Effects of phthalate esters on actin cytoskeleton of Py1a rat osteoblasts

We evaluated, by confocal laser scanning microscopy, the actin cytoskeleton of immortalized rat Py1a osteoblasts treated with phthalate esters (butyl benzyl phthalate, BBP and dibutyl phthalate, DBP), endocrine disruptors with estrogenic activity. We observed some peculiar modifications of actin cytoskeleton and cells changing from a spindle shape to a rounded form. In particular, F-actin formed thick bundles around the cell membrane but only a weak labeling was observed in rounded cells. Also influence on apoptosis and short-term effects on FGF-2 were studied. It was found that BBP and DBP exert their action in a similar way, act in a transient manner and do not induce apoptosis.     

Effects of three phthalate esters on the life-table demography of freshwater rotifer <Emphasis Type="Italic">Brachionus calyciflorus</Emphasis> Pallas

Phthalate esters (PAEs) are mainly used in the polymer industry as external plasticizers in PVC, and tend to migrate slowly out of the plastic, either into the air by volatilization or into water or other solvents by dissolution. Di-n-butyl phthalate (DBP), butyl benzyl phthalate (BBP) and di-2-ethylhexyl phthalate (DEHP) are three members of PAEs, identified as priority controlled hazardous substances by the United States Environmental Protection Agency, and have been shown to have potential for endocrine disrupting effects on vertebrates and humans. The effects of DBP, BBP and DEHP on survival and reproduction of the freshwater rotifer Brachionus calyciflorus were studied using life-table demographic methods. The results showed that all the life-table demographic parameters of B. calyciflorus were markedly affected by DBP and BBP, but not by DEHP. The net reproductive rate representing the output of reproduction was more affected than all the other parameters representing population growth, development or survival of the rotifers. Compared to the solvent control, DBP and BBP, both at 500 μg l⁻¹, significantly increased the net reproductive rate, and prolonged the generation time and the life expectancy at hatching of the rotifers. DBP at 50 μg l⁻¹ markedly decreased the intrinsic rate of population increase of the rotifers, but the reverse was true for BBP at 50 and 500 μg l⁻¹. Among all the parameters, the intrinsic rate of population increase was the most sensitive to DBP and BBP. The levels of PAEs in water from all the studied rivers and lakes in the world did not affect the population growth of rotifers.     

Nuclear localization of basic fibroblast growth factor is mediated by heparan sulfate proteoglycans through protein kinase C signaling

Understanding the process of wound healing will provide valuable insight for the development of new strategies to treat diseases associated with improper regeneration, such as blindness induced by corneal scarring. Heparan sulfate proteoglycans (HSPG) are not normally expressed in the corneal stroma, but their presence at sites of injury suggests their involvement in the wound healing response. Primary cultured corneal stromal fibroblasts constitutively express HSPG and represent an injured phenotype. Recently, nuclear localization of HSPG was shown to increase in corneal stromal fibroblasts plated on fibronectin (FN), an extracellular matrix protein whose appearance in the corneal stroma correlates with injury. One possible role for the nuclear localization of HSPG is to function as a shuttle for the nuclear transport of heparin-binding growth factors, such as basic fibroblast growth factor (FGF-2). Once in the nucleus, these growth factors might directly modulate cellular activities. To investigate this hypothesis, cells were treated with (125)I-labelled FGF-2 under various conditions and fractionated. Our results show that nuclear localization of FGF-2 was increased in cells plated on FN compared to those on collagen type I (CO). Interestingly, FGF-2-stimulated proliferation was increased in cells plated on FN compared to CO and this effect was absent in the presence of heparinase III. Furthermore, pre-treatment with heparinase III decreased nuclear FGF-2, and CHO cells defective in the ability to properly synthesize heparan sulfate chains showed reduced nuclear FGF-2 indicating that the heparan sulfate chains of HSPG are critical for this process. HSPG signaling, particularly through the cytoplasmic tails of syndecans, was investigated as a potential mechanism for the nuclear localization of FGF-2. Treatment with phorbol 12-myristate-13-acetate (PMA), under conditions that caused downregulation of protein kinase Calpha (PKCalpha), decreased nuclear FGF-2. Using pharmacological inhibitors of specific PKC isozymes, we elucidated a potential mode of regulation whereby PKCalpha mediates the nuclear localization of FGF-2 and PKCdelta inhibits it. Our studies suggest a novel mechanism in which FGF-2 translocates to the nucleus in response to injury.     

Nuclear activities of basic fibroblast growth factor: potentiation of low-serum growth mediated by natural or chimeric nuclear localization signals

Human basic fibroblast growth factor (FGF-2) occurs in four isoforms: a low molecular weight (LMW FGF-2, 18 kDa) and three high molecular weight (HMW FGF-2, 22, 22.5, and 24 kDa) forms. LMW FGF-2 is primarily cytoplasmic and functions in an autocrine manner, whereas HMW FGF-2s are nuclear and exert activities through an intracrine, perhaps nuclear, pathway. Selective overexpression of HMW FGF-2 forms in fibroblasts promotes growth in low serum, whereas overexpression of LMW FGF-2 does not. The HMW FGF-2 forms have two functional domains: an amino-terminal extension and a common 18-kDa amino acid sequence. To investigate the role of these regions in the intracrine signaling of HMW FGF-2, we produced stable transfectants of NIH 3T3 fibroblasts overexpressing either individual HMW FGF-2 forms or artificially nuclear-targeted LMW FGF-2. All of these forms of FGF-2 localize to the nucleus/nucleolus and induce growth in low serum. The nuclear forms of FGF-2 trigger a mitogenic stimulus under serum starvation conditions and do not specifically protect the cells from apoptosis. These data indicate the existence of a specific role for nuclear FGF-2 and suggest that LMW FGF-2 represents the biological messenger in both the autocrine/paracrine and intracrine FGF-2 pathways.     

Nuclear accumulation of fibroblast growth factor receptors is regulated by multiple signals in adrenal medullary cells.

In an effort to determine the localization of fibroblast growth factor (FGF) receptors (FGFR) that could mediate the intracellular action of FGF-2, we discovered the presence of high-affinity. FGF-2 binding sites in the nuclei of bovine adrenal medullary cells (BAMC). Western blot analysis demonstrated the presence of 103-, 118-, and 145-kDa forms of FGFR1 in nuclei isolated from BAMC. 125I-FGF-2 cross-linking to nuclear extracts followed by FGFR1 immunoprecipitation showed that FGFR1 can account for the nuclear FGF-2 binding sites. Nuclear FGFR1 has kinase activity and undergoes autophosphorylation. Immunocytochemistry with the use of confocal and electron microscopes demonstrated the presence of FGFR1 within the nuclear interior. Nuclear subfractionation followed by Western blot or immunoelectron microscopic analysis showed that the nuclear FGFR1 is contained in the nuclear matrix and the nucleoplasm. Agents that induce translocation of endogenous FGF-2 to the nucleus (forskolin, carbachol, or angiotensin II) increased the intranuclear accumulation of FGFR1. This accumulation was accompanied by an overall increase in FGF-2-inducible tyrosine kinase activity. Our findings suggest a novel mode for growth factor action whereby growth factor receptors translocate to the nucleus in parallel with their ligand and act as direct mediators of nuclear responses to cell stimulation.     

Endocrine Disruption: Computational Perspectives on Human Sex Hormone-Binding Globulin and Phthalate Plasticizers

Phthalates are a class of high volume production chemicals used as plasticizers for household and industrial use. Several members of this chemical family have endocrine disrupting activity. Owing to ubiquitous environmental distribution and exposure of human population at all stages of life, phthalate contamination is a continuous global public health problem. Clinical and experimental studies have indicated that several phthalates are associated with adverse effects on development and function of human and animal systems especially the reproductive system and exposures during pregnancy and early childhood are by far of utmost concern. Sex hormone-binding globulin (SHBG) is a plasma carrier protein that binds androgens and estrogens and represents a potential target for phthalate endocrine disruptor function in the body. In the present study, the binding mechanism of the nine phthalates i.e. DMP, DBP, DIBP, BBP, DNHP, DEHP, DNOP, DINP, DIDP with human SHBG was delineated by molecular docking simulation. Docking complexes of the nine phthalates displayed interactions with 15–31 amino acid residues of SHBG and a commonality of 55–95% interacting residues between natural ligand of SHBG, dihydrotestosterone, and the nine phthalate compounds was observed. The binding affinity values were more negative for long chain phthalates DEHP, DNOP, DINP, and DIDP compared to short chain phthalates such as DMP and DBP. The Dock score and Glide score values were also higher for long chain phthalates compared to short chain phthalates. Hence, overlapping of interacting amino acid residues between phthalate compounds and natural ligand, dihydrotestosterone, suggested potential disrupting activity of phthalates in the endocrine homeostasis function of SHBG, with long chain phthalates expected to be more potent than the short chain phthalates.     

Integrative nuclear FGFR1 signaling (INFS) pathway mediates activation of the tyrosine hydroxylase gene by angiotensin II,depolarization and protein kinase C

The integrative nuclear FGFR1 signaling (INFS) pathway functions in association with cellular growth, differentiation, and regulation of gene expression, and is activated by diverse extracellular signals. Here we show that stimulation of angiotensin II (AII) receptors, depolarization, or activation protein kinase C (PKC) or adenylate cyclase all lead to nuclear accumulation of fibroblast growth factor 2 (FGF-2) and FGFR1, association of FGFR1 with splicing factor-rich domains, and activation of the tyrosine hydroxylase (TH) gene promoter in bovine adrenal medullary cells (BAMC). The up-regulation of endogenous TH protein or a transfected TH promoter-luciferase construct by AII, veratridine, or PMA (but not by forskolin) is abolished by transfection with a dominant negative FGFR1TK-mutant which localizes to the nucleus and plasma membrane, but not by extracellularly acting FGFR1 antagonists suramin and inositolhexakisphosphate (IP6). Mechanism of TH gene activation by FGF-2 and FGFR1 was further investigated in BAMC and human TE671 cultures. TH promoter was activated by co-transfected HMW FGF-2 (which is exclusively nuclear) but not by cytoplasmic FGF-1 or extracellular FGFs. Promoter transactivation by HMWFGF-2 was accompanied by an up-regulation of FGFR1 specifically in the cell nucleus and was prevented FGFR1(TK-) but not by IP6 or suramin. The TH promoter was also transactivated by co-transfected wild-type FGFR1, which localizes to both to the nucleus and the plasma membrane, and by an exclusively nuclear, soluble FGFR1(SP-/NLS) mutant with an inserted nuclear localization signal. Activation of the TH promoter by nuclear FGFR1 and FGF-2 was mediated through the cAMP-responsive element (CRE) and was associated with induction of CREB- and CBP/P-300-containing CRE complexes. We propose a new model for gene regulation in which nuclear FGFR1 acts as a mediator of CRE transactivation by AII, cell depolarization, and PKC.     

Growth factor regulation of cell growth and proliferation in the nervous system

This article discusses a novel intracrine mechanism of growth-factor action in the nervous system whereby fibroblast growth factor-2 (FGF-2) and its receptor accumulate in the cell nucleus and act as mediators in the control of cell growth and proliferation. In human and rat brain the levels and subcellular localization of FGF-2 differ between quiescent and reactive astrocytes. Quiescent cells express a low level of FGF-2, which is located predominantly within the cytoplasm. In reactive astrocytes, the expression of FGF-2 increases and the proteins are found in both the cytoplasm and nucleus. In glioma tumors, FGF-2 is overexpressed in the nuclei of neoplastic cells. Similar changes in FGF-2 expression and localization are found in vitro. The nuclear accumulation of FGF-2 reflects a transient activation of the FGF-2 gene by potentially novel transactivating factors interacting with an upstream regulatory promoter region. In parallel with FGF-2, the nuclei of astrocytes contain the high-affinity FGF-2 receptor, FGFR1. Nuclear FGFR1 is full length, retains kinase activity, and is localized within the nuclear interior in association with the nuclear matrix. Transfection of either FGF-2 or FGFR1 into cells that do not normally express these proteins results in their nuclear accumulation and concomitant increases in cell proliferation. A similar regulation of nuclear FGF-2 and FGFR1 is observed in neural crest-derived adrenal medullary cells and of FGF-2 in the nuclei of cerebellar neurons. Thus, the regulation of the nuclear content of FGF-2 and FGFR1 could serve as a novel mechanism controlling growth and proliferation of glial and neuronal cells.     

Ligand dependent and independent internalization and nuclear translocation of fibroblast growth factor (FGF) receptor 1

Basic fibroblast growth factor (FGF-2) is one of the prototype members of a rapidly expanding family of polypeptides. FGF-2 acts on cells via a dual-receptor system consisting of high-affinity tyrosine kinase receptors (FGFR) and low-affinity receptors comprised of heparan sulfate proteoglycans. Following ligand binding and subsequent internalization, both FGF-2 and FGFR1 are translocated to the nucleus where they have activities distinct from those expressed at the cell surface. Despite the growing number of growth factors and receptors shown to translocate to the nucleus, little is known about the mechanisms of internalization and translocation and how these processes are regulated. In the studies reported in this paper, we examined the roles of clathrin-dependent and -independent endocytosis in the uptake of FGFR1 and one of its ligands, FGF-2. While the uptake of FGF-2 occurred at least partly by a caveolar-dependent mechanism, that of FGFR1 was independent of both caveolae and coated pits. Surprisingly, neither the uptake of FGF-2 nor FGFR1 required the activity of the receptor tyrosine kinase. In addition, we identified a cell cycle-dependent pathway of FGFR1 nuclear translocation that appears to be independent of ligand binding.     

Syndecan-1 and FGF-2, but Not FGF Receptor-1, Share a Common Transport Route and Co-Localize with Heparanase in the Nuclei of Mesenchymal Tumor Cells

Syndecan-1 forms complexes with growth factors and their cognate receptors in the cell membrane. We have previously reported a tubulin-mediated translocation of syndecan-1 to the nucleus. The transport route and functional significance of nuclear syndecan-1 is still incompletely understood. Here we investigate the sub-cellular distribution of syndecan-1, FGF-2, FGFR-1 and heparanase in malignant mesenchymal tumor cells, and explore the possibility of their coordinated translocation to the nucleus. To elucidate a structural requirement for this nuclear transport, we have transfected cells with a syndecan-1/EGFP construct or with a short truncated version containing only the tubulin binding RMKKK sequence. The sub-cellular distribution of the EGFP fusion proteins was monitored by fluorescence microscopy. Our data indicate that syndecan-1, FGF-2 and heparanase co-localize in the nucleus, whereas FGFR-1 is enriched mainly in the perinuclear area. Overexpression of syndecan-1 results in increased nuclear accumulation of FGF-2, demonstrating the functional importance of syndecan-1 for this nuclear transport. Interestingly, exogenously added FGF-2 does not follow the route taken by endogenous FGF-2. Furthermore, we prove that the RMKKK sequence of syndecan-1 is necessary and sufficient for nuclear translocation, acting as a nuclear localization signal, and the Arginine residue is vital for this localization. We conclude that syndecan-1 and FGF-2, but not FGFR-1 share a common transport route and co-localize with heparanase in the nucleus, and this transport is mediated by the RMKKK motif in syndecan-1. Our study opens a new perspective in the proteoglycan field and provides more evidence of nuclear interactions of syndecan-1.     

Analysis of endogenous and exogenous nuclear translocation of fibroblast growth factor-1 in NIH 3T3 cells.

Nuclear localization of fibroblast growth factors (FGF) have been reported by many laboratories. We demonstrate here that FGF-1, the precursor for acidic FGF contains a putative nuclear translocation sequence (NTS) NYKKPKL, which is able to direct the expression of the bacterial beta galactosidase (beta gal) gene to the nucleus of transfected NIH 3T3 cells. However, this NTS is unable to target either FGF-1 itself or a FGF-1-beta gal fusion protein into the nucleus, suggesting that FGF-1 may contain an additional sequence which prevents endogenously expressed FGF-1 from being translocated into the nucleus. Indeed, when FGF-1 was fused to the NTS derived from the yeast histone 2B gene, the chimeric construct also failed to be transported into the nucleus either by itself or as a beta gal fusion protein. Interestingly, when 125I-FGF-1 was used to stimulate quiescent NIH 3T3 cells, a significant amount of internalized 125I-FGF-1 (approximately 10%) was found within the nucleus and the nuclear localization of FGF-1 through the exogenous pathway could be significantly reduced by suramin, an inhibitor of the interaction of FGF-1 with its receptor. These data suggest that while FGF-1 contains a NTS, nuclear translocation requires an exogenous and not an endogenous pathway.     

IL-6 promoter is modulated by the 24 kDa FGF-2 isoform fused to the hormone binding domain of the oestrogen receptor

Fusion proteins consisting of the 24 kDa nuclear form of basic fibroblast growth factor (FGF-2), associated with the hormone binding domain of oestrogen receptor (HBD), convey oestrogen inducibility to FGF-2. When stable HBD-FGF-2 HeLa cell lines were transiently transfected with an interleukin 6 (IL-6) construct, the IL-6 promoter activity was downregulated by the addition of oestradiol. Moreover, in these cell lines, the function of the FGF-2 nuclear localisation sequence was abolished by its fusion to HBD, while addition of oestradiol restored the location of the chimera to the nucleus.     

Uncoupling of cell proliferation and differentiation activities of basic fibroblast growth factor.

FGF-2 exerts its pleiotropic effects on cell growth and differentiation by interacting with specific cell surface receptors. In addition, exogenously added FGF-2 is translocated from outside the cell to the nucleus during G1-S transition. In this study, we show that a single point mutation in FGF-2 (substitution of residue serine 117 by alanine) is sufficient to drastically reduce its mitogenic activity without affecting its differentiation properties. The FGF-2(S117A) mutant binds to and activates tyrosine kinase receptors and induces MAPK and p70S6K activation as strongly as the wild-type FGF-2. We demonstrate that this mutant enters NIH3T3 cells, is translocated to the nucleus, and is phosphorylated similar to the wild-type growth factor. This suggests that FGF-2 mitogenic activity may require, in addition to signaling through cell surface receptors and nuclear translocation, activation of nuclear targets. We have previously shown that, in vitro, FGF-2 directly stimulates the activity of the casein kinase 2 (CK2), a ubiquitous serine/threonine kinase involved in the control of cell proliferation. We report that, in vivo, FGF-2(WT) transiently interacts with CK2 and stimulates its activity in the nucleus during G1-S transition in NIH3T3 cells. In contrast, the FGF-2(S117A) mutant fails to interact with CK2. Thus, our results show that FGF-2 mitogenic and differentiation activities can be dissociated by a single point mutation and that CK2 may be a new nuclear effector involved in FGF-2 mitogenic activity.-Bailly, K., Soulet, F., Leroy, D., Amalric, F., Bouche, G. Uncoupling of cell proliferation and differentiation activities of basic fibroblast growth factor (FGF-2).     

Nuclear and nucleolar localization of 18-kDa fibroblast growth factor-2 is controlled by C-terminal signals

Members of high (22-, 22.5-, 24-, and 34-kDa) and low (18-kDa) molecular mass forms of fibroblast growth factor-2 (FGF-2) regulate cell proliferation, differentiation, and migration. FGF-2s have been previously shown to accumulate in the nucleus and nucleolus. Although high molecular weight forms of FGF-2 contain at least one nuclear localization signal (NLS) in their N-terminal extension, the 18-kDa FGF-2 does not contain a standard NLS. To determine signals controlling the nuclear and subnuclear localization of the 18-kDa FGF-2, its full-length cDNA was fused to that of green fluorescent protein (GFP). The fusion protein was primarily localized to the nucleus of COS-7 and HeLa cells and accumulated in the nucleolus. The subcellular distribution was confirmed using wild type FGF-2 and FGF-2 tagged with a FLAG epitope. A 17-amino acid sequence containing two groups of basic amino acid residues separated by eight amino acid residues directed GFP and a GFP dimer into the nucleus. We systematically mutated the basic amino acid residues in this nonclassical NLS and determined the effect on nuclear and nucleolar accumulation of 18-kDa FGF-2. Lys(119) and Arg(129) are the key amino acid residues in both nuclear and nucleolar localization, whereas Lys(128) regulates only nucleolar localization of 18-kDa FGF-2. Together, these results demonstrate that the 18-kDa FGF-2 harbors a C-terminal nonclassical bipartite NLS, a portion of which also regulates its nucleolar localization.     

Metabolism of butyl benzyl phthalate by Gordonia sp. strain MTCC 4818

The microbial degradative characteristics of butyl benzyl phthalate (BBP) were investigated by the Gordonia sp. strain MTCC 4818 isolated from creosote-contaminated soil. The test organism can utilize a number of phthalate esters as sole sources of carbon and energy, where BBP was totally degraded within 4 days under shake culture conditions. High performance liquid chromatography profile of the metabolites isolated from spent culture indicated the accumulation of two major products apart from phthalic acid (PA), which were characterized by gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy as mono-n-butyl phthalate (MBuP) and monobenzyl phthalate (MBzP). Neither of the metabolites, MBuP, MBzP or PA, supported growth of the test organism, while in resting cell transformation, the monoesters were hydrolyzed to PA to a very minor extent, which was found to be a dead-end product in the degradation process. On the other hand, the test organism grew well on benzyl alcohol and butanol, the hydrolyzed products of BBP. The esterase(s) was found to be inducible in nature and can hydrolyze in vitro the seven different phthalate diesters tested to their corresponding monoesters irrespective of their support to the growth of the test organism.     

Urinary concentration of endocrine-disrupting phthalates and breast cancer risk in Indian women: A case-control study with a focus on mutations in phthalate-responsive genes

BackgroundPhthalates are known endocrine-disrupting chemicals used indiscriminately as constituents in consumer products including food processing, and packaging, cosmetics, personal care and household items. Although, few studies have assessed the risk of breast cancer on exposure to phthalates, their association with breast cancer risk in Indian women have not yet been evaluated.MethodsWe conducted a case-control study involving 171 participants. Urinary concentrations of six phthalate dieters; DMP (Dimethyl phthalate), DEP (Diethyl phthalate), DBP (Dibutyl phthalate), BBP (benzyl butyl phthalate), DEHP (Di-2-ethyl-hexyl phthalate), DINOP (Di-n-octyl phthalate) were estimated by GC-MS and geometric means were calculated. Univariate and multivariable logistic regression was performed to assess breast cancer risk on exposure to phthalates. Genes responsive to phthalates were identified through literature search and matched with NGS data, and gene-enrichment analysis was performed.ResultsSignificant associations were observed between urinary phthalate concentrations and increased risk of breast cancer for di-butyl phthalate (OR=1.5, 95% CI; 1.06, 2.11, p = 0.002) and di-2-ethyl-hexyl phthalate (>median vs ≤ median; OR=2.97, 95% CI; 1.18, 7.47, p = 0.005) in multivariable analyses. We also found several phthalate-responsive gene mutations in paired breast tumor tissues, which include PTPRD (76.19%), AR (42.86%), CYP1A1 (42.86%), CYP19A1 (23.81%), AHRR (19.05%), PIK3CA (19.05%), CYP1B1 (9.52%), RB1 (9.52%) and MMP9 (9.52%). Gene-enrichment analysis revealed that these genes form a major part of ER/PR, PPAR and HIF-1α-TGF-β signaling cascades involved in breast cancerConclusionAlthough the sample size is small, in this first case-control study from India, DBP and DEHP were found to be associated with increased risk of invasive breast cancer and tumor tissues revealed mutations in several phthalate-responsive genes. It is, therefore suggested that human biomonitoring in India and larger studies evaluating the early life genetic and epigenetic alterations on phthalates exposure are required to establish their role in breast carcinogenesis.     

Elevated fibroblast growth factor-2 increases tumor necrosis factor-alpha induced endothelial cell death in high glucose

Glucose and tumor necrosis factor-alpha (TNFalpha) concentrations are elevated in diabetes. Both of these factors correlate with diabetic vasculopathy and endothelial cell apoptosis, yet their combined effects have not been measured. We have previously shown that the angiogenic growth factor fibroblast growth factor-2 (FGF-2), which is generally protective against endothelial cell death, is similarly elevated in high glucose conditions. We therefore investigated the effect of TNFalpha on endothelial cell death under normal and elevated glucose conditions, with a particular focus on FGF-2. Porcine aortic endothelial cells were cultured in 5 and 30 mM glucose and stimulated with TNFalpha, together with FGF-2 or a neutralizing FGF-2 antibody. Cell death was measured via cell counts or an annexin apoptotic assay, and cell cycle phase was determined by propidium iodide labeling. TNFalpha-induced endothelial cell death increased for cells in high glucose, and cell death was enhanced with increasing FGF-2 exposure and negated by a neutralizing FGF-2 antibody. Endothelial cells were most susceptible to TNFalpha-induced cell death when stimulated with FGF-2 18 h prior to TNFalpha, corresponding to cell entry into S phase of the proliferative cycle. The FGF-2 associated increase in TNFalpha-induced cell death was negated by blocking cell entry into S phase. Endothelial cell release of FGF-2 in high glucose leads to cell cycle progression, which makes cells more susceptible to TNFalpha-induced cell death. These data suggest that growth factor outcomes in high glucose depend on secondary mediators such as cytokines and stimulation cell cycle timing.