Growth factors and apoptosis in development. The role of insulin like growth factor I and TGFbeta1 in regulating cell growth and cell death in a human teratocarcinoma derived cell line.

The balance between different cell populations in the developing organism is controlled by regulating the rates of multiplication, differentiation or death of its constituent cells. The human teratocarcinoma derived cell line Tera 2, which in several aspects mirrors early embryonic cells, can be induced to undergo programmed cell death (apoptosis) by depriving cell cultures of serum. This study demonstrates that this process can be reversed by replacing serum with physiological concentrations of insulin like growth factor I (IGF I). As a result, IGF I enhances the rate of Tera 2 cell proliferation in serum free medium. In contrast, Transforming Growth Factor beta1 did not exert any effect on growth or apoptosis in Tera 2 cells. The results indicate that one effect of growth factors on pluripotential cells is to regulate the balance between cell proliferation and cell death.

Growth factors and apoptosis.

Apoptosis is nowadays recognized as an important mechanism by which cells can be eliminated from the organism. In particular its role in tissue modelling during embryogenesis has been highlighted. The human teratoma cell line Tera 2, which in several respects acts as a human embryonic stem cell, can be induced to undergo apoptosis by reducing the serum content of the tissue culture medium. We report here that this process can be reversed by replacing serum with the heparin-binding growth factors, acidic FGF and basic FGF. In contrast, neither of the mammalian transforming growth factors (TGF-beta 1-3) managed to exert any effect on growth or apoptosis in Tera 2 cells.

Transcriptional regulation and biological significance of the insulin like growth factor II gene.

The insulin like growth factors I and II are the most ubiquitous in the mammalian embryo. Moreover they play a pivotal role in the development and growth of tumours. The bioavailability of these growth factors is regulated on a transcriptional as well as on a posttranslational level. The expression of non-signalling receptors as well as binding proteins does further tune the local concentration of IGFs. This paper aims at reviewing how the transcription of the IGF genes is regulated. The biological significance of these control mechanisms will be discussed.

Regulation of a promoter from the mouse insulin like growth factor II gene by glucocorticoids.

We have microinjected constructs containing the murine IG II P3 promoter linked to different flanking sequences and a luciferase reporter gene into mouse pronuclei to establish transgenic lines of mice. The offspring was used as a source of embryonic fibroblast cultures and the effect of exogenous addition of glucocorticoids on transgene expression was analysed. It was found that both dexamethasone and hydrocortisone gave rise to a functional stimulation of the IGF II P3 promoter when the construct also contained other elements. This study demonstrates for the first time that there is an effect of glucocorticoids on the activation of an embryonic IGF II promoter, thus providing a molecular rationale for previous findings that glucocorticoids can under certain circumstances give rise to an increased transcriptional activity of the IGF II gene.

Differentiation associated modulation of K-FGF expression in a human teratocarcinoma cell line and in primary germ cell tumours.

The human teratocarcinoma cell line Tera 2 can be induced to differentiate in vitro after exposure to retinoic acid. We show in this paper that whereas the K-FGF oncogene is expressed in undifferentiated cells, addition of retinoic acid rapidly (less than 60 min) downregulates the expression of this gene. However, when cells are cultured in RA for an extended period of time (greater than 15 days) K-FGF transcripts reappear. We also report that K-FGF is expressed in approximately one-third of primary human germ cell tumours but not in the corresponding normal testicular tissue.

Bradykinin blocks the action of EGF, but not PDGF, on fibroblast division.

Quiescent fibroblasts derived from human fetal lung can be stimulated to reinitiate DNA synthesis by sequential addition of 3 nM IGF-1 and a low concentration (8 pM) of EGF or by continuous exposure to 10% fetal calf serum or 10 ng/ml PDGF. Bradykinin blocks the IGF-1 and EGF-dependent signals without affecting the response to serum or PDGF. It activates protein kinase C and its anti-mitogenic effect is abolished after this kinase has been down-regulated. Bradykinin has no effect on the binding affinity of the EGF receptor whereas phorbol ester induces its 'transmodulation' to low affinity.

Concentration-dependent modulation of basic fibroblast growth factor action on multiplication and locomotion of human teratocarcinoma cells.

A human teratoma cell line (Tera 2) was grown in serum-free medium, and the population multiplication was stimulated by the addition of 1-10 ng basic fibroblast growth factor (bFGF)/ml. The bFGF-effect was abrogated by the addition of protamine sulphate. When high concentrations of bFGF were added, a preferential effect on cell locomotion was observed. 100 ng bFGF/ml stimulated cell movement but only exerted a marginal effect on cell multiplication. These observed exogenous requirements for multiplication and locomotion were complemented by the expression of bFGF receptors. Scatchard analysis of binding data suggests the existence of a high-affinity and a low-affinity class of receptors.

The effects of leukemia inhibitory factor (lif) on cell multiplication and locomotion in human teratocarcinoma cells.

The human teratocarcinoma cell line (Tera 2) could be stimulated to a moderate increase in cell number in serum-free medium by addition of 5 ng leukemia inhibitory factor (LIF)/ml. However this effect was only observed in short term (24 h) cultures. By comparing cell numbers with thymidine incorporation data and proportion intact cell nuclei, we concluded that this short term increase in cell number was due to enhanced cell survival rather than a real increase in the proportion of cells traversing the cell cycle. When increased concentrations of LIF were added a preferential effect on clonal cell locomotion was observed. 50-200 ng of LIF stimulated cell movement but exerted no effect on Tera 2 cell proliferation at any time interval studied.

Growth activation of resting cells: induction of balanced and imbalanced growth.

Little is known about how mitogenic factors generate growth regulatory signals and how these are mediated within the cell. We have developed three different types of mitogenic stimuli in order to identify the possible existence of common denominators in a complex and perhaps pleiotypic signal-response system. 3T3 cells, starved to quiescence by reducing the serum content of the culture medium 100-fold, can be irreversibly committed to undergo DNA replication and mitosis in a serum-free medium after an initial exposure of short duration to (a) serum factors and cholesterol, (b) a relative excess of glutamine, or (c) alkaline pH. Cells stimulated by any of these procedures, and subsequently incubated in serum-free Dulbecco's modified Eagle's medium (DMEM) undergo DNA replication and mitosis in the absence of concomitant cellular enlargement (imbalanced growth). However, cellular enlargement is induced after the initial mitogenic stimuli (a, b, and c, as described previously) if the cells are subsequently incubated in DMEM containing greater than or equal to 0.5% serum, supraphysiological concentrations of insulin, or normal concentrations of somatomedin C.

Phylogenetical aspects on islet hormone families: a minireview with particular reference to insulin as a growth factor and to the phylogeny of PYY and NPY immunoreactive cells and nerves in the endocrine and exocrine pancreas.

A common feature in the phylogeny of the four islet hormones (insulin, somatostatin, glucagon, PP) is that they do not seem to occur in the most primitive metazoan animals investigated so far, namely the coelenterates. However, already in the earliest protostomian invertebrates, such as flatworms and annelids, somatostatin and PP immunoreactive nerve fibres were found. In highly developed forms of protostomian invertebrates, such as insects, all the four islet hormones are represented as immunoreactive nerve cells and nerve fibres in the brain. In deuterostomian invertebrates a brain-gut-axis has evolved as regards somatostatin and PP, whereas insulin and glucagon now seem to occur exclusively as cells of open type in the gut mucosa. This brain-gut-axis for somatostatin and PP persists in all the vertebrates. The insulin cells, however, leave the gut mucosa already in the earliest forms of vertebrates and then appear only as cells in the islet parenchyma and in the mucosa of the bile duct (Agnatha) or in the pancreatic ducts (Gnathostomi). To some extent, glucagon islet cells evolve in a similar manner; here, however, cells immunoreactive with the precursor hormone, glicentin (enteroglucagon), persist in the gastrointestinal tract mucosa. A few PYY immunoreactive cells have been found in the pancreatic islet parenchyma of reptiles and mammals, often as disseminated cells in the acinar tissue. In the pancreas of these phyla NPY only occurs in neurons and nerve fibres. In pilot studies the effects of hagfish insulin as a growth factor have been compared with those of pig insulin on Swiss 3T3 mouse embryonic fibroblasts.

Urine B2-microglobulin in patients with macroglobulinemia Waldenström.

The level of B2-microglobulin in the urine was determined by a radioimmunoassay technique in six patients with Waldenström's macroglobulinemia. In 50% of the patients the B2-microglobulin in the urine was significantly increased. In none of the analyzed patients any glomerular malfunction, as measured by serum creatinine and creatinine clearance, was observed.

Urinary excretion of beta 2-microglobulin in myeloma patients.

The levels of beta 2-microglobulin in urine and serum were determined in 39 patients with myelomatosis. In 25 patients the serum beta 2-microglobulin was elevated, and in seven of the patients with increased serum beta-microglobulin the urinary excretion of the protein was also increased. It was concluded that the increased urine beta-microglobulin indicates a renal tubular disorder.

Dual effects of four members of the fibroblast growth factor member family on multiplication and motility in human teratocarcinoma cells in vitro.

The effects of four recently discovered members of the Fibroblast Growth Factor family, FGF-10, FGF-16, FGF-17 and FGF-18, on the human embryonal carcinoma derived cell line Tera 2 were examined. It was found that all four FGF:s enhanced the survival rate of Tera-2 cells by counteracting apoptosis at concentrations in the interval 1-10 ng/ml. When higher concentrations of either of the four FGF:s were added, a preferential effect on cell motility was observed. The observed requirements for externally supplied FGF:s were complemented by the expression of all four FGF-receptors.

The effects of glycosylation inhibitors on the proliferation of a Wilms tumour cell line.

We have examined the effects of different glycosylation inhibitors on the proliferation of a human Wilms tumour derived cell line WCCS-1. It was found that two compounds that specifically inhibit distal steps in the glycosylation chain (swainsonone and castanospermine) only exerted marginal effects on cell multiplication and survival. In contrast, a proximal inhibitor (tunicamycin) efficiently increased necrosis in a dose dependent fashion. It is shown that this cell death was accompanied by a marked decrease in the incorporation of glucosamine, but rather unexpectedly, only caused a limited inhibition of de novo protein synthesis. Moreover, the entrance into S-phase was virtually unchanged in the cells surviving the exposure to tunicamycin. The effects of tunicamycin on cell multiplication and survival could not be reversed by concomitant addition of mevalonate as has been shown in other cell lines. Taken together this data suggests that tunicamycin does not operate in a cell cycle specific manner in Wilms tumour cells.

Expression of the 3-hydroxy-3-methylglutaryl coenzyme A-reductase and LDL-receptor genes in human embryonic tumours and in normal foetal tissues.

The expression of the HMG-CoA-reductase and LDL-receptor genes was examined in primary human embryonic tumours and in normal human first trimester foetal tissues. All embryonic and extra-embryonic tissues examined contained RNA that hybridized with the HMG-CoA-reductase and LDL-receptor cDNA-probes. However, the transcript levels varied considerably between organs. By using Northern blotting, we confirmed that normal foetal tissues contained HMG-CoA-reductase and LDL-receptor transcripts of normal size (4.2 kB and 5.3 kB, respectively). All human developmental tumours examined were characterized by high levels of HMG-Co-A-reductase RNA. However, when the quality of this RNA was examined in greater detail it was found that the 4.2 kB band only accounted for a minor portion of the RNA. Instead we found a range of other transcripts (3.1, 1.1., 0.95, 0.78 and 0.55 kB) that hybridized with the probe. There was a qualitative difference in that only the non-seminomas contained high levels of the three smaller transcripts (0.95, 0.78 and 0.55 kB). In contrast, only one tumor--an embryonal carcinoma--contained increased levels of LDL-receptor transcript. This was found to be of the expected size (5.3 kB).

The biological effects of a high molecular weight form of IGF II in a pluripotential human teratocarcinoma cell line.

The human teratoma cell line Tera 2 synthesizes and secretes insulin like growth factors into the culture medium. Size fractionation of conditioned medium by acidic gel filtration chromatography showed that the medium contains the canonical 7 kD IGF II, as well as a large IGF II variant, immunologically crossreactive with canonical IGF II. Amino acid analysis of the Tera 2 secreted large IGF II variant has shown that it is biochemically distinct from previously isolated high molecular weight variants of IGF II. Both species of IGF II support cell multiplication of Tera 2 cultures, albeit with different potency. In spite of the resulting potential for autocrine growth of Tera 2, we failed to observe such a situation. We propose that one reason for this failure to observe such a situation. We propose that one reason for this failure is the co-secretion of the 29 kD IGF binding protein type 1 with IGF II, which we have demonstrated to inhibit the biological effects of the growth factor on Tera 2 cells.

Expression of c-myc in canine mammary tumours.

The expression of the protooncogene c-myc was examined in twelve canine mammary tumours of different histological type. Only in one tumour--a poorly differentiated carcinoma--was the amount of RNA that hybridized with our cDNA probe for the myc gene significantly increased. Restriction fragment length analysis of genomic DNA from this tumour did not reveal any amplification or other rearrangements in this gene locus.

Expression of the myc protooncogene in canine rhabdomyosarcoma.

The expression of the protooncogene c-myc in a canine rhabdomyosarcoma was examined. It was found that this highly malignant tumour contained vast quantities of RNA that hybridized with a cDNA probe for c-myc. Restriction fragment length analysis after endonuclease digestion of tumour DNA did not reveal any rearrangements in this gene locus. The potential role of this oncogene in the development of canine rhabdomyosarcoma is discussed.

Local tolerance to spider silks and protein polymers in vivo.

Spider silks were implanted subcutaneously in pigs for a study of the tolerance against this material. Four types of spider silks of high purity and cleanliness were implanted: (i) major ampullate dragline silk reeled from the golden silk spider Nephila clavipes, (ii) native (unsterilised) silk reeled from a Brachypelma spider, (iii) native silk taken from this spider's web and (iv) its web silk thermally treated at 80 degrees C. For comparison we used fibrous silk analogue protein polymers and four already marketed wound dressings (polyurethane film, collagen dressings, gauze pads). All materials were applied epicutaneously to split skin wounds. The implants were examined macroscopically as well as by light microscopy. Superficially, all sites healed rapidly. There were marked inflammatory reactions in all sites with lympho-plasmacellular infiltrations, evidence of phagocytosis and granuloma formation as indicated by the appearance of giant cells. However there was a marked absence of epitheloid cells indicating that the observed reaction was a foreign body granuloma. Furthermore, the histopathological images recorded after 14 days revealed no marked differences between the dressings. Polyurethane films, however, seemed to be superior with respect to the duration of the wound healing process.

Role of fibroblast growth factors in elicitation of cell responses.

Fibroblast growth factors (FGFs) are signalling peptides that control important cell processes such as proliferation, differentiation, migration, adhesion and survival. Through binding to different types of receptor on the cell surface, these peptides can have different effects on a target cell, the effect achieved depending on many features. Thus, each of the known FGFs elicits specific biological responses. FGF receptors (FGFR 1-5) initiate diverse intracellular pathways, which in turn lead to a variety of results. FGFs also bind the range of FGFRs with a series of affinities and each type of cells expresses FGFRs in different qualitative and quantitative patterns, which also affect responses. To summarize, cell response to binding of an FGF ligand depends on type of FGF, FGF receptor and target cell, all interacting in concert. This review aims to examine properties of the FGF family and its members receptors. It also aims to summarize features of intracellular signalling and highlight differential effects of the various FGFs in different circumstances.