Epigenetic regulation of the Igf2/H19 gene cluster.

Igf2 (insulin-like growth factor 2) and H19 genes are imprinted in mammals; they are expressed unevenly from the two parental alleles. Igf2 is a growth factor expressed in most normal tissues, solely from the paternal allele. H19 gene is transcribed (but not translated to a protein) from the maternal allele. Igf2 protein is a growth factor particularly important during pregnancy, where it promotes both foetal and placental growth and also nutrient transfer from mother to offspring via the placenta. This article reviews epigenetic regulation of the Igf2/H19 gene-cluster that leads to parent-specific expression, with current models including parental allele-specific DNA methylation and chromatin modifications, DNA-binding of insulator proteins (CTCFs) and three-dimensional partitioning of DNA in the nucleus. It is emphasized that key genomic features are conserved among mammals and have been functionally tested in mouse. 'The enhancer competition model', 'the boundary model' and 'the chromatin-loop model' are three models based on differential methylation as the epigenetic mark responsible for the imprinted expression pattern. Pathways are discussed that can account for allelic methylation differences; there is a recent study that contradicts the previously accepted fact that biallelic expression is accompanied with loss of differential methylation pattern.

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.

The role of scaffold proteins in JNK signalling.

This paper summarises how scaffold proteins affects and regulate the JNK signalling pathway. We believe that some of these scaffold proteins, by virtue of their anchoring and catalytic properties contribute to a high degree of specificity of intra cellular signalling pathways that regulate the progression through the cell cycle.

Major ampullate spidroins from Euprosthenops australis: multiplicity at protein, mRNA and gene levels.

Spider dragline silk possesses extraordinary mechanical properties. It consists of large fibrous proteins called spidroins that display modular structures. It is known to consist of two proteins: the major ampullate spidroin (MaSp) 1 and MaSp2. This study analyses MaSp sequences from the nursery-web spider Euprosthenops australis. We have identified a previously uncharacterized MaSp2 sequence and a new MaSp-like spidroin, which display distinct homogenous submotifs within their respective Gly-rich repeats. Furthermore, a group of MaSp1 cDNA clones show unexpected heterogeneity. Genomic PCR identified several MaSp1 gene variants within individual spiders, which suggests the presence of a gene cluster in E. australis. Finally, the evolution of spidroin genes is discussed in relation to phylogenetic analysis of nonrepetitive C-terminal domains from diverse species.

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.

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.

Expression of the insulin-like growth factor II gene in polychlorinated biphenyl exposed female mink (Mustela vison) and their fetuses.

AIM: To study how polychlorinated biphenyls (PCBs) affect fetal growth and the expression of the insulin-like growth factor (IGF II) gene in the mink (Mustela vision). METHODS: Ten female mink were each exposed to 0.65 or 1.3 mg Clophen A50/day, respectively, during the reproductive season. The numbers and sizes of viable fetuses were recorded. The expression of the IGF II gene was studied by northern blotting using a mink specific IGF II cDNA probe. RESULTS: The number of viable fetuses decreased after PCB exposure in a dose dependent fashion. Expression of the IGF II gene in adult livers from PCB exposed animals was decreased, compared with control animals, in a dose dependent fashion. In contrast, IGF II expression in placentas and fetuses was unaltered. Furthermore, the maternal excretion of urinary cortisol increased in both exposed groups during the implantation period. CONCLUSIONS: Expression of the IGF II gene is downregulated by PCB exposure in the adult liver. There is also an indication that the regulation of the expression of this gene differs between adult and fetal life.

A conserved structural element in horse and mouse IGF2 genes binds a methylation sensitive factor.

The equine IGF2 gene has been cloned and characterised. It spans a 9 kb region, which is substantially less than the corresponding human gene. Three coding exons and three untranslated leader exons, all highly homologous to those in other species, were identified. Downstream of the polyadenylation site in exon 6, a dinucleotide repeat sequence was identified. Three putative promoters (P1-P3) were localised in the 5' region of the gene. RNase protection analysis revealed two active promoters in fetal tissues, P2 and P3, whereas P3 was the only promoter active in adult tissues. This represents a transcriptional pattern different from that in humans or rodents. A novel structural element, an inverted repeat, is predicted in the 3' region of the IGF2 gene. This repeat is conserved between species and located in a region which is differentially methylated in the human and mouse genes and might therefore be involved in the imprinting mechanism. The inverted repeat acquires a stem-loop structure in vitro with a hybrid A/B-DNA conformation in the stem area. Both in horse and mouse, a methylation-sensitive protein binds this structure with a strong requirement for the loop area. Furthermore, the protein might be developmentally regulated.

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.

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.

Cloning and sequencing of an equine insulin-like growth factor I cDNA and its expression in fetal and adult tissues.

A cDNA for equine insulin-like growth factor I (IGF I) has been isolated by reverse transcriptase-polymerase chain reaction and subsequently sequenced. The sequenced fragment contained 465 bp including the coding regions for the signal peptide, the entire mature protein, and 4 amino acids into the E-peptide. Like its human counterpart, the mature equine IGF I peptide contains 70 amino acids and was 100% homologous between horse and man. The 49-amino-acid signal peptide had the threonine in position 26 of the human signal peptide substituted by isoleucine. The nucleotide homology across the entire clone was 96.3% between horse and man and 91.6% between horse and rat. The isolated cDNA hybridized to the same transcripts in fetal and adult tissues.

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.

Insulin-like growth factor II in the horse: determination of a cDNA nucleotide sequence and expression in fetal and adult tissue.

Horse cDNA for insulin-like growth factor II (IGF II) has been isolated. cDNA was synthesized from bulk mRNA and subsequently PCR-amplified and sequenced. Like its human counterpart, the mature horse IGF II peptide contains 67 amino acids with only two substitutions, isoleucine instead of valine in position 35 and asparagine instead of serine in position 36. The nucleotide homology was 92.1% between horse and human and 87.8% between horse and mouse. The isolated cDNA hybridized to multiple transcripts in fetal and adult tissues, thus confirming earlier reports on developmental expression of this gene in the horse.

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.