Pituitary adenylyl cyclase activating polypeptide inhibits gli1 gene expression and proliferation in primary medulloblastoma derived tumorsphere cultures.
Pituitary adenylyl cyclase activating polypeptide inhibits gli1 gene expression and proliferation in primary medulloblastoma derived tumorsphere cultures.
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DOI：
10.1186/1471-2407-10-676
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发表时间：
2010-12-09
期刊：
影响因子：
3.8
通讯作者：
Waschek JA
中科院分区：
文献类型：
--
作者：
Cohen JR;Resnick DZ;Niewiadomski P;Dong H;Liau LM;Waschek JA
在小脑发育过程中，Hedgehog （HH）信号对于外颗粒层（EGL）内颗粒神经元前体（GNPs）的扩张至关重要。GNPs中异常的HH信号被认为会导致成神经管细胞瘤（MB），这是最常见的儿童恶性脑肿瘤。无脊椎动物和脊椎动物的证据表明，环amp依赖性蛋白激酶A （PKA）拮抗HH信号传导。垂体腺苷酸环化酶激活多肽（PACAP，基因名ADCYAP1）的特异性受体在GNPs中表达。PACAP已被证明可保护GNPs免于体外凋亡，并与HH信号相互作用以调节GNP增殖。与ptch1小鼠相比，PACAP/ptch1双突变小鼠的MB发病率增加，表明PACAP可能调节HH通路介导的MB发病机制。从13只ptch1+/-/p53+/-双突变小鼠中制备原代MB肿瘤球培养物，并使用平滑化（SMO）激动剂purmorphamine、SMO拮抗剂sant1、神经肽PACAP、PKA激活剂forskolin和PKA抑制剂H89进行处理。分别评估gli1和[3H]-胸苷结合基因表达，以确定药物对HH通路活性和增殖的影响。细胞提取物中PKA活性通过Western blotting测定，采用磷酸PKA底物抗体。在无血清条件下培养1周的原代肿瘤细胞生长为肿瘤球，并发现表达PAC1受体转录物。Gli1基因的表达在santo -1、PACAP和forskolin的作用下明显降低，而在purmorphamine的作用下则不受影响。PKA抑制剂H89可以逆转PACAP对gli1基因表达的抑制作用，同时阻断PKA的激活。用PACAP、福斯克林和st -1处理肿瘤球培养24或48小时可减少增殖。来源于ptch1+/-/p53+/-小鼠的原发肿瘤球表现出组成性HH通路活性。PACAP以PKA拮抗剂H89阻断的方式拮抗这些细胞中的HH信号。PACAP和PKA的药理激活也能抑制细胞增殖。我们的数据表明，通过PACAP/PKA信号调节HH信号可能为治疗MB提供了一种替代SMO抑制的方法。
Hedgehog (HH) signaling is critical for the expansion of granule neuron precursors (GNPs) within the external granular layer (EGL) during cerebellar development. Aberrant HH signaling within GNPs is thought to give rise to medulloblastoma (MB) - the most commonly-observed form of malignant pediatric brain tumor. Evidence in both invertebrates and vertebrates indicates that cyclic AMP-dependent protein kinase A (PKA) antagonizes HH signalling. Receptors specific for the neuropeptide pituitary adenylyl cyclase activating polypeptide (PACAP, gene name ADCYAP1) are expressed in GNPs. PACAP has been shown to protect GNPs from apoptosis in vitro, and to interact with HH signaling to regulate GNP proliferation. PACAP/ptch1 double mutant mice exhibit an increased incidence of MB compared to ptch1 mice, indicating that PACAP may regulate HH pathway-mediated MB pathogenesis. Primary MB tumorsphere cultures were prepared from thirteen ptch1+/-/p53+/- double mutant mice and treated with the smoothened (SMO) agonist purmorphamine, the SMO antagonist SANT-1, the neuropeptide PACAP, the PKA activator forskolin, and the PKA inhibitor H89. Gene expression of gli1 and [3H]-thymidine incorporation were assessed to determine drug effects on HH pathway activity and proliferation, respectively. PKA activity was determined in cell extracts by Western blotting using a phospho-PKA substrate antibody. Primary tumor cells cultured for 1-week under serum-free conditions grew as tumorspheres and were found to express PAC1 receptor transcripts. Gli1 gene expression was significantly reduced by SANT-1, PACAP and forskolin, but was unaffected by purmorphamine. The attenuation of gli1 gene expression by PACAP was reversed by the PKA inhibitor H89, which also blocked PKA activation. Treatment of tumorsphere cultures with PACAP, forskolin, and SANT-1 for 24 or 48 hours reduced proliferation. Primary tumorspheres derived from ptch1+/-/p53+/- mice exhibit constitutive HH pathway activity. PACAP antagonizes HH signalling in these cells in a manner blocked by the PKA antagonist H89. PACAP and pharmacological activation of PKA also inhibited proliferation. Our data suggests that regulation of HH signaling by PACAP/PKA signaling may provide an alternative to SMO inhibition for the treatment of MB.
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影响因子：
2
作者：
Cohen, MM
通讯作者：
Cohen, MM
影响因子：
64.8
作者：
DONEHOWER, LA;HARVEY, M;BRADLEY, A
通讯作者：
BRADLEY, A
影响因子：
4.6
作者：
Palma, V;Lim, DA;Altaba, ARI
通讯作者：
Altaba, ARI
影响因子：
5.3
作者：
KINZLER, KW;VOGELSTEIN, B
通讯作者：
VOGELSTEIN, B
影响因子：
4.8
作者：
Hahn, H;Christiansen, J;Wainwright, B
通讯作者：
Wainwright, B