Action of multiplication-stimulating activity on [3H]thymidine incorporation in rabbit and human fetal chondrocytes in vitro

The mitogenic action of multiplication-stimulating activity (MSA) on normal mammalian chondrocytes has been examined. Addition of MSA (NIH, PkII-MSA, 2.5-500 ng/ml or Collaborative Research, CR-MSA, 50-250 ng/ml) to primary suspensions of chondrocytes prepared by enzymic digestion of costal and articular cartilage of rabbits (356-481 g body wt) resulted in a dose-dependent increase in [3H]thymidine incorporation into the trichloroacetic acid-precipitated cell contents. CR-MSA (50-250 ng/ml) also had a significant stimulatory effect on [3H]thymidine incorporation into human fetal chondrocytes (22 weeks of gestation) prepared by enzymic digestion. When PkII-MSA was added in the presence of 1.25% of a standard adult or cord plasma to either rabbit or human fetal (18 weeks) chondrocytes, the increase in [3H]thymidine incorporation appeared to be synergistic. The mitogenic action of MSA can thus be demonstrated on primary suspensions of mammalian chondrocytes. The action of MSA on human chondrocytes has not previously been reported.     

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.     

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.     

Growth factor control of rat thyroid follicular cell proliferation

We have investigated the proliferative responses of rat thyroid follicular cells in serum-free culture to a range of growth factors including TSH, epidermal growth factor, and insulin, added singly or in combination. Follicles released from normal thyroids by collagenase/dispase digestion were cultured in suspension in agarose-coated microtiter plates to prevent monolayer formation. Growth responses were measured by [3H] thymidine incorporation and by autoradiography over successive 24- or 36-h periods. Insulin, even in the absence of other growth factors, stimulated [3H]thymidine incorporation in a concentration-dependent manner, rising from basal levels of 486 +/- (SE) 18 cpm to 4222 +/- 367 cpm/5 X 10(4) cells at 8 micrograms/ml. In contrast, TSH alone had no effect. In the presence of threshold levels (0.08 micrograms/ml) of insulin, however, there was a highly significant (P less than 0.001) response to TSH, [3H]thymidine incorporation rising from 1089 +/- 163 cpm in the absence of TSH to a maximum of 7548 +/- 585 with 1 mU/ml TSH. There was a synergistic interaction between insulin and TSH over the concentrations tested. Epidermal growth factor either alone or in combination with insulin failed to produce a significant response. Parallel autoradiographic studies were concordant with the [3H]thymidine incorporation data. We conclude that whereas in the absence of other growth factors TSH is unable to stimulate DNA synthesis in isolated rat thyroid follicles, the inclusion of just a single growth factor, insulin, permits a marked response. These observations emphasize the need for inclusion of appropriate permissive growth factor(s) when assessing the in vitro effect of a suspected tissue-specific mitogen.     

Effects of triiodothyronine (T3) and identification of specific nuclear T3-binding sites in cultured human fetal epiphyseal chondrocytes.

The effects of T3 on cultured human fetal epiphyseal chondrocytes were assessed by studying its effects on DNA synthesis and alkaline phosphatase activity. DNA synthesis was evaluated as follows: after 48-h incubation in Ham's F-12 serum-free medium, cultured chondrocytes were incubated with or without T3 (0.1-100 nM) in MCDB-104 serum-free medium for different periods of time (2-10 days), with the addition of [3H]thymidine (5 microCi/mL) for the last 24 h. Confluent cultured chondrocytes in 25-cm2 tissue culture flasks were incubated in Ham's F-12 serum-free medium for up to 9 days with or without T3 (0.1-100 nM); the cellular cytoplasmic fraction was obtained, and alkaline phosphatase activity was evaluated using paranitrophenylphosphate as a substrate. No significant effects of T3 (0.1-100 nM) on DNA-[3H]thymidine incorporation were observed in any experiment (n = 17) for any gestational age (12-39 weeks) or for any incubation period studied (2-10 days). However, a significant (P less than 0.025 or more) stimulatory effect of T3 (0.1-100 nM) on alkaline phosphatase activity was observed after 9 days of incubation. This effect was highest for 5 nM T3 and was present in cultured chondrocytes from human fetuses of all ages studied (13-40 weeks). Cultured human fetal epiphyseal chondrocytes from human fetuses 12-40 weeks old (n = 8) showed specific nuclear binding sites for T3. The binding capacity was 27.14 +/- 2.84 fmol/100 micrograms DNA, and the Kd was 0.66 +/- 0.14 x 0.1 nM (mean +/- SEM), with no significant differences among fetal ages. In conclusion, our results show that T3 elicits a biological response in cultured human fetal epiphyseal chondrocytes and has specific nuclear binding sites. Since alkaline phosphatase is closely related to the mineralization of epiphyseal cartilage, these results suggest that thyroid hormones could regulate this process.     

Evaluation of the rat thyroid cell strain FRTL-5 as an in-vitro bioassay system for thyrotrophin

The cyclic AMP response to bovine TSH was characterized in a strain of rat thyroid follicular cells ( FRTL -5) maintained in continuous culture. Significant stimulation of intracellular cyclic AMP was attained at a TSH dose of 5 muu./ml. Cyclic AMP accumulation continued to increase, at higher TSH doses, with no evidence for attainment of a maximum level at the highest dose tested (5 mu./ml). The precision of TSH measurement was better than 10% over the range 50-5000 muu./ml, comparing favourably with that observed with analogous assays based on human cells, tissue slices or membrane preparations. Using sequential subcultures of FRTL -5 cells, the between-assay variation in response to a single dose of a standard preparation of bovine TSH (53/11; 370 muu./ml) was of the order of 20% which compared favourably with the between-assay variation observed with different cultures of human thyroid cells. Prolongation of the incubation of FRTL -5 cells with TSH to 3 h revealed a progressive increase in the extracellular accumulation of cyclic AMP. Addition of TSH to resting FRTL -5 cells resulted in a stimulation of inorganic iodide uptake with pronounced bell-shaped dose-response characteristics. Thus a maximum uptake was observed at a TSH dose of 100 muu./ml with a significant reduction at higher doses. Acute stimulation of cells with TSH (100 muu./ml) resulted in a rapid and marked alteration in cell morphology, with evidence of cellular retraction and surface ruffling.     

Effects of pregnancy hormones on pancreatic islets in organ culture

Pregnancy is associated with hyperinsulinaemia and decreased glucose tolerance which may lead to gestational diabetes. Sera from pregnant women in the last trimester were found to stimulate insulin secretion of mouse pancreatic islets maintained in organ culture suggesting a direct effect of pregnancy hormones and/or metabolites on the endocrine pancreas. Previous studies have shown that progesterone and human placental lactogen (hPL) had direct effects on isolated islets and in the present study the effects of combined addition of these hormones to the cultured islets were evaluated. hPL, 1 microgram/ml was found to prevent the decrease in the islet insulin content due to progesterone 100 ng/ml, and hPL induced an increase in the DNA-content compared with the progesterone treated islets. The effect of the hormones on the DNA synthesis was evaluated by incorporation of [3H]thymidine into newborn rat islets. Progesterone in a high concentration, 1 microgram/ml, inhibited while 0.1 and 1 microgram/ml hPL stimulated the [3H]thymidine incorporation. These results indicate that progesterone may contribute to the increased glucose-induced insulin release during pregnancy and hPL to the beta-cell hyperplasia. Since progesterone and hPL counteracted each other with regard to both insulin content and DNA-synthesis in the islets, the direct interaction between these two hormones on the beta-cell may be of importance for the changes in glucose metabolism during pregnancy.     

Regulation of amino acid uptake and deoxyribonucleic acid synthesis in isolated human fetal fibroblasts and myoblasts: effect of human placental lactogen, somatomedin-C, multiplication-stimulating activity, and insulin

We compared the abilities of human placental lactogen (hPL), somatomedin-C/insulin-like growth factor I (SM-C/IGF-I), multiplication-stimulating activity (MSA), and insulin to induce a rapid anabolic event, the uptake of the nonmetabolizable amino acid [3H]alpha-aminoisobutyric acid ([3H] AIB) or the more long term action of increasing [3H]thymidine incorporation, as a measure of DNA synthesis, in isolated human fetal fibroblasts and myoblasts. Myoblasts were derived from skeletal muscle and fibroblasts from skin explants removed from human fetuses delivered between 12 and 19 weeks gestation after prostaglandin-induced abortion. Each of the four peptides caused a dose-dependent increase in [3H]AIB uptake by both fibroblasts and myoblasts, with mean half-maximal concentrations (ED50) ranging from 0.9-1.9 nM. The concentration of each peptide required to stimulate [3H]thymidine uptake was significantly greater, with the exception of insulin, which was inactive. For myoblast cultures, the mean ED50 values were: hPL, 7.9 nM; SM-C/IGF-I, 2.0 nM; and MSA, 2.2 nM. For fibroblast cultures, the mean ED50 values were: hPL, 2.3 nM; SM-C/IGF-I, 3.3 nM; and MSA, 4.3 nM. Insulin did not stimulate [3H]thymidine incorporation into either cell type at concentrations up to 6.9 nM. Incubation in the presence of monoclonal antibody against SM-C/IGF-I abolished the ability of SM-C/IGF-I to stimulate either [3H]thymidine or [3H]AIB uptake into fetal fibroblasts. The antibody substantially inhibited the incorporation of [3H]thymidine by these cells in response to hPL, but was less effective in blocking hPL-stimulated [3H]AIB uptake. It did not inhibit the uptake of either radioisotope in response to MSA or [3H]AIB uptake in response to insulin. The actions of SM-C/IGF-I and hPL on thymidine incorporation were additive at submaximal concentrations, but not so at maximal individual concentrations. Their actions on AIB uptake were additive at both submaximal and maximal concentrations. The results suggest that hPL as well as the SMs may contribute to the growth stimulus in human fetal connective tissues. Since incubation with SM-C/IGF-I antibody reduced the mitogenic response of fetal cells to hPL, the actions on DNA synthesis may be partially mediated by local release of SM. However, the similar ED50 values with which these peptides stimulated [3H]AIB uptake during a short incubation, and their additive effects at maximal individual concentrations, suggest that hPL may also have direct actions.     

Comparison of the effect of interleukin 6 and interleukin 1 on collagenase and proteoglycan production by chondrocytes

In order to determine whether interleukin 6 (IL-6) is involved in the pathogenesis of the cartilage destruction observed in arthritis, the effect of human recombinant IL-6 on collagenase production and proteoglycan synthesis by bovine articular chondrocytes was examined. Addition of IL-6 (1.0 to 1000 U/ml) to the culture medium did not stimulate collagenase production or alter proteoglycan secretion. Whereas human purified interleukin 1 (IL-1) (20 U/ml) stimulated collagenase production and inhibited proteoglycan synthesis. Furthermore addition of IL-1 (20 U/ml) to chondrocyte cultures did not stimulate the chondrocytes to produce IL-6. Under our experimental conditions, IL-6 did not stimulate chondrocytes to proliferate as measured by [3H] thymidine incorporation. This would suggest that IL-6 is not involved in mediating cartilage loss.     

Repeatedly passed FRTL-5 rat thyroid cells can develop insulin and insulin-like growth factor-I-sensitive cyclooxygenase and prostaglandin E2 isomerase-like activities together with altered basal and thyrotropin-responsive thymidine incorporation into DNA

Repeatedly passed or aged rat FRTL-5 thyroid cells develop a high level of basal [3H]thymidine incorporation into DNA and a reduced response to TSH in medium containing 5% serum and insulin (5H medium). The basal [3H]thymidine incorporation into DNA of aged cells can exceed the TSH-induced increase in earlier passages of the same cell line (fresh cells) and the TSH response decreases from more than 10-fold above basal in fresh cells to less than 2-fold in aged cells. This change is not associated with a loss of the diploid karyotype, a change in basal cAMP levels, or a change in dependence on TSH for cell growth. Attenuation of the TSH response in the [3H]thymidine incorporation assay is more evident than the reduced effect of TSH on cAMP levels or iodide transport; moreover, the TSH effect on cAMP levels does not correlate with that on [3H] thymidine incorporation as a function of hormone concentration. The high basal activity in [3H]thymidine incorporation into DNA in aged cells is due to an increased responsiveness to insulin, insulin-like growth factor-I (IGF-I), or serum. Thus, removal of serum and insulin from the medium eliminates the high basal [3H]thymidine incorporation into DNA, and this activity is restored by insulin or IGF-I in a concentration-dependent manner. The increased responsiveness of aged cells to insulin or IGF-I is inhibited by indomethacin or hydrocortisone and is associated with insulin or IGF-I, but not TSH, stimulation of cyclooxygenase and prostaglandin E2 (PGE2) isomerase-like activity. Fresh cells, in contrast, require TSH plus insulin or IGF-I to increase these activities. Increased responsiveness of cyclooxygenase activity to insulin or IGF-I in aged cells reflects at least in part an increase in cyclooxygenase mRNA levels. We suggest that insulin/IGF-I stimulation of PGE2 production leads to the high basal thymidine incorporation into DNA in aged cells maintained in TSH-depleted (5H) medium; the reduced stimulation by TSH of cAMP content or iodide uptake may reflect PG inhibition (negative feedback regulation) of cAMP production.     

Interaction of insulin-like growth factor II with rat chondrocytes: receptor binding, internalization, and degradation

We have characterized the interaction of insulin-like growth factor II (IGF-II) with its plasma membrane receptor(s) on cultured rat chondrocytes. Our studies, paralleling those already reported for IGF-I, demonstrate that [125I]IGF-II binds to these receptors with a high degree of affinity and that this process is reversible, specific, and time, temperature, and concentration dependent. At 4 C, unlabeled IGF-II causes half-maximal displacement of the labeled ligand at a concentration of 22 ng/ml, whereas IGF-I is approximately 1/200th as potent, and insulin does not displace [125I]IGF-II even at a concentration of 10 micrograms/ml. Maximum binding to chondrocytes (44% of added radioactivity) occurred after 4-5 h of incubation at 15 C. Compared to [125I]IGF-I binding, this value is 7-fold higher and is consistent with an affinity constant (Ka = 3.8 X 10(8) M-1) approximately 1 order of magnitude greater. Photoaffinity labeling studies disclose that IGF-II binds primarily to the type II IGF receptor, which has an apparent mol wt of 220K when electrophoresed under nonreducing conditions and 270K under reducing conditions. Nanomolar concentrations of IGF-II stimulated the synthesis of DNA and RNA in a dose-related manner, and micromolar concentrations of insulin demonstrated an additive effect with respect to the incorporation of [3H]thymidine into DNA, but not [3H]uridine into RNA. Preincubation of rat chondrocytes with increasing concentrations of insulin caused a marked dose-related increase in [125I]IGF-II binding, a phenomenon previously reported in several other cell types. In addition to defining the binding characteristics of IGF-II, we used the lysosomotropic agents chloroquine and ammonium chloride to demonstrate that its ligand-receptor complex, like that of IGF-I, is internalized and degraded partially via the lysosomal pathway.     

Insulin and insulin-like growth factors stimulate deoxyribonucleic acid synthesis in PC12 pheochromocytoma cells

The effects of insulin and insulin-like growth factors (IGFs) on the replication of PC12 pheochromocytoma cells were investigated. Incubation of PC12 cells for 2-3 days in low (0.3%) serum medium decreased [3H]thymidine incorporation into PC12 cell DNA to approximately 30% of that in control (15% serum) medium. Incubation of the cells in low serum medium also slowed the growth of the cultures and increased the percentage of cells in the G0/G1 phase of the cell cycle. Addition of insulin to cells in low serum medium increased [3H]thymidine incorporation into the cells, increased the number of cells in PC12 cultures, and decreased the percentage of cells in the G0/G1 phase of the cell cycle. IGF-I and IGF-II also increased [3H]thymidine incorporation into PC12 cells incubated in low serum medium. IGF-I (EC50, approximately 0.3 nM) was a more potent stimulus of [3H]thymidine incorporation than was insulin (EC50, approximately 3.5 nM). These data suggest that insulin and IGFs are growth factors for PC12 cells, and that the growth-promoting effects of these agents may be mediated by a type I IGF receptor on PC12 cells.     

Proliferative response to cyclic AMP elevation of thyroid epithelium in suspension culture

The role of cyclic AMP (cAMP) as an intracellular growth signal in thyroid epithelial (follicular) cells has been studied using primary suspension cultures of rat thyroid follicles closely resembling the in vivo state. TSH at 0.1 mU/ml in the presence of insulin (0.08 microgram/ml) induced a 9.6-fold increase in [3H]thymidine incorporation with a peak after 48-72 h. The response to the cAMP elevating agent cholera toxin (10 ng/ml) was identical both in timing, magnitude and dependence on insulin. A lower amplitude response occurred with forskolin. The data further support the conclusion that elevation of intracellular cAMP concentration is a major, if not the only, signal required for the proliferative response of the follicular cell to its physiological mitogen, TSH.     

Somatomedin C-binding and action in fibroblasts from aged and progeric subjects

Aging is associated with diminished cell growth, which has been ascribed in part to decreased cellular responsiveness to serum mitogens. To investigate whether there is an age-related loss of responsiveness to somatomedin-C (SM-C), we studied SM-C binding and action in early passage fibroblasts from normal donors, aged 7-96 yr, and one progeric subject. SM-C stimulated [3H]thymidine incorporation 4- to 16-fold in young cells, 4- to 17-fold in aged cells, and 4- to 11-fold in progeric cells. SM-C was synergistic with 0.25% human hypopituitary serum in stimulating [3H]thymidine incorporation in all cell lines. Dose-response curves for SM-C stimulation of thymidine incorporation were not significantly altered in aged or progeric cells. Half-maximal responses occurred at 5-15 ng/ml SM-C for all cell lines. [3H]Thymidine incorporation results were supported by cell replication studies. In addition, binding of [125I] SM-C was virtually identical in all cell lines, with 50% displacement at 2-5 ng/ml SM-C. Thus, in vivo aging does not appear to be associated with either an alteration in SM-C receptors or a diminished cellular responsiveness to SM-C's mitogenic effects.     

Effects of tri-iodothyronine and insulin-like growth factor-I (IGF-I) on alkaline phosphatase activity, [3H]thymidine incorporation and IGF-I receptor mRNA in cultured rat epiphyseal chondrocytes.

The effects of tri-iodothyronine (T3) and insulin-like growth factor-I (IGF-I) on [3H]thymidine incorporation, alkaline phosphatase (ALP) activity and IGF-I receptor mRNA levels were studied in rat epiphyseal chondrocytes cultured in monolayer. Chondrocytes from enzymatically digested rat tibia epiphyseal growth plates were seeded in monolayer culture and precultured for 7-14 days in Ham's F-12 medium supplemented with 10% (v/v) newborn calf serum and 1% (v/v) of a serum substitute. After preculture the medium was changed to Ham's F-12 medium containing 1% (v/v) serum from hypophysectomized rats, and the effects of T3 and/or IGF-I on DNA synthesis ([3H]thymidine incorporation), ALP activity (a late marker of differentiated epiphyseal chondrocytes) and IGF-I receptor mRNA levels were studied. ALP activity was increased by T3 in a dose-dependent manner with a maximal response at 10 micrograms T3/l (678 +/- 86% compared with control culture). The increase in ALP activity was accompanied by a concomitant decrease in [3H]thymidine incorporation (52 +/- 14% compared with control culture). Human GH (hGH; 50 micrograms/l) and IGF-I (25 micrograms/l) had no stimulatory effect on ALP activity. However IGF-I (10 micrograms/l) exerted an inhibition on the T3 (10 micrograms/l)-induced increase in ALP activity (64 +/- 9% compared with T3-treated culture). T3 (3 micrograms/l) inhibited the increase in [3H]thymidine incorporation caused by 25 micrograms IGF-I/l (51 +/- 13% compared with IGF-I-treated culture). Furthermore, IGF-I receptor mRNA levels were increased by 10 micrograms T3/l (137 +/- 4.2% compared with control culture) while no effect of hGH (50 micrograms/l) or IGF-I (25 micrograms/l) was demonstrated. Both T3 and IGF-I were shown to interact with epiphyseal chondrocytes and both substances seemed to affect cell proliferation and maturation and therefore longitudinal bone growth. Furthermore, the results indicated that IGF-I is important for proliferation of the cells while T3 initiates the terminal differentiation of epiphyseal chondrocytes.     

Inhibition of thyrotropin-induced growth of rat thyroid cells, FRTL-5, by immunoglobulin G from patients with primary myxedema

We studied thyroid growth-blocking activity in immunoglobulin G (IgG) fractions of serum from 24 patients with primary myxedema, 24 patients with goitrous Hashimoto's thyroiditis, and 18 normal subjects by measuring the ability of their IgG to inhibit TSH-induced [3H]thymidine incorporation into DNA in a rat thyroid cell line, FRTL-5. Both groups of patients were receiving T4 when studied. [3H]Thymidine incorporation induced by 0.1 mU/ml bovine TSH was significantly inhibited by the addition of 2 mg/ml IgG from patients with primary myxedema (P less than 0.01), while it was not affected by IgG from the normal subjects or 23 of the 24 patients with goitrous Hashimoto's thyroiditis. IgG from patients with primary myxedema also inhibited the [3H]thymidine incorporation induced by Graves' IgG, but not that induced by forskolin, cholera toxin, (Bu)2cAMP or phorbol-12-myristate-13-acetate. The inhibition of TSH-induced [3H]thymidine incorporation by IgGs from patients with primary myxedema was significantly correlated with their inhibitory activities against both TSH-induced cAMP generation and TSH binding (P less than 0.001). These data indicate that these growth-blocking antibodies are directed against the TSH receptor and might be one of the causes of the thyroid atrophy in patients with primary myxedema.     

Hormonal stimulation of DNA synthesis in primary cultures of adult rat hepatocytes.

Adult rat hepatocytes have been previously isolated and maintained in monolayer culture, but attempts to stimulate DNA synthesis have been unsuccessful. Hormonal conditions are now described which induce DNA synthesis in cultured hepatocytes from partially hepatectomized rats. DNA synthesis was determined autoradiographically by the incorporation of [3H]thymidine into nuclei of morphologically distinct hepatocytes. Insulin (4-4000 nM) or epidermal growth factor (10 ng/ml) alone caused significant increases in the labeling index. The two hormones together acted synergistically to produce labeling indices of 35-50% on the third day of culture, compared with 2-7% in control cultures. The addition of glucagon (400 nM) further increased the labeling indes. Dexamethasone (80 ng/ml) inhibited DNA synthesis but, under certain conditions, enhanced cell attachment. Growth hormone and triiodothyronine had no significant effect on DNA synthesis. The mixture of epidermal growth factor, insulin, and glucagon also stimulated incorporation of [3H]thymidine into phenol-extracted DNA. Although DNA synthesis was stimulated, cell division occurred infrequently. These data suggest a prominent role for epidermal growth factor in promoting hepatic DNA synthesis by acting in concert with insulin and glucagon.     

Monoclonal antibodies to the insulin receptor mimic metabolic effects of insulin but do not stimulate receptor autophosphorylation in transfected NIH 3T3 fibroblasts.

The metabolic actions of insulin and anti-insulin receptor monoclonal antibodies were compared with their effects on insulin receptor phosphorylation in mouse NIH 3T3 fibroblasts transfected with human insulin receptor cDNA. In serum-starved NIH 3T3 HIR3.5 cells, uptake of 2-deoxy-[3H]glucose was stimulated up to 2-fold after 30 min with insulin, with a half-maximal effect at 0.1 nM insulin. Incorporation of [3H]thymidine was stimulated approximately 12-fold after a 16-hr preincubation with insulin, with a half-maximal effect at 2 nM insulin. Phosphorylation of insulin receptor beta-subunit in cells prelabeled with [32P]phosphate was increased 10- to 20-fold within 5 min of adding insulin, with a half-maximal effect at approximately 3 nM insulin. Monoclonal antibodies reacting with four different epitopes on the insulin receptor mimicked the effect of insulin on 2-deoxyglucose uptake. These antibodies also stimulated thymidine incorporation, although the maximum stimulation was only approximately 30% that of insulin. Two antibodies (25-49 and 83-14) showed a similar concentration dependence to insulin in their metabolic effects and in the inhibition of 125I-labeled insulin binding to cells. The other two antibodies (83-7 and 18-44) were somewhat less potent and did not inhibit insulin binding. None of the antibodies significantly increased insulin receptor phosphorylation at concentrations up to 100 nM, which at least in the case of 25-49 and 83-14 was sufficient for full receptor occupancy. It is concluded that the insulin-like metabolic effects of antibodies involve a mechanism of receptor activation that is independent of autophosphorylation and hence that receptor autophosphorylation is not an essential step in triggering at least some events in the insulin signaling pathway.     

Thyrotropin modulates EGF receptor function in porcine thyroid follicle cells

Porcine thyroid follicle cells in monolayer cultures were shown to contain one single class of high-affinity EGF receptors with Kd = 4.5 X 10(-10) M and approximately 20 000-25 000 receptors per cell. Suspension cultures of aggregated follicle cells, exposed to TSH for 3 days, showed a 3-fold increase in [125I]EGF binding. Scatchard analysis demonstrated that this was due to an increase in receptor number. Other cAMP-elevating agents (cholera toxin, dibutyryl cAMP, forskolin) induced a similar effect. In suspension cultures, preincubation with TSH or cholera toxin for 2 days reduced the subsequent [3H]thymidine incorporation. This inhibition was overcome by a low concentration of EGF (0.1 ng/ml). At higher concentrations of EGF (1-10 ng/ml) the incorporation of [3H]thymidine was potentiated 2-3-fold in cultures preexposed to TSH or cholera toxin. The results demonstrate the presence of a high-affinity EGF receptor in porcine thyroid follicle cells. Receptor expression, as well as responsiveness to the mitogenic action of EGF, is modulated in vitro by TSH, through a cAMP-dependent process.     

Somatomedin and analogues of cyclic AMP increase the number of cells synthesizing DNA in cartilage from hypophysectomized rats

The effects of somatomedin and certain nucleotides on nuclear labelling of cartilage cells with [3H]thymidine were determined by autoradiography. Segments of costal cartilage from hypophysectomized rats were incubated for 24 h in a basal medium with or without additions and then pulsed for 2 h with [3H]thymidine in the basal medium. Both somatomedin (0.1 U/ml) and Bt2cAMP (10(-4)M) increased the number of labelled nuclei, and the combined effects were more than additive. A parallelism between the effects of these agents on nuclear labelling and their effects on total thymidine incorporation into DNA was demonstrated. The 8-bromated derivative of cAMP (10(-4)M) also enhanced chondrocyte nuclear labelling, but neither 8-Br-5'-AMP (10(-4)7) nor 8-Br-cGMP (10(-4)M) exhibited actions of the cAMP analogues. It is concluded that in cartilage obtained from hypophysectomized rats and incubated under the specified conditions (1) both somatomedin and cAMP analogues increase the number of cells synthesizing DNA as well as total thymidine incorporation into DNA, (2) the effects of the hormone and cyclic nucleotide in combination are synergistic, and (3) the increased incorporation of labelled thymidine into DNA reflects increased DNA synthesis and not merely an alteration of the specific activity of the intracellular thymidine nucleotide pool.