Glioblastoma growth inhibited in vivo by a dominant-negative Flk-1 mutant

Angiogenesis, the sprouting of capillaries from pre-existing blood vessels, is a fundamental process in the formation of the vascular system during embryonic development. In adulthood, angiogenesis takes place during corpus luteum formation and in pathological conditions such as wound healing, diabetic retinopathy, and tumor-igenesis. Vascularization is essential for solid tumour growth and is thought to be regulated by tumour cell-produced factors, which have a chemotactic and mitogenic effect on endothelial cells. Vascular endothelial growth factor (VEGF), a homodimeric glycoprotein of relative molecular mass 45,000, is the only mitogen, however, that specifically acts on endothelial cells, and it may be a major regulator of tumour angiogenesis in vivo. Its expression has been shown to be upregulated by hypoxia, and its cell-surface receptor, Flk-1, is exclusively expressed in endothelial cells. Here we investigate the biological relevance of the VEGF/Flk-1 receptor/ligand system for angiogenesis using a retrovirus encoding a dominant-negative mutant of the Flk-1/VEGF receptor to infect endothelial target cells in vivo, and find that tumour growth is prevented in nude mice. Our results emphasize the central role of the Flk-1/VEGF system in angiogenesis in general and in the development of solid tumours in particular.     

Growth suppression mediated by transfection of wild-type hMLH1 in human cancer cells expressing endogenous truncated hMLH1 protein

Eosinophils contribute to the inflammatory process in a variety of chronic inflammatory bowel diseases. Ketotifen is beneficial in experimental models of colitis and in patients with eosinophilic gastroenteritis. Therefore, we investigated the efficacy of ketotifen therapy for the treatment of active ulcerative colitis. Children with newly or previously diagnosed ulcerative colitis with mild-moderate disease activity were treated with ketotifen at a dosage of 4 mg daily for eight weeks. Efficacy was determined by a physician disease severity index and by endoscopic and histologic examinations. Ten patients were enrolled. Symptoms improved in four patients and resolved completely in one patient. There was endoscopic improvement in three patients and histologic improvement in one. Increased eosinophils on rectal biopsy at entry were present in two of the responders. Five patients withdrew due to a lack of symptomatic improvement. No adverse events were identified. Low-dose ketotifen offers a limited therapeutic advantage in active ulcerative colitis that may be enhanced in the subgroup of patients with a high eosinophil count in the colonic mucosa. Further study of therapeutic efficacy with increased dosages of the mast cell stabilizer for acute and maintenance therapy is warranted.     

Inhibition of gap junctional intercellular communication by tumor promoters in connexin43 and connexin32-expressing liver cells: cell specificity and role of protein kinase C

Nur77 is a member of the steroid receptor superfamily and is known to be expressed in animals under stress. We studied the role of nur77 in the regulation of the hypothalamic-pituitary-adrenal (HPA) axis during the stress response using a murine pituitary corticotrope cell line, AtT-20. Corticotropin-releasing hormone (CRH), a stress mediator in the HPA axis, induced the expression of nur77 transiently in AtT-20 cells. Gel shift assay showed that nur77 bound to negative glucocorticoid responsive element (nGRE) in the promoter of the human proopiomelanocortin (POMC) gene and the formation of the nur77-nGRE complex increased after treatment of the cells with CRH. Negative GRE is known to be necessary for the negative regulation by glucocorticoid of the POMC gene expression. In stable transformants of AtT-20 cells expressing a human homolog of nur77, NAK-1, at a high level, glucocorticoid-mediated inhibition of both POMC mRNA induction and ACTH secretion was significantly lower than that in the NAK-1-non-expressing cells (P < 0.001). These results strongly suggest that nur77 antagonizes the negative feedback effect of glucocorticoid on the synthesis and secretion of ACTH in pituitary corticotropes. This suggests that nur77 plays an important role in the pituitary gland in the biological adaptation to overcome stress.     

A role of the transforming growth factor-beta in suppression of B-cell blastogenesis mediated by erythroid cells

The bio-psycho-social model was used in the presented study to observe the course of chronic diseases. An attempt was undertaken to correlate the psychosocial situation of 165 women with no presence of disease following breast cancer or genital cancer with their present organic status. The open discussion, a half standardized interview and the gynecological-oncological physical examination were implemented using predetermined parameters. The results of the general well-being scores from the interview and the results of organic status for all 165 patients were related using a rank-correlation between the objective and subjective score. A discrepancy between the psychosocial situation and the objective physical finding was found in the majority of women. The goal of the treatment of chronically ill patients especially following oncological tumors should thus have equal emphasis on the psychosocial and organic dimensions of the disease.     

Macrophage inhibition of lymphocyte and tumor cell growth is mediated by 25-hydroxycholesterol in the cell membrane

A cDNA clone encoding corticotropin-releasing factor (CRF) type 1 (CRF-R1) has been isolated from the tree shrew Tupaia belangeri with a PCR-based approach. The full-length cDNA encoded a 415-amino-acid protein with highest sequence identity (approximately 98%) to human CRF-R1 and slightly less identity to rat or mouse CRF-R1 (approximately 97%). Only eight amino acids (residues 3, 4, 6, 35, 36 and 39 in the N-terminus, residue 232 in transmembrane domain 4 and residue 410 in the C-terminus) differed between tree shrew CRF-R1 (tCRF-R1) and human CRF-R1 (hCRF-R1). tCRF-R1 mRNA was detected by semiquantitative RT-PCR and RNase protection analysis in the pituitary and in brain areas such as amygdala, brainstem, cerebellum, cortex, olfactory bulb, and striatum. In peripheral organs, only weak expression of tCRF-R1 mRNA was observed in ovary, testis, and adrenal gland. Binding studies using human embryonic kidney 293 (HEK293) cells stably transfected with tCRF-R1 showed that the CRF agonists ovine CRF (KD = 1.28 nM), human/rat CRF (KD = 1.09 nM), urocortin (KD = 0.37 nM) and sauvagine (KD = 0.77 nM), respectively, were bound with significantly higher affinities than the CRF antagonist astressin (KD = 12.4 nM). In agreement with the binding data half maximum effective EC50 values of 0.83 nM (human/rat CRF), 1.41 nM (ovine CRF), 1.25 nM (rat urocortin) and 0.71 nM (sauvagine) were calculated when the cAMP production in HEK293 cells stably transfected with tCRF-R1 was stimulated with the four CRF analogues. These data underline the close relationship between human and tree shrew CRF-R1.     

Skeletal muscle cell-derived insulin-like growth factor (IGF) binding proteins inhibit IGF-I-induced myogenesis in rat L6E9 cells

The insulin-like growth factors (IGFs) stimulate the differentiation of skeletal muscle cells. IGF binding proteins (IGFBPs), which are expressed by skeletal muscle cells, may enhance or inhibit IGF actions. To explore the role of skeletal muscle-derived IGFBPs in IGF-induced myogenesis, we compared the differentiation-inducing effects of IGF-I and des(1-3)IGF-I in rat L6E9 skeletal myoblasts. Des(1-3)IGF-I is a naturally occurring IGF-I analog with markedly reduced affinity for IGFBPs but with an affinity for the IGF-I receptor that is comparable to that for native IGF-I. We find that rat L6E9 cells produce principally IGFBP-4 and BP-6, with a minor component of IGFBP-5. Both IGFBP-4 and BP-6 accumulate during differentiation and increase further in response to IGF-I or des(1-3)IGF-I treatment. We find that an IGF-I analog with reduced affinity for IGFBPs is significantly more potent than native IGF-I in stimulating myogenesis (as assessed by myogenin messenger RNA abundance and muscle creatine kinase activity), indicating that IGFBPs expressed by skeletal muscle cells inhibit differentiation induced by IGF-I. In view of the relative abundance of IGFBP-4, its relatively high affinity for IGF-I and the low affinity of IGFBP-6 for IGF-I, it is likely that the inhibitory effect of rat skeletal muscle-derived IGFBPs on IGF-I-induced myogenesis is mediated principally by IGFBP-4.     

Neurite outgrowth stimulated by neural cell adhesion molecules requires growth-associated protein-43 (GAP-43) function and is associated with GAP-43 phosphorylation in growth cones

The mechanisms whereby cell adhesion molecules (CAMs) promote axonal growth and synaptic plasticity are poorly understood. Here we show that the neurite outgrowth stimulated by NCAM-mediated fibroblast growth factor (FGF) receptor activation in cerebellar granule cells is associated with increased GAP-43 phosphorylation on serine-41. In contrast, neither NCAM nor FGF was able to stimulate neurite outgrowth in similar neurons from mice in which the GAP-43 gene had been deleted by homologous recombination. Integrin-mediated neurite outgrowth was unaffected by GAP-43 deletion. Both neurite outgrowth and rapid phosphorylation of GAP-43 in isolated growth cones required the first three Ig domains of a NCAM-Fc chimera and were stimulated maximally at 5 micrograms/ml (approximately 50 nM). Likewise, GAP-43 phosphorylation in isolated growth cones also was stimulated by an L1-Fc chimera. Both neurite outgrowth and NCAM-stimulated GAP-43 phosphorylation were inhibited by antibodies to the FGF receptor and a diacylglycerol lipase inhibitor (RHC80267) that blocks the production of arachidonic acid in response to activation of the FGF receptor. Direct activation of the FGF receptor and the arachidonic acid cascade with either basic FGF or melittin also resulted in increased GAP-43 phosphorylation. These data suggest that the stimulation of neurite outgrowth by NCAM requires GAP-43 function and that GAP-43 phosphorylation in isolated growth cones occurs via an FGF receptor-dependent increase in arachidonic acid.     

Expression of wild-type and mutant rat liver CTP: phosphocholine cytidylyltransferase in a cytidylyltransferase-deficient Chinese hamster ovary cell line

The strain 58 Chinese hamster ovary (CHO) mutant defective in CTP:phosphocholine cytidylyltransferase was characterized as an expression system for exogenous cytidylyltransferase. Strain 58 cells express less than 5% of the wild-type level of cytidylyltransferase protein at the permissive temperature even though the steady-state messenger RNA levels were found to be similar to those in the parental CHO-K1 cell line. A point mutation from arginine to histidine at amino acid 140 was identified in the strain 58 protein. Rat liver cytidylyltransferase was stably expressed in strain 58 cells and shown to be active, targeted to the nucleus, phosphorylated, and activated by methylethanolamine supplementation or phospholipase C treatment. Thus, the mechanisms by which cytidylyltransferase is processed and regulated in CHO-K1 cells are intact in strain 58 cells. The heterologously expressed protein complemented the strain 58 defects in both temperature-sensitive growth and phosphatidylcholine biosynthesis, consistent with a single lesion in the structural gene for cytidylyltransferase being responsible for both phenomena. Overexpression of cytidylyltransferase activity at levels up to eightfold higher than those in CHO-K1 cells did not appreciably affect phosphatidylcholine metabolism. A putative casein kinase II phosphorylation site was altered by site-directed mutagenesis and expressed in the strain 58 cells. Alteration of this site did not affect expression and regulation of cytidylyltransferase activity.     

Basic fibroblast growth factor-stimulated endothelial cell movement is mediated by a pertussis toxin-sensitive pathway regulating phospholipase A2 activity

Basic fibroblast growth factor (bFGF) stimulates migration and proliferation of vascular endothelial cells (EC). Fibroblast growth factor (FGF) receptors have an intrinsic tyrosine kinase activity involved in FGF-mediated proliferation, but the signal transduction pathway(s) responsible for movement is not known. We compared the role of signal-transducing G-proteins in migratory and proliferative responses of bovine aortic EC in vitro. Pertussis toxin, which ADP-ribosylates susceptible G-proteins, reduced bFGF-stimulated EC migration by 80%. The toxin did not block serum-stimulated movement, indicating that structural components required for motility were not impaired. The toxin did not inhibit bFGF-stimulated EC proliferation, showing that distinct intracellular signaling mechanisms are involved in the migratory and proliferative responses to bFGF. Three experimental approaches indicated that promigratory responses were due to release of arachidonic acid: (i) of the second messengers induced by G-protein-coupled effectors, only arachidonic acid over-came the pertussis toxin block, (ii) bFGF stimulated arachidonic acid release from EC in a pertussis toxin-sensitive manner, and (iii) phospholipase A2 inhibitors blocked the EC migration in response to bFGF. These data provide evidence that the migratory response of vascular EC to bFGF may be mediated by a G-protein-coupled phospholipase A2 activity.     

Insulin-like growth factor-binding protein (IGFBP-3) predisposes breast cancer cells to programmed cell death in a non-IGF-dependent manner

Insulin-like growth factor (IGF) -independent growth inhibition of human breast cancer cells, Hs578T, by IGF-binding protein-3 (IGFBP-3) has previously been demonstrated. Cell growth is a balance between proliferation and programmed cell death (apoptosis). We have investigated whether IGFBP-3 can affect apoptosis of Hs578T cells. As no induction of apoptosis was found, we also investigated its effect on the response to ceramide, an intracellular second messenger that mediates the signal for apoptosis. Using the cell permeable ceramide analogue, C2, induction of apoptosis was established by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) assay, trypan blue uptake, morphological criteria, and flow cytometry. Incubation of cells with non-glycosylated IGFBP-3 (ngIGFBP-3; 0.5-100 ng/ml) resulted in no growth inhibition or increase in apoptosis; whereas, C2 (1-30 microM) resulted in a dose-dependent induction of apoptosis. Addition of IGFs to the cells, alone or with C2, elicited no response in terms of proliferation or survival, respectively. When the cells were preincubated with ngIGFBP-3 before addition of C2 (2-5 microM), apoptosis was accentuated in a dose-dependent manner (at 100 ng/ml IGFBP-3, apoptosis increased from 11 to 88%). In conclusion, we found that IGFBP-3 had no direct inhibitory effect on Hs578T cells but could accentuate apoptosis induced by the physiological trigger ceramide in an IGF-independent manner.     

Insulin-stimulated cell growth in insulin receptor substrate-1-deficient ZR-75-1 cells is mediated by a phosphatidylinositol-3-kinase-independent pathway

The projections of enteric neurons to the circular muscle of the guinea pig gastric corpus were investigated systematically by using the retrogradely transported fluorescent carbocyanine dye 1,1'-didodecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (DiI), applied to the muscle layer or myenteric plexus in vitro. DiI-labeled motor neuron cell bodies were located up to 6.3 mm aboral, 17 mm oral, and up to 20 mm circumferential to the DiI application site. Labeled nerve fibers ran for long distances from the DiI application site toward the greater and lesser curvatures, where they coursed parallel to the bundles of the "gastric sling" muscle. The majority of labeled cells were located toward the lesser curvature of the stomach. Nerve cell bodies that were aboral to the DiI application site were usually small, immunoreactive for choline acetyltransferase, and, thus, were likely to be excitatory motor neurons. Neurons that were located orally were larger, fewer in number, and immunoreactive for nitric oxide synthase and, thus, were likely to be inhibitory motor neurons. Application of DiI directly to the myenteric plexus filled neurons up to 15 mm aborally and up to 21 mm orally but labeled few neurons circumferentially. All nerve cells that were filled from either the circular muscle or the myenteric plexus had Dogiel type I morphological features. These results demonstrate a clear polarity of projection of inhibitory and excitatory motor neurons and a functionally continuous innervation of the circular and gastric sling muscle layers. Nonmotor neurons in the myenteric plexus were demonstrated, but neurons with Dogiel type II morphological features are apparently absent.     

Isolated rat hepatocytes bind lactoferrins by the RHL-1 subunit of the asialoglycoprotein receptor in a galactose-independent manner

Isolated rat hepatocytes bind and internalize the iron-binding protein lactoferrin (Lf) by a set of high-affinity, recycling, Ca2+-dependent binding sites. We have purified a 45-kDa membrane protein (p45) from rat hepatocytes that exhibits Ca2+-dependent receptor activity. In this study, we found p45 to be identical to the major subunit (RHL-1) of the rat asialoglycoprotein receptor. Two tryptic fragments of p45 showed 100% identity with RHL-1 internal sequences (Leu121 --> Lys126 and Phe198 --> Lys220), and monospecific antisera against p45 and RHL-1 cross-reacted equally well with each protein. Molar excesses of anti-p45 IgG, anti-RHL-1 IgG, asialoorosomucoid, and asialofetuin competitively blocked the binding of 125I-Lf to isolated rat hepatocytes at 4 degrees C. Similarly, either excess anti-p45 or Lf blocked the binding of 125I-asialoorosomucoid to cells at 4 degrees C. We did not detect the minor subunits of the rat asialoglycoprotein receptor (RHL-2/3) in p45 preparations from Triton X-100 extracts of hepatocytes and 125I-Lf bound to purified RHL-1 but not to RHL-2/3 immobilized on nitrocellulose. Nonetheless, anti-RHL-2/3 IgG reduced the binding of 125I-Lf to hepatocytes at 4 degrees C. Exoglycosidases were used to remove terminally-exposed N-acetylneuraminyl, alpha- and beta-galactosyl, and N-acetylhexosaminyl sugars from human and bovine Lf glycans, and lectin blotting confirmed that glycosidase-treated Lfs lacked detectable terminal galactosyl sugars. Unexpectedly, these deglycosylated Lfs exhibited no loss in their ability to compete with unmodified Lfs for binding to isolated hepatocytes. In addition, molar excess of beta-lactose but not sucrose competitively blocked the binding of 125I-Lf to cells, indicating that Lf bound at or very near the carbohydrate-recognition domain of RHL-1. We conclude that RHL-1 is the Ca2+-dependent Lf receptor on hepatocytes and that it binds Lf at its carbohydrate-recognition domain yet in a galactose-independent manner.     

Insulin-like growth factor binding proteins localize to discrete cell culture compartments in periosteal and osteoblast cultures from fetal rat bone

Insulin-like growth factor (IGF)-I and IGF-II are expressed at biologically effective levels by bone cells. Their stability and activity are modulated by coexpression of IGF binding proteins (IGFBPs). Secreted IGFBPs may partition to soluble, cell-associated, and matrix-bound compartments. Extracellular localization may sequester, store, or present IGFs to appropriate receptors. Of the six IGFBPs known, rat osteoblasts synthesize all but IGFBP-1. Of these, IGFBP-3, -4, and -5 mRNAs are induced by an increase in cAMP. Little is known about extracellular IGFBP localization in bone and nothing about IGFBP expression by nonosteoblastic periosteal bone cells. We compared basal IGFBP expression in periosteal and osteoblast bone cell cultures and assessed the effects of changes in cAMP-dependent protein kinase A or protein kinase C. Basal IGFBP gene expression differed principally in that more IGFBP-2 and -5 occurred in osteoblast cultures, and more IGFBP-3 and -6 occurred in periosteal cultures. An increase in cAMP enhanced IGFBP-3, -4, and -5 mRNAand accordingly increased soluble IGFBP-3, -4, and -5 and matrix-bound IGFBP-3 and -5 in both bone cell populations. In contrast, protein kinase C activators suppressed IGFBP-5 mRNA, and its basal protein levels remained very low. We also detected low Mr bands reactive with antisera to IGFBP-2, -3, and -5, suggesting proteolytic processing or degradation. Our studies reveal that various bone cell populations secrete and bind IGFBPs in selective ways. Importantly, inhibitory IGFBP-4 does not significantly accumulate in cell-associated compartments, even though its secretion is enhanced by cAMP. Because IGFBPs bind IGFs less tightly in cell-bound compartments, they may prolong anabolic effects by agents that increase bone cell cAMP.     

Platelet-derived growth factor-B increases colon cancer cell growth in vivo by a paracrine effect

PDGF-B released from colon tumor cells regulated tumor growth in athymic mice in a paracrine manner by inducing blood vessel formation. A positive correlation was found between expression of PDGF B-chain in cells grown in vitro and the number of factor VIII-positive blood vessels in tumors induced by three classes of colon carcinoma cell lines. Elevated expression of PDGF-B was also correlated with tumor size. Each cell line had the same mutations in the colon cancer genes APC, DCC, and p53 and had wild type c-K-ras genes (Huang et al. [1994] Oncogene, 9:3701-3706.) eliminating the possibility that any differences in tumor blood vessel formation were due to mutations and/or deletions in these genes. Colon carcinoma cells released biologically active PDGF capable of stimulating the growth of NIH3T3 cells, which was inhibited by neutralizing antisera to PDGF-AB chains. An inverse correlation was found between induction of factor VIII-positive blood vessels and expression of vascular endothelial growth factor (VEGF), while no correlation was seen with expression of either TGF alpha or k-FGF. Basic fibroblast growth factor (FGF) expression was not detected in these tumor cells. TGF beta 1 was capable of inducing PDGF-B expression in the undifferentiated U9 colon carcinoma cell line, but this sensitivity was not seen in differentiated cells. In contrast, TGF beta 1 inhibited VEGF expression in both undifferentiated cells and differentiated colon cancer cells. Thus, TGF beta 1 has two roles in the growth of undifferentiated U9 colon carcinoma cells in vivo: direct stimulation of cell proliferation as we have showed in earlier studies, and an increase in angiogenesis by inducing PDGF-B.     

beta-Lactamase activity in mycobacteria including Mycobacterium avium and suppression of their growth by a beta-lactamase-stable antibiotic

beta-Lactamase has been reported from only a few mycobacteria. It is widely assumed that Mycobacterium avium strains do not contain the enzyme, but earlier assays were done using insensitive methods. Thus the beta-lactamase activity in cell-free extracts of ten selected strains of mycobacteria, including four strains of M. avium, was determined using a highly sensitive spectrophotometric method. The results showed that all the mycobacteria tested possess the enzyme, which explains their resistance to beta-lactam antibiotics. However, some of the bacteria differed from others in the action of the inhibitors, clavulanate, sulbactam and tazobactam against their beta-lactamases. Growth of the mycobacteria was suppressed by novel combinations of the beta-lactam/beta-lactamase-inhibitors, and by a new beta-lactamase-stable cephalosporin, Cefepime (aminothiazolyl methoxyimino cephalosporin). The results presented, as well as reports of previous studies in vivo, suggest that the intracellular growth of the bacilli or the high partition coefficient of a beta-lactamase inhibitor such as sulbactam does not impede the antimycobacterial action of these compounds.