Effects of insulin-like growth factor-binding protein 2 and an IGF-type I receptor-blocking antibody on apoptosis in human teratocarcinoma cells in vitro.

Human teratocarcinoma cells (Tera-2) deprived of serum undergo programmed cell death which can be counteracted by simultaneous addition of IGF-I or IGF-II. This protective effect of IGFs was specific in the sense that both addition of IGF-binding protein-2, and blocking of the IGF-type I receptor by a specific antibody, both resulted in an increased apoptotic rate.

Insulin-like growth factors I and II induce cell death in Wilms's tumour cells.

AIM: To study the effects of insulin-like growth factors (IGFs) on the growth phenotype of a Wilms's tumour cell line (WCCS-1). METHODS: WCCS-1 cells were cultured in vitro and exposed to IGF-I and IGF-II, as well as their antagonists, IGF binding protein 2 and the type I receptor blocking antibody IGF-IRalpha. The effects on proliferation and cell cycle parameters were assayed by assessing cell numbers, autoradiography after labelling with tritiated thymidine, and flow cytometry after double staining with fluorescein isothiocyanate (FITC) labelled annexin V and propidium iodide. RESULTS: The addition of IGF-I as well as IGF-II in physiological doses induced cell death in Wilms's tumour cells. Cell numbers decreased most dramatically on the fifth to sixth day after growth factor addition. The occurrence of apoptosis as well as necrosis was confirmed by annexin-V staining of cell cultures. S-phase indices were comparable, irrespective of whether the cells were exposed to IGFs or not, which suggests that WCCS-1 cells undergo cell death at random during the cell cycle rather that from the prereplicative phase. To exclude any influences of the IGF binding proteins (IGFBPs), all results were repeated with Des(1-3)IGF-I, which is unable to bind to any of the IGFBPs. However, this peptide was equally potent in inducing cell death. Finally, the addition of IGFBP-2 or the type 1 receptor blocking antibody IGF-IRalpha partly abrogated the death inducing effects of IGF-I and IGF-II. CONCLUSIONS: Insulin like growth factors induce cell death--apoptosis as well as necrosis--in cultured Wilms's tumour cells. Furthermore, it is proposed that this effect is mediated by the type 1 receptor.

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.

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.

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.

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.

Insulin-like growth factor II prevents apoptosis in a human teratoma derived cell line.

Aim-To study how insulin-like growth factor II (IGF-II) affects the behaviour of human teratoma cells.Methods-The human pluripotential teratoma cell line Tera 2 was cultured under serum-free conditions in the presence or absence of IGF-II. Effects on cell proliferation and apoptosis as well as on the expression of the proto-oncogene c-myc were studied.Results-In this study we show that Tera 2 cells deprived of serum undergo programmed cell death (apoptosis). The onset of nuclear fragmentation was observed 12 hours after serum withdrawal. The morphological changes of the Tera 2 cell nuclei were confirmed by the occurrence of a nucleosome ladder. However, the constitutive expression of the proto-oncogene c-myc was not decreased in parallel with initiation of apoptosis. The apoptotic response to serum withdrawal could be counteracted by simultaneous addition of IGF-II. In addition it was found that human testicular tumours (seminoma and embryonal carcinoma) contain raised levels of insulin-like growth factors.Conclusions-The precise roles of IGF-I and IGF-II have been unclear, and there is overwhelming evidence against these factors as primarily transforming agents. The finding that IGF-II apparently counteracts apoptosis in vitro may well explain its effects on tumours in vivo.

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.

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.

Developmental regulation of insulin like growth factor II expression in the horse.

The expression of the insulin like growth factor (IGF) II gene has been examined in the developing equine fetus. It was found that IGF II transcripts were present in abundant quantities in third trimester embryonic and extraembryonic tissues as for example the placenta. The expression of the IGF II gene was high in the fetal liver where two prominent transcripts--4.6 and 4.1--kB were produced. However, these transcripts could not be traced in the adult liver. Instead we found two different transcripts with the sizes of 4.0 and 2.9 kB in the adult liver. These findings taken together with the demonstration of heterologous IGF II cDNA hybridizing to equine DNA-digests suggests that the IGF II gene is under developmental control in the horse, with the possible existence of different promoters.

Heparin binding growth factors and the control of teratoma cell proliferation.

The role of basic fibroblast growth factor (bFGF) in the control of teratoma cell proliferation was examined. It was found that bFGF stimulates proliferation at low concentrations but induces cell migration at higher doses. These effects could be efficiently counteracted by addition of protamine sulphate. Moreover the bFGF gene is actively transcribed in primary human testicular embryonal carcinomas but was not expressed in any other embryonic tumour examined. The biological implications of these findings will be discussed.

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.

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.