Cycling in a crowd: Coordination of plant cell division,growth, and cell fate

The reiterative organogenesis that drives plant growth relies on the constant production of new cells, which remain encased by interconnected cell walls. For these reasons, plant morphogenesis strictly depends on the rate and orientation of both cell division and cell growth. Important progress has been made in recent years in understanding how cell cycle progression and the orientation of cell divisions are coordinated with cell and organ growth and with the acquisition of specialized cell fates. We review basic concepts and players in plant cell cycle and division, and then focus on their links to growth-related cues, such as metabolic state, cell size, cell geometry, and cell mechanics, and on how cell cycle progression and cell division are linked to specific cell fates. The retinoblastoma pathway has emerged as a major player in the coordination of the cell cycle with both growth and cell identity, while microtubule dynamics are central in the coordination of oriented cell divisions. Future challenges include clarifying feedbacks between growth and cell cycle progression, revealing the molecular basis of cell division orientation in response to mechanical and chemical signals, and probing the links between cell fate changes and chromatin dynamics during the cell cycle.

Plant cell cycle and division are linked to specific cell fates and respond to growth-related cues, such as metabolic state, cell size, cell shape, and mechanical stress.     

Two distinct mechanisms for inhibition of cell growth in human prostate carcinoma cells with antioxidant enzyme imbalance.

The purpose of the present study was to determine whether manganese superoxide dismutase (MnSOD) overexpression in DU145 human prostate carcinoma cells affected cell reduction-oxidation state (cell redox) and to correlate changes in cell redox status with cell cycle progression and plating efficiency. One MnSOD-overexpressing cell line had no change in other antioxidant enzymes (AEs) (nonadapted clone), whereas a second MnSOD-overexpressing cell line studied had an increase in catalase (CAT) activity (adapted clone). Correlation of biochemical studies with cell cycle studies suggested that heteroploidy observed in the nonadapted MnSOD-overexpressing cell line may be due to increased intracellular peroxides with resultant disruption of the microtubule network, while a decreased mitotic rate was associated with decreased ATP levels in mitosis. In contrast, the decrease in cell growth in the adapted cell line was demonstrated to be due to a decrease in plating efficiency. Our results demonstrate complex effects of AE imbalance on cell growth of DU145 prostate cancer cells.     

Hybrid cellular automaton modeling of nutrient modulated cell growth in tissue engineering constructs

Mathematic models help interpret experimental results and accelerate tissue engineering developments. We develop in this paper a hybrid cellular automata model that combines the differential nutrient transport equation to investigate the nutrient limited cell construct development for cartilage tissue engineering. Individual cell behaviors of migration, contact inhibition and cell collision, coupled with the cell proliferation regulated by oxygen concentration were carefully studied. Simplified two-dimensional simulations were performed. Using this model, we investigated the influence of cell migration speed on the overall cell growth within in vitro cell scaffolds. It was found that intense cell motility can enhance initial cell growth rates. However, since cell growth is also significantly modulated by the nutrient contents, intense cell motility with conventional uniform cell seeding method may lead to declined cell growth in the final time because concentrated cell population has been growing around the scaffold periphery to block the nutrient transport from outside culture media. Therefore, homogeneous cell seeding may not be a good way of gaining large and uniform cell densities for the final results. We then compared cell growth in scaffolds with various seeding modes, and proposed a seeding mode with cells initially residing in the middle area of the scaffold that may efficiently reduce the nutrient blockage and result in a better cell amount and uniform cell distribution for tissue engineering construct developments.     

Effects of nitrogen on mesophyll cell division and epidermal cell elongation in tall fescue leaf blades

Leaf elongation rate (LER) in grasses is dependent on epidermal cell supply (number) and on rate and duration of epidermal cell elongation. Nitrogen (N) fertilization increases LER. Longitudinal sections from two genotypes of tall fescue (Festuca arundinacea Schreb.), which differ by 50% in LER, were used to quantify the effects of N on the components of epidermal cell elongation and on mesophyll cell division. Rate and duration of epidermal cell elongation were determined by using a relationship between cell length and displacement velocity derived from the continuity equation. Rate of epidermal cell elongation was exponential. Relative rates of epidermal cell elongation increased by 9% with high N, even though high N increased LER by 89%. Duration of cell elongation was approximately 20 h longer in the high- than in the low-LER genotype regardless of N treatment. The percentage of mesophyll cells in division was greater in the high- than in the low-LER genotype. This increased with high N in both genotypes, indicating that LER increased with cell supply. Division of mesophyll cells adjacent to abaxial epidermal cells continued after epidermal cell division stopped, until epidermal cells had elongated to a mean length of 40 micrometers in the high-LER and a mean length of 50 micrometers in the low-LER genotype. The cell cycle length for mesophyll cells was calculated to be 12 to 13 hours. Nitrogen increased mesophyll cell number more than epidermal cell number: in both genotypes, the final number of mesophyll cells adjacent to each abaxial epidermal cell was 10 with low N and 14 with high N. A spatial model is used to describe three cell development processes relevant to leaf growth. It illustrates the overlap of mesophyll cell division and epidermal cell elongation, and the transition from epidermal cell elongation to secondary cell wall deposition.     

Three-Dimensional Numerical Model of Cell Morphology during Migration in Multi-Signaling Substrates

Cell Migration associated with cell shape changes are of central importance in many biological processes ranging from morphogenesis to metastatic cancer cells. Cell movement is a result of cyclic changes of cell morphology due to effective forces on cell body, leading to periodic fluctuations of the cell length and cell membrane area. It is well-known that the cell can be guided by different effective stimuli such as mechanotaxis, thermotaxis, chemotaxis and/or electrotaxis. Regulation of intracellular mechanics and cell’s physical interaction with its substrate rely on control of cell shape during cell migration. In this notion, it is essential to understand how each natural or external stimulus may affect the cell behavior. Therefore, a three-dimensional (3D) computational model is here developed to analyze a free mode of cell shape changes during migration in a multi-signaling micro-environment. This model is based on previous models that are presented by the same authors to study cell migration with a constant spherical cell shape in a multi-signaling substrates and mechanotaxis effect on cell morphology. Using the finite element discrete methodology, the cell is represented by a group of finite elements. The cell motion is modeled by equilibrium of effective forces on cell body such as traction, protrusion, electrostatic and drag forces, where the cell traction force is a function of the cell internal deformations. To study cell behavior in the presence of different stimuli, the model has been employed in different numerical cases. Our findings, which are qualitatively consistent with well-known related experimental observations, indicate that adding a new stimulus to the cell substrate pushes the cell to migrate more directionally in more elongated form towards the more effective stimuli. For instance, the presence of thermotaxis, chemotaxis and electrotaxis can further move the cell centroid towards the corresponding stimulus, respectively, diminishing the mechanotaxis effect. Besides, the stronger stimulus imposes a greater cell elongation and more cell membrane area. The present model not only provides new insights into cell morphology in a multi-signaling micro-environment but also enables us to investigate in more precise way the cell migration in the presence of different stimuli.     

Construction of a cultivation system of a yeast single cell in a cell chip microchamber

A novel single cell screening system was constructed using a yeast cell chip in combination with the yeast cell surface engineering [NanoBiotechnology 2005, 1, 105-111]. Enzymes or functional proteins displayed on a yeast cell surface can be used as a protein cluster. To achieve high-throughput screening of protein libraries on the cell surface, a catalytic reaction by a single cell-surface-engineered yeast cell was successfully carried out in the microchamber on the yeast cell chip. After screening, to replicate a target cell for use in measuring of activity, DNA sequencing, and preservation, a novel single cell cultivation system in the yeast cell chip was constructed. To avoid damage of the rapid dry up of medium in the microchamber array, the yeast cell chip was modified with a protection sheet, so that the modified chip was like a micro-culture tank constructed on the yeast cell chip microchamber. As a result, single yeast cell cultivation in the yeast cell chip microchamber was observed, and the modified yeast cell chip was evaluated to be good for a single cell selection. The improvement showed that the single cell screening system coupled with the single cell cultivation using the modified yeast cell chip may be superior to that by a cell sorter for the isolation of a target cell and its practical use.     

Cell motility in a new single-cell wound model

Summary  Until now researchers have used a monolayer of cultured cells to investigate cell motility toward an injured cell. However, we suspect that, when using this method, adjacent cells move to the free space due to relief of contact inhibition. The current study was designed to investigate the cell motility nearby an injured cell in varying cell connectivity. A lowpower laser beam was used to damage one cell selectively with the silver coating beads. After injury, we observed the cell motility in three different cell types: (1) those immediately adjacent to the injured cell, 92) those removed from the injured cell by interposition of another cell, and (3) those removed from the injured cell by free space. The cells that are in direct contact with the injured cell moved toward the injured cell within 1.5–3.0 h. Indirectly connected cells and cells with no contact, on the other hand, showed no significant movement toward the injured cell. This suggests that the cell motility toward the cell injury is not only due to relief of contact inhibition but might also be caused by cell-to-cell signaling via cell connection. The current method will provide a tool to create a cell injury without damaging adjacent cells.     

Effects of surface substances of untransformed fibroblastic cells on the adhesion and proliferation of neighboring cells: a study using fixed confluent cell sheets

To study the effects of surface materials of cells on the behavior of other neighboring cells in a crowded culture, confluent sheets of rat 3Y1 fibroblasts were fixed and then 3Y1 cells were seeded on to them. Among confluent sheets unfixed, fixed with formalin and fixed with ethanol and an empty plastic dish surface, the substrate activity to permit cell adhesion was compared. After confluent 3Y1 cells (mainly composed of cells with a G1-DNA content) were reseeded with fresh medium on to these substrates, the capacity to initiate DNA synthesis per attached cell was also compared. The substrate activity of the ethanol-fixed cell sheet to permit cell adhesion was as high as that of the empty dish surface, whereas that of the unfixed cell sheet and that of the formalin-fixed cell sheet were low. When the ethanol-fixed cell sheet and the empty dish surface were coated with the ethanol extract of the unfixed cell sheet, the substrate activity diminished, indicating that during the fixation process with ethanol an adhesion-inhibitory factor (s) was removed. The capacity to initiate DNA synthesis of each cell that had completed adhesion and spreading on the cell sheets unfixed, fixed with formalin, and fixed with ethanol was lower compared to the cell that had adhered to the empty dish surface. We conclude that factors over the 3Y1 cell surface inhibit the overlapping cell adhesion and the proliferation of cells contacting each other, resulting in the ordered cell configuration in the confluent culture.     

Increased G2 delay in radiation-resistant cells obtained by transformation of primary rat embryo cells with the oncogenes H-ras and v-myc

Cell cycle perturbation after irradiation was studied in five cell lines transfected with oncogenes. Two immortalized, radio-sensitive cell lines with D0s of 1.06 and 1.08 Gy were compared to three radioresistant cell lines with D0s of 1.68-2.17 Gy. The sensitive cell lines were transfected with the v-myc or c-myc oncogenes, the resistant cell lines with the v-myc plus H-ras oncogenes. Exponentially growing populations were exposed to 5, 10, or 15 Gy of orthovoltage radiation. The percentage of cells in each phase of the cell cycle was determined at various times after irradiation using flow cytometry. All cell lines underwent a dose-dependent arrest in G2 phase after irradiation, but the resistant cell lines underwent a significantly longer arrest in G2 phase after irradiation than did the sensitive cell lines. In conjunction with other results from our laboratories, we suggest that this difference in G2 arrest may be the basis for the increased resistance of cells transfected with oncogenes to irradiation.     

Properties of a nuclear protein marker of human myeloid cell differentiation

The Mr 55,000 nuclear antigen present in the human promyelocytic cell line HL-60 is a basic protein that is extracted from nuclei or chromatin by 0.35 M NaCl. The antigen is confined to the nucleus of the interphase HL-60 cell as judged by immunocytochemical localization but disperses throughout the cell during mitosis. The antigen was not detected in leukemic cell lines with blast cell properties or in cell lines representing other lineages. Additional cell lines (ML-1, ML-2, and U937) with myeloid cell characteristics similar to those of the HL-60 cells, which also differentiate in vitro, express the antigen. The presence of antigen in normal human myeloid cells in peripheral blood and bone marrow is consistent with its proposed role in nuclear events associated with normal human myeloid cell differentiation.     

Ontogeny of B lymphocyte function. XV. A role for thymus cells in the maturation of the capacity of a subpopulation of B cells to re-express surface immunoglobulin after treatment with anti-immunoglobulin

The maturation of the C57BL/6 B cell population to be able to re-express surface immunoglobulin (sIg) after its removal by treatment with rabbit antimouse Ig (RAMIg) was studied in a cell transfer system. It was found that thymus cells were required for the maturation of a subset of the B cell population to be able to re-express sIg. The B cell population of irradiated, thymectomized mice reconstituted with spleen cells from donors under 2 wk of age remained deficient in their ability to re-express sIg even after 4 wk residence in the cell transfer recipient. In contrast, if adult thymus cells were transferred together with the immature B cells, the B cell population matured to be able to re-express sIg after treatment with RAMIg. Approximately one-third of the B cell population appears to require thymus cells for this maturation. The maturation of the thymus cell population to be capable of mediating this maturation of the B cell population occurs in two steps: between 2 and 3 and between 3 and 4 wk of age. This timing corresponds to the age at which the B cell population of C57BL/6 mice normally acquires the capacity to re-express sIg, which we have previously shown to also occur in two steps. Thymus cells from 3-wk-old donors can mediate the first step in B cell maturation to be able to re-express sIg, but cannot mediate the second step in this maturation of the B cell population. Thymus cells from 4-wk-old donors can mediate both steps in the maturation of the B cell population. The results suggest that thymus cells are involved in regulating some aspects of B cell differentiation.     

Clonal expansion of adult rat hepatic stem cell lines by suppression of asymmetric cell kinetics (SACK)

Adult stem cells have potential use for several biomedical applications, including cell replacement therapy, gene therapy, and tissue engineering. However, such applications have been limited due to difficulties encountered in expanding functional adult stem cells. We have developed a new approach to the problem of adult stem cell expansion based on the suppression of asymmetric cell kinetics (SACK). We postulated that asymmetric cell kinetics, required for adult stem cell function, were a major barrier to their expansion in culture. As such, conversion of adult stem cells from asymmetric cell kinetics to symmetric cell kinetics would promote their exponential expansion and longterm propagation in culture. The purine nucleoside xanthosine (Xs), which promotes guanine ribonucleotide biosynthesis, can be used to reversibly convert cells from asymmetric cell kinetics to symmetric cell kinetics. We used Xs supplementation to derive clonal epithelial cell lines from adult rat liver that have properties of adult hepatic stem cells. The properties of two Xs-derived cell lines, Lig-8 and Lig-13, are described in detail and compared to properties of adult rat hepatic cell lines derived without Xs supplementation. The Xs-derived cell lines exhibit Xs-dependent asymmetric cell kinetics and Xs-dependent expression of mature hepatic differentiation markers. Interestingly, Lig-8 cells produce progeny with properties consistent with hepatocyte differentiation, while Lig-13 progeny cells have properties consistent with bile duct epithelium differentiation. A stable adult cholangiocyte stem cell line has not been previously described. Consistent with the principles of their derivation, the SACK-derived hepatic cell lines exhibit neither senescence nor tumorigenic properties, and their differentiation properties are stable after longterm culture. These characteristics of SACK-derived stem cell lines underscore asymmetric cell kinetics as an essential adult stem cell property with potential to be the basis for a general approach to expansion and propagation of diverse adult stem cells.     

Traction in smooth muscle cells varies with cell spreading

Changes in cell shape regulate cell growth, differentiation, and apoptosis. It has been suggested that the regulation of cell function by the cell shape is a result of the tension in the cytoskeleton and the distortion of the cell. Here we explore the association between cell-generated mechanical forces and the cell morphology. We hypothesized that the cell contractile force is associated with the degree of cell spreading, in particular with the cell length. We measured traction fields of single human airway smooth muscle cells plated on a polyacrylamide gel, in which fluorescent microbeads were embedded to serve as markers of gel deformation. The traction exerted by the cells at the cell-substrate interface was determined from the measured deformation of the gel. The traction was measured before and after treatment with the contractile agonist histamine, or the relaxing agonist isoproterenol. The relative increase in traction induced by histamine was negatively correlated with the baseline traction. On the contrary, the relative decrease in traction due to isoproterenol was independent of the baseline traction, but it was associated with cell shape: traction decreased more in elongated than in round cells. Maximum cell width, mean cell width, and projected area of the cell were the parameters most tightly coupled to both baseline and histamine-induced traction in this study. Wide and well-spread cells exerted larger traction than slim cells. These results suggest that cell contractility is controlled by cell spreading.     

Possible role of cell cycle-dependent morphology, geometry, and mechanical properties in tumor cell metastasis.

Studies that examine the shear- and abrasion-sensitivity of proliferating cells are important in order to understand the behavior of hybridoma cells in bioreactor culture and metastasizing cancer cells in the bloodstream. Little is known about the link between morphology, structure, and mechanical properties of a given cell line, especially with respect to variations throughout the cell cycle. In our experiments with GAP A3 hybridoma cells, distinct cell morphologies were identified and correlated with phases of the cell cycle by video microscopic observation of synchronized cells, and of individual cells that were followed throughout their cell cycle. Micropipet manipulation was used to measure the geometrical (cell volume) and mechanical (apparent cell viscosity) properties of single cells. As the cell cycle progressed at 37 degrees C, an increase in cell volume from 1400 microns 3 to 5700 microns 3 was accompanied by an increase in apparent cell viscosity from 430 poise to 12,000 poise, consistent with an accumulation of more cytoplasmic material in the "older" cells. Hybridomas are representative of the various leukemias derived from hemopoietic cells, and even though as a whole, they appeared to be rather shear-insensitive, the wide range of property values demonstrates that a given cell line cannot be characterized by a single value for any one property, and that properties must be related to the cell cycle when considering proliferating cells. It is interesting to see if distinct stages in the metastatic sequence of events might correlate with any of these physical features of the cell cycle, irrespective of cell type or cell line. For example, the cytokinetic doublet could represent a fragile structure that may fail and produce cell death under fluid-shear conditions that would not affect the cells at any other stage in the cell cycle. Identifying such cell cycle-dependent features in metastasizing cancer cells could lead to a better understanding of the metastatic process and to possible clinical treatments directed at making cells more shear- and abrasion-sensitive, and therefore, more likely to be killed by the natural hydrodynamic forces of the circulatory system.     

Action potential conduction between a ventricular cell model and an isolated ventricular cell.

We used the Luo and Rudy (LR) mathematical model of the guinea pig ventricular cell coupled to experimentally recorded guinea pig ventricular cells to investigate the effects of geometrical asymmetry on action potential propagation. The overall correspondence of the LR cell model with the recorded real cell action potentials was quite good, and the strength-duration curves for the real cells and for the LR model cell were in general correspondence. The experimental protocol allowed us to modify the effective size of either the simulation model or the real cell. 1) When we normalized real cell size to LR model cell size, required conductance for propagation between model cell and real cell was greater than that found for conduction between two LR model cells (5.4 nS), with a greater disparity when we stimulated the LR model cell (8.3 +/- 0.6 nS) than when we stimulated the real cell (7.0 +/- 0.2 nS). 2) Electrical loading of the action potential waveform was greater for real cell than for LR model cell even when real cell size was normalized to be equal to that of LR model cell. 3) When the size of the follower cell was doubled, required conductance for propagation was dramatically increased; but this increase was greatest for conduction from real cell to LR model cell, less for conduction from LR model cell to real cell, and least for conduction from LR model cell to LR model cell. The introduction of this "model clamp" technique allows testing of proposed membrane models of cardiac cells in terms of their source-sink behavior under conditions of extreme coupling by examining the symmetry of conduction of a cell pair composed of a model cell and a real cardiac cell. We have focused our experimental work with this technique on situations of extreme uncoupling that can lead to conduction block. In addition, the analysis of the geometrical factors that determine success or failure of conduction is important in the understanding of the process of discontinuous conduction, which occurs in myocardial infarction.     

Cell cycle transition in early embryonic development of Xenopus laevis

This article reviews cell cycle changes that occur during midblastula transition (MBT) in Xenopus laevis based on research carried out in the authors' laboratory. Blastomeres dissociated from the animal cap of blastulae, as well as those in an intact embryo, divide synchronously with a constant cell cycle duration in vitro, up to the 12th cell cycle regardless of their cell sizes. During this synchronous cleavage, cell sizes of blastomeres become variable because of repeated unequal cleavage. After the 12th cell cycle blastomeres require contact with an appropriate protein substrate to continue cell division. When nucleocytoplasmic (N/C) ratios of blastomeres reach a critical value during the 13th cycle, their cell cycle durations lengthen in proportion to the reciprocal of cell surface areas, and cell divisions become asynchronous due to variations in cell sizes. The same changes occur in haploid blastomeres with a delay of one cell cycle. Thus, post-MBT cell cycle control becomes dependent not only on the N/C relation but also on cell surface activities of blastomeres. Unlike cell cycle durations of pre-MBT blastomeres, which show monomodal frequency distributions with a peak at about 30 min, those of post-MBT blastomeres show polymodal frequency distributions with peaks at multiples of about 30 min, suggesting 'quantisement' of the cell cycle. Thus, we hypothesised that MPF is produced periodically during its unit cycle with 30 min period, but it titrates, and is neutralized by, an inhibitor contained in the nucleus in a quantity proportional to the genome size; however, when all of the inhibitor has been titrated, excess MPF during the last cycle triggers mitosis. At MBT, cell cycle checkpoint mechanisms begin to operate. While the operation of S phase checkpoint to monitor DNA replication is initiated by N/C relation, the initiation of M phase checkpoint operation to monitor chromosome segregation at mitosis is regulated by an age-dependent mechanism.     

Rapid signaling to B cells by antigen-specific T cells requires CD18/CD54 interaction.

This study reports early B and T cell signaling events during cognate interactions between a human B cell line pulsed with peptide and an Ag-specific T cell clone. As has been previously reported, peptide in the context of the appropriate class II molecule stimulated a rise in intracellular calcium [Ca2+]i in the Ag-specific T cell clone. The activation of the T cell clone was associated with a reciprocal rise in [Ca2+]i in the B cells. Engagement of receptors on the B cell surface by the T cell also was associated with inositol phospholipid turnover comparable to that elicited by stimulation through sIg. Early signaling events in B cells can therefore be stimulated in cognate interactions with Ag-specific T cells, without the direct engagement of Ig receptors. A class II deficient B lymphoblastoid mutant, 6.1.6, which was incapable of presenting peptide to the T cell clone, could be stimulated to produce a rise in [Ca2+]i if the T cell clone was activated by monoclonal antibodies to CD3. Therefore, the interaction of class II molecules on the B cell with the TCR and/or the CD4 accessory molecule was not essential for T-dependent B cell activation. However, T-dependent signalling of B cells was profoundly inhibited by mAb to CD18 (beta-chain of LFA-1) on the T cell or CD54 (ICAM-1) on the B cell, demonstrating the importance of this pair of adhesion molecules in early T-B cell interactions.     

Digital Holographic Microscopy for Non-Invasive Monitoring of Cell Cycle Arrest in L929 Cells

Digital holographic microscopy (DHM) has emerged as a powerful non-invasive tool for cell analysis. It has the capacity to analyse multiple parameters simultaneously, such as cell- number, confluence and phase volume. This is done while cells are still adhered and growing in their culture flask. The aim of this study was to investigate whether DHM was able to monitor drug-induced cell cycle arrest in cultured cells and thus provide a non-disruptive alternative to flow cytometry. DHM parameters from G1 and G2/M cell cycle arrested L929 mouse fibroblast cells were collected. Cell cycle arrest was verified with flow cytometry. This study shows that DHM is able to monitor phase volume changes corresponding to either a G1 or G2/M cell cycle arrest. G1-phase arrest with staurosporine correlated with a decrease in the average cell phase volume and G2/M-phase arrest with colcemid and etoposide correlated with an increase in the average cell phase volume. Importantly, DHM analysis of average cell phase volume was of comparable accuracy to flow cytometric measurement of cell cycle phase distribution as recorded following dose-dependent treatment with etoposide. Average cell phase volume changes in response to treatment with cell cycle arresting compounds could therefore be used as a DHM marker for monitoring cell cycle arrest in cultured mammalian cells.     

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.