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.     

Genetic analysis of p73 localized at chromosome 1p36.3 in primary neuroblastomas

BACKGROUND: Human p73, a novel homolog of p53, has recently been cloned and mapped at chromosome 1p36.3, the locus for putative tumor suppressor gene(s) of neuroblastoma (NBL) and other cancers. p73, like p53, inhibits growth and induces apoptosis in neuroblastoma and osteosarcoma cell lines. PROCEDURE: To test the hypothesis that p73 is a NBL suppressor gene, we examined expression, allelo-typing, and mutation of the p73 gene in primary human neuroblastomas. Loss of heterozygosity (LOH) for p73 was performed in 272 primary NBLs using a CT repeat polymorphic marker, which we found in intron 9 of the p73 gene. RESULTS: p73 LOH was observed in 28 out of 151 (19%) informative cases. The high frequency of p73 LOH was significantly associated with sporadic neuroblastomas (P< 0.001), MYCN amplification (P< 0.001), and advanced stages (P< 0.05). Mutational analyses by PCR-SSCP (single strand conformation polymorphism) revealed two mis-sense mutations in 140 NBLs, one somatic and one germline. CONCLUSION: Thus, the present results have shown that mutation of p73 is infrequent in NBLs, although the p73 locus is frequently lost in advanced stage tumors. These suggest that p73 may not be a tumor suppressor in the classic Knudson manner.     

肾母细胞瘤患儿p73基因的表达及其甲基化研究

目的：探讨肾母细胞瘤患儿血液中p73基因的转录表达、启动子甲基化状态及二者之间关系。方法：收集45例肾母细胞瘤患儿为病例组,以健康体检或因其他原因就诊的性别、年龄匹配的15例（排除肿瘤等恶性疾病）儿童为对照组。采集两组儿童外周血,运用实时荧光定量PCR（qRT-PCR）和甲基化特异性PCR (MSP)法检测p73基因转录表达水平及其启动子甲基化状态,并分析病例组中p73基因的表达及甲基化与临床资料的关系,及p73基因的甲基化对其转录表达的影响。结果：病例组中p73 mRNA的相对表达量（3.2±0.9）高于对照组（1.6±1.1）,差异有统计学意义（P<0.01）;病例组p73基因甲基化阳性率（20%）低于对照组 (73%),差异有统计学意义（P<0.01）。病例组中甲基化p73 mRNA的相对表达量大于非甲基化p73 mRNA的相对表达量（P<0.01）,且大于对照组中甲基化p73 mRNA的相对表达量（P<0.01）;非甲基化p73 mRNA的相对表达量在两组间差异无统计学意义（P=0.810）。结论：肾母细胞瘤外周血中p73基因启动子的异常甲基化是其基因表达调节方式之一,并与肾母细胞瘤的发生发展有关;发生甲基化的p73基因在肾母细胞瘤中有可能充当癌基因的角色,转录水平的过度表达与其甲基化状态有关。     

甲基化抑制剂对CEM细胞EphB4基因表达及细胞增殖与凋亡的影响

目的：探讨甲基化抑制剂5-氮杂-2'脱氧胞苷（5-aza-2'-deoxycytidine,5aza-CdR）对人急性淋巴细胞白血病细胞株CEM中抑癌基因EphB4的转录调控作用及对CEM细胞增殖凋亡的生物学影响,为寻找肿瘤治疗新靶点提供实验依据。方法：采用亚硫酸氢盐修饰后测序法检测EphB4基因甲基化水平,荧光定量PCR法(Q-PCR)和Western blot方法检测5-aza-CdR处理前后EphB4基因mRNA及蛋白表达水平;MTS法检测不同剂量(1.0、2.5、5.0 μmol/L) 的5-aza-CdR作用于CEM细胞0 h、24 h、48 h、72 h、96 h对CEM细胞存活率的影响,流式细胞仪分析5-aza-CdR作用96 h对CEM细胞周期及凋亡的影响。结果：CEM细胞中存在EphB4基因的甲基化,甲基化水平为31.4%;不同浓度5-aza-CdR作用后CEM细胞甲基化水平均下降。5-aza-CdR作用使CEM细胞中EphB4 基因的mRNA和蛋白表达上升;5-aza-CdR明显抑制CEM细胞增殖,并与药物浓度及作用时间呈正相关。5-aza-CdR处理 96 h后,早期凋亡由4.1%上升为24.8%。用药96 h后,CEM细胞G1期由62.4% 降至46.8%,G2期由2.1%升至16.2%,细胞阻滞于G2期。结论：特异性甲基化转移酶抑制剂5-aza-CdR能使CEM细胞中沉默的EphB4基因重新表达,并可抑制白血病细胞增殖和诱导其凋亡。     

Effects of standard chemotherapy on tumor growth and regulation of multidrug resistance genes and proteins in childhood rhabdomyosarcoma

The prognosis of rhabdomyosarcoma (RMS) in advanced stages is still sobering. Therapy is limited due to local tumor recurrence, development of metastases and multidrug resistance. The aim of this study was to investigate the development of multidrug resistance in cell lines and in xenografts of alveolar and embryonal RMS treated according to the German Soft Tissue Sarcoma Study (CWS). Alveolar and embryonal RMS cell lines were treated with Vincristine, Topotecan, Carboplatin, Actinomycin D, or Ifosfamide. Expression levels of resistance-associated genes were assessed using Real time-PCR. Nude mice (NMRI nu/nu, n = 10 per group) underwent xenotransplantation of human embryonal or alveolar RMS. Animals were treated with standard chemotherapeutic drugs Vincristine, Topotecan, Carboplatin, Actinomycin D, or Ifosfamide according to treatment schedules of the CWS-study. Tumor sizes were measured and relative tumor volumes were calculated. Animals were sacrified after 20 days and standard histology, Real-time-PCR for MDR1-, MRP-, LRP- and MDM2-gene as well as immunhistochemistry for MDR1-, LRP-, and MRP-protein were performed. In the cell lines, an up-regulation of MDR-1 gene was found in alveolar rhabdomyosarcoma. In embryonal rhabdomyosarcoma, an up-regulation of LRP and MRP was found. Standard chemotherapy of alveolar rhabdomyosarcoma resulted in a significant reduction of tumor growth (P < 0.05) in all groups. In embryonal rhabdomyosarcoma strongest effects were found after treatment with Ifosfamide, Vincristine and Carboplatin (P < 0.05). RT-PCR revealed a MDR1-dependent mechanism in alveolar rhabdomyosarcoma. In embryonal rhabdomyosarcoma, MDR1 occurred to a lower degree. Immunhistochemistry revealed correlating expression levels of multidrug resistance-associated proteins. The use of established chemotherapy on human RMS in vivo had strong effects on xenografts compared to their controls. In all cases, there was only a reduction of tumor growth, but not a complete eradication of the tumors. Chemotherapy seemed to upregulate the expression of resistance-associated genes in vitro and in vivo. The mechanism of multidrug resistance depends on the tumor subtype. Therefore, further investigations will be required to evaluate multidrug resistance in patients and to investigate new modalities for a reversal of multidrug resistance.     

Induction of Drug Resistance in Human Rhabdomyosarcoma Cell Lines Is Associated with Increased Maturation: Possible Explanation for Differentiation in Recurrences?

In rhabdomyosarcoma (RMS) of childhood and adolescence very little is known about interactions of cytotoxic drugs and tumor cells. In recurrent RMS the tumor cells are often more mature than in the primary tumor. The biological properties of these cells are still a subject of controversy. We investigated two human (RD2 and TE 671) cell lines by cultivating them with doxorubicin, cisplatinum, and etoposide. Degree of differentiation and proliferation rate were estimated morphologically and by means of immunohistochemistry and a monolayer proliferation assay. Both morphological and immunohistochemical maturation was measurable in most resistant cell lines. An increase in myosin expression was most marked in the etoposide- and doxorubicin-resistant RD cell lines. The proliferation rate was decreased in almost all resistant cell lines. Nevertheless, the resistant cell lines tolerated high-dose levels of cytotoxic drugs at a higher proliferation rate than parental cell lines cultivated under similar conditions. The maturation seen in some recurrent tumors of RMS can be simulated in vitro by cultivating cell lines with cytotoxic drugs at sublethal doses. Interestingly, the resistance-associated induced maturation was not accompanied by p170 expression. After comparing these in vitro results with the maturation seen in RMS specimens after chemotherapy, we conclude that chemotherapy-induced differentiation in vivo might be a morphological sign of chemoresistance. Received August 6, 2001; accepted December 3, 2001.     

环磷酰胺和吡柔比星对横纹肌肉瘤RD细胞株CXCR4表达的影响

目的观察敏感剂量下环磷酰胺（CTX）和吡柔比星（THP）对体外培养人横纹肌肉瘤RD细胞株CXCR4基因表达的影响。方法体外培养RD细胞株;MTT试验明确CTX和THP对RD细胞的效应剂量;细胞划痕实验检测RD细胞的迁移能力;RT—PCR及Westernblot法检测RD细胞CXCR4表达。结果（1）敏感剂量下CTX（30mmol／L）和THP（1000ng／mL）均可抑制RD细胞的迁移（P〈0．05）,两药联合应用对细胞迁移抑制作用增强,较对照组差异有非常显著性（P〈0．01）;（2）各药物处理组（CTX,THP,CTX＋THP）药物作用于RD细胞24h后CXCR4蛋白的表达较对照组减少,其差异均有显著性（P〈0．05）;其中CTX＋THP组较CTX组CXCR4蛋白表达减少更明显（P〈0．05）,但与THP组比较,差异无显著性（P〉0．05）;（3）同样条件下,各药物处理组（CTX．THP,CTX＋THP）与对照组比较,RD细胞CXCR4mRNA的表达均减少,差异均有显著性（P〈0．05）;CTX＋THP组较CTX组CXCR4mRNA表达减少明显（P〈0．05）,但较THP组差异无显著性（P〉0．05）。结论化疗药物CTX和THP均能抑制人横纹肌肉瘤RD细胞的增殖;敏感剂量下的两种药物均能抑制RD细胞的迁移及下调RD细胞CXCR4的表达,提示两种化疗药物发挥作用的机制可能涉及到CXCR4基因。     

TRAIL/Apo2L ligands induce apoptosis in malignant rhabdoid tumor cell lines

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) is a potent inducer of apoptosis in various cancer cells, whereas normal cells are not sensitive to TRAIL-mediated apoptosis. Four TRAIL/Apo2L receptors (DR4, DR5, DcR1, and DcR2) have been identified. DR4 and DR5 have a death domain, whereas DcR1 and DcR2 are called decoy receptors because of their incomplete or lack of a death domain. Malignant rhabdoid tumor (MRT) is an aggressive neoplasm showing a poor prognosis because of its resistance to chemotherapeutic agents. In this study, we examined whether TRAIL could induce apoptotic cell death in MRT cell lines. We found that although half of the MRT cell lines examined were sensitive to TRAIL/Apo2L, Western blot analysis revealed that the expression of DcR2 was low in TRAIL-sensitive MRT cells. We examined the effect of doxorubicin on the expression levels of TRAIL receptors and its enhancement on the susceptibility of MRT cell lines to TRAIL. Western blot and flow cytometric analyses revealed that doxorubicin significantly increased the expression of DR5, and somewhat up-regulated the expression of DR4 and DcR2. Moreover, doxorubicin, NF-kappaB inhibitor (SN50), and PI3-kinase/Akt inhibitor (wortmannin, LY294002) enhanced the susceptibility of MRT cell lines to TRAIL/Apo2L-induced apoptosis. These results suggest that TRAIL/Apo2L may provide the basis for clinical trials of TRAIL-based treatment to improve the outcome of MRT patients.     

药物相关性分子靶标检测在指导Ⅲ-Ⅳ期儿童神经母细胞瘤治疗中的初步经验

目的 探讨药物相关性分子靶标检测在儿童神经母细胞瘤个体化治疗中的作用和意义.方法 2011年4月至2013年4月间在上海交通大学医学院附属新华医院诊治的13例儿童神经母细胞瘤患儿,取其手术肿瘤及外周血标本共31份(手术/二次手术标本共15份,外周血标本16份).通过免疫组化,酶联免疫吸附试验,荧光原位杂交,PCR扩增测序等方法检测常见肿瘤药物的相关分子靶标.共检测12种常用化疗药物和1种靶向药物的相关性基因的表达或突变.结果 共检测肿瘤标本分子靶点7个,包括:DNA拓扑异构酶ⅡA(TOPOⅡA),微管蛋白β3(Tubulinβ3),人切除修复交叉互补基因(ERRC 1),DNA拓扑异构酶Ⅰ(TOPO Ⅰ),DNA修复蛋白-O6甲基鸟嘌呤-DNA-甲基转移酶(MGMT),二氢叶酸还原酶(DHFR),胸苷酸合成酶(TS);外周血标本分子靶点2个,包括:CYP2C9*3和二氢叶酸还原酶DHFR(C829T)基因多态性测序.根据靶标对应的相关药物进行总结分析,研究结果提示:氟尿嘧啶类,长春碱类,环磷酰胺和甲氨蝶呤四类化疗药物敏感性较高;而紫杉醇类、拓扑替康/伊立替康、替莫唑胺/卡莫斯汀/司莫斯汀、蒽环类/依托泊苷和铂类的药物敏感性较低.研究共检测肿瘤标本和外周血标本涉及的靶向药物基因2个:血管内皮生长因子受体(VEGFR-2)和细胞间粘附分子1(ICAM-1).结果提示:贝伐单抗敏感性高.依据上述检测结果,我们及时调整治疗策略,优化化疗方案,取得了良好的效果,并为部分患儿创造了手术机会.结论 本研究在儿童神经母细胞瘤的多学科治疗过程中引入药物相关性分子靶标的检测,对于开展规范化治疗基础上的个体化治疗方案的选择,提供了依据和经验.     

Loss of caspase-8 expression in neuroblastoma is related to malignancy and resistance to TRAIL-induced apoptosis

Background and Procedures NB-derived cell lines were tested for their sensitivity to apoptosis induced by the tumor-selective apoptotic ligand TRAIL. Noninvasive S-type cell lines are highly sensitive to TRAIL, whereas invasive N-type cell lines are resistant. RESULTS: Although both S- and N-type cell lines express TRAIL-R2, FADD, and caspases-3 and -10, only S-type cells express caspase-8. Reduced levels of caspase-8 protein were also observed in a stage IV NB tumor when compared to a ganglioneuroma. The caspase-8 gene is not deleted in either N-type NB cell lines or high-stage tumors, and expression can be induced by demethylation. CONCLUSIONS: Therefore, caspase-8 expression is silenced in malignant NB, which correlates to tumor severity and resistance to TRAIL-induced apoptosis.     

Hypermethylation as a potential prognostic factor and a clue to a better understanding of the molecular pathogenesis of medulloblastoma--results of a genomewide methylation scan

BACKGROUND: The molecular mechanisms controlling initiation and progression of medulloblastomas are largely unclear. Changes in DNA methylation of promoter regions have been shown to disturb the expression of growth regulatory genes. PATIENTS AND METHODS: We evaluated DNA methylation patterns in 17 medulloblastomas, 5 stPNETs and 5 medulloblastoma cell lines using Restriction Landmark Genomic Scanning (RLGS), a method displaying up to 2.000 potential gene loci in a single gene. To test whether previously characterized tumor suppressor genes are affected by hypermethylation we performed MS-PCR for p15INK4B, p16INK4A, VHL, TP53 and E-cadherin. RESULTS: The analysis of RLGS profiles from tumors revealed an abundance of hypermethylation in primary tumors and cell lines. Extrapolated to the human genome with its approximately 36,000 genes a total of 420 loci become hypermethylated in the tumor genomes. The previously characterized medulloblastoma breakpoint cluster in 17p11.2 appears to be a hotspot for aberrant methylation. Cox regression analysis of survival data identified seven CpG islands for which hypermethylation is suggestive of a poor prognosis. MS-PCR analysis of known genes demonstrated hypermethylation of p16INK4A in a limited number of tumors. The pattern of DNA hypermethylation was similar in medulloblastomas and stPNETs. However, some CpG islands were shown to be specific for a tumor type, while others were shared targets. CONCLUSIONS: Hypermethylation is a common abnormality in primary medulloblastomas and supratentorial PNETs. Several hundreds of CpG islands are potential targets for methylation in medulloblastomas including the breakpoint cluster in 17p11.2. The methylation status of certain gene sequences appears to be associated with the clinical outcome. Promoter hypermethylation has an outstanding potential as a marker for the identification of novel tumor suppressors as well as diagnostic and therapeutic targets in medulloblastomas.     

Ewing sarcoma family of tumors express adenovirus receptors and are susceptible to adenovirus-mediated oncolysis

PURPOSE: Attenuated viruses derived from adenoviruses (Ad) that kill tumor cells (oncolysis) are currently in clinical trials for selected cancers. Some cancers have proven resistant to Ad infection due to low expression of viral receptors. The authors sought to determine whether members of the Ewing sarcoma family of tumors (ESFTs) express Ad receptors and are sensitive to Ad-mediated oncolysis. METHODS: Using flow cytometry, the authors tested a panel of cell lines derived from ESFTs for expression of both the Ad receptor, coxsackie-adenovirus receptor (CAR), and the cellular mediator of Ad uptake, alpha(v)-integrins, as well as for Ad-mediated gene transduction. Cell survival assays were used to assess the sensitivity to Ad-mediated oncolysis. Immunohistochemistry was used to assess CAR expression in primary tumors. mRNA levels of CAR in cell lines and tumor samples were also queried from a cDNA expression database. RESULTS: The ESFT cell lines expressed CAR and alpha(v)-integrins, showed high levels of gene transduction, and were highly sensitive to viral oncolysis. Primary tumor samples were positive for CAR expression by immunohistochemistry. Microarray analysis confirmed CAR expression in ESFT cell lines and tumors. CONCLUSIONS: Ewing sarcoma cells express the Ad receptors and are sensitive to Ad oncolysis. Treatment of Ewing sarcoma using conditionally replicative adenoviruses should be explored.     

p53 mutations and loss of p53 function confer multidrug resistance in neuroblastoma

BACKGROUND: Neuroblastomas often acquire sustained drug resistance during therapy. Sensitivities to carboplatin, etoposide, or melphalan were determined for 18 neuroblastoma cell lines; eight were sensitive and ten were resistant. As p53 mutations are rare in neuroblastomas studied at diagnosis, we determined if acquired p53 mutations and loss of function conferred multidrug resistance. RESULTS: Loss of p53 function (p53-LOF), defined as a failure to induce p21 and/or MDM2 in response to melphalan, was seen in 1/8 drug-sensitive and 6/10 drug-resistant cell lines. In four cell lines p53-LOF was associated with mutations in the DNA binding region of p53, while three cell lines with LOF and four cell lines with functional p53 had no evidence of p53 muta-tions. Nonfunctional and mutated p53 was detected in one resistant cell line, while a sensitive cell line derived from the same patient prior to treatment had functional and wild type (wt) p53. We transfected HPV 16 E6 (which mediates degradation of p53, causing LOF) into two drug-sensitive neuroblastoma cell lines with functional p53. LC(90) values of HPV 16 E6 transfected cell lines were 3-7-fold (melphalan), 8-109-fold (carboplatin), and 2-158-fold (etoposide) greater than that of LXSN-transfected controls. CONCLUSIONS: These data suggest that some neuroblastomas acquire p53 mutations during therapy, which is associated with a loss of p53 function, and can confer high-level multidrug resistance.     

小儿常见恶性实体肿瘤耐药性分析

目的 通过对小儿常见的神经母细胞瘤、肾母细胞瘤、横纹肌肉瘤和恶性畸胎瘤的药敏检测,讨论各种常用化疗方案的治疗价值。方法 用17例肿瘤标本,其中神经母细胞瘤5例,化疗方案CCSG。肾母细胞瘤4例,化疗方案为DD方案。横纹肌肉瘤5例,CYVADIC:方案。恶性畸胎瘤3例,PVB方案。用流式细胞术荧光标记法,测定化疗药物敏感性。分析化疗疗效与耐药性。 结果 神经母细胞瘤对烷化剂类(CTX,IFO)、抗代谢药物(ACD)、VM26、TAL敏感性较高(P<0.05),化疗方案与药敏符合率约50％。肾母细胞瘤敏感的药物较多,化疗方案与药敏结果符合率约66.7％。以上两者疗效较好。横纹肌肉瘤耐药性较强。恶性畸胎瘤药敏对化疗方案符合率仪33_3％。两者疗效较差。结论 小儿实体恶性肿瘤现行化疗方案与肿瘤实际的药敏结果有一定的差距。用流式细胞仪测定肿瘤药物敏感性对预测化疗疗效及个体化化疗有指导意义     

神经母细胞瘤组织及裸鼠荷瘤细胞RECK和MMP-14的表达及临床意义

目的检测RECK及膜型基质金属蛋白酶-14（MMP-14）在神经母细胞瘤患者蜡块标本中的表达,进一步检测将患者新鲜肿瘤标本原代培养建立的细胞系接种于裸鼠后自建的裸鼠原位荷瘤及转移瘤细胞系中二者的表达,比较裸鼠两种细胞中二者的表达差异及临床意义。方法首先采用PV-6000免疫组化方法检测临床及病理资料齐全的存档神经母细胞瘤蜡块标本36例及节细胞神经瘤蜡块标本10例中RECK及MMP-14蛋向的表达。其次应用RT-PCR法检测自建裸鼠原位荷瘤及转移瘤细胞中RECK及MMP-14mRNA的表达。结果RECK蛋白在神经母细胞瘤组织中低表达（16．7％）,并随肿瘤浸润深度的加深及远处转移的发生而降低（P=0．015）。MMP-14蛋白在神经母细胞瘤组织中高表达（58．3％）,并随肿瘤浸润深度的加深及远处转移的发生而增高（P=0．002）。裸鼠原位荷瘤细胞系中RECKmRNA的表达较裸鼠转移瘤细胞系中明碾增高（t=3．012,P〈0．05）,MMP-14mRNA表达则明显降低（t=2．802,P〈0．05）。结论本研究提示,RECK及MMP-14蛋白和基因可能与神经母细胞瘤的浸润、转移有关,RECK及MMP-14可能在小儿神经母细胞瘤的发生、发展中起重要作用。