Ascorbate potentiates serum-induced S phase entry of quiescent 3T3 cells

Summary Arrested BALB/c 3T3 cells were induced to the G₀-G₁ transition by fetal calf serum (FCS) and S phase entry was measured by [³H]thymidine incorporation as an index of DNA synthesis. [³H]Thymidine uptake was proportional to FCS concentration. Ascorbate (ASC) itself was unable to increase DNA synthesis in these cells but potentiated it in the presence of both 1% and 10% FCS. [³H]Thymidine uptake profile was similar with and without ASC, and showing at 24 h an ASC stimulation of 69% in the presence of 1% FCS and 58% with 10% FCS. These data are discussed in reference to the participation of ASC on plasma membrane energization for membrane translocations and transport.Abbreviations ASC ascorbate - FCS fetal calf serum     

Manipulation of medium conditions and differentiation in the rat myogenic cell line L6

Myoblasts of the L6 rat cell line were grown in Ham's F12 nutrient medium containing 10% fetal calf serum (F12 + FCS). Although the cells were confluent by 6 days in culture, fusion was not observed even if cultures were maintained for 10–14 days. At least 80% of the cells in such confluent unfused cultures were in the G₁ phase of the cell cycle and less than 5% of the cells in confluent cultures synthesized DNA during a 4-day period. The synthesis of muscle-specific proteins (α-actin, β-tropomyosin, and myosin light chains LC1_emb and LC2_F) was negligible when compared to fused cultures of L6 cells grown for a similar time in Dulbecco's medium with 10% FCS (DME + FCS). When the unfused cultures were shifted from F12 + FCS to DME + FCS, DNA synthesis could be demonstrated in more than 95% of the cells and fusion occurred, indicating that neither proliferative nor myogenic capacity had been irreversibly lost. Raising the levels of calcium, varying the serum concentration from 0 to 20%, or the addition of medium components (present in DME but reduced or absent in F12) all failed to induce fusion in the L6 cells grown in F12. However, L6 cells will fuse in mixtures of F12 + FCS and DME + FCS. Fusion will also occur if L6 cells are grown at clonal density in F12 + FCS supplemented with calcium. While it has not been possible to determine why F12 + FCS is nonpermissive for L6 cells in confluent mass cultures, the results demonstrate that prolonged residence in the G₁ phase of the cell cycle is not a sufficient condition for L6 myoblast differentiation to occur.     

Protein synthesis and secretion and RNA and DNA synthesis in human skin fibroblasts in a medium with a low serum content

Human skin fibroblasts, both postnatal and embryonic, were cultured in the stationary phase of growth for 6-10 days in the DMEM with bovine serum (BS), 0.1-0.5% fetal calf serum (FCS) or 1% human serum (HS). On the day 4 of culturing, a considerable increase was observed in the synthesis and secretion of protein by postnatal fibroblasts in the Eagle medium with 0.1-0.5% FCS, or with 0.5% BS, and in medium 199 with 0.1-0.5 BS, or with 0.1 FCS. Maximum synthesis and secretion of 14C-proline labeled protein was observed on day 2 of culturing of cells in the DMEM medium with 1% HS. In the DMEM medium with low serum content, protein synthesis being virtually unchanged, 75-80% of protein was secreted by cells into the culture medium with BS on days 2-4; in the medium with FCS such a high secretion of protein was observed only on day 4. High synthesis of protein by fetal fibroblasts in the DMEM medium with 0.1% BS and high protein secretion in all the media with 0.1% BS or 0.5% FCS were observed. The maximum level of secretion of protein by fibroblasts coincided with a considerable increase in both RNA and DNA syntheses. The data obtained suggest that cells in deep resting state actively react to the composition of the medium as well as to the quality and quantity of the serum. It may also be suggested that the mechanism of protein secretion has an important role in maintenance of the constant level of intracellular proteins in resting cells.     

Differential response of cultured rabbit articular chondrocytes (RAC) to transforming growth factor beta (TGF-beta)-evidence for a role of serum factors

We show that addition of TGF-beta (0.01-10 ng/ml) to proliferating rabbit articular chondrocytes in presence of low level of fetal calf serum (FCS, 2%) results in a sustained decrease of cell number and DNA synthesis up to 72 h. In contrast, incubation with high serum concentration (10% FCS) induces a transient increase of cell number after 48 h without elevation of DNA synthesis. Moreover, when the factor is added in 10% FCS-containing medium, a differential effect is observed at 48 h (either increase or decrease of cell number) depending on the serum level (2 or 10%) present between 24 and 48 h. Recruitment of cells in late S-phase occurred under TGF-beta-treatment in both 2 and 10% FCS. These arrested cells may then be released by further exposure to 10% FCS-containing medium. The data show that factor(s) from the serum modulate(s) the action of TGF-beta on chondrocyte proliferation. Addition of epidermal growth factor (EGF) to the cultures in presence of 2% FCS mimicks the effects observed with 10% serum, suggesting that the serum component(s) involved in the mechanism could be of EGF type.     

Uterine epithelial cells in primary culture: a model system to study cell proliferation

Guinea-pig uterine glandular epithelial cells were grown in primary culture. During the 4-day initial culture period, a 6.7 fold increase in DNA synthesis and a doubling time of approximately 30 hours were observed. Then the cells were submitted to serum depletion (60 hours) and the quiescent cells were stimulated with 15% fetal calf serum (FCS). The control cells were submitted to 1% heated and dextran-coated charcoal stripped FCS. In stimulated cells, the DNA synthesis increased and peaked between the 12th and 24th hour. In these cells, c-fos mRNAs increased rapidly, within 30 min., peaked at 75 min. (ratio to the control = 2.5), and returned to basal level within 90 min. These results prove that uterine epithelial cells in primary culture are able to respond to unspecific mitogen by both rapid expression of c-fos gene and DNA synthesis, suggesting that this cell culture system will be useful in studying the growth regulation in endometrium.     

Induction of (2'-5')oligoadenylate synthetase in serum-starved HeLa S3 cells by growth factors and its role in growth regulation

When serum-starved HeLa S3 cells were stimulated to proliferate by addition of fetal calf serum (FCS), (2'-5')oligoadenylate synthetase (2-5A synthetase) activity was induced. Although no interferon (IFN) activity was detectable in the HeLa S3 cell-conditioned culture medium after growth stimulation, addition of anti-IFN-beta monoclonal antibody inhibited both the expression of the 2-5A synthetase gene and the production of the enzyme, suggesting that endogenous IFN-beta was involved in 2-5A synthetase induction. Purified preparations of three growth factors, epidermal growth factor, platelet-derived growth factor, and insulin, also induced 2-5A synthetase through IFN-beta. When serum-starved HeLa S3 cells were treated with FCS, DNA synthesis was initiated synchronously, with peaks after 12 and 32 h, although the level of 2-5A synthetase reached a maximum after the first peak of DNA synthesis. Inhibition of 2-5A synthetase induction by anti-IFN-beta antibody enhanced the second, but not the first cycle of DNA synthesis. These results suggested that in HeLa S3 cells, after stimulation with growth factors the IFN/2-5A synthetase system played a role in cell growth negative regulatory mechanisms.     

Adeno-associated virus DNA replication complexes in herpes simplex virus or adenovirus-infected cells.

A complex which is active in in vitro synthesis of adeno-associated virus (AAV) DNA was solubilized from Vero cells that were co-infected with AAV and either adenovirus (Ad5) or a herpes simplex virus type 1 (HSV-1) as the helper virus. The complexes from the Ad5 and HSV-1-infected cells sedimented at 23 S and 28 S, respectively. The optimal conditions for in vitro DNA synthesis for the two types of complex using the endogenous AAV template and the endogenous DNA polymerase, differed with respect to the effect of KCl and K2SO4 concentration. In addition the complex from HSV-1-infected cells, but not that from Ad5-infected cells, was inhibited by phosphonoacetic acid. Thus, the two complexes appear to contain different DNA polymerase activities. This was verified by phosphocellulose chromatography of the DNA polymerases solubilized from the isolated complexes. The major activity in the complex from HSV-1 infected cells was the HSV-induced DNA polymerase with lesser amounts of cellular DNA polymerase alpha and gamma or both. The complex from the Ad5-infected cells contained mainly a cellular DNA polymerase gamma.     

Microenvironment of endosomal aqueous phase investigated by the mobility of microparticles using fluorescence correlation spectroscopy

Temporal observation of the dynamic behavior of molecules in cells gives information about the physiological environment at the region of interest. Here we report the direct measurement of the mobility of rhodamine-labeled microparticles (14 and 35 nm in diameter) ingested in endosomes of cultured bovine aortic endothelial cells using fluorescence correlation spectroscopy (FCS). The fluctuation of fluorescent signals from microparticles were measured by FCS. Obtained autocorrelation functions (FAFs) were analyzed by the 2-D multicomponent model according to an evaluation procedure we newly developed. It was found that microparticles moved freely in endosomes with average diffusion coefficients of 4.3 x 10(-8) and 2.7 x 10(-8) cm2 s(-1) for 14 and 35 nm, which were 45% slower than in water. This result implies that the endosomal aqueous phase is homogeneous with the viscosity about 2.2 times of water. Our study also proposes the new use of FCS for investigation of the internal space of organelles.     

Human purified interleukin-1 inhibits DNA synthesis and cell growth of osteoblastic cell line (MC3T3-E1), but enhances alkaline phosphatase activity in the cells

We examined the effects of human purified interleukin-1 (IL-1) on DNA synthesis, cell growth, and alkaline phosphatase activity in the osteoblastic cell line MC3T3-E1 under both preconfluent and confluent culture conditions. Addition of IL-1 to the cells markedly inhibited their DNA synthesis and growth over the range 1-10 U/ml. Such significant inhibitory effects were observed in cells cultivated in 1 or 5% fetal calf serum (FCS)-containing alpha modification Eagle's medium (alpha-MEM), but not in alpha-MEM containing 10% FCS. In contrast, alkaline phosphatase activity was enhanced significantly by IL-1 in the cell line cultivated in 1% FCS-containing alpha-MEM. These results demonstrate that human purified IL-1 is effective in inducing the differentiation of osteoblastic cell MC3T3-E1.     

Growth and interferon-γ production in batch culture of CHO cells

The relationship between growth and interferon- (IFN-) production in the recombinant cell line CHO 320 was studied by varying the foetal calf serum (FCS) concentration. The specific growth rate varied with the initial FCS concentration in a manner which could be well fitted by the Monod model. TheK _s and _max values were found to be 0.771% (v/v) serum and 0.031 h^–1 respectively. The average specific IFN- production rates during the exponential phase increased with increasing FCS concentration. A good correlation between specific production rate and specific growth rate was found in all phases of the culture except the lag phase and it was clearly demonstrated that IFN- production was growth associated. Specific glucose and glutamine utilisation rates were inversely related to specific growth rates.     

In vitro lead-induced cell toxicity and cytoprotective activity of fetal calf serum in human fibroblasts

The underlying mechanisms by which lead ions produce their deleterious effects prior to the onset of clinical symptoms are incompletely understood. This study aimed to assess lead-induced cell toxicity mechanisms by focusing on the effects of the metal on cell growth, DNA synthesis, cellular ATP, intracellular hexosaminidase activity and lysosomal function, and examine the possible cytoprotective role of fetal calf serum (FCS). Several human dermal cultured fibroblast lines were exposed to Pb (400 M) for 1–6 days with 2, 5, and 10% FCS. The earliest toxic effect of Pb was significant inhibition of DNA synthesis after 24 h direct exposure; this harmful effect was not progressive during the first 3 days, but worsened clearly on the 4th day regardless of the FCS concentration. A time-dependent depletion of intracellular ATP content was also caused by ionic lead, thereby compromising the cell energy charge which precedes cell death. Fibroblast growth was progressively and significantly inhibited from day 2 onwards; the greatest noxious effect was observed in the presence of 2% FCS: 49% reduction in cell proliferation after 5 days. Lead salts produced loss of cell adhesion to the culture dish which worsened from the 2nd day and was more pronounced when FCS in growth medium was decreased. Toxic actions on lysosomal membrane integrity provoked a decrease in neutral red uptake (NRU) which was exposure time-dependent and more marked with 2% FCS. In contrast, increased relative NRU (to 20% at 4 days), suggestive of endocytosis-induced lysosome enlargement, was observed in Pb-exposed cells. Intracellular hexosaminidase activity was not negatively affected until 5 days after exposure to Pb salts. FCS had a significant cytoprotective effect on Pb-induced toxicity.     

Regulatory role of endogenous regucalcin in the enhancement of nuclear deoxyribonuleic acid synthesis with proliferation of cloned rat hepatoma cells (H4-II-E)

The role of endogenous regucalcin in the regulation of deoxyribonuleic acid (DNA) synthesis in the nuclei of the cloned rat hepatoma cells (H4-II-E) with proliferative cells was investigated. Cells were cultured for 6-96 h in a alpha-minimum essential medium (alpha-MEM) containing fetal bovine serum (FBS; 1 or 10%). Cell number was significantly increased between 24 and 96 h after culture with 10% FBS; cell proliferation was markedly stimulated by culture with 10% FBS as compared with that of 1% FBS. In vitro DNA synthesis activity in the nuclei of cells was significantly elevated 6 h after culture with 10% FBS and its elevation was remarkable at 12 and 24 h after the culture. Nuclear DNA synthesis activity was significantly reduced in the presence of various protein kinase inhibitors (PD98059, staurosprine, or trifluoperazine) in the reaction mixture containing the nuclei of cells cultured for 12 and 24 h with FBS (1 and 10%). The addition of regucalcin (10(-7) and 10(-6)M) in the reaction mixture caused a significant inhibition of nuclear DNA synthesis activity. The presence of anti-regucalcin monoclonal antibody (25-100 ng/ml) in the reaction mixture containing the nuclei of cells cultured for 24 h with 10% FBS resulted in a significant increase in nuclear DNA synthesis activity. This increase was completely blocked by the addition of regucalcin (10(-6) M). The effect of anti-regucalcin antibody (100 ng/ml) in increasing nuclear DNA synthesis activity was significantly inhibited in the presence of various protein kinase inhibitors. DNA synthesis activity was significantly enhanced in the presence of anti-regucalcin antibody (100 ng/ml) in the reaction mixture containing the nuclei of cells cultured for 24 h with 10% FBS in the presence of Bay K 8644 (2.5 x 10(-6) M). Culture with Bay K 8644 did not cause a significant increase in DNA synthesis activity in the absence of anti-regucalcin antibody. The present study demonstrates that endogenous regucalcin plays a suppressive role in the enhancement of nuclear DNA synthesis with proliferative cells.     

The mitogenic effect of 15- and 12-hydroxyeicosatetraenoic acid on endothelial cells may be mediated via diacylglycerol kinase inhibition

15-Hydroxyeicosatetraenoic acid (15-HETE), a major lipoxygenase metabolite of arachidonic acid in fetal bovine aortic endothelial cells, was a mitogen for these cells, stimulating both cell proliferation and DNA synthesis in the presence of serum and serum-deprived cells. In [14C]arachidonic acid-labeled confluent endothelial cell monolayers, 15-HETE (30 microM) caused an elevation of [14C]diacylglycerol (DAG) with a concomitant decrease in cellular [14C]phosphatidylinositol (PI) in both unstimulated and stimulated cells. 1-Oleoyl-2-acetylglycerol, a synthetic DAG analog, stimulated endothelial cell DNA synthesis in a concentration-dependent manner. In [3H]inositol-labeled cells, 15-HETE also caused a decrease in cellular PI content under both basal and stimulated conditions. 15-HETE, however, had no effect on either isolated phospholipase C activity or phosphoinositide turnover in lithium chloride-treated cells. In intact cells, 15-HETE (30 microM) inhibited the synthesis of [3H]PI from [3H]inositol (80% inhibition, p less than 0.001). In human red cell membranes, the production of phosphatidic acid from endogenous DAG was inhibited by 15-HETE in a concentration-dependent manner with an IC50 of 41 microM. Although 12-HETE had effects similar to those of 15-HETE, the parent compound arachidonic acid did not affect DNA synthesis or DAG kinase activity. Our study thus demonstrates that the mitogenic activity of 15- and 12-HETE on endothelial cells may be mediated via DAG kinase inhibition with the concomitant accumulation of cellular DAG.     

Prostaglandin D2 lowers nuclear DNA polymerase activity in cultured mastocytoma cells

Prostaglandin D2 strongly inhibited growth of cultured mastocytoma P-815, 2-E-6 cells, which were established and cloned from mouse mast tumor cells. The inhibition was dose-dependent (IC50=2.09 x 10-5 M). Prostaglandin D2 also inhibited the DNA synthesizing activity of the cells dose-dependently. We next measured the activities of endogenous DNA polymerases extracted from untreated and prostaglandin D2-treated cells. Prostaglandin D2-treated cells were the same suggesting there was no gross change in the size of the enzyme. Prostaglandin D2 pretreatment of the cells reduced endogenous DNA polymerase beta activity to 68% of the control value; the sedimentation coefficients of the enzymes from treated and untreated cells were both 3.5 S. Interestingly, prostaglandin D2 had no direct inhibitory effect on the activity of either DNA polymerase alpha or beta. Our results indicate that the activities of DNA polymerase alpha and beta are lower in prostaglandin D2-treated mastocytoma cells. This finding accounts for the lower level of DNA synthesis in these cells.     

Stimulation of heparan sulfate proteoglycan synthesis and secretion during G1 phase induced by growth factors and PMA

Fetal calf serum (FCS) and PMA (phorbol 12-myristate-13-acetate) specifically stimulate the synthesis of heparan sulfate proteoglycan in endothelial cells. Staurosporine and n-butanol, kinase inhibitors, abolish the PMA effect. Forskolin and 8-bromo adenosine 3′:5′-cyclic monophosphate, activators of, respectively, adenylate cyclase and protein kinase A cannot reproduce the PMA effect. The kinetics of cell entry into S phase of the endothelial cells was determined by DNA synthesis ([³H]-thymidine and Br-dU incorporation), and flow cytometry. The mitogenic effect of fetal calf serum is abolished by PMA. Also, PMA pre-treatment inhibits the enhanced synthesis of heparan sulfate proteoglycan after a second PMA exposure. Remarkably, the stimulation of heparan sulfate proteoglycan synthesis by fetal calf serum and PMA seems to be mainly restricted to G₁ phase. Therefore fetal calf serum and PMA cause an enhanced synthesis of heparan sulfate proteoglycan, and PMA causes a cell cycle block at G₁ phase. J. Cell. Biochem. 70:563–572, 1998. © 1998 Wiley-Liss, Inc.     

The effects of ionizing radiation on the pulmonary endothelial cell uptake of alpha-aminoisobutyric acid and synthesis of prostacyclin

The effects of gamma irradiation (150-3000 rad) on prostacyclin synthesis (PGI2) and Na+-dependent amino acid uptake (alpha-aminoisobutyric acid, AIB) were assessed in vitro in bovine pulmonary artery endothelial cells grown in plastic culture dishes. A dose-dependent increase in both PGI2 synthesis and AIB was found 24 h after irradiation at exposure levels greater than 600 rad. The increase in PGI2 synthesis [297% of sham-irradiated values at 3000 rad, P less than 0.01] was due to an increase in release of arachidonic acid from plasma membrane stores as well as stimulation of cyclooxygenase and/or prostacyclin synthetase enzymes. The increase in AIB uptake (75% increase at 3000 rad compared to sham-exposure values) correlated with the increased synthesis of PGI2 (r = 0.94). There was also a dose-dependent increase in the number of cells that became detached from the culture dishes during the 24-h period after irradiation. The changes in PGI2 synthesis and AIB uptake induced by gamma irradiation differed if the endothelial cells were grown on cover slips, indicating that the endothelial response to irradiation may be dependent on the interaction between the endothelial cell and its extracellular basement membrane matrix.     

IGF-I is a mitogen involved in differentiation-related gene expression in fetal rat brown adipocytes

Fetal rat brown adipocytes at time zero of culture constitute a population of cells of broad spectrum, as estimated by cell size, endogenous fluorescence and lipid content, and show an intrinsic mitogenic competence. They express constitutively early growth-related genes such as c-myc, c-fos, and beta-actin, tissue specific-genes such as the uncoupling protein (UCP) and the lipogenic marker malic enzyme (ME). Fetal brown adipocytes bear a high expression of insulin-like growth factor receptor (IGF-IR), and show a high affinity IGF-I specific-binding to its receptor, and a high number of binding sites per cell. After cell quiescence, insulin-like growth factor I (IGF-I) was as potent as 10% FCS in inducing DNA synthesis, cell number increase, and the entry of cells into the cell-cycle. In addition, IGF- I or 10% FCS for 48 h increased the percentage of [3H]thymidine-labeled nuclei as compared to quiescent cells. Single cell autoradiographic microphotographs show typical multilocular fat droplets brown adipocytes, resulting positive to [3H]thymidine-labeled nuclei in response to IGF-I. IGF-I increased mRNA expression of the early- response genes c-fos (30 min), c-myc (2 and 24 h), and H-ras (4 and 24 h). 10% FCS also increased c-fos and c-myc, but failed to increase H- ras as an early event. IGF-I or 10% FCS, however, similarly increased the mRNA late expression of c-myc, H-ras, c-raf, beta-actin, and glucose 6-phosphate dehydrogenase (G6PD) at 72 h, as compared to quiescent cells. IGF-I or FCS maintained at 24 h or increased at 48 and 72 h UCP mRNA expression. The results demonstrate that IGF-I is a mitogen for fetal rat brown adipocytes, capable of inducing the expression of early and late growth-regulated genes, and of increasing the lipogenic marker ME and the tissue-specific gene UCP, suggesting the involvement of IGF-I in the differentiation as well as in the proliferation processes.     

Cell Cycle-Specific Requirement for Mevalonate, but Not for Cholesterol, for DNA Synthesis in Glial Primary Cultures

The requirement for the sterol biosynthetic pathway for the occurrence of DNA synthesis in glial cells and, in particular, the relative roles of cholesterol and of mevalonate have been studied. Primary cultures of developing glial cells were synchronized by reducing the content of fetal calf serum (FCS) in the culture medium from 10% to 0.1% (vol/vol) for 48 h between days 4 and 6 in culture. Reversal of the resulting quiescent state by the return of the cultures to 10% serum caused after 24 h a marked increase in DNA synthesis, and this increase was prevented by the simultaneous addition of mevinolin, a specific inhibitor of the sterol biosynthetic pathway at the 3-hydroxy-3-methylglutaryl coenzyme A reductase step, at the time of serum repletion. A dose-dependent reversal of the mevinolin inhibition of DNA synthesis occurred with simultaneous addition of mevalonate to the culture medium. The induction of DNA synthesis by serum repletion, its inhibition by mevinolin, and the reversal of the inhibition by mevalonate were unaffected by a 95% reduction in exogenous cholesterol produced by utilization of lipoprotein-poor serum (LPPS) rather than FCS. Similarly, return of quiescent cultures to 10% LPPS containing mevinolin and sufficient low-density lipoprotein (LDL) to raise the cholesterol concentration 80-fold failed to restore DNA synthesis. In addition, reversal of the mevinolin inhibition of DNA synthesis by mevalonate occurred despite the continuous presence of mevinolin if mevalonate was added as late as 12 h after serum repletion, but not if added after 16 h or more.(ABSTRACT TRUNCATED AT 250 WORDS)     

The response of small vessel endothelial cells from fetal rat lung to growth factors

Small vessel pulmonary endothelial cells were obtained from rat fetal lung at day 20 of gestation, and were maintained in culture to passage three for study. Endothelial cells grown on a collagen matrix with Dulbecco's minimal essential medium: Ham's F12 medium (1:1, v/v) supplemented with 20 ml/l fetal bovine serum, bovine pituitary extract (50 mg/l), endothelial cell growth supplement (100 mg/l), hydrocortisone (1 mg/l) and an increased (10 mmol/l) magnesium concentration retained the characteristic endothelial cell marker factor VIII antigen during the third passage in culture. The factors responsible for small vessel growth in the developing fetal lung are unknown. To test the hypothesis that small vessel pulmonary endothelial cells would respond to autocrine or paracrine growth factors the effects of conditioned media from fetal lung endothelial cells, fibroblasts and pneumocytes from lungs of the same gestational age were studied in vitro. None of the tested conditioned media had any effect on endothelial cell DNA synthesis in the presence of 20 ml/l fetal bovine serum. Since no paracrine or autocrine effects of conditioned media were observed, the effect of other growth factors that could be derived from the circulation, or from storage sites in subcellular matrix, were studied for effect. When endothelial cells were studied in the presence of 20 ml/l fetal bovine serum and 100 mg/l endothelial cell growth supplement they had enhanced DNA synthesis in response to the progression-type growth factors insulin (5 mg/l), insulin-like growth factor-I and insulin-like growth factor-II (20 micrograms/l) and epidermal growth factor (10 micrograms/l). In the absence of serum or endothelial growth supplement endothelial cell DNA synthesis was enhanced by the competence-type growth factors acidic and basic fibroblastic growth factors at 100 micrograms/l and platelet derived growth factor at 10 micrograms/l. In the absence of exogenous competence-type growth factors neutralizing antibodies to basic fibroblast growth factor reduce DNA synthesis. Of various cytokines tested only interleukin-1 (1 x 10(3) U/l) and tumor necrosis factor (25 x 10(4) U/l) had an effect on endothelial cell DNA synthesis. Endothelial cell division during fetal lung development may be controlled by progression growth factors present in serum, and by either autocrine release of the competence factor basic fibroblast growth factor or paracrine release of platelet-derived growth factor by other cell types.