Loss to follow-up among youth accessing outpatient HIV care and treatment services in Kisumu, Kenya.

Youth are particularly vulnerable to acquiring HIV, yet reaching them with HIV prevention interventions and engaging and retaining those infected in care and treatment remains a challenge. We sought to determine the incidence rate of loss to follow-up (LTFU) and explore socio-demographic and clinical characteristics associated with LTFU among HIV-positive youth aged 15-21 years accessing outpatient care and treatment clinics in Kisumu, Kenya. Between July 2007 and September 2010, youth were enrolled into two different HIV care and treatment clinics, one youth specific and the other family oriented. An individual was defined as LTFU when absent from the HIV treatment clinic for ≥ 4 months regardless of their antiretroviral treatment status. The incidence rate of LTFU was calculated and Cox regression analysis used to identify factors associated with LTFU. A total of 924 youth (79% female) were enrolled, with a median age of 20 years (IQR 18-21). Over half, (529 (57%)), were documented as LTFU, of whom 139 (26%) were LTFU immediately after enrolment. The overall incidence rate of LTFU was 52.9 per 100 person-years (p-y). Factors associated with LTFU were pregnancy during the study period (crude HR 0.68, 95% CI 0.53-0.89); CD4 cell count >350 (adjusted hazard ratios (AHR) 0.59, 95% CI 0.39-0.90); not being on antiretroviral therapy (AHR 4.0, 95% CI 2.70-5.88); and non-disclosure of HIV infection status (AHR 1.43, 95% CI 1.10-1.89). The clinic of enrolment, age, marital status, employment status, WHO clinical disease stage and education level were not associated with LTFU. Interventions to identify and enrol youth into care earlier, support disclosure, and initiate ART earlier may improve retention of youth and need further investigation. Further research is also needed to explore the reasons for LTFU from care among HIV-infected youth and the true outcomes of these patients.

Complete sequence of a novel protein containing a femtomolar-activity-dependent neuroprotective peptide.

The vulnerability of neurons and the irreversibility of loss make discoveries of neuroprotective compounds fundamentally important. Here, the complete coding sequence of a novel protein (828 amino acids, pI 5.99), derived from mouse neuroglial cells, is revealed. The sequence contained (1) a neuroprotective peptide, NAPVSIPQ, sharing structural and immunological homologies with the previously reported, activity-dependent neurotrophic factor; (2) a glutaredoxin active site; and (3) a zinc binding domain. Gene expression was enriched in the mouse hippocampus and cerebellum and augmented in the presence of the neuropeptide vasoactive intestinal peptide, in cerebral cortical astrocytes. In mixed neuron-astrocyte cultures, NAPVSIPQ provided neuroprotection at subfemtomolar concentrations against toxicity associated with tetrodotoxin (electrical blockade), the beta-amyloid peptide (the Alzheimer's disease neurotoxin), N-methyl-D-aspartate (excitotoxicity), and the human immunodeficiency virus envelope protein. Daily NAPVSIPQ injections to newborn apolipoprotein E-deficient mice accelerated the acquisition of developmental reflexes and prevented short-term memory deficits. Comparative studies suggested that NAPVSIPQ was more efficacious than other neuroprotective peptides in the apolipoprotein E-deficiency model. A potential basis for rational drug design against neurodegeneration is suggested with NAPVSIPQ as a lead compound. The relative enrichment of the novel mRNA transcripts in the brain and the increases found in the presence of vasoactive intestinal peptide, an established neuroprotective substance, imply a role for the cloned protein in neuronal function.

Density-dependent inhibition of mouse embryo fibroblast growth: involvement of IGFBP-3.

Up until now the phenomenon of density dependent inhibition of growth has remained unexplained; one hypothesis suggests that autocrine growth inhibitory molecules are secreted in the medium of dense cultures and inhibit cell growth. From medium conditioned by mouse fibroblasts we purified a 45-kDa inhibitory factor which is an insulin-like growth factor binding protein (IGFBP-3). Based on different results we assumed that IGFBP-3 is a bifunctional molecule and has inhibitory function independent of its known function of binding IGF. In the present publication we attempted to verify whether IGFBP-3 is involved in DDI of growth. IGFBP-3 was secreted by mouse embryo fibroblasts (MEF). Its concentration in the medium increased with the cell density of the culture and was large at saturation density when DNA synthesis was minimum. Medium conditioned (CM) by dense culture was inhibitory compared to fresh medium and this inhibition disappeared when CM was preincubated with anti-IGFBP-3 IgG. Addition of FGFb to MEF dense cultures increased but transiently DNA synthesis which decreased as soon as 24 h after growth factor addition. By contrast accumulation of IGFBP-3 in the medium increased with time and was large at the time when DNA synthesis was minimum. Our results suggest that the rapid decrease of DNA synthesis in stimulated dense culture was the result of both depletion of the medium (particularly of FGFb) and the increase in concentration of inhibitory molecules like IGFBP-3. Addition of FGFb and preincubation of CM with anti-IGFBP-3 IgG were able to greatly reduce the inhibition.

In vivo proteolysis of serum insulin-like growth factor (IGF) binding protein-3 results in increased availability of IGF to target cells.

IGF Binding Protein-3 (IGFBP-3), the major IGF carrier in the blood, undergoes limited proteolysis which reduces its affinity for IGFs, thus facilitating dissociation. The functional effects of this at the cellular level were studied by comparing two serum pools, one from healthy adults, one from women during late pregnancy when IGFBP-3 proteolysis is increased. Sera were mixed to yield identical IGF-I and IGF-II concentrations in the two pools. Western ligand and immunoblotting gave the characteristic IGFBP patterns for the two types of serum. Both pools dose-dependently stimulated DNA synthesis in cultured chick embryo fibroblasts. Stimulation by pregnancy serum was twice that by normal serum at 0.05-0.2% concentrations (P < 0.001). In the presence of excess monoclonal anti-IGF-I and -II antibodies, stimulation by both (0.1-0.2%) pools was 70-80% reduced and residual stimulation was similar. Addition of recombinant human (rh) IGFBP-3 dose-dependently depressed both pools' activity, more so for normal serum at 25 and 50 ng/ml, equally for each at 100 ng/ml. At the latter concentration, slight proteolysis of the rhIGFBP-3 was detectable in the presence of 0.2% pregnancy serum, but at 25 ng/ml, proteolysis was absent. These results suggest that IGFs are released more readily from pregnancy serum, accounting for the weaker inhibitory effect of low rhIGFBP-3 concentrations. For identical IGF concentrations, pregnancy serum's greater biological activity therefore reflects greater IGF availability to the cells. This study demonstrates the functional consequences at cellular level of serum IGFBP-3 proteolysis, underlining its significance in regulating serum IGF bioavailability.

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)

IGF binding protein-3 secreted by the prostate adenocarcinoma cells (PC-3): differential effect on PC-3 and normal prostate cell growth.

Deregulation of growth in malignant cells has been suggested to be the result of increased secretion by these cells, of autocrine growth factors; alternatively, this deregulation has been assumed to be related to loss of sensitivity by malignant cells to secreted inhibitory molecules. The results of the present publication lend new support to both hypotheses. We recently showed that human prostate adenocarcinoma cells (PC-3 cells) secreted insulin-like growth factor binding proteins (IGFBP) of 45, 34 and 25 kDa. From medium conditioned by dense cultures of PC-3 cells, we have now purified two IGFBPs of M(r) 45 kDa and 34 kDa. The N-amino terminal sequences were determined, and it was shown that they are IGFBP-3. IGFBP-34 appeared to be a deglycosylated form of IGFBP-45. The two IGFBPs had more affinity for IGF-II than for IGF-I. IGFBP-45 and IGFBP-34 were growth-inhibitory factors of chick embryo fibroblasts (CEF): they totally inhibited DNA synthesis stimulated by serum in CEF. Our results point to a clear difference between the effects of these IGFBPs upon growth of normal prostate cells and malignant PC-3 cells. At a concentration of 150 ng/ml, they inhibited growth of normal prostate cells even in the presence of 1 microgram/ml insulin. This suggests that such inhibition was not simply the result of a decrease by the IGFBP of stimulation induced by serum IGF or IGF secreted by the cells. At a concentration of 150 ng/ml, IGFBP did not modify the growth of PC-3 cells. In contrast, it stimulated growth of PC-3 cells when added at a concentration lower than 50 ng/ml (about 1 nM). Our results thus provide new insight concerning the regulation of growth in PC-3 cells.

Insulin-like growth factor binding protein (IGFBP-3), an inhibitor of serum growth factors other than IGF-I and -II.

Our results show that an insulin-like growth factor binding protein, IGFBP-3, purified from rat serum, is an inhibitor of chick embryo fibroblast (CEF) growth. It abolished DNA synthesis in CEF stimulated by IGF-I as well as by human serum. Rat IGFBP-3 and IDF45 (an inhibitory diffusible factor secreted by mouse cells) had the same activities, confirming that they have an intrinsic capacity to inhibit serum stimulation and may be considered as growth inhibitors. Our data show that inhibition by IGFBP-3 of serum stimulation was not simply the result of its inhibition of IGF present in the serum: 1) While anti-IGF-I IgG was able to completely inhibit stimulation induced by added IGF-I, it did not decrease stimulation induced by 1% human serum. Anti-IGF-II IgG inhibited the stimulation induced by added IGF-II, but only 25% decreased the stimulation induced by 0.7% serum. The percent inhibition was not significantly increased when the concentration of serum was decreased to 0.2%, which induced 140% stimulation of DNA synthesis; 2) stimulation by 0.2% serum was much more inhibited by IGFBP-3 than by IgG anti IGF-II; 3) after separation of IGF-I and IGF-II from serum by chromatography of acidified serum proteins on BioGel P150, the remaining serum proteins (with a molecular mass greater than 45 kDa) which were depleted in IGF-I and -II (verified by RIA determination) still stimulated DNA synthesis, and this stimulation was 80% inhibited by IGFBP-3.

Synthesis and secretion by mouse 3T3 cells of an inhibitor of cell growth (mIGFBP-3): correlation with cell proliferation.

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.

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.

An IGF binding protein is an inhibitor of FGF stimulation.

We purified to homogeneity a growth inhibiting diffusible factor (IDF45) secreted by dense cultures of mouse 3T3 cells and which was able to inhibit 100% of DNA synthesis stimulated by serum in chick embryo fibroblasts (CEF) (Blat et al., 1989a). We then demonstrated that this factor was an IGF-binding protein (Blat et al., 1989b). Indeed, its N-terminal amino acid sequence was homologous to that of rat IGFBP-3. Our present results show that basic fibroblast growth factor (bFGF) induced, respectively, a fivefold and threefold increase in DNA synthesis in mouse embryo fibroblasts (MEF) and CEF. IDF-45 inhibited the stimulation induced by bFGF by about 65%, while stimulation induced by insulin, PDGF, or EGF was only weakly or not at all inhibited by IDF45. When bFGF stimulation was determined in the presence of a high concentration of insulin in conditions which minimize the effect of endogenous IGF-I or -II, this stimulation was decreased by about 50% in the presence of IDF45. This result suggests that addition of bFGF stimulates IGF secretion, thereby resulting in partial loss of inhibition, by IDF45, of bFGF stimulation.

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.

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.

Differences in inhibition by IDF45 (an inhibitory diffusible factor) of early RNA synthesis stimulation induced by pp60 v-src and various mitogens.

Factors inhibiting cell growth have been isolated from different cell types. However, little information is available concerning their mode of action. A novel growth inhibitory factor of 45 kDa (IDF45) was recently purified to homogeneity from medium conditioned by 3T3 cells. This molecule was able to inhibit DNA synthesis and the growth of chick embryo fibroblasts (CEF) in a reversible manner. By contrast, DNA synthesis stimulated by v-src expression in CEF was poorly inhibited by IDF45. In order to gain further insight into the IDF45 mode of action in normal and transformed CEF, we compared the effects of IDF45 on early stimulation of RNA synthesis induced in CEF by different mitogenic factors and by v-src gene expression. Stimulation, by serum, of RNA synthesis was inhibited by IDF45; however, inhibition increased when cells were preincubated with IDF45 before addition of serum and cell labeling for 2 h. IDF45 was also able to inhibit partially the stimulation of RNA synthesis induced by PMA and PDGF but was unable to inhibit stimulation of RNA synthesis induced by insulin and v-src expression. By contrast, stimulation of RNA synthesis induced by IGF-I was rapidly 100% inhibited by IDF45. The effect of IDF45 on DNA synthesis stimulated by the different mitogens was also determined and was correlated with the effect of IDF45 on RNA synthesis. These results suggest that the modes of action of IDF45 on stimulation of RNA synthesis by v-src and by insulin are similar. Our present results agree with others showing the bifunctional activity of IDF45 as an IGF-binding protein and as an inhibitory molecule in DNA stimulation induced by serum.

Inhibitory diffusible factor 45 bifunctional activity. As a cell growth inhibitor and as an insulin-like growth factor I-binding protein.

From medium conditioned by 3T3 cells, we had previously purified to apparent homogeneity a novel inhibitory diffusible factor of 45 kDa (IDF45), and then determined the amino-terminal sequence. IDF45 prevented reversibly the growth of chick embryo fibroblast (CEF). In these cells, DNA synthesis stimulated by 1% serum was 50% inhibited in the presence of 45 ng/ml (1 nM) IDF45. In the present article, we show that, in CEF, DNA synthesis stimulated by IGF-I was 100% inhibited in the presence of purified IDF45. Furthermore, the 45-kDa protein (IDF45) was, after Western blotting, able to bind IGF-I. The inhibitory effect of IDF45 upon serum stimulation did not seem to be the result of its inhibitory activity upon IGF-I stimulation, since stimulation by IGF-I and serum were additive. Moreover, it was possible to dissociate the two inhibitory effects: when added to v-src transformed CEF, IDF45 was able to 100% inhibit stimulation induced by IGF-I and was unable to significantly decrease stimulation induced by serum, as was previously observed. Taken together, our results strongly suggest that IDF45 has two distinct functions, one of which was to bind IGF-I and the other to inhibit serum stimulation. Indeed, it was impossible to separate the two functions when IDF45 was purified by cation exchange fast protein liquid chromatography, a method very different from reverse-phase fast protein liquid chromatography previously used for purification to apparent homogeneity of IDF45. On the other hand, if the IGF binding activity and inhibitory activity effect upon serum stimulation were carried by two different proteins, the presence of IGF-I (in conditions where most of the 45-kDa proteins were bound to IGF-I) should not have affected the activity of the molecule inhibiting serum stimulation. However, we observed the contrary: when IDF45 was bound to IGF-I, it lost its inhibitory effect upon stimulation induced by serum. This suggests that the two activities occurred on the same protein and that IDF45 is a bifunctional protein.

Isolation and amino-terminal sequence of a novel cellular growth inhibitor (inhibitory diffusible factor 45) secreted by 3T3 fibroblasts.

A growth inhibitory protein named inhibitory diffusible factor 45 (IDF45) has been purified to homogeneity from medium conditioned by dense cultures of mouse 3T3 cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of fast protein liquid chromatography-purified bioactive material showed a single band corresponding to a protein of 45 kDa. The molecule is a monomer. The pI of the molecule, as determined by isoelectric focusing, is about 6.5. The amino-terminal sequence of the protein was established as Ser-Ala-Gly-Ala-Val-Gly-Ala-Gly-Pro-Val-Val-Arg. The protein inhibits DNA synthesis in chicken embryo fibroblasts in a dose-dependent manner (ED50: 40 ng/ml, approximately 1 nM). The chemical properties of IDF45, i.e. molecular weight and amino-terminal sequence, clearly distinguish it from other known growth inhibitory proteins.

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

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.

Growth inhibitory factor diffusing from chick embryo fibroblasts.

Density-dependent inhibition (DDI) of growth is assumed to be the result of diffusion in the medium of growth inhibitory molecules. In this work, we demonstrate the presence of inhibitory molecules (IDFc: chicken inhibitory diffusible factor) in the medium of chick embryo fibroblasts (CEF) cultures. IDFc partially purified by Bio-Gel P150 chromatography followed by reverse phase FPLC. The dose-response curve showed that 250 ng/ml IDFc inhibited 50% DNA synthesis. IDFc was also able to inhibit the growth of sparse cultures of CEF; this inhibition was reversible. IDFc was unable to prevent the DNA synthesis in cells transformed by v-src gene expression. These results suggest that IDFc is involved in the DDI of CEF growth.