Comparative effects of somatomedin C and insulin on the metabolism and growth of cultured human fibroblasts

At concentrations of 25 ng/ml in serum-free medium, somatomedin C (SM-C) and insulin stimulated 3H-thymidine incorporation in adult human fibroblasts 4- and 1.5-fold, respectively. The presence of 0.25% human hypopituitary serum (HHS), which by itself had little effect, enhanced the mitogenicity of both SM-C and insulin. Furthermore, 10(-7)M dexamethasone dramatically potentiated SM-C stimulation (70-fold) and insulin stimulation (28-fold) of 3H-thymidine incorporation. With dexamethasone and 0.25% HHS, significant stimulation of DNA synthesis was seen at 2.5 ng/ml for both SM-C and insulin. The effects of SM-C and insulin on 3H-thymidine incorporation were additive. These 3H-thymidine incorporation results were clearly supported by cell replication studies. On the other hand, SM-C and insulin had equivalent, nonadditive effects on RNA and protein synthesis and protein degradation. Half-maximal effects were seen for both peptides on all three metabolic processes at 2-5 ng/ml. In contrast to their synergism with SM-C in the stimulation of DNA synthesis and cell replication, HHS and dexamethasone did not enhance SM-C stimulation of RNA or protein synthesis or protein degradation. These data indicate that SM-C and insulin stimulate DNA, RNA, and protein synthesis, protein degradation, and cell replication in adult human fibroblasts at nanomolar concentrations, suggesting that each peptide is capable of acting through its own receptor. Both SM-C and insulin are also capable of synergism with low concentrations of serum and dexamethasone in the stimulation of DNA synthesis and cell replication. It is proposed that SM-C and insulin both participate in the regulation of cell growth and metabolism in vivo.     

Insulin-like growth factors, IGF-1, IGF-2 and somatomedin C trigger cell proliferation in mammalian epithelial cells cultured in a serum-free medium

A single insulin-like growth factor which constitutes part of a defined serum-free medium is sufficient to stimulate DNA synthesis and mitosis in mammalian lens epithelial cells. Rabbit lenses were cultured in KEI-4, a medium which mimics rabbit aqueous humor, or in KEI-4 containing insulin growth factor I (IGF I), insulin growth factor II (IGF II) or somatomedin C. The magnitude of DNA synthesis and mitosis was evaluated on whole mount preparations of the epithelium at various times of culture. IGF I and II, the most highly purified of the insulin-like growth factors, and somatomedin C were equipotent lens mitogens, were active at the ng level, were more mitogenic toward lens epithelial cells than insulin, and initiated cell proliferation throughout the normally amitotic central region of the lens epithelium. The time course of the mitotic response elicited by the insulin-like growth factors was identical to that noted in lenses cultured in medium supplemented with serum or insulin. The present results, coupled with those of other investigators, suggest that insulin-like factors may regulate cell division in the mammalian lens in vivo.     

Response of Lens Epithelial Cells to Hydrogen Peroxide Stress and the Protective Effect of Caloric Restriction

Hydrogen peroxide (H₂O₂) has been reported to be present at significant levels in the lens and aqueous humor in some cataract patients and suggested as a possible source of chronically inflicted damage to lens epithelial (LE) cells. We measured H₂O₂effects on bovine and mouse LE cells and determined whether LE cells from old calorically restricted mice were more resistant to H₂O₂-induced cellular damage than those of same age ad libitum fed (AL) mice. Bovine lens epithelial cells were exposed to H₂O₂at 40 or 400 μM for 2 h and then allowed to recover from the stress. The cells were assayed for DNA damage, DNA synthesis, cell viability, cell morphology, response to growth stimuli, and proliferation potential. Hydrogen peroxide-treated cells showed an increased DNA unwinding 50% greater than that for untreated controls. These DNA strand breaks appeared to be almost completely rejoined by 30 min following removal of the cells from a 2-h exposure. The 40 μM exposure did not produce a significantly lower DNA synthesis rate than the control, it responded to growth factor stimuli, and it replicated as did the control cells after removal of H₂O₂. The 400 μM H₂O₂severely affected DNA synthesis and replication, as shown by increased cell size and by markedly reduced clonal cell growth. The cells did not respond to growth stimulation by serum or growth factors and lost irreversibly the capacity to proliferate. The responses of LE cells from old adlib diet (AL) and calorically restricted (CR) mice to H₂O₂were significantly different. Exposure of LE cells to 20, 40, or 100 μM H₂O₂for 1 h induces a significant loss of cellular proliferation in cells from old AL mice. LE cells from long-term CR mice of the same strain and age were more resistant to oxidative damage at all three concentrations of H₂O₂than those of both old and young AL mice and showed a significantly higher proliferation potential following treatment. It is concluded that CR results in superior resistance to reactive oxygen radicals in the lens epithelium.     

Insulin/insulin-like growth factor-I and estrogen cooperate to stimulate cyclin E-Cdk2 activation and cell Cycle progression in MCF-7 breast cancer cells through differential regulation of cyclin E and p21(WAF1/Cip1)

Estrogens and insulin/insulin-like growth factor-I (IGF-I) are potent mitogens for breast epithelial cells and, when co-administered, induce synergistic stimulation of cell proliferation. To investigate the molecular basis of this effect, a MCF-7 breast cancer cell model was established where serum deprivation and concurrent treatment with the pure estrogen antagonist, ICI 182780, inhibited growth factor and estrogen action and arrested cells in G(0)/G(1) phase. Subsequent stimulation with insulin or IGF-I alone failed to induce significant S-phase entry. However, these treatments increased cyclin D1, cyclin E, and p21 gene expression and induced the formation of active Cdk4 complexes but resulted in only minor increases in cyclin E-Cdk2 activity, likely due to recruitment of the cyclin-dependent kinase (CDK) inhibitor p21(WAF1/Cip1) into these complexes. Treatment with estradiol alone resulted in a greater increase in cyclin D1 gene expression but markedly decreased p21 expression, with a concurrent increase in Cdk4 and Cdk2 activity and subsequent synchronous entry of cells into S phase. Co-administration of insulin/IGF-I and estrogen induced synergistic stimulation of S-phase entry coincident with synergistic activation of high molecular mass (approximately 350 kDa) cyclin E-Cdk2 complexes lacking p21. To determine if the ability of estrogen to deplete p21 was central to these effects, cells stimulated with insulin and estradiol were infected with an adenovirus expressing p21. Induction of p21 to levels equivalent to those following treatment with insulin alone markedly inhibited the synergism between estradiol and insulin on S-phase entry. Thus the ability of estradiol to antagonize the insulin-induced increase in p21 gene expression, with consequent activation of cyclin E-Cdk2, is a central component of the synergistic stimulation of breast epithelial cell proliferation induced by simultaneous activation of the estrogen and insulin/IGF-I signaling pathways.     

Identification of the fibroblast growth factor receptor of Swiss 3T3 cells and mouse skeletal muscle myoblasts

Two distinct fibroblast growth factors (FGF) were purified to homogeneity from bovine brain on the basis of their ability to stimulate skeletal muscle myoblast proliferation. These growth factors are also mitogenic for Swiss 3T3 cells and appear to be closely related to or identical with previously isolated anionic and cationic fibroblast growth factors. The half-maximum concentrations (EC50) for stimulation of myoblast DNA synthesis by the anionic and cationic growth factors were 30pM and 1pM, respectively. In contrast, an EC50 of 45 pM was observed for stimulation of 3T3 cell DNA synthesis by both growth factors. Binding of 125I-labeled anionic FGF was saturable with apparent Kd values of 45 pM and 11 pM and approximately 60 000 and 2000 receptor sites per cell for 3T3 cells and MM14 murine myoblasts, respectively. Unlabeled anionic and cationic FGF equally displaced 125I-labeled anionic FGF from 3T3 cells while cationic FGF was more potent than anionic FGF for displacement from skeletal muscle myoblasts, demonstrating that a single receptor binds the two distinct growth factors. Binding was specific for these factors since platelet-derived growth factor, insulin, insulin-like growth factor 1, epidermal growth factor, and nerve growth factor were unable to displace bound 125I-labeled anionic FGF from Swiss 3T3 cells. Chemical cross-linking of specifically bound 125I-labeled anionic FGF to 3T3 cells and MM14 myoblasts identified a single detergent-soluble FGF receptor with an apparent molecular weight of 165 000.     

Substance P primes human neutrophil activation: a mechanism for neurological regulation of inflammation

The neuropeptide substance P (SP), a member of the tachykinin family, has stimulatory effects on various cell types at nanomolar concentrations. SP has also direct effects on polymorphonuclear leukocytes (PMNs). However, unlike other cells, stimulation of PMNs requires extremely high concentrations of the peptide (greater than 10 microM), suggesting that direct PMN activation by SP is not physiologically relevant. By measuring primed stimulation of PMNs, we now demonstrate potent synergistic effects of nanomolar doses of SP on the migratory and cytotoxic functions of human PMNs stimulated by fMLP and C5a. This synergism between SP and chemotactic peptides reveals a new regulatory activity of SP and suggests that neurogenic stimuli may prepare neutrophils for an exaggerated inflammatory response to other phlogistic mediators.     

Receptors for insulin and epidermal growth factor: relation to synthesis of DNA in cultured rabbit lens epithelium.

Epidermal growth factor (EG factor) and insulin stimulate the incorporation of thymidine into contact-inhibited rabbit lens epithelial cells in culture. The maximal stimulation observed with EG factor is greater than with insulin. Half-maximal stimulation by EG factor is observed at 6 × 10^?10m and for insulin at 1 × 10^?9m. [¹²⁵I]-labeled EG factor (2000 Ci/mmol, about 1 g atom of iodine per mol) is equipotent with native EG factor in stimulating DNA synthesis. Both insulin and EG factor bind to distinct high-affinity sites in intact lens cell monolayers; half-maximal binding is observed at about 10^?9m for both polypeptides. A maximum of approximately 8 × 10⁴ insulin molecules and 4 × 10⁴ EG factor molecules are bound per cell. These observations indicate that cultured rabbit lens cells possess receptors for insulin and EG factor by biological and physicochemical criteria and raise the possibility that both peptides may play a role in lens growth and development.     

Initiation of DNA synthesis in mammary epithelium and mammary tumors by lithium ions

The growth promoting effects of lithium and insulin on cultures of mammary gland epithelium and CZF mouse mammary tumor cells were investigated. Lithium chloride exerts a 450-fold increase in the rate of DNA synthesis in mammary epithelium from mid-pregnant mice in organ culture or monolayer culture. There is an increase in both the percentage of cells initiating DNA synthesis and the net accumulation of DNA. The most effective lithium concentration is 10 mM, and the maximally effective rate of stimulation is reached 48 hours after addition. The magnitude of response to lithium varies with the physiological state of the mammary epithelial cell donor: epithelium from non-pregnant or lactating mice is less responsive than that from mid-pregnant mice. In combination, insulin and lithium produce either a synergistic or an additive effect on the growth of epithelium dependent upon the physiological state of the donor animal. Lithium also promotes the growth of mammary tumor cells in the absence of serum or other mitogens. The action of lithium on DNA synthesis appears to be a direct effect on the epithelial cells.     

Induction of DNA synthesis in dog thyrocytes in primary culture: synergistic effects of thyrotropin and cyclic AMP with epidermal growth factor and insulin

We have investigated the growth effects of thyrotropin (TSH) (mimicked by forskolin and acting through cyclic AMP), epidermal growth factor (EGF), serum (10%) and insulin on quiescent dog thyroid epithelial cells in primary culture in a serum-free defined medium. These cells were previously shown to retain the capacity to express major thyroid differentiation markers. In the presence of insulin and after a similar prereplicative phase of 18 +/- 2h, TSH, EGF, and serum promoted DNA synthesis in such quiescent cells only a minority of which had proliferated in vitro before stimulation. The combination of these factors induced more than 90% of the cells to enter S phase within 48 h and near exponetial proliferation. Analysis of the cell cycle parameters of the stimulated cells revealed that the G1 period duration was similar to the length of the prereplicative phase of quiescent thyroid cells; this might indicate that they were in fact in an early G1 stage rather than in G0 prior to stimulation. TSH and EGF action depended on or was potentiated by insulin. Strikingly, nanomolar concentrations of insulin were sufficient to support stimulation of DNA synthesis by TSH, while micromolar concentrations of insulin were required for the action of EGF. This suggests that insulin supported the action of TSH by acting on its own high affinity receptors, whereas its effect on EGF action would be related to its somatomedinlike effects at high supraphysiological concentrations. Insulin stimulated the progression in the prereplicative phase initiated by TSH or forskolin. In addition, in some primary cultures TSH must act together with insulin to stimulate early events of the prereplicative phase. In the presence of insulin, EGF, and forskolin, an adenylate cyclase activator, markedly synergized to induce DNA synthesis. Addition of forskolin 24 h after EGF or EGF 24 h after forskolin also resulted in amplification of the growth response but with a lag equal to the prereplicative period observed with the single compound. This indicates that events induced by the second factor can no longer be integrated during the prereplicative phase set by the first factor. These findings demonstrate the importance of synergistic cooperation between hormones and growth factors for the induction of DNA synthesis in epithelial thyroid cells and support the proposal that essentially different mitogenic pathways--cyclic AMP-dependent or independent--may coexist in one cell.     

Proliferation of guinea-pig uterine epithelial cells in serum-free culture conditions: effect of 17 beta-estradiol, epidermal growth factor and insulin

The effects of 17 beta-estradiol (E2), epidermal growth factor (EGF) and insulin, alone or in association on guinea-pig uterine epithelial cell proliferation were examined in serum-free culture conditions. Primary cultures of epithelial cells were made quiescent by serum depletion, then incubated in a chemically defined medium. In this medium, insulin increased DNA synthesis but not in a dose-dependent manner for concentrations ranging from 0.2 to 10 micrograms/ml. A significant effect of EGF was found only for the highest concentration tested (100 ng/ml). E2 alone or in the presence of insulin (1 microgram/ml) had no effect whatsoever on the concentration tested (10(-10)-10(-5)M). Insulin (10 micrograms/ml) plus EGF (100 ng/ml) exerted on DNA synthesis and cell proliferation a significant additive effect which was identical to the growth stimulation induced by 10% fetal calf serum. The effects of insulin plus EGF were not modified by the addition of E2. These findings suggest that E2 is not directly mitogenic for uterine epithelial cells in defined culture conditions and that the mitogenic response to optimal concentration of insulin plus EGF is independent of E2.     

Control of DNA synthesis and ornithine decarboxylase activity by hormones and amino acids in primary cultures of adult rat hepatocytes

The conditions for stimulation of ornithine decarboxylase (ODC) and DNA synthesis in primary monolayer cultures of non-growing, highly differentiated hepatocytes from adult rats were compared. The syntheses of ODC and DNA were not stimulated by hormones on the 1st day of culture, but they were induced markedly by insulin (10⁻⁸ M) and epidermal growth factor (EGF, 0.1 μg/ml) in cells cultured for 40 h. The effects of insulin and EGF were synergistic, and the ODC activity as well as the DNA synthesis in the presence of these hormones was comparable to that of cultured hepatocytes from partially hepatectomized liver. Other factors had different effects on the two processes. Dexamethasone induced ODC slightly, but it inhibited DNA synthesis strongly. Putrescine inhibited ODC activity, but it had no effect on DNA synthesis. Asparagine and glutamine induced ODC activity, but they inhibited DNA synthesis; their inhibitory effects on DNA synthesis were specific to primary cultured liver cells and were not seen in an established rat liver cell line or in mouse L cells. These results show that although there is some correlation between ODC induction and DNA synthesis, the former is not essential for cell growth. There was no indication of cell division under conditions where maximal ODC induction and DNA synthesis were observed. Cytofluorometry of cells treated with insulin and EGF showed that the DNA content increased from 2 N to 4 N, and to 8 N in some cells. Therefore, under the present culture conditions, mature liver cells could enter G2 phase through S phase, but could not enter M phase.     

Early stimulation of ATP turnover induced by growth factors : Synergistic effect of EGF and insulin and correlation with DNA synthesis

Addition of a mixture of EGF + insulin to quiescent cell cultures synergistically stimulates the cells to reinitiate DNA synthesis and cell division. We have previously demonstrated that this mixture rapidly increases ATP turnover in quiescent cells. The present work shows that each of the two growth factors, EGF and insulin, when added separately to quiescent cells was able to stimulate the phosphorylation of the organic acid-soluble compounds (Po) pool and ATP turnover. The stimulation of ATP turnover was closely correlated with the increase in phosphorylation of the Po pool which suggests that Po labelling reflects the ATP turnover. In many experiments, the synergy between the two growth factors on the early increase in phosphorylation of the Po pool was clearly shown. Doubling the concentration of EGF (12-24 ng/ml) or insulin (50-100 ng/ml) did not increase early stimulation of phosphorylation of the Po pool, whereas simultaneous addition of the two growth factors induced a greater stimulation than that of each growth factor separately added. The augmentation in Po labelling after addition of EGF or insulin alone was transient. The synergistic effect of the two growth factors was more significant when determined 150 or 300 min after growth-factor addition. In our experimental conditions, each of the two growth factors, EGF and insulin, was able to induce a stimulation of DNA synthesis. However, the best stimulatory effect was observed with the mixture of the two which synergistically increased DNA synthesis determined between 6 and 24 h after growth-factor addition. The comparison between DNA replication and Po labelling suggests a correlation between the increase in DNA replication and in the total ATP synthesized in the first 5 h after cell stimulation by growth factors added separately or in combination.     

Presence of epidermal growth factor receptors and influence of epidermal growth factor on proliferation and aging in cultured smooth muscle cells.

Epidermal growth factor (EGF) at nanomolar concentrations stimulated DNA synthesis in confluent, serum-starved cultures of calf aorta and human uterine smooth muscle cells. Stimulation of DNA synthesis in lens epithelial cells was studied for comparison. L and D-ascorbic acid potentiated the effect of serum and EGF on DNA synthesis in calf aorta cells. In contrast L-ascorbic acid had minimal potentiating effect with serum and no effect with EGF present along with serum on DNA synthesis in human uterine smooth muscle and rabbit lens epithelial cells. EGF and ascorbic acid increased cell number when added to stationary phase cultures. Specific binding of 125I-labelled EGF to smooth muscle cells was demonstrated. Receptor concentration in calf-aorta smooth muscle cells was higher in dense cultures compared to sparse cultures. The time course of binding and dissociation of 125I-labelled EGF was similar in "dense" and "sparse" cultures. Human uterine smooth muscle cells in culture exhibited a finite lifespan. There was no stimulation of DNA synthesis in response to serum and EGF in cells of high population doubling level (PDL); although 125I-labeled EGF binding was higher in old cells (high PDL) compared to young cells (low PDL). This increase in binding was shown to be due to changes in the concentration of receptors without changes in their affinity for EGF.     

The influence of epidermal growth factor and insulin on proliferation and DNA synthesis in ciliates Tetrahymena pyriformis

The influence of the epidermal growth factor (EGE) (10(-8) M), insulin (10(-6) M) and EGF (10(-8) M) in combination with insulin (10(-6) M) on proliferation and DNA synthesis in the nuclei of ciliates Tetrahymena pyriformis GL was studied. Insulin and EGF, known to stimulate growth of many types of mammalian cells revealed a mitogen influence on the unicellular eukaryotes. This effect involves stimulation of DNA synthesis, rising synchronization of cell division (upon the influence of EGF), and increase in cell number during the exponential growth. The mitogen effect may be evoked by cell progression in G1-phase under the action of growth factors and, consequently by earlier entry of cells into S-phase of the first cell cycle. Insulin repressed division of cells that entered into the generative cycle. These cells were delayed in late S-phase and G2-phase of the cycle. Part of these cells perished, while other cells could successively overcome the cell block to start their division by the 4th hours of cultivation. A collateral cytotoxic effect of insulin was found, being most prominent in early periods of Tetrahymena cultivation.     

Rat transformation-associated proteins (TAP) induced by Moloney murine sarcoma virus interact with specific receptors on normal rat kidney cells

In this report, data are presented to show that transformation-associated proteins (TAP) secreted from the transformed 6M2 cells have mitogenic activities in the stimulation of DNA synthesis and proliferation of normal rat kidney (NRK-2) cells and of nonpermissively grown 6M2 cells. TAP also bound specifically to NRK-2 cells with a binding dissociation constant (Kd) of 1.4 pM. Approximately 2 X 10(5) binding sites per cell were found. Therefore, TAP may represent a set of virally-induced growth stimulatory factors.     

Mouse blastocysts respond metabolically to short-term stimulation by insulin and IGF-1 through the insulin receptor

Insulin specifically stimulates protein synthesis in compacted mouse embryos on days 3 and 4 after fertilization, with an EC50 of 0.5 pM (Harvey and Kaye, 1988). The identity of the receptor mediating this short-term effect of insulin was further examined by dose-response studies with IFG-1 and by using a specific anti-insulin receptor antiserum that has no appreciable cross-reaction with IGF-1 receptors. IGF-1 caused a maximum 40% stimulation of protein synthesis after 4 h exposure (similar to the response to insulin) with an EC50 of 150 pM IGF-1. The insulin receptor-specific antiserum, or IgGs isolated from it, also stimulated protein synthesis at dilutions as high as 1:1,000 to the same degree as insulin (approximately 40%). This agonistic action of the insulin receptor antiserum, the EC50 of 150 pM for IGF-1, and the previously established EC50 of 0.5 pM for insulin, all with similar maximal stimulation, strongly support the conclusion that the short-term metabolic stimulation of mouse blastocysts by insulin is mediated by insulin receptors. Immunosurgical isolation of inner cell masses before and after exposure to 1.7 pM insulin (sufficient to stimulate only the insulin receptor) showed that insulin stimulates protein synthesis in these cells as well as in the trophectoderm cells of the blastocyst. This finding suggests that in intact blastocysts, insulin may travel across the trophectoderm to the inner cell mass, acting anabolically on both tissues. Analysis of the agonistic effect of the B-10 antiserum showed there was no evidence of an unresponsive subpopulation of embryos.     

Modulation of the mitogenic response of an epidermal growth factor-dependent keratinocyte cell line by dexamethasone, insulin, and transforming growth factor-beta

The stimulation of DNA synthesis by epidermal growth factor (EGF) has been studied for a cell line having properties useful for investigating the mechanism of action of EGF in epithelial cell populations. These studies employ a mouse keratinocyte cell line (MK), isolated by Weissman and Aaronson (1983), which is stringently dependent on exogenous EGF for growth in serum containing medium. The studies reported here characterize the compliment of EGR receptors present on the surface of MK cells and demonstrate the regulatory influence of other hormones on the capacity of EGF to stimulate DNA synthesis. Up-regulated MK cells contain approximately 22,000 EGF receptors per cell, but when the cells are grown in the presence of EGF the receptor number is reduced to about 4,000. It is estimated that only a small number of high-affinity receptors (less than 500) are required for EGF-dependent cell proliferation. In contrast to its action in fibroblastic cells, dexamethasone is a strong inhibitor of EGF-stimulated DNA synthesis of MK cells. Insulin at high concentrations, or insulin-like growth factors I or II (IGF-I, IGF-II) at physiological concentrations, synergistically enhance the EGF response. Interestingly, insulin or IGF-I or II are also able to reverse most of the dexamethasone inhibition of DNA synthesis. Transforming growth factor-beta (TGF-beta) inhibits, in reversible manner, the EGF stimulation of DNA synthesis and this inhibition is not overcome by insulin. TGF-beta receptors have been measured in MK cells and Scatchard analysis indicates approximately 20,000 receptors per cell. None of the modulatory hormones (insulin, dexamethasone, TGF-beta) significantly altered 125I-EGF binding characteristics in MK cells, suggesting a point of action distal to 125I-EGF binding.     

Mode of fibroblast growth enhancement by human interleukin-1

Previous studies have demonstrated that interleukin-1 (IL-1) stimulates fibroblast growth (Schmidt, J. A., S. B. Mizel, D. Cohn, and I. Green. 1982. J. Immunol. 128:2177-2182) and binds to specific, high affinity receptors of BALB/c3T3 cells (Bird, T. A., and J. Saklatval. 1986. Nature (Lond.). 324:263-265, 266-268). We have investigated the mechanism of fibroblast growth stimulation by IL-1. Addition of fibroblast growth factor derived from platelets (PDGF) to a quiescent culture of BALB/c3T3 cells produced 8-10-fold increase in DNA synthesis during 24-h incubation. The cellular action of PDGF was mediated through competence induction and required synergistic action of plasma-derived factors for full mitogenic activity. When tested at a wide range of concentrations (0.1-100 pM), natural IL-1 or recombinant IL-1 produced only a maximum of 5-10% of DNA synthesis elicited in response to PDGF or serum. Induction of DNA synthesis required continuous presence of IL-1 and did not exhibit synergism with plasma. Competence induction and mitogenic stimulation by PDGF was associated with early induction of proteins P32, P38, P46-48, P75, and changes in cytoskeletal organization. Examination of these early cellular changes showed that IL-1 did not produce similar induction of cellular proteins and the morphological changes associated with growth stimulation. These results suggest that the mode of IL-1 action on BALB/c3T3 was not through competence induction. When IL-1 was added to cells rendered competent by brief exposure to PDGF, 10-15% additional DNA synthesis occurred during the first 24 h. Extended incubation of PDGF-treated cells in the presence of IL-1 revealed that the stimulation by IL-1 occurred predominantly during the subsequent cycle of DNA replication, wherein DNA synthesis reached three- to fivefold higher than the untreated cultures. We conclude (a) IL-1 alone is not a potent mitogen for BALB/c3T3 cells, and does not bring cells out of the growth arrest Go phase, (b) treatment with PDGF renders the cells more responsive to IL-1, (c) part of the IL-1 action on competent cells may be characterized as progression inducing activity, further, (d) our results indicate that action of IL-1 on PDGF-treated cells produces sustained DNA synthesis for an extended period, perhaps by preventing the entry of cells into growth arrest Go phase.     

Conditional responsiveness of a chemically transformed cell line to growth factor stimulated DNA synthesis

BP3T3, a clonal benzo(a)pyrene-transformed BALB/c-3T3 cell line, is conditionally responsive to growth factor stimulation. Density arrested cell populations deprived of growth factors by pretreatment with 0.5% platelet-poor plasma synthesized DNA both in response to ng/ml concentrations of PDGF, EGF, and somatomedin C, and in response to insulin, plasma, and serum. The above agents acted singly to induce DNA synthesis, but synergism is suggested because a higher percentage of cells were stimulated to enter the S phase when the growth factors were added in combination. Desensitization to growth factors occurred when cultures were pretreated with the high concentration of growth factors present in 10% serum (or plasma). In desensitized cultures none of the above agents, added singly or in combination, stimulated DNA synthesis. This effect appears to be global because pretreatment with one growth factor (e.g., insulin) inhibited the action of another (e.g., PDGF). Cell density appears to play a critical role in regulating DNA synthesis. Unlike nontransformed BALB/c-3T3 cells whose density is regulated by the serum concentration, the density of BP3T3 cells reached a plateau when cultures were grown in a serum (or plasma) concentration of 3% or greater. Such density arrested cultures were growth factor unresponsive; however, the cells rapidly responded to growth factors by synthesizing DNA and replicating when reseeded at a lower cell density. Thus the growth of BP3T3 cells is regulated by both growth factors and cell density.     

Rat hepatoma cell variants resistant to insulin-diphtheria toxin A fragment conjugates. Genetic evidence for the separate pathways for insulin receptor-mediated mitogenic and hormonal stimulation

The rat hepatoma H35 cells in serum-free medium produce tyrosine aminotransferase (TAT) and initiate DNA synthesis and cell division upon exposure to 10(-9)-10(-10) M insulin. This insulin-dependent hormonal and mitogenic stimulation is through the insulin receptors and not through the receptors for insulin-like growth factor type I. We have isolated genetic variants of H35 cells which are resistant to a cytotoxic insulin-diphtheria toxin A fragment conjugate. These variants showed different degrees of insulin binding capacity and exhibited different sensitivities to insulin-stimulated TAT and DNA synthesis. Variant DTaI-b had a slight decrease in number of insulin receptors but completely lost sensitivity to both TAT and DNA stimulation. Variant 11-1 had a reduced number of insulin receptors but retained both TAT and DNA inducibilities. Variant 14-1, which had a high number of insulin receptors, was not responsive to DNA synthesis but was responsive to TAT induction. The beta-subunit of insulin receptors in these cell variants had different sensitivities to their insulin-dependent autophosphorylation. The rat hepatoma HTC cells used as a control showed very low insulin binding, no stimulation of TAT and DNA synthesis by insulin, and no detectable insulin-enhancement of beta-subunit phosphorylation. These characteristics provide genetic evidence for the divergence of the insulin receptor-mediated mitogenic and hormonal signals during the post-receptor pathways which is apparently regulated by the insulin-dependent phosphorylation.