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L Harel

Publications and source records attributed to L Harel.

At least 91 records · Page 5Linked to original sources
PubMed1985

Early stimulation of ATP turnover by EGF + insulin. Relation to external pH and Na+/H+ exchange system.

We previously shown a rapid increase in ATP turnover after addition of epidermal growth factor and insulin to quiescent 3T3 cell cultures. Here, the relationship between this increase in ATP turnover and the activation by growth factors of Na+/H+ and Na+/K+ exchange systems was studied. Our results show that alkalinization of the medium enhances ATP turnover but they do not support the assumption that stimulation by growth factors of the Na+/H+ exchange induces an increase in ATP turnover since this increase was not inhibited by amiloride. Conversely, when ATP synthesis was abolished, the increase, in intracellular pH, by growth factors, was significantly decreased.

Adenosine Triphosphate↗
PubMed1985

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

Alpha-Globulins↗
PubMed1985

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.

Adenosine Triphosphate↗
PubMed1985

[Changes induced by expression of the v-src gene in the regulation of cell proliferation. Hypothesis and preliminary results].

Similarities between the mode of action of growth factors and the oncogene product (pp 60 src protein) of Rous Sarcoma virus have been described. However, a major difference is that addition of growth factors does not induce a malignant transformation of cells. The present work proposes a hypothesis concerning this difference. Various data suggest that density-dependent inhibition (DDI) of growth in non-transformed cells is due to the diffusion of growth inhibitory molecules. Inhibitory factors of 45 K (IDF 45) and 12 K have been fractionated. We assume that the stimulation of DNA synthesis induced by growth factor addition to dense quiescent cultures of non-transformed cells leads to an increase in the activity of autocrine inhibitory molecules in such a manner that the growth factor stimulatory effect is only transient, and cells re-enter the Go phase. On the contrary, the stimulation of DNA synthesis by v-src transformation would not be counterbalanced by inhibitory diffusing factors and cells would not enter Go phase. We present preliminary results which support this assumption. Dense quiescent cultures of chick embryo fibroblasts infected by Ny 68 virus (ts mutant for transformation of Rous Sarcoma virus) were stimulated to proliferate either by addition of growth factors in cultures maintained at 41 degrees C or by expression of transformation (by the cell transfer from 41 to 37 degrees C, the permissive temperature for expression of transformation). Stimulation of DNA synthesis by growth factors was totally inhibited by the inhibitory diffusing factors of 45 K (IDF45) whereas the stimulation of DNA synthesis produced by transformation was reproducibly not decreased by IDF45.

Alpha-Globulins↗
PubMed1984

Density-dependent inhibition of growth: inhibitory diffusible factors from 3T3- and Rous sarcoma virus (RSV)-transformed 3T3 cells.

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.

Animals↗
PubMed1984

Early release of the density-dependent inhibition of phosphate uptake and ATP synthesis after src gene expression in chick embryo fibroblasts.

Our results showed that the expression of the src gene in chick embryo fibroblasts (CEF) released the density-dependent inhibition (DDI) of phosphate metabolism (phosphate uptake and phosphorylation of small organic compounds). With increasing cell density, phosphate metabolism decreased by 58% in normal CEF and, in contrast, increased by 20% in Rous sarcoma virus (RSV)-transformed CEF. The same change in the DDI was observed in CEF infected by NY68 (a ts mutant for transformation of RSV) and maintained at the permissive temperature (37 degrees C) instead of the restrictive temperature (41.5 degrees C) for the expression of transformation. An interesting feature was that the release of the DDI of phosphate metabolism was an early event in the process of transformation, since it was almost concomitant with the stimulation of the pp60 src kinase activity following the shift from 41.5 to 37 degrees C of NY68 CEF. The phosphorylation of small organic compounds (Po) was more strongly increased by the change in temperature than was 32Pi accumulation. Furthermore, the percentage increases of Po and adenosine triphosphate (ATP) labelling with 32P were similar, suggesting that the expression of src gene enhanced ATP synthesis. In glucose-free medium, the stimulation of Po-labelling was still observed but was decreased. Therefore the activation of glycolytic activity is not an absolute requirement, but is necessary for the maximum effect of transformation on the release of DDI of phosphate metabolism. Oligomycin added in complete medium did not prevent the increase in Po-labelling. From these results, we assumed that ATP turnover was stimulated as a consequence of enhanced ATP degradation. We verified that the stimulation of Po phosphorylation was not a consequence of increased ATP utilization for RNA or protein synthesis. The stimulation of Po labelling was specifically abolished by quercetin. This drug inhibited the transformed cells more strongly than the non-transformed cells.

Adenosine Triphosphate↗
PubMed1984

Inhibition by quercetin of the release of density dependent-inhibition of cell growth in RSV-transformed chicken cells.

The expression of src gene in dense cultures of chick embryo fibroblasts (CEF) infected by a thermosensitive mutant (NY68) of RSV released density-dependent inhibition of growth and induced in these cells a large increase in DNA, RNA and protein synthesis. This stimulation of cellular metabolism was abolished in the presence of quercetin. Furthermore, quercetin added to the culture medium also inhibited the stimulation of pp60src kinase due to the expression of transformation.

Animals↗
PubMed1983

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.

Animals↗
PubMed1983

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.

Adenosine Triphosphate↗
PubMed1983

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

Different data have suggested that the density-dependent inhibition of growth in normal 3T3 cells is due to the release of inhibitory factor(s) into the medium. The present results provide more evidence supporting this hypothesis. From conditioned medium of dense cultures of 3T3 cells, we fractionated a thermolabile inhibitory diffusible factor (I.D.F.) with a molecular weight of about 40.000 This factor decreased the [11C]-inosine incorporation into purine nucleotides and into nucleic acids of stimulated 3T3 cells. DNA synthesis, determined by [11C]-thymidine incorporation into nucleic acids, and protein synthesis (determined by the protein content of the cultures maintained in the presence or absence of I.D.F.), were also decreased. The possibility that I.D.F. is involved in density-dependent inhibition of growth is discussed.

Animals↗
PubMed1981

Altered RNA/protein ratio associated with the induction of differentiation of Friend erythroleukemia cells.

The synthesis and accumulation of RNA in Friend erythroleukemia (FL) cells induced to differentiate by treatment with the aminonucleoside of puromycin (AMS) or inhibited from differentiating by the addition of inosine to the medium were studied. When FL cells were grown in the presence of AMS, RNA synthesis was substantially inhibited. This effect could not be attributed solely to the inhibition of de novo purine synthesis because the biosynthesis of purine nucleotides from labeled inosine was much less depressed. The ratios of ATP to protein and of GTP to protein were slightly modified as compared to the untreated controls. However, the RNA/protein ratio was decreased. Thus, the RNA content of the cells was reduced 30-40%, but the protein content was not significantly affected. When the cells were treated with AMS together with inosine at a concentration that inhibits AMS-induced differentiation, the RNA/protein ratio was increased as compared with that found in cells treated with AMS alone and approached the level of the ratio in untreated control cells. Adenosine had a similar effect in overcoming the inhibition of RNA synthesis by AMS. Because the RNA/protein ratio of FL cells treated with dimethyl sulfoxide or sodium butyrate, two other potent inducers, was decreased by 44%, our results suggest that a correlation exists between the RNA content of the cells and the triggering of differentiation by inducers.

Adenosine↗
PubMed1980

Induction of differentiation of murine erythroleukemia cells by aminonucleoside of puromycin and inhibition of this induction by purines and purine derivatives.

The effect of the aminonucleoside of puromycin (AMS) on Friend erythroleukemia cells in culture was investigated, because purines and purine analogues are known to act as inducers of differentiation. After treatment with 20-30 micro M AMS for 4 days, the cultures contained between 80 and 90% benzidine-positive cells. Stimulation of hemoglobin synthesis was dose and time dependent. Inosine had no stimulatory activity; however, when it was added to the medium together with AMS, erythroid differentiation was almost completely inhibited. The inhibitory effect of inosine on this potent inducer was also dose and time dependent. No cytotoxicity was observed with either compound, alone or in combination. Inhibition of AMS stimulation of erythroid differentiation was also observed in the presence of inosine monophosphate and poly(inosinic acid). Hypoxanthine had a dual effect. At high concentrations (500 microgram/ml) it acted as an inducer, but when added at low concentrations (20 microgram/ml) together with AMS it inhibited differentiation. These findings suggest there is a link between purine biosynthesis and the event(s) required to trigger differentiation. Agonist-antagonist activity of closely related biological compounds has thus been revealed in the erythroleukemia cells.

Animals↗
PubMed1978

Diffusible factor(s) controlling density inhibition of 3T3 cell growth: a new approach.

In 3T3 Swiss mouse fibroblasts, incorporation of phosphate into cells and phosphorylation of small organic compounds were increased by shaking dense cultures. This response was not obtained with SV40 transformed Swiss 3T3 cells (SV-3T3). It appeared likely that these results could be accounted for by an inhibitor released from 3T3 cells but not from SV-3T3 cells. Our new method of co-incubation of sparse and dense cultures allowed us to demonstrate inhibition of growth and phosphate metabolism in sparse 3T3 cultures which were shaken in the presence of dense cultures. The inhibition was much less when the cultures were co-cultivated but not shaken. The inhibition of phosphate incorporation in acid-soluble and acid-insoluble fractions of sparse cultures was observed as early as 20 minutes of co-incubation in the presence of dense cultures, so this inhibition is not the result of depletion of growth factors in the medium. Our experiments suggest that an inhibitor(s) was released from dense cultures of 3T3 cells.

Cell Count↗