Roles of insulinlike growth factor 1 (IGF-1) and the IGF-1 receptor in epidermal growth factor-stimulated growth of 3T3 cells.

BALB/c3T3 cells are exquisitely growth regulated and require platelet-derived growth factor, epidermal growth factor (EGF), and insulinlike growth factor 1 (IGF-1) for growth. When BALB/c3T3 cells are transfected with plasmids constitutively expressing both EGF and the human IGF-1 receptor mRNAs, the cells are capable of growing in serum-free medium without the addition of any exogenous growth factor. These cells, called p5 cells, can grow for prolonged periods in serum-free medium. BALB/c3T3 cells transfected with only the IGF-1 receptor expression plasmid (p6 cells) do not grow in serum-free medium but do grow if IGF-1 (or insulin in supraphysiological concentrations) is added. p6 cells also grow in response to EGF, confirming that the combination of EGF and an overexpressed IGF-1 receptor is sufficient for the growth of 3T3 cells. We have found that in EGF-stimulated p6 cells there is an increase in the expression of IGF-1 mRNA, that IGF-1 is secreted into the medium, and that the growth of p5 cells and EGF-stimulated p6 cells is inhibited by exposure to antisense oligodeoxynucleotides to IGF-1 receptor RNA. Finally, while cells constitutively expressing both EGF and EGF receptor RNAs grow, albeit modestly, in serum-free medium, their growth is also inhibited by an antisense oligodeoxynucleotide to IGF-1 receptor RNA. In contrast, in cells overexpressing the IGF-1 receptor, IGF-1-mediated cell growth occurs independently of the platelet-derived growth factor and EGF receptors (Z. Pietrzkowski, R. Lammers, G. Carpenter, A. M. Soderquist, M. Limardo, P. D. Phillips, A. Ullrich, and R. Baserga, Cell Growth Differ. 3:199-205, 1992, and this paper). These data indicate that an important role for EGF is participation in the activation of an autocrine loop based on the IGF-1-IGF-1 receptor interaction, which is obligatory for the proliferation of 3T3 cells.     

Growth effects of lithium chloride in BALB/c 3T3 fibroblasts and madin-darby canine kidney epithelial cells

The ability of LiCl to initiate DNA synthesis was studied in Madin-Darby canine kidney (MDCK) cells, and mouse BALB/c 3T3 fibroblasts. In a defined culture medium lacking serum, LiCl increased DNA synthesis in BALB/c 3T3 cells 100–200% over control values. Maximum DNA synthesis was observed with concentrations of LiCl between 10 and 25 mM and increases from 40–50% over control were observed with concentrations as low as 1 mM. Exposure of BALB/c 3T3 cultures to LiCl resulted in an increase in the percentage of cells initiating DNA synthesis, total DNA content and cell number. Lithium chloride, in combination with insulin or epidermal growth factor (EGF), had either an additive or synergistic effect upon the growth of BALB/c 3T3 fibroblasts. MDCK cells proved refractory to the growth actions of LiCl, although they responded to EGF and insulin with increased DNA synthesis. Lithium chloride appears to have a direct effect on cell proliferation in some but not all cell types.     

Malignant transformation of mouse BALB/3T3 cells by polyoma middle T antigen requires epidermal growth factor receptor expression.

The mouse cell line MO-5, which is defective in receptor-binding activity of epidermal growth factor (EGF), is very poorly transformed by polyoma middle T antigen or v-src gene, but activated c-H-ras and v-mos gene can induce the transformation (M. Ono, M. Yakushinji, K. Segawa, and M. Kuwano, Mol. Cell. Biol., 8: 4190-4196, 1988). We established clones of MO-5 expressing a functional EGF receptor (EGF-R) after introduction of the human EGF-R complementary DNA into MO-5 (MNER23 and MNER31), and we also established a clone (BNER4) expressing human EGF-R from the parental cell line, BALB/3T3. MNER23, MNER31, and BNER4 expressed EGF-R activity at about 2- to 6-fold higher levels than did control BALB/3T3 cells. A marked increase in DNA synthesis in response to EGF was observed in these BNER4, MNER23, and MNER31 cell lines compared to BALB/3T3 cells; however, there was little if any increase in DNA synthesis of MO-5 in the presence of EGF. Introduction of the polyoma middle T antigen gene into BALB/3T3, BNER4, MNER23, and MNER31 resulted in the appearance of transformation foci, but MO-5 again showed little response. We purified clones B4-mT-2, M23-mT-1, M23-mT-2, M23-mT-3, and M31-mT-13 from transformation foci of BNER4, MNER23, and MNER31 cells, which were respectively transfected with the middle T antigen. All of the middle T antigen-positive transfectants demonstrated abilities to form both colonies in soft agar and tumors in nude mice. The presence of EGF-R appears to be indispensable for malignant transformation by polyoma middle T antigen.     

Epidermal growth factor, insulin, and estrogen stimulate development of prolactin-secreting cells in cultures of GH3 cells

Pituitary tumor GH3 cells synthesize and secrete both growth hormone (GH) and prolactin (PRL). Morphological and functional changes of GH3 cells induced by epidermal growth factor (EGF, 10 nM), insulin (300 nM), and estradiol-17beta (E2, 1 nM) were studied. Treatment of cultures of GH3 cells for 6 days with EGF, insulin, or E2 alone, and with EGF plus E2 did not affect the total number of GH3 cells, but a combination of EGF, insulin, and E2 decreased the total number of GH3 cells compared with control treatment. DNA-synthesizing cells were detected by monitoring 5-bromo-2'-deoxyuridine (BrdU) uptake. EGF, E2, or a combination of EGF, insulin, and E2 significantly decreased the proportion of BrdU-labeled cells (21.1+/-1.7%, 21.0+/-1.4%, 18.2+/-1.3%; P<0.05, P<0.05, P<0.01, respectively) compared with control treatment (28.6+/-1.5%), but insulin did not (31.4+/-2.4%). Immunocytochemical analysis of GH3 cells cultured in 5% fetal calf serum-supplemented medium (control) showed that about 70% of all GH3 cells were GH-immunoreactive cells (GH-ir cells), apparently containing only GH, and 14% were mammosomatotrophs (MS cells), containing both GH and PRL, while PRL-immunoreactive cells (PRL-ir cells), containing only PRL, were not detected. No GH or PRL immunoreactivity could be detected in the remaining cells (15%). EGF decreased the proportion of GH-ir cells. The effects of EGF were enhanced by simultaneous exposure to insulin and E2; this decreased the proportion of GH-ir cells to about 20% of the total GH3 cells and significantly increased the proportion of MS cells to 300% of controls. Treatment with EGF plus insulin, EGF plus E2, or a combination of EGF, insulin, and E2 all stimulated the appearance of PRL-ir cells. Exposure to EGF caused a significant decrease in GH mRNA (P<0.01) and a significant increase in PRL mRNA (P<0.05). These observations suggest that EGF is closely involved in differentiation of PRL-ir cells from GH-ir cells via MS cells in GH3 cell cultures. Cytosine arabinoside (10(-7) M), an inhibitor of cell division, did not affect the changes in proportion of the three cell types induced by treatment with a combination of EGF, insulin, and E2. It is therefore probable that the transdifferentiation does not require mitosis of the GH3 cells.     

Nuclear transport in 3T3 fibroblasts: effects of growth factors, transformation, and cell shape

Nucleocytoplasmic transport of fluorescent-labeled macromolecules was investigated in transformed and nontransformed 3T3 fibroblasts. Insulin and epidermal growth factor enhanced transport three-fold after 1-2-h incubation with nontransformed adhering fibroblasts; no enhancement of transport was observed for spherical unattached fibroblasts. The concentration of growth factor for maximal enhancement was 3-10 nM. Nuclear transport for Kirsten murine sarcoma virus-transformed BALB/c 3T3 fibroblasts, however, was maximally enhanced before addition of growth factors; addition of insulin or epidermal growth factor causes no additional transport enhancement. Transformation also minimizes cell shape effects on macromolecular nuclear transport. These results provide evidence that protein growth factors and oncogenic transformation may use a similar mechanism for activation of nuclear transport.     

Loss of epidermal growth factor receptors and release of transforming growth factors do not correlate with sarcoma virus-transformation in clonally-related NIH/3T3-derived cell lines.

Transformation of NIH/3T3 cells by Kirsten murine sarcoma virus (MSV) caused a dramatic reduction in the number of cell-surface receptors for epidermal growth factor (EGF). However, the number of EGF receptors remained at a very low level in a non-tumourigenic revertant cell line isolated from the virus-transformed cells, indicating that an increase in EGF receptors is not a requirement for the phenotypic reversion of Kirsten MSV-transformed 3T3 cells. Serum-free conditioned medium from normal and virus-transformed cell lines contained similar amounts of cell growth-promoting activity as assayed by the ability to stimulate DNA synthesis in quiescent Swiss 3T3 cell cultures. However, the concentrated conditioned medium from these cell lines showed no evidence of beta-transforming growth factor (TGF) activity as assayed by promotion of anchorage-independent growth of untransformed normal rat kidney (NRK) fibroblasts in agarose. The cellular release of alpha-TGF activity was assayed by measuring the ability of concentrated conditioned medium to inhibit the binding of 125I-EGF to Swiss 3T3 cells. Conditioned medium protein from the virus-transformed cell line inhibited 125I-EGF binding but only to the same extent as conditioned medium protein prepared from the untransformed cell line. The alpha-TGF secretion by these cell lines was estimated to be 30-45-fold lower than the level of alpha-TGF released by a well-characterized alpha-TGF-producing cell line (3B11). These results suggest that the induction of TGF release is not a necessary event in the transformation of NIH/3T3 cells by Kirsten MSV.     

Development of insulin and epidermal growth factor receptors during the differentiation of rat preadipocytes in primary culture

An homogeneous cell population isolated from the inguinal tissue of 3-day-old rats is able to proliferate in primary culture. In the presence of a physiological concentration of insulin (1.5 nM) it converts into cells exhibiting the morphology and the biochemical characteristics of adipocytes. Insulin and epidermal growth factor (EGF) receptors were studied during both the exponential growth and the adipose conversion phases of these cells. Binding experiments with 125I-labelled peptides were performed directly in the culture dishes. The number of high affinity insulin binding sites increased, during the entire culture period studied, reaching 18 days after plating the value of 10,600 x 2360. Control cells (cultured in the presence of anti-insulin antibody) exhibited an increase of the concentration of insulin binding sites from no more than 500 sites/cell to 6880 +/- 1710 sites/cell between dat 0 and 9 (corresponding to the exponential growth phase); this increase was followed by a rapid reduction in insulin receptors during the stationary phase. The density of EGF binding sites increased between day 0 and 4 (one cell cycle), whether the cells were maintained or not with insulin, and plateaued thereafter. Mature adipocytes freshly isolated from the inguinal tissue of 3-day-old rats had no detectable EGF binding sites, but their content in high affinity binding sites for insulin was similar to that of cells after complete adipocyte conversion in primary culture.     

Epidermal growth factor controls smooth muscle alpha-isoactin expression in BC3H1 cells

We have examined the effects of epidermal growth factor (EGF), platelet-derived growth factor, and insulin on the differentiation of a mouse vascular smooth muscle-like cell line, the BC3H1 cells. On the basis of cell morphology and smooth muscle alpha-isoactin synthesis, we demonstrate that EGF at physiological concentrations prevents the differentiation of these cells, whereas platelet-derived growth factor has no apparent effect. The induction of alpha-isoactin synthesis by serum deprivation is inhibited by EGF in a dose-dependent manner with a half-maximal effect at 3-5 ng/ml and a maximal inhibition at approximately 30 ng/ml. Northern analysis also shows that EGF blocks the accumulation of alpha-isoactin mRNA normally observed during cell differentiation. Addition of EGF to differentiated cells results in a repression of alpha-isoactin synthesis, a stimulation of beta- and gamma-isoactin synthesis, and the stabilization of the nonmuscle isoactins. The synthesis of creatine phosphokinase, a muscle-specific noncontractile protein, is also regulated by EGF in a similar fashion. Modulation by EGF of alpha-isoactin expression is not affected by aphidicolin and is therefore independent of its mitogenic effect on these cells. Insulin is not required for observation of the EGF-dependent effects but instead seems to promote differentiation. Our results show that EGF can replace serum in controlling the differentiation of BC3H1 cells.     

Mitogenic response to epidermal growth factor (EGF) modulated by platelet-derived growth factor in cultured fibroblasts

The mitotic effects of epidermal growth factor (EGF) were investigated in two cultured fibroblast lines, BALB/c-3T3 and C3H 10T1/2 cells. EGF (30 ng/ml) added to quiescent 3T3 cells in medium containing either platelet-poor plasma or 10(-5) M insulin caused only minimal increases in the percentage of cells stimulated to initiate DNA synthesis. In contrast, EGF acted synergistically with either insulin or plasma to stimulate DNA synthesis in quiescent cultures of 10T1/2 cells, although the maximum effects of EGF were measured at concentrations several-fold greater than those found in either serum or plasma. In either 3T3 or 10T1/2 cells a transient preexposure to platelet-derived growth factor (PDGF) caused over a 10-fold increase in the sensitivity to the mitogenic effects of EGF. It is therefore possible that a primary action of PDGF is to increase the sensitivity of fibroblasts to EGF, independent of whether EGF alone is found to be mitogenic.     

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.     

Insulin-like growth factor 1 and insulin reduce epidermal growth factor binding to Swiss 3T3 cells by an indirect mechanism that is apparently independent of protein kinase C

Insulin-like growth factor 1 and insulin reduced the binding of 125I-labelled epidermal growth factor (125I-EGF) to Swiss 3T3 cells by 15-20% at 37 degrees C, but not at 4 degrees C. Scatchard analysis indicated that IGF-1 and insulin affected the higher-affinity component of EGF binding, an effect previously associated with the activation of protein kinase C. However, the inhibition of 125I-EGF binding by IGF-1 and insulin was increased, not reduced, when the cells were treated with high concentrations of phorbol esters to down-modulate protein kinase C. We suggest that IGF-1 and insulin activate a protein kinase with similar or overlapping specificity to that of protein kinase C.     

Platelet-derived growth factor-induced alterations in vinculin and actin distribution in BALB/c-3T3 cells

Exposure of BALB/c-3T3 cells (clone A31) to platelet-derived growth factor (PDGF) results in a rapid time- and dose-dependent alteration in the distribution of vinculin and actin. PDGF treatment (6-50 ng/ml) causes vinculin to disappear from adhesion plaques (within 2.5 min after PDGF exposure) and is followed by an accumulation of vinculin in punctate spots in the perinuclear region of the cell. This alteration in vinculin distribution is followed by a disruption of actin-containing stress fibers (within 5 to 10 min after PDGF exposure). Vinculin reappears in adhesion plaques by 60 min after PDGF addition while stress fiber staining is nondetectable at this time. PDGF treatment had no effect on talin, vimentin, or microtubule distribution in BALB/c-3T3 cells; in addition, exposure of cells to 5% platelet-poor plasma (PPP), 0.1% PPP, 30 ng/ml epidermal growth factor (EGF), 30 ng/ml somatomedin C, or 10 microM insulin also had no effect on vinculin or actin distribution. Other competence-inducing factors (fibroblast growth factor, calcium phosphate, and choleragen) and tumor growth factor produced similar alterations in vinculin and actin distribution as did PDGF, though not to the same extent. PDGF treatment of cells for 60 min followed by exposure to EGF (0.1-30 ng/ml for as long as 8 h after PDGF removal), or 5% PPP resulted in the nontransient disappearance of vinculin staining within 10 min after EGF or PPP additions; PDGF followed by 0.1% PPP or 10 microM insulin had no effect. Treatment of cells with low doses of PDGF (3.25 ng/ml), which did not affect vinculin or actin organization in cells, followed by EGF (10 ng/ml), resulted in the disappearance of vinculin staining in adhesion plaques, thus demonstrating the synergistic nature of PDGF and EGF. These data suggest that PDGF-induced competence and stimulation of cell growth in quiescent fibroblasts are associated with specific rapid alterations in the cellular organization of vinculin and actin.     

Transforming growth factor-beta and retinoic acid modulate phenotypic transformation of normal rat kidney cells induced by epidermal growth factor and platelet-derived growth factor

In this study we have investigated the ability of epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and transforming growth factor-beta (TGF beta) together with retinoic acid (RA) at saturating concentrations to induce phenotypic transformation of normal rat kidney (NRK) cells in a growth factor-defined medium. This medium contains serum in which all growth factor activity has been chemically inactivated, thereby eliminating the effects of growth factors from serum in the assay. It is shown that neither TGF eta nor a ligand binding to the EGF receptor is essential for phenotypic transformation of NRK cells, since anchorage-independent growth is also induced by EGF in combination with RA and by PDGF in combination with RA and TGF beta. Our data indicate strong similarities between TGF beta and RA in their ability to act as modulators for phenotypic transformation. In addition, both agents enhance the number of EGF receptors in NRK cells, without affecting the number of PDGF receptors. On the other hand, TGF beta has mitogenic effects on a number of non-transformed cell lines, such as Swiss 3T3 fibroblasts, particularly when assayed in the absence of insulin, whereas RA is mitogenic for these cells only in the presence of insulin. These data demonstrate that phenotypic transformation of NRK cells requires specific combinations of polypeptide growth factors and modulating agents, but that this process can be induced under many more conditions than previously described. Moreover, our data point toward both parallels and differences in the activities of TGF beta and RA.     

Dexamethasone inhibits feedback regulation of the mitogenic activity of tumor necrosis factor, interleukin-1, and epidermal growth factor in human fibroblasts

Tumor necrosis factor (TNF), interleukin-1 (IL-1), and epidermal growth factor (EGF) were mitogenic for human diploid FS-4 fibroblasts. Dexamethasone amplified the growth-stimulating action of all three agents. Amplification of the growth-stimulating action was maximal when dexamethasone was added along with TNF or EGF; no amplification was seen if the addition of dexamethasone was delayed for more than 3 hr. Prolonged simultaneous treatment with TNF and EGF resulted in less growth stimulation than treatment with EGF alone. Dexamethasone abolished this apparent antagonistic interaction between TNF and EGF. Dexamethasone also inhibited the antiviral action of TNF against encephalomyocarditis (EMC) virus in FS-4 cells. TNF and IL-1 increased the steady state level of interferon (IFN)-beta 2 mRNA but failed to induce detectable levels of IFN-beta 1 mRNA in FS-4 cells. Dexamethasone inhibited the increase of IFN-beta 2 mRNA levels by IL-1 or TNF. Inhibition of IFN-beta synthesis is likely to be responsible for the inhibition of the TNF-induced antiviral state by dexamethasone. Since IFNs suppress cell growth, inhibition of endogenous IFN-beta synthesis may also be responsible for the amplification by dexamethasone of the growth-stimulating action of TNF and IL-1. Amplification of the mitogenic action of EGF by dexamethasone appears to be mediated by different mechanism.     

Epidermal growth factor (EGF) and somatomedin C regulate G1 progression in competent BALB/c-3T3 cells

The activity in platelet-poor plasma that allowed density-arrested BALB/c-3T3 cells rendered competent by a transient exposure to platelet-derived growth factor (PDGF) to traverse G1 and enter the S phase has been termed progression activity. Epidermal growth factor (EGF) and somatomedin C-supplemented medium was shown to be capable of replacing the progression activity of 5% platelet-poor plasma (PPP) for competent density-inhibited BALB/c-3T3 cells. Exposure of competent cells to medium supplemented with EGF and somatomedin C reduced the 12 h minimum G1 lag time found in plasma-supplemented medium by 2 h. It is suggested that the reduction in the minimum time required for progression through G1 is due to the availability of free, unbound somatomedin C. Complete G1 traverse required both EGF and somatomedin C; however, the traverse of the last 6 h of G1 and entry into the S phase required only somatomedin C. Though EGF and somatomedin C could replace the G1 phase progression activity of plasma, medium supplemented with EGF and somatomedin C did not support complete cell cycle traverse or growth of sparse cultures of BALB/c-3T3 cells.     

Neural cell adhesion molecule mediates contact-dependent inhibition of growth of near-diploid mouse fibroblast cell line m5S/1M

A near-diploid mouse fibroblast cell line m5S/1M used in this study shows a high sensitivity to contact-dependent inhibition of growth, and the addition of EGF causes both morphological change and loss of contact-dependent inhibition of growth. The m5S/1M cell and its transformants obtained by x-ray irradiation have been used to search for the cell surface glycoproteins that are responsible for the growth regulation via cell-cell interactions. Lectin blotting analyses showed that the expression of the cell surface glycoprotein of 140 kD (140KGP) is highly sensitive to the transformation induced either by x-ray irradiation or by the EGF stimulation. We purified the 140KGP and found that it was composed of two glycoproteins. The major component of 140KGP was identified as neural cell adhesion molecule (NCAM) by amino acid sequence analyses of the peptide fragments and by the cross-reactivity with anti-NCAM mAb, clone H28.1.2.3. Monoclonal antibody against 140KGP (clone LN-10) recognizes all three isoforms of NCAM expressed on m5S/1M cell and showed that the expression of NCAM was highly sensitive to the transformation. Furthermore, the immobilized LN-10 strongly inhibited the growth of actively proliferating m5S/1M cells and the LN-10 in a soluble form showed a significant growth-stimulating effect on the confluent quiescent cultures of m5S/1M cells. The results show that NCAM plays a major role in the contact-dependent inhibition of growth of m5S/1M, and that NCAM might be involved in the regulation of cell growth during embryogenesis and formation of nervous systems.     

Effect of certain growth factors on proliferation in serum-free collagen gel culture of vaginal epithelial cells from prepuberal mice exposed neonatally to diethylstilbestrol

Neonatal treatment with diethylstilbestrol (DES) induces ovary-independent vaginal epithelial changes in mice. The response of vaginal epithelial cells from intact prepuberal BALB/cCrgl mice treated neonatally with 2 micrograms of DES for 5 days to growth-stimulatory and -inhibitory factors was studied using a serum-free collagen gel culture system that sustains the growth of normal vaginal epithelial cells. Cells from control and DES-exposed mice at 21 days of age showed about a 5-fold increase in number during 10 days in a serum-free medium supplemented with transferrin, bovine serum albumin fraction V, insulin, and epidermal growth factor. Epidermal growth factor and insulin stimulated dose-related proliferation of vaginal epithelial cells from both control and DES-exposed mice; however, cells from DES-exposed mice showed a reduced growth response to epidermal growth factor and an increased growth response to insulin, compared with control cells. Insulin-like growth factor I (1-100 ng/ml) tested in the absence of insulin failed to stimulate cell growth. Transforming growth factor-beta (0.05-5 ng/ml) consistently inhibited cell growth in a dose-dependent manner.     

Synergistic actions of cytokines and growth factors in enhancing porcine granulosa cell growth.

In our studies of the growth-promoting effect of a cytokine, interleukin-1 (IL-1), on cultured porcine granulosa cells, we found that the potency of IL-1 action correlated with the serum concentration in the culture medium and that IL-1 acted synergistically with insulin to increase the number of cells in the presence of low serum concentrations (0.1-1%). With granulosa cells maintained in a quiescent state under serum-free conditions, we therefore examined the effects of combined treatment with IL-1 and peptide growth factors, including insulin, on [3H]thymidine incorporation by these cells. IL-1 by itself enhanced [3H]thymidine incorporation in a concentration-dependent manner. Moreover, IL-1 acted synergistically with insulin, epidermal growth factor (EGF), or fibroblast growth factor (FGF) to enhance [3H]thymidine incorporation. Combinations of maximally effective concentrations of insulin (1 micrograms/ml), EGF (1 ng/ml), or FGF (50 ng/ml) with the maximally effective concentration of IL-1 (10 ng/ml) increased the levels of [3H]thymidine incorporation to 10-, 22-, and 20-fold, respectively, over the control values. Whereas IL-2 (0.1-100 ng/ml) did not affect [3H]thymidine incorporation, tumor necrosis factor alpha (TNF alpha) stimulated [3H]thymidine incorporation by itself and reproduced the actions of IL-1 to act synergistically with insulin, EGF, or FGF. When IL-1 and TNF alpha were added together in relatively low concentrations (1 ng/ml each), the combination had synergistic effects in enhancing [3H]thymidine incorporation. The present study demonstrates that cytokines and peptide growth factors act synergistically to markedly enhance porcine granulosa cell growth in vitro.     

Stimulation by insulin-like growth factors is required for cellular transformation by type beta transforming growth factor

Medium conditioned by BRL-3A cells, a known source of insulin-like growth factor II (IGF-II), induced phenotypic transformation (anchorage-independent proliferation) of mouse BALB/c 3T3 fibroblasts but not rat NRK-49F fibroblasts, in the presence of 10% calf serum. A specific radioreceptor assay and a bioassay indicated that BRL-3A conditioned medium contained 0.5-1 ng/ml of type beta transforming growth factor (beta TGF). Purified IGF-II and beta TGF acting together reconstituted the transforming activity of BRL-3A conditioned medium on BALB/c 3T3 cells. Insulin was 5-10% as potent as IGF-II in supporting the transforming action of beta TGF on BALB/c 3T3 cells. NRK-49F cells were phenotypically transformed by beta TGF in the presence of EGF and 10% calf serum as the sole source of IGFs. However, transformation of NRK-49F cells under these conditions was inhibited by addition of purified IGF-binding protein. Addition of an excess of IGF-II prevented the inhibitory action of IGF-binding protein. The different sensitivity of the two cell lines to IGFs was correlated with lower levels of type I IGF receptor and higher levels of type II IGF receptor in NRK-49F cells as compared with BALB/c 3T3 cells. The results suggest that cellular stimulation by IGFs is a prerequisite for transformation of rodent fibroblasts by beta TGF. We propose that transformation of fibroblasts by beta TGF requires concomitant stimulation by the set of growth factors that support normal cell proliferation.     

Domains of laminin with growth-factor activity

Laminin and fragments (1, 1-4) containing the inner rod-like segments from its short arms, which consist of cysteine-rich, "EGF-like" repeats, stimulated thymidine incorporation in cultured cells possessing EGF receptors but had no effect on a cell line lacking this receptor. The response was comparable to that of EGF concerning effective concentrations, magnitude, time dependence, and synergistic enhancement by insulin. Other fragments (4 and 8) were inactive. Laminin and its active fragments could not compete with the binding of EGF to cells. There was no correlation between growth promotion and attachment of cells to a high affinity binding site present on laminin fragment 8. The data indicate that mitogenic effects induced by laminin and EGF proceed in some steps via related pathways and that different domains of laminin are involved in growth promotion and in adhesion and spreading of cells.