Enhanced DNA Synthesis Research Articles

Regulation of insulinoma cell proliferation and insulin accumulation by peptides and second messengers.

The regulation of clonal rat insulinoma (RINm5F) cell proliferation and hormone accumulation was investigated with the aim of identifying putative compounds capable of inducing differentiation, i.e. decreased growth and increased insulin accumulation, by the tumor cells. In particular, interest was focused on the role of a number of peptides as well as pharmacological probes modulating various signal transduction systems and which have been shown to regulate normal beta-cell proliferation and insulin accumulation. Growth hormone stimulated insulin accumulation and inhibited DNA synthesis, whereas galanin and insulin-like growth factor I caused a moderate suppression of insulin accumulation but did not affect proliferation, while epidermal growth factor, transforming growth factor beta, platelet-derived growth factor, acidic and basic fibroblast growth factor, bradykinin and somatostatin were virtually inactive on all parameters tested. Exogenous prostaglandins E2 and F1 alpha were inactive, while the cycloxygenase inhibitor indomethacin slightly suppressed insulin accumulation. The cytokine IL-1 beta caused a significant decrease in both beta-cell mitogenesis and insulin accumulation, effects that were mediated through nitric oxide generation. The vitamin A derivative retinyl acetate slightly inhibited serum-stimulated DNA synthesis, but did not affect insulin accumulation. The vitamin E alpha-tocopherol significantly enhanced insulin release but did not affect mitogenesis. By contrast, gamma-tocopherol was inactive on both these parameters. The alpha-adrenergic agonist clonidine evoked a slight inhibition of serum-stimulated DNA synthesis, without influencing insulin accumulation, whereas phenylephrine did not affect any of these parameters. Carbamylcholine increased insulin accumulation, but not cell proliferation, whereas the adenylyl cyclase activator forskolin suppressed mitogenesis but did not affect insulin accumulation. Inhibition of protein kinase C with staurosporine or prolonged treatment with phorbol ester suppressed DNA synthesis, as did the tyrosine kinase inhibitor genistein. Stimulating Ca2+ influx by closing ATP-dependent K+ channels with glibenclamide enhanced DNA synthesis, while opening of these channels with diazoxide suppressed cell growth. Conversely, preventing Ca2+ influx by the Ca2+ channel antagonist D-600, chelating intracellular Ca2+ by fura-2 AM or inhibiting the Ca2+/calmodulin-dependent protein kinase by calmidazol resulted in a decreased DNA synthesis. On the other hand, uncontrolled influx or mobilization of Ca2+ by ionomycin or thapsigargin resulted in an arrested DNA synthesis. The present paper shows that RINm5F insulinoma cell proliferation and insulin accumulation can be modulated by various peptidergic and pharmacological agents regulating certain signal transduction pathways. However, mitogenesis in the insulinoma cells seemingly is controlled in a vastly different manner in comparison to that in normal beta-cells. The most spectacular finding in this screening study, i.e. that growth hormone, contrarily to its effect on normal beta-cells, suppresses insulinoma cell growth, merits further elucidation of the underlying mechanisms. Possibly the hormone might become of utility in a clinical setting in the treatment of patients with insulin-producing tumors.

Regulation of mesothelial cell mitogenesis by antisense oligonucleotides for the urokinase receptor.

The association of urokinase-type plasminogen activator (uPA) with its receptor (uPAR) influences various biologic functions, including cell migration, angiogenesis, differentiation, and wound healing. Expression of uPAR at the mesothelial surface could, therefore, influence cellular responses in the pleural space. We found that a line of cultured human mesothelial cells (MeT5A) expressed specific and saturable binding sites for uPA that increased on stimulation with PMA. Ligand blotting studies showed that the mesothelial receptor is a 50 kD protein similar to that in other cell lines. Binding of active and intact, but not amino terminal or low molecular weight fragment, uPA to mesothelial cells enhanced DNA synthesis and cell proliferation, and antibodies against either the active site of uPA or uPAR abrogated this effect. We reasoned that regulation of uPAR expression could control uPA-induced mitogenesis and tested this hypothesis with antisense oligonucleotides complementary to uPAR mRNA. Phosphorothioate-modified antisense oligonucleotides inhibited uPA-mediated mesothelial cell proliferation in a concentration-dependent manner. These effects were associated with decreased binding of 125I-uPA and reduced expression of the uPAR gene product. The results indicate that uPAR is involved in signal transduction pathways that control uPA-mediated mesothelial cell proliferation, a process implicated in the pathogenesis of mesothelial inflammation and pleural neoplasia. Antisense oligonucleotides to uPAR suppress mesothelial cell mitogenesis in vitro and offer a potential means of regulating the process in vivo.

Endothelin synthesis and receptors in human endometrium throughout the normal menstrual cycle

This study was undertaken to investigate the presence of messenger RNA (mRNA) for prepro-endothelin-I (ET-1) and the known receptor subtypes (ETA and ETB) in human endometrium at different stages of the menstrual cycle obtained at hysterectomy. Northern blot analysis revealed expression of ET-1 mRNA in human endometrium during the normal menstrual cycle. The concentration of ET-1 mRNA in endometrial tissue was greater during the menstrual and proliferative phases than during the ovulatory and secretory phases. Immunoreactive ET-1 was secreted into the medium of isolated endometrial stromal cells. Oestradiol and progesterone significantly attenuated ET-1 release in endometrial stromal cells cultured for 6 days. ETA and ETB mRNA were also present in endometrial tissue of the normal cycle. The concentration of ETA receptor mRNA was greater in the proliferative phase than in the secretory phase, whereas expression of ETB mRNA increased in menstrual phase. ET-1 significantly increased extracellular accumulation of cyclic AMP (cAMP), intracellular generation of inositol phosphates and significantly enhanced DNA synthesis in cultured endometrial stromal cells from the proliferative phase. Our results showed that human endometrial cells synthesized and released ET-1, and contained ETA and ETB receptors which were functionally coupled to phosphoinositide breakdown and to adenylate cyclase with the increase of cAMP by ET-1 stimulation. Our findings suggest that ET-1 may have a potential autocrine and/or paracrine function in human endometrial stromal cells.

Exogenously expressed granulocyte colony-stimulating factor (G-CSF) receptor on K562 cells can transduce G-CSF-triggered growth and differentiation signals.

Human granulocyte colony-stimulating factor (G-CSF) receptor cDNA was introduced into the erythroleukemic cell line K562, which normally does not express the receptor, using lipofection transfection of a G-CSF receptor expression plasmid vector. Transfected cells expressed the receptor with a dissociation constant of 130 pmol/L, and a maximum of 11,800 binding sites per cell. Culture of G-CSF receptor-expressing cells (GR-K562) in the presence of G-CSF enhanced DNA synthesis and a stimulation index of 4.61 was obtained in a 3H-thymidine uptake assay. Furthermore, flow cytometric studies revealed induced expression of CD11b (29% from 7%), and enhanced expression of CD13 (57% from 27%) on GR-K562 cultured in the presence of G-CSF. Our findings indicate that the exogenously expressed G-CSF receptor can deliver signals and function efficiently on these immature cells on which the receptor is not normally expressed, which suggests the presence of intact intracellular signal transduction pathways that can be used by the ectopically expressed receptor.

Facilitation of Autonomous Phenotype Acquisition in Androgen‐dependent Shionogi Carcinoma 115 Cells by Transfection of Androgen‐induced Growth Factor Expression Vector

Androgen‐induced growth factor (AIGF) is an autocrine growth factor for androgen‐dependent SC‐3 cells, which is induced by androgen stimuli. To elucidate the mechanism of the progression from hormone‐dependent to ‐independent tumor, we transfected an expression vector of cDNA encoding AIGF into SC‐3 cells and established a stable transfectant (Al) expressing AIGF. Al cells showed enhanced DNA synthesis. This enhanced DNA synthesis was blocked by exposing the cells to AIGF autisense oligonucleotides, heparin, or suramin, indicating that enforced AIGF expression is responsible for the increase in DNA synthesis. However, Al cells did not grow in serum‐free medium unless stimulated with androgen. Recloning from Al cells in semi‐solid agar supplemented with fetal calf serum but without androgen quickly generated an autonomous subline that was able to grow rapidly in the serum‐free medium irrespective of androgen stimulus. Mock‐transfected SC‐3 cells failed to form any colony under identical conditions. These results suggest that stable expression of AIGF alone is not sufficient for, but facilitates the conversion of SC‐3 cells from androgen‐dependent to ‐independent phenotype.

In vivo and in vitro effects of insulin-like growth factor-I (IGF-I) on femoral mRNA expression in old rats

The in vivo response of bone to IGF-I infusion in a marrow ablation model and the effect of IGF-I on bone marrow stromal cells in vitro was evaluated. IGF-I (25 ng/day), infused directly into femur, stimulated the expression of alkaline phosphatase, procollagen ∝ 1 (I) and osteopontin mRNA, while osteocalcin mRNA was not affected. The dose dependency to IGF-I was bi-phasic, with stimulation at 25 and 50 ng but not at 150 ng/day. The effect of IGF-I was observed in the aged but not in the adult rat femur. However, the elevated mRNA levels in old bones with IGF-I treatment were still below those observed in adult bones. The effect of IGF-I was also examined in cultured stromal cells. IGF-I (50 ng/ml) stimulates the expression of alkaline phosphatase, procollagen ∝ 1 (I), osteopontin and osteocalcin mRNA in stromal cells from both adult and old rats. These results suggest that the lack of response of adult bone to IGF-I in vivo was not due to the impaired response of the stromal cells to IGF-I. Differences in the responses of stromal cells from adult and old animals were noted. In the presence of serum (10%), stromal cells from adult rats were stimulated to synthesize DNA at lower levels of IGF-I than stromal cells from old animals. Our results show that IGF-I can stimulate mRNA expression of osteoblast markers in vivo in aged rats in a marrow ablation model and enhance DNA synthesis and gene expression in cultured marrow stromal cells from old rats. Thus, it is possible that exogenous IGF-I could be beneficial in treating age-associated osteopenia.

Differential actions of prostaglandins in separate cell populations from fetal rat bone.

Prostaglandins (PGs) may stimulate or inhibit bone cell replication and protein synthesis. These disparities may be concentration or time dependent, or occur in discrete cell types or by different second signals. Cell populations that express progressive degrees of osteoblast-like activity can be obtained by serial collagenase digestion of fetal rat parietal bone. The first (population 1) appears less differentiated, whereas the later (populations 3-5) exhibit biochemical features characteristic of osteoblasts. Within 24 h of treatment, three separate PGs increased DNA synthesis in population 1 with relative potencies of PGE1 < PGE2 < PGF2 alpha. By contrast, PGE1 and PGE2 (both strong cAMP inducers) inhibited basal DNA synthesis in population 3-5. These differences were paralleled by analogous changes in collagen and noncollagen synthesis in each population. The mitogenic effect in population 1 persisted for 72 h, and at later times was sensitive to indomethacin. These changes were unlikely to be cAMP dependent, as PGF2 alpha did not induce cAMP production, and the cAMP inducer forskolin was inhibitory. Moreover, phorbol ester treatment enhanced DNA synthesis to a greater extent in population 1 than in populations 3-5, and cotreatment with H-8 (at Km, approximately 10 microM) and staurosporine (at Km, approximately 0.01 microM) decreased the mitogenic effect of PGs in population 1, consistent with a reduction in protein kinase-C activation. These studies suggest that PGs activate less differentiated bone cells by a protein kinase-dependent event, whereas cAMP (induced by PGE1 and PGE2) decreases DNA and protein synthesis in more differentiated bone cells and tempers the increase in cellular activation found in population 1. Consequently, agents or events that increase the synthesis of specific PGs could differentially regulate, in positive and negative ways, biochemical activities in discrete bone cell populations.

Effects of inorganic arsenicals on DNA synthesis in unsensitized human blood lymphocytes in vitro

Effects of inorganic arsenicals on DNA synthesis in unsensitized human blood lymphocytes were biphasic: The chemicals at very low concentrations enhanced DNA synthesis, whereas higher concentrations inhibited DNA synthesis. The concentrations of arsenicals at which the maximum stimulating effect was found were 1 x 10(-5) M, 1 x 10(-6) or 2 x 10(-6) M, and 0.8 x 10(-6) or 1 x 10(-6) M for sodium arsenite exposure of 1 h, 3 d, and 6 d, respectively; for sodium arsenate, 1 x 10(-5) M, 1 x 10(-5) M, and 2 x 10(-6) or 5 x 10(-6) M, respectively. Arsenicals must be present for the entire 6-d culture period to produce maximum stimulation of DNA synthesis in human lymphocytes. The longer exposure of the lymphocytes to arsenicals, the lower the concentrations of arsenicals at which the maximum stimulating effect on DNA synthesis was found. Stimulating effect of trivalent arsenic (sodium arsenite) on DNA synthesis was stronger than pentavalent arsenic (sodium arsenate), and the stronger the effect of trivalent arsenic than pentavalent, the longer exposure of the cells to the chemicals. Both sodium arsenite and sodium arsenate stimulated DNA synthesis in human lymphocytes to a lower degree than phytohemagglutinin (PHA).

Human eosinophils stimulate DNA synthesis and matrix production in dermal fibroblasts.

Fibrosis is frequently found in diseases exhibiting tissue eosinophilia, such as some parasitic worm infections, eosinophilia-myalgia syndrome, and eosinophilic fasciitis. Previously, eosinophil extracts have been shown to induce proliferation in neonatal foreskin fibroblasts in vitro. To determine if living eosinophils can induce synthesis of DNA and components of the extracellular matrix in dermal fibroblasts, we cultured purified human eosinophils for 2 or 7 days in the presence of the eosinophil-active cytokines granulocyte/macrophage colony stimulating factor, interleukin-3, or interleukin-5, and added eosinophil-conditioned medium to cultures of dermal fibroblasts. Using flow cytometry, we found that eosinophil-conditioned medium increased by two-fold the percentage of fibroblasts in S-phase. This stimulation of fibroblast DNA synthesis was corroborated using a standard tritiated thymidine assay and the two methods were shown to correlate well with each other. Eosinophil-conditioned medium stimulation of DNA synthesis was dose dependent and conditioned medium from eosinophils treated with any one of the three cytokines induced increased DNA synthesis. Treatment of fibroblasts with cytokines alone did not induce enhanced DNA synthesis. Eosinophil-conditioned medium also affected fibroblast matrix production. Eosinophil-conditioned medium induced a two-fold increase in soluble and cell-associated fibroblast glycosaminoglycan production and a 76% increase in collagen production. These observations support the concept that eosinophils may be active contributors to the pathophysiology of eosinophil-associated fibrotic disease.

Distinct roles of erythropoietin, insulin-like growth factor I, and stem cell factor in the development of erythroid progenitor cells.

Erythropoietin (EP), insulin-like growth factor I (IGF-I) and stem cell factor (SCF) each reduce apoptosis of human erythroid progenitor cells. To determine if these growth factors have additional roles in stimulating erythropoiesis, the proliferation, maturation, and survival of highly purified human erythroid colony-forming cells (ECFCs) were studied during the application of different combinations of these growth factors in a serum-free liquid culture. EP maintained cell viability and supported heme synthesis during erythroid maturation, with little increase in viable cell number or stimulation of DNA synthesis. The addition of SCF with EP resulted in a substantial increase in DNA synthesis, which was greater than that seen with the addition of EP and was associated with a large expansion in the number of ECFCs. Thus EP, by itself, produces little increase in cell proliferation, and expansion of the number of erythroid cells depends upon the presence of SCF with EP. The addition of IGF-I with EP led to enhanced heme synthesis and moderate cellular proliferation, but also greatly enhanced nuclear condensation and enucleation in the late erythroblasts. Thus EP, by itself, is not sufficient for complete end-terminal nuclear condensation/enucleation and the presence of IGF-I is necessary for this complete process. While EP greatly reduced apoptosis during 16 h of incubation at 37 degrees C, the addition of SCF and IGF-I with EP had little additional effect, but these additions enhanced DNA synthesis > 3.4-fold. Thus SCF may have an additional role in directly stimulating proliferation through a process that is distinct from apoptosis. Our observations indicate that EP prevents apoptosis and maintains erythroid cell viability and development. IGF-I enhances erythroid maturation and proliferation, but the proliferation of erythroid progenitors is mainly controlled by the addition of SCF with EP, independent of an effect on apoptosis.

Oxidation of low density lipoprotein by mesangial cells may promote glomerular injury

Low density lipoprotein (LDL) deposition and local oxidation play a key role in the pathogenesis of atherosclerosis and may likewise contribute to glomerular injury. These studies were designed to determine whether cultured human mesangial cells oxidize homologous LDL and to compare the effects of unmodified and oxidized lipoprotein on cell proliferation, viability and eicosanoid production. Cell-mediated lipoprotein oxidation was demonstrated and could be suppressed by oxygen free radical scavengers and inhibitors of arachidonic acid metabolism. When incubated with cells, oxidized LDL (Ox-LDL) at concentrations up to and including 100 micrograms/ml reduced 3H-thymidine incorporation without causing cytotoxicity as assessed by lactate dehydrogenase release. Under the same conditions there was a concentration-dependent increase in the synthesis of prostaglandins E2,6-keto-PGF1 alpha and thromboxane B2. In contrast, unmodified LDL enhanced DNA synthesis at concentrations less than 40 micrograms/ml and had little effect on eicosanoid production. These results demonstrate that exogenous oxidized LDL inhibits mesangial cell proliferation and increases eicosanoid synthesis. Unmodified lipoprotein can be directly oxidized by these cells through mechanisms that involve generation of oxygen free radicals.

Modulation of scatter factor/hepatocyte growth factor activity by cell-substratum adhesion.

Scatter factor/hepatocyte growth factor (SF/HGF) is a multifunctional growth and motility factor whose activities vary with cell type. Here, the composition of the substratum was found to profoundly alter the scattering activities of SF/HGF, but not its mitogenetic effects, in MDCK cells. Whereas enhancement of DNA synthesis and induction of cell flattening by SF/HGF were independent of substratum composition (i.e. occurred on both fibronectin and vitronectin surfaces), colony dispersion as a result of cell separation fails to occur or is markedly reduced on surfaces where vitronectin is the major adhesive ligand. Prolonged exposure of non-scattering cultures to SF/HGF resulted in cells at colony margins producing long protrusions, which indicate that the motility of these cells is stimulated but 'frustrated' by the lack of breakdown of cell-cell adhesion. Scattering therefore appears to comprise two major components: increased motility and breakdown of cell-cell adhesion. The pathway leading to the breakdown of cell-cell contacts is modulated by downstream signals from extracellular matrix receptors. When cultured on immobilised fibronectin, vitronectin or a surface containing both, colony dissociation correlates with the presence of fibronectin, suggesting that positive signals from fibronectin receptors are required for SF/HGF-induced cell separation. Comparison of the findings in this study with those of a recent report on the modulation of SF/HGF-induced tubulogenesis by ECM (Santos, O. F. P. and Nigam, S. K. (1993) Dev. Biol. 160, 293-302), where vitronectin in type-1 collagen gels alters the pattern of SF/HGF-induced MDCK tubule formation from highly branched to long and unbranched, suggests that cell motility enhancement leads to tubule formation whereas the breakdown of cell-cell adhesion is required for tubule branching.

Signaling Mechanisms of Basic Fibroblast Growth Factor in Arterial Cells From Genetically Hypertensive Rat

The mechanisms of vascular structural alterations in hypertension were studied in cultured adventitial fibroblasts isolated from aortas of spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats. Basic fibroblast growth factor (bFGF)-, epidermal growth factor (EGF)-, or platelet-derived growth factor (PDGF)-induced DNA synthesis and phospholipase C activity were estimated by determining 3H-thymidine incorporation and 3H-inositol phosphate production, respectively. The role of protein tyrosine kinases was assessed by stimulating the cells in the presence of tyrphostin, a protein tyrosine kinase inhibitor. Both the mitogenic potency of bFGF, EGF, and PDGF and the phospholipase C activity elicited by these factors were increased markedly in SHR (v WKY) fibroblasts. SHR fibroblasts were significantly less sensitive to tyrphostin inhibition of bFGF-induced 3H-thymidine incorporation than WKY fibroblasts, whereas when the cells were stimulated with EGF, PDGF, or 5% serum, SHR and WKY fibroblasts were equally sensitive to tyrphostin inhibition. At doses that abolished bFGF-induced 3H-thymidine incorporation, tyrphostin did not affect bFGF-induced 3H-inositol phosphate production. These results indicate that in aortic fibroblasts phospholipase C activation is not sufficient for bFGF-induced DNA synthesis. They suggest that tyrosine kinase activation is a necessary step in the transduction of bFGF mitogenic signal and plays an important role in the enhanced DNA synthesis exhibited by SHR (v WKY) cells. Therefore, one may envisage that bFGF contributes, through paracrine/autocrine mechanisms, to the vascular smooth muscle hyperplasia/hypertrophy in SHR.

TNF accelerates the S-phase of the cell cycle in tumor cells

The reduction of glucose supply induced the killing of tumor cells by tumor necrosis factor (TNF) in vivo and in vitro. In contrast, normal cell lines were resistant to TNF regardless of the presence or absence of glucose. Epidermal growth factor (EGF) did not exert a cytotoxic effect on tumor cells in the absence of glucose. Therefore, the killing mechanism of TNF under conditions of reduced glucose supply was investigated. Flow cytometry experiments and studies of kinetics revealed that the S-phase of the cell cycle was prolonged in the absence of glucose. After TNF treatment, the S-phase was found to be shortened and the rate of 3H-thymidine incorporation into DNA was increased, whereas EGF failed to exert such an effect. DNA synthesis and entry into mitosis are known to be regulated by cyclin A. In serum-starved tumor cells (HeLa) we have observed increased cyclin A synthesis within 10 hr, in parallel with enhancement of DNA synthesis and shortening of the S-phase after TNF treatment. We conclude that, under conditions of low glucose supply, TNF can assume the role of a growth factor in transformed cells.

Hepatocyte Growth Factor Immobilized onto Culture Substrates through Heparin and Matrigel Enhances DNA Synthesis in Primary Rat Hepatocytes

Newly prepared phosphatidylethanolamine (PE) conjugates of glycosaminoglycans can be immobilized to solid phase through hydrophobic interaction. Primary rat hepatocytes were cultured on type I collagen or laminin substrates containing heparin-PE and assayed for DNA synthesis initiated by hepatocyte growth factor (HGF). Hepatocytes responded to HGF that had been added to culture media by active DNA synthesis and pronounced cell spreading, regardless of the presence of heparin-PE on the substrates. The preincubation with HGF, but not with epidermal growth factor, of the substrates containing heparin-PE significantly enhanced DNA synthesis and cell spreading in the absence of these mitogens in culture media. The enhancements were abolished by omitting heparin-PE or washing the HGF-treated substrates with 1 M NaCl, suggesting the immobilization of HGF to substrates through heparin. These phenomena were more evident with laminin substrates than with type I collagen substrates. Chondroitin sulfate-PE only partly substituted for heparin-PE, but Matrigel gave results similar to those of the substrates containing heparin-PE. Both type I collagen and laminin substrates containing heparin-PE bound higher amounts of HGF than the respective control substrates, whereas neither of the substrates containing chondroitin sulfate-PE did. However, the increase in HGF binding to the substrates was not as evident as the increase in DNA synthesis, suggesting that the latter is not simply due to the former but due to a concerted action of the immobilized HGF and heparin. Taken together, these results suggest that HGF can be trapped in extracellular matrix, probably through heparan sulfate in vivo, thereby acting as a mitogen for hepatocytes in cooperation with heparan sulfate.

Glutamic acid potentiates hepatocyte response to mitogens in primary culture.

Adult rat hepatocytes in primary culture responded to epidermal growth factor (EGF) by increased DNA synthesis. When hepatocytes were cultured in Leibovitz L-15 medium, their response to EGF was low compared with that in Williams' medium E or Koga's medium L. Furthermore, female rat hepatocytes showed almost no response to the mitogenic action of EGF compared with male rat hepatocytes in L-15 medium. Addition of glutamic acid (1-20 mM) to EGF-containing L-15 medium not only enhanced DNA synthesis > tenfold in both male and female hepatocytes, but eliminated the sex differences in DNA synthesis. Aspartic acid, glutamine, or ornithine at 20 mM did not replace the glutamic acid effect on DNA synthesis. Proline also enhanced EGF-induced DNA synthesis, although it was less effective than glutamic acid. Therefore, this effect may be specific to a high concentration of glutamic acid. Glutamic acid by itself did not stimulate DNA synthesis at any concentrations tested. In the presence of glutamic acid, EGF showed a dose-dependent (0.5-20 ng/ml) stimulation of DNA synthesis with a maximal effect at 10 ng/ml. Almost the same effect was obtained with transforming growth factor alpha (0.5-20 ng/ml). Glutamic acid also induced an expansion of the mitogenic action of angiotensin II. Since glutamic acid did not affect [125I]EGF binding to hepatocytes or its processing, the effect may occur internal to the receptor. These results suggest that glutamic acid modulates the sensitivity of the hepatocyte response to mitogens.

Antiproliferative effect of prostaglandin E2 in cultured guinea pig tracheal smooth muscle cells.

The respiratory epithelium plays an important role in modulating airway smooth muscle responsiveness to contractile agonists, and damage of the epithelium may be involved in the pathogenesis of bronchial hyperresponsiveness. This study was undertaken to determine whether prostaglandin E2 (PGE2), a relaxant agent synthesized and released by airway epithelial cells, could exert long-term effects on airway smooth muscle by regulating cell proliferation. Incubation of growth-arrested tracheal smooth muscle cells with serum for 24 h stimulated DNA synthesis in a concentration-dependent manner, as determined by [3H]thymidine incorporation. PGE2 and forskolin, a direct activator of adenylate cyclase, inhibited serum-induced cell proliferation, and the effects were dose dependent. PGE2 and forskolin also stimulated adenosine 3',5'-cyclic monophosphate accumulation. An inhibitor of cyclooxygenase, indomethacin, enhanced DNA synthesis induced by serum. These results indicate that exogenous PGE2 exerts an antiproliferative effect on smooth muscle cells in culture by activation of adenylate cyclase and suggest a role for the epithelium in modulating airway smooth muscle growth.

Keratinocyte Growth Factor and Fibroblast Growth Factor Action on DNA Synthesis in Rat and Human Hepatocytes: Modulation by Heparin

Keratinocyte growth factor (KGF/FGF-7) is a member of the fibroblast growth factor (FGF) superfamily. Unlike other members of the family, the biological activity of KGF appears to he restricted to epithelial cells. Here we have tested the activity of KGF, acidic fibroblast growth factor (aFGF), and basic fibroblast growth factor (bFGF) on normal adult rat and human hepatocytes and their modulation by heparin. Although more modest than the growth response to epidermal growth factor (EGF) and hepatocyte growth factor (HGF), recombinant KGF enhanced DNA synthesis in rat hepatocytes by two- to threefold. This stimulation occurred in the absence of serum and of other exogenous growth factors. Addition of heparin inhibited the KGF response. Although basic FGF showed little activity on rat hepatocytes, acidic FGF stimulated DNA synthesis by approximately twofold and was substantially enhanced by heparin. In contrast to rat cells, human hepatocytes consistently failed to respond to KGF, aFGF, or bFGF with or without heparin, under conditions where EGF and HGF stimulated DNA synthesis up to sixfold. These results indicate that KGF is capable of acting as a complete mitogen for rat hepatocytes in culture and that the activity is consistent with expression by these cells of a type II FGF receptor subtype, the KGF receptor. These observations suggest that KGF/aFGF together with proteoglycans may help regulate rat but not human liver growth.

Comitogenic effects of estrogens on DNA synthesis induced by various growth factors in cultured female rat hepatocytes

Ethinyl estradiol is a weak complete carcinogen and potent tumor promoter. In vivo, ethinyl estradiol causes a rapid but transient increase in liver growth, whereas in cultured female hepatocytes it enhances DNA synthesis induced by epidermal growth factor and is thus classified as a comitogen. The objectives of this study were to determine: (a) whether estradiol also has comitogenic activity; (b) whether the comitogenic effects of estrogen extend to complete hepatic mitogens other than epidermal growth factor and (c) whether inhibition of hepatocyte DNA synthesis by transforming growth factor-beta can be blocked by estradiol. Female rat hepatocytes in primary culture were exposed to estradiol with and without various growth factors, and we determined DNA synthesis by measuring [3H]thymidine incorporation into extracted DNA or by determining the nuclear labeling index by means of autoradiography. The results show that estradiol, like ethinyl estradiol, has comitogenic activity for epidermal growth factor, although it is somewhat less potent. Four complete hepatic mitogens showed different abilities to stimulate DNA synthesis, with hepatocyte growth factor > transforming growth factor-alpha > epidermal growth factor > acidic fibroblast growth factor. Estradiol enhanced DNA synthesis occurring in response to all four of these complete hepatic mitogens. This finding suggests that the mechanism of estrogen comitogenesis may involve effects at a point where the different signal-transduction pathways leading from the growth factors converge. The level of estrogen-mediated enhancement of DNA synthesis was similar for all four mitogens, ranging from 1.5 to 2.2-fold, depending on the experiment. Furthermore, determination of the nuclear labeling index showed that estrogen enhancement of DNA synthesis was associated with an increase in the percentage of hepatocytes that responded to the growth factors. Finally, estradiol did not specifically block the growth-inhibitory effects of transforming growth factor-beta.

Lymphokine-Activated Killer Cells Induce Differentiation in MCF-7 Breast Carcinoma Nodules but Not in Mastosis Nodules Maintained in Three-Dimensional Culture

In order to better understand the interaction between activated lymphocytes and breast carcinoma cells, we studied the degree of infiltration, the membrane contacts established and their cytostatic and cytolytic effects in MCF-7 nodules maintained in three-dimensional culture. A comparison was made with nodules of a nonmalignant, immortalized mastosis cell line. Histological, immunohistochemical and electron microscopical observations were performed as well as DNA synthesis measurements in the two components of the coculture. The lymphokine-activated killer (LAK) cells adhered more frequently to the carcinoma nodules than to the mastosis nodules. They actively penetrated into both of them. The penetration remained peripheral, and only a few cells migrated more deeply. The LAK cells established close cell-to-cell contacts with the two types of nodules, and intercellular gaps were formed: damaged cells could be seen near the activated killer cells. In MCF-7 nodules, a 5-fold inhibition of proliferation occurred, and extensive necrotic zones developed; this was accompanied by a general tendency for glandular redifferentiation. In mastosis nodules, necrosis also developed but no cell differentiation occurred and proliferation was less inhibited (2 times). Interleukin-2 alone enhanced DNA synthesis in mastosis nodules but had no effect on MCF-7 nodules, and no extending necrosis could be seen in both types of nodules. The cytolytic effects of LAK cells combined with their redifferentiating effect in MCF-7 breast carcinoma nodules may be a useful indication for further breast cancer therapy research.