Biomedical subjects

J H Resau

Publications and source records attributed to J H Resau.

At least 37 records · Page 2Linked to original sources

PubMed1997

Met and hepatocyte growth factor/scatter factor expression in human gliomas.

Using double immunofluorescence staining and quantitative confocal laser scan microscopy, we show that the intensity of hepatocyte growth factor/scatter factor (HGF/SF) and Met staining in human primary brain tumors increases with the grade of malignancy and is prevalent in both the infiltrating tumor cells and endothelial hyperplastic areas. HGF/SF and Met also are expressed in vitro in glioblastoma multiforme cell lines as well as in normal human astrocyte (NHA) cells. Moreover, HGF/SF stimulates tyrosine phosphorylation of Met in both glioma cell lines and NHA cells, but only the glioma cell lines proliferate and become motile and invasive in response to HGF/SF, whereas the NHA cells are nonresponsive. These results implicate autocrine/paracrine Met-HGF/SF signaling in glioma tumorigenesis and suggest that HGF/SF signaling through Met is negatively regulated in NHA cells.

3T3 Cells↗

PubMed1997

The inducibly expressed GTPase localizes to the endoplasmic reticulum, independently of GTP binding.

The inducibly expressed GTPase (IGTP) is representative of a newly identified group of interferon gamma-inducible GTPases, whose functions are currently unknown. We have begun to address the cellular function of IGTP by examining its subcellular distribution and its guanine nucleotide binding status. Using immunofluorescence, electron microscopy, and subcellular fractionation, IGTP was localized predominantly to the endoplasmic reticulum of both RAW 264. 7 macrophages and C127 fibroblasts. In the immunostaining experiments, staining of discrete cytoplasmic structures on the periphery of the endoplasmic reticulum was also evident. Using polyethyleneimine-cellulose thin layer chromatography, the guanine nucleotides that complexed to immunoprecipitated IGTP, in both control and interferon gamma-stimulated cells, were 90-95% GTP and 5-10% GDP, suggesting that the protein was in an active state. A mutant IGTP protein was created that had no detectable complexed GTP, and in both subcellular fractionation and IGTP-green fluorescent protein fusion studies, this mutant also localized to the endoplasmic reticulum. These results suggested that the GTP binding status of IGTP is independent of its capacity to localize to the endoplasmic reticulum. Given these results, we propose that IGTP is representative of a new family of endoplasmic reticulum GTPases that may be involved in protein processing or trafficking.

Amino Acid Sequence↗

PubMed1997

Endocytosis of gastrin in cancer cells expressing gastrin/CCK-B receptor.

Endocytosis of gastrin was studied in a number of gastrin-receptor-expressing cell lines by confocal laser scanning microscopy (CLSM) with the aid of a biologically active fluorescent derivative, rhodamine green heptagastrin. Rapid clustering (within 4-7 min) and internalization of fluorescent ligand upon binding at room temperature and 37 degrees C were observed in the rat pancreatic acinar carcinoma cell line AR42J, human gastric carcinomas AGS-P and SIIA, human colon carcinomas HCT116 and HT29, and in NIH/3T3 cells transfected with human and rat gastrin/cholecystokinin-B receptor cDNA. Internalization was inhibited by hypertonic medium. Fluorescent heptagastrin and transferrin colocalized in the same endocytic vesicles at different stages of internalization suggesting that endocytosis occurred predominantly through a clathrin-dependent mechanism. At 37 degrees C partial colocalization with the lysosomal marker neutral red was detected by CLSM, implying that internalized gastrin accumulated in the lysosomes. Immunoelectron microscopy studies with antibodies against gastrin revealed the presence of the internalized hormone in multivesicular vesicles and endosomes. Almost no hormone was detected in lysosomes with the antibodies to gastrin, suggesting that the degradation of the peptide is rapid in those vesicles. Continuous accumulation of fluorescent label was observed by CLSM in the presence of the protein synthesis inhibitor cycloheximide, suggesting that the gastrin receptor is recycled back to the cell membrane after hormone delivery to intracellular compartments. An estimated average recycling time for the receptor molecules was 1 h in NIH/3T3 cells.

3T3 Cells↗

PubMed1997

Transforming growth factor-beta1 in adult human microglia and its stimulated production by interleukin-1.

Ameboid microglia express human immunodeficiency virus 1 (HIV-1) more frequently than do ramified microglia. These two microglial subtypes might also differ in the frequency with which they express transforming growth factor-beta1 (TGF-beta1), a cytokine that regulates HIV-1 expression in monocytes. Results described here show that ameboid and ramified microglia express TGF-beta1. In brain tissues from HIV-1-infected individuals as compared with seronegative controls, ameboid rather than ramified microglia more frequently expressed TGF-beta1. Ameboid microglia, isolated and cultured from postmortem adult human brain more frequently expressed TGF-beta1 in presence of interleukin-1(IL-1), a cytokine that is elevated in brains of HIV-1-infected individuals when compared with seronegative controls. The stimulation of TGF-beta1 by IL-1 was dose and time dependent, occurring with ameboid microglia isolated from either frontal cortex or globus pallidus but not midbrain pons. Ameboid microglia are similar to the RCA-1-positive cells that form clusters, called microglial nodules, in the brain of HIV-1-infected individuals. Pathologic conditions, such as disseminated microglial nodules, are associated with HIV-1 encephalitis, direct infection of the brain, and moderate to severe neurologic impairment. TGF-beta1 expression in ameboid microglia may play a role in HIV-1 neuropathogenesis.

Acquired Immunodeficiency Syndrome↗

PubMed1996

Co-localization of the TSC2 product tuberin with its target Rap1 in the Golgi apparatus.

Tuberin is the protein product of the tuberous sclerosis-2 (TSC2) gene, which is associated with tuberous sclerosis (TSC), a human genetic syndrome characterized by the development of tumors in a variety of tissues. We have previously shown that tuberin is a widely expressed 180 kDa protein which exhibits specific GTPase activating activity in vitro towards the Ras-related Rap1 protein. In this study we have used affinity-purified antibodies against tuberin to analyse its expression in human and rat tissues and to examine its subcellular localization. Tuberin expression was detected in all adult human tissues tested, with the highest levels found in brain, heart and kidney, organs that are commonly affected in TSC patients. By contrast, in adult rats the highest levels of tuberin were found in brain, liver and testis. Indirect immunofluorescence of tuberin in various cultured cell lines revealed a punctate, mostly perinuclear staining pattern. Double-indirect immunofluorescence analysis with anti-tuberin sera and antisera against known Golgi markers (mannosidase-II and furin) revealed that the staining of tuberin was consistent with its localization in the stacks of the Golgi apparatus. In support of this, treatment of cells with brefeldin A, a drug known to cause disassembly of the Golgi apparatus, abolished the perinuclear staining of tuberin. Moreover, conventional and confocal immunofluorescence demonstrated co-localization of tuberin with Rap1, which has previously been localized to the Golgi apparatus. The co-localization of tuberin and Rap1 in vivo strengthens the likelihood that the in vitro catalytic activity of tuberin toward Rap1 plays a physiologically relevant role in the tumor suppressor function of tuberin.

Animals↗

PubMed1996

In vivo expression of inducible nitric oxide synthase in cerebellar neurons.

In the CNS, nitric oxide (NO) functions as both neuromodulator and neurotoxic agent. In vivo neuronal expression of NO synthase (NOS) has been attributed to constitutive NOS--both the neuronal and the endothelial types. The other class of NOS--the inducible NOS (iNOS)--is known to mediate toxic effects of NO in various tissues. In this study, we show for the first time that direct intracerebellar injection of endotoxin and cytokine (lipopolysaccharide and interferon-gamma) induced in vivo neuronal expression of the iNOS gene, as demonstrated by fluorescent in situ hybridization and immunohistochemical staining analyzed by confocal laser-scanning microscopy. This raises the possibility that neuronal iNOS might contribute significantly to the vulnerability of the brain to various insults.

Animals↗

PubMed1996

Effects of Helicobacter pylori vacuolating cytotoxin on primary cultures of human gastric epithelial cells.

BACKGROUND: Many Helicobacter pylori strains produce a cytotoxin that induces cytoplasmic vacuolation in various types of eukaryotic cells. In contrast with the marked cell vacuolation that occurs in vitro in response to this cytotoxin, comparatively little epithelial vacuolation has been observed in the gastric mucosa of H pylori infected persons. AIMS: Experiments were performed to determine the susceptibility of human gastric epithelial cells in vitro to H pylori vacuolating cytotoxin activity. METHODS: Human gastric epithelial cells, harvested from upper gastrointestinal endoscopic biopsy specimens, were incubated overnight with broth culture supernatants from either a wild type cytotoxin producing (tox+) H pylori strain or an isogenic mutant strain that lacks cytotoxin activity. RESULTS: Prominent cytoplasmic vacuolation occurred in response to tox+ supernatant, but not supernatant from the isogenic mutant strain. Primary human gastric epithelial cells were significantly more sensitive to H pylori vacuolating cytotoxin activity than were either HeLa or AGS cells. Exposure of human gastric epithelial cells to high concentrations of tox+ supernatant for 48 hours caused lethal cell injury. CONCLUSIONS: These studies indicate that primary human gastric epithelial cells are highly sensitive to H pylori vacuolating cytotoxin activity.

Bacterial Proteins↗

PubMed1996

Conditioned medium from a rat ureteric bud cell line in combination with bFGF induces complete differentiation of isolated metanephric mesenchyme.

Differentiation of metanephric mesenchyme is triggered by an inductive signal(s) from the epithelial ureteric bud. As a result of this induction, most of the metanephric mesenchyme converts into epithelium of a nephron. We have developed and characterized an explant culture system, in which metanephric mesenchyme can grow and completely differentiate in vitro in the absence of an inductive tissue. When separated 13 dpc rat metanephric mesenchymes were cultured in serum-free conditioned medium from a rat ureteric bud cell line (RUB1) in the presence of bFGF and TGFalpha, they were induced to differentiate into nephron epithelia and glomeruli-like structures. The nephric type of differentiation was confirmed by both morphological and molecular criteria and paralleled the developmental changes of nephron differentiation in vivo. Expression patterns of brush-border antigen as well as molecular markers of kidney differentiation Wt1, Lim1, Hgf and c-met, c-ret, Shh, Wnt4, Wnt7b, and Wnt11 were analyzed in explants by whole mount and tissue section in situ hybridization following 1-9 days in culture. The expression of secreted patterning molecules Bmp7 and Wnt7b, but not Shh or Wnt11, were demonstrated by RT-PCR and northern blot hybridization with RNA from the RUB1 cells. Our culture system lends itself to examining the relevance of these and other signaling molecules required for nephron differentiation.

Animals↗

PubMed1996

Hepatocyte growth factor/scatter factor-Met signaling induces proliferation, migration, and morphogenesis of pancreatic oval cells.

Hepatocyte growth factor/scatter factor (HGF/SF) is a pleiotropic effector for cells expressing the Met tyrosine kinase receptor. In this investigation, we show that pancreatic oval cells express Met and exhibit a proliferative response to HGF/SF. Additionally, we found that oval cells treated transiently with this factor become "scattered," whereas those exposed to HGF/ SF for extended periods of time form branching tubular structures. These structures possess true lumens, which are lined by cells with ductal features, including apical microvilli, well-developed intercellular junctions, interdigitation of plasma membranes, and abundant cytoplasmic organelles. Interestingly, these ductal structures are formed by HGF/SF-treated cells cultured on plastic dishes in the absence of exogenous extracellular matrix components. Consistent with their ability to form ductal structures in vitro, we found that pancreatic oval cells form ductal adenocarcinomas in nude mice. This study supports the involvement of HGF/SF-Met signaling in the growth, migration, and morphogenesis of pancreatic oval cells and may have important implications for the expansion and morphogenic differentiation of these cells during developmental, regenerative, and neoplastic growth.

Adenocarcinoma↗

PubMed1995

Mechanism of action of bisimidazoacridones, new drugs with potent, selective activity against colon cancer.

Antitumor bisimidazoacridones are bifunctional DNA binders which have recently been shown to selectively target human colon carcinoma cells in vitro and in vivo and appear to be excellent candidates for clinical development. We have studied the mechanism of action of one bisimidazoacridone, WMC26, which is 1,000-10,000 times more toxic to human colon carcinoma cells (HCT116) than to melanoma cells (SKMEL2) in vitro. Plasmid DNA exposed to WMC26 showed enhanced digestion by DNase I at A-T-rich sites, suggesting alterations in DNA conformation upon drug binding. These results led us to investigate whether WMC26 was selectively toxic due to a specific recognition of DNA bends by repair excinucleases, as has been demonstrated with the DNA bisintercalator, ditercalinium. Both prokaryotic and eukaryotic cells with intact repair capacity were shown to be selectively sensitive to WMC26, strongly indicating that excision repair plays a role in its toxicity. Confocal microscopy studies utilizing fluorescence of the WMC26 chromophore showed compound localization in the perinuclear cytoplasmic area, as had been previously noted for ditercalinium, indicating that cytoplasmic DNA could be the target. This irreversible accumulation of compound was gradually followed by vacuolization of the cytoplasm and cell death. Cell cycle analysis of both lines treated with WMC26 or with ditercalinium showed that, while the latter induced HCT116 growth arrest at G1-G0, WMC26 also blocked the cell cycle at G2-M; SKMEL2 cells did not undergo any changes in cell cycle as a result of either treatment. Our data show that WMC26 is 10-100 times more cytotoxic than ditercalinium in vitro. Like ditercalinium, WMC26 appears to exert its toxicity via cytoplasmic elements, through a mechanism involving excision repair processes. However, its highly selective cytotoxicity may stem from additional undefined targets in sensitive colon cancer cells.

Aminoacridines↗

PubMed1995

Met proto-oncogene product is overexpressed in tumors of p53-deficient mice and tumors of Li-Fraumeni patients.

Inappropriate expression of Met, the receptor for hepatocyte growth factor/scatter factor, has been implicated in sarcomagenesis via an autocrine mechanism. Sarcomas occur at high frequency in individuals with Li-Fraumeni syndrome as well as in p53-deficient mice. Here we show that these tumors express high levels of Met. Moreover, late passage fibroblast cell lines established from p53-deficient animals overexpress Met and can be tumorigenic in athymic nude mice, suggesting that progression occurs in vitro. The tumor explants display increased hepatocyte growth factor/scatter factor expression and Met turnover, indicating that autocrine Met activation contributes to tumor progression. Thus, the loss of wild-type p53 appears to greatly enhance the opportunity for inappropriate Met expression. Loss of p53 function does not by itself cause transformation, but inappropriate Met expression may be an important factor in sarcomagenesis.

3T3 Cells↗

PubMed1995

Human endometrial carcinoma cells release factors which inhibit the growth of normal epithelial cells in culture.

Autocrine and paracrine interactions between cells are important homeostatic mediators in normal tissues. Alterations to growth factor signalling pathways are likely to play a role in multistep carcinogenesis. In this study normal human endometrial epithelial cells (NHEC) after 3 days in culture were treated with serum-free medium conditioned for 24 h by log phase or confluent cultures of established RL95-2, HEC1A, or AN3CA endometrial carcinoma (EC) cell lines. By day 4, NHEC treated with either log phase or confluent conditioned medium (CM) showed a significant decrease (approximately 50-90% of control) in [3H]thymidine ([3H]TdR) incorporation. DNA synthesis was inhibited more by confluent than by log phase CM. By day 7, NHEC treated with CM exhibited fewer colonies per culture, fewer cells per colony, and an increased percentage of single cells. Several growth-regulatory gene products found in the nucleus or at the cell membrane have been shown to be expressed differently in normal and transformed cells. We selected the p53 and c-Ha-ras p21 proteins to further investigate the mechanism of alteration of proliferation in cells treated with carcinoma CM. Thus, by day 7, the percentage of NHEC with nuclear localization of wild type p53 (wt p53) was elevated by treatment with CM. In contrast, CM-treated EC cells continued to proliferate, and showed a decrease in the percentage of cells expressing nuclear wt p53 and an increase in the cytoplasmic expression of c-Ha-ras p21. Our studies show that EC cell lines release factors which inhibit the proliferation of NHEC, thus favoring the proliferation of EC cells.

Adenocarcinoma↗

PubMed1995

Evidence of a functional VIP receptor in cultured human retinal pigment epithelium.

The effect of VIP on the intracellular cyclic AMP of human retinal pigment epithelium cultures has been studied. Functional VIP receptor has been demonstrated in cultures from eyes given by five normal donors (age 16-64) (N-HRPE). But it has been found to be absent from high passage number cultures obtained from a retinitis pigmentosa eye of an 84-year-old patient (RP-HRPE). After 3 min of reaction with 1 x 10(-6) M VIP, the intracellular cyclic AMP level has increased to 5-15-fold over the basal level. The maximal effect of VIP (20-fold over the basal level) has been observed at 1 x 10(-7) M VIP. The half maximal activity of VIP is 3-5 x 10(-8) M. The present study also demonstrates the inducibility of the VIP responsiveness in RP-HRPE cultures after they have been treated with butyrate. Curr. Eye Res. 14: 1009-1014, 1995.

Adolescent↗

PubMed1994

Invasiveness and metastasis of NIH 3T3 cells induced by Met-hepatocyte growth factor/scatter factor autocrine stimulation.

The met protooncogene product, Met, is the tyrosine kinase growth factor receptor for hepatocyte growth factor/scatter factor (HGF/SF). NIH 3T3 cells express HGF/SF endogenously and become tumorigenic in nude mice via an autocrine mechanism when murine Met is expressed ectopically (Metmu cells) or when human Met and human HGF/SF are coexpressed (HMH cells). Here, we show that Metmu and HMH cells are invasive in vitro and display enhanced protease activity necessary for the invasive phenotype. In experimental and spontaneous metastasis assays, Metmu or HMH cells metastasize to the lung, but lower numbers of subcutaneously injected Metmu and HMH cells produced invasive tumors in the heart, diaphragm, salivary gland, and retroperitoneum. It has been reported elsewhere that Met expression increased with tumor passage in athymic nude mice, and these tumor explants show enhanced activity in the metastasis assays. Autocrine-mediated Met-HGF/SF signal transduction in NIH 3T3 mesenchymal cells may provide an important system for understanding the biological process of metastasis.

3T3 Cells↗

PubMed1994

The Met proto-oncogene mesenchymal to epithelial cell conversion.

Coexpression of the human Met receptor and its ligand, hepatocyte growth factor/scatter factor (HGF/SF), in NIH 3T3 fibroblasts causes the cells to become tumorigenic in nude mice. The resultant tumors display lumen-like morphology, contain carcinoma-like focal areas with intercellular junctions resembling desmosomes, and coexpress epithelial (cytokeratin) and mesenchymal (vimentin) cytoskeletal markers. The tumor cells also display enhanced expression of desmosomal and tight-junction proteins. The apparent mesenchymal to epithelial conversion of the tumor cells mimics the conversion that occurs during embryonic kidney development, suggesting that Met-HGF/SF signaling plays a role in this process as well as in tumors that express both epithelial and mesenchymal markers.

3T3 Cells↗

PubMed1993

Met expression and sarcoma tumorigenicity.

The met protooncogene tyrosine kinase receptor (Met) and its ligand, hepatocyte growth factor/scatter factor (HGF/SF), ordinarily constitute a paracrine signaling system in which cells of mesenchymal origin produce the ligand, which binds to the receptor that is predominantly expressed in cells of epithelial origin. However, mouse NIH/3T3 fibroblasts overexpressing Met induce tumor formation in nude mice via an autocrine mechanism (S. Rong et al., Mol. Cell. Biol., 12: 5152-5158, 1992). In this study, we report that human cell lines established from various sarcomas express high levels of activated Met receptor. HGF/SF is also detected in the human sarcoma cell lines but at a reduced level when compared to primary fibroblasts. These properties, high Met expression and reduced ligand levels, are indistinguishable from the properties of NIH/3T3 tumor explant cells overexpressing Met (S. Rong et al., Mol. Cell. Biol., 12: 5152-5158, 1992; S. Rong et al., Cell Growth & Differ., 4: 563-569, 1993). Moreover, paraffin-embedded sections of primary tumors from human osteosarcomas, chondrosarcomas, and leiomyosarcoma stain intensely for Met and/or HGF/SF and display extensive tumor cell heterogeneity with regard to both paracrine and autocrine stimulation. On the basis of these findings, we propose that Met-HGF/SF autocrine signaling may contribute to the tumorigenic process in human sarcomas.

3T3 Cells↗

PubMed1993

Adherence of Helicobacter pylori to cultured human gastric epithelial cells.

Experiments were performed to demonstrate that adherence of Helicobacter pylori to gastric epithelial cells causes alterations in the cell cytoskeleton. H. pylori intimately attached to cultured human gastric epithelial cells on small cellular projections, while there was no intimate association of H. pylori with cultured human esophageal epithelial cells. Fluorescein-conjugated phalloidin staining of gastric epithelial cells showed that H. pylori adherence stimulated actin polymerization; this stimulation was not observed with esophageal cells. Also, this organism's selectivity for gastric mucosa was supported by rare binding of bacteria to esophageal epithelial cells and gastric fibroblasts.

Actins↗

PubMed1993

Tumorigenesis induced by coexpression of human hepatocyte growth factor and the human met protooncogene leads to high levels of expression of the ligand and receptor.

We have previously shown that, in mouse NIH/3T3 cells, it is necessary to coexpress the gene for human hepatocyte growth factor/scatter factor (HGF/SFhu) with its receptor, the human met protooncogene (methu), to activate the transforming activity of the receptor (S. Rong, M. Bodescot, D. Blair, T. Nakamura, K. Mizuno, M. Park, A. Chan, S. Aaronson, and G. F. Vande Woude, Mol. Cell. Biol., 12: 5152-5158, 1992). In this study, we report that exceptionally high levels of the ligand and its receptor are expressed in tumor cell explants after several tumor passages through nude mice. Confluent tumor cells explanted after the second passage in nude mice can express 1700 units/ml/10(6) cells/72 h of scatter activity as determined in Madin-Darby canine kidney cell scatter assays. The motogenic factor produced by these cells is easily purified by heparin-Sepharose chromatography, and the purified factor efficiently induces tyrosine phosphorylation of Methu in YaOvBix2NMA human ovarian carcinoma cells. To account for the unusually high level of HGF/SFhu and Methu expression, we propose that normal levels of Methu receptor are inefficient at transducing the signal(s) required for transformation of mouse cells. Therefore, high levels of Methu receptor are required for tumorigenesis, and corresponding high levels of the ligand are required to induce the signal. Consistent with this model, endogenous mouse scatter factor is not detected in conditioned medium from cells transformed by overexpression of the Metmu receptor.

3T3 Cells↗