[Studies on a bacterial Hemophilus-X-factor. II. Distribution of the growth factor in heterotrophic bacteria].
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Bacteriological Techniques↗
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Epidermal growth factor-like transforming growth factor. II. Interaction with epidermal growth factor receptors in human placenta membranes and A431 cells.
A 125I-labeled derivative of rat epidermal growth factor-like transforming growth factor (125I-eTGF) that retains the chemical and biological properties of native eTGF has been prepared to characterize the interaction of eTGF with epidermal growth factor (EGF) membrane receptors. Binding of 125I-eTGF and 125I-labeled mouse EGF (125I-EGF) to membrane preparations from human placenta and the A431 human epidermoid carcinoma cell line was competed for to the same extent and with the same potency by either unlabeled eTGF or EGF. Components of placenta membranes and A431 membranes (145-170 kDa) were the major species affinity-labeled by cross-linking with membrane-bound 125I-eTGF or 125I-EGF. Affinity labeling of these receptor species was fully inhibited by both native eTGF and native EGF when present in excess during incubation of membranes with 125I-eTGF or 125I-EGF. The binding affinity and capacity of receptors in A431 membranes and placenta membranes were strikingly similar for eTGF and EGF. However, binding of 125I-eTGF to membrane receptors was highly sensitive to pH, being optimal only within the pH 8.0-8.5 range whereas EGF binding was optimal at pH 7.0-9.0. eTGF binding and EGF binding were modified in parallel by exposure of placental membranes to phytohemagglutinin, concanavalin A, or wheat germ agglutinin. Prolonged exposure of A431 cells to eTGF or EGF induced the same extent of down-regulation of both eTGF binding and EGF binding. eTGF-induced down-regulation of receptors in A431 cells followed the same kinetics as EGF-induced receptor down-regulation. The data indicate that despite the limited sequence homology between eTGF and EGF, these two ligands exhibit a remarkably similar mode of interaction with a common eTGF/EGF membrane receptor type.
Carcinoma, Squamous Cell↗
B cell helper factors. II. Synergy among three helper factors in the response of T cell- and macrophage-depleted B cells.
The concanavalin A- (Con A) stimulated supernatant of normal spleen cells (normal Con A SN) was shown to contain a set of helper factors sufficient to allow T cell- and macrophage- (M phi) depleted murine splenic B cells to produce a plaque-forming cell response to the antigen sheep red blood cells (SRBC). The activity of normal Con A SN could be reconstituted by a mixture of three helper factor preparations. The first was the interleukin 2- (IL 2) containing Con A SN of the T cell hybridoma, FS6-14.13. The second was a normal Con A SN depleted of IL 2 by extended culture with T cell blasts from which the 30,000 to 50,000 m.w. factors were isolated (interleukin X, IL X). The third was a SN either from the M phi tumor cell line P388D1 or from normal M phi taken from Corynebacterium parvum-immune mice. The combination of all three helper factor preparations was required to equal the activity of normal Con A SN; however, the M phi SN had the least overall effect. The M phi SN and IL 2 had to be added at the initiation of the culture period for a maximal effect, but the IL X preparation was most effective when added 24 hr after the initiation of culture. These results indicate that at least three nonspecific helper factors contribute to the helper activity in normal Con A SN.
Animals↗
Comparison of the half-lives and regeneration rates of blood clotting factors II, VII, and X in anticoagulant-resistant and susceptible Norway rats (Rattus norvegicus Berk.).
The half-lives and regeneration rates of clotting factors II, VII, and X in the plasma of anticoagulant-resistant and susceptible rats were determined. There is little or no difference in the half-lives of factors II and X in anticoagulant-resistant rats compared to susceptible rats, but the half-life of factor VII is longer in anticoagulant-resistant rats. In anticoagulant-resistant rats critical clotting factors appear to be carboxylated in preference to factor II, whereas the opposite occurs in susceptible rats; this may contribute to an animal's resistance status.
Animals↗
Posttranscriptional regulation of insulin-like growth factor II mRNA.
The insulin-like growth factor II (IGF-II) gene generates multiple mature transcripts with different 5' untranslated regions (5'UTR) but identical coding regions and 3'UTRs. We have analysed the translational regulation and decay of the transcripts. Human IGF-II mRNAs provide a provocative example of translational discrimination in which only the minor 4.8 kb mRNA is actively engaged in protein synthesis while the major 6.0 kb mRNA is present in a 100S RNP particle. The 6.0 kb mRNA exhibits a structured 5'UTR of 1170 nucleotides that function as a cis-acting translational attenuator. IGF-II transcripts are processed by endonucleolytic cleavage 1209 and 2183 nucleotides downstream from the translation termination codons in the rat and human, respectively. The cleavage site is situated in a highly conserved and structured domain that exhibits two large hairpins and an intramolecular guanosine quadruplex. The structural elements may provide binding sites for trans-acting factors and ensure that the cleavage site is not sequestered in stable RNA structures. Since expression of IGF-II is initiated from minimal promoters and finished by constitutive secretion from the cell, regulatory events governing IGF-II production are likely to be implemented between the transcriptional and the posttranslational level. The combined effect of translational discrimination and endonucleolysis may therefore play an important role in the regulation of IGF-II expression.
Animals↗
Lower birth-weight in neonates of mothers carrying factor V G1691A and factor II A(20210) mutations.
BACKGROUND AND OBJECTIVES: Inherited thrombophilia has been associated with fetal and maternal complications of pregnancy. It is reasonable to suppose that an imbalance of maternal hemostasis could lead to decreased fetal growth. DESIGN AND METHODS: We retrospectively investigated the birth-weight of neonates in relation to the presence of factor V G1691A and factor II A(20210) mutations in the mothers. Overall, 755 women (194 with a history of unexplained recurrent pregnancy loss, 202 with gestational hypertension with or without proteinuria, 359 with at least one uneventful pregnancy) and 1100 alive neonates were considered. RESULTS: Among 980 neonates from mothers without mutations, 136 (13.9%) weighed <2500 grams, whereas 34 out 123 (27.6%) neonates from mothers carrying the factor V G1691A or factor II A(20210) mutation were under this birth-weight (OR: 2.4, 95%CI: 1.5- 3.7). Adjusting for diagnosis, parity, and age, the risk of having a baby <2500 grams was 2.0 (95%CI: 1.1-3.6) in women carrying factor V G1691A or factor II A(20210) mutation. When we analyzed all the neonates according to growth centiles and the presence of a thrombophilic mutation in the mothers, we found 142 (14.5%) and 28 (22.8%) neonates under the 10th centile from mothers without and with thrombophilic mutations, respectively (OR: 1.7, 95%-CI: 1.1-2.7). Adjusting for confounding variables (diagnosis, parity and age), the association between thrombophilic mutations and <10th growth centile did not change (OR: 1.7, 95% CI: 1.0-3.0). INTERPRETATION AND CONCLUSIONS: Mothers carrying the factor V G1691A or factor II A(20210) mutation have a significantly higher risk of delivering neonates with a lower birth-weight.
5' Untranslated Regions↗
Insulin-like growth factor II (IGF-II) and the IGF-II/mannose-6-phosphate receptor: the myth continues.
Insulin-like growth factor II (IGF-II) is a polypeptide hormone with structural and functional homology with IGF-I and pro-insulin. It is now thought that IGF-II acts as a growth factor during fetal life and development. In rats, IGF-II levels in the circulation are high in the fetus and decline rapidly after birth. IGF-II mRNA expression in many tissues including the liver and the choroid plexus is also high during fetal life and low thereafter. Targeted disruption of the IGF-II gene in mice leads to a deficiency in their growth. Alternatively, it has been proposed that IGF-II could act as a growth and differentiation factor in the central nervous system. Administration of IGF-II into the central nervous system in rats leads to increased food intake and altered feeding behaviour. In muscle cells and in colon epithelial cells, IGF-II might also serve as an important regulator of differentiation. A key role for IGF-II as a paracrine or autocrine growth factor in certain tumours has been proposed. The IGFs exert their effects by binding to high-affinity membrane receptors that are expressed in many cells and tissues. The IGF-I receptor, which binds IGF-I with the highest affinity and which is very similar to the insulin receptor, is thought to mediate most, if not all, of the IGF-induced biological functions. The IGF-II/mannose-6-phosphate (M6P) receptor is a bifunctional glycoprotein with no homology to the insulin receptor. This receptor binds IGF-II and lysosomal enzymes bearing the M6P recognition marker at distinct binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Insulin-like growth factor II (IGF-II) inhibits insulin-like growth factor binding protein I (IGFBP-1) production in luteinized human granulosa cells with a potency similar to insulin-like growth factor I (IGF-I) and higher than insulin.
Insulin-like growth factor binding protein I (IGFBP-I) is produced by human granulosa cells and may be important in follicular development. Production of IGFBP-I in human granulosa cells is under inhibitory control of insulin and insulin-like growth factor-I (IGF-I). It had not been known if IGF-II affected IGFBP-I production in these cells. We examined the effect of IGF-II on IGFBP-I production in human granulosa cells and compared this effect of IGF-II to similar effects of insulin and IGF-I. Human granulosa cells were obtained during in vitro fertilization and plated at a density of 10(5) cells/mL in McCoy 5A tissue culture medium supplemented with 10% fetal calf serum. After 48 h of preincubation at 37 C, 90% humidity, 5% CO2, the medium was removed, and serum-free medium was added. After 24 h of incubation in the serum-free medium, the medium was removed, cells were washed, and new serum-free medium supplemented with various concentrations of insulin, IGF-I, or IGF-II was added. After 24 h of incubation with insulin, IGF-I, or IGF-II, the medium was removed and frozen at-20 C until assayed for IGFBP-I. IGFBP-I was measured using kits from Diagnostic System Laboratories, Webster, Texas. Concentrations of IGFBP-I in the medium were 4.4 +/- 1.7 ng/mL for control cells; 3.0 +/- 0.8, 3.0 +/- 0.7, 3.1 +/- 0.7, 1.8 +/- 0.5, and 1.1 +/- 0.3 ng/mL for 1, 10, 10(2), 10(3), and 10(5) ng/mL insulin respectively; 1.4 +/- 0.6, 2.1 +/- 0.5, 0.3 +/- 0.1, 0.1 +/- 0.02, and 0.01 +/- 0.02 ng/mL for 0.5, 1, 5, 10, and 10(2) ng/mL of IGF-I, respectively; and 2.3 +/- 2.2, 1.2 +/- 0.8, 0.9 +/- 0.3, 0.7 +/- 0.2, and 0.04 +/- 0.02 ng/mL for 0.5, 1, 5, 10, and 10(2) ng/mL of IGF-II, respectively. Concentration of IGFBP-I in the medium collected from cells incubated with hormones was statistically lower than that for control cells starting at 10(3) ng/mL of insulin (P < 0.05), 0.5 ng/mL of IGF-I (P < 0.05) and 1 ng/mL of IGF-II (P < 0.05). Within the range of hormone concentrations tested, the P-values (independent groups t-test) never reached less than 0.01 levels for insulin, but reached this level at 5 ng/mL for both IGF-I and IGF-II. We conclude that IGF-II inhibits IGFBP-I production in luteinized human granulosa cells in a dose-dependent manner and with potency similar to IGF-I and higher than insulin. Further studies are needed to determine the physiological significance of this and other effects of IGF-II in the human ovary in both normal and pathological states.
Culture Media, Conditioned↗
Feeding of butylated hydroxytoluene to rats caused a rapid decrease in blood coagulation factors II (prothrombin), VII, IX and X.
Male Sprague-Dawley rats were fed a diet containing 1.2% butylated hydroxytoluene (BHT) for 1-7 days, and blood coagulation factors II, VII, VIII, IX and X, and platelet aggregation were measured. The plasma concentrations of factors II, VII, IX and X were significantly reduced in a time-dependent fashion when BHT was administered for 2-7 days and haemorrhages in epididymis were found in rats given BHT for 4-7 days. On the contrary, thrombin-induced and calcium-required aggregation of washed platelets was unchanged throughout the experiment. These results suggest that factors II, VII, IX and X rapidly decrease immediately after the administration of BHT, but hypoaggregability of washed platelets reported previously may be a secondary defect caused by bleeding.
Animals↗
Elements required for activation of the major promoter of the human insulin-like growth factor II gene.
The human insulin-like growth factor II (IGF-II) gene contains four promoters, P1-P4. In fetal liver promoter P3 is the major promoter, which consists of a proximal region that supports general transcription, and a cell-specific region located more upstream. In addition to the TATA box, the proximal region contains four binding sites for nuclear proteins, designated PE3-1 to PE3-4. To determine the influence of the proteins binding to these elements, the transcriptional activity of the proximal region of P3 was investigated. Promoter P3 was analyzed in Hep3B cells, which express high levels of IGF-II mRNA derived from this promoter, and in HeLa cells, that have an inactive IGF-II gene. By analysis of 5'-deletion constructs in an in vitro transcription system and in transient expression assays, and by competition with specific oligonucleotides it was shown that the factors binding to the elements PE3-4, PE3-2 and PE3-1 play an important role in the regulation of promoter P3.
Base Sequence↗
Insulin-like growth factor II mRNA expression in human breast cancer.
Insulin-like growth factor II is a growth factor important in fetal development. Several cancer tissues and cell lines have been reported to express IGF-II and rat IGF-II is mitogenic for breast cancer cell lines. Using Northern analysis and ribonuclease protection assays, IGF-II mRNA was detected in normal fibroblasts and in the established breast cancer cell line, T47D. In this cell line, steady state levels of IGF-II message were increased by treatment with estradiol. 10 nM IGF-II, purified from human serum, was mitogenic for breast cancer cell lines. In vitro, IGF-II may act as an autocrine growth factor for some cell lines. RNA derived from breast cancer, pathologically normal breast tissue, and benign breast disease also contained IGF-II mRNA. When paired samples of normal and cancer tissue were obtained from the breast of the same patient, the level of IGF-II mRNA expression in the normal tissue was at least that found in the cancer. This is consistent with previous observations that show IGF-II is expressed in mesenchyme. These findings suggest that in breast cancer IGF-II is produced by stromal tissue elements and potentially by the malignant epithelial cells. Therefore, IGF-II may function as an autocrine or a paracrine growth factor in different breast tumors.
Blotting, Northern↗
Autocrine inhibition of chemotherapy response in human liver tumor cells by insulin-like growth factor-II.
Insulin-like Growth Factor (IGF)-II is frequently overexpressed in experimental and human hepatocellular carcinomas (HCCs) and has been correlated with increased tumor growth. We have analyzed, whether IGF-II affects chemotherapy response and apoptosis in human liver tumor cells. Three liver tumor cell lines highly expressed IGF-II and supported their growth in an autocrine manner by secreting excessive amounts of IGF-II. Neutralization of IGF-II significantly increased response to the chemotherapeutic agents cisplatin and etoposide especially at lower, cytostatic doses. While blocking of IGF-II did not increase spontaneous cell death in exponentially growing cultures, increased cell death was found under conditions of confluent growth and chemotherapy. Thus in HCC cells, IGF-II is a relevant protumorigenic growth factor that significantly reduces susceptibility to apoptosis and chemotherapeutic treatment. Therefore interference with IGF-II activity may improve response of HCCs to otherwise inefficient chemotherapeutic agents.
Antineoplastic Agents↗
Affinity for the insulin-like growth factor-II (IGF-II) receptor inhibits autocrine IGF-II activity in MCF-7 breast cancer cells.
We have investigated the autocrine regulation of insulin-like growth factor-II (IGF-II) signaling by the insulin-like growth factor-I receptor (IGF-IR) and the insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-IIR) in MCF-7 breast cancer cells, employing retroviruses encoding both IGF-I, IGF-II, and IGF-I and II mutants with reductions in affinity for either the IGF-IR or the IGF-IIR. These studies revealed reciprocal roles for IGF-IR and IGF-IIR affinity in the regulation of autocrine IGF-II activity. IGF-IR affinity was required for serum-free proliferation but also for efficient IGF-II secretion. In contrast, cellular proliferation, receptor tyrosine kinase-dependent signaling, and extracellular IGF-II protein accumulation were all reduced in the presence of IGF-IIR affinity. Inhibition of IGF-II signaling appeared to be the sole consequence of IGF-IIR affinity, as no cellular responses attributable to selective IGF-IIR binding by a reduced IGF-IR affinity IGF-II mutant could be detected. By operating as an IGF-II antagonist, the IGF-IIR has tumor suppressor-like properties, a suggestion consistent with reports of loss of heterozygosity at the IGF-IIR locus in a variety of human malignancies.
Amino Acid Sequence↗
Structure and activity dependence of recombinant human insulin-like growth factor II on disulfide bond pairing.
The complete peptide map of purified folded recombinant human insulin-like growth factor II (rhIGF-II) was determined to verify its sequence and disulfide bonding scheme. Each peptide generated by digestion with pepsin was purified and characterized by amino acid analysis, amino acid sequence analysis, and fast atom bombardment/mass spectrometry. Some peptides were also sequenced using tandem mass spectrometry. The rhIGF-II peptide map was compared to that of rat insulin-like growth factor II and to that of a disulfide-bonded isomer of rhIGF-II. The data obtained in these studies are consistent with the conclusion that the rhIGF-II obtained from Escherichia coli has the correct amino acid composition, sequence, and the native disulfide-bonded structure. The binding affinities of these forms of recombinant IGF-II for IGF carrier proteins were measured in an IGF binding protein assay. The disulfide isomer of rhIGF-II was 160-fold less potent than native rhIGF-II in the competitive protein binding assay. These studies illustrate the need to characterize recombinant polypeptides containing disulfide bonds to allow the native structure to be verified before characterizing the biological properties of such molecules in hopes of elucidating their physiologic functions.
Amino Acid Sequence↗
The rate of internalization of the mannose 6-phosphate/insulin-like growth factor II receptor is enhanced by multivalent ligand binding.
The cation-independent mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF-II receptor) undergoes constitutive endocytosis, mediating the internalization of two unrelated classes of ligands, mannose 6-phosphate (Man-6-P)-containing acid hydrolases and insulin-like growth factor II (IGF-II). To determine the role of ligand valency in M6P/IGF-II receptor-mediated endocytosis, we measured the internalization rates of two ligands, beta-glucuronidase (a homotetramer bearing multiple Man-6-P moieties) and IGF-II. We found that beta-glucuronidase entered the cell approximately 3-4-fold faster than IGF-II. Unlabeled beta-glucuronidase stimulated the rate of internalization of 125I-IGF-II to equal that of 125I-beta-glucuronidase, but a bivalent synthetic tripeptide capable of occupying both Man-6-P-binding sites on the M6P/IGF-II receptor simultaneously did not. A mutant receptor with one of the two Man-6-P-binding sites inactivated retained the ability to internalize beta-glucuronidase faster than IGF-II. Thus, the increased rate of internalization required a multivalent ligand and a single Man-6-P-binding site on the receptor. M6P/IGF-II receptor solubilized and purified in Triton X-100 was present as a monomer, but association with beta-glucuronidase generated a complex composed of two receptors and one beta-glucuronidase. Neither IGF-II nor the synthetic peptide induced receptor dimerization. These results indicate that intermolecular cross-linking of the M6P/IGF-II receptor occurs upon binding of a multivalent ligand, resulting in an increased rate of internalization.
Dimerization↗
Insulin-like growth factor-II/mannose 6 phosphate receptors facilitate the matrix effects of latent transforming growth factor-beta1 released from genetically modified keratinocytes in a fibroblast/keratinocyte co-culture system.
This study was conducted to explore the mechanism of activation of transforming growth factor-beta1 (TGF-beta1) which is critical to its role in many physiological and pathological conditions. To date, almost all reports concerning TGF-beta1 activation delineated that release of mature TGF-beta1 from latency associated protein (LAP) is required for its activation. We report that latent TGF-beta1 (LTGF-beta1) released from TGF-beta1 genetically modified keratinocytes grown in the top chamber of a co-culture system functions as a fibrogenic factor through interaction with insulin-like growth factor-II/mannose-6-phosphate (IGF-II/M6P) receptors of human dermal fibroblasts grown in the lower chamber of this system. Following successful transduction, the pLin-LTGF-beta1 vector was amplified in PA31 7 packaging cells which possess viral structural proteins for vector in the presence of neomycin. Conditioned medium derived from packaging cells containing competent viral particles was then used to transduce either keratinocytes or fibroblasts grown in the upper chamber of a co-culture system, in which a 0.4 microm porous membrane separates the two chambers. In this way, LTGF-beta1 produced by transduced cells in the upper chamber is released and diffuses into the lower chambers where dermal fibroblasts are grown. Conditioned medium from the lower chamber was removed 3 days later and used to evaluate the latency and bioactivity of TGF-beta1 using enzyme-linked immunosorbent assay (ELISA) and mink lung (Mv1 Lu) epithelial growth inhibition assay. Cells were also harvested and used for RNA extraction. The results of these experiments showed that 1) the TGF-beta1-LAP complex, which was latent in traditionally used mink lung growth inhibition assay, directly modulated the expression of collagenase, type I, and type III collagen mRNA by dermal fibroblasts; 2) this stimulation was inhibited by M6P in a dose-dependent manner; 3) the TGF-beta1-LAP inhibits Mv1Lu epithelial cells only when this complex was incubated with cell membranes isolated from dermal fibroblasts; and 4) LTGF-beta1 activation seems to occur through a conformational alteration rather than by release of the mature TGF-beta1 from LAP in our co-cultured system. This conformational alteration seems to occur through the interaction of the TGF-beta1-LAP complex with the IGF-II/M6P receptors. Thus, the quantity of IGF-II/M6P receptors is important in cellular response to LTGF-beta1 in any physiological and pathological conditions.
Autocrine Communication↗
Characterization of the P4 promoter region of the human insulin-like growth factor II gene.
The human insulin-like growth factor II (IGF-II) gene contains four promoters (P1, P2, P3 and P4). In order to determine the mechanism by which the P4 promoter is controlled, the human IGF-II P4 promoter was analyzed in cell lines. DNA sequence analysis of the human IGF-II P4 promoter gene showed that the P4 promoter region contains a TATA-like sequence and several G+C rich regions which are essential for transcription. Analysis of the transcription initiation site by S1 nuclease mapping revealed two transcription start sites; both are located immediately behind TATA-like sequence. To determine the location of sites that may be important for the function of the human IGF-II P4 promoter, we constructed chimeric genes of the human IGF-II P4 promoter fused to the coding region for chloramphenicol acetyltransferase (CAT). These constructs were transfected into HepG2, PLC/PRF/5, G401 and A549 cells, and were examined for CAT activity. All transfected cells showed a similar profile of CAT activity. Sequences responsible for putative enhancer and silencer regions were identified and the 5' flanking sequences of the human IGF-II P4 promoter contain negative regulatory regions (-213 to -174). The 53-base pair fragment located between 111 and 59 base pairs upstream of the start site contains positive regulatory activity. Gel mobility shift assay showed that Sp1 and another proteins might be involved in positive regulation of the human IGF-II P4 promoter.
Animals↗
Stimulation of bone formation in vivo by insulin-like growth factor-II in rats.
Insulin-like growth factor-II (IGF-II) plays an important role in skeletal remodeling, however, little is known about its effect on bone formation in vivo. In our study of the stimulation of bone formation in vivo by IGF II we injected recombinant human IGF-II into the parietal bones of neonatal rats once a day for 12 days. The bone mineral density measured by dual energy X-ray absorptiometry and the thickness of IGF-II-injected parietal bones increased in a dose-dependent manner. The layers of osteoblasts were observed along the IGF-II-injected side.
Animals↗