Inhibition of Replication in Functional Mouse Adrenal Tumor Cells by Adrenocorticotropic Hormone Mediated by Adenosine 3′:5′-Cyclic Monophosphate

Adrenocorticotropic hormone (ACTH) inhibited replication in functional adrenal tumor cells with a concomitant stimulation of steroidogenesis and a characteristic change of morphology from a flattened to a spherical type. [(3)H]Thymidine incorporation into DNA was inhibited by about 50% 6 hours after ACTH treatment. Both cyclic AMP and dibutyryl cyclic AMP inhibited [(3)H]thymidine incorporation and caused the characteristic morphological change noted with ACTH. The extent of stimulation of steroidogenesis and the amount of inhibition of [(3)H]thymidine incorporation in response to various doses of ACTH were closely related and both were in parallel with the concentration of cyclic AMP in the cells. Cyclic GMP and cyclic IMP did not inhibit [(3)H]thymidine incorporation significantly, and did not change the morphology of the cells. AMP inhibited [(3)H]thymidine incorporation into DNA and caused the characteristic morphological change. However, AMP did not increase the cyclic AMP content of the cells. CMP, GMP, and UMP showed a significant inhibition of [(3)H]thymidine incorporation into DNA, but the extent of the inhibition was much less than that with AMP. These nucleotides did not change the morphology of the cells.     

Cigarette smoke impairment of human lymphocyte function by inhibition of transglutaminase

The in-vitro and in-vivo effects of cigarette smoke were studied in human peripheral blood lymphocytes by applying a method for the capping of beta 2-microglobulin- or phytohaemagglutinin (PHA)-stimulated lymphocyte transformation (measured as (3H)thymidine incorporation) involving the transglutaminase pseudosubstrate monodansylthiacadaverine (MDTC), whose presence resulted in significantly reduced capping and (3H)thymidine incorporation in a concentration-dependent manner. The addition of dimethyl sulphoxide-soluble particles from cigarette smoke to lymphocytes in vitro significantly reduced the capping ability and the PHA-induced (3H)thymidine incorporation. Whereas no significant change in MDTC-dependent capping inhibition was seen in lymphocytes from smokers after 10 d abstinence from smoking. there was a marked decrease in (3H)thymidine incorporation in lymphocytes from smokers after smoking three cigarettes following 10 h abstinence. The tentative conclusion is that exposure to cigarette smoke, or smoke extract, impairs MDTC-dependent capping inhibition and PHA-stimulated lymphocyte transformation by transglutaminase inhibition.     

An antisense oligodeoxynucleotide to growth hormone messenger ribonucleic acid inhibits lymphocyte proliferation

The role of GH in lymphocyte proliferation was studied by examining the effect of an antisense oligodeoxynucleotide (ODN) complementary to GH mRNA. The results of these studies showed that antisense GH ODN treatment inhibits lymphocyte production of immunoreactive GH (irGH). Lymphocytes treated with the GH antisense ODN produced less irGH than did lymphocytes treated with control sense GH ODN. Antisense GH ODN-mediated inhibition of irGH production resulted in a decrease in lymphocyte proliferation. Cells with the antisense GH ODN had less (87%) incorporation of [3H]thymidine [( 3H]TdR) in both resting and Concanavalin-A-stimulated lymphocytes, whereas the incorporation of [3H]TdR in cells treated with a control ODN was not significantly affected. The effect of the antisense ODN on [3H]TdR incorporation was specific, since it could be reversed by hybridization competition with a complementary GH sense ODN or by the addition of exogenous rat GH. Collectively, the data indicate that lymphocytes synthesize and secrete irGH and that irGH produced by these cells can stimulate proliferation, suggesting that GH may play an autocrine/paracrine role in lymphocyte replication.     

Dexamethasone and 8-bromo-cyclic AMP depress the incorporation of [3H]thymidine into mouse condylar cartilage by different pathways

We examined whether cyclic AMP (cAMP) affects the incorporation of [3H]thymidine into cartilage cells and, if so, whether this action could be related to the inhibitory effect of glucocorticoid hormones on the growth of ossifying cartilage. Incorporation of [3H]thymidine into trichloroacetic acid-precipitable material by mouse cartilage was measured concomitantly with the concentration of cAMP. Dexamethasone (1 mumol/l) significantly (P less than 0.05) depressed the incorporation of [3H]thymidine. The cAMP analogue 8-bromo-cAMP (0.01-1 mmol/l) also depressed the incorporation of the radionucleotide in a dose-dependent fashion. When various concentrations of 8-bromo-cAMP were added with dexamethasone (1 mumol/l), no apparent changes took place compared with the effect of dexamethasone alone. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.2-1 mmol/l) elicited an inhibitory effect on [3H]thymidine incorporation and a stimulatory influence on cartilage cAMP concentrations. Dexamethasone, at doses (0.01-1 mumol/l) causing significant inhibition of [3H]thymidine incorporation, failed to increase cartilage levels of cAMP. It seems, therefore, that the depressive effect of dexamethasone on [3H]thymidine incorporation in condylar cartilage is not mediated through an increase of cAMP in the tissue.     

Alteration of Nucleoside Transport of Chinese Hamster Cells by Dibutyryl Adenosine 3′:5′-Cyclic Monophosphate

Cultured Chinese hamster ovary cells showed no significant change in generation time or fraction in the S-phase in the presence of 1 mM N(6),O(2')-dibutyryl adenosine 3':5'-cyclic monophosphate. Growth continued for at least two generations after expression of the morphological transformation induced by this cyclic AMP analog. Despite identical growth rates, apparent rates of DNA and RNA synthesis (incorporation of [(3)H]-thymidine or [(3)H]uridine) were reduced up to 15-fold in log phase by 1 mM cyclic nucleotide. [(3)H]Deoxycytidine incorporation was much less sensitive to dibutyryl cyclic AMP. Uptake studies with [(3)H]thymidine demonstrated an inhibition of transport rate dependent on the concentration of dibutyryl cyclic AMP in the growth medium. The rate of thymidine uptake at 1 degrees was decreased 21-fold by 1 mM cyclic nucleotide; half-maximal inhibition occurred at 6 muM. At 37 degrees , the pool size of acid-soluble thymidylate was strongly reduced by 1 mM cyclic nucleotide, and synergistic reduction of the pool size was found with 0.5 mM aminophylline. Phosphorylation of the acid-soluble intracellular label was unaffected by dibutyryl cyclic AMP. Inhibition of thymidine uptake is attributed to an observed decrease in thymidine kinase activity caused by growth in 1 mM dibutyryl cyclic AMP, and possibly to a simultaneous alteration in membrane permeability. Kinase-facilitated uptake of other metabolites may be regulated in a similar fashion by cyclic AMP.     

In vitro stimulation of lymphocytes of donors seronegative for Epstein-Barr virus by preparations of inactivated Epstein-Barr virus.

Heat-inactivated preparations of Epstein-Barr virus stimulated human lymphocytes as assayed by incorporation of [(3)H]thymidine. The inactivated Epstein-Barr virus stimulated the lymphocytes of all five seropositive donors, 11 of 14 seronegative donors (aged eight to 26 years), and none of 15 neonates. Control antigens prepared from a human lymphoid cell line devoid of the Epstein-Barr virus genome did not stimulate the lymphocytes of seronegative donors. Fetal calf serum at the concentration used for suspension of Epstein-Barr virus did not stimulate or only minimally stimulated the lymphocytes of seronegative donors. The reactivity of the histocompatibility antigens found on human lymphocytes was abolished by procedures used for inactivation of the virus.     

Enumeration of Activated Thymus-Derived Lymphocytes by the Virus Plaque Assay

Lymphocytes activated by antigens or mitogens acquire the capacity to replicate viruses, and the number of activated lymphocytes can be estimated by the virus plaque assay. Concanavalin A and pokeweed mitogen produced 33-fold and 17-fold increases in virus plaqueforming cells (V-PFC), respectively, above background, while lipopolysaccharide produced only a 2- to 3-fold increase. T (thymus-derived lymphocyte)-depleted lymphocyte populations, derived from anti-theta-treated or nude (arthymic) mouse spleens, failed to produce V-PFC after culture with concanavalin A or pokeweed mitogen. The present studies thus demonstrate that the virus plaque assay measures activated T-lymphocytes.A dissociation between the V-PFC response and cell proliferation was previously observed in antigen-stimulated cells cultured in the presence of mitotic inhibitors. In the present studies, while stimulation of CBA (H2(k)) lymphocytes by DBA/2 (H2(d)) cells produced high levels of thymidine incorporation, lymphocyte target-cell cytotoxicity, and V-PFC, stimulation of BALB/c (H2(d)) lymphocytes against DBA/2 (H2(d)) cells resulted in even higher levels of thymidine incorporation with a virtual absence of cytotoxic lymphocytes or V-PFC. These results indicate that proliferation is not a sufficient condition for permitting lymphocytes either to exert cytotoxicity on target cells or to replicate viruses, and suggest that there may be a correlation between the development of V-PFC and cytotoxic lymphocytes. They are consistent with the view that there are at least two functional subpopulations of T-lymphocytes.     

Activation of vascular smooth muscle cells by TNF and PDGF: overlapping and complementary signal transduction mechanisms

OBJECTIVE: Because tumor necrosis factor-alpha (TNF) has been implicated in the pathogenesis of vein graft neointimal hyperplasia, we sought to determine mechanisms by which TNF could induce proliferative and migratory responses in smooth muscle cells (SMCs). METHODS AND RESULTS: In rabbit jugulocarotid interposition vein grafts, SMCs expressed TNF as early as four days postoperatively. In rabbit aortic SMCs, TNF and platelet-derived growth factor (PDGF) elicited comparable migration (1.7-fold/basal), and their effects were partially additive. In contrast, while TNF failed to promote SMC [(3)H]thymidine incorporation alone, it doubled the [(3)H]thymidine incorporation observed with PDGF alone. To gain mechanistic insight into these phenomena, we found that TNF and PDGF each activated p38(mapk) equivalently in SMCs, but that PDGF was two to three times more efficacious than TNF in activating SMC extracellular signal-regulated kinases (ERK) 1 and 2 and phosphoinositide 3-kinase. However, only TNF activated NF kappa B. SMC [(3)H]thymidine incorporation that depended on TNF, but not PDGF, was abolished by overexpression of a dominant-negative I kappa B alpha mutant. Inhibition of ERK activation by U0126 reduced SMC migration stimulated only by PDGF (by 35%, P<0.05), but not by TNF. Inhibition of phosphoinositide 3-kinase by LY294002, however, significantly reduced both TNF- and PDGF-stimulated chemotaxis (by 38-54%, P<0.05). In contrast, both U0126 and LY294002 abolished SMC [(3)H]thymidine incorporation induced by either TNF, PDGF, or both agonists. CONCLUSIONS: In primary rabbit SMCs, TNF promotes migration and mitogenesis through signaling mechanisms that are both distinct from and overlapping with those employed by PDGF. TNF-induced SMC mitogenesis requires complementary co-stimulation with other growth factors.     

Distribution and virogenic effects of 5-bromodeoxyuridine in synchronized rat embryo cells.

Rat embryo cell cultures were synchronized by a double thymidine block. The DNA replication phase (S) was divided into an early, middle, and late period. Cell cultures in the early, middle, or late S phase were pulsed with 0.1 muM 5-bromo[(3)H]deoxyuridine (BrdU) or equimolar [(3)H]dT. DNA-DNA reassociation experiments of each sample revealed that [(3)H]BrdU was more concentrated in the intermediate repetitive than the repetitive or unique DNA sequences of the early and middle S phase. In contrast, [(3)H]dT was nearly uniformly jistributed throughout all nucleotide sequences during the entire S phase. synchronized rat cells were pulsed during various portions of the S phase with unlabeled 0.1 mM or 0.1 muM BrdU and examined for sytoplasmic immumofluorescence against the 30,000 molecular weight group-specific antigen (p30) of Friend mouse leukemia virus. Equally strong fluorescence was detected 12 hr later in cells treated with each concentration of BrdU. Furthermore, incorporation of BrdU during late S phase was suffieient to elicit maximal antigen expression.     

Inhibition of growth and guanylate cyclase activity of an undifferentiated prostate adenocarcinoma by an extract of the balsam pear (Momordica charantia abbreviata).

We have recently described the presence of a guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2] inhibitor (GCI) in an aqueous extract of the balsam pear (Momordica charantia abbreviata). Because the guanylate cyclase-cyclic GMP system is though to be involved in cell growth, DNA and RNA synthesis, and possible malignant transformation, we examined the effect of the aqueous extract containing GCI on an undifferentiated adenocarcinoma of the rat prostate and concanavalin-A-stimulated [3H]thymidine incorporation into cultured splenic lymphocytes, a process thought to be mediated by cyclic GMP. The results demonstrate that the extract of the balsam pear blocks both the growth of the rat prostatic adencarcinoma in vitro and [3H]thymidine incorporation into DNA. DNA histograms from flow cytometry indicated that the extract containing GCI inhibited in the G2 + M phase of the cell cycle, a presumed locus of cyclic GMP effects. In addition, guanylate cyclase activity was significantly greater in the tumor than normal prostate tissue and was decreased by the extract containing GCI. Cyclic GMP levels in the tumor in culture wer also decreased by addition of the extract. It remains to be determined whether or not the anti-tumor agent and GCI are the same substance.     

Membrane potential changes during mitogenic stimulation of mouse spleen lymphocytes

By monitoring differences in accumulation of the lipophilic cation [³H]tetraphenylphosphonium in media containing low or high potassium concentrations [Lichtshtein, D., Kaback, H. R. & Blume, A. J. (1979) Proc. Natl. Acad. Sci. USA 76, 650-654], the membrane potential of lymphocytes from various sources has been estimated. On the basis of this method, the potential of normal mouse spleen lymphocytes (T and B cells) is -65 ± 2 mV (mean ± SEM, interior negative). During the course of mitogenic stimulation by concanavalin A, lipopolysaccharide, or fetal calf serum, the membrane potential of murine spleen lymphocytes changes systematically according to the following pattern: (i) early depolarization lasting 2-3 hr, (ii) repolarization over the next 7 hr, and (iii) a final hyperpolarization phase during the last 24-48 hr. During repolarization and hyperpolarization, moreover, there is a direct correlation between the membrane potential and DNA synthesis, as judged by [³H]thymidine incorporation. By using isolated T and B cells, it is observed that concanavalin A depolarizes T cells only, whereas lipopolysaccharide depolarizes B cells only. Thus, both mitogens exhibit the same specificity for depolarization as for mitogenic stimulation. On the basis of these observations, it is suggested that the transition of lymphocytes from a resting state to mitotic activity is initiated by depolarization of the plasma membrane.     

An Inhibitor of Cell Proliferation Released by Cultures of Macrophages

Culture fluids from mouse peritoneal exudate cells inhibited [(3)H]thymidine incorporation by, and proliferation of, EL-4 leukemia cells, 3T3 cells, and mitogen-stimulated spleen lymphocytes. Inhibited EL-4 leukemia cells recovered their normal proliferative capacity when washed and incubated in normal medium. The inhibitory activity resided in a low-molecular-weight substance that could be absorbed by incubation with the tumor cells. This substance was dialyzable and resistant to tryptic digestion and phosphodiesterase treatment. The mononuclear phagocytes in the peritoneal exudate seemed to be the source of the inhibitor. The inhibitory material was found in the same amounts in exudates of normal mice or mice injected with peptone or infected with Listeria monocytogenes; spleen cells adherent to plastic released the inhibitor but in lesser amount. We suggest that this inhibitor may contribute to the deleterious effects found when various cells, including neoplastic ones, are cultured in the presence of macrophages.     

周期素激酶抑制剂p27在肿瘤坏死因子-α诱导系膜细胞增生中的作用

目的 研究周期素激酶抑制剂p27在肿瘤坏死因子－α（TNF－α）诱导系膜细胞（MC）增生中的作用。方法 采用蛋白印迹（Western杂交）方法测定MC裂解液p27蛋白水平。[^3H]胸腺嘧啶核苷（[^3H]TdR）测定MC增生的情况,观察p27反义寡核苷酸（ODN）转染对TNF-α刺激MC中的p27水平及其对增生程度的影响。结果 TNF－α（200000U/L）可使无血清培养24h的MC中的p27水平降低（P＜0.01）,同时使[^3H]TdR掺入增加（P＜0.01）;p27反义ODN转染可降低TNF－α刺激24h的MC中的p27水平（P＜0.01）,同时可使[^3H]TdR掺入增加更为明显（P＜0.05）。结论 p27水平降低可能在TNF－α诱导MC增生中起重要作用。     

Liver DNA synthesis promoter activity detected in human plasma from subjects with hepatitis

A liver DNA synthesis promoter activity was detected in human plasma from subjects with hepatitis. The assay procedure consisted of intraperitoneal injection into mice of aliquots of plasma, previously chromatographed on Sephadex G-25. After 24 hr, [3H]thymidine was injected and its incorporation into liver DNA measured. The increase in [3H]thymidine uptake of injected mice was not detected in those administered plasma from normal subjects (basal [3H]thymidine incorporation was that corresponding to saline-injected mouse values). At a maximal effective dose (0.3 mg protein per mouse), plasma from subjects with hepatitis increased the mitotic index of mouse liver hepatocytes; at the same dose, plasma from normal subjects had no effect. This DNA synthesis promoter activity appears to be a protein, as it is sensitive to trypsin digestion and heat.     

Influence of reproductive activity, sex steroids, and seasonality on epigonal organ cellular proliferation in the skate (Leucoraja erinacea)

In elasmobranchs, the epigonal organ, a unique leukopoietic immune tissue, is associated with the gonads. As the ovaries increase in size during reproductive activity, the overall mass of the epigonal organ does not change. However, immunohistochemistry (proliferating cell nuclear antigen Ab) demonstrated more proliferative activity and extravasation of epigonal leukocytes from blood vessels in reproductively active (RA) skates (Leucoraja erinacea) than in non-reproductively active (NRA) skates. In addition, [(3)H]thymidine incorporation was greater in epigonal leukocytes from RA skates than in leukocytes from NRA skates. Plasma from RA skates, but not from NRA skates, increased proliferation of epigonal leukocytes in vitro, an effect that was not seen using steroid-free plasma. In contrast to the stimulatory effect of plasma on leukocyte proliferation, addition of steroids (estrogen, progesterone, testosterone, and dexamethasone) in vitro decreased [(3)H]thymidine incorporation. While the inhibitory response to steroids was seasonally variable, (3)[H]thymidine incorporation was always highest in RA animals, in which plasma steroid levels were also consistently highest. These studies suggest functional interactions between reproductive and immune tissues in the skate, and that cellular turnover in epigonal tissue may be influenced by gonadal activity.     

Monoclonal antibody to the type I insulin-like growth factor (IGF-I) receptor blocks IGF-I receptor-mediated DNA synthesis: clarification of the mitogenic mechanisms of IGF-I and insulin in human skin fibroblasts.

Insulin and insulin-like growth factor type I (IGF-I) stimulate an overlapping spectrum of biological responses in human skin fibroblasts. Although insulin and IGF-I are known to stimulate the incorporation of [3H]thymidine into DNA in these cells, the identity of the receptor(s) that mediates this effect has not been fully clarified. The mouse anti-human IGF-I receptor antibody alpha IR-3 binds with specificity to IGF-I but not to insulin receptors in human placental membranes; it also specifically inhibits the binding of 125I-labeled IGF-I but not 125I-labeled insulin to suspensions of human skin fibroblasts in a dose-dependent manner. alpha IR-3 competitively inhibits IGF-I-mediated stimulation of [3H]thymidine incorporation into DNA. This inhibition is dependent on the concentration of alpha IR-3 and in the presence of a fixed antibody concentration can be partially overcome by high concentrations of IGF-I. In contrast, at concentrations of less than 1 microgram/ml, the effect of insulin to stimulate [3H]thymidine incorporation is not inhibited by alpha IR-3. However, the incremental effects of higher concentrations (greater than 1 microgram/ml) of insulin on [3H]thymidine incorporation are inhibited by alpha IR-3. alpha IR-3 is a highly specific antagonist of IGF-I receptor-mediated mitogenesis in human skin fibroblasts. By using this antibody, it is shown directly that insulin can act through the IGF-I receptor to stimulate DNA synthesis but can also activate this effect through the insulin receptor itself.     

Induction of Mitochondrial DNA Synthesis in Monkey Cells Infected by Simian Virus 40 and (or) Treated with Calf Serum

Infection of confluent monolayer cultures of African green monkey kidney cells with simian virus 40 results in an enhanced synthesis of nuclear and mitochondrial DNA. This is demonstrated both by an increased rate of incorporation of [(3)H]thymidine into mitochondrial DNA and by detection of increased amounts of mitochondrial DNA in infected cells. With monkey BSC-1 cells, where SV40 infection does not result in a stimulation of nuclear DNA synthesis, no stimulation of mitochondrial DNA synthesis is observed. SV40 infection of mouse 3T3 cells also stimulates nuclear and mitochondrial DNA synthesis.     

Increased thymidine incorporation into fetal rat cartilage in vitro in the presence of human somatomedin, epidermal growth factor and other growth factors

The incorporation of [3H]thymidine by rat costal cartilage in vitro was studied at different fetal and postnatal ages and the effect of partially purified human somatomedin, mouse epidermal growth factor, platelet secretion products, insulin and growth hormone on thymidine uptake by fetal cartilage was examined. Thymidine uptake in plasma-free medium was many times greater in late fetal life than after birth. The incorporation of [3H]thymidine into costal cartilage from 21-day fetuses was significantly (P less than 0.05) increased above control values in the presence of 10 micrograms somatomedin/1, and when cartilage was incubated in medium containing somatomedin and diluted human plasma there was a synergistic action. Epidermal growth factor at a concentration of 1 ng/l was a potent stimulator of thymidine uptake. Secretion products from human platelets after their aggregation by thrombin stimulated [3H]thymidine uptake at a concentration of 2% (v/v), but were inhibitory at high concentrations. High concentrations of platelet secretion products stimulated the incorporation of [35S]sulphate by cartilage. A pharmacological concentration of 10 mu. insulin/ml stimulated [3H]thymidine uptake, but not concentrations of 1 or 100 mu./ml. Growth hormone had no effect. The results showed that fetal cartilage had a greater endogenous mitogenic activity than postnatal cartilage. While somatomedins may be important in the regulation of fetal body growth, other protein growth factors also stimulate fetal skeletal tissues.     

Tri-iodothyronine induces proliferation in cultured bovine thyroid cells: evidence for the involvement of epidermal growth factor-associated tyrosine kinase activity

The effects of the tri-iodothyronine (T(3)) secreted by thyroid cells on the growth of the thyrocyte are poorly known. In this study we analyzed the effects of T(3) on the proliferation of bovine thyroid follicles in primary culture previously depleted of endogenous T(3). Cellular deoxiribonucleic acid (DNA) synthesis, determined by [(3)H]thymidine incorporation, was stimulated by T(3) (0.1-5.0 nM) for 24 h in a concentration-dependent fashion with a maximal effect at 1.0 nM T(3) (P<0.01). This T(3) action was time-dependent when assayed from 12 to 72 h. The induction of mitogenic activity was corroborated by the increase in proliferating cell nuclear antigen (PCNA) measured by Western blot analysis. PCNA increased after treatment with T(3) (0.1-5.0 nM) in a concentration-dependent manner. Since T(3) modifies the activity of growth factors whose actions are mainly mediated by tyrosine kinase (TK) activation in diverse cellular types, we assayed the effects of genistein, a general TK inhibitor, and tyrphostin A25, a specific epidermal growth factor (EGF)-receptor (EGFR)-dependent TK activity inhibitor, on the proliferative effects of T(3). The T(3)-induced [(3)H]thymidine incorporation was inhibited by both agents in a concentration-dependent manner. A significant increase in the total TK activity measured in cellular protein extracts was induced by 0.5 and 1.0 nM T(3) (P<0.001). Tyrosine phosphorylation of the EGFR was also stimulated by T(3) (P<0.001) with no change in the EGFR expression as determined by Western blot analysis. Both, the T(3)-stimulated [(3)H]thymidine incorporation and the TK activity were inhibited by a anti-mouse EGF antibody. These results lead us to propose that T(3) could operate as a proliferative agent in bovine thyroid cells through a mechanism involving an autocrine/paracrine EGF/EGFR-dependent regulation.     

Influence of growth factors on an insulin-producing cell line (RINm5F)

The influence of IGF-I, insulin and epidermal growth factor (EGF) as well as glucose as control was studied on both growth and function of RINm5F cells, an insulin-producing cell line derived from rat insulinoma tissue. [3H]thymidine incorporation and DNA content (fluorometrically) were measured for estimating cell growth, insulin release and biosynthesis [( 3H]leucine incorporation) as parameters for cell function. There was a significant increase in both [3H] thymidine incorporation and DNA content into RIMm5F cells by glucose already at very low concentrations. IGF-I and high concentrations of insulin too were able to stimulate cell growth (insulin also in additional experiments with isolated rat islets). EGF, however, was without growth effect on this cell line (in contrast to previous own results in isolated islets). Insulin secretion and biosynthesis were also increased by very low glucose concentrations. IGF-I and EGF were ineffective regarding these functional parameters in RINm5F cells. Besides for glucose, our results demonstrate a role for IGF-I and insulin, but not for EGF, in regulating growth of these cells.