Cell and Molecular Genetic Analysis of Pediatric Tumors

小儿肿瘤的细胞和分子遗传学分析

• 批准号: 7292027
• 负责人: Carol J Thiele
• 金额: --
• 依托单位: DIVISION OF CLINICAL SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7292027
• 关键词: Cell; Molecular; Genetic; Analysis; Pediatric

The focus of the CMBS is pediatric neuroectodermal tumors including neuroblastoma, brain tumors and Ewings sarcoma.Differentiation and NeuroblastomaSeveral clinical and biologic attributes of pediatric neuroectodermal tumors indicate that alterations in differentiation programs contribute to tumorigenesis. We have utilized the retinoid induced differentiation of neuroblastoma tumor cell lines as a model to define the signal transduction paths that mediate growth control, differentiation and cell death. Through a variety of molecular approaches we have identified a number of genes that are important in neuroblastoma differentiation, localize to cytogenetic regions altered in Neuroblastoma and/or have prognostic significance. Our studies are aimed at assessing how these signaling paths control neuroblastoma differentiation and affect tumor cell biology. Clinically relevant strategies are employed to activate or inhibit signaling paths in order to translate these findings into new therapeutic approaches. Trks and NB Biology In NB tumors, Trks serve as tumor markers; TrkA is expressed in good prognosis tumors. We have found that the differential activation of these signal transduction pathways in NB may alter their growth, invasiveness, chemosensitivity and cell survival. Recently we published that NGF activation of TrkA causes a transcriptional decrease in N-myc levels via activation of the MAPkinase path. Furthermore the decrease in N-myc causes a decrease in cell cycle progression that is mediated by decreases in Cyclin E dependent kinase activity, cdk6 levels and E2F transcription factors expression as well as an increase in p27. By analyzing the key components of several mechanisms required for arresting growth of NB cells we have identified that decreases in N-myc and cdk6 and increases in p27 are key control elements in regulating NB cell cycle progression. Neuroblastoma (NB) tumors expressing high levels of BDNF and TrkB are associated with poor 5-year survival outcomes. Our previous studies indicated that BDNF blocked the cytotoxic effects of vinblastine on NB cells. We evaluated the ability of BDNF to decrease the chemosensitivity of NB cells to a number of common chemotherapeutic agents and mapped the BDNF signaling system that mediates the chemoprotective effect. Two SH-SY5Y NB cell lines (TB3, TB8) expressing TrkB under the control of a tetracycline (Tet)-repressible promoter element were isolated, and used to assess apoptosis resulting from treatment with cisplatin (Cis), doxorubicin (Doxo), etoposide (Etop), and vinblastine (Vbl). BDNF treatment of high TrkB-expressing TB8 (Tet-) and TB3 (Tet-) cells blocked drug-induced cell death in a dose-dependent manner. Only high dose BDNF blocks the effects of chemotherapy in cell with low TrkB expressioin while even low doses of BDNF affect the sensitivity of cells expressing high levels of TrkB. This indicates that the environmental level of TrkB lignads may differentially affect the chemosensitivity of NB cells. The inability of NGF to protect cells indicated that activation of p75 alone was not responsible for the chemoprotective effect. BDNF's ability to rescue the cells is TrkB dependent since the selective Trk tyrosine kinase inhibitor K252a blocks it. The PI3-kinase inhibitors LY294002 and Wortmannin but not the MEK inhibitor PD98059 or the PLC-gamma inhibitor U73122 block the ability of BDNF to rescue cells from etoposide and cisplatin induced cell death. BDNF also protects NGP and KCNR NB cells expressing endogenous TrkB receptors from chemotherapy induced death and inhibition of the PI3-kinase path abrogates this effect. These results indicate that BDNF activation of TrkB via the PI3-kinase path protects NB cells from chemotherapy and that by specifically inhibiting the TrkB TK and/or PI3-kinase paths one may improve the chemosensitivity of NB cells.

CMB的重点是儿科神经皮质肿瘤，包括神经母细胞瘤，脑肿瘤和ewings肉瘤。差异和神经细胞酶的临床临床和生物学属性以及儿科神经外皮瘤的生物学属性，表明分化程序的变化对分化的肿瘤造成了肿瘤的变化。我们利用了类维生素类动物诱导的神经母细胞瘤细胞系的分化为定义介导生长控制，分化和细胞死亡的信号转导路径的模型。通过多种分子方法，我们已经确定了许多在神经母细胞瘤分化中很重要的基因，这些基因位于神经母细胞瘤中改变的细胞遗传学区域和/或具有预后意义。我们的研究旨在评估这些信号通路如何控制神经母细胞瘤分化并影响肿瘤细胞生物学。采用临床上相关的策略来激活或抑制信号通路，以将这些发现转化为新的治疗方法。 TRK和NB生物学 在NB肿瘤中，TRK充当肿瘤标记。 TRKA以良好的预后肿瘤表示。我们发现，NB中这些信号转导途径的差异激活可能会改变其生长，侵入性，化学敏感性和细胞存活。最近，我们发表了TRKA的NGF激活通过MAPIKINASE路径的激活导致N-MYC水平的转录降低。此外，N-MYC的降低会导致细胞周期进程的降低，这是由细胞周期蛋白E依赖性激酶活性，CDK6水平和E2F转录因子表达以及P27的增加所介导的。通过分析阻止NB细胞生长所需的几种机制的关键组成部分，我们已经确定N-MYC和CDK6的降低以及P27的增加是调节NB细胞周期进程的关键控制元素。表达高水平BDNF和TRKB的神经母细胞瘤（NB）肿瘤与5年生存结果较差有关。我们先前的研究表明，BDNF阻断了长文碱对NB细胞的细胞毒性作用。我们评估了BDNF降低NB细胞对许多常见化学治疗剂的化学敏度的能力，并映射了介导化学保护作用的BDNF信号系统。分离了两种SH-SY5Y NB细胞系（TB3，TB8），它们在四环素（TET）控制下表达TRKB（TET） - 可抑制启动子元件，并用于评估用顺铂（CIS），doxorubibicin（doxo），Etoposides，Etoposoposide，et poposide，Etopoposide，e eToposide Conse trkb的凋亡。 （ETOP）和Vinblastine（VBL）。 BDNF对高TRKB表达TB8（TET-）和TB3（TET-）细胞的治疗以剂量依赖性方式阻断了药物诱导的细胞死亡。只有高剂量BDNF阻止了TRKB表达低的细胞中化学疗法的影响，而即使低剂量的BDNF也会影响表达高水平TRKB的细胞的敏感性。这表明TRKB木质木质的环境水平可能会差异地影响NB细胞的化学敏感性。 NGF无法保护细胞的能力表明，仅p75的激活并不负责化学保护作用。 BDNF拯救细胞的能力取决于TRKB，因为选择性TRK酪氨酸激酶抑制剂K252A阻止了它。 PI3-激酶抑制剂LY294002和WORTMANNIN，但没有MEK抑制剂PD98059或PLC-GAMMA抑制剂U73122阻止BDNF从依托泊苷和顺铂诱导的细胞死亡中拯救细胞的能力。 BDNF还保护NGP和KCNR NB细胞表达内源性TRKB受体免受化学疗法诱导的死亡和抑制PI3-激酶路径的抑制作用，从而消除了这种作用。这些结果表明，通过PI3-激酶路径的BDNF激活TRKB可以保护NB细胞免受化学疗法的影响，并通过专门抑制TRKB TK和/或PI3-激酶路径可以改善NB细胞的化学敏度。