Transforming growth factor beta as a potential tumor progression factor among hyperdiploid glioblastoma cultures: evidence for the role of platelet-derived growth factor

Among early-passage, near-diploid gliomas in vitro, transforming growth factor type beta (TGF beta) has been previously shown to be an autocrine growth inhibitor. In contrast, hyperdiploid (> or = 57 chromosomes/metaphase) glioblastoma multiforme (HD-GM) cultures were autocrinely stimulated by the TGF beta. The mechanism of this 'conversion' from autocrine inhibitor to mitogen is not understood; previous studies have suggested that platelet-derived growth factor (PDGF) might be modulated by TGF beta. The similar expression of TGF beta types 1-3, PDGF-AA; -BB, as well as the PDGF receptor alpha and beta subunits (a/beta PDGFR) between biopsies of the HD-GM and near-diploid, TGF beta-inhibited glioblastomas (GM) by immunohistochemistry did not explain the discrepancy in their regulatory responses. Flow cytometry demonstrated that TGF beta's mitogenic effect was selective for the aneuploid subpopulations of two of three selected HD-GM cultures, while the diploid cells were inhibited. Among the HD-GM, TGF beta 1 induced the RNA of PDGF-A, c-sis and TGF beta 1. The amount of PDGF-AA secreted following TGF beta treatment was sufficient to stimulate the proliferation of a HD-GM culture. Antibodies against PDGF-AA, -BB, -AB, alpha PDGFR and/or beta PDGFR subunits effectively neutralized TGF beta's induction of DNA synthesis among the HD-GM cell lines, indicating that PDGF served as the principal mediator of TGF beta's growth stimulatory effect. By comparison, TGF beta induced only the RNA of PDGF-A and TGF beta 1 among the near-diploid GM, c-sis was not expressed at all. However, the amount of PDGF-A which was secreted in response to TGF beta 1 was insufficient to prevent TGF beta's arrest of the near-diploid cultures in G1 phase. Thus, the emergence of hyperdiploidy was associated with qualitative and quantitative differences in TGF beta's modulation of PDGF-A and c-sis, which provided a mechanism by which the aneuploid glioma cells might achieve 'clonal dominance'. We hypothesize that TGF beta may serve as an autocrine promoter of GM progression by providing a selective advantage to the hyperdiploid subpopulation through the loss of a tumor suppressor gene which mediates TGF beta's inhibitory effect.     

The role of nerve growth factor in a model of visceral inflammation

There is growing evidence that nerve growth factor may be an important mediator of the sensory disorders associated with inflammation. This hypothesis was tested in a rat model of cystitis. In this model, an experimental inflammation is created in anaesthetized rats with an irritant chemical. Within 1 h, bladder reflexes, activated by the sensory innervation of this viscus, become exaggerated, mimicking the disorders seen in humans with chronic cystitis. The development of this hyper-reflexia following experimental inflammation was quantified using the technique of repeated cystometrograms. By several measures, bladder reflex excitability increased about three-fold after 5 h. Firstly, the study investigated whether inflammatory changes can be prevented by pharmacological antagonism of nerve growth factor. A synthetic fusion protein was used, consisting of the extracelluar domain of the high-affinity nerve growth factor receptor, trkA, coupled to the Fc portion of an immunoglobulin. Previous work has shown that this molecule can sequester nerve growth factor and reduce its bioavailability both in vitro and in vivo. Treatment of animals with the fusion molecule at 1 mg/kg, immediately before inflammation of the bladder, largely, and very significantly, prevented the expected increases in reflex excitability of this organ. Pretreatment with a related fusion protein, capable of sequestering the neurotrophin brain-derived neurotrophic factor and neurotrophin-4/5, but not nerve growth factor, was without effect. Similarly, a control fusion molecule, without neurotrophin-sequestering capacity, did not reduce the inflammation-induced hyper-reflexia. Systemic treatment with the nerve growth factor-sequestering molecule, but not control molecules, partially and significantly reversed established inflammatory changes, by about 30-60%, depending on outcome measure. The nerve growth factor-sequestering protein had no significant effects on bladder reflex excitability in the uninflamed state. It was also without significant effect on capsaicin-induced contractions of the urinary bladder. Administration of exogenous nerve growth factor into the lumen of the urinary bladders of normal anaesthetized rats produced a rapid and marked bladder hyper-reflexia similar to that seen with experimental inflammation. These findings are consistent with other circumstantial evidence that nerve growth factor may interact with visceral sensory systems. Together, the data strongly suggest that nerve growth factor produced in inflamed tissues is a critical mediator of the sensory disorders associated with inflammation.     

Phosphorylation of serum response factor by casein kinase II: evidence against a role in growth factor regulation of fos expression

With the sequencing of the first complete eukaryotic chromosome, III of yeast (YCIII) of length 315 kb, several types of questions concerning chromosomal organization and the heterogeneity of eukaryotic DNA sequences can be approached. We have undertaken extensive analysis of YCIII with the goals of: (1) discerning patterns and anomalies in the occurrences of short oligonucleotides; (2) characterizing the nature and locations of significant direct and inverted repeats; (3) delimiting regions unusually rich in particular base types (e.g., G+C, purines); and (4) analyzing the distributions of markers of interest, e.g., delta (delta) elements, ARS (autonomous replicating sequences), special oligonucleotides, close repeats and close dyad pairings, and gene sequences. YCIII reveals several distinctive sequence features, including: (i) a relative abundance of significant local and global repeats highlighting five genes containing substantial close or tandem DNA repeats; (ii) an anomalous distribution of delta elements involving two clusters and a long gap; (iii) a significantly even distribution of ARS; (iv) a relative increase in the frequency of T runs and AT iterations downstream of genes and A runs upstream of genes; and (v) two regions of complex repetitive sequences and anomalous DNA composition, 29000-31000 and 291000-295000, the latter centered at the HMRa locus. Interpretations of these findings for chromosomal organization and implications for regulation of gene expression are discussed.     

Expression of fetal kidney growth factors in a kidney tumor line: role of FGF2 in kidney development

To identify growth factors which may play a role in kidney organogenesis, we have analyzed culture supernatants from the pediatric kidney tumor cell line G401. G401 cells were found to secrete fibroblast growth factor 2 (FGF2), a potent mitogen for mesenchymal cells, OP-1/BMP7, an epithelial cell growth inhibitor, and midkine (MK). Northern blotting confirmed expression of FGF2, OP-1/BMP7 and MK mRNA, as well as Wnt5A mRNA in G401 cells. In situ hybridization and immunocytochemistry on human fetal kidney demonstrated FGF2 expression in epithelial cells of the branching ureteric bud epithelium, nephron precursors ("S-shaped bodies"), proximal tubule epithelium and the parietal epithelium of the glomerulus. FGF2 protein in condensed "caps" of induced mesenchymal cells was also detected by immunocytochemistry. FGF2 protein was found to be concentrated in nuclei, particularly in proximal tubule epithelial cells. Recombinant FGF2 was found to act as a mitogen on primary mouse fetal kidney cell cultures. The results demonstrate G401 cells secrete a variety of fetal kidney growth factors and that FGF2 may act as a mitogen for fetal kidney cells and thus could play a role in the morphogenesis of the kidney.     

Targeted disruption of fibroblast growth factor (FGF) receptor 2 suggests a role for FGF signaling in pregastrulation mammalian development

We disrupted the fibroblast growth factor (FGF) receptor 2 (FGFR2) gene by introducing a neo cassette into the IIIc ligand binding exon and by deleting a genomic DNA fragment encoding its transmembrane domain and part of its kinase I domain. A recessive embryonic lethal mutation was obtained. Preimplantation development was normal until the blastocyst stage. Homozygous mutant embryos died a few hours after implantation at a random position in the uterine crypt, with collapsed yolk cavity. Mutant blastocysts hatched, adhered, and formed a layer of trophoblast giant cells in vitro, but after prolonged culture, the growth of the inner cell mass stopped, no visceral endoderm formed, and finally the egg cylinder disintegrated. It follows that FGFR2 is required for early postimplantation development between implantation and the formation of the egg cylinder. We suggest that FGFR2 contributes to the outgrowth, differentiation, and maintenance of the inner cell mass and raise the possibility that this activity is mediated by FGF4 signals transmitted by FGFR2. The role of early FGF signaling in pregastrulation development as a possible adaptation to mammalian (amniote) embryogenesis is discussed.     

Hepatocyte growth factor expression in the developing myocardium: evidence for a role in the regulation of the mesenchymal cell phenotype and urokinase expression

BACKGROUND: No previous studies have examined the extent to which correctional facilities in the United States screen for and treat hepatitis C (HCV) infection. METHODS: Medical directors of state correctional facilities responded to a survey assessing the degree to which prisons screen for and treat hepatitis C. To estimate numbers of inmates eligible for interferon treatment and to examine costs associated with HCV management, we constructed a feasibility model that incorporated screening criteria used in California and Rhode Island. RESULTS: Thirty-six states and Washington, DC, responded, resulting in a survey response rate of 73%, representing 77% of all inmates in state facilities nationwide. Colorado alone reported routine screening. Only California reported conducting a systematic seroprevalence study, which found that 39.4% of male inmates were hepatitis C antibody positive in 1994. Seventy-three percent of the respondents sometimes consider treating with interferon. Four states follow a standard protocol. The feasibility model suggests that treating suitably screened inmates is a reasonable expenditure for correctional systems. CONCLUSION: Prison may be an appropriate setting for treatment of hepatitis C. If accompanying substance abuse issues are addressed, instituting HCV treatment for certain eligible incarcerated individuals may be a worthy target for public health dollars.     

Identification in vitro of a post-translational regulatory site in the hinge 1 region of Arabidopsis nitrate reductase

Nitrate reductase (NR) is rapidly inactivated by phosphorylation of serine residues in response to loss of light or reduction in CO2 levels. To identify sites within NR protein that play a role in this post-translational regulation, a heterologous expression system and an in vitro inactivation assay for Arabidopsis NR were developed. Protein extracts containing NR kinases and inhibitor proteins were prepared from an NR-defective mutant that had lesions in both the NIA1 and NIA2 NR genes of Arabidopsis. Active NR protein was produced in a Pichia pastoris expression system. Incubation of these two preparations resulted in a Mg-ATP-dependent inactivation of NR that was reversed with EDTA. Mutant forms of NR were constructed, produced in P. pastoris, and tested in the in vitro inactivation assay. Six conserved serine residues in the hinge 1 region of NR, which separates the molybdenum cofactor and heme domains, were specifically targeted for mutagenesis because they are located in a potential regulatory region identified as a target for NR kinases in spinach. A change in Ser-534 to aspartate was found to block NR inactivation; changes in the other five serines had no effect. The aspartate that replaced Ser-534 did not appear to mimic a phosphorylated serine but simply prevented the NR from being inactivated. These results identify Ser-534, located in the hinge 1 of NR and conserved among higher plants NRs, as an essential site for post-translational regulation in vitro.     

Regression of vessels in the tunica vasculosa lentis is initiated by coordinated endothelial apoptosis: a role for vascular endothelial growth factor as a survival factor for endothelium

This review is concerned with the application of the method of differential scanning calorimetry (DSC) to structural and functional studies of myosin and actin--the main two proteins of muscles and many other systems of biological motility. The domain organization of these proteins as revealed by DSC is considered. Data are presented on the conformational changes which occur in the myosin head and in F-actin due to the formation of the ternary complexes with ADP and Pi analogs (such as orthovanadate, beryllium fluoride, or aluminum fluoride). Recent data on the application of DSC to studies on the interaction of F-actin with myosin heads and with tropomyosin are also considered. It is concluded that DSC offers a new and promising approach to probe the structural changes which occur in the myosin head and in F-actin during ATP hydrolysis and due to interaction of these proteins with each other.     

Cloning of a human platelet-activating factor receptor gene: evidence for an intron in the 5''-untranslated region

Twenty-four chickens were randomly assigned to one of three treatments (ketamine, 30 mg/kg; thiopental, 20 mg/kg; saline, 0.8 mL). Baseline data (heart rate, respiratory rate, systolic blood pressure, and cloacal temperature) were recorded before ulnar intraosseous cannulation and administration of drug treatment and for 30 minutes after administration. One investigator, unaware of the treatment administered, assessed the reaction to cannulation, number of attempts per cannulation, reaction to injection, time to induction and recovery, and quality of induction and recovery. Respiratory rate increased significantly (p < .05) from baseline after thiopental. Other parameters did not vary within groups or between groups. Most birds did not react or had a mild reaction to cannulation and injection, and on average fewer than two attempts were necessary. Quality of recovery was significantly (p < .05) better after thiopental. Time to recovery was significantly (p < .05) shorter after thiopental. No major histopathologic changes were noted in bone marrow samples from the injection site. This study demonstrates that the intraosseous route may be used to induce anesthesia in chickens, and that minimal changes in the variables studied were produced by ketamine and thiopental.     

Wnt-4 is a mesenchymal signal for epithelial transformation of metanephric mesenchyme in the developing kidney

Development of the mammalian kidney is initiated by ingrowth of the ureteric bud into the metanephric blastema. In response to signal(s) from the ureter, mesenchymal cells condense, aggregate into pretubular clusters, and undergo epithelialisation to form simple epithelial tubules. Subsequent morphogenesis and differentiation of the tubular epithelium lead to the establishment of a functional nephron. Here we demonstrate that Wnt-4, a secreted glycoprotein which is required for tubule formation, is sufficient to trigger tubulogenesis in isolated metanephric mesenchyme, whereas Wnt-11 which is expressed in the tip of the growing ureter is not. Wnt-4 signaling depends on cell contact and sulphated glycosaminoglycans and is only required for triggering tubulogenesis but not for later events. The Wnt-4 signal can be replaced by other members of the Wnt gene family including Wnt-1, Wnt-3a, Wnt-7a and Wnt-7b. Further, dorsal spinal cord, which has been thought to mimic ureteric signaling in tubule induction induces Wnt-4 mutant as well as wild-type mesenchyme suggesting that spinal cord derived signal(s) most likely act by mimicking the normal mesenchymal action of Wnt-4. These results lend additional support to the notion that Wnt-4 is a key auto-regulator of the mesenchymal to epithelial transformation that underpins nephrogenesis adding another level of complexity in the hierarchy of molecular events mediating tubulogenesis.     

Keratinocyte growth factor (KGF/FGF-7) has a paracrine role in canine prostate: molecular cloning of mRNA encoding canine KGF

BACKGROUND: Recent studies have demonstrated the usefulness of gene-modified tumor cells for immunotherapy. Using the tumorigenic murine fibrosarcoma, MCA 106, we investigated the effects of localized interferon-gamma (IFNg) secretion on tumorigenicity and on long-term memory. METHODS: The murine IFNg (MuIFNg) gene was introduced into tumor cells. High and low IFNg-secreting clones were isolated. C57BL/6 mice were injected subcutaneously (s.c.) with either parental (P), high or low IFNg-secreting (H- or L-IFNg) cells, and tumor growth was assessed weekly. Spleens were harvested on different days postinjection (p.i.) to assess in vitro cytolytic activity. In parallel, tissues from injection sites were stained with macrophage-, CD4-, and CD8-detecting antibodies. Mice were injected s.c. with H-IFNg MCA106 tumor. After 150 days the animals were rechallenged s.c. with MCA106P in one leg and with irrelevant syngeneic tumor in the other. RESULTS: Both P- and L-IFNg cells had similar growth, whereas the H-IFNg cells never grew. Only splenocytes from the H-IFNg animals showed in vitro CTL activity persisting until day 30 p.i. Histological data revealed a macrophage and CD4+ infiltrate much earlier in the H-IFNg group compared with the P group. Only the irrelevant, syngeneic tumor grew in animals previously injected with H-IFNg cells, whereas both P and irrelevant syngeneic tumors grew in controls. CONCLUSIONS: Transduction of MCA106 cells with the MuIFNg gene diminished in vivo tumorigenicity in proportion to the amount of IFNg secreted. Immunization with H-IFNg cells elicited a host response characterized by macrophages and CD4+ cells. Long-term tumor-specific memory was seen after immunization with H-IFNg cells.     

Nerve growth factor is a potent inducer of proliferation and neuronal differentiation for adult rat chromaffin cells in vitro

Adult rat chromaffin cells in vitro show a large proliferative response to NGF, followed by neuronal differentiation. Infection of replicating chromaffin cells with a retrovirus carrying the Escherichia coli beta-galactosidase (beta-gal) gene demonstrates beta-gal expression in cells that continue to multiply, that differentiate into neurons, and that become static. The effects of NGF on proliferation and differentiation are abolished by the protein kinase inhibitors K252a and staurosporine, and by cholera toxin, an activator of adenylate cyclase. They are diminished, but not abolished, by high concentrations of dexamethasone. Both cholera toxin alone and phorbol myristate acetate (PMA), an activator of protein kinase C, elicit small and inconsistent mitogenic responses. The responses to PMA cannot be shown to be additive with the effects of NGF. NGF is a known mitogen and neuritogen for chromaffin cells from neonatal rats, but has not previously been believed to affect similarly chromaffin cells from adults. The present findings suggest that portions of NGF signaling pathways might continue to be involved in regulating proliferation of adult rat chromaffin cells in vivo, and might be constitutively activated in PC12 cells and other adrenal medullary tumors. They further suggest that rat chromaffin cells might be propagated in vitro to obtain large numbers of sympathetic neurons expressing normal or exogenous genes.     

A Gly1127Ser mutation in an EGF-like domain of the fibrillin-1 gene is a risk factor for ascending aortic aneurysm and dissection

Ascending aortic disease, ranging from mild aortic root enlargement to aneurysm and/or dissection, has been identified in 10 individuals of a kindred, none of whom had classical Marfan syndrome (MFS). Single-strand conformation analysis of the entire fibrillin-1 (FBN1) cDNA of an affected family member revealed a G-to-A transition at nucleotide 3379, predicting a Gly1127Ser substitution. The glycine in this position is highly conserved in EGF-like domains of FBN1 and other proteins. This mutation was present in 9 of 10 affected family members and in 1 young unaffected member but was not found in other unaffected members, in 168 chromosomes from normal controls, and in 188 chromosomes from other individuals with MFS or related phenotypes. FBN1 intragenic marker haplotypes ruled out the possibility that the other allele played a significant role in modulating the phenotype in this family. Pulse-chase studies revealed normal fibrillin synthesis but reduced fibrillin deposition into the extracellular matrix in cultured fibroblasts from a Gly1127Ser carrier. We postulate that the Gly1127Ser FBN1 mutation is responsible for reduced matrix deposition. We suggest that mutations such as this one may disrupt EGF-like domain folding less drastically than do substitutions of cysteine or of other amino acids important for calcium-binding that cause classical MFS. The Gly1127Ser mutation, therefore, produces a mild form of autosomal dominantly inherited weakness of elastic tissue, which predisposes to ascending aortic aneurysm and dissection later in life.     

Macrophage migration inhibitory factor production by Leydig cells: evidence for a role in the regulation of testicular function

Macrophage migration inhibitory factor (MIF), described originally as a product of activated T lymphocytes, recently has been found to be released by monocytes/macrophages and the anterior pituitary gland. Immunohistochemical studies of the adult rat testis using an affinity-purified polyclonal antimurine MIF antibody demonstrated strong staining for MIF in Leydig cells and their putative precursors. Peritubular myoid cells and the seminiferous epithelium were negative for MIF staining; however, a weak reaction around the heads of elongated spermatids also was observed. The expression of MIF messenger RNA and protein in whole rat testis was demonstrated by Northern blot and Western blot analyses, respectively. Both MIF messenger RNA and protein immunoreactivity in Leydig cells was observed in testes obtained from long term hypophysectomized rats. Significant concentrations of intracellular MIF were detected in lysates of the TM3 Leydig cell line (7.23 +/- 2.6 pg/microgram protein), and testicular interstitial fluid contained 14.7 +/- 1.6 ng/ml MIF protein, as measured by MIF-specific enzyme-linked immunosorbent assay. To gain insight into the possible biological role of MIF in the testis, cultures of adult rat seminiferous tubules and purified Leydig cells were incubated together with recombinant murine MIF (rMIF). Neither rMIF (50 ng/ml) nor a neutralizing anti-MIF antiserum was found to affect basal or LH-stimulated Leydig cell steroidogenesis in vitro. However, a dose-dependent decrease in the secretion of inhibin by the seminiferous tubules was observed at rMIF concentrations ranging from 10-100 ng/ml. Taken together, these data indicate that Leydig cells produce MIF in vivo and suggest an important regulatory role for this newly discovered mediator of testicular function.     

Regulation of RAR beta 2 mRNA expression: evidence for an inhibitory peptide encoded in the 5'-untranslated region

Regulation of mRNA translation and stability plays an important role in the control of gene expression during embryonic development. We have recently shown that the tissue-specific expression of the RAR beta 2 gene in mouse embryos is regulated at the translational level by short upstream open reading frames (uORFs) In the 5'-untranslated region (Zimmer, A., A.M. Zimmer, and K. Reynolds. 1994. J. Cell Biol. 127:1111-1119). To gain insight into the molecular mechanism, we have performed a systematic mutational analysis of the uORFs. Two series of constructs were tested: in one series, each uORF was individually inactivated by introducing a point mutation in its start codon; in the second series, all but one ORF were inactivated. Our results indicate that individual uORFs may have different functions. uORF4 seems to inhibit translation of the major ORF in heart and brain, while uORFs 2 and 5 appear to be important for efficient translation in all tissues. To determine whether the polypeptide encoded by uORF4 or the act of translating it, is the significant event, we introduced point mutations to create silent mutations or amino acid substitutions in uORF4. Our results indicate that the uORF4 amino acid coding sequence is important for the inhibitory effect on translation of the downstream major ORF.     

Akt activation by growth factors is a multiple-step process: the role of the PH domain

The protein kinase encoded by the Akt proto-oncogene is activated by phospholipid binding, membrane translocation and phosphorylation. To address the relative roles of these mechanisms of Akt activation, we have employed a combination of genetic and pharmacological approaches. Transient transfection of NIH3T3 cells with wild-type Akt, pleckstrin homology (PH) domain mutants, generated on the basis of a PH domain structural model, and phosphorylation site Akt mutants provided evidence for a model of Akt activation consisting of three sequential steps: (1) a PH domain-dependent, growth factor-independent step, marked by constitutive phosphorylation of threonine 450 (T450) and perhaps serine 124 (S124), that renders the protein responsive to subsequent activation events; (2) a growth factor-induced, PI3-K-dependent membrane-translocation step; and (3) a PI3-K-dependent step, characterized by phosphorylation at T308 and S473, that occurs in the cell membrane and is required for activation. When forced to translocate to the membrane, wild-type Akt and PH domain Akt mutants that are defective in the first step become constitutively active, suggesting that the purpose of this step is to prepare the protein for membrane translocation. Both growth factor stimulation and forced membrane translocation, however, failed to activate a T308A mutant. This, combined with the finding that T308D/S473D double mutant is constitutively active, suggests that the purpose of the three-step process of Akt activation is the phosphorylation of the protein at T308 and S473. The proposed model provides a framework for a comprehensive understanding of the temporal and spatial requirements for Akt activation by growth factors.