed when NHP were extracted from non-infected CEF maintained either at 41 or 37 °C. The increase in endogenous phosphorylation of NHP does not seem to be entirely due to a change in the density-dependent inhibition of cell metabolism in transformed cells, as an increase in protein phosphorylation was observed when the ts cultures were in both exponential growth phase and plateau phase. The urea-polyacrylamide gel electrophoresis of 32P-phosphorylated proteins showed that the increase in phosphorylation observed when growing ts cells were maintained at 37 instead of 41 °C was mostly due to the stimulation of one fraction (fraction d), as if the increase in phosphorylation of this fraction was linked to the expression of transformation. When the ts cultures were in stationary phase, the enhancement of the phosphorylation of proteins observed when cells were maintained at 37 instead of 41 °C was due to stimulation of the phosphorylation of all protein fractions, and particularly of fractions d and b. When phosvitin was used as a substrate, the kinase activity of NHP extracted from ts-infected cells was increased 300% when the cells were cultivated at 37 instead of 41 °C. On the other hand, the phosvitin kinase activity of NHP extracted from non-infected cells decreased when the cells were maintained at 37 instead of 41 °C. Thus, the transformation of cells induced an increase in the phosvitin kinase activity of nuclear NHP. When ts cells were cultivated at 41 °C until saturation density and then transferred to 37 °C for 3 h before extraction of proteins, a 37% increase in phosvitin kinase activity of NHP was observed, as compared with the activity of proteins extracted from cells left at 41 °C. Thus, the change in kinase activity is an early event in the process of transformation.

Purification from transformed mouse fibroblast of a cell growth inhibitor which is an IGF-binding protein.

From medium conditioned by 3T3 cells, we had previously purified an inhibitory factor of Mr 45 kDa which we termed IDF45 (inhibitory diffusible factor). The protein was able to 100% inhibit stimulation induced in CEF by 1% calf serum and to reversibly prevent cell growth. We then demonstrated that IDF45 was an IGF-binding protein. Our results suggested that IDF45 was a bifunctional molecule able to bind IGF and to inhibit DNA synthesis stimulated by this hormone, but also to inhibit stimulation of DNA synthesis induced by another growth factor in serum. Indeed, its N terminal amino acid sequence has great homology with that of IGFBP-3 and IDF45 is now proposed to be named IGFBP-3 (mouse IGF binding protein). Present results show that Ha-ras transfected 3T3 cells (EJ cells), like 3T3 cells, secrete a mIGFBP-3 molecule. In addition, transfected cells secrete a doublet of an IGF-binding protein (IGFBP-28) of Mr 28 kDa which is not secreted by untransformed 3T3 cells. IGFBP-28 has been purified and characterized in this work. Various results suggest that IGFBP-28 is not a degradation product of mIGFBP-3. Its N terminal amino acid sequence was different from that of mIGFBP-3. IGFBP-28 inhibited DNA synthesis stimulated by IGF-I, but much more IGFBP-28 protein than mIGFBP-3 was required to prevent this stimulation. In agreement with this result, IGFBP-28 has low affinity for IGF-I. In contrast, IGFBP-28 has high affinity for IGF-II. Like mIGFBP-3, IGFBP-28 was able to inhibit the stimulation induced by serum in CEF and to reversibly prevent growth, though with a specific activity lower than that of mIGFBP-3. It has also the capacity to inhibit stimulation of DNA synthesis induced by high molecular weight serum proteins depleted in IGF-I and II. In conclusion we have shown that transformation of 3T3 cells with Ha-ras induced the synthesis of a new IGF binding protein in medium conditioned by normal 3T3 cells. Our results suggest that IGFBP-28 like mIGFBP-3 is a bifunctional protein able to inhibit stimulation induced by IGF and by serum proteins different from IGFs.

Inhibitory diffusible factor IDF45, a G1 phase inhibitor

An inhibitory diffusible factor of 45 kDa (IDF45) was isolated from medium conditioned by dense cultures of 3T3 cells. The procedure involved Bio‐Gel PI 50 chromatography and 2 reverse‐phase FPLC. After the final step of purification, 60 of IDF45 inhibited 50% of α‐globulin‐stimulated DNA synthesis. It was shown that IDF45 acted in the G1 phase of the cell cycle. When added for 8 h in the G1 phase of the cell cycle, it was able to inhibit DNA synthesis in the S phase which followed this G1 phase. Furthermore, IDF45 inhibited the early stimulation of RNA synthesis induced by α‐globulin.

Difference in biological effects between insulin-like growth factor binding protein 1 and 3.

Insulin-like growth factor binding proteins 1 and 3 are essentially known as regulators of IGF bioactivity. However, we previously showed that IGFBP-3 was able, in chick embryo fibroblast (CEF), to 100% inhibit DNA synthesis stimulated by calf serum, while the maximal inhibition found with IGFBP-1 was 60%, suggesting a difference between the two IGFBPs in their biological functions. Results of the present work agree with this assumption: (a) Recombinant human IGFBP-3, like rat IGFBP-3, was able to 100% inhibit DNA synthesis stimulation induced by human serum, while this stimulation was 75% decreased by IGFBP-1. However, the most striking difference was observed when the effects of the two IGFBPs were compared for stimulation induced by a serum growth factor (SGF) fraction depleted in IGFs. Stimulation induced by the SFG fraction was more significantly decreased (p < 0.001) by IGFBP-3 than by IGFBP-1. The mean percent inhibition +/- SEM was 67.1 +/- 2.5 in the presence of IGFBP-3 (200 ng/ml) and 29.3 +/- 2.7 and 34.2 +/- 4 in the presence of 200 and 400 ng/ml IGFBP-1 respectively. Inhibition by 200 ng/ml IGFBP-1 and inhibition by 6 ng/ml IGFBP-3 were additive. However, inhibition by IGFBP-3 and that by IGFBP-1 were no longer additive at high concentrations of IGFBP-3, which might thus replace IGFBP-1. (b) FGF stimulation of CEF was similarly inhibition (65% and 70%) by IGFBP-1 and IGFBP-3. (c) TGF beta stimulation of CEF was more strongly decreased by IGFBP-3 (90%) than by IGFB-1 (60%).(ABSTRACT TRUNCATED AT 250 WORDS)

Rous sarcoma virus-induced changes in the pattern of phosphorylation of non-histone nuclear proteins

We here studied the protein kinase activity and in vitro phosphorylable sites of non-histone nuclear proteins, 0.4 M NaCl extracts (mostly chromosomal proteins) from chick embryo fibroblasts (CEF), infected or not with a Schmidt Ruppin strain subgroup A of Rous sarcoma virus (RSV). The infection and transformation of chick fibroblasts by RSV induced an increase in kinase activity and endogenous phosphorylation of non-histone chromosomal (NHC) proteins. The stimulation, by a change of medium, of the proliferation of dense cultures of normal chick fibroblasts also induced an increase in the kinase activity and endogenous phosphorylation of NHC proteins. However, two-dimensional gel electrophoresis of the 32P-phosphorylated proteins showed that stimulation due to a change of medium and that due to the expression of transformation were very different. The stimulation by a change of medium increased to a greater or lesser extent the phosphorylation of the different NHC proteins, with no fundamental variations in the pattern of protein phosphorylation. In contrast, RSV infection induced significant changes in the pattern of protein phosphorylation. One of the most striking feature was the large increase of amount and phosphorylation of high molecular weight (HMW) proteins in particular of phosphoproteins having an evaluated molecular weight (MW) of 78 K and 82 K and pI greater than 8.2. The percent of phosphotyrosine residues in NHC proteins was clearly increased when the proteins were extracted from transformed cells instead of normal cells. But the alkaline treatment of two-dimensional gel electrophoresis indicated that the 80 K phosphoproteins did not contain phosphotyrosine residues, and thus cannot be considered as substrates for pp60src kinase.

Phosphoproteins des membranes plasmiques isolees de foie de rat. Phosphorylation par le γ-32P] ATP

Abstract Recently we have characterized in phosphoproteins of plasma membranes from rat-liver cells three different sorts of phosphorylated groups: alkali-unstable phosphoserine and phosphothreonine, acid-unstable phosphohistidine and acylphosphate group unstable in hydroxylamine. It is shown here that the three groups were phosphorylated when the membranes were incubated with [γ- 32 P]ATP, during 30 sec. The degree of phosphorylation depended on conditions of incubation: 90% of the incorporated radioactivity was in acylphosphate phosphoproteins when the membranes were incubated at 0° with a low concentration of Mg 2+ . When the membranes were incubated at 25° with Mg 2+ (5 mM) only 43% of the incorporated radioactivity was hydroxylamine-unstable, 41% was acid-unstable and 16% alkali-unstable. Chase experiments have shown that the turnover of phosphate of the acyl-phosphate group was faster than that of phosphate of phosphohistidine. In contrast the phosphate of phosphoserine and phosphothreonine appeared to be relatively stable. Phosphorylation of acylphosphate and phosphohistidine phosphoproteins was especially Na + -, K + -dependent and was inhibited by ouabain as is (Na + K + )-dependent ATPase of the membranes. Our observations are consistent with the hypothesis that acylphosphate-bound phosphoproteins are related in the Na + -K + active transport across cell membranes. Our results suggest that ATP may not be the immediate precursor of phosphate of phosphoserine and phosphohistidine. In this case phosphorylation of these groups would be indirectly dependent on K + and ouabain.

Synthesis and secretion by mouse 3T3 cells of a inhibitor of cell growth (mIGFBP-3): Correlation with cell proliferation

Summary— Our results show that stimulation by serum of dense cultures of 3T3 cells rapidly induced increased synthesis of a growth inhibitor (mIGFBP‐3) capable of binding IGF. mIGFBP‐3 was secreted by stimulated cells immediately after its synthesis, and accumulated in the medium. Accumulation of mIGFBP‐3 in the medium increased, as a function of growth factor (bFGF, PDGF, insulin) concentrations and time. bFGF was the best stimulatory factor for both DNA synthesis and accumulation of mIGFBP‐3 in the first 24 h of incubation. DNA synthesis was arrested after 48 h of incubation with bFGF when accumulation of mIGFBP‐3 was maximal. Since we showed that mIGFBP‐3 is able to inhibit bFGF stimulation of DNA synthesis in mouse fibroblasts, it is possible that the accumulation of mIGFBP‐3 induces a feedback regulation of cell growth.