Phenotype Of Glioblastoma Multiforme Research Articles

Candidate genes for sensitivity and resistance of human glioblastoma multiforme cell lines to erlotinib

The authors have previously reported that erlotinib, an EGFR tyrosine kinase inhibitor, exerts widely variable antiproliferative effects on 9 human glioblastoma multiforme (GBM) cell lines in vitro and in vivo. These effects were independent of EGFR baseline expression levels, raising the possibility that more complex genetic properties form the molecular basis of the erlotinib-sensitive and erlotinib-resistant GBM phenotypes. The aim of the present study was to determine candidate genes for mediating the cellular response of human GBMs to erlotinib. Complementary RNA obtained in cell lines selected to represent the sensitive, somewhat responsive, and resistant phenotypes were hybridized to CodeLink Human Whole Genome Bioarrays. Expression analysis of 814 prospectively selected genes involved in major proliferation and apoptosis signaling pathways identified 19 genes whose expression significantly correlated with phenotype. Functional annotation analysis revealed that 2 genes (DUSP4 and STAT1) were significantly associated with sensitivity to erlotinib, and 10 genes (CACNG4, FGFR4, HSPA1B, HSPB1, NFATC1, NTRK1, RAC1, SMO, TCF7L1, and TGFB3) were associated with resistance to erlotinib. Moreover, 5 genes (BDNF, CARD6, FOSL1, HSPA9B, and MYC) involved in antiapoptotic pathways were unexpectedly found to be associated with sensitivity. Gene expressions were confirmed by quantitative polymerase chain reaction. Based on an analysis of gene expressions in cell lines with sensitive, somewhat responsive, and resistant phenotypes, the authors propose candidate genes for GBM response to erlotinib. The 10 gene candidates for conferring GBM resistance to erlotinib may represent therapeutic targets for enhancing the efficacy of erlotinib against GBMs. Five additional genes warrant further investigation into their role as putative cotargets of erlotinib.

Yes and PI3K Bind CD95 to Signal Invasion of Glioblastoma

Invasion of surrounding brain tissue by isolated tumor cells represents one of the main obstacles to a curative therapy of glioblastoma multiforme. Here we unravel a mechanism regulating glioma infiltration. Tumor interaction with the surrounding brain tissue induces CD95 Ligand expression. Binding of CD95 Ligand to CD95 on glioblastoma cells recruits the Src family member Yes and the p85 subunit of phosphatidylinositol 3-kinase to CD95, which signal invasion via the glycogen synthase kinase 3-beta pathway and subsequent expression of matrix metalloproteinases. In a murine syngeneic model of intracranial GBM, neutralization of CD95 activity dramatically reduced the number of invading cells. Our results uncover CD95 as an activator of PI3K and, most importantly, as a crucial trigger of basal invasion of glioblastoma in vivo.

Molecular markers of extracellular matrix remodeling in glioblastoma vessels: Microarray study of laser‐captured glioblastoma vessels

Glioblastoma multiforme (GBM) are the most malignant and vascularized brain tumors. The aberrant vascular phenotype of GBM could be exploited for diagnosis or therapeutic targeting. This study identified new molecular markers of GBM vessels, using a combination of laser capture microdissection (LCM) microscopy, RNA amplification, and microarray analyses to compare vessels from nonmalignant human brain and GBM tumors. Forty-two genes were differentially expressed in GBM vessels compared to nonmalignant brain vessels. Validation of differentially expressed genes was performed by literature mining, Q-PCR, and immunohistochemistry. Among the differentially expressed genes, only 64% were previously associated with vessels, angiogenesis, gliomas, and/or cancer. The upregulation of genes encoding secreted extracellular proteins IGFBP7 and SPARC was confirmed by Q-PCR in LCM-captured vessels. Whereas SPARC and IGFBP7 protein were absent in nonmalignant brain vessels, a distinct immunoreactivity patterns were observed in GBM sections whereby SPARC was strongly expressed in perivascular cells adjacent to GBM vessels while GBM endothelial cells were immunostained for IGFBP7. IGFBP7 immunoreactivity was also detected on the abluminal side of GBM vessels deposited between strands of vascular basal lamina. The study discerns unique molecular characteristics of GBM vessels compared with nonmalignant brain vessels that could potentially be used for diagnostic or therapeutic purposes.

Radiation Enhances the Invasive Potential of Primary Glioblastoma Cells via Activation of the Rho Signaling Pathway

Glioblastoma multiforme (GBM) is among the most treatment-refractory of all human tumors. Radiation is effective at prolonging survival of GBM patients; however, the vast majority of GBM patients demonstrate progression at or near the site of original treatment. We have identified primary GBM cell lines that demonstrate increased invasive potential upon radiation exposure. As this represents a novel mechanism by which radiation-treated GBMs can fail therapy, we further investigated the identity of downstream signaling molecules that enhance the invasive phenotype of irradiated GBMs. Matrigel matrices were used to compare the extent of invasion of irradiated vs. non-irradiated GBM cell lines UN3 and GM2. The in vitro invasive potential of these irradiated cells were characterized in the presence of both pharmacologic and dominant negative inhibitors of extracellular matrix and cell signaling molecules including MMP, uPA, IGFR, EGFR, PI-3K, AKT, and Rho kinase. The effect of radiation on the expression of these signaling molecules was determined with Western blot assays. Ultimately, the in vitro tumor invasion results were confirmed using an in vivo 9L GBM model in rats. Using the primary GBM cell lines UN3 and GM2, we found that radiation enhances the invasive potential of these cells via activation of EGFR and IGFR1. Our findings suggest that activation of Rho signaling via PI-3K is required for radiation-induced invasion, although not required for invasion under physiologic conditions. This report clearly demonstrates that radiation-mediated invasion is fundamentally distinct from invasion under normal cellular physiology and identifies potential therapeutic targets to overcome this phenomenon.

Inverse correlation of epidermal growth factor receptor messenger RNA induction and suppression of anchorage-independent growth by OSI-774, an epidermal growth factor receptor tyrosine kinase inhibitor, in glioblastoma multiforme cell lines.

Quantitative and qualitative alterations in the epidermal growth factor receptor (EGFR) commonly occur in many cancers in humans, including malignant gliomas. The aim of the current study was to evaluate molecular and cellular effects of OSI-774, a novel EGFR tyrosine kinase inhibitor, on nine glioblastoma multiforme (GBM) cell lines. The effects of OSI-774 on expression of EGFR messenger (m)RNA and protein, proliferation, anchorage-independent growth, and apoptosis were examined using semiquantitative reverse transcription-polymerase chain reaction, immunocytochemical analysis, Coulter counting, soft agar cloning, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling/fluorescence-activated cell sorting, respectively. All p53 genes were completely and bidirectionally sequenced. Suppression of anchorage-independent growth by OSI-774 was inversely correlated to the induction of EGFR mRNA during relative serum starvation (r = -0.74) and was unrelated to p53 status. Overall, suppression of anchorage-independent growth was a considerably stronger effect of OSI-774 than inhibition of proliferation. The extent of OSI-774-induced apoptosis positively correlated with both proliferation and anchorage-independent growth of GBM cell lines (r = 0.75 and 0.79, respectively). In a single cell line derived from a secondary GBM, exposure to concentrations of greater than or equal to 1 micromol/L resulted in a substantial net cell loss during proliferation studies. The induction of EGFR mRNA may constitute a cellular mechanism to counteract the inhibitory effect of OSI-774 on the anchorage-independent growth of GBM cells. In contrast, no considerable correlation could be established between baseline expression levels of EGFR (both mRNA and protein) in GBM cell lines and their biological response to OSI-774. The OSI-774 induced greater (p53-independent) apoptosis in more malignant GBM phenotypes and may be a promising therapeutic agent against secondary GBM.

Inverse correlation of epidermal growth factor receptor messenger RNA induction and suppression of anchorage-independent growth by OSI-774, an epidermal growth factor receptor tyrosine kinase inhibitor, in glioblastoma multiforme cell lines

Object Quantitative and qualitative alterations in the epidermal growth factor receptor (EGFR) commonly occur in many cancers in humans, including malignant gliomas. The aim of the current study was to evaluate molecular and cellular effects of OSI-774, a novel EGFR tyrosine kinase inhibitor, on nine glioblastoma multiforme (GBM) cell lines. Methods The effects of OSI-774 on expression of EGFR messenger (m)RNA and protein, proliferation, anchorage-independent growth, and apoptosis were examined using semiquantitative reverse transcription–polymerase chain reaction, immunocytochemical analysis, Coulter counting, soft agar cloning, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling/fluorescence-activated cell sorting, respectively. All p53 genes were completely and bidirectionally sequenced. Suppression of anchorage-independent growth by OSI-774 was inversely correlated to the induction of EGFR mRNA during relative serum starvation (r = −0.74) and was unrelated to p53 status. Overall, suppression of anchorage-independent growth was a considerably stronger effect of OSI-774 than inhibition of proliferation. The extent of OSI-774–induced apoptosis positively correlated with both proliferation and anchorage-independent growth of GBM cell lines (r = 0.75 and 0.79, respectively). In a single cell line derived from a secondary GBM, exposure to concentrations of greater than or equal to 1 μmol/L resulted in a substantial net cell loss during proliferation studies. Conclusions The induction of EGFR mRNA may constitute a cellular mechanism to counteract the inhibitory effect of OSI-774 on the anchorage-independent growth of GBM cells. In contrast, no considerable correlation could be established between baseline expression levels of EGFR (both mRNA and protein) in GBM cell lines and their biological response to OSI-774. The OSI-774 induced greater (p53-independent) apoptosis in more malignant GBM phenotypes and may be a promising therapeutic agent against secondary GBM.

FLUDARABINE PHOSPHATE SELECTIVELY INHIBITS GROWTH AND MODIFIES THE ANTIGENIC PHENOTYPE OF HUMAN GLIOBLASTOMA-MULTIFORME CELLS EXPRESSING A MULTIDRUG RESISTANCE PHENOTYPE

Fludarabine phosphate (FLU), the 2-fluro derivative of Ara-A, 9-beta-D-arabino-furanosyl-2-fluoroadenine, has been shown to display both in vitro and in vivo antiproliferative activity toward a variety of murine tumors and human lymphoid malignancies. In the present study, we have determined the effect of FLU, alone and in combination with recombinant human fibroblast interferon (IFN-B), on in vitro growth, gene expression and the antigenic phenotype of human glioblastoma multiforme (GBM) cells displaying a multidrug sensitive and a multidrug resistant (MDR) phenotype. FLU exhibited a marked differential toxicity toward GBM-MDR cells versus the multidrug sensitive GBM parental cell line. Growth of GBM-MDR cells for seven days in 2.5 to 7.5 muM FLU resulted in a dose-dependent reduction or elimination of growth which persisted after removal of this agent. In contrast, recovery from FLU-induced growth suppression was observed in parental multidrug sensitive GBM cells. Acquisition of increased FLU sensitivity in GBM-MDR cells did not appear to result from selection for a subset of sensitive cells or an artifact associated with the DNA-transfection process. This conclusion is supported by the similar pattern of FLU resistance in GBM-18 clones isolated after transfection with a cloned hygromycin resistance gene and selection for resistance to hygromycin. The antiproliferative and toxic effect of FLU was increased in GBM-MDR cells by simultaneous growth in IFN-B and the toxic effect of FLU could be blocked in a dose-dependent manner by the simultaneous addition of deoxycytidine. In contrast, the toxicity of FLU toward GBM-MDR cells was not altered when cells were grown in the presence or absence of colchicine or by the administration of verapamil, which can reverse the MDR phenotype in GBM-MDR cells. The selective toxicity of FLU toward GBM-MDR versus GBM-18 cells was not associated with a consistent differential change in all of the GBM-18 MDR clones in the steady-state mRNA levels of a number of genes, including mdr-1, c-myc, c-fos, JunB, C-jun, proliferative cell nuclear antigen (PCNA), interferon stimulated gene-15 (ISG-15), fibronectin, tenascin, Class I HLA antigen, intercellular adhesion molecule I (ICAM-1), beta-actin or GAPDH. A common change observed in both parental GBM-18 cells and MDR GBM-18 clones exposed to FLU was an increase in the steady-state mRNA levels of deoxycytidine kinase (DCT). Analysis of the antigenic phenotype in GBM and GBM-MDR cells by fluorescence activated cell sorter (FACS) analysis using specific monoclonal antibodies (MoAbs) recognizing ICAM-1, Class I HLA antigen and a high molecular weight-melanoma associated antigen (HMW-MAA) indicated that FLU was generally more active as an immunomodulating agent in MDR versus non-MDR GBM cells. Although the mechanism underlying the differential effect of FLU toward GBM-MDR versus GBM cells is not presently known, the present findings indicate that the growth inhibitory and immunomodulatory effects of FLU are enhanced in cells expressing an MDR phenotype resulting from overexpression of a cell membrane localized 170,000 M(r) glycoprotein (P-glycoprotein).