Vanadium ions stimulate DNA synthesis in Swiss mouse 3T3 and 3T6 cells.

Vanadyl sulfate and sodium orthovanadate in the concentration range between 5 and 50 microM are shown to be mitogenic for quiescent cultures of Swiss mouse 3T3 and 3T6 cells. The compounds caused a striking shift in the dose-response for the effect of serum on [3H]thymidine incorporation and DNA synthesis. In the absence of serum the effect of vanadium was greatly potentiated by insulin. Vanadium ions produced no more than additive increases in [3H]thymidine incorporation when combined with epidermal growth factor, cholera toxin, or phorbol 12-myristate 13-acetate. Both vanadium compounds stimulated ouabain-inhibitable 86Rb+ uptake, indicating that the vanadium ions increase, rather than inhibit, Na+/K+ pump activity in the intact cell. Neither vanadium compound had any effect on cellular cAMP under a variety of different conditions. The mitogenic effect of the vanadium compounds was similar to that of colchicine. Taxol, which stabilizes cytoplasmic microtubules, prevented the stimulation of DNA synthesis by vanadium.     

Effects of adenine nucleotides on the proliferation of aortic endothelial cells.

The effects of adenine nucleotides and adenosine on DNA synthesis and cell growth have been studied in bovine aortic endothelial cells (BAECs). ATP produced a small but significant (+44%) increase of the fraction of BAECs whose nuclei are labeled by [3H]thymidine. This mitogenic effect was mimicked by ADP, the phosphorothioate analogues ATP gamma S and ADP beta S, and the nonhydrolyzable analogue adenosine 5'-(beta, gamma-imido)triphosphate (APPNP), whereas adenosine 5'-(alpha, beta-methylene)triphosphate (APCPP), a selective agonist of P2x-purinoceptors, had no effect at 10 microM and a small one at 100 microM; this profile is consistent with the involvement of P2y-receptors. Adenosine induced a mitogenic response of a magnitude similar to that of ATP. This effect was not reproduced by R-phenylisopropyl adenosine, by 5'-N-ethylcarboxamide adenosine, or by 2',5'-dideoxyadenosine, selective ligands of the A1- and A2-receptors and the P site, respectively, nor was it inhibited by 8-phenyltheophylline, an antagonist of both A1- and A2-receptors. The mechanism of this adenosine action thus remains unclear. ATP and ATP gamma S did not enhance the proliferation of BAECs cultured in the presence of fetal calf serum concentrations ranging from 0.5% to 10%. They inhibited the growth-promoting effect of basic fibroblast growth factor; among the various nucleotides tested, APCPP was the least effective to reproduce the action of ATP, suggesting the possible involvement of P2y-receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclic AMP: a mitogenic signal for Swiss 3T3 cells.

Addition of cholera toxin (100 ng/ml) to quiescent cultures of Swiss 3T3 cells acts synergistically with serum (2-4%), insulin, phorbol esters, epidermal growth factor, and fibroblast-derived growth factor to stimulate DNA synthesis. In the presence of insulin, cholera toxin caused a dose-dependent increase in cumulative [3H]thymidine incorporation into acid-insoluble material and in the intracellular cyclic AMP (cAMP) level. The dose--response curves for the two processes were similar. Furthermore, addition of 1-methyl-3-isobutylxanthine (15--500 microM) or of 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (5--100 microM), both of which are potent inhibitors of cyclic nucleotide phosphodiesterase which are potent inhibitors of cyclic nucleotide phosphodiesterase activity, stimulated DNA synthesis and increased cAMP levels in Swiss 3T3 cells. These compounds strikingly potentiated the effect of cholera toxin on DNA synthesis and on cAMP levels. When quiescent Swiss 3T3 cells were exposed to cholera toxin (100 ng/ml) and insulin at 10 micrograms/ml (4- to 7-fold increase in cAMP level) or to these agents and 1-methyl-3-isobutyl xanthine at 50 microM (35-fold increase in cAMP level), DNA synthesis began after a lag of 16 hr. These results indicate that cAMP acts as a mitogenic signal for Swiss 3T3 cells and differ from the widely held view that cyclic AMP inhibits the proliferation of fibroblast cells.     

Adenosine 3',5'-monophosphate mediates both the mitogenic effect of thyrotropin and its ability to amplify the response to insulin-like growth factor I in FRTL5 cells

In previous studies we have demonstrated that bovine TSH (bTSH) and insulin-like growth factor I (IGF-I) independently stimulate both the incorporation of [3H]thymidine into DNA and replication in quiescent FRTL5 cells. In the case of TSH, evidence was presented that these responses are cAMP mediated. In addition, responses of thymidine incorporation are greatly amplified when particular concentrations of the two agents are added together, but this effect diminishes as the concentration of either bTSH or IGF-I is increased. The present experiments were undertaken to obtain further information concerning the mechanism of the independent mitogenic effects of bTSH and IGF-I and to explore the nature of the biphasic synergistic interaction with respect to thymidine incorporation that occurs when bTSH and IGF-I are added together. Verification that the increases in [3H] thymidine incorporation induced by bTSH and IGF-I, alone and together, are truly reflective of increases in DNA synthesis was obtained in experiments in which labeled nuclei were counted in cultures of cells grown in the presence of one or both mitogenic agents to which [3H]thymidine had been added. In these studies the number of cells with labeled nuclei was increased markedly by each of the two agents, and the response when the two mitogens were added together was far greater than the sum of their individual effects. Over a range of concentrations which included those that elicit a mitogenic response in FRTL5 cells, IGF-I, unlike bTSH, failed to increase cAMP generation when added alone. Moreover, IGF-I did not significantly enhance the cAMP response to varying concentrations of bTSH. A concentration-dependent increase in the incorporation of [3H]thymidine into DNA was induced by culturing cells in the presence of the cAMP analog (Bu)2cAMP (Bt2cAMP), the phosphodiesterase inhibitor isobutylmethylxanthine, and the stimulator of adenylate cyclase forskolin. When increasing concentrations of these agents were added together with IGF-I, a biphasic pattern of response of DNA synthesis, mimicking that produced by the combination of IGF-I and increasing concentrations of bTSH, was observed. Further evidence that cAMP mediates the mitogenic response to bTSH was the observation that adenosine inhibited the stimulation of both cAMP generation and DNA synthesis that bTSH produced. Although preincubation of quiescent FRTL5 cells for 24 h in the presence of bTSH resulted in only a small increase in DNA synthesis, measured during the last 3 h of a subsequent 24-h incubation carried out in the absence of bTSH, it greatly amplified the response to IGF-I added alone during the second incubation.(ABSTRACT TRUNCATED AT 400 WORDS)     

The epidermal growth factor-like domain of recombinant human thrombomodulin exhibits mitogenic activity for Swiss 3T3 cells

Thrombomodulin (TM) is an anticoagulant endothelial cell surface glycoprotein containing six tandem epidermal growth factor (EGF)-like structures. We prepared a recombinant TM peptide (rTME1-6, from R214GHWA to DSGK466 of native TM) composed of these six EGF-like structures and investigated the effect of rTME1-6 peptide on the growth of the Swiss 3T3 fibroblast cell line. It was found that rTME1-6 induced proliferation of Swiss 3T3 cells and accelerated [3H]thymidine uptake into their DNA. [3H]Thymidine uptake increased in a dose- dependent manner, plateauing at 50 ng/mL rTME1-6, which was 1.8 times the control level. rTME1-6 peptide (50 ng/mL) also accelerated the DNA synthesis of human dermal fibroblasts (HDFs), A549 (a human lung cancer cell line), HepG2 (a human hepatocarcinoma cell line), and U937 cells (a human monocytic cell line) to 1.5, 1.6, 1.4, and 1.2 times the control level, respectively. The magnitude of the acceleration of DNA synthesis in Swiss 3T3 induced by rTME1-6 was approximately 20% of that of EGF on a molar basis. The uptake of [3H]thymidine was accelerated synergistically by coculture of the cells with rTME1-6 and insulin, similar to the coculture with EGF and insulin. The effects of rTME1-6 were abolished by addition of polyclonal antihuman TM IgG, whereas the actions of insulin and EGF were not influenced. Glucose uptake in Swiss 3T3 cells also increased 1.6 times over control levels by culture with 50 ng/mL rTME1-6 (1.25 nmol/L), compared with 2.7 times by 10 ng/mL EGF (1.66 nmol/L). Binding of [125I]EGF (0.5 ng/mL, 0.083 nmol/L) by the cells was inhibited by about 60% by addition of an eight-fold molar excess of nonlabeled EGF (0.664 nmol/L), whereas no inhibition of [125I]EGF binding was observed, even in the presence of a 1,000-fold molar excess (83 nmol/L) of rTME1-6. Specific binding of [125I]rTME1-6 on the cells showed a saturation curve, and the apparent concentration of rTME1-6 required for half maximum binding of the peptide on the cells was calculated to be 31.5 ng/mL. Thus, the overall results indicated that the rTME1-6 peptide had mitogenic activity for Swiss 3T3 cells, accelerated DNA synthesis and glucose uptake, and that the mitogenic activity might be mediated by binding of the peptide to a specific site different from the EGF receptor.     

Growth inhibitory and stimulatory effects of retinoic acid on murine 3T3 cells.

All-trans-beta-retinoic acid (RA) has both comitogenic and antiproliferative effects on murine Swiss 3T3 cells. Treatment of quiescent 3T3 cells for less than 24 hr with micromolar concentrations of RA potentiates subsequent mitogenic response of those cells to phorbol 12-myristate 13-acetate. Longer exposures of 3T3 cells to RA result in inhibition of DNA replication as measured by [3H]thymidine incorporation and decreased growth rates and saturation densities for cells grown in either 2% or 10% serum. Both the comitogenic and antiproliferative activities of RA for 3T3 cells are RA-dose dependent. RA-induced decreases in the 3T3 cell saturation density are reversible only after resuspension of cells by trypsinization and replating. Treatment of 3T3 cells for 48 hr with RA inhibits the rate of [3H]thymidine incorporation by 35--50%, while autoradiographic data show that labeling indices are similar to control values. Equal percentages of control and 48-hr RA-treated quiescent 3T3 cells respond to subsequent stimulation with 10% serum as determined by autoradiographic and flow cytometric analyses. However, the progression of RA-treated cells through the S phase of the cell cycle is slowed. These data suggest that inhibition of 3T3 cell proliferation by RA is established after a minimum 24-hr treatment and that this inhibition is the result of a decreased rate of DNA replication in S-phase cells.     

Vasopressin stimulation of mouse 3T3 cell growth.

Vasopressin is shown to be a potent mitogen for Swiss 3T3 cells. The hormone (1--10 ng/ml) causes a striking shift of the dose--response curve for the effect of serum on thymidine incorporation by cultures of 3T3 cells arrested in the G1/G0 phase of the cell cycle. In the absence of added serum, the effect of vasopressin on DNA synthesis is greatly potentiated by insulin, epidermal growth factor, and a factor isolated from medium conditioned by simian virus 40-infected baby hamster kidney cells. The mitogenic effect of vasopressin is dependent on time and hormone concentration. In the presence of insulin, the half-maximal effect elicited by the peptide is obtained at 0.6 ng/ml. [Arg]Vasopressin and [Lys]vasopressin are equally potent. The vasopressins are 10(3)-fold more potent than oxytocin. In the presence of a low (2.5%) concentration of serum, vasopressins stimulate cell proliferation.     

Interaction of receptors for insulin-like growth factor I, platelet-derived growth factor, and fibroblast growth factor in rat aortic cells

Serum contains various growth factors which regulate the proliferation of cells. We investigated the growth of cultured arterial smooth muscle cells under the influence of insulin-like growth factor I (IGF I), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF), and examined the effect of these growth factors on the binding of [125I] IGF I and on the binding of [125I]PDGF to these cells. IGF I, FGF, and PDGF stimulated [6-3H]thymidine incorporation into DNA of confluent cultures of cells which were incubated in modified Dulbecco's modified Eagle medium. However, the effect of these growth factors on DNA synthesis was much more potent in Dulbecco's modified Eagle medium with 1% fetal calf serum. FGF and PDGF potentiated the growth-promoting effect of IGF I. The binding of [125I]IGF I to the cells was increased after a preincubation with FGF and PDGF. The binding was potently increased by FGF (100 ng/ml) after a preincubation time of 30 min. There was an increase in binding during the first 3 h of preincubation followed by a decrease after 4-5 h. PDGF (10-1000 ng/ml) stimulated [125I]IGF I binding only after 2 h of preincubation. The stimulation was dose dependent. Maximal stimulation of the binding was observed after 3 h of preincubation followed by a decrease after 4-5 h of preincubation. Specific binding sites for PDGF on smooth muscle cells could be demonstrated too. A preincubation of confluent cells with IGF I caused a dose-dependent increase in [125I]PDGF binding. These results support the hypothesis that the regulation of the binding of a specific growth factor by a second growth factor is important for the control of cell growth.     

脂质过氧化对血管内皮细胞源性生长因子合成和基因表达的影响

本研究以氢过氧化枯烯作为脂质过氧化反应的引发剂，作用于培养的牛主动脉内皮细胞，制备内皮细胞条件培养液，测定基对Ｓｗｉｓｓ３Ｔ３细胞ＤＮＡ合成的影响；并以抗血小板源性生长因子抗体中和实验和Ｎｏｒｔｈｅｒｎｂｌｏｔ分析测定了脂质过氧化作用对内皮细胞ＰＤＧＦ产生的影响。     

Adenosine has divergent effects on deoxyribonucleic acid synthesis in FRTL5 cells: inhibition of thyrotropin-stimulated and potentiation of insulin-like growth factor-I-stimulated thymidine incorporation

Adenosine inhibits TSH-stimulated [3H]thymidine incorporation into DNA in FRTL5 thyroid follicular cells by both inhibiting cAMP generation and acting at a locus beyond adenylate cyclase. On the other hand, adenosine markedly potentiates DNA synthesis in FRTL5 stimulated by insulin-like growth factor-I (IGF-I). The mechanisms of this latter effect are unknown, but require the coincubation of adenosine and IGF-I and not mediated by an increase in intracellular cAMP concentration. Adenosine increases the maximal response of FRTL5 to [3H]thymidine incorporation stimulated by IGF-I and increases the sensitivity of FRTL5 to IGF-I. These effects of adenosine are reflected by an increase in nuclear labeling as well as by an increase in [3H]thymidine incorporation into DNA. Adenosine also plays a role as an autocrine growth factor in FRTL5, since adenosine deaminase increases the response of these cells to TSH. The effects of adenosine on both TSH- and IGF-I-stimulated DNA synthesis are shared by guanosine and inosine, although with different potencies for the various guanine nucleosides. Inosine potentiates IGF-I-stimulated DNA synthesis, but inhibits TSH-stimulated DNA synthesis only weakly. Adenosine interacts with multiple receptors and with multiple postreceptor pathways in FRTL5 to produce divergent effects on the control of cell replication by two growth factors (TSH and IGF-I) that act through different postreceptor pathways.     

Alterations in adipogenic and mitogenic activity of porcine serum in response to hypophysectomy

The importance of the pituitary in postnatal regulation of peripheral preadipocyte proliferation and differentiation was examined by hormone supplementation of hypophysectomized pig serum in primary cultures of preadipocyte and stromal-vascular cells derived from rat inguinal adipose tissue. Hypophysectomized pig serum promoted at least 25% less preadipocyte proliferation, less differentiation of sn-glycerol-3-phosphate dehydrogenase activity, and less histochemical differentiation than serum from intact pigs. Porcine GH supplementation of hypophysectomized serum-stimulated [3H]thymidine incorporation by preadipocytes and stromal cells and also histochemical differentiation of preadipocytes, but not enzymatic differentiation. Insulin-like growth factor I (IGF-I) stimulated [3H]thymidine incorporation by preadipocytes and stromal cells. Enzyme differentiation by developing cells was stimulated by IGF-I. Hydrocortisone supplementation of hypophysectomized serum inhibited [3H]thymidine incorporation and stimulated enzymatic differentiation. Thyroid hormones (T3 and T4) stimulated [3H]thymidine incorporation by preadipocytes in a dose-responsive manner when supplemented to hypophysectomized serum. Thyroid hormones stimulated differentiation of enzyme activity at the lowest concentrations examined. The mitogenic effects of GH, IGF-I, and T4 were not specific to the preadipocyte population, since the stromal-vascular cells responded in a similar manner. However, hypophysectomy resulted in a specific reduction in preadipocyte proliferation while stimulating multiplication of stromal-vascular cells. These results suggest that these hormones are nonspecific mitogens in adipose tissue, while unidentified factors of pituitary origin may be important for the specific regulation of proliferation of preadipocytes. Additionally, hypophysectomy appears to remove mitogenic inhibitors that are specific for the stromal-vascular cells.     

Insulin-treated 3T3-L1 adipocytes and cell-free extracts derived from them incorporate 32P into ribosomal protein S6.

Differentiated 3T3-L1 preadipocytes preincubated for 60 min with 32Pi incorporate 32P into ribosomal protein S6 within 5 min after exposure to 0.1-1.0 nM insulin. Undifferentiated 3T3-L1 cells, which possess only 3-5% of the high-affinity cell surface insulin receptors present on the differentiated cells, are less sensitive to this stimulation. Under the same conditions used for insulin, epidermal growth factor, antibody to the insulin receptor, and a combination of isoproterenol and 1-methyl-3-isobutylxanthine also promote 32P incorporation into S6 in the differentiated cells although less effectively than insulin. Cell-free extracts derived from cells treated for 5-10 min with either physiological concentrations of insulin or epidermal growth factor (0.1 microgram/ml) reflect intact cells and catalyze the incorporation of 32P from exogenous [gamma-32P]ATP into ribosomal protein S6.     

Microinjected pBR322 stimulates cellular DNA synthesis in Swiss 3T3 cells.

When pBR322 is manually microinjected into the nuclei of quiescent Swiss 3T3 cells it stimulates the incorporation of [3H]thymidine into DNA. The evidence clearly shows that this increased incorporation that is detected by in situ autoradiography in microinjected cells represents cellular DNA synthesis and not DNA repair or plasmid replication. The effect is due to pBR322 and not due to impurities, mechanical perturbances due to the microinjection technique, or aspecific effects. This stimulation is striking in Swiss 3T3 cells. Some NIH 3T3 cells show a slight stimulation, but hamster cells, derived from baby hamster kidney (BHK) cells, are not stimulated when microinjected with pBR322. The preliminary evidence seems to indicate that the integrity of the pBR322 genome is important for the stimulation of cellular DNA synthesis in quiescent Swiss 3T3 cells. These results, although of a preliminary nature, are of interest because they indicate that a prokaryotic genome may alter the cell cycle of mammalian cells. From a practical point of view the stimulatory effect of microinjected pBR322 on cellular DNA synthesis has a more immediate interest, because pBR322 is the vector most commonly used for molecular cloning and 3T3 cells are very frequently used for gene transfer experiments.     

Platelet-derived growth factor and multiplication-stimulating activity II, but not multiplication-stimulating activity III-2, stimulate [3H]thymidine and [35S]sulphate incorporation by fetal rat costal cartilage in vitro

The actions of partially purified porcine platelet-derived growth factor (PDGF) and highly purified multiplication-stimulating activity (MSA) II and MSA III-2, which are somatomedins, were investigated on the incorporation of [3H]thymidine and [35S]sulphate by fetal rat costal cartilage in vitro. This was compared with their effects in the presence of 1% fetal calf serum (FCS) on the uptake of thymidine by growth-arrested fetal rat fibroblasts. Platelet-derived growth factor at concentrations of 0.21-21 micrograms/l enhanced the incorporation of both isotopes by fetal cartilage in the presence of 1% FCS, but had an inconsistent action on thymidine uptake and no significant action on sulphate uptake in serum-free medium. Platelet-derived growth factor promoted thymidine uptake by growth-arrested, isolated fetal rat fibroblasts. Multiplication-stimulating activity II (10-100 micrograms/l) stimulated the uptake of thymidine and sulphate by fetal cartilage in medium containing 1% FCS but had no consistent action in serum-free medium, although MSA II and PDGF had a synergistic effect on thymidine uptake in the absence of serum. Multiplication-stimulating activity III-2 had no consistent action on thymidine or sulphate incorporation by fetal cartilage in either serum-free or serum-supplemented medium. However, the same preparation of MSA III-2 stimulated the uptake of [3H]thymidine into fetal rat fibroblasts with a half-maximal response at a concentration of 5-10 micrograms/l. The results identify PDGF as a possible mitogenic agent for fetal rat connective tissues in vitro and show a differential sensitivity of fetal cartilage to MSA peptides.     

Platelet-derived growth factor stimulates tyrosine-specific protein kinase activity in Swiss mouse 3T3 cell membranes.

Platelet-derived growth factor (PDGF) stimulates the incorporation of 32P from [gamma-32P]ATP into a Mr approximately 170,000 protein by an endogenous tyrosine-specific protein kinase in membrane preparations of Swiss mouse 3T3 cells. Epidermal growth factor (EGF), but not fibroblast growth factor (FGF) or insulin, stimulates limited incorporation of 32P into a protein of similar molecular weight. The ligand concentration required for half-maximal activity (S0.5) for PDGF stimulation of phosphorylation is 50 ng/ml; saturation is achieved at 300 ng/ml. The S0.5 for ATP is 15 microM. Mg2+ or Mn2+ is required for protein kinase activity. Stimulation of PDGF results in the preferential phosphorylation of tyrosine residues in this Mr approximately 170,000 membrane protein. The Mr approximately 170,000 protein can be resolved into Mr approximately 180,000 and 160,000 components in 4% NaDodSO4 gels. PDGF stimulates 32P incorporation preferentially into the Mr approximately 180,000 and less extensively into the Mr approximately 160,000 protein. EGF stimulates 32P incorporation predominantly into a protein of Mr approximately 160,000. The similarity of PDGF and EGF in stimulating phosphotyrosine-specific protein kinase activity and the stimulation of a similar activity by viral transformation (src) genes suggest that a common mechanism may exist for the phenotypic expression of increased DNA synthesis and cell growth stimulated by these separate factors.     

Glucagon-like peptide 2 stimulates intestinal epithelial proliferation in vitro

Glucagon-like peptide 2 (GLP-2) is a potent intestine-specific growth factor. To determine whether GLP-2 has a direct effect on cellular proliferation, we examined the effects of GLP-2 on three different intestinal epithelial cell lines: Caco-2, T84, and IEC-6. Proliferation was assessed by measuring [³H]thymidine incorporation into cellular DNA. The expression of the cell cycle regulators cyclins D1, E, and A was determined by western blotting. GLP-2 stimulated [³H]thymidine incorporation in Caco-2 and T84 cells but not in IEC-6 cells. In Caco-2 cells, increased [³H]thymidine incorporation was detectable by day 1 and was maximal at day 5 following initiation of daily GLP-2 administration. Increased [³H]thymidine incorporation persisted for three days following treatment with a single dose of GLP-2. GLP-2 administration resulted in the induction of cyclins D1, E, and A in Caco-2 cells. These results suggest that GLP-2 acts directly on human intestinal epithelial cells to stimulate their proliferation.     

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.     

Schwannoma-derived growth factor must be transported into the nucleus to exert its mitogenic activity.

Schwannoma-derived growth factor (SDGF) is a mitogen and neurotrophic protein which belongs to the epidermal growth factor (EGF) family. There are two basic amino acid clusters in the SDGF molecule which are homologous to the nuclear targeting signal of the simian virus 40-encoded large tumor antigen. Mutational analysis of these clusters showed that they function as nuclear targeting signals, and a gel retardation assay showed that SDGF binds to A+T-rich DNA sequences. Both the wild-type SDGF and a mutant defective in the nuclear targeting signals activate the immediate early genes NGFI-A and c-fos. The wild-type SDGF is a mitogen for Swiss mouse 3T3 fibroblasts, but the mutant defective in the nuclear targeting signals is not mitogenic. Moreover, wild-type SDGF potentiates [3H]thymidine incorporation in NIH mouse 3T3 cells bearing an EGF receptor defective in the kinase domain, whereas the mutant SDGF does not stimulate DNA synthesis. These results suggest that transport into the nucleus is required for SDGF to induce a mitogenic response.     

Enhancement of [3H-methyl]thymidine incorporation and replication of rat chondrocytes grown in tissue culture by plasma, tissue extracts and vasopressin.

A pituitary mitogenic peptide, which stimulates cellular replication of a variety of cells maintained in tissue culture, has been identified by other investigators. To study this mitogenic substance, we developed an assay to measure mitogenic substances utilizing fetal rat chondrocytes grown in monolayer culture. Mitogenic activity of added test substances was determined by [3H-methyl]thymidine incorporation into trichloroacetic acid insoluble cell products and increase in total cell number after 24 h exposure. Extracts of whole pituitary glands were more potent in stimulating these cellular indices than either those of liver or muscle, confirming that the chondrocytes are sensitive to the described mitogen. Identically prepared extracts of either anterior or posterior pituitary lobes were mitogenic indicating the presence of two or more mitogenic substances in crude pituitary extracts. Synthetic lysine vasopressin and a beef pitressin concentrate stimulated thymidine incorporation into chondrocytes in the absence of calf serum and this effect was additive to that of calf serum, suggesting that the mitogenic substance of posterior pituitary extracts was vasopressin. The maximum effective dose of vasopressin leading to an increase in either thymidine incorporation or total cell number was between 100 to 500 pg/ml, and as little as 50 pg/ml of hormone elicited an increase in total cell number. The mitogenic effect of both vasopressin and calf serum on chondrocytes was partially inhibited by 1 X 10(-4)M N, O'dibutryl cyclic adenosine 3',5' monophosphate suggesting that cell division of chrondrocytes may be under tonic control by the andenylyl cyclase system. We conclude that vasopressin is a potent mitogen for chondrocytes maintained in tissue culture and its presence must be rigorously excluded in evaluating mitogenic activity of pituitary or serum concentrates.     

Antitubulin agents enhance the stimulation of DNA synthesis by polypeptide growth factors in 3T3 mouse fibroblasts.

Colchicine and other antitubulin agents markedly enhanced the stimulation of DNA synthesis by combinations of various growth factors such as epidermal growth factor, insulin, fibroblast-derived growth factor, and vasopressin in serum-free cultures of several quiescent 3T3 mouse fibroblast cell lines. Enhancing effects were observed based on continuous incorporation of [3H]thymidine into DNA as well as by autoradiographic labeling of cell nuclei. The concentration of colchicine and podophyllotoxin required to produce half-maximal enhancement of DNA synthesis stimulated by epidermal growth factor and insulin was 25-50 nM. Lumicolchicine did not produce enhancing effects. The disassembly of microtubules resulting from the action of colchicine, Colcemid, and vinblastine did not inhibit the stimulation of DNA synthesis in quiescent Swiss 3T3 fibroblasts by fetal bovine serum. We conclude that the cytoplasmic microtubule network in 3T3 mouse fibroblasts does not exert a positive regulatory function in the initiation of DNA synthesis but rather can produce a constraint on the initial action of the peptide growth factors in serum-free media.