应用Alamar blue法检测TRAIL对神经母细胞瘤细胞的细胞毒作用

目的:神经母细胞瘤(neuroblastoma,NB)细胞对TRAIL的杀伤效应不敏感与其Caspase 8表达的缺失有关.本研究中我们用γ-干扰素(Interferon γ,IFNγ)诱导NB细胞株CHP212和SH-SY5Y(SY5Y)Caspase 8表达,观察表达Caspase 8的NB细胞是否对TRAIL的细胞毒作用敏感,从而探讨Alamar blue法用于测定细胞体外增殖及细胞毒实验的可行性.方法:应用Western-blot方法检测IFNγ作用前后NB细胞Caspase 8蛋白的表达;应用Alamar blue还原率测定IFNγ、TRAIL、IFNγ+TRAIL、Caspase 8抑制剂+TRAIL对NB细胞生长的影响.结果:对TRAIL敏感的CHP212细胞表达Caspase 8且经IFNγ处理后Caspase 8表达水平逐步增加;对TRAIL不敏感的SY5Y细胞不表达Caspase 8,IFNγ作用后其Caspase 8蛋白表达明显增加.CHP212细胞还原率随TRAIL浓度的增加和作用时间的延长而降低,各实验组与对照组比较有显著差别(P<0.05).SY5Y对TRAIL耐药,而IFNγ+TRAIL组的SY5Y细胞还原率与单独用IFNγ或TRAIL组比较,显著降低(P<0.01).结论:Alamar blue还原率测定结果表明CHP212细胞对TRAIL的细胞毒作用敏感,存在时间和剂量依赖性;经IFNγ诱导表达Caspase 8的SYSY细胞对TRAIL的细胞毒作用亦敏感.本研究结果证实Alamar blue法是一种简便、敏感、安全的方法,可用于体外测定细胞增殖及化疗药的细胞毒作用.     

IFNγ对TRAIL诱导神经母细胞瘤细胞凋亡的调节作用

目的:探讨IFNγ(γ-干扰素)对TRAIL(肿瘤坏死因子相关凋亡诱导配体)诱导神经母细胞瘤细胞株SH-SY5Y凋亡的影响及其发生机制.方法:应用RT-PCR方法检测IFNγ作用前后SY5Y细胞Caspase 8的表达;应用四甲基偶氮唑蓝(MTT)比色法及流式细胞仪(FCM)检测IFNγ、TRAIL、IFNγ TRAIL及IFNγ Caspase 8抑制剂 TRAIL对SY5Y细胞生长及凋亡的影响;应用透射电镜对凋亡细胞进行形态学观察.结果:SY5Y细胞不表达Caspase 8,IFNγ作用48h后的SY5Y细胞Caspase 8表达明显增加;SY5Y细胞对TRAIL不敏感,经IFNγ诱导表达Caspase 8的SY5Y细胞对TRAIL敏感,且与TRAIL浓度有关,Caspase 8抑制剂可明显降低TRAIL对表达Caspase 8的SY5Y细胞的杀伤作用;透射电镜可见到典型的细胞凋亡特征.结论:IFNγ可逆转SY5Y细胞对TRAIL诱导凋亡的耐受,其发生机制可能是IFNγ通过上调SY5Y细胞Caspase 8表达而实现的.     

维拉帕米与长春新碱联合应用对神经母细胞瘤细胞的作用

探讨维拉帕米能否提高神经母细胞瘤细胞对长春新碱的敏感性。方法对２３例神经母细胞瘤组织进行分离获得肿瘤细胞,采用原代细胞培养及ＭＴＴ检测药敏的方法,测定了长春新碱及其与维拉帕米合用时对肿瘤细胞的抑制率。结论化疗对长春新碱敏感性降低的神经母细胞瘤患者,化疗时加用维拉帕米可望提高化疗结果。     

IFN-γ联合阿霉素或依托泊苷增强TRAIL对神经母细胞瘤细胞的诱导凋亡作用

  目的  探讨半胱-天冬氨酸蛋白酶8(Caspase 8)和死亡受体(DR)在肿瘤坏死因子相关凋亡诱导配体(TRAIL)诱导神经母细胞瘤(NB)细胞凋亡中的作用。  方法  应用RT-PCR方法检测γ干扰素(IFN-γ)作用前后NB细胞Caspase 8 mRNA的表达; 应用Western blot方法检测Caspase 8、DR4和DR5蛋白表达; 应用四甲基偶氮唑蓝(MTT)比色法及流式细胞术检测IFN-γ, TRAIL, IFN-γ+TRAIL, Caspase 8抑制剂+TRAIL及IFN-γ+阿霉素/依托泊苷+TRAIL对NB细胞株生长及凋亡的影响。  结果  IFN-γ在NB细胞株SKNDZ中诱导了Caspase 8mRNA及蛋白表达。SY5Y细胞对TRAIL不敏感, 而IFN-γ与TRAIL或阿霉素, 依托泊苷联用对SY5Y细胞有明显诱导凋亡作用。IFN-γ+TRAIL组SY5Y细胞早期凋亡率(23.09+2.35)%高于TRAIL组[(6.15±0.54)%(P< 0.01)], 但低于IFN-γ+阿霉素/依托泊苷+TRAIL组[(43.41±6.46)%/(38.86±7.29)%, P< 0.01]。阿霉素或依托泊苷可以诱导NB细胞株DR5蛋白表达, 但未诱导DR4蛋白表达。IFNγ诱导后表达Caspase 8的SKNDZ细胞对TRAIL的诱导凋亡作用仍不敏感, 而阿霉素或依托泊苷处理后同时表达DR5的SKNDZ细胞对TRAIL的诱导凋亡作用相对敏感。IFN-γ+阿霉素/依托泊苷+TRAIL组SKNDZ细胞早期凋亡率(11.54±2.49)%/(13.38±1.65)%高于IFN-γ+TRAIL组(P< 0.01)。  结论  IFN-γ上调Caspase 8表达及化疗药阿霉素或依托泊苷诱导DR5表达可以恢复NB细胞对TRAIL的敏感性, Caspase 8和DR5在TRAIL诱导NB细胞凋亡中起着十分关键的作用。      

神经母细胞瘤中Schwann细胞抗肿瘤作用研究进展

神经母细胞瘤(Neuroblastoma, NB)包括两个细胞群:神经母/神经节细胞和Schwann细胞.     

CIK、DC-CIK细胞对神经母细胞瘤细胞杀伤作用的研究

目的：研究细胞因子诱导的杀伤细胞（ CIK）与树突状细胞（ DC）共培养后对神经母细胞瘤（ neuro-blastoma,NB）细胞株的杀伤作用。方法：取健康人和肿瘤患者外周血单个核细胞（ PBMC）,加入不同的细胞因子分别诱导出DC和CIK细胞,用流式细胞术测定诱导培养前后DC和CIK细胞的表型,MTT法测定不同组CIK细胞对NB细胞株的杀伤活性。结果：流式细胞仪检测健康人PBMC培养后CD3＋CD56＋淋巴细胞百分比以及对NB细胞株的杀伤活性均显著高于肿瘤患者（ P〈0.05）。此外,与单纯CIK细胞相比,DC-CIK细胞具有更强的杀伤NB细胞株的活性（ P〈0.05）。结论：DC-CIK细胞是一种细胞毒作用高于单纯CIK细胞的免疫活性细胞。健康人和肿瘤患者的PBMC经诱导培养获得的CIK细胞有显著差别,为临床进一步提高CIK细胞的治疗效果提供了实验依据。     

IFNγ、TRAIL及化疗药联合诱导神经母细胞瘤细胞凋亡的研究

目的探讨γ-干扰素(Interferon,IFNγ)、肿瘤坏死因子相关凋亡诱导配体(tumornecrosisfactorre-latedapoptosisinducingligand,TRAIL)及化疗药联合诱导神经母细胞瘤(neuroblastoma,NB)细胞株SH-SY5Y(SY5Y)凋亡的作用及其发生机制。方法应用Western-blot方法检测IFNγ作用前后SY5Y细胞Caspase8蛋白的表达;应用四甲基偶氮唑蓝(MTT)比色法及流式细胞仪(FCM)检测IFNγ、TRAIL、IFNγ TRAIL、IFNγ Caspase8抑制剂(zIETD-FMK) TRAIL及IFNγ 化疗药 TRAIL对SY5Y细胞生长及凋亡的影响。结果SY5Y细胞不表达Caspase8,IFNγ作用48h后的SY5Y细胞Caspase8蛋白表达明显增加;SY5Y细胞对TRAIL不敏感,经IFNγ诱导表达Caspase8的SY5Y细胞对TRAIL敏感;zIETD-FMK可明显抑制TRAIL对表达Caspase8的SY5Y细胞的杀伤作用;化疗药可增强SY5Y细胞对TRAIL的敏感性。结论IFNγ可通过上调SY5Y细胞Caspase8表达而逆转SY5Y细胞对TRAIL诱导凋亡的耐受,化疗药可增强TRAIL对SY5Y细胞的诱导凋亡作用。     

节细胞神经母细胞瘤1例

患者男,59岁.因左腹、腰部胀痛,逐渐加重1个月于1998年1月8日入院.平素健康.查体:左上腹触及一6cm×5cm质硬肿块,固定、轻压痛.B超检查:左肾前上极探及一8.0cm×7.8cm,低回声不均质肿块,包膜完整、增厚.CT扫描示左侧腹膜后肿瘤,与胰、左肾关系密切.X线胸片未见心肺异常.CEA<20μg/ml.经手术证实,肿瘤位于腹膜后、胰体尾部及左肾上极之间,并侵犯左肾门及肾蒂血管,血供丰富.肿瘤为灰红色,卵圆形9cm×7cm×6cm大小,切面见大片出血、坏死,部分囊性变,伴有鱼肉样组织.肿瘤免疫组化结果:NSE( )、FN(-)、GFAP(-)、S_(100)节细胞( )、小细胞(-).病理诊断为节细胞神经母细胞瘤.     

节细胞神经母细胞瘤1例

患者,男,13岁.因右侧腰痛伴低热3天,在襄樊市中医院做双肾CT示右肾上腺肿瘤于2003年6月入我院.     

TRAIL诱导神经母细胞瘤细胞凋亡中 Caspase 8活性的变化

目的：探讨肿瘤坏死因子相关凋亡诱导配体（TRAIL）诱导神经母细胞瘤细胞株CHP212细胞凋亡过程中Caspase 8活性的变化.方法：应用流式细胞仪检测TRAIL、Caspase 8抑制剂（zIETD-FMK）＋TRAIL对CHP212细胞的诱导凋亡作用 应用比色法测定Caspase 8相对活性 应用透射电镜对凋亡细胞进行形态学观察.结果：CHP212细胞对TRAIL的诱导凋亡作用敏感，存在剂量依赖性.随TRAIL作用时间的延长，Caspase 8活性逐步升高，于作用16h达高峰 不同浓度TRAIL处理组Caspase 8相对活性随浓度增加而递增.zIETD-FMK能阻断Caspase 8的活化而抑制TRAIL对CHP212细胞的诱导凋亡作用.透射电镜可见到典型的细胞凋亡特征.结论：TRAIL通过Caspase 8信号传导通路诱导CHP212细胞凋亡并伴随Caspase 8活性增高.     

Alamar Blue法用于体外培养细胞活性检测的方法研究

目的在比较A lam ar B lue法与MTT法的基础上探讨A lam ar B lue法用于测定细胞体外增殖及细胞毒实验的可行性。方法采用A lam ar B lue法及MTT法分别测定人肝癌细胞株SMMC-7721的体外增殖以及肿瘤坏死因子相关凋亡诱导配体、顺铂、羟基喜树碱对该细胞株体外增殖的影响。结果使用A lam ar B lue法,接种人肝癌SMMC-7721细胞数在3.12×102~2×104/孔范围内时,细胞数与A lam ar B lue还原率呈线性相关(r=0.9989),高、中、低三种细胞浓度组内差异分别为1.41%、3.88%、4.87%。使用MTT法接种人肝癌SMMC-7721细胞数在3.12×102~2×104/孔范围内时,细胞数与OD490~620值呈线性相关(r=0.9981),高、中、低三种细胞浓度组内差异分别为1.44%、4.07%、8.27%。结论A lam ar B lue法是一种简便、敏感、安全、经济的方法,可用于体外测定细胞增殖及化疗或免疫制剂的细胞毒作用。     

Differential modulation of the TRAIL receptors and the CD95 receptor in colon carcinoma cell lines

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and CD95 ligand (CD95L) are potent inducers of apoptosis in various tumour cell types. Death receptors DR4 and DR5 can induce and decoy receptors DcR1 and DcR2 can inhibit TRAIL-mediated apoptosis. The study aim was to investigate whether anticancer agents can modulate similarly TRAIL-receptor and CD95 membrane expression and TRAIL and CD95L sensitivity.Three colon carcinoma cell lines (Caco-2, Colo320 and SW948) were treated with 5-fluorouracil (5-FU), cisplatin or interferon-gamma. TRAIL-receptor and CD95 membrane expression was determined flow cytometrically. Sensitivity to TRAIL or CD95L agonistic anti-CD95 antibody was determined with cytotoxicity and apoptosis assays. SW948 showed highest TRAIL sensitivity. The protein synthesis inhibitor cycloheximide decreased FLICE-like inhibitory protein levels in all cell lines, and the TRAIL-resistant cell lines Caco-2 and Colo320 became sensitive for TRAIL. Exposure of the cell lines to 5-FU, cisplatin and interferon-gamma left TRAIL-receptor membrane expression and TRAIL sensitivity unaffected. CD95 membrane expression and anti-CD95 sensitivity was, however, modulated by the same drugs in all lines. Cisplatin and interferon-gamma raised CD95 membrane levels 6-8-fold, interferon-gamma also increased anti-CD95 sensitivity. These results indicate that the CD95 and TRAIL pathways use different mechanisms to respond to various anticancer agents. Induced CD95 membrane upregulation was associated with increased anti-CD95 sensitivity, whereas no upregulation of TRAIL-receptor membrane expression or TRAIL sensitisation could be established. For optimal use of TRAIL-mediated apoptosis for cancer therapy in certain tumours, downregulation of intracellular inhibiting factors may be required.     

Schedule-dependent response of neuroblastoma cell lines to combinations of etoposide and cisplatin

The growth inhibitory effects of cisplatin and etoposide on neuroblastoma cell lines were investigated in several scheduled combinations. Results were analyzed using median effect and combination index analyses. In all schedules in which cisplatin was administered prior to etoposide a synergistic effect was observed. Conversely, an antagonistic effect was seen in all schedules where etoposide was administered before cisplatin.     

Synergistic antitumor activity of TRAIL combined with chemotherapeutic agents in A549 cell lines in vitro and in vivo

Purpose To investigate the synergistic cytotoxicity of TRAIL in combination with chemotherapeutic agents in A549 cell lines, we systematically evaluated the cytotoxicity of TRAIL alone and TRAIL in combination with cisplatin, paclitaxel (Taxol) or actinomycin D in A549 cell lines in vitro and in vivo, and whether the sensitivity was correlated with the expression level of TRAIL receptors.Methods We investigated the cytotoxicity of TRAIL alone and the synergistic antitumor effects of TRAIL in combination with chemotherapeutic agents in A549 cells by crystal violet staining and FACS in vitro. The expression levels of DR4, DR5, DcR1 and DcR2 were measured in TRAIL-treated and chemotherapeutic agent-treated A549 cells by Western blotting. The growth inhibition of tumors was evaluated in terms of incidence, volume and weight in a A549-implanted nude mice model.Results Chemotherapeutic agents cisplatin (5.56 g/ml), Taxol (10 and 30 g/ml) or actinomycin D (9.26, 83.3 and 750 ng/ml) augmented the cytotoxicity of TRAIL in A549 cell lines within a range of concentrations of TRAIL (1.98–160 ng/ml) in vitro. The expression levels of DR4 and DR5 were not significantly different and the expression of DcR2 was slightly downregulated, but the expression of DcR1 was not detected in non-treated, TRAIL-treated and chemotherapeutic agent-treated A549 cells. The rates of tumor inhibition following treatment with TRAIL alone (15 mg/kg per day, daily for 10 days) and TRAIL/cisplatin (15 mg/kg per day TRAIL, daily for 10 days; 1.5 mg/kg per day cisplatin, daily for 10 days with 7-day intervals) were 28.3% and 76.8% by tumor weight (P<0.05 for TRAIL alone versus control, P<0.05 for TRAIL/cisplatin versus cisplatin alone and TRAIL alone) on day 65 in vivo.Conclusion TRAIL in combination with chemotherapeutic agents cisplatin, Taxol or actinomycin D exerted synergistic antitumor effects in A549 cell lines in vitro and TRAIL/cisplatin demonstrated synergistic antitumor effects in vivo. The expression levels of TRAIL receptors suggested that the synergistic effects of TRAIL in combination with chemotherapeutic agents are not at the receptor level in A549 cell lines.Abbreviations TNF Tumor necrosis factor - TRAIL TNF-related apoptosis-inducing ligand - NSCLC Non-small cell lung cancer - DR Death receptor - DcR Decoy receptor - IB Inclusion body - FACS Fluorescence activated cell sorter - PI Propidium iodide - IR Rate of inhibition - PMSF Phenyl methyl sulfonyl fluoride     

Retinoids and the control of growth/death decisions in human neuroblastoma cell lines

Cell proliferation, the balance between mitosis and apoptosis is the result of the continuous integration of a number of different signal transduction pathways stimulated in a cell at any given point in its life. Neuroblastoma cells regulate the switch between mitosis and death, according both to intrinsic factors and extrinsic factors, such as growth factor withdrawal and action of the vitamin A derivative, retinoic acid. In this review, we describe the balance of some factors regulating growth and death of human neuroblastoma cells in vitro. These dynamic studies are necessarily performed on cell lines, which offer controlled conditions enabling the disection of the complex stimuli mediating survival and growth (IGF, trk, BDNF) and death (transglutaminase, free radicals, Bcl-2). Although the conclusions drawn may therefore not be directly applicable to tumour cells in vivo, the results herein discussed are of sufficient significance to warrant in vivo relevance.     

ＴＲＡＩＬ联合蛋白酶抑制剂ＭＧ１３２诱导肺癌Ａ５４９细胞凋亡的实验研究

目的　观察ＴＲＡＩＬ对肺癌Ａ５４９细胞的体外生长抑制及诱导凋亡作用,并探讨ＴＲＡＩＬ与蛋白酶抑制剂ＭＧ１３２联合用药诱导细胞凋亡的作用及其机制。方法　采用ＭＴＴ法检测不同浓度ＴＲＡＩＬ对肺癌Ａ５４９细胞增殖的影响;应用流式细胞仪技术检测ＴＲＡＩＬ、蛋白酶抑制剂ＭＧ１３２及其两药联合干预２４ｈ对肺癌Ａ５４９细胞凋亡率的影响;采用Ｗｅｓｔｅｒｎｂｌｏｔｔｉｎｇ检测药物干预前后细胞凋亡相关蛋白Ｃａｓｐａｓｅ-８、Ｃａｓｐａｓｅ-９以及核转录因子ＮＦ-κＢ的表达情况。结果　ＭＴＴ结果显示不同浓度ＴＲＡＩＬ对肺癌Ａ５４９细胞的体外抑制作用与剂量效应正相关,２４ｈＩＣ５０为９６.３２２μｇ／ｍｌ,４８ｈＩＣ５０为４３.５９μｇ／ｍｌ;流式细胞术显示ＴＲＡＩＬ与ＭＧ１３２联合用药后肺癌Ａ５４９细胞凋亡率明显增高（平均６７.６５％）,与ＴＲＡＩＬ（平均２３.５５％）、ＭＧ１３２（平均２５.９１％）单药组比较,有显著性差异（Ｐ＝０.０１６）;Ｗｅｓｔｅｒｎｂｌｏｔｔｉｎｇ结果显示,与ＴＲＡＩＬ单药组比较,两药联合后细胞凋亡相关蛋白Ｃａｓｐａｓｅ-８、Ｃａｓｐａｓｅ-９表达明显增强（Ｐ＝０.００６）,抑制细胞凋亡因子ＮＦ-κＢ表达则明显减弱（Ｐ＝０.０４）。结论　ＴＲＡＩＬ可抑制Ａ５４９细胞的增殖,呈浓度依赖关系;ＴＲＡＩＬ与蛋白酶抑制剂ＭＧ１３２联合应用可以明显提高细胞凋亡率,可能与蛋白酶抑制剂ＭＧ１３２降低ＮＦ-κＢ的活性,增强细胞对ＴＲＡＩＬ诱导凋亡的敏感性,促进凋亡相关蛋白Ｃａｓｐａｓｅ-８及Ｃａｓｐａｓｅ-９的表达有关。     

High delta-like 1 expression in a subset of neuroblastoma cell lines corresponds to a differentiated chromaffin cell type

The childhood tumour neuroblastoma originates from neural crest-derived progenitors of the sympathetic nervous system. By Serial Analysis of Gene Expression (SAGE), we previously identified the Drosophila Delta homologue Dlk1 as one of the genes most highly expressed in the neuroblastoma cell line SK-N-FI. The Delta-Notch pathway controls many differentiation steps in Drosophila and man. We analysed expression of 21 genes of this pathway in 21 neuroblastoma cell lines. Dlk1 expression was very high in 5 cell lines, while another subset expressed Notch3. The imprinting of Dlk1 was faithfully preserved in neuroblastomas. The single paternal allele can therefore produce over 0.5% of all cellular mRNAs. Dlk1 maps to 14q32, a region that exhibited LOH in 31/170 (18%) tumours. The random parental origin of the deleted alleles excluded Dlk1 as target of the LOH. In addition, Dlk1 was not amplified, rearranged or mutated in neuroblastoma cell lines and tumours. We therefore analysed whether high Dlk1 expression marks a specific differentiation stage of the sympatho-adrenal lineage. Many neuroblastomas arise in the adrenal medulla, which predominantly consists of chromaffin cells. Normal adrenal medulla exhibited equally high Dlk1 levels as the SK-N-FI cell line. Chromaffin cells in young children are marked by noradrenalin production, which is mediated by dopamine-beta-hydroxylase (DBH). DBH expression in the neuroblastoma cell lines almost perfectly corresponded to Dlk1 expression. Neuroblastoma cell lines with high Dlk1 expression are therefore arrested in a relatively late stage of chromaffin lineage differentiation, while Notch3 expression might correspond to earlier precursor stages or to an alternative developmental fate.     

Reactivation of death receptor 4 (DR4) expression sensitizes medulloblastoma cell lines to TRAIL

Object Apoptosis, a key cellular response to therapeutic agents is often inactivated in tumor cells. In this study, we evaluated the expression of the tumor necrosis family of death receptors, DR4 and DR5, in medulloblastoma tumor samples and cell lines to determine if epigenetic modulation of gene expression could sensitize tumor cell lines to TRAIL-mediated apoptosis. Methods Human medulloblastoma samples and cell lines were analyzed for DR4 and DR5 expression by quantitative PCR and immunofluorescence assays. Cell lines with downregulated expression of one or both genes were treated with the histone deacetylase inhibitor, MS-275, and the expression of DR4 and DR5 measured by quantitative PCR, Western blotting, flow cytometry and chromatin immunoprecipitation assays. Induction of apoptosis in the presence of MS-275 was evaluated by TUNEL assay and its ability to augment TRAIL-mediated cytotoxicity was determined by MTT assays, Western blotting and flow cytometry. Results Compared to normal cerebellum, DR4, but not DR5 expression was consistently downregulated in medulloblastoma tumor samples and in Daoy and D283 cell lines. Interestingly, MS-275 decreased cell growth and induced apoptosis in Daoy and D283 cells. In Daoy cells, this coincided with increased histone H3 and H4 acetylation at the DR4 promoter and enhanced DR4 gene and protein expression as well as elevated Caspase-8 activity. The involvement of DR4 in the cellular response to MS-275 was further confirmed by the observation that knockdown of DR4 and FADD abrogated apoptosis. Further, addition of TRAIL to MS-275 treated cells resulted in an enhancement of apoptosis, suggesting that the upregulated death receptors were functional. Conclusion Our study provides an understanding of the role of DR4 in apoptosis of medulloblastoma cell lines and suggests a potential contribution of aberrant histone deacetylation to the resistance of medulloblastoma cells to therapeutic death. This work was supported in part by Award P50CA127001 from the National Cancer Institutes and Grants from the National Brain Tumor Foundation and the American Cancer Society-IRG to VG.     

Search for neuroblastoma loci: characterization of tumor cell lines that could facilitate their positional cloning

Specific chromosomal aberrations might indicate the position of genes responsible for a particular disease. Neuroblastoma is characterized by frequent deletions and/or rearrangements of the subtelomeric 1p region which, accordingly, is believed to host one or more oncosuppressor gene(s) directly or indirectly involved in the development of this and other tumors. Identification of these genes could be facilitated if cell lines with well characterized interstitial deletions or reciprocal translocations could be available for application of positional cloning strategies.In the present report we present additional and novel molecular data on three well established neuroblastoma cell lines (NLF, NMB and NGP). In one of these we have identified two sites that might be good candidates for hosting oncosuppressor genes; one of these is flanked by the D1S47 and ENO1 loci while the other is distal to the A12M2 locus.