Growth hormone improves cardiac function in rats with experimental myocardial infarction

Glioblastoma multiforme is a highly invasive primary brain tumor, which is known to strongly express the CD44 cell adhesion receptor. A number of experimental studies suggest that the interaction of this receptor with extracellular matrix (ECM) proteins such as hyaluronic acid may in part mediate human glioma cell adhesion and invasion of brain tissue. Although the expression of CD44 and its spliced variants in brain tumors have been extensively studied, there have been no reports localizing its expression to the invasive margin of the tumor. The authors used immunoelectron microscopy to investigate the expression patterns of CD44 in an in vitro organotypic invasion assay. Tumor spheroids initiated from the U373 MG human glioblastoma line were confronted with fetal rat brain aggregates in a spheroid coculture system. The CD44 expression appeared at the interface between glioblastoma tumor spheroids and brain tissue, as well as in the spheroid itself. CD44 immunoreactivity was not detectable in mature 21-day fetal brain aggregates. The findings provide direct evidence that CD44 is expressed at the confrontational invasive border between glioblastomas and brain tissue, further supporting its role in glioma cell-ECM recognition and attachment.     

Reduction in surface urokinase receptor forces malignant cells into a protracted state of dormancy

Considerable evidence links urokinase plasminogen activator (uPA) bound to its surface receptor (uPAR) with enhanced invasiveness of cancer cells. By blocking uPAR expression in human epidermoid carcinoma cells (HEp3), we have now identified an additional and novel in vivo function for this receptor by showing that receptor-deficient cells enter a state of dormancy reminiscent of that observed in human cancer metastasis. Its main characteristic is survival without signs of progressive growth. Five clones transfected with a vector expressing uPAR antisense RNA under the beta-actin promoter were isolated and shown to have uPAR (at the mRNA and protein levels) reduced by 50 to 80%; four clones, transfected with vector alone and having uPAR levels similar to those of parental cells, served as controls. In confirmation of our previous results, reduced uPAR always coincided with a significantly reduced invasiveness. Each of the control clones produced rapidly growing, highly metastatic tumors within 2 wk of inoculation on chorioallantoic membranes (CAMs) of chick embryos. In contrast, each of the clones with low surface uPAR, whose proliferation rate in culture was indistinguishable from controls, remained dormant for up to 5 mo when inoculated on CAMs. Thus, the reduction in uPAR altered the phenotype of HEp3 tumor cells from tumorigenic to dormant. Although protracted, tumor dormancy was not permanent since in spite of maintaining low uPAR levels, each of the in vivo-passaged antisense clones eventually reemerged from dormancy to initiate progressive growth and to form metastases at a level of 20 to 90% of that of fully malignant control. This observation suggested that other factors, whose expression is dependent on cumulative and prolonged in vivo effects, can compensate for the lack of a full complement of surface uPAR required for the expression of malignant properties. These "reemerged," uPAR-deficient clones were easily distinguishable from the vector-transfected controls by the fact that after only 1 wk in culture, the invasion of CAM by all five clones and tumorigenicity of four of the five clones were reduced back to the values observed before in vivo maintenance. In contrast, dissociated and in vitro-grown cells of control tumors were fully invasive and produced large, metastatic tumors when reinoculated on CAMs. Quantitation of the percent of apoptotic and S-phase cells in vivo, in the control and uPAR-deficient, dormant clones, showed that the mechanism responsible for the dormancy was a diminished proliferation.     

Cloning of a highly conserved human protein serine-threonine phosphatase gene from the glioma candidate region on chromosome 19q13.3

Allelic loss studies have suggested that a glioma tumor suppressor gene resides in a 425-kb region of chromosome 19q, telomeric to D19S219 and centromeric to D19S112. Exon amplification of a cosmid contig spanning this region yielded four exons with high homology to a rat protein serine-threonine phosphatase from a cosmid approximately 100 kb telomeric to D19S219. Isolation of a near full-length cDNA from a human fetal brain cDNA library revealed a protein serine-threonine phosphatase with a tetratricopeptide motif, almost identical to human PPP5C (PP5) and highly homologous to rat PPT. Northern blotting demonstrated expression in most tissues, including brain. Primary and cultured gliomas were studied for genetic alterations in this gene using pulsed-field gel electrophoresis, routine Southern blots, and genomic DNA-and RNA-based single-strand conformation polymorphism analysis. Genomic alterations were were not detected in any of the gliomas, and all studied gliomas expressed the gene, suggesting that this phosphatase is not the putative chromosome 19q glioma tumor suppressor gene.     

Interleukin-6-mediated autocrine growth promotion in human glioblastoma multiforme cell line U87MG

Human glioblastoma multiforme cell lines, brain tumor biopsy tissue, and normal human fetal brain synthesize interleukin (IL)-6 and IL-6 receptor (IL-6R). Neither of these is expressed in human neurons or neuroblastoma cell lines in culture. Astrocytes from fetal brain grown in culture retain the ability to synthesize IL-6 but do not express IL-6R as inferred from RT-PCR and Southern blot studies. Coexpression of IL-6 and IL-6R in the glioblastoma cell line U87MG is confirmed by immunofluorescence staining. Both specific monoclonal antibodies against IL-6 and IL-6R and antisense oligonucleotide to IL-6 mRNA inhibit the growth of U87MG cells in culture, suggesting the existence of a functional autocrine growth loop. Anti-IL-6 antibodies also inhibit the growth of glioblastoma cell lines U373 and U118. The expression of IL-6 by human fetal astrocytes in culture is highly suggestive of its role as an oncofetal protein responsible for rapid proliferation of fetal and tumor cells but not cells of adult brain.     

Recall and attitudes in patients with prostate cancer

Urokinase-type plasminogen activator (uPA) and its receptor (uPAR) are important in the regulation of tumor tissue progenesis, cell differentiation, tumor cell motility, and tumor cell invasiveness. We have recently reported that the levels of uPA and uPAR were higher in malignant astrocytomas than in low-grade gliomas. In the present study, we measured the levels of uPA and uPAR during the growth of glioblastomas in nude mice. Using fibrin zymography, densitometry, and an enzyme-linked immunosorbent assay, we found that the enzyme activity and content of uPA were increased 4- to 10-fold during tumor formation. Using a receptor assay and an enzyme linked immunosorbent assay, we found the numbers and content of uPAR were increased 5- to 15-fold during tumor formation. In addition, immunohistochemical staining for uPA and uPAR revealed strong immunoreactivity in tumor cells with the staining more intense on day 28 than on day 14. These results suggest that the upregulation of uPA and uPAR plays a major role in the formation of gliomas.     

Stimulation of extracellular matrix components in the normal brain by invading glioma cells

Malignant gliomas are characterized by an extensive invasion of tumor cells into the normal brain parenchyma. A substantial amount of data indicates that cell movement in general is regulated by specific interactions between extracellular matrix components and specific cell-surface receptors. In the present work, multicellular spheroids from 4 human glioma cell lines (U-373Mg, A-172Mg, U-251Mg and HF-66) were confronted with normal rat brain cell aggregates in vitro, which resulted in a progressive invasion of tumor cells into the brain aggregates. The co-cultures were then sectioned and immuno-stained for specific extracellular matrix components (laminin, fibronectin and collagen type IV) and for specific cell-surface receptors which bind to these components (integrins beta1, beta4, alpha3, alpha6). In addition, flow-cytometric measurements and Northern blot analyses showed expression of several different integrins within the cell lines. The alpha3 subunit was expressed strongly in all cell lines. Whereas the beta1 subunit was expressed weakly in exponentially growing monolayer cultures, it showed a pronounced expression in multicellular spheroids, indicating that the integrin expression may vary depending on the micro-environment within a tumor. Furthermore, normal brain tissue was able to produce laminin when confronted with the glioma cells, which also was observed for fibronectin and collagen type IV. The relevance of our observations to the in vivo situation was investigated further by immuno-staining 5 human glioma biopsy samples for laminin. In some areas of the tumors, specific deposits of laminin were observed. In conclusion, we have shown that normal brain tissue has the ability to produce extracellular matrix components, such as laminin, collagen type IV and fibronectin, when confronted with invading glioma cells. Our results show that the glioma cells express specific integrins which can interact with these extracellular matrix components. Such interactions may facilitate tumor cell migration and invasion.     

Overexpression of the EGF receptor and p53 mutations are mutually exclusive in the evolution of primary and secondary glioblastomas

Glioblastoma multiforme, the most malignant human brain tumor, may develop de novo (primary glioblastoma) or through progression from low-grade or anaplastic astrocytoma (secondary glioblastoma). We present further evidence that primary and secondary glioblastomas constitute distinct disease entities which develop through the acquisition of different genetic alterations. We analyzed p53 mutations, p53 protein accumulation and epidermal growth factor receptor (EGFR) overexpression in 49 biopsies classified as primary or secondary glioblastoma according to clinical and histopathologic criteria. Patients with primary glioblastoma were selected on the basis of a clinical history of less than 3 months and histopathologic features of glioblastoma at the first biopsy (19 cases; mean age, 55 years). The diagnosis of secondary glioblastomas required at least two biopsies and clinical as well as histologic evidence of progression from low grade or anaplastic astrocytoma (30 cases; mean age, 39 years). DNA sequence analysis showed that p53 mutations were rare in primary glioblastomas (11%) while secondary glioblastomas had a high incidence of p53 mutations (67%), of which 90% were already present in the first biopsy. The incidence of p53 protein accumulation (nuclear immunoreactivity to PAb 1801) was also lower in primary (37%) than in secondary glioblastomas (97%). In contrast, immunoreactivity for the EGF receptor prevailed in primary glioblastomas (63%) but was rare in secondary glioblastomas (10%). Only one out of 49 glioblastomas showed EGFR overexpression and a p53 mutation. These data indicate that overexpression of the EGF receptor and mutations of the p53 tumor suppressor gene are mutually exclusive events defining two different genetic pathways in the evolution of glioblastoma as the common phenotypic endpoint.     

Variable efficiency of the thymidine kinase/ganciclovir system in human glioblastoma cell lines: implications for gene therapy

The gene therapy strategy using the hsvl-thymidine kinase gene (TK) and ganciclovir (GCV) injections that has been used for treating human glioblastomas has not been as effective as expected after the first animal experiments. A better understanding of the different steps involved in this treatment, like gene transfer, gene expression, and sensitivity of the recipient cells, is needed. After proposing sensitivity criteria for the TK/GCV system and for the bystander effect, based on the levels of GCV that can be reached in vivo, we studied seven human glioblastoma cell lines (U87, U118, U251, SNB19, SNB75, SF295, SF539) for their sensitivity to the TK/GCV system. We also studied their in vitro bystander effect and their in vitro transfectability using LipofectAMINE as a transfection enhancer. Among six human glioblastoma cell lines stably transfected with the TK gene, five were sensitive to TK/GCV, and two had a good in vitro bystander effect. The in vitro transfectability of the cell lines tested was low (< or = 1%) compared to that of an established animal cell line, C6 rat glioma, in which 20-30% of the cells can be transfected routinely. According to this in vitro analysis, most of the glioblastoma cell lines should be sensitive to the TK/GCV system, but there is an urgent need for agents to increase transfection efficiency.     

Migratory pattern of fetal rat brain cells and human glioma cells in the adult rat brain

The migratory behavior of two human glioma cell lines (D-54MG and GaMG) and fetal rat brain cells grafted into the adult rat brain was studied. To trace the implanted cells, they were stained with the carbocyanine vital dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate before injecting them into the white matter above the corpus callosum. The animals were sacrificed 2 h and 7 and 21 days after injection, and the brains were removed and cryosectioned. Fluorescence microscopy showed that both the 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate-stained fetal and tumor cells had the same migratory pattern. Implanted cells were found along myelinated fibers in the corpus callosum and in the perivascular space. After immunostaining for several extracellular matrix (ECM) components (laminin, fibronectin, collagen type IV, and chondroitin sulfate), laminin deposits were observed in the border zone between the host tissue and implanted tumor cells as well as fetal cells. By using two different types of antibodies against fibronectin, it is shown that the fibronectin expression observed in the tumor matrix may be host derived. This was further supported by the fact that tumor spheroids obtained from the two glioma cell lines were negative when immunostained for these ECM components. Several of the ECM components may be host derived. This can be caused by neovascularization and repair synthesis or by a local production of guiding substrates which are important for tumor cell locomotion. The present data suggest that the migratory patterns of fetal and glioma cells are indistinguishable when transplanted into the adult rat brain. Thus, glioma cells may be routed by the same ECM components that play a major role during brain development.     

Preproinsulin I and II mRNAs and insulin electron microscopic immunoreaction are present within the rat fetal nervous system

Insulin-like substance has been found within the nervous system. In the rat, preproinsulin II mRNA was shown within the brain and preproinsulin I mRNA within the retina. The present study demonstrates the presence of preproinsulin mRNAs within the 15, 17 and 19 day gestational age fetal rat brain, spinal cord and dorsal root ganglia (DRG), employing RNA template-specific polymerase chain reaction (RS-PCR), semi-nested PCR and RNase protection assay. Preproinsulin I mRNA was present in the 17 and 19 day gestational age brain, spinal cord and DRG, and only in the brain of the 15 day gestational age brain. Preproinsulin II mRNA was present in all the gestational ages studied in the brain, spinal cord and DRG. The RS-PCR and the semi-nested PCR demonstrated products that co-migrated with the pancreatic control. The semi-nested products were characterized as preproinsulin I and II by restriction enzyme digestion and sequence. RNase protection assay using specific cRNA for preproinsulin I and II showed a band that co-migrated with pancreatic preproinsulin I and II mRNAs, and confirmed the PCR results. In addition, insulin receptor mRNA was detected by RS-PCR. Ultrastructural studies showed insulin immunoreaction within the endoplasmic reticulum, Golgi apparatus, cytoplasm, axon, dendrites, and in relation to the synapses. Thus, we demonstrated the presence of preproinsulin I and II mRNA, insulin receptor mRNA and insulin immunoreaction within the rat fetal central and peripheral nervous system.     

Guidance of circumferentially growing axons by netrin-dependent and -independent floor plate chemotropism in the vertebrate brain

BACKGROUND: A strong positive correlation exists between the breast cancer tissue content of either urokinase-plasminogen activator (uPA) or plasminogen activator, inhibitor type I (PAI-1), quantified in the tissue extracts by immunoassays, and the survival of patients with breast cancer. Furthermore, several studies assign to the urokinase-type plasminogen activator receptor (uPAR) a pivotal role in triggering the proteolytic activity of the urokinase pathway involved in tumor stroma degradation, tumor spread and metastasis. However, the pattern of distribution of uPAR in normal and cancerous human tissue and the pattern of coexpression of activators and inhibitors that occurs in breast cancer tissues is not completely known. METHODS: The immunohistochemical localization of uPAR, uPA, tPA) and PAI-1 was evaluated by using the avidin-biotin immunoperoxidase technique and affinity-purified monoclonal antibodies from American Diagnostica Inc. Studies were performed in formalin fixed, paraffin-embedded tissue prepared from 23 surgically excised non-neoplastic breast tissues and 18 ductal breast carcinomas. RESULTS: While the expression of uPAR protein represents a constant feature of invasive ductal breast cancer, it was also observed in most of the breast tissue samples, including the normal breast tissues. The staining for uPAR was mainly localized on normal or tumoral epithelial cells, even if the co-expression of uPAR in stromal cells was frequently observed in adjacent slides. A semiquantitative analysis of immunohistochemical results showed that uPAR and PAI-1 were overexpressed in invasive breast cancer in comparison with normal and benign breast tissues. In addition, uPA was higher in both invasive breast carcinomas and benign breast lesions with respect to normal breast tissues. CONCLUSIONS: We showed that overexpression of uPAR, uPA, and its main inhibitor, PAI-1, is a constant feature of invasive ductal breast carcinomas. However, the expression of the above fibrinolytic reactants is not specific for breast cancer since positive staining for these molecules was frequently observed in benign breast lesions as well as in normal breast tissues. The combined increased expression of uPA and its cellular receptor, uPAR on the surface of tumor epithelial cells may account for the activation of the proteolytic system which occurs in breast cancer.     

Blockade of NMDA receptors and apoptotic neurodegeneration in the developing brain

Programmed cell death (apoptosis) occurs during normal development of the central nervous system. However, the mechanisms that determine which neurons will succumb to apoptosis are poorly understood. Blockade of N-methyl-D-aspartate (NMDA) glutamate receptors for only a few hours during late fetal or early neonatal life triggered widespread apoptotic neurodegeneration in the developing rat brain, suggesting that the excitatory neurotransmitter glutamate, acting at NMDA receptors, controls neuronal survival. These findings may have relevance to human neurodevelopmental disorders involving prenatal (drug-abusing mothers) or postnatal (pediatric anesthesia) exposure to drugs that block NMDA receptors.     

Pretreatment with antisense oligodeoxynucleotides directed against the NMDA-R1 receptor enhances survival and behavioral recovery following traumatic brain injury in rats

Treatment with N-methyl-D-aspartate (NMDA) receptor antagonists limits tissue damage following CNS ischemia or trauma, supporting the hypothesis that NMDA receptors participate in the pathophysiology of such injuries. An alternative approach for evaluating this hypothesis is to examine the effects of selective inhibition of NMDA receptor synthesis, using antisense oligodeoxynucleotides. In the present studies, the effects of antisense oligodeoxynucleotides directed at NMDA-R1 receptor subunit, administered intracerebroventricularly (i.c.v.) prior to injury, were evaluated in a well-defined traumatic brain injury model in rats. Outcome measures included survival, motor recovery, and histological changes. Administration of antisense oligodeoxynucleotides (15 nmol/ml twice daily x 2 days) did not alter physiological variables or motor function prior to trauma. However, such treatment significantly decreased mortality and improved behavioral recovery at 2 weeks after trauma as compared to animals treated with the corresponding sense oligodeoxynucleotides. Although cell counts in hippocampus did not differ between treatment groups, astrocyte activation as reflected by glial fibrillary astrocytic protein (GFAP) immunocytochemistry was significantly reduced in antisense treated animals. These findings provide additional evidence that NMDA receptors contribute to secondary injury after brain trauma and may suggest an alternative treatment approach.     

Predicting age at menopause

Macrophages in the tissues have been shown to express receptor for urokinase-type plasminogen activator (uPAR) on their cell surface which plays an important role in cell invasion and attachment. We examined the effects of inflammatory mediators on the expression of uPAR employing U937 cells which have monocyte/macrophage-like characteristics. U937 cells were incubated with various mediators such as interleukins (IL), tumor necrosis factors (TNF), dexamethasone, thrombin, fibrin fragment D, bradykinin, complement C5a, and components of the extracellular matrix. The uPAR expression on the cell surface was then analyzed by radio-ligand binding assay using 125I-scuPA. The strongest enhancement of uPAR was observed in the cells stimulated by TNF alpha and TNF beta. IL-1 beta, IL-6, and C5a also increased the uPA binding sites with various patterns of affinity change. Dexamethasone decreased the uPA binding sites without changing the affinity. Fibrin fragment D and IL-3 reduced the affinity without changing the number of receptors. These findings suggest that the expression of uPAR in inflammatory cells could be modulated by various inflammatory mediators.     

Use of subunit-specific antisense oligodeoxynucleotides to define developmental changes in the properties of N-methyl-D-aspartate receptors

Antisense oligodeoxynucleotides were used to determine whether alterations in the expression of N-methyl-D-aspartate (NMDA) receptor subunit mRNA are responsible for developmental changes in the sensitivity of receptors to agonists and antagonists. Xenopus laevis oocytes were injected with mRNA prepared from neonatal and adult rat cerebral cortex, and the effects of agonists and antagonists were determined under voltage-clamp conditions. Glycine-site antagonists like 7-chlorokynurenate and glutamate-site antagonists like CGP-39653 were more potent at NMDA receptors expressed from mRNA from adult rat cerebral cortex than those expressed from mRNA from 1-day-old rat. NMDA receptors from 1-day-old rat cerebral cortex were more sensitive to activation by glycine than were receptors from adult rat cerebral cortex. 7-Chlorokynurenate and CGP-39653 were more potent inhibitors of responses seen with heteromeric NR1/NR2A receptors than with NR1/ NR2B receptors. Conversely, heteromeric NR1/NR2B receptors were more sensitive to activation by glycine than were NR1/NR2A receptors. We previously described a delay in the expression of the NR2A subunit in developing rat brain. Anti-sense oligodeoxynucleotides were used to determine whether the delayed expression of the NR2A subunit underlies changes in pharmacological properties observed during development. The properties of receptors seen when adult brain mRNA was coinjected with antisense oligodeoxynucleotides against the NR2A subunit were similar to those found in receptors from 1-day-old rat brain. These data suggest that changes in the sensitivity of NMDA receptors to antagonists and to glycine seen during development are a result of alterations in the expression of different species of NR2 subunit mRNA.     

The urokinase receptor (CD87) facilitates CD11b/CD18-mediated adhesion of human monocytes

Urokinase receptors (uPAR; CD87) from complexes with complement receptor 3 (CR3) (CD11b/CD18), a beta2 integrin. In this study, we sought to determine if this association modulates the adhesive function of CR3. Both CR3 and uPAR concentrate at the ventral surface of fibrinogen-adherent human monocytes, and CR3-uPAR coupling increases substantially upon adhesion to fibrinogen. Pretreatment with anti-uPAR monoclonal antibody reduced adhesion to CR3 counterligands (fibrinogen and keyhole limpet hemocyanin) by 50%, but did not affect adhesion to fibronectin, a beta1 integrin counterligand. Antisense (AS) oligonucleotides were used to determine if selectively suppressing uPAR expression also modulates CR3 adhesive function. AS-uPAR oligo reduced CR3-dependent adhesion by 43+/-9% (P<0.01), but did not affect CR3-independent adhesion. To determine if the effects of uPAR are mediated through its ligand, monocytes were pre-treated with AS oligo to block uPA expression. Unlike the effects of blocking uPAR expression, AS-uPA oligo increased adhesion by 46% (P<0.005), and exogenous intact uPA, but not uPA fragments, reversed this effect. We conclude that complex formation with uPAR facilitates the adhesive functions of CR3. This function of uPAR is not dependent upon its occupancy with uPA, which negatively influences adhesion.