Primitive Neuroectodermal Tumor Cell Lines Research Articles

Comparison of Aggressive Pattern in Ewing Sarcoma and Primitive Neuroectodermal Tumor Cell Lines

Comparison of Aggressive Pattern in Ewing Sarcoma and Primitive Neuroectodermal Tumor Cell Lines

Abstract A19: Epigenetic drug combination induces genome-wide demethylation and altered gene expression in neuro-ectodermal tumor-derived cell lines

Abstract Background: Epigenetic alterations are inherent to cancer cells, and epigenetic drugs are currently primarily used to treat hematological malignancies. Pediatric neuro-ectodermal tumors originate from neural crest cells and also exhibit epigenetic alterations involving e.g. apoptotic pathways, which suggests that also these tumors may be sensitive to epigenetic drugs. This notion prompted us to assess molecular and functional effects of low dosage epigenetic drugs in neuro-ectodermal tumor-derived cell lines of pediatric origin. Results: In 17 neuroblastoma (NBL) and 5 peripheral primitive neuro-ectodermal tumor (PNET) cell lines a combination treatment of 5-aza-2′-deoxycytidine (DAC) and Trichostatin A (TSA) at nanomolar dosages was found to reduce proliferation and to induce wide-spread DNA demethylation, accompanied by major changes in gene expression profiles. Approximately half of the genes that were significantly up-regulated upon treatment exhibited a significant demethylation in their promoter regions. In the NBL cell lines, almost every cellular pathway (193/200) investigated showed expression alterations after treatment, especially a marked up-regulation of genes in the p53 pathway. The combination treatment also resulted in up-regulation of known epigenetically regulated genes such as X-chromosomal genes, tissue-specific genes and a limited number of imprinted genes, as well as known tumor suppressor genes and oncogenes. Conclusions: Nanomolar dosages of epigenetic drugs have a dramatic impact on the genomes of neuro-ectodermal tumor derived cell lines, including alterations in DNA methylation and concomitant alterations in gene expression. Citation Format: Max M. van Noesel, Floor A M Duijkers, Renee X. de Menezes, Dominique J. P. M. Stumpel, Pieter Admiraal, Rob Pieters, Jules P P Meijerink. Epigenetic drug combination induces genome-wide demethylation and altered gene expression in neuro-ectodermal tumor-derived cell lines. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A19.

Expulsion of micronuclei containing amplified genes contributes to a decrease in double minute chromosomes from malignant tumor cells

Double minute chromosomes (DMs) are a hallmark of gene amplification. The relationship between the formation of DMs and the amplification of DM-carried genes remains to be clarified. The human colorectal cancer cell line NCI-H716 and human malignant primitive neuroectodermal tumor cell line SK-PN-DW are known to contain many DMs. To examine the amplification of DM-carried genes in tumor cells, we performed Affymetrix SNP Array 6.0 analyses and verified the regions of amplification in NCI-H716 and SK-PN-DW tumor cells. We identified the amplification regions and the DM-carried genes that were amplified and overexpressed in tumor cells. Using RNA interference, we downregulated seven DM-carried genes, (NDUFB9, MTSS1, NSMCE2, TRIB1, FAM84B, MYC and FGFR2) individually and then investigated the formation of DMs, the amplification of the DM-carried genes, DNA damage and the physiological function of these genes. We found that suppressing the expression of DM-carried genes led to a decrease in the number of DMs and reduced the amplification of the DM-carried genes through the micronuclei expulsion of DMs from the tumor cells. We further detected an increase in the number of γH2AX foci in the knockdown cells, which provides a strong link between DNA damage and the loss of DMs. In addition, the loss of DMs and the reduced amplification and expression of the DM-carried genes resulted in a decrease in cell proliferation and invasion ability.

Epigenetic drug combination induces genome-wide demethylation and altered gene expression in neuro-ectodermal tumor-derived cell lines

Epigenetic alterations are inherent to cancer cells, and epigenetic drugs are currently primarily used to treat hematological malignancies. Pediatric neuro-ectodermal tumors originate from neural crest cells and also exhibit epigenetic alterations involving e.g. apoptotic pathways, which suggests that these tumors may also be sensitive to epigenetic drugs. This notion prompted us to assess molecular and functional effects of low dosage epigenetic drugs in neuro-ectodermal tumor-derived cell lines of pediatric origin. In 17 neuroblastoma (NBL) and 5 peripheral primitive neuro-ectodermal tumor (PNET) cell lines a combination treatment of 5-aza-2'-deoxycytidine (DAC) and Trichostatin A (TSA) at nanomolar dosages was found to reduce proliferation and to induce wide-spread DNA demethylation, accompanied by major changes in gene expression profiles. Approximately half of the genes that were significantly up-regulated upon treatment exhibited a significant demethylation in their promoter regions. In the NBL cell lines, almost every cellular pathway (193/200) investigated showed expression alterations after treatment, especially a marked up-regulation of genes in the p53 pathway. The combination treatment also resulted in up-regulation of known epigenetically regulated genes such as X-chromosomal genes, tissue-specific genes and a limited number of imprinted genes, as well as known tumor suppressor genes and oncogenes. Nanomolar dosages of epigenetic drugs have a dramatic impact on the genomes of neuro-ectodermal tumor-derived cell lines, including alterations in DNA methylation and concomitant alterations in gene expression.

Increased unsaturation of lipids in cytoplasmic lipid droplets in DAOY cancer cells in response to cisplatin treatment

Increases in 1H nuclear magnetic resonance spectroscopy (NMR) visible lipids are a well-documented sign of treatment response in cancers. Lipids in cytoplasmic lipid droplets (LDs) are the main contributors to the NMR lipid signals. Two human primitive neuroectodermal tumour cell lines with different sensitivities to cisplatin treatment were studied. Increases in NMR visible saturated and unsaturated lipids in cisplatin treated DAOY cells were associated with the accumulation of LDs prior to DNA fragmentation due to apoptosis. An increase in unsaturated fatty acids (UFAs) was detected in isolated LDs from DAOY cells, in contrast to a slight decrease in UFAs in lipid extracts from whole cells. Oleic acid and linoleic acid were identified as the accumulating UFAs in LDs by heteronuclear single quantum coherence spectroscopy (HSQC). 1H NMR lipids in non-responding PFSK-1 cells were unchanged by exposure to 10 μM cisplatin. These findings support the potential of NMR detectable UFAs to serve as a non-invasive marker of tumour cell response to treatment.

Characteristics of human Ewing/PNET sarcoma models

Ewing/PNET (peripheral neuroepithelioma) tumors are rare aggressive bone sarcomas occurring in young people. Rare-disease clinical trials can require global collaborations and many years. In vivo models that as accurately as possible reflect the clinical disease are helpful in selecting therapeutics with the most promise of positive clinical impact. Human Ewing/PNET sarcoma cell lines developed over the past 45 years are described. Several of these have undergone genetic analysis and have been confirmed to be those of Ewing/PNET sarcoma. The A673 Ewing sarcoma line has proven to be particularly useful in understanding the biology of this disease in the mouse. The chromosomal translocation producing the EWS/FLI1 fusion transcript characterizes clinical Ewing sarcoma. Cell lines that express this genetic profile are confirmed to be those of Ewing sarcoma. The A673 Ewing sarcoma line grows in culture and as a xenograft in immunodeficient mice. The A673 model has been used to study Ewing sarcoma angiogenesis and response to antiangiogenic agents. Many Ewing sarcoma clinical specimens express the cell surface protein endosialin. Several Ewing sarcoma cell lines, including the A673 line, also express cell surface endosialin when grown as subcutaneous tumor nodules and as disseminated disease; thus the A673 is a useful model for the study of endosialin biology and endosialin-directed therapies. With the advent of tools that allow characterization of clinical disease to facilitate optimal treatment, it becomes imperative, especially for rare tumors, to develop preclinical models reflecting disease subsets. Ewing PNET sarcomas are a rare disease where models are available.

Expression of podoplanin in human bone and bone tumors: New marker of osteogenic and chondrogenic bone tumors

Podoplanin is known as a lymphatic marker because its expression is detected in lymphatic but not vascular endothelium. Podoplanin is also expressed in several normal tissues including osteocytes or osteoblasts. A systematic examination of the podoplanin expression was conducted in normal skeletal tissues and some bone tumor cell lines, and the diagnostic value determined in primary bone tumors. Podoplanin mRNA was expressed at a high level in bone marrow tissue and cartilage, and was upregulated with differentiation to osteoblasts in bone marrow cells. Strong podoplanin expression was seen in osteocytes, chondrocytes, and osteoblasts on immunohistochemistry. Podoplanin mRNA was expressed at a high level in several osteosarcoma and chondrosarcoma cell lines, whereas podoplanin was expressed at a low level in a Ewing's/primitive neuroectodermal tumor cell line. In the clinical samples, osteosarcomas (22/26) expressed podoplanin at various levels. In small cell osteosarcomas (2/2), podoplanin was expressed strongly, although the tissue samples included few diagnostic osteoids. Chondrosarcomas (10/10) expressed podoplanin strongly, and chondroblastomas (5/5) expressed podoplanin moderately, while podoplanin was absent or expressed at low levels in Ewing's sarcomas (0/5), chordomas (0/6) and giant cell tumors of bone (1/7). Therefore, podoplanin may be a sensitive immunohistochemical marker of osteogenic and chondrogenic bone tumors.

Antitumor effect in medulloblastoma cells by gefitinib: Ectopic HER2 overexpression enhances gefitinib effects in vivo

The effects of the epidermal growth factor receptor (EGFR) inhibitor gefitinib on cell growth and signaling were evaluated in three medulloblastoma (MB) cell lines (D283, D341, Daoy), one supratentorial primitive neuroectodermal tumor cell line (PFSK), and four MB primary cultures. Cell lines showed diverse expression of EGFR and human epidermal receptor 2 (HER2), with high levels of constitutively activated HER2 in the HER2-overexpressing D341 and D283 cells. Gefitinib sensitivity varied across lines and was not related to expression of HER receptors or receptor baseline activation. Gefitinib induced G(0)/G(1) arrest in all lines, whereas apoptosis was dose-dependently induced only in D283 and D341 cells. The molecular response to gefitinib was investigated in Daoy and D341 lines, which showed a higher (half-maximal inhibitory concentration [IC(50)], 3.8 microM) and lower (IC(50), 6.6 microM) sensitivity to the agent, respectively. Gefitinib inhibited constitutive and EGF-triggered EGFR phosphorylation in both lines but was ineffective in constitutive activation of HER2 in D341 cells. Phosphorylated AKT inhibition paralleled that of phosphorylated EGFR, suggesting the presence of an autocrine gefitinib-sensitive EGFR/AKT pathway. On the whole, EGF-dependent signaling was less responsive to ligand stimulation and gefitinib inhibition in D341 cells, which correlated with the lower sensitivity to gefitinib's antiproliferative effect of this line. In vivo, the growth of D341 and Daoy xenografts treated with gefitinib at 150 mg/kg per day was inhibited by approximately 50%. Ectopically overexpressed HER2 in Daoy cells significantly increased sensitivity to gefitinib's antitumor effects in vivo (tumor volume inhibition = 78%). Our data indicate that gefitinib might be a molecularly targeted agent for the treatment of MB.

Interaction between ERK and GSK3β Mediates Basic Fibroblast Growth Factor-induced Apoptosis in SK-N-MC Neuroblastoma Cells

The Ewing's sarcoma family of tumors (ESFT) includes Ewing's sarcoma (ES), Askin's tumor of the chest wall, and peripheral primitive neuroectodermal tumor. Basic fibroblast growth factor (FGF2) suppresses the growth of ESFT cells and causes their apoptosis. The underlying mechanism is unclear. Using a human peripheral primitive neuroectodermal tumor cell line, SK-N-MC, we demonstrated FGF2 stimulated phosphorylation of ERK1 and ERK2 (pERK1/2) and GSK3beta (pGSK3beta(Tyr-216)), all of which were primarily retained in the cytoplasm. FGF2 promoted the association between ERK and pGSK3beta(Tyr-216). Inhibitors for GSK3beta (TDZD and LiCl) and ERK (PD98059) protected cells from FGF2-induced apoptosis. On the other hand, inhibitors of GSK3beta, but not PD98059 decreased ERK/pGSK3beta(Tyr-216) association and caused a nuclear translocation of pERK1/2. Similarly, expression of a kinase-deficient (K85R) GSK3beta or GSK3beta-small interfering RNA inhibited FGF2-regulated ERK/pGSK3beta(Tyr-216) association and translocated pERK to the nucleus. Both K85R GSK3beta and small interfering RNA offered protection against FGF2-induced cell death. In contrast, overexpression of wild-type GSK3beta sensitized cells to FGF2 cytotoxicity. Hydrogen peroxide and ethanol enhanced FGF2-stimulated pGSK3beta(Tyr-216), ERK/pGSK3beta(Tyr-216) association, and cytoplasmic retention of pERK1/2. As a result, they potentiated FGF2-induced cell death. Taken together, our results suggested that FGF2-induced accumulation of pERK1/2 in the cytoplasm is toxic for SK-N-MC cells. The formation of an ERK.GSK3beta complex retained pERK1/2 in the cytoplasm. In contrast, disruption of the ERK.GSK3beta complex resulted in nuclear translocation of pERK1/2 and offered protection.

RASSF1A promoter is highly methylated in primitive neuroectodermal tumors of the central nervous system

Although cancer is rare in children, primary brain tumors constitute the most frequent location of solid tumors in childhood. Primitive neuroectodermal tumors (PNET) of the central nervous system can be divided into infratentorial PNET or medulloblastoma (MB), and supratentorial (sPNET) tumors. Although MB and sPNET are histologically similar, clinical evolution differs, sPNET being more aggressive than MB. Some studies have suggested that MB and sPNET present different molecular genetic aberrations. The RASSF1A (Ras Association Domain Family Protein 1) gene, located at 3p21.3, is highly methylated in multiple primary tumor samples, including neuroblastoma. In order to define whether there are genetic differences in the methylation frequency of RASSF1A between MB and sPNET, we analyzed 32 PNET paraffin-embedded samples (23 MB and 9 sPNET) by methylation specific polymerase chain reaction (MSP). We also analyzed RASSF1A expression by reverse transcription polymerase chain reaction in five PNET cell lines. All PNET cell lines showed lack of RASSF1A expression that was correlated with RASSF1A promoter hypermethylation. RASSF1A methylation was detected in 19 of 21 MB cases (91%) and in five of six sPNET samples (83%). Although the methylation frequency found in MB was slightly higher than in sPNET, no statistical differences were found for the RASSF1A hypermethylation frequency (P > 0.05) presented at MB versus sPNET. Therefore, the inactivation of the RASSF1A gene seems to be an important step in the tumorigenesis of PNET of the central nervous sytem. More studies should be performed in order to determine genetic differences between MB and sPNET.

High promoter hypermethylation frequency of p14/ARF in supratentorial PNET but not in medulloblastoma

Medulloblastoma (MB) is the most common primitive neuroectodermal tumour (PNET) of the central nervous system. Although supratentorial PNET (sPNET) and MB are histologically similar, their clinical behaviour differs, sPNET being more aggressive than MB. The aim of this study was to determine whether sPNET and MB are genetically different entities. We investigated 32 PNET primary tumour samples (23 MB and nine sPNET) and four PNET cell lines, for the presence of CDKN2A homozygous deletions at exon 1-alpha of p16/INK4 and exon 1-beta of p14/ARF, and promoter hypermethylation of both genes. No homozygous deletion of either p16/INK4 or p14/ARF was demonstrated in any of the PNET primary tumour samples. Methylation of p16/INK4 was found in one of six sPNET and in one of 23 MB, while p14/ARF methylation was observed in three of six sPNET and in three of 21 MB. No methylation of p16/INK4 or p14/ARF was found in any of the PNET cell lines analysed. The three MB cell lines did not show p16/INK4 expression, and only the MB Daoy cell line (homozygously deleted at CDKN2A) presented loss of p14/ARF expression. Our results in this limited series of central PNET show that p14/ARF is frequently involved in PNET carcinogenesis, with a higher frequency, but not statistically significant, for sPNET than for MB.

PTEN and DMBT1 homozygous deletion and expression in medulloblastomas and supratentorial primitive neuroectodermal tumors

Medulloblastoma, which accounts for 20-25% of all childhood brain tumors, is defined as a primitive neuroectodermal tumor (PNET) located in the cerebellum. Supratentorial PNET are less frequent than medulloblastoma. But their clinical outcome is worse than in medulloblastomas. Chromosome 10q contains at least 2 tumor suppressor genes that might play a role in brain tumor development: PTEN and DMBT1. The aim of this study was to compare the status of homozygous deletion and expression of PTEN and DMBT1 genes in PNET primary tumor samples and cell lines. Homozygous deletions of PTEN and DMBT1 were studied in 32 paraffin-embedded PNET samples (23 medulloblastomas and 9 supratentorial PNET) and in 7 PNET cell lines, by differential PCR and by FISH. PTEN homozygous losses were demonstrated in 7 medulloblastomas (32%) and in no supratentorial PNET, while homozygous deletions of DMBT1 appeared in 1 supratentorial PNET (20%) and in 7 medulloblastomas (33%). No homozygous deletion of PTEN or DMBT1 was detected in any of the PNET cell lines either by differential PCR or by FISH. Expression study of the 2 genes was performed in the 7 PNET cell lines by RT-PCR. One PNET cell line lacked PTEN and DMBT1 expression, while 2 medulloblastoma cell lines did not express DMBT1. Our results add some positive data to the hypothesis that supratentorial PNETs and medulloblastomas might be genetically different.

Fenretinide cytotoxicity for Ewing's sarcoma and primitive neuroectodermal tumor cell lines is decreased by hypoxia and synergistically enhanced by ceramide modulators.

Patients with disseminated Ewing's family of tumors (ESFT) often experience drug-resistant relapse. We hypothesize that targeting minimal residual disease with the cytotoxic retinoid N-(4-hydroxyphenyl) retinamide (4-HPR; fenretinide) may decrease relapse. We determined the following: (a) 4-HPR cytotoxicity against 12 ESFT cell lines in vitro; (b) whether 4-HPR increased ceramide species (saturated and desaturated ceramides); (c) whether physiological hypoxia (2% O(2)) affected cytotoxicity, mitochondrial membrane potential (DeltaPsi(m)) change, or ceramide species or reactive oxygen species levels; (d) whether cytotoxicity was enhanced by l-threo-dihydrosphingosine (safingol); (e) whether physiological hypoxia increased acid ceramidase (AC) expression; and (f) the effect of the AC inhibitor N-oleoyl-ethanolamine (NOE) on cytotoxicity and ceramide species. Ceramide species were quantified by thin-layer chromatography and scintillography. Cytotoxicity was measured by a fluorescence-based assay using digital imaging microscopy (DIMSCAN). Gene expression profiling was performed by oligonucleotide array analysis. We observed, in 12 cell lines tested in normoxia (20% O(2)), that the mean 4-HPR LC(99) (the drug concentration lethal to 99% of cells) = 6.1 +/- 5.4 microm (range, 1.7-21.8 microm); safingol (1-3 microm) synergistically increased 4-HPR cytotoxicity and reduced the mean 4-HPR LC(99) to 3.2 +/- 1.7 microm (range, 2.0-8.0 microm; combination index < 1). 4-HPR increased ceramide species in the three cell lines tested (up to 9-fold; P < 0.05). Hypoxia (2% O(2)) reduced ceramide species increase, DeltaPsi(m) loss, reactive oxygen species increase (P < 0.05), and 4-HPR cytotoxicity (P = 0.05; 4-HPR LC(99), 19.7 +/- 23.9 microm; range, 2.3-91.4). However, hypoxia affected 4-HPR + safingol cytotoxicity to a lesser extent (P = 0.04; 4-HPR LC(99), 4.9 +/- 2.3 microm; range, 2.0-8.2). Hypoxia increased AC RNA expression; the AC inhibitor NOE enhanced 4-HPR-induced ceramide species increase and cytotoxicity. The antioxidant N-acetyl-l-cysteine somewhat reduced 4-HPR cytotoxicity but did not affect ceramide species increase. We conclude the following: (a) 4-HPR was active against ESFT cell lines in vitro at concentrations achievable clinically, but activity was decreased in hypoxia; and (b) combining 4-HPR with ceramide modulators synergized 4-HPR cytotoxicity in normoxia and hypoxia.

Telomere maintenance in childhood primitive neuroectodermal brain tumors

Primitive neuroectodermal tumors (PNETs), including medulloblastoma (PNET/MB) and supratentorial PNET (sPNET), are the most common malignant brain tumors of childhood. The stabilization of telomere lengths by telomerase activation is an important step in carcinogenesis and cell immortalization. Epigallocatechin gallate (EGCG), the major polyphenol in green tea, is a telomerase inhibitor with antiproliferative and anticarcinogenic effects against different types of cancer. In this study, we used real-time reverse transcriptase-polymerase chain reaction to measure the mRNA expression of the human telomerase reverse transcriptase (hTERT) in 50 primary PNET samples (43 PNET/MB, 7 sPNET), 14 normal human brain samples, and 6 human PNET cell lines. Compared to normal human cerebellum, 38/50 (76%) primary PNET samples had >or= 5-fold upregulated hTERT mRNA expression. We then examined PNET cell lines for telomerase activity using a quantitative telomeric repeat amplification protocol (TRAP), and for telomere length using terminal restriction fragment analysis. While a positive correlation between hTERT mRNA expression and telomerase activity was detected in PNET cell lines, no correlation was found between telomerase activity and telomere length. Treatment of PNET cell lines with EGCG resulted in a dose-dependent inhibition of telomerase activity at micromolar levels. Although EGCG displayed strong proliferation inhibitory effects against TRAP-positive PNET cell lines, it had no significant effect against TRAP-negative D425 cells. These results provide evidence for a possible role of telomerase in the pathogenesis of most PNETs and indicate that subsets of PNETs maintain telomere length by alternative mechanisms. Inhibition of telomerase function represents a novel experimental therapeutic strategy in childhood PNETs that warrants further investigation.

Specific alternative HOX11 transcripts are expressed in paediatric neural tumours and T-cell acute lymphoblastic leukaemia

HOX11 is a proto-oncogene, which is silent in normal mature T-cells, while being aberrantly activated in T-cell acute lymphoblastic leukaemia (T-ALL) by translocations t(10;14)(q24;q11) or t(7;10)(q35;q24). Although many oncogenes are expressed in alternative forms in cancer, thus far, only one form of the human HOX11 transcript has been reported. We describe here the identification of three alternative transcripts of the HOX11 proto-oncogene, expressed in primary T-ALL specimens. Using rapid amplification of cDNA ends (RACE) and targeted RT-PCR, we have sequenced 23 individual cDNA clones characterising these novel transcripts. Northern hybridisation identified particular novel exons expressed in T-ALL, which are not expressed in normal T-cells. To date, aberrant expression of HOX11 has only been associated with leukaemia. Our survey of a range of neuroblastoma and primitive neuroectodermal tumour (PNET) cell lines demonstrated the expression of these novel HOX11 transcripts in tumours of neural origin, while their expression was not detected in normal brain tissues. Strikingly, the dominant transcript in these neural tumour cell lines is more than 1 kb larger than the dominant transcript in T-ALL. These observations, combined with sequence data from several EST clones derived from medulloblastoma cDNA libraries, support a new hypothesis that HOX11 may also function as a neural oncogene or brain tumour marker.

Genomic structure and mutational analysis of the human KIF1Bα gene located at 1p36.2 in neuroblastoma

KIF1a is a member of the kinesin superfamily proteins that are microtubule-dependent molecular motors involved in important intracellular functions such as organelle transport and cell division. We previously determined the structure of the human KIF1Bbeta gene, which was found to be a homologue of the murine Kif1bbeta, and demonstrated that the human KIF1Bbeta is a causative gene of Charcot-Marie-Tooth disease type 2A although we did not prove that it is a tumor suppressor gene of neuroblastoma. Here, we identified another isoform of the human KIF1B gene, KIF1Balpha. The KIF1Balpha and KIF1Bbeta are alternative splicing products of the KIF1B gene located on 1p36.2. The KIF1Balpha is distinct from KIF1Bbeta in the C-terminal cargo-binding domain; however, they have the same N-terminal motor domain. We found that the transcript of approximately 7.8 kb of KIF1Balpha was expressed in several tissues, especially in skeletal muscle, by Northern blot analysis. To determine whether this gene is one of the candidate tumor suppressor genes for neuroblastoma (NB) or other pediatric solid tumors, we performed mutational screening of KIF1Balpha in 25 NB, 9 rhabdomyosarcoma, 12 Ewing sarcoma and 24 other pediatric solid tumor cell lines. Using RT-PCR single-strand conformation polymorphism analysis and direct sequencing we detected a missense mutation (M807I) in 1 NB cell line (SK-N-SH), 3 silent mutations in 2 NB cell lines and 1 primitive neuroectodermal tumor cell line, respectively. RT-PCR analysis revealed that KIF1Balpha was obviously expressed in almost all of the tumor cell lines examined except NB-1. Furthermore, real-time quantitative RT-PCR showed that there was no significant difference in KIF1Balpha expression between 14 early-stage (stage I and II) and 14 advanced-stage (stage III and IV) NB fresh tumor specimens. These results suggest that KIF1Ba in addition to KIF1Bbeta may not be a candidate tumor suppressor gene for NB.

Ewing’s sarcoma and peripheral primitive neuroectodermal tumor cells produce large quantities of bioactive tumor necrosis factor-α (tnf-α) after radiation exposure

Purpose In the present study, we examined human Ewing’s sarcoma (ES) and peripheral primitive neuroectodermal tumor (pPNET) cell lines that are able to produce TNF-α as part of the response to irradiation. Radiation-induced tumor cell production of TNF-α may enhance irradiation efficacy and improve the effect of local tumor irradiation. On the other hand, radiation-induced tumor cell production of TNF-α may adversely affect the normal tissue. Methods and materials Twelve different ES/pPNET cell lines were investigated in vitro and (after establishment as tumor xenografts in athymic nude mice) in vivo for their TNF-α mRNA expression (real-time quantitative reverse transcriptase polymerase chain reaction) and TNF-α protein production ( in vitro: enhanced amplified sensitivity immunoassay; in vivo: immunohistochemistry) after exposure to different irradiation doses (2, 5, 10, 20, 30, or 40 Gy) and after different time intervals (1, 3, 6, 12, 24, 48, or 72 h after irradiation). The bioactivity of the TNF-α protein was evaluated in chromogenic cytotoxicity and neutralization assays. Results Nine out of 12 ES/pPNET cell lines express constitutively significant quantities of bioactive TNF-α in vitro. ES/pPNET cells originating from primary tumors secreted higher TNF-α levels than cells derived from metastatic lesions. In 5 of the 9 TNF-α–producing cell lines, TNF-α mRNA and protein levels were upregulated after irradiation exposure in a time- and dose-dependent manner. After establishment of the ES/pPNET cell lines in athymic nude mice, the radiation-induced TNF-α release could be demonstrated also in the xenograft tumors in vivo (analogous to the in vitro experiments). Using the same methods for quantitative analysis, it was determined that the TNF-α expression of the radiation-responsive tumor cells was up to 2000-fold higher compared to the maximal radiation-induced TNF-α release in normal lung tissue measured during the pneumonic phase. Conclusion Certain ES/pPNET cell lines produce extremely large quantities of bioactive TNF-α after radiation exposure in a time- and dose-dependent manner. Radiation-induced TNF-α production of tumor cells may be of paramount importance in respect to not only tumor behavior, but also to potential damage to normal tissue and the clinical status of the host.

Overexpression of cyclooxygenase-2 (COX-2) in human primitive neuroectodermal tumors: effect of celecoxib and rofecoxib

In this study the role of cyclooxygenase-2 (COX-2) in primitive neuroectodermal tumor (PNET) the most malignant brain tumors of childhood was investigated. COX-2 expression in human brain tumor biopsy samples (seven/seven) was about 6–8-fold higher than normal brain tissue and several PNET cell lines also express COX-2. The effect of selective COX-2 inhibitors, celecoxib and rofecoxib on the growth of two PNET cell lines (DAOY and PFSK) was determined. Celecoxib was more potent than rofecoxib in suppressing cell growth. Growth inhibition by celecoxib and rofecoxib was independent of Bcl-2 expression. Celecoxib suppressed the expression of Akt and activated the caspase-3 in DAOY and PFSK, whereas rofecoxib did not have such an effect.

Loss of caspase-8 mRNA expression is common in childhood primitive neuroectodermal brain tumour/medulloblastoma

Upon binding of tumour necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL), the agonistic TRAIL receptors DR4 and DR5 activate caspase-8 leading to apoptosis. In primitive neuroectodermal brain tumour (PNET) cell lines, TRAIL-induced apoptosis was recently shown to correlate with caspase-8 mRNA expression (Grotzer MA, Eggert A, Zuzak TJ, et al. Oncogene 2000, 19, 4604–4610). In this study, we analysed the expression of the TRAIL death pathway in 27 primary PNET/medulloblastoma. As shown by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR), all PNET/medulloblastoma evaluated expressed DR5, the adapter protein FADD and caspase-3, but only 48% expressed caspase-8. The mRNA expression of caspase-8 was significantly lower in primary PNET/medulloblastoma compared with normal brain samples. PCR revealed >75% methylation of the caspase-8 promoter region in three of seven PNET cell lines and in 55% of the primary PNET/medulloblastoma evaluated. In the PNET cell lines, the methylation status correlated with the caspase-8 mRNA expression. We conclude that loss of caspase-8 gene expression is common in PNET/medulloblastoma suggesting that suppression of death receptor induced apoptosis may play an important role in the pathogenesis of this common childhood brain tumour.

Expression of matrix metalloproteinases and the metastasis-associated gene S100A4 in human neuroblastoma and primitive neuroectodermal tumor cells

Background: Matrix metalloproteinases (MMPs) and their endogenous inhibitors (tissue inhibitors of MMPs; TIMPs) have been shown to correlate with in vitro invasiveness and clinical outcome in several adult malignancies. The importance of MMP and TIMP expression in neuroblastoma (NB) and primitive neuroectodermal tumors (PNET) is incompletly understood. The aim of the current study was to relate in vitro invasion of NB and PNET cell lines with MMP and TIMP expression and evaluate the effect of a synthetic MMP inhibitor. Furthermore, S100A4 levels were determined because recent reports have suggested a possible associaton between MMPs, TIMPs, and the metastasis-associated gene S100A4.Methods: Expression of MMPs, TIMPs, and S100A4 was evaluated at both mRNA and protein levels in 2 human NB and 2 PNET cell lines. In vitro invasion and effects of the synthetic MMP inhibitor Marimastat were assessed in the Transwell chamber assay. Results: The most invasive cells expressed the highest levels of MMPs and S100A4. Marimastat reduced invasion by 30%. Conclusions: In vitro invasion correlated with MMP and S100A4 expression. The fact that Marimastat reduced in vitro invasion is encouraging for further studies on a possible therapeutic application for proteinase inhibitors. J Pediatr Surg 36:1040-1044. Copyright © 2001 by W.B. Saunders Company.