Prevention of EGF-modulated adhesion of tumor cells to matrix proteins by specific EGF receptor inhibition

The authors compared patients in a geropsychiatric unit who showed marked cognitive decline during hospitalization with those who did not. Patients who declined in cognitive function were older, were more likely to have a diagnosis of dementia, and were more anergic on admission. These patients were also discharged to more restrictive living environments. The subgroup of demented patients who declined in cognitive function were also older and improved less on anxiety and depression.     

Hypertension mediated by the activation of the rat brain 5-hydroxytryptamine receptor sites

5-Hydroxytryptamine (5-HT) administered intraventricularly (i.vent.) in rats produced hypertension without considerable changes in heart rate. After transsection of the spinal cord or i.vent. administration of methysergide, 5-HT failed to produce the pressor effect. Thus, the hypertension results from the activation of 5-HT receptor sites of the rat brain.     

Overexpressed human CD44s promotes lung colonization during micrometastasis of murine fibrosarcoma cells: facilitated retention in the lung vasculature

Normally nonmetastatic murine sis-transformed BALB/c 3T3 cells, transfected with human CD44s gene (hCD44s), acquire spontaneous metastatic capacity to the lung. The mechanism(s) of this facilitated micrometastasis was analyzed in an experimental metastasis model. Human CD44s overexpression promoted the earliest stages severalfold (initial implantation and subsequent stabilization of tumor cells) but was irrelevant for later stages (subsequent outgrowth) of lung experimental micrometastasis. By injecting mixed populations of parental (nonmetastatic) and CD44s-transfected cells, it was shown that cell-cell adhesion between tumor and parental cells was not promoted by hCD44s but that promotion of cell-cell adhesion to lung endothelium or specifically between transfected cells (via hyaluronan) are likely mechanisms. Results obtained with hCD44s-negative primary tumor cells and hCD44s-positive or -negative variants of lung micrometastatic cells (after s.c. injection of transfectants) confirmed the importance of CD44s overexpression for early but not late stages of experimental lung metastasis. Therefore, CD44s represents a metastasis-facilitating molecule that is irrelevant for primary tumor outgrowth but that promotes micrometastasis to the lungs at the very earliest stages.     

Neutrophil adhesion to histamine stimulated cultured endothelial cells is primarily mediated via activation of phospholipase C and nitric oxide synthase isozymes

OBJECTIVE AND DESIGN: In order to understand the underlying mechanism of histamine stimulated inflammatory responses, histamine receptor subtypes and signal transduction pathways by which histamine mediates the stimulation of neutrophil adhesion to endothelial cells has been studied in vitro. MATERIAL: Human neutrophils and human umbilical vein endothelial cells. TREATMENT: Confluent human endothelial cell layer were incubated with histamine (1 mM), H1, H2 or H3 receptor agonists: fluorophenylhistamine (10 microM), amthamine (10 microm), methylhistamine (10 microM), respectively. Ten minutes prior to histamine (1 mM) stimulation H1, H2 or H3 receptor antagonists (dimethindene, 100 microM; famotidine, 1OO microM, thioperamid 100 microM, respectively) were added. Histamine stimulated signal transduction pathways were inhibited by adding phospholipase C inhibitor 2-nitro-4-carboxyphenyl N,N-diphenylcarbamat (200 microM), adenylate cyclase inhibitor 9-(2 tetrahydrofuryl)adenine (80 microM), nitric oxide synthase isozymes inhibitor S-ethylisothiourea (1 microM) or guanylate cyclase inhibitor (LY 83583; 10 microM). Neutrophil adhesion was monitored at 30, 60, 90, 120, 150, 180 and 210 min. METHODS: Neutrophil adhesion to endothelial cells was quantified by analysing alkaline phosphatase activity. RESULTS: Histamine stimulation of endothelial cells resulted in a biphasic time and concentration dependent pattern of neutrophil adhesion. This pattern of neutrophil adhesion was mimicked by stimulation of endothelial cells with H1 or H2 agonists. Stimulation of endothelial cells with an H3 agonist had no effect on neutrophil binding. Inhibition of phospholipase C (PLC), nitric oxide synthase isozymes (NOS) or guanylate cyclase (GC) resulted in a significant decrease of neutrophil binding to histamine or agonist stimulated endothelial cells. An increase of neutrophil binding to unstimulated or to agonist stimulated endothelial cells was observed during inhibition of adenylate cyclase. CONCLUSIONS: Our results suggest that histamine stimulated neutrophil adhesion is due to H1 and H2 receptor mediated activation of PLC, NOS and GC. Increase of cAMP concentration seems to mediate an inhibitory effect on PMN adhesion to endothelial cells.     

A dual mechanism for impairment of GABAA receptor activity by NMDA receptor activation in rat cerebellum granule cells

It has been estimated that over three million workers in the USA are potentially exposed to silica or other mineral dusts. Results of epidemiological studies evaluating whether silica or glass fibers increase lung cancer risk to the exposed workers are inconclusive. Detection of DNA damage in cells exposed to genotoxic agents is being used to assess the carcinogenic potential of environmental agents. The alkaline (pH > 13) single cell gel/comet (SCG) assay was used to determine and compare DNA damage in cultured Chinese hamster lung fibroblasts (V79 cells) and human embryonic lung fibroblasts (Hel 299 cells) exposed to crystalline silica (Min-U-Sil 5), amorphous silica (Spherisorb), carbon black, and glass fibers (AAA-10). V79 or Hel 299 cells were exposed to these mineral dusts for 3 h at various concentrations. Min-U-Sil 5 and AAA-10, at almost all concentrations tested, caused a significant increase in DNA migration measured as tail length in both V79 and Hel 299 exposed cells. However, the increase was much higher in V79 then in Hel 299 cells for Min-U-Sil 5. Tail length was also increased relative to controls after amorphous silica treatment, but not to the same extent as that induced by crystalline silica. Exposure to carbon black did not induce DNA migration at any of the concentrations tested. These results indicate that silica and glass fibers, but not carbon black, can induce DNA damage in mammalian cells, and that crystalline silica has a higher DNA-damaging activity than amorphous silica. For glass fibers, induction of DNA damage in both V79 and Hel 299 cells was observed even at a concentration 10 times lower than silica and the response was similar in both cell lines. These results suggest that the SCG/comet assay is useful for the detection of DNA damage caused by occupationally related dusts/particles.     

Alcohol-induced expression of the CD14 endotoxin receptor protein in rat Kupffer cells

Gut-derived endotoxins (lipopolysaccharide, LPS) are believed to contribute to alcohol-induced liver disease (ALD) by stimulating Kupffer cells, the resident liver macrophages, to release proinflammatory cytokines. This activation is largely mediated by CD14, a high-affinity membrane-anchored receptor for LPS. We observed, by chemiluminescence-enhanced detection, an increase in immunoreactive CD14 protein in Kupffer cells isolated from rats treated with ethanol for 2 weeks. Immunocytofluorescence experiments confirmed that this increase was confined to the membranes of Kupffer cells from the alcohol-treated rats. The increase was regulated pretranslationally: a 3-fold elevation (p < 0.01) in the hepatic level of CD14 mRNA was observed. The marked increase in CD14 expression suggests a new mechanism by which alcohol increases the LPS-mediated cytokine signaling by the liver macrophages, thus promoting the interaction between alcohol and endotoxins in the development of liver damage.     

Heparin-binding properties of selenium-containing thioredoxin reductase from HeLa cells and human lung adenocarcinoma cells

Mammalian selenocysteine-containing thioredoxin reductase (TR) isolated from HeLa cells and from human lung adenocarcinoma cells was separated into two major enzyme species by heparin-agarose affinity chromatography. The low-affinity enzyme forms that were not retained on heparin agarose showed strong crossreactivity in immunoblot assays with anti-rat liver TR polyclonal antibodies, whereas the high-affinity enzyme forms that were retained by the heparin column were not detected. Both low and high heparin-affinity enzyme forms contained FAD, were indistinguishable on SDS/PAGE analysis, and exhibited similar catalytic activities in the NADPH-dependent DTNB [5,5'-dithiobis(2-nitrobenzoate)] assay. The C-terminal amino acid sequences of 75Se-labeled tryptic peptides from lung adenocarcinoma low- and high heparin-affinity enzyme forms were identical to the predicted C-terminal sequence of human placental TR. These two determined peptide sequences were -Ser-Gly-Ala-Ser-Ile-Leu-Gln-Ala-Gly-Cys-Secys-(Gly). Occurrence of the Se-carboxymethyl derivative of radioactive selenocysteine in the position corresponding to TGA in the gene confirmed that UGA is translated as selenocysteine. The presence of cysteine followed by a reactive selenocysteine residue in this C-terminal region of the protein may explain some of the unusual properties of the mammalian TRs.     

Transforming growth factor-alpha promotes mammary tumorigenesis through selective survival and growth of secretory epithelial cells

Transforming growth factor (TGF)-alpha stimulates the growth and development of mammary epithelial cells and is implicated in the pathogenesis of human breast cancer. In this report we evaluate the consequences of overexpressing TGF-alpha in the mammary gland of transgenic mice and examine associated cellular mechanisms. When operating on a FVB/N genetic background (line MT100), TGF-alpha induced the stochastic development of mammary adenomas and adenocarcinomas f secretory epithelial origin in 64% of multiparous females. In contrast, tumors were exceedingly rare in virgin MT100 females, MT100 males, and multiparous FVB/N females. In MT100 females multiple foci of hyperplastic secretory lesions preceded the development of frank tumors; these initial lesions appeared during the involution period after the first lactation. Serial transplantation of these hyperplasias indicated an absence of proliferative immortality. Nevertheless, they gave rise to tumors at a low frequency and after a prolonged latency in virgin hosts; in multiparous hosts, tumors developed earlier and at a high incidence. The TGF-alpha transgene was highly expressed in hyperplasias and tumors but not in virgin and nonlesion-bearing tissue, suggesting that TGF-alpha overexpression provides a selective growth advantage. TGF-alpha also induced at lactation a 6.4-fold increase in DNA synthesis in MT100 epithelial cells, many of which were binucleated. MT100 mammary tissue experienced an obvious delay in involution, resulting in the postlactational survival of a significant population of unregressed secretory epithelial cells. In contrast, another line of transgenic mice on a CD-1 genetic background (MT42), in which TGF-alpha overexpression induced liver but not mammary tumors, failed to demonstrate postlactational epithelial cell survival. These data show that TGF-alpha promotes mammary tumorigenesis in multiparous MT100 mice by stimulating secretory epithelial cell proliferation during lactation and prolonging survival during involution. These points support the notion that TGF-alpha can act as a mitogen and also as a differentiation factor in mammary epithelium.     

Isolation and characterization of rat primary lung cells

Lung cell culture may be useful as an in vitro alternative to study the susceptibility of the lung to various toxic agents. Lungs from female Wistar rats were enzymatically digested by recirculating perfusion through the pulmonary artery with a sequence of solutions containing deoxyribonuclease, chymopapain, pronase, collagenase, and elastase. Lung tissue was microdissected and resuspended and the cells obtained were washed by centrifugation. By this isolation method, 2 x 10(8) cells per rat lung were obtained with an average viability of 97%. Lung cells cultured in medium containing antibiotics and serum maintained a viability of > 70% for 5 d. Rat primary lung cells were exposed to various toxic agents and their viability was assessed by formazan production capacity after 18 h of incubation. Compared to rat and mouse hepatocyte cultures (EC50 = 5.8 mM), rat primary lung cells were much more susceptible to hydrogen peroxide (EC50 = 0.6 mM). All cell types were equally sensitive to the more potent toxicant tert-butylhydroperoxide (EC50 = 0.1 mM). Paraquat was more toxic to lung cells (EC50 = 0.03 mM) than to rat (EC50 = 2.8 mM) and mouse (EC50 = 0.2 mM) hepatocytes. In contrast, rat lung cells were less sensitive to sodium nitroprusside (EC50 = 2.6 mM) compared to rat (EC50 = 0.2 mM) and mouse (EC50 = 0.03 mM) hepatocytes. Nitrofurantoin and menadione (at EC50 = 0.04 mM and 0.006 mM, respectively) were more toxic to rat lung and liver cells than to murine hepatocytes (EC50 = 0.2 mM and 0.04 mM, respectively). Our findings demonstrate the applicability of this rat primary lung cell culture for studying the effects of lung toxicants.     

Entry of alphaherpesviruses mediated by poliovirus receptor-related protein 1 and poliovirus receptor

A human member of the immunoglobulin superfamily was shown to mediate entry of several alphaherpesviruses, including herpes simplex viruses (HSV) 1 and 2, porcine pseudorabies virus (PRV), and bovine herpesvirus 1 (BHV-1). This membrane glycoprotein is poliovirus receptor-related protein 1 (Prr1), designated here as HveC. Incubation of HSV-1 with a secreted form of HveC inhibited subsequent infection of a variety of cell lines, suggesting that HveC interacts directly with the virus. Poliovirus receptor (Pvr) itself mediated entry of PRV and BHV-1 but not of the HSV strains tested. HveC was expressed in human cells of epithelial and neuronal origin; it is the prime candidate for the coreceptor that allows both HSV-1 and HSV-2 to infect epithelial cells on mucosal surfaces and spread to cells of the nervous system.     

Effect of tumour necrosis factor-alpha on proliferation, activation and protein synthesis of rat hepatic stellate cells

BACKGROUND/AIMS: Hepatic stellate cells represent the principal matrix-synthesising cells of damaged liver and are targets of a number of cytokines currently under investigation. The study analyses the effects of tumour necrosis factor-alpha and interferon-gamma on proliferation, "activation" and protein synthesis of hepatic stellate cells. METHODS: Primary cultures of hepatic stellate cells were exposed to tumour necrosis factor-alpha and interferon-gamma. Cell proliferation was studied by 3H-thymidine and bromo-deoxy-uridine incorporation. Protein synthesis was analysed using immunoprecipitation, Western- and Northern blotting techniques. RESULTS: Proliferation of hepatic stellate cells was reduced by tumor necrosis factor-alpha and interferon-gamma, while "activation" of hepatic stellate cells as assessed by expression of smooth muscle alpha-actin and of TGF-beta/activin type I receptor was induced by tumour necrosis factor-alpha but downregulated by interferon-gamma. Tumour necrosis factor-alpha increased the synthesis of distinct extracellular matrix proteins, particularly of fibronectin and tenascin, but decreased collagen type III expression. In contrast, interferon-gamma reduced the synthesis of all connective tissue proteins tested. Among the protease inhibitors, interferon-gamma induced C1-esterase inhibitor synthesis, while tumour necrosis factor-alpha stimulated plasminogen activator inhibitor type 1 production. CONCLUSIONS: Tumour necrosis factor-alpha and interferon-gamma decrease proliferation of hepatic stellate cells, while "activation" of hepatic stellate cells and synthesis of proteins involved in matrix metabolism are regulated in a differential, cytokine-specific manner, suggesting that both cytokines play an important role in liver repair.     

Nerve growth factor promotes activation of the alpha, beta and gamma isoforms of protein kinase B in PC12 pheochromocytoma cells

The activation of phosphatidylinositol (PtdIns) 3-kinase is considered to be a key event occurring after stimulation of cells with growth factors. The proto-oncogenic protein kinase B (PKB; also known as RAC protein kinase or Akt) has recently been shown to be a downstream target of PtdIns 3-kinase and may be involved in cell survival. We therefore asked whether stimulation of neuronal cells with nerve growth factor (NGF), on which certain types of neurons are dependent for survival, causes activation of PKB. Stimulation of serum-starved PC12 rat pheochromocytoma cells with NGF caused an increase of up to 14-fold in PKB activity. This activation was detected within 1 min of stimulation and occurred at NGF concentrations that are consistent with TrkA-mediated signaling. PKB activation was accompanied by a decrease in electrophoretic mobility of the kinase, which is characteristic of phosphorylation. Both PKB activation and mobility changes were prevented by wortmannin, indicating the upstream involvement of PtdIns 3-kinase in these events. Analyses employing isoform-specific antibodies for immunoprecipitation suggested that all three isoforms of PKB (alpha, beta and gamma) are activated in response to NGF. G-protein-coupled-receptor agonists, lysophosphatidic acid (lyso-PtdH) and thrombin, which induce rapid neurite retraction, neither stimulated PKB activity, nor affected NGF-induced or insulin-induced kinase activation. Wortmannin treatment did not prevent neurite retraction induced by lyso-PtdH or thrombin. These data suggest that PtdIns 3-kinase and PKB are not involved in cytoskeletal changes mediated by the small GTPase Rho.     

Role for Bcl-xL in the regulation of apoptosis by EGF and TGF beta 1 in c-myc overexpressing mammary epithelial cells

We previously showed that TGF alpha synergizes with c-myc in mammary tumorigenesis through inhibition of Myc-induced apoptosis. We therefore examined the effects of growth factors on apoptosis induction in several cell lines from MMTV-myc mammary tumors. When EGF was withdrawn or TGF beta 1 was added, cells became apoptotic after 15 h (by ELISA and morphology). Northern and Western analysis revealed high levels of Bax and p53, and low or undetectable levels of Bcl-2 and Bcl-xS under all treatment condition. In contrast, Bcl-xL expression was highest in the presence of EGF or TGF alpha, with a significant reduction upon removal of EGF or exposure to TGF beta. In mouse mammary tumors, the relative Bcl-xL/Bax ratio was higher in TGF alpha/Myc double transgenics than in Myc single transgenics, in agreement with the in vitro data. Our results suggest a role for Bcl-xL in the regulation of apoptosis by EGF and TGF beta in mammary epithelial cells.