Hypoglycaemia associated with the production of insulin-like growth factor II and insulin-like growth factor binding protein 6 by a haemangiopericytoma.

Non-islet-cell tumour-induced hypoglycaemia (NICTH) is, in most cases, attributable to tumour production of insulin-like growth factor II (IGF-II). Tumour-derived IGF-II has a higher than normal molecular weight (big 'IGF-II') and an impaired ability to form the normal ternary 150 kD complex with IGF binding protein-3 (IGFBP-3) and the acid-labile subunit (ALS). Consequently, tumoral IGF-II circulates mainly in smaller binary complexes which have a higher bioavailability than the ternary complex. We had the opportunity to analyze IGFs and IGF-related factors in both pre- and post-operative blood, tumour tissue and tumour cyst fluid from a patient with a disseminated haemangiopericytoma and severe hypoglycaemia. In addition, the effect of serum and tumour cyst fluid on autophosphorylation of the insulin receptor was examined. Patient serum contained low levels of IGF-I, IGFBP-3 and ALS, while the concentrations of IGFBP-2 and IGFBP-6 were markedly elevated. The total level of circulating IGF-II was within the normal range, but Biogel P-60 gel filtration of patient serum revealed that 77% of the IGF-II was present in high molecular weight forms (normal: 10-15%), which decreased to 53% after partial removal of the tumour. Most of the IGF-II immunoreactivity in pre- and post-operative patient serum was associated with 50-60 kD complexes with only a minimal contribution (<10%) from the 150 kD complex. Tumour cyst fluid contained excessive amounts of both big IGF-II and IGFBP-6. Northern blot analysis of total mRNA isolated from the tumour demonstrated high expression of the IGF-II gene and abundant 1.1 kb IGFBP-6 transcript, while the genes encoding IGFBP-3, -4 and -5 were only weakly expressed and mRNA of IGFBP-1, -2 and IGF-I could not be detected. mRNAs for the IGF type II receptor could be easily demonstrated, whereas those for the insulin- and IGF type I receptor were hardly detectable. In contrast to patient serum tumour cyst fluid strongly stimulated the insulin receptor in vitro. The present study suggests an important role of the simultaneous production of IGF-II and IGFBP-6 in the pathophysiology of tumour-induced hypoglycaemia.

Expression of insulin-like growth factors (IGFs), their receptors and IGF binding protein-3 in normal, benign and malignant smooth muscle tissues.

To assess the role of insulin-like growth factors (IGFs) in growth and transformation of normal (myometrium) and tumorous smooth muscle cell (SMC) tissues, in situ hybridization (ISH) analysis for insulin-like growth factor I and II (IGF-I and IGF-II) mRNAs was combined with detection of IGF peptides, their receptors and IGF binding protein-3 (IGFBP-3). mRNAs for both IGFs were detected in smooth muscle cells in normal, benign and malignant SMC tissues, together with the IGF peptides, both IGF receptors and IGFBP-3. This suggests an autocrine role for both IGFs. Leiomyomas had higher IGF-I peptide levels and higher levels of type I IGF receptors than myometrium, supporting the idea that IGFs play a role in the growth and transformation of these tumours. Low-grade leiomyosarcomas contained more IGF-II mRNAs than myometrium and leiomyoma, fewer type II IGF/mannose 6-phosphate receptors and less IGFBP-3 than myometrium and, in addition, fewer IGF-I mRNAs and type I IGF receptors than leiomyoma. Intermediate- and high-grade leiomyosarcomas had intermediate levels of IGF-II mRNAs and peptide, ranging between those in myometrium and low-grade leiomyosarcomas. Thus, growth and transformation of leiomyosarcomas may be regulated by IGF-II, although more markedly in low-grade than in high-grade leiomyosarcomas. In conclusion, the various categories of SMC tissues are associated with a distinct expression pattern of the IGF system. This suggests that each category of SMC tumours arises as a distinct entity and that there is no progression of transformation in these tissues.

Histamine-stimulated expression of insulin-like growth factors in human glioma cells.

Glioma tumour growth is associated with the expression of insulin-like growth factors I and II (IGFs) and of both type I and type II IGF receptors. It has also been shown that IGFs can stimulate proliferation of cultured glioma cells. We previously reported that histamine too can stimulate the growth of glioma cells in vitro. In this report, we study whether the histamine-induced growth of G47 glioma cells is mediated by the IGFs. We found that histamine stimulates the expression of both IGF-I and IGF-II mRNAs, as determined by a semiquantitative in situ hybridization analysis. Furthermore, incubation of G47 cells with histamine also induced cellular immunostaining for IGF-II. It could be shown that IGF-I-stimulated proliferation is inhibited by IGFBP-3, which decreases the availability of IGFs for binding to the IGF receptors, and by beta-galactosidase, which may decrease IGF binding to the type II IGF receptor, but is not inhibited by the anti-type I IGF receptor monoclonal antibody alphaIR3. However, neither IGFBP-3 nor beta-galactosidase nor alphaIR3 inhibited the histamine-induced proliferation. These results show that the growth-stimulatory effect of histamine is accompanied by the induction of IGFs. This histamine-induced growth stimulation is not mediated by activation of cell surface IGF receptors, although intracrine activation of type II IGF receptors may be involved.

Modulation of insulin-like growth factor (IGF) action by IGF-binding proteins in normal, benign, and malignant smooth muscle tissues.

The insulin-like growth factor (IGF) system is involved in the growth of uterine leiomyomas (L), as these tumors have higher IGF-II messenger ribonucleic acid levels, type I IGF receptor levels, and IGF-I peptide concentrations than myometrium (M). Furthermore, cultured L smooth muscle cells (SMC) respond with greater efficiency to IGF-I than M SMC. Here we investigate a possible modulating role of the binding proteins for the IGFs (IGFBPs) on the actions of IGFs. IGFBP-3 is the most predominant IGFBP in conditioned medium from SMC, with levels ranging from 13-288 ng/mL. Incubation of SMC cultures with IGF-I and the IGF-I analogs long-R3IGF-I and des(1-3)-IGF-I, which have decreased affinity for IGFBPs, revealed a facilitating effect of IGFBPs on the growth-stimulating activity of a high concentration of IGF-I in cell lines with high IGFBP-3 levels. Both a decreased level of IGFBP-3 and a low concentration of the growth factors added were a disadvantage for the facilitating effect. In M and L tissue sections, IGFBP-3 was found exclusively bound to the constituting cells, not in the extracellular matrix. This suggests that a negative modulating role of IGFBP-3 due to sequestration of IGF-I, as occurs in culture medium, is less relevant in vivo. In leiomyosarcoma sections, IGFBP-3 levels are decreased, indicating a decreasing, role for this binding protein in malignant smooth muscle tissues.

Growth advantage of human leiomyoma cells compared to normal smooth-muscle cells due to enhanced sensitivity toward insulin-like growth factor I.

Human uterine leiomyomas exhibit increased IGF-I binding compared to myometrium, while both tissues show IGF-I gene expression. In this study we have examined the functional importance of these findings by testing the presence of IGF-I in 15 leiomyoma biopsies and in 18 myometrium biopsies and the capacity of smooth-muscle cells cultured from these tissues to react to IGF-I. The mean IGF-I peptide concentration in leiomyomas was 3 times higher than in myometrium. This resulted from increased IGF-I uptake in leiomyomas rather than from increased synthesis, as these tissues contain higher concentrations of type-I IGF receptors, as detected by immunohistochemistry, and equal levels of IGF-I mRNA. Blocking IGF-I transport with cytochalasin-B and with the type-I IGF receptor blocking antibody alpha IR3 in cultured cells induced decreased immunostaining intensity for IGF-I in most myometrium and leiomyoma cultures, indicating that the detected IGF-I is internalized. Depending on the culture conditions, IGF-I administration yielded increased survival or a higher proliferation rate in leiomyoma cultures than in myometrium cultures, indicating the increased importance of exogenous IGF-I for the growth of transformed smooth-muscle cells. We conclude that the increased concentrations of type-I IGF receptors in leiomyoma compared to myometrial smooth-muscle cells are functional with respect to the enhanced internalization of IGF-I and that they provide these tumor cells with a growth advantage compared to their normal counterparts.

The root epidermis-specific pea gene RH2 is homologous to a pathogenesis-related gene.

Two-dimensional gel electrophoresis of pea root and root hair proteins revealed the existence of at least 10 proteins present at elevated levels in root hairs. One of these, named RH2, was isolated and a partial amino acid sequence was determined from two tryptic peptides. Using this sequence information oligonucleotides were designed to isolate by PCR an RH2 cDNA clone. In situ hybridization studies with this cDNA clone showed that rh2 is not only expressed in root hairs, but also in root epidermal cells lacking these tubular outgrowths. During post-embryonic development the gene is switched on after the transition of protoderm into epidermis and since rh2 is already expressed in a globular pea embryo in the protoderm at the side attached to the suspensor, we conclude that the expression of rh2 is developmentally regulated. At the amino acid level RH2 is 95% homologous to the pea PR protein I49a. These gene encoding I49a is induced in pea pods upon inoculation with the pathogen Fusarium solani [12]. We postulate that rh2 contributes to a constitutive defence barrier in the root epidermis. A similar role has been proposed for chalcone synthase (CHS) and chitinase, pathogenesis-related protein that are also constitutively present in certain epidermal tissues.

An avaII restriction fragment length polymorphism in the insulin-like growth factor II gene and the occurrence of smooth muscle tumors.

To detect a previously described AvaII restriction fragment length polymorphism (RFLP) in the human insulin-like growth factor II (IGF-II) gene we used the polymerase chain reaction (PCR) and genomic sequencing. The RFLP is located in exon 9 of the IGF-II gene at nucleotide 820 (GenBank accession number X07868) as a C-->T transition. Digestion with AvaII reveals a two-allele polymorphism, an a allele in which the AvaII site is not present, and a b allele. In healthy Dutch persons (n = 26), the frequency of the a allele was 62%. A similar a allele frequency was found in groups of Japanese (53%, n = 65) and Chinese (54%, n = 84), while in a French group the frequency was significantly lower (25%, n = 52). In Dutch individuals that had developed benign (n = 11; all women) and malignant (n = 9; 2 women and 7 men) smooth muscle tumors, a significantly higher frequency of 83% for the a allele was found. Since there was no difference between the presence of the a and b alleles in normal and tumor tissue of the same individual, the higher a allele frequency was not due to mutation in the IGF-II gene or loss of heterozygosity. There was no correlation between the presence of the a allele and expression of the IGF-II gene. The data reveal a correlation between homozygosity for the a allele and the occurrence of smooth muscle tumors. Women homozygous for the IGF-II a allele are more prone to develop a leiomyoma than women who are heterozygous or homozygous for the b allele. Furthermore, in both women and men the risk for leiomyosarcomas seems to be higher in a allele homozygotes.

Expression and CpG methylation of the insulin-like growth factor II gene in human smooth muscle tumors.

Previously we have shown that expression of the insulin-like growth factor II (IGF-II) gene in 36 normal smooth muscle tissues (myometria) and 26 benign smooth muscle tumors (leiomyomas) was detectable by Northern blot analysis but that the RNA levels were low. In 9 of 20 malignant smooth muscle tumors (leiomyosarcomas) IGF-II gene expression was also low or absent, while in 11 of 20 the IGF-II gene was abundantly expressed. In 32 of these tissues we have now studied the DNA methylation state of the IGF-II gene. For the analysis of overall methylation of the gene the restriction endonucleases HpaII and MspI were used. In normal smooth muscle and in leiomyomas the IGF-II gene appeared to be methylated. In leiomyosarcomas with low IGF-II gene expression the DNA was partly demethylated. In leiomyosarcomas with abundant IGF-II gene expression overall methylation of the DNA tended to be low. In addition, we have studied the methylation state of one particular CpG site in the IGF-II gene with the restriction endonuclease AvaII. The results of the latter analysis confirm the analysis with HpaII and MspI. In conclusion, in malignant smooth muscle tumors the data indicate an inverse correlation between CpG methylation and expression of the IGF-II gene.

Insulin-like growth factor gene expression in human smooth muscle tumors.

A role for insulin-like growth factors (IGF) in autocrine or paracrine growth stimulation of tumor cells has been proposed for tumors of different origins. We have studied IGF gene expression in human uterus smooth muscle (myometrium) and in a panel of benign (leiomyoma) and malignant (leiomyosarcoma) smooth muscle tumors. Using RNA transfer blot analysis we could demonstrate that in smooth muscle tissue and tumors IGF genes are differentially expressed. The mRNA species detected had the same size as reported for IGF mRNAs from other tissues. However, the abundance of the IGF gene transcripts varied from tissue to tissue. The amounts of IGF mRNAs detected in smooth muscle tumors were compared to the levels found in normal smooth muscle. The IGF-I gene was expressed at high levels in normal myometrium and in leiomyomas but appears to be repressed in leiomyosarcomas. Also the IGF-I peptide was detected in myometrium and in leiomyomas, but in leiomyosarcomas the level was substantially lower. The IGF-II gene was expressed at low levels in normal myometrium and leiomyomas but is activated in leiomyosarcomas. With increasing malignancy from the two major IGF-II mRNA species, 6.0 and 4.8 kilobases, in particular the 6.0-kilobase mRNA is produced at higher levels. In conclusion, these data suggest that for IGF-I a role in tumor cell growth is not likely, but probably IGF-II is involved in malignant smooth muscle tumor growth progression.

The ENOD12 gene product is involved in the infection process during the pea-Rhizobium interaction.

The pea cDNA clone pPsENOD12 represents a gene involved in the infection process during Pisum sativum L.-Rhizobium leguminosarum bv. viciae symbiosis. The ENOD12 protein is composed of pentapeptides containing two hydroxyprolines. The expression of the ENOD12 gene is induced in cells through which the infection thread is migrating, but also in cells that do not yet contain an infection thread. Soluble compounds from Rhizobium are involved in eliciting ENOD12 gene expression. Rhizobium common and host-specific nodulation genes are essential for the production of these compounds. Two ENOD12 genes are expressed in nodules and in stem tissue of uninoculated plants. The gene represented by the cloned ENOD12 mRNA is also expressed in flowers, but a different transcription start may be used.

Involvement of Rhizobium leguminosarum nodulation genes in gene expression in pea root hairs.

The mRNA population in pea root hairs was characterized by means of in vitro translation of total root hair RNA followed by 2-dimensional gel electrophoresis of the translation products. Root hairs contain several mRNAs not detectable in total RNA preparations from roots. Most of these root hair-specific mRNAs occur in elongating root hairs at higher levels than in mature root hairs. The expression of some genes in pea root hairs is typically affected by inoculation with Rhizobium leguminosarum. One gene, encoding RH-42, is specifically induced while the expression of another gene, encoding RH-44, is markedly enhanced. Using R. leguminosarum mutants it was shown that the nodC gene is required for the induction and enhancement of expression of the RH-42 and RH-44 genes, respectively, while the Rhizobium chromosomal gene pss1, involved in exopolysaccharide synthesis, is not essential. After induction of the nod genes with apigenin the bacteria excrete into the culture medium a factor that causes root hair deformation. This deformation factor stimulates the expression of the RH-44 gene but does not induce the expression of the gene encoding RH-42.

Characterization of cDNA for nodulin-75 of soybean: A gene product involved in early stages of root nodule development.

Establishment of a nitrogen-fixing root nodule is accompanied by a developmentally regulated expression of nodulin genes, only some of which, the so-called early nodulin genes, are expressed in stages preceding actual nitrogen fixation. We have isolated soybean cDNA clones representing early nodulin genes and have studied clone pENOD2 in detail. The cDNA insert of this clone hybridizes to nodule-specific RNA of 1200 nucleotides in length. The RNA that was hybrid-selected by the cloned ENOD2 DNA was in vitro translated to produce two nodulins with an apparent M(r) of 75,000, the N-75 nodulins. These two nodulins differ slightly in charge and one does not contain methionine. The amino acid sequence deduced from the DNA sequence shows that proline accounts for 45% of the 240 residues in these nodulins and the sequence contains at least 20 repeating heptapeptide units. The amino acid composition of none of the (hydroxy)proline-rich (glyco)proteins described in plants resembles the composition of the N-75 nodulins, especially with respect to the high glutamic acid and the low serine content. This suggests that the N-75 nodulins belong to a hitherto unidentified class of presumably structural proteins. The genes encoding the N-75 nodulins were found to be expressed in nodule-like structures devoid of intracellular bacteria and infection threads, indicating that these nodulins do not function in the infection process but more likely function in nodule morphogenesis.

Nodulin gene expression during soybean (Glycine max) nodule development.

In vitro translation products of total RNA isolated from soybean nodules at successive stages of nodule development were analyzed by two-dimensional gel electrophoresis. In that way the occurrence of over 20 mRNAs specifically transcribed from nodulin genes was detected. The nodulin genes could be divided into two classes according to the time of expression during nodule development. Class A comprises at least 4 nodulin mRNAs which are found when a globular meristem is present in the root cortex. These class A nodulin genes have a transient expression. Class B nodulin genes are expressed when the formation of a nodule structure has been completed. Bradyrhizobium japonicum nod (+) fix(-)mutants, with large deletions spanning the nif H,DK region, still induced nodules showing normal expression of all nodulin genes, indicating that the nif H,DK region is not involved in the induction of nodulin genes. In nodules induced by Bradyrhizobium japonicum nod (+) fix(-)mutant HS124 the bacteria are rarely released from the infection thread and the few infected cells appear to be collapsed. All class A and class B nodulin genes are expressed in HS124 nodules with the exception of 5 class B genes.

Expression of plant genes during the development of pea root nodules.

The expression of plant genes involved in the pea-Rhizobium symbiosis was studied by analysing mRNA from root nodules. The RNA was translated in vitro and the translation products were separated by two-dimensional gel electrophoresis. The results show differential expression of nodulin genes during root nodule development. One gene encoding N-40' is expressed at a significant level 5 days before the leghemoglobin genes. Most other nodulin genes are expressed more of less concomitantly with the leghemoglobin genes whereas the N-21 mRNA is only present late during the development. In the development of ineffective root nodules induced by infection with different nod+fix- mutants of R. leguminosarum all nodulin genes are expressed except for the N-21 gene. The results suggest that neither bacteroid development, heme excretion nor nitrogen fixation are essential for the induction of nodulin gene expression in the host plant. Further, it appears that the amount of leghemoglobin in ineffective nodules is regulated at a post-transcriptional level.