Human keratinocyte sensitivity towards inflammatory cytokines varies with culture time

Proliferating keratinocyte cultures have been reported to synthesize higher concentrations of prostaglandin (PG) E than confluent ones. As interleukin-1 (IL-1) stimulates keratinocyte PGE synthesis we investigated whether the degree of confluency of the keratinocyte culture modified the response of the cells to IL-1. It was found that IL-1alpha (100 U/ml) stimulated PGE(2) synthesis by proliferating (7 days in culture) but not differentiating (14 days in culture) keratinocytes. Similar effects were observed using tumour necrosis factor-alpha. Both arachidonic acid (AA) and the calcium ionophore A23187 stimulated PGE(2) synthesis by 7 and 14 day cultures although the increase was greatest when 7 day cultures were used. Our data indicate that there is a specific down-regulation of the mechanism(s) by which some inflammatory cytokines stimulate keratinocyte eicosanoid synthesis as cultured keratinocytes begin to differentiate.     

Role of phospholipases A(2) in growth-dependent changes in prostaglandin release from 3T6 fibroblasts

Previously, we reported a growth-dependent change in prostaglandin production as a consequence of a marked growth-dependent alteration in arachidonic acid (AA) mobilization from phospholipids. Our present results show that fetal calf serum (FCS) and 4 beta-phorbol-12-myristate acetate (PMA) caused an enhancement of phospholipase A(2) (PLA(2)) activity in the membrane fraction of non-confluent cells allowing PLA(2) access to its substrate and the release of AA. Western blot analysis has shown that FCS and PMA increased secreted PLA(2) (sPLA(2)) expression in non-confluent 3T6 fibroblast cultures. Moreover, FCS and PMA induced dithiothreitol-sensitive and bromoenol lactone-sensitive PLA(2) activities in cytosol and membrane fraction. However, these stimuli did not modify significantly the PLA(2) activity in both fractions when 3T6 fibroblasts reached a high cell density. This could be associated with the impairment of AA mobilization in these cell culture conditions. On the other hand, we observed that FCS and PMA induced the same prostaglandin H synthase-2 induction in non-confluent and confluent culture conditions. Moreover, the prostaglandin E(2) levels reached in cell culture supernatants were independent of the degree of confluence when AA was added exogenously. These results suggest that the changes of intracellular distribution of PLA(2) activity of sPLA(2) and iPLA(2) stimulated by exogenous stimuli may be controlled by cell density conditions which constitute an important mechanism in the regulation of prostaglandin release.Copyright 2001 Wiley-Liss, Inc.     

The mechanisms of preterm labour; the interaction between amnion cells and leukocytes in the metabolism of arachidonic acid.

Chorioamnionitis is frequently associated with preterm labour. We have used a cell culture model system to examine the effects of leukocytes upon the metabolism of endogenous arachidonic acid from within amnion cells. We have demonstrated that activated leukocytes release substances which increase the overall release and metabolism of endogenous arachidonic acid within amnion cells causing an increase in prostaglandin E2 production as well as a smaller increase in non-cyclo-oxygenase metabolism. When amnion cells and leukocytes are cultured together, in addition to prostaglandin E2 production by amnion cells, arachidonic acid released by the amnion cells appears to be metabolised by leucocytes to prostaglandin F2 alpha, prostacyclin and thromboxane A2. Prostaglandins E2 and F2 alpha are the principal cyclo-oxygenase products of this interaction. We postulate that chorioamnionitis stimulates preterm labour not only by causing an increase in prostaglandin E2 synthesis by amnion cells but by metabolism of amnion derived arachidonic acid to the powerfully oxytocic prostaglandin F2 alpha by leukocytes.     

Synthesis and distribution of cytoskeletal elements in endothelial cells as a function of cell growth and organization

Confluent cultures of adult bovine aortic endothelial (ABAE), correal endothelial (BCE), and fetal bovine heart endothelial (FBHE) cells form a monolayer of highly flattened, closely apposed, and nonoverlapping cells. In ABAE and BCE cultures, this is associated with a 50-fold decrease in the rate of DNA synthesis and correlates with a 14-fold decrease in protein synthesis. In contrast, in confluent FBHE cultures only partial decreases in the rates of DNA synthesis (6-fold) and protein synthesis (3-fold) are observed. FBHE cells therefore fulfill the morphological, but not the biochemical, criteria for confluent cultured endothelial cell monolayers. The appearance of the cytoskeletal elements actin, tubulin, and vimentin in sparse and confluent cultures of endothelial cells has been analyzed by two-dimensional gel electrophoresis and immunofluorescence. Sparse versus confluent ABAE, FBHE, and BCE cultures showed no changes in their relative rates of synthesis or cellular content of tubulin. Actin behaved similarly to tubulin in FBHE and BCE cultures, while in ABAE cultures a small increase (3-fold) in its relative rate of synthesis was observed in confluent versus sparse cultures. BCE cultures showed no change in the rate of synthesis of vimentin, but the cellular content of vimentin was markedly increased when cultures reached confluence. When the distribution of vimentin in both sparse and confluent BCE cultures was analyzed by immunofluorescence, in both cases it appeared distributed throughout the cytoplasm as thin fibers and bundles of fibers. In confluent ABAE cultures, both the relative amount and biosynthetic rate of vimentin increased by 15-fold. This increase in the intracellular accumulation of vimentin correlated with its immunofluorescent distribution within the cells. While in sparse cultures, vimentin appeared to be distributed as thin fibers, in confluent cultures thick curl-like fibrous bundles could be seen distributed throughout the cytoplasm and organized in a perinuclear ring. In contrast, in FBHE cultures no significant changes in the distribution and organization of rate of synthesis of vimentin were observed.     

Microtubule disruption enhances prostaglandin E2 production in osteoblastic cells

Prostaglandins have been implicated in the response of bone to mechanical stimuli. To explore the potential role of the cytoskeleton in the control of prostaglandin production, we examined the effect of cytoskeleton disrupting agents on arachidonic acid metabolism in rat calvaria osteoblastic cells. We found that microtubule disrupting agents increase prostaglandin E production 4-5-fold. Stimulation was first detectable at 4 h and rose sharply between 4 and 8 h. 2 h exposure to 1 microM colchicine was sufficient to produce the maximum effect. Cytochalasin B at concentrations which caused marked shape changes had no effect on prostaglandin E production or on its stimulation by colchicine. Taxol, a stabilizer of microtubules, reduced the colchicine effect. The increase in prostaglandin E production was associated with enhanced conversion of arachidonic acid to prostaglandin E2 rather than enhanced release of arachidonic acid from phospholipids. This increase in enzymatic activity was not abolished by cycloheximide treatment at concentrations which inhibited 90% of protein synthesis in the cells.     

Synthesis of prostaglandins and cyclic AMP by cultured embryonic palate mesenchyme at various population densities

During embryonic development, facial and palate mesenchymal cells exhibit differential growth rates. Normal palatal growth is regulated in part by hormones and growth factors. Because hormonal responsiveness of some cells correlates with their cell density, we have investigated the relationship between embryonic palate mesenchymal cell population density and their ability to synthesize prostaglandins (PGs) and cyclic AMP. Primary cultures of palate mesenchymal cells exhibited typical lag, log, and stationary phases of growth with a doubling time of 32-34 hrs. The ability of cells to produce PGE2 in response to a calcium ionophore (A23187), an activator of phospholipase A2 (melittin), arachidonic acid, or serum was maximal during the period of early exponential growth. Prostaglandin F2 alpha synthesis in response to A23187 or arachidonic acid showed a similar transient increase also corresponding temporally to the period of early exponential growth. The ability to synthesize PGF2 alpha in response to melittin, however, failed to diminish after early exponential growth. The pattern of cAMP synthesis in response to isoproterenol and PGE1 was different from that seen for induced prostaglandin synthesis. A transient increase in sensitivity to isoproterenol and PGE1 was seen that corresponded temporally to the period of late exponential growth just prior to attainment of confluency. Decreased sensitivity to stimulation of either prostaglandin or cAMP production as the cells became confluent was shown to be a density-dependent phenomenon; confluent cultures that were subcultured to reestablish logarithmic growth exhibited density-dependent hormonal responses identical to those seen in primary cultures. The ability of palate mesenchymal cells to synthesize both prostaglandins and cAMP, thought to be critical for proper palatal development, might thus be related to local differential craniofacial growth rates.     

Oxidative stress mediates synthesis of cytosolic phospholipase A2 after UVB injury

UVB irradiation has previously been shown to significantly increase phospholipase activity and prostaglandin synthesis. Because UVB irradiation is a potent oxidative stress, the role of active oxygen species in regulating UV-induced cPLA₂ synthesis and phosphorylation was examined. In the present study, irradiation produced a 3-fold increase in synthesis within 6 h following irradiation. Phosphorylation of cPLA₂ was also increased to a similar extent. UVB-induced synthesis and phosphorylation of cPLA₂ could be inhibited by pretreatment with the antioxidants 2,2,5,7,8-pentamethyl-6-hydroxychromane (50 μM) or N-acetylcysteine (10 mM). Treatment of unirradiated cultures with the potent oxidant tert-butyl hydroperoxide (500 μM) also increased cPLA₂ synthesis and phosphorylation, suggesting that oxidative injury is an important regulator of cPLA₂ synthesis. Increased synthesis of cPLA₂ correlated well with increased [³H]arachidonic acid release, PGE₂ synthesis and lipid peroxidation in epidermis after oxidant or UVB treatment. The results indicate that UVB-induced upregulation of cPLA₂ synthesis is mediated by UVB-induced formation of free radicals.     

Decreased prostaglandin production in cultured smooth muscle cells from atherosclerotic rabbit aorta

Prostaglandin synthesis in aortic smooth muscle cells originating from healthy an atherosclerotic rabbits was studied by incubating [14C]arachidonic acid with intact confluent cells and cell homogenates. In spite of a reduced 6-keto prostaglandin F1 alpha formation, no potentiating effect on the prostaglandin E2 generation occurred. Indeed, both cyclooxygenase and prostaglandin I2 synthetase activities appear to be reduced. These results suggest that an impaired arachidonic acid utilisation in aortic smooth muscle cells may be involved in the course of the atherosclerotic process.     

Linoleate metabolites enhance the in vitro proliferative response of mouse mammary epithelial cells to epidermal growth factor

Linoleic acid, arachidonic acid, prostaglandin E1, and prostaglandin E2 stimulated the proliferation of mammary epithelial cells in serum-free primary cultures only in the presence of epidermal growth factor. Linoleate-stimulated growth was manifest later in culture when proliferation, initiated by epidermal growth factor only, reached a plateau while linoleate-supplemented epidermal growth factor cultures continued to proliferate. The cultures in the plateau phase of growth could be restimulated to grow by adding either linoleic acid or prostaglandin E2 to the media. While the linoleate response could be abolished by the cyclooxygenase inhibitor, indomethacin, prostaglandin E2-stimulated growth remained unaffected. Linoleic acid was metabolized to arachidonic acid and prostaglandin E2, both in the growing and resting cultures. Proliferating cells metabolized linoleate and prostaglandin E2 extensively so that neither the fatty acid nor prostaglandin E2 accumulated in large quantities in the proliferating cultures. The concentrations of prostaglandin E2 in growing cultures supplemented with linoleic acid were much higher than in cultures without it. These results suggest that the metabolism of linoleic acid leading to prostaglandin production, not its contribution to membrane polyunsaturation, is necessary for sustained growth of mammary epithelial cells in the presence of epidermal growth factor.     

Dual effects of staurosporine on arachidonic acid metabolism in rat peritoneal macrophages

Staurosporine is a microbial anti-fungal alkaloid having a most potent inhibitory activity on protein kinase C and is recently found as a non-12-O-tetradecanoylphorbol-13-acetate (non-TPA)-type tumor promoter of mouse skin, although tumor promotion induced by a TPA-type tumor promoter teleocidin is suppressed by staurosporine. When rat peritoneal macrophages were incubated in the medium containing various concentrations of staurosporine, prostaglandin E2 production and release of radioactivity from [3H]arachidonic acid-labeled macrophages were stimulated at concentrations of 1 and 10 ng/ml. But higher concentrations of staurosporine such as 100 and 1000 ng/ml showed no stimulative effect on prostaglandin E2 production although cytoplasmic free calcium levels were increased in a dose-dependent manner. Staurosporine-induced stimulation of prostaglandin E2 production was inhibited by treatment with cycloheximide, suggesting that a certain protein synthesis is prerequisite for the stimulation of arahcidonic acid metabolism. At higher concentrations (100 and 1000 ng/ml), staurosporine inhibited TPA-type tumor promoter (TPA, teleocidin and aplysiatoxin)-induced stimulation of arachidonic acid metabolism probably due to the inhibition of protein kinases. Tumor promotion activity and anti-tumor promotion activity of staurosporine might be explained by the fact that the lower concentrations of staurosporine stimulate arachidonic acid metabolism and the higher concentrations of staurosporine inhibit the tumor promoter-induced arachidonic acid metabolism, respectively.     

Effect of fatty acid modification on prostaglandin production by cultured 3T3 cells

We have investigated the extent to which modifications in the essential fatty acid content of mammalian cells can affect prostaglandin production. Swiss mouse 3T3 cells stimulated with the calcium ionophore A23187 produced 1.7 to 7 times more prostaglandin E(2) (PGE(2)) when the cultures were supplemented with linoleic acid. Increases in PGE(2) production as a result of linoleic acid supplementation occurred under all culture conditions except during the first 24 hr after attachment, when prostaglandin production was very high. Arachidonic acid supplementation produced a similar enhancement in the capacity of the cells to produce PGE(2), but no appreciable increase occurred when the cultures were supplemented with oleic acid. The phospholipids of the cells exposed to the linoleate-enriched medium contained 4 times more arachidonic acid and twice as much linoleic acid as compared with the corresponding controls. The choline phosphoglycerides were most highly enriched in arachidonic acid, but 2- to 3-fold increases also occurred in the inositol and ethanolamine phosphoglycerides. When cultures initially enriched with linoleic acid were transferred to an unsupplemented medium, the fatty acid composition as well as the capacity of the cells to produce PGE(2) reverted almost to control values. The amount of exogenous arachidonic acid converted to PGE(2) as measured by radioimmunoassay also was greater when the cells were enriched with linoleic acid. Studies with radioactive arachidonic acid indicated that the distribution of prostaglandin metabolites was not affected appreciably by linoleic acid enrichment. These findings suggest that at least two factors contribute to the increased capacity of the cultures supplemented with linoleate to produce PGE(2). One is enrichment of the phospholipid substrate pools with arachidonic acid. The other is an increased ability of the cells to synthesize PGE(2) from unesterified arachidonic acid, perhaps because the prostaglandin-forming enzymes are more active.-Denning, G. M., P. H. Figard, and A. A. Spector. Effect of fatty acid modification on prostaglandin production by cultured 3T3 cells.     

Tocopherol analogs suppress arachidonic acid metabolism via phospholipase inhibition.

alpha-Tocopherol and three derivatives in which the phytol chain is modified or deleted were examined for their effect on cultured keratinocyte arachidonic acid metabolism. 2,2,5,7,8-Pentamethyl-6-hydroxychromane (PMC), in which the phytol chain is replaced by a methyl group, inhibited basal, bradykinin (BK)- and A23187-stimulated prostaglandin E2 (PGE2) synthesis with an apparent Ki of 1.3 microM. The Ki of the analogue with six carbon atoms in the side chain (C6) was 5 microM while that of the C11 analogue was 10 microM. No effect of alpha-tocopherol was observed. The mechanism of inhibition was studied using PMC. The effect of PMC on phospholipase and cyclooxygenase activity was assayed using stable isotope mass measurements of PGE2 formation, which assesses arachidonate release and cyclooxygenase metabolism simultaneously. BK-stimulated formation of PGE2, derived from endogenous phospholipid, was decreased 60% by 5 microM PMC and eliminated by 50 microM PMC, compared with controls. No difference in PGE2 formed from exogenous arachidonic acid was observed, indicating no effect of PMC on cyclooxygenase activity. In contrast, no effect of 5 microM PMC was observed on BK-stimulated [3H]arachidonic acid release from prelabeled cultures. The capacity of PMC to inhibit phospholipase activity in vitro was also assessed. PMC inhibited hydrolysis of phospholipid substrate by up to 60%. These results suggest that alpha-tocopherol analogues with alterations in the phytol chain inhibit eicosanoid synthesis by preferential inhibition of phospholipase.     

Radioimmunologic identification of prostaglandins produced by serum-stimulated mouse embryo palate mesenchyme cells

High-performance liquid chromatography and radioimmunoassay were used to identify the prostaglandins synthesized by mouse embryo palate mesenchyme cells. Serum stimulated the release of several different metabolites of arachidonic acid including 6-ketoprostaglandin F1 alpha (the stable product of prostacyclin, prostaglandin I2), prostaglandin E2 and prostaglandin F2 alpha. Compared to control cells, the serum-stimulated cells produce elevated levels of prostaglandin E2 (36-fold), 6-ketoprostaglandin F1 alpha (15-fold) and prostaglandin F2 alpha (7-fold). The acetylenic analogue of arachidonic acid, 5,8,11,14-eicosatetraynoic acid prevented this accelerated synthesis.     

Arachidonic acid is preferentially metabolized by cyclooxygenase-2 to prostacyclin and prostaglandin E2

The two cyclooxygenase isoforms, cyclooxygenase-1 and cyclooxygenase-2, both metabolize arachidonic acid to prostaglandin H2, which is subsequently processed by downstream enzymes to the various prostanoids. In the present study, we asked if the two isoforms differ in the profile of prostanoids that ultimately arise from their action on arachidonic acid. Resident peritoneal macrophages contained only cyclooxygenase-1 and synthesized (from either endogenous or exogenous arachidonic acid) a balance of four major prostanoids: prostacyclin, thromboxane A2, prostaglandin D2, and 12-hydroxyheptadecatrienoic acid. Prostaglandin E2 was a minor fifth product, although these cells efficiently converted exogenous prostaglandin H2 to prostaglandin E2. By contrast, induction of cyclooxygenase-2 with lipopol- ysaccharide resulted in the preferential production of prostacyclin and prostaglandin E2. This shift in product profile was accentuated if cyclooxygenase-1 was permanently inactivated with aspirin before cyclooxygenase-2 induction. The conversion of exogenous prostaglandin H2 to prostaglandin E2 was only modestly increased by lipopolysaccharide treatment. Thus, cyclooxygenase-2 induction leads to a shift in arachidonic acid metabolism from the production of several prostanoids with diverse effects as mediated by cyclooxygenase-1 to the preferential synthesis of two prostanoids, prostacyclin and prostaglandin E2, which evoke common effects at the cellular level.     

On the mechanism of elevated prostaglandin E2 production in 3T3 fibroblasts transformed by polyoma virus

Polyoma-virus-transformed 3T3 fibroblasts (py 3T3 cells) produce considerably more prostaglandin E2 than regular 3T3 cells during growth in cell culture. Incubations with exogenous arachidonic acid showed no increase in prostaglandin-producing capacity in the transformed cells. The rates of degradation of prostaglandin E2 were similar in the two lines. After labeling of cells with [1-14C]arachidonic acid, py 3T3 cultures continuously released radioactivity while the release by regular 3T3 cells was almost completed after 3 h. Prostaglandin E2 production during short incubations in buffer at various times after medium change was constantly higher in the transformed cells. Furthermore, hydrocortisone completely inhibited prostaglandin synthesis by the transformed cells. These results suggest that the increased formation of prostaglandin by py 3T3 cells is due to continuously elevated activity of phospholipase A2 or another acyl hydrolase.     

Calcium dependence of prostaglandin release from the guinea pig Taenia coli

We studied the calcium dependency of the stimulation of prostaglandin synthesis which occurs when perfusing strips of guinea pig Taenia coli with potassium-free media. Stimulation was rapidly reversed by removal of extracellular Ca from the bathing solution. The Ca ionophore A23187 markedly stimulated prostaglandin E2 synthesis, an effect that is dependent on the presence of extracellular Ca. Prostaglandin E2 production in strips in potassium-deficient media was also sensitive to increases in extracellular Ca, and was augmented at concentrations of 7-15 mM. In strips which had been incubated with [3H]arachidonic acid, exposure to potassium-free media caused an increased release of [3H]arachidonic acid and [3H]prostaglandin E2. Release of these labeled compounds with the strips in potassium-free media was further augmented by increasing extracellular [Ca2+] from 2.5 to 10 mM. Treatment with the Ca antagonist agent verapamil did not influence activation of prostaglandin synthesis by potassium-deficient media. The presence of Mn2+ of Ba2+ had similar effects on prostaglandin synthesis, although they had opposite effects on mechanical activity. We conclude that a plasma membrane associated Ca pool is involved in activation of phospholipid metabolism which results in release of esterified arachidonic acid and subsequent prostaglandin synthesis. This Ca pool is in rapid equilibrium with extracellular Ca, is not influenced by cytoplasmic Ca, and is not related to Ca involved in Ca gating in the surface membrane. These data also indicate dissociation between processes involved in muscle contraction and activation of prostaglandin synthesis.     

Flurbiprofen: highly potent inhibitor of prostaglandin synthesis.

Flurbiprofen, 2-(2-fluoro-4-biphenylyl)propionic acid, inhibited the formation of prostaglandin E2 from arachidonic acid by bovine seminal vesicular microsomes. It was found that flurbiprofen was an approx. 12.5-fold better inhibitor than indomethacin by comparison of their I50 values. It was suggested that the inhibition of prostaglandin synthesis by flurbiprofen might be due to the inhibition of the endoperoxygenase which catalyzed conversion of arachidonic acid to cyclic endoperoxide. Other carboxylic acid compounds such as aspirin, ibuprofen and indomethacin showed the same type of inhibition as flurbiprofen. In contrast, phenylbutazone which was a pyrozolone derivative inhibited the formation of prostaglandin E2, but not affected the endoperoxygenase reaction. The kinetic studies for inhibition of prostaglandin E2 synthetase indicated that flurbiprofen competitively inhibited prostaglandin E2 synthesis, just like indomethacin. The Ki values were estimated to be 0.128 micron for flurbiprofen and 3.18 micron for indomethacin.     

Solubilization and partial purification of prostaglandin endoperoxide synthetase of rabbit kidney medulla.

The microsomes of rabbit kidney medulla converted arachidonic acid into prostaglandin E2 in the presence of hemoglobin, tryptophan and glutathione as activators. When themicrosomal suspension was treated with 1% Tween 20, a solubilized enzyme was obtained which catalyzed the conversion of arachidonic acid to prostaglandins G2 and H2. The solubilized enzyme was adsorbed to and then eluted from an omega-aminooctyl Sepharose 4B column, resulting in about 10-fold purification over the microsomes. The partially purified enzyme produced predominantly prostaglandin G2 in the presence of hemoglobin, while prostaglandin H2 was produced in the presence of both hemoglobin and tryptophan. The stimulation of prostaglandin endoperoxide formation was also observed with other heme and aromatic compounds. Prostaglandin H2 synthesis was inhibited by a variety of compounds including non-steroidal anti-inflammatory drugs, thiol compounds and prostaglandin analogues with a thiol group(s).     

Arachidonic acid stimulates internalisation of leptin by human placental choriocarcinoma (BeWo) cells

Arachidonic acid at 100 nM stimulated internalisation of 125I-leptin in human placental choriocarcinoma (BeWo) cells by 3-fold compared with controls. In contrast, eicosapentaenoic acid at similar concentration decreased internalisation of leptin by 2-fold. Use of ibuprofen and indomethacin (inhibitors of prostaglandin synthesis) inhibited the stimulatory effect of arachidonic acid. Prostaglandin E(2), a cyclooxygenase metabolite of arachidonic acid, stimulated internalisation of leptin by these cells. All these data demonstrate that stimulation of leptin internalisation by arachidonic acid in placental trophoblasts may be mediated via prostaglandin E(2).     

Behavior of confluent endothelial cells after irradiation. Modulation of wound repair by heparin and acidic fibroblast growth factor

Image analysis was used to study the repair process of a circular mechanical lesion of confluent human endothelial cells in culture after irradiation (10 Gy) prior to wounding. Coverage of denuded areas 48 and 96 h after injury of endothelial cells was identical in control and irradiated cultures, although the labeling index was lowered by 80 to 95% in irradiated cultures. The cell density of non damaged irradiated areas was decreased by 50%. When cultures were submitted to increasing doses of radiation (5.0-30 Gy), the labeling index of the cells diminished rapidly between 0 and 5.0 Gy and reached a plateau at 10 Gy. The decrease in cell density (50% of control at 96 h) was identical at each dose of radiation. Thus cell migration alone could be sufficient for the repair of the lesion, while cell proliferation would mainly maintain the original cell density. The addition of heparin to the culture medium slowed down cell migration and proliferation, but the speed of repair was identical in irradiated and non-irradiated cultures. Acidic fibroblast growth factor plus heparin accelerated equally the repair process whether the cultures were irradiated or not. In irradiated cultures the presence of acidic fibroblast growth factor and heparin maintained cell density in confluent areas at a level similar to that in non-irradiated damaged control cultures without addition of mitogens. Thus heparin and acidic fibroblast growth factor play a role in cell proliferation, in the maintenance of the cell monolayer integrity and in restoring a continuous layer by rapid cell migration and elongation after irradiation.