Sensitivity to cisplatin in primary cell lines derived from human glioma correlates with levels of EGR-1 expression

<p>Abstract</p> <p>Background</p> <p>Less than 30% of malignant gliomas respond to adjuvant chemotherapy. Here, we have asked whether variations in the constitutive expression of early-growth response factor 1 (EGR-1) predicted acute cytotoxicity and clonogenic cell death <it>in vitro</it>, induced by six different chemotherapics.</p> <p>Materials and methods</p> <p>Cytotoxicity assays were performed on cells derived from fresh tumor explants of 18 human cases of malignant glioma. In addition to EGR-1, tumor cultures were investigated for genetic alterations and the expression of cancer regulating factors, related to the p53 pathway.</p> <p>Results</p> <p>We found that sensitivity to cisplatin correlates significantly with levels of EGR-1 expression in tumors with wild-type <it>p53/INK4a/p16 </it>status.</p> <p>Conclusion</p> <p>Increased knowledge of the mechanisms regulating EGR-1 expression in wild-type <it>p53/INK4a/p16 </it>cases of glioma may help in the design of new chemotherapeutic strategies for these tumors.</p

Morphology demonstrates similar autophagy alterations in neurodegeneration and brain tumors

Malignant glioma are the most malignant brain tumors. Frequent genetic alterations involve PTEN (Phosphatase homolog deleted on chromosome Tensin and Ten), a lipid phosphatase that after mutation is not able to convert phosphatidylinositol (3,4,5)-triphosphate (PIP3) into phosphatidylinositol (4,5)-bisphosphate (PIP2) and thereby inhibiting AKT, which in turn activates the apoptotic factors, mutations of p53 and retinoblastoma, both responsible of controlling the phase transition G1 / S of cell cycle under physiological conditions and prevent the replication of DNA when the cell is altered. Recently a dramatic uptake of the amino acid glutamine was reported in malignant glioma. This amino acid produces a marked inhibition of the autophagy pathway. Consistently, autophagy is defective in human glioblastoma similar to neurodegeneration. Autophagy is the main clearing system to remove damaged or potentially harmful organelles and misfolded proteins. Autophagy progresses through several stages: (i) the phagophore (ii) the autophagosome (iii) the amphisomes; (iv) the autophagolysosome and (v) the autophagoproteasome. The last stages involves the fusion of amphisome with lysosome which originates autophagolysosome that contains the elements to be removed and lytic enzymes necessary for this degradation process, while the autophagoproteasome derives from the fusion with component of the proteasome. The autophagy machinery can be measured by several specific markers such as beclin1 and LC3, the occurrence of stagnant autophagy vacuoles. In the present study we characterized the consistency and relevance of autophagy failure in glioblastoma by using human cell lines, primary human cell cultures and in vivo human glioblastoma cells implanted in the brain of nude mice. In baseline conditions we observed in cells obtained from surgery of human patients a marked inhibition of the autophagy pathway. This was associated with an increase in autophagy substrates overlapping with neurodegenerative disorders. In keeping with the ongoing autophagy inhibition we found that activation of the autophagy pathway reduced cell proliferation and promoted cell differentiation dose-dependently in vitro while the systemic administration of a powerful autophagy activator reduced the volume of brain tumor in vivo by 96.6%. These data indicate a relevant role of autophagy failure in glioblastoma and suggest potential approaches to contrast tumor growth

Rapamycin dose-dependently promotes differentiation and cell death in an in vitro model of malignant gliomas

Glioblastoma (GB, grade IV astrocytoma) is the most common and lethal brain tumor characterized by increased proliferation and resistance to chemotherapy and radiotherapy. GB infiltrates the brain, always relapses and leads to death within 2 years from diagnosis. At cellular level, relapse and infiltration are correlated with the presence of GB precursors stem-like cells. At molecular level, relapse and infiltration are correlated with upregulation of the mammalian target of rapamycin, mTOR which constantly characterizes malignant gliomas. By definition mTOR is strongly inhibited by rapamycin and rapalogs. Several studies were carried out to evaluate the effects of rapamycin in experimental models of GB. However, it remains controversial whether rapamycin and rapalogs produce cell differentiation or cell death. Therefore, in the present study we used a wide range of doses of rapamycin to address the issue of differential effects obtained by rapamycin in various experimental setting. In particular, rapamycin was administered ranging from 1 nM up to 1microM to assess cell viability in GB cell lines (U87MG). All the experiments were carried out exposing the cells for 24 h to rapamycin. We found that rapamycin produced dose dependently cell death which was significant for doses starting at 100 nM and reaches a plateau at 1 microM (50% cell death). Interestingly, when using rapamycin at doses between 1 nM and 100 nM we described only slight cell death while the prominent effect induced by rapamycin consisted of cell differentiation. In detail, beta 3 tubulin increased, while nestin decreased under rapamycin exposure. These markers were related to phenotypic changes consisting of an increased number and length of cell processes along with the loss of fusiform cell shape. In the process of characterizing the effects of mTOR up-regulation and its pharmacological inhibition we also measured the accumulation of an mTOR-dependent protein such as alpha-synuclein. In fact, mTOR up-regulation is known to inhibit the autophagy pathway which removes alpha-synuclein. Consistently, in U87MG cells we found accumulation of alpha-synuclein which was removed dose dependently by rapamycin exposure. Our data show that inhibiting mTOR with rapamycin is a powerful tool to induce cell differentiation in U87MG cells, whereas cell death significantly occurs only at the highest doses

Inhibition of cell growth by EGR-1 in human primary cultures from malignant glioma

BACKGROUND: The aim of this work was to investigate in vitro the putative role of EGR-1 in the growth of glioma cells. EGR-1 expression was examined during the early passages in vitro of 17 primary cell lines grown from 3 grade III and from 14 grade IV malignant astrocytoma explants. The explanted tumors were genetically characterized at the p53, MDM2 and INK4a/ARF loci, and fibronectin expression and growth characteristics were examined. A recombinant adenovirus overexpressing EGR-1 was tested in the primary cell lines. RESULTS: Low levels of EGR-1 protein were found in all primary cultures examined, with lower values present in grade IV tumors and in cultures carrying wild-type copies of p53 gene. The levels of EGR-1 protein were significantly correlated to the amount of intracellular fibronectin, but only in tumors carrying wild-type copies of the p53 gene (R = 0,78, p = 0.0082). Duplication time, plating efficiency, colony formation in agarose, and contact inhibition were also altered in the p53 mutated tumor cultures compared to those carrying wild-type p53. Growth arrest was achieved in both types of tumor within 1–2 weeks following infection with a recombinant adenovirus overexpressing EGR-1 but not with the control adenovirus. CONCLUSIONS: Suppression of EGR-1 is a common event in gliomas and in most cases this is achieved through down-regulation of gene expression. Expression of EGR-1 by recombinant adenovirus infection almost completely abolishes the growth of tumor cells in vitro, regardless of the mutational status of the p53 gene

The relationship between frequency of carbohydrates intake and dental caries: a cross-sectional study in Italian teenagers

Objective: To propose an approach for investigation of the relationship between the frequency of carbohydrates intake and dental caries in real-life conditions. Design. The frequency of separate eating events (with the exclusion of lunch and dinner) and their sugars and starch content were assessed on the basis of diaries collected for seven consecutive days. The total number of decayed, missing and filled teeth (DMFT) was assessed through clinical examination. The interrelation between DMFT and frequency of eating events was analysed through Pearson correlation coefficient and stepwise forward linear regression analysis. Setting: Italy. Subjects: One hundred and ninety-three students (males and females, mean age 16 years) of a secondary school. Results: Individual DMFT varied between 0 (24% of subjects) and 12, with a mean of 3. Once lunch and dinner were excluded, the mean number of separate eating events was 2.9. A statistically significant relationship was found between DMFT and eating frequency thus defined. Correlations were calculated considering only eating events containing a proportion of sugars or starch higher than different cut-off levels. In a 'high sugars and high stepwise multiple regression model for DMFT, the frequency of starch events' accounted for 8% of the DMFT variance. Overall frequency of separate eating events defined according to their content of both sugars and starch accounted for 18% of the DMFT variance. A stronger correlation was found among males only. Conclusions: The precise characterisation of eating events in terms of their sugars and starch content allows us to explain a consistent percentage of the variability in DMFT

Ependymoma with neuropil-like islands: a case report with diagnostic and histogenetic implications

We describe a case of ependymoma with neuronal differentiation in form of neuropil-like islands. A 6-year-old boy presented at clinical examination for a short history of headaches and vomiting. Brain computed tomography showed a large, partially cystic, parieto-occipital lesion. The tumor was composed by glial fibrillary acidic protein-positive round cells with a perivascular arrangement and scattered neuropil-like islands, showing intense positivity for synaptophysin. Despite radiotherapy, the tumor recurred, showing frank features of anaplasia, but lacking the neuropil-like islands. The histological features of the tumor are discussed in the light of the concept that neuronal differentiation can occur occasionally in gliomas of different lineage without affecting the expected biological behavior

Caveolin-1 expression in diffuse gliomas: correlation with the proliferation index, epidermal growth factor receptor, p53, and 1p/19q status

Caveolin-1 (cav-1) has been proposed as an immunohistochemical marker able to distinguish astroglial from oligodendroglial tumors. In addition, it has been suggested that the reduction of caveolin-1 expression in glioblastoma cells increases their proliferative and invasive potential. Accordingly, the present study investigates caveolin-1 immunoexpression and correlation with the 1p/19q status, histologic grade, proliferation index, epidermal growth factor receptor, and p53 expression in a series of 73 diffuse gliomas. A membranous and cytoplasmic immunolabeling for caveolin-1 was detected in neoplastic cells of 60% of cases. No significant differences in terms of caveolin-1 expression were observed between astrocytomas, oligodendrogliomas, and oligoastrocytomas. In addition, caveolin-1 expression was not correlated with 1p/19q status in oligodendrogliomas and mixed oligoastrocytomas. Caveolin-1 was expressed in most high-grade (World Health Organization III and IV) gliomas. Low caveolin-1 expression correlated with a higher Ki-67 labeling index and the absence of p53 overexpression in glioblastomas. and it was significantly associated with epidermal growth factor receptor overexpression in anaplastic astrocytomas. In conclusion, the present study indicates that caveolin-1 is not useful as diagnostic marker to differentiate grade 11 astrocytomas from oligodendrogliomas. (C) 2009 Elsevier Inc. All rights reserved