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G M Brodeur

Publications and source records attributed to G M Brodeur.

At least 37 records · Page 2Linked to original sources
PubMed2000

Implications of EPHB6, EFNB2, and EFNB3 expressions in human neuroblastoma.

Neuroblastoma (NB) is a common pediatric tumor that exhibits a wide range of biological and clinical heterogeneity. EPH (erythropoietin-producing hepatoma amplified sequence) family receptor tyrosine kinases and ligand ephrins play pivotal roles in neural and cardiovascular development. High-level expression of transcripts encoding EPHB6 receptors (EPHB6) and its ligands ephrin-B2 and ephrin-B3 (EFNB2, EFNB3) is associated with low-stage NB (stages 1, 2, and 4S) and high TrkA expression. In this study, we showed that EFNB2 and TrkA expressions were associated with both tumor stage and age, whereas EPHB6 and EFNB3 expressions were solely associated with tumor stage, suggesting that these genes were expressed in distinct subsets of NB. Kaplan-Meier and Cox regression analyses revealed that high-level expression of EPHB6, EFNB2, and EFNB3 predicted favorable NB outcome (P<0.005), and their expression combined with TrkA expression predicted the disease outcome more accurately than each variable alone (P<0.00005). Interestingly, if any one of the four genes (EPHB6, EFNB2, EFNB3, or TrkA) was expressed at high levels in NB, the patient survival was excellent (>90%). To address whether a good disease outcome of NB was a consequence of high-level expression of a "favorable NB gene," we examined the effect of EPHB6 on NB cell lines. Transfection of EPHB6 cDNA into IMR5 and SY5Y expressing little endogenous EPHB6 resulted in inhibition of their clonogenicity in culture. Furthermore, transfection of EPHB6 suppressed the tumorigenicity of SY5Y in a mouse xenograft model, demonstrating that high-level expressions of favorable NB genes, such as EPHB6, can in fact suppress malignant phenotype of unfavorable NB.

Animals↗
PubMed2000

Resistance to TRAIL-induced apoptosis in primitive neuroectodermal brain tumor cells correlates with a loss of caspase-8 expression.

TNF-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in adult malignant glioma and various other human solid tumor models but not in normal tissues. To characterize the TRAIL death pathway in childhood primitive neuroectodermal brain tumor (PNET), 8 human PNET cell lines were tested for TRAIL-induced apoptosis. TRAIL-sensitivity of the PNET cell lines was correlated with mRNA expression levels of TRAIL, its agonistic (TRAIL-R1, TRAIL-R2) and antagonistic (TRAIL-R3, TRAIL-R4) receptors, cellular FLICE-like inhibitory protein (cFLIP), caspase-3 and caspase-8. Three of 8 PNET cell lines tested were susceptible to TRAIL-induced apoptosis. Sensitivity to TRAIL-induced apoptosis did not correlate with mRNA expression of TRAIL receptors or cFLIP. However, all TRAIL-sensitive PNET cell lines expressed caspase-8 mRNA and protein, while none of the five TRAIL-resistant PNET cell lines expressed caspase-8 protein. Treatment with the methyltransferase inhibitor 5-aza-2'-deoxycytidine restored mRNA expression of caspase-8 and TRAIL-sensitivity in formerly TRAIL-resistant PNET cells, suggesting that gene methylation inhibits caspase-8 transcription in these cells. We conclude, that loss of caspase-8 mRNA is an important mechanism of TRAIL-resistance in PNET cells. Treatment with recombinant soluble TRAIL, possibly in combination with methyltransferase inhibitors, represents a promising therapeutic approach for PNET that deserves further investigation.

Adult↗
PubMed2000

Molecular dissection of TrkA signal transduction pathways mediating differentiation in human neuroblastoma cells.

Activation of the neurotrophin receptor TrkA by its ligand nerve growth factor (NGF) initiates a cascade of signaling events leading to neuronal differentiation in vitro and might play an important role in the differentiation of favorable neuroblastomas (NB) in vivo. To study TrkA signal transduction pathways and their effects on differentiation in NB, we stably expressed wild-type TrkA and five different TrkA mutants in the NGF unresponsive human NB cell line SH-SY5Y. Resulting clones were characterized by TrkA mRNA and protein expression, and by autophosphorylation of the receptor. Introduction of wild-type TrkA restored NGF responsiveness of SH-SY5Y cells, as demonstrated by morphological differentiation, activation of mitogen-activated protein kinases (MAPK) and induction of immediate-early genes. Expression of TrkA in the absence of NGF resulted in growth inhibition of transfectants compared to parental cells, whereas NGF-treatment increased their proliferation rate. Analysis of downstream signal transduction pathways indicated that NGF-induced differentiation was dependent on TrkA kinase activity. Our data suggest that several redundant pathways are present further downstream, but activation of the RAS/MAPK signaling pathway seems to be of major importance for NGF mediated differentiation of NB cells. Our results also show that the signaling effector SH2-B is a substrate of NGF-mediated Trk signaling in NB, whereas it is not activated by NGF in rat pheochromocytoma PC12 cells. This might explain the differences we observed in TrkA signaling between neuroblastoma and PC12 cells. Further insight into TrkA signaling may suggest new options for the treatment of NB.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Identification of a 1-megabase consensus region of deletion at 1p36.3 in primary neuroblastomas.

BACKGROUND: Deletion of the distal short arm of chromosome 1 occurs frequently in neuroblastoma. In addition, neuroblastoma has been described in children with constitutional deletions within 1p36, supporting the existence of one or more neuroblastoma suppressor genes within this region. PROCEDURE: We have pursued a 1p36 tumor suppressor gene identification strategy that has included deletion mapping of 566 primary neuroblastomas and 46 neuroblastoma-derived cell lines, and have determined the parental origin of the deleted 1p homologue in 44 cases to determine whether there is evidence for genomic imprinting within this region. RESULTS AND CONCLUSIONS: We have identified a 1-Mb consensus region of deletion within 1p36.3 defined by primary tumor deletions, constructed a physical map of the region that is being sequenced to completion, and have identified and prioritized candidate genes within this region for further analyses.

Alleles↗
PubMed2000

Localization of a hereditary neuroblastoma predisposition gene to 16p12-p13.

BACKGROUND: Hereditary predisposition to develop neuroblastoma segregates as an autosomal dominant Mendelian trait. PROCEDURE: We have performed linkage analysis on 10 families with neuroblastoma to localize a hereditary neuroblastoma predisposition gene (HNB1). RESULTS: A single genomic interval at chromosome bands 16p12-p13 was consistent with linkage (lod = 3.46), and identification of informative recombinants defined a 25.9-cM critical region between D16S748 and D16S3068. Loss of heterozygosity was identified in 5/12 familial (42%) and 55/259 nonfamilial (21%) neuroblastomas at multiple 16p polymorphic loci. A 12.8-cM smallest region of overlap of deletions was identified within the interval defined by linkage analysis (tel-D16S764-D16S412-cen). CONCLUSIONS: Taken together, these data suggest that HNB1 is located at 16p12-p13 and that inactivation of this gene may contribute to the pathogenesis of nonfamilial neuroblastomas.

Child↗
PubMed2000

Deletion of 11q23 is a frequent event in the evolution of MYCN single-copy high-risk neuroblastomas.

BACKGROUND: Deletions of the long arm of chromosome 11 are frequently identified in human neuroblastomas. PROCEDURE: We screened 394 primary neuroblastomas and 52 tumor-derived cell lines with a panel of 11q and 11p polymorphic markers to determine the frequency of chromosome 11 allelic deletion, and to differentiate partial deletions of chromosome 11q (unb[11q] LOH) from whole chromosome loss. RESULTS: Allelic deletion occurred most frequently at cytogenetic band 11q23 and was detected in 161 primary neuroblastomas (41%) and 18 cell lines (35%). Eighty-seven tumors (22%) had unb[11q] LOH with a heterogeneous distribution of deletion breakpoints. Unb[11q] LOH was highly correlated with age > 1 year at diagnosis (P = 0.008), stage 4 disease (P = 0.001), unfavorable Shimada histopathology (P < 0.001), and assignment to a high-risk therapeutic protocol (P < 0.001), and was inversely correlated with MYCN amplification (P = 0.018). Patients whose tumors showed unb[11q] LOH were less likely to survive (P < 0.001), but there was only a trend towards an independent prognostic influence in multivariate analyses. CONCLUSIONS: Thus, structural rearrangements resulting in unb[11q] LOH commonly occur during the malignant evolution of high-risk neuroblastomas with single-copy MYCN.

Alleles↗
PubMed2000

BIN1 inhibits colony formation and induces apoptosis in neuroblastoma cell lines with MYCN amplification.

BACKGROUND: MYCN amplification and overexpression occurs in 25% of neuroblastomas and independently predicts for poor prognosis disease, an effect thought to be mediated by its role as a transcriptional activator of growth promoting genes. However, in many mammalian cells, deregulated expression of MYC family genes (including MYCN) induces apoptosis. We hypothesized that BIN1, a MYC interacting protein capable of inducing apoptosis, may be an important regulator of MYCN in neuroblastoma. RESULTS: BIN1 expression was found to be reduced in MYCN-amplified cell lines. Further, forced expression of BIN1 markedly reduced colony formation in MYCN-amplified, but not single-copy, cell lines. This effect appeared to be caused by an increase in apoptosis, and was augmented by serum deprivation and concurrent cytotoxic drug therapy in cell culture CONCLUSION: BIN1 inactivation may be necessary for MYCN overexpression to lead to cellular proliferation rather than programmed cell death in neuroblastomas with MYCN amplification.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Expression of neurotrophin receptor TrkA inhibits angiogenesis in neuroblastoma.

BACKGROUND: Mechanisms regulating the expression of angiogenic factors in tumor cells are largely unknown. High expression of the neurotrophin receptor TrkA in neuroblastomas (NB) is associated with favorable prognosis, whereas TrkB is expressed on aggressive, MYCN-amplified NB. PROCEDURE: To investigate the biological effects of TrkA and TrkB expression on angiogenesis in NB, we examined the expression of angiogenic factors in the human NB cell line SY5Y and its TrkA and TrkB transfectants. RESULTS: In comparison to parental SY5Y cells, mRNA and protein levels of angiogenic factors were significantly reduced in SY5Y-TrkA cells, whereas SY5Y-TrkB cells did not demonstrate a significant change. Conditioned medium (CM) of parental SY5Y and SY5Y-TrkB cells induced endothelial cell proliferation, but this effect was completely absent in SY5Y-TrkA cells. TrkA expression also resulted in severely impaired tumorigenicity in a mouse xenograft model, and was associated with reduced angiogenic factor expression and less vascularization of tumors, as determined by immunohistochemistry and an in vivo Matrigel assay.

Cell Division↗
PubMed2000

p75 mediated apoptosis in neuroblastoma cells is inhibited by expression of TrkA.

BACKGROUND: Neurotrophins mediate their effects by binding to members of the Trk family of receptor tyrosine kinases and to the low-affinity nerve growth factor receptor p75. Nerve growth factor (NGF) has been demonstrated to support survival and differentiation of neuroblastoma (NB) cells by activation of the TrkA receptor. The p75 receptor belongs to the tumor necrosis factor (TNF) family of death receptors and has been suggested as a receptor that mediates apoptosis in neuronal and NB cells. PROCEDURE: To investigate the effect of p75 expression in NB, we transfected the p75 cDNA into SH-SY5Y cells, an NB cell line lacking expression of both p75 and TrkA. RESULTS: Cell clones expressing elevated levels of p75 showed a high degree of apoptosis even in 10% serum-supplemented medium. Apoptotic signaling by p75 was ligand-independent and only partly caspase-dependent. The level of apoptosis correlated directly with the expression level of the receptor, indicating that p75 may activate the cell death program directly. However, additional transfection of TrkA into SY5Y-p75 cells resulted in a significantly reduced rate of apoptosis even in the absence of NGF. CONCLUSIONS: Thus, expression of the TrkA receptor itself inhibits p75 mediated apoptosis in NB cells.

Apoptosis↗
PubMed2000

Schwann cell-conditioned medium inhibits angiogenesis in vitro and in vivo.

BACKGROUND: Neuroblastomas are biologically heterogeneous tumors that consist of two main cell populations: neuroblastic/ganglionic cells and Schwann cells. The amount of Schwannian stroma strongly impacts prognosis. Low tumor vascularity, localized stage, and favorable outcome are associated with tumors that are Schwannian stroma-rich/stroma-dominant. PROCEDURE: To investigate if Schwann cells play a role in inhibiting angiogenesis in neuroblastoma tumors, we examined the ability of human Schwann cell-conditioned medium to affect bFGF- and VEGF-induced endothelial cell proliferation and migration, and in vivo angiogenesis. RESULTS: Schwann cell-conditioned medium significantly inhibited bFGF- and VEGF-induced endothelial cell proliferation and migration. This effect appears to be specific for endothelial cells as smooth muscle cell and fibroblast proliferation were not inhibited by this medium. Schwann cell-conditioned medium also inhibited in vivo angiogenesis in rat corneal assays. CONCLUSIONS: Schwann cells produce a potent inhibitor(s) of angiogenesis that may be responsible for the low level of vascularity and more benign clinical behavior of Schwannian stroma-rich/stroma-dominant neuroblastoma tumors. Studies to identify the inhibitor(s) are ongoing.

Cell Division↗
PubMed2000

Resistance to TRAIL-induced apoptosis in neuroblastoma cells correlates with a loss of caspase-8 expression.

BACKGROUND: Disruption of apoptotic pathways may be involved in tumor formation, regression, and treatment resistance of neuroblastoma (NB). TNF-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in cancer cell lines. PROCEDURE: In this study we analyzed the expression and function of TRAIL, its agonistic and antagonistic receptors, and important intracellular signaling elements in 18 NB cell lines. RESULTS: Semiquantitative RT-PCR revealed that TRAIL-R2 and TRAIL-R3 are the main TRAIL-receptors used by NB cells. Sensitivity to TRAIL-induced apoptosis did not correlate with mRNA expression of TRAIL receptors or cFLIP. Surprisingly, caspase-8 and caspase-10 mRNA was detected in only 5 of 18 NB cell lines. Interestingly, only these five NB cell lines were susceptible to TRAIL-induced apoptosis in a time- and dose-dependent manner. CONCLUSIONS: Treatment with 5-aza-2'-deoxycytidine restored mRNA expression of caspase-8 and -10 and TRAIL sensitivity of resistant cell lines, suggesting that gene methylation is involved in caspase inactivation. Since many cytotoxic drugs induce caspase-dependent apoptosis, failure to express caspase-8 and/or caspase-10 might be an important mechanism of resistance to chemotherapy in NB.

Apoptosis↗
PubMed2000

Different effects of TrkA expression in neuroblastoma cell lines with or without MYCN amplification.

BACKGROUND: Expression of the neurotrophin receptor TrkA is associated with a favorable prognosis in neuroblastoma (NB) and promotes growth inhibition and neuronal differentiation. Aggressive, MYCN-amplified NB tumors express little or no TrkA mRNA, suggesting that MYCN overexpression may inhibit TrkA expression. PROCEDURE: To study the interactions of TrkA expression and MYCN amplification in NB, we stably expressed the TrkA receptor in the MYCN single copy cell lines SH-SY5Y and NB69 as well as in the MYCN amplified cell lines CHP134 and IMR5. RESULTS: All four transfected cell lines demonstrated high TrkA expression and similar activation of the TrkA receptor and of mitogen-activated protein kinases as well as induction of immediate-early genes in response to nerve growth factor (NGF). Introduction of TrkA restored NGF responsiveness of SH-SY5Y and NB69 cells, as demonstrated by morphologic differentiation, growth inhibition, and enhanced survival in serum-free medium. However, no morphologic, growth, or survival responses to NGF were detected in MYCN-amplified CHP134 and IMR5 TrkA transfectants. CONCLUSIONS: Thus, transfection of TrkA into MYCN amplified NB cell lines only partly restored the TrkA/NGF signaling pathway, suggesting additional inhibitory effects of MYCN overexpression on TrkA signaling.

Cell Division↗
PubMed2000

Prognostic significance of EPHB6, EFNB2, and EFNB3 expressions in neuroblastoma.

BACKGROUND: EPH family receptor tyrosine kinases and their ligand ephrins play pivotal roles in development. High-level expression of transcripts encoding EPHB6 receptors (EPHB6), its ligands ephrin-B2 and ephrin-B3 (EFNB2, EFNB3) is predictive of favorable disease outcome of neuroblastoma (NB). When combined with TrkA expression, the expression of EPHB6, EFNB2, or EFNB3 predicts more accurately the disease outcome than each of the four variables alone. PROCEDURE: Cox regression and Kaplan-Meier analyses were used to assess the prognostic significance of EPHB6, EFNB2, EFNB3, and TrkA expressions in NB without MYCN amplification. RESULTS: High-level expression of EFNB3 or TrkA predicted favorable NB outcome of NB without MYCN amplification (p < 0.03). As found in the general NB population, EPHB6, EFNB2, or EFNB3 expression in combination with TrkA expression was significantly predictive of the disease outcome of normal MYCN NB (p < 0.01). CONCLUSIONS: EPHB6, EFNB2, and EFNB3 expressions may permit further refinement of the prognostic stratification of NB into favorable and unfavorable groups.

Child↗
PubMed2000

Gene amplification in PNETs/medulloblastomas: mapping of a novel amplified gene within the MYCN amplicon.

OBJECTIVES: The pathological entity of primitive neuroectodermal tumour/medulloblastoma (PNET/MB) comprises a very heterogeneous group of neoplasms on a clinical as well as on a molecular level. We evaluated the importance of DNA amplification in medulloblastomas and other primitive neuroectodermal tumours (PNETs) of the CNS. METHOD: Restriction landmark genomic scanning (RLGS), a method that allows the detection of low level amplification, was used. RLGS provides direct access to DNA sequences circumventing positional cloning efforts. Furthermore, we analysed several samples by CGH. DESIGN: Twenty primary medulloblastomas, five supratentorial PNETs, and five medulloblastoma cell lines were studied. RESULTS: Although our analysis confirms that gene amplification is generally a rare event in childhood PNET/MB, we found a total of 17 DNA fragments that were amplified in seven different tumours. Cloning and sequencing of several of these fragments confirmed the previous finding of MYC amplification in the cell line D341 Med and identified novel DNA sequences amplified in PNET/MB. We describe for the first time amplification of the novel gene, NAG, in a subset of PNET/MB. Despite genomic amplification, NAG was not overexpressed in the tumours studied. We have determined that NAG maps less than 50 kb 5' of DDX1 and approximately 400 kb telomeric of MYCN on chromosome 2p24. CONCLUSION: We found a similar but slightly higher frequency of amplification than previously reported. We present several DNA fragments that may belong to the CpG islands of novel genes amplified in a small subset of PNET/MB. As an example we describe for the first time the amplification of NAG in the MYCN amplicon in PNET/MB.

Blotting, Northern↗
PubMed2000

Favorable biology and outcome of stage IV-S neuroblastoma with supportive care or minimal therapy: a Children's Cancer Group study.

PURPOSE: Stage IV-S neuroblastoma is a metastatic disease associated with spontaneous regression and good survival, but 10% to 20% of infants die from early complications. The purpose of this study was to evaluate outcome and prognostic factors in infants with stage IV-S neuroblastoma treated prospectively with supportive care only or, in symptomatic patients, with low-dose cytotoxic therapy. PATIENTS AND METHODS: Eighty eligible infants were studied for response and survival with supportive care or, for symptomatic patients, cyclophosphamide 5 mg/kg/d for 5 days with or without hepatic radiation of 4.5 Gy over 3 days. Staging was reviewed centrally, and MYCN gene copy number, Shimada histopathologic classification, serum ferritin levels, and bone marrow immunocytology were determined. RESULTS: Stage IV-S and International Neuroblastoma Staging System stage 4S were 98% concordant. MYCN was not amplified in any of the tumors tested (n = 58), and Shimada histopathologic classification was favorable in 96% (n = 68/71). The 5-year event-free survival (EFS) rate for all infants was 86% and the survival rate was 92%. Supportive care was the only treatment provided for 44 (55%) of 80 infants, and their 5-year survival rate was 100%, compared with 81% survival for those requiring cytotoxic therapy for symptoms (P =.005). Five of six deaths were in infants younger than 2 months of age at diagnosis and were due to complications of extensive abdominal involvement with respiratory compromise or disseminated intravascular coagulation. Although age </= 3 months at diagnosis was significant for EFS (P =. 043), it was less significant for survival (P =.077). The only other significant factor predictive for improved survival was favorable Shimada histopathologic classification. Sites of metastatic involvement (liver, skin, or bone marrow) and surgical resection of the primary tumor were not significant for survival. CONCLUSION: This study confirms the favorable biologic features and excellent survival of infants with stage IV-S neuroblastoma with minimal therapy. Infants younger than 2 months old at diagnosis with rapidly progressive abdominal disease may benefit from earlier and more intensive treatment.

Adrenal Gland Neoplasms↗
PubMed2000

Biologic factors determine prognosis in infants with stage IV neuroblastoma: A prospective Children's Cancer Group study.

PURPOSE: A prospective Children's Cancer Group study, CCG-3881, has been completed to determine if a more accurate prediction of prognosis by biologic features can identify subgroups of infants with stage IV neuroblastoma (NBL) who require differing intensities of treatment. PATIENTS AND METHODS: One hundred thirty-four infants were registered from June 1989 to August 1995, with a median follow-up of 47.1 months (range, 0 to 88 months). The biologic factors examined were tumor MYCN copy number, Shimada histopathologic classification, serum ferritin, and bone marrow immunocytology (sensitivity, one tumor cell per 10(5) bone marrow cells). Patients treated on CCG-3881 (n = 116) received four-drug chemotherapy for 9 months (cisplatin, cyclophosphamide, doxorubicin, and etoposide), with surgery and local radiation to residual disease. After January 1991, all subsequent infants with tumor MYCN amplification (n = 18) were transferred after one cycle of therapy to the high-risk CCG-3891 protocol (open January 1991 to April 1996) for more intensive treatment. RESULTS: The 3-year event-free survival (EFS) and overall survival (mean +/- SD) for the 134 infants were 63% +/- 5% and 71% +/- 5%, respectively. Patients whose tumors were without MYCN amplification had a 93% +/- 4% 3-year EFS, whereas those with amplified MYCN had a 10% +/- 7% 3-year EFS (P <. 0001). Each of the other biologic features studied had prognostic significance in univariate analysis but not after stratifying by MYCN copy number. CONCLUSION: Infants less than 1 year of age at diagnosis with stage IV NBL have a much improved outcome compared with children >/= 1 year of age. Nonamplified MYCN tumors identify a group of infants with a 93% +/- 4% EFS, whereas amplified MYCN copy number clearly identifies patients who are unlikely to survive despite intensive chemotherapy.

Biological Factors↗
PubMed2000

Loss of heterozygosity at 1p36 independently predicts for disease progression but not decreased overall survival probability in neuroblastoma patients: a Children's Cancer Group study.

PURPOSE: To determine the independent prognostic significance of 1p36 loss of heterozygosity (LOH) in a representative group of neuroblastoma patients. PATIENTS AND METHODS: Diagnostic tumor specimens from 238 patients registered onto the most recent Children's Cancer Group phase III clinical trials were assayed for LOH with 13 microsatellite polymorphic markers spanning chromosome band 1p36. Allelic status at 1p36 was correlated with other prognostic variables and disease outcome. RESULTS: LOH at 1p36 was detected in 83 (35%) of 238 neuroblastomas. There was a correlation of 1p36 LOH with age at diagnosis greater than 1 year (P = .026), metastatic disease (P<.001), elevated serum ferritin level (P<.001), unfavorable histopathology (P<.001), and MYCN oncogene amplification (P<.001). LOH at 1p36 was associated with decreased event-free survival (EFS) and overall survival (OS) probabilities (P<.0001). For the 180 cases with single-copy MYCN, 1p36 LOH status was highly correlated with decreased EFS (P = .0002) but not OS (P = .1212). Entering 1p36 LOH into a multivariate regression model suggested a trend toward an independent association with decreased EFS (P = .0558) but not with decreased OS (P = .3687). Furthermore, allelic status at 1p36 was the only prognostic variable that was significantly associated with decreased EFS in low-risk neuroblastoma patients (P = .0148). CONCLUSION: LOH at 1p36 is independently associated with decreased EFS, but not OS, in neuroblastoma patients. Determination of 1p36 allelic status may be useful for predicting which neuroblastoma patients with otherwise favorable clinical and biologic features are more likely to have disease progression.

Child↗