Patterns of oncogene activation in human neuroblastoma cells

The authors report 7 cases of intestinal carcinoids. They examine the clinical aspects and describe and discuss both surgical and medical treatment strategies. They also critically evaluate the value of monitoring some oncological markers and their prognostic significance. Each patient underwent an in-depth evaluation of tumour evolution (CAT, ultrasonography, NMR, angiography) and urinary 5HIAA and platelet 5HT were monitored. Surgery took the form of ileal or ileocolic resection, gastric resection, exeresis of the tumour using a transanal route, ligature of the right branch of the hepatic artery afferent to the metastasised lobe of the liver. Five patients were treated using chemotherapy and three, also suffering from carcinoid syndrome, with octreotide. On the basis of their personal experience the authors underline the limited value of the study of 5HT and 5HIAA tumour markers in the diagnosis of small carcinoid tumours. This is compensated by the outstanding role of these markers in the diagnosis of the hepatic and/or lymphoglandular diffusion of the tumour. These markers were not influenced by octreotide treatment in cases in which longastatin was successfully used to combat carcinoid syndrome. Their behaviour allowed useful information to be acquired regarding the tumour evolution following surgery.     

Induction of differentiation in mouse neuroblastoma cells

In the presence of 1--2% dimethyl sulfoxide (DMSO), mouse neuroblastoma cells are induced to differentiate morphologically as well as electrically. In addition, treatment of neurolbastoma cells with 2% DMSO results in a marked increase in the veratridine-activated K+ or Rb+ efflux. At 4% DMSO, neurite outgrowth is completely repressed and electrical activity is poorly developed. However, at this concentration, the cells have a relatively high resting potential which suggested that membrane components determining passive and active permeability properties are not necessarily under coordinated control. Induction of differentiation by 2% DMSO is also accompanied by an increase in a heavier molecular form of acetylcholinesterase sedimenting at 10.5S. The effect of other agents on the growth and differentiation of neuroblastoma cells is also presented.     

Divergent pathways regulate ligand-independent activation of ER alpha in SK-N-BE neuroblastoma and COS-1 renal carcinoma cells

OBJECTIVE: To evaluate our experience with primary carcinomas of the female urethra, by analysing the impact of tumour variables and treatment on overall, disease-specific, local recurrence- and metastasis-free survival. PATIENTS AND METHODS: Between 1958 and 1994, 72 women (median age 60 years, mean 59, range 21-84) with primary urethral carcinoma were identified. They were followed for a median (range) of 85 (0-384) months. The patients were stratified by stage, nodal status, histology, treatment, type of surgery, site of disease, year of diagnosis and smoking habit. RESULTS: In a univariate analysis, stage, nodal status, type of surgery and site of the disease were important factors for survival and recurrence. In a multivariate analysis, primary stage, nodal status and site of disease were independent predictors of survival. CONCLUSION: Current modalities of treatment are ineffective for local control and survival; new treatment strategies are needed for female urethral cancers.     

The morphological differentiation of murine neuroblastoma NIE-115 cells

Characters and kinesis of mouse neuroblastoma (MN) cells morphological differentiation affected by non-serum, hypo- and hyperosmotic media and salt solution were studied. The nature of morphological differentiation was found not to depend on the inductors, while kinesis varies significantly. Morphological differentiation speed is directly proportional to the extent of non-specific action and probability of the following cell death. on the other hand, the number of irreversibly differentiated cells is inversely proportional to the action strength. For the induction of morphological differentiation a minimal nutrition media do not containing serum is sufficient and a common growth media changed once in 3-5 days according to how it acidifies is better to use for the prolonged maintenance of it. Universality of neuronal morphological differentiation is under discussion according tot he data obtained from MN cells and cultured mollusk isolated neurons.     

Gamma radiation-induced differentiation on human neuroblastoma cells in culture

We have studied the effect of gamma radiation on differentiation in neuroblastoma cell lines AF8 and SJ-N-KP. Growth inhibition and morphological and biochemical differentiation have been examined following radiation exposure to 1-10 Gy. Gamma radiation induced marked growth inhibition and morphological differentiation in a dose-and time-dependent manner in both cell lines, and induced biochemical differentiation in AF8 cells.     

Expression of the protein tyrosine phosphatase beta2 gene in mouse erythroleukemia cells induces terminal erythroid differentiation

We have cloned cDNA for protein tyrosine phosphatase beta2, which had been implicated in erythroid differentiation of mouse erythroleukemia cells. Expression of cDNA constructs, in which beta2 cDNA is placed under the control of mouse metallothionein-I promoter, by ZnCl2 converted a significant portion (20 to 38%) of the cells to erythroid-like cells, which is 25-50% of the erythroid differentiation efficiency observed by conventional erythroid-inducing agents. Furthermore, introduction and expression of altered protein tyrosine phosphatase beta2 cDNA constructs designed to produce the enzyme lacking the phosphatase activity inhibited erythroid differentiation by 100-20%, depending upon the concentration of erythroid-inducing agents employed. These results strongly suggest that protein tyrosine phosphatase beta2 is involved in triggering erythroid differentiation in mouse erythroleukemia cells.     

The delta-opioid receptor regulates activity of ryanodine receptors in the human neuroblastoma cell line SK-N-BE

Recent studies have demonstrated that opioid agonists affect the cytosolic Ca2+ concentration ([Ca2+]i) either by regulating plasma membrane Ca(2+)-channel activity or by mobilizing intracellular Ca2+ stores. The present report documents the [Ca2+]i increase induced by opioid agonists in a human neuroblastoma cell line, SK-N-BE, expressing delta-opioid receptors. In the presence, as well as in the absence, of extracellular Ca2+, opioid agonists enhanced significantly [Ca2+]i, whereas carbachol, known to mobilize specifically inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ stores, acted only in the presence of extracellular Ca2+. The opioid-induced increase in [Ca2+]i was not affected by treatments modifying the trimeric Gl, Go, and Gs protein transduction mechanisms or the activity of adenylyl cyclase. The Ca(2+)-ATPase pump-inhibiting sesquiterpene lactone, thapsigargin, did not modify the opioid-induced [Ca2+]i response, whereas it abolished the effects of carbachol. The Ryana speciosa alkaloid, ryanodine, at concentrations known to block endoplasmic reticulum ryanodine receptors, decreased significantly the response to opioids without affecting the effects of carbachol. Thus, our results suggest that, in SK-N-BE cells, delta-opioid receptors mobilize Ca2+ from intracellular ryanodine-sensitive stores and the mechanism involved is independent of Gl/Go Gs proteins and protein kinase A activation.     

Hinokitiol induces differentiation of teratocarcinoma F9 cells

Hinokitiol, a constituent of the wood of Chamaecyparis taiwanensis, was found to induce differentiation of teratocarcinoma F9 cells. When examined by the agar-overlay method, in which expression of plasminogen activator as a differentiation marker protein was detected, this compound exhibited a dose- and time-dependent induction. Induction of differentiation by hinokitiol occurred irreversibly and required its addition for more than 12h. Among its structure-related compounds tested, tropolone and two colchicine-related compounds exerted potent activities comparable to that of hinokitiol. These findings indicate that free tropolone structure in the molecules plays an essential role in inducing differentiation of F9 cells. Hinokitiol showed a strong inhibitory effect of DNA synthesis in very early stages of culture, suggesting that this effect may be responsible for triggering differentiation of F9 cells.     

Evidence that the MYCN oncogene regulates MRP gene expression in neuroblastoma

We have recently shown that expression of the multidrug resistance-associated protein (MRP) gene is a powerful prognostic indicator in childhood neuroblastoma and have suggested that the MYCN oncogene may regulate MRP gene expression. To address this hypothesis, we have examined the relationship between MYCN and MRP gene expression in neuroblastoma tumours and cell lines. MYCN and MRP gene expression were highly correlated in 60 primary untreated tumours both with (P = 0.01) and without MYCN gene amplification (P < 0.0001). Like MRP, high MYCN gene expression was significantly associated with reduced survival, both in the overall study population and in older children without MYCN gene amplification (relative hazards = 13.33 and 19.61, respectively). Inhibition of MYCN, through the introduction of MYCN antisense RNA constructs into human neuroblastoma cells in vitro, resulted in decreased MRP gene expression, determined both by RNA-PCR and Western analysis. The data are consistent with MYCN influencing neuroblastoma outcome by regulating MRP gene expression.     

Neurite differentiation is modulated in neuroblastoma cells engineered for altered acetylcholinesterase expression

Previous observations from several groups suggest that acetylcholinesterase (AChE) may have a role in neural morphogenesis, but not solely by virtue of its ability to hydrolyze acetylcholine. We tested the possibility that AChE influences neurite outgrowth in nonenzymatic ways. With this aim, antisense oligonucleotides were used to decrease AChE levels transiently, and N1E.115 cell lines were engineered for permanently altered AChE protein expression. Cells stably transfected with a sense AChE cDNA construct increased their AChE expression 2.5-fold over the wild type and displayed significantly increased neurite outgrowth. Levels of the differentiation marker, tau, also rose. In contrast, AChE expression in cell lines containing an antisense construct was half of that observed in the wild type. Significant reductions in neurite outgrowth and tau protein accompanied this effect. Overall, these measures correlated statistically with the AChE level (p < 0.01). Furthermore, treatment of AChE-overexpressing cells with a polyclonal antibody against AChE decreased neurite outgrowth by 43%. We conclude that AChE may have a novel, noncholinergic role in neuronal differentiation.     

Leukemia inhibitory factor induces apoptosis and proliferation of human carcinoma cells through different oncogene pathways

The aim of this study was to investigate the effects of a high ambient pressure of He on vascular contraction induced by noradrenaline and to distinguish the effects of ambient pressure per se from those of increased pressure of inert gas. Rings of thoracic aorta were isolated from male Wistar rats. Isometric tension was measured in preparations exposed to 7.1 MPa (absolute pressure) of He. Dose-response curves for noradrenaline and contractions elicited by 120 mM KCl were compared with time-matched experiments performed at atmospheric pressure. The same protocol was also carried out under 7.1 MPa of N2. At the high pressure of He, the contraction elicited by noradrenaline was increased with no change in the response to K(+)-evoked depolarization. The tension developed in response to noradrenaline also increased under 7.1 MPa of N2 but the effects were less marked than during the He experiments. Moreover, the response to KCl was reduced in this circumstance. Hyperbaric conditions enhance the noradrenaline-induced contraction of rat aorta in vitro. This effect probably results from an action of pressure per se on activation of adrenoceptors. However, the hyperbaric-induced increase in vascular smooth muscle contraction is partially counteracted by high pressures of inert gases (N2, but also probably He), which impair the efficiency of the contractile machinery.     

Measles virus infection induces terminal differentiation of human thymic epithelial cells

Measles virus infection induces a profound immunosuppression that may lead to serious secondary infections and mortality. In this report, we show that the human cortical thymic epithelial cell line is highly susceptible to measles virus infection in vitro, resulting in infectious viral particle production and syncytium formation. Measles virus inhibits thymic epithelial cell growth and induces an arrest in the G0/G1 phases of the cell cycle. Moreover, we show that measles virus induces a progressive thymic epithelial cell differentiation process: attached measles virus-infected epithelial cells correspond to an intermediate state of differentiation while floating cells, recovered from cell culture supernatants, are fully differentiated. Measles virus-induced thymic epithelial cell differentiation is characterized by morphological and phenotypic changes. Measles virus-infected attached cells present fusiform and stellate shapes followed by a loss of cell-cell contacts and a shift from low- to high-molecular-weight keratin expression. Measles virus infection induces thymic epithelial cell apoptosis in terminally differentiated cells, revealed by the condensation and degradation of DNA in measles virus-infected floating thymic epithelial cells. Because thymic epithelial cells are required for the generation of immunocompetent T lymphocytes, our results suggest that measles virus-induced terminal differentiation of thymic epithelial cells may contribute to immunosuppression, particularly in children, in whom the thymic microenvironment is of critical importance for the development and maturation of a functional immune system.     

Papillary thyroid carcinoma oncogene (RET/PTC) alters the nuclear envelope and chromatin structure

Current evidence suggests the papillary thyroid carcinoma oncogene (RET/PTC) generates papillary thyroid carcinomas in one genetic step. We tested a resulting prediction that RET/PTC expression in thyroid epithelium should be sufficient to cause the changes in nuclear morphology diagnostic of this tumor. Primary cultures of human thyroid epithelial cells were infected with a RET/PTC retroviral construct. Morphological scoring by two independent cytopathologists shows RET/PTC expression by immunohistochemistry to be highly associated (p < 0.0001) with an irregular nuclear contour and a euchromatic appearance compared with non-expressing cells in the same cultures. The altered nuclear morphology is not due to gene transfer or transformation per se as primary thyroid cell cultures infected with a retroviral H-RAS construct differ from RET/PTC-infected cells by showing round nuclear envelopes and coarser chromatin, as determined by the independent scoring of two cytopathologists (p < 0.0001). In addition, RET/ PTC-transfected cells appear to disperse, whereas RAS-transfected cells grow as discrete colonies. The results provide additional support for the hypothesis that RET/PTC is sufficient to cause papillary thyroid carcinomas. A signaling pathway downstream of RET/ PTC leads to restructuring of the nuclear envelope and chromatin, and the signal does not depend entirely, if at all, on a RAS pathway.     

Long-term exposure to retinoic acid induces the expression of IRK1 channels in HERG channel-endowed neuroblastoma cells

T-cell types are important in maintaining immune homeostasis in the lung and their imbalance may be associated with several diseases. We examined the relationship between bronchoalveolar lavage (BAL) T-cell subset profiles and the clinical course of 46 patients with idiopathic pulmonary fibrosis (IPF). A flow cytometry cell sorter (FACS) was used to analyse the T-cell subsets. Pulmonary function tests (PFT) were performed at baseline and 6-12 months later. Patients were divided into two groups according to their CD4/CD8 ratio: CD4/CD8 >1 (group 1, n=21); and CD4/CD8 <1 (group 2, n=25). A lower percentage of lymphocytes, a higher percentage of CD8/S6F1 cells (cytotoxic T-lymphocytes) and a higher percentage of neutrophils were found in the BAL in group 2 compared to group 1 (11+/-7.5% versus 19+/-13.2%; p=0.024 and 29.8+/-17.6% versus 13.3+/-6.9%; p=0.068, respectively for lymphocytes and cytotoxic T-lymphocytes; and 8+/-11% versus 29+/-27%; p=0.003 for neutrophils). Inversely, in the peripheral blood, the distribution of CD8/S6F1 cells was lower in group 1 than in group 2 (8.3+/-6.9% versus 33.4+/-16.5%; p=0.0048). The patients were followed over a period of 1 yr in order to test whether those findings could determine efficacy of therapy. The baseline transfer factor of the lung for carbon monoxide (TL,CO) capacity in group 1 and group 2 was 59+/-22% and 51+/-21%, respectively (p=0.29), but only in group 1 was the TL,CO capacity improved significantly in response to steroids treatment after 6-12 months. IPF patients with a higher percentage of lymphocytes, a lower percentage of neutrophils, CD4/CD8 >1 and a low percentage of CD8/S6F1 may have a more benign course of disease. These parameters may identify an early stage of reversible disease responsive to therapy. We conclude that these measurements may be a useful tool in monitoring response to treatment in patients with idiopathic pulmonary fibrosis.     

Epidermal growth factor induces terminal differentiation in human epidermoid carcinoma cells

In this study, spermatogenesis in the adult Djungarian hamster is described. Undifferentiated spermatogonia topographically arranged as Asingle (A(s)), Apaired (Apr), and Aaligned (Aal) spermatogonia were observed, as were six generations of differentiating spermatogonia (A1, A2, A3, intermediate, B1, and B2). The differentiating spermatogonia divided at regular intervals during the cycle of the seminiferous epithelium. Mitosis of these cells was observed at the transition from stage IX to stage X (mitosis of A1 into A2 spermatogonia), at the transition from stage XII to stage I, at the transition from stage II to stage III, at the transition from stage IV to stage V, at the end of stage VI, and at approximately the middle of stage VII. Cellular associations in the cycle of the seminiferous epithelium are described. The seminiferous epithelium was divided into 12 stages, based upon the developmental steps in spermiogenesis, and the frequency of these stages was determined. The duration of the cycle of the seminiferous epithelium, determined by [3H]thymidine incorporation, was shown to be 7.90 +/- 0.01 (mean +/- SEM) days.     

Integration of endothelial cells in multicellular spheroids prevents apoptosis and induces differentiation

Single endothelial cells (EC) seeded in suspension culture rapidly undergo apoptosis. Addition of survival factors, such as VEGF and FGF-2, does not prevent apoptosis of suspended EC. However, when cells are allowed to establish cell-cell contacts, they become responsive to the activities of survival factors. These observations have led to the development of a three-dimensional spheroid model of EC differentiation. EC spheroids remodel over time to establish a differentiated surface layer of EC and a center of unorganized EC that subsequently undergo apoptosis. Surface EC become quiescent, establish firm cell-cell contacts, and can be induced to express differentiation antigens (e.g., induction of CD34 expression by VEGF). In contrast, the unorganized center spheroid cells undergo apoptosis if they are not rescued by survival factors. The responsiveness to the survival factor activities of VEGF and FGF-2 was not dependent on cell shape changes since it was retained after cytochalasin D treatment. Taken together, these findings characterize survival factor requirements of unorganized EC and indicate that polarized surface EC differentiate to become independent of exogenous survival factors. Furthermore, they demonstrate that spheroid cell culture systems are useful not just for the study of tumor cells and embryonic stem cells but also for the analysis of differentiated functions of nontransformed cells.