Functional substance P receptors on a human astrocytoma cell line (U-373 MG)

[125I]Bolton Hunter conjugate of substance P ([125I]BHSP) can bind to human astrocytoma membranes in a monophasic and saturable manner with a Kd of 0.57 +/- 0.17 nM and a Bmax of 67.8 +/- 5.5 fmol/mg protein. The rank order of potency of tachykinins and related analogues as inhibitors of [125I]BHSP binding to astrocytoma membranes and intact cells correlated with their relative abilities to stimulate uridine incorporation into nucleic acid. The observed specificity pattern conformed to that reported for the NK1 tachykinin receptor with SP much greater than eledoisin greater than neurokinin A greater than neurokinin B and [Glp6, L-Pro9]SP(6-11) much greater than [Glp6, D-Pro9]SP(6-11).     

人CD80基因转染U251瘤株细胞的实验研究

目的 构建编码人CD80基因的真核表达载体，转染神经胶质瘤细胞株U251，并检测CD80基因在U251中的表达。方法 PCR法扩增人类CD80基因的开放阅读框（ORF）全长，酶切法连接入真核表达载体pcDNA3．1。构建为重组质粒pcDNA3．1／CD80，双酶切及测序鉴定。利用脂质体法将pcDNA3．1／CD80转染U251细胞株，应用RT-PCR、流式细胞术、免疫细胞化学法、Western blotting等技术从细胞及分子水平检测人CD80分子在U251细胞表面的表达情况。结果 重组质粒pcDNA3．1／CD80双酶切可切出约900bp目的片段。测序结果和NCBI检索CD80序列吻合；RT-PCR检测到pcDNA3．1／CD80转染后的U251细胞中CD80mRNA表达；荧光显微镜下观察可见大量免疫荧光标记的绿色荧光细胞，检测转染效率约31．8％；Western blotting可检测到约60kD蛋白条带。结论 本实验成功构建了pcDNA3．1／CD80真核表达载体，并实现了在神经胶质瘤细胞株U251中的表达，为后续研究CD80的表达对肿瘤细胞免疫原性的影响以及制备神经胶质瘤肿瘤疫苗提供了重要的实验材料。     

Mobilization of intracellular calcium by substance p in a human astrocytoma cell line (U-373 MG)

Variations in intracellular free calcium concentration (Δ[Ca²⁺]_i) were measured in intact and isolated human astrocytoma cells (U373 MG) loaded with fura-2 acetoxymethylester. Microperfusion of 50 nM substance P (SP), applied for 1 s, increased [Ca²⁺]_i by 351 nM from a stable basal level of [Ca²⁺]_i of 26 nM. The peak Δ[Ca²⁺]_i induced by SP was dose dependent with a threshold of 10_-3 nM, an ED₅₀ of 1.3 nM and a maximal effect for concentrations of SP greater than 100 nM. The NKI receptor agonist, [Sar⁹Met(O₂)¹¹]SP, mimicked the effect of SP, while the NK₂ and NK₃ selective receptor agonists, [N1¹⁰]NKA(4-10) and senktide, respectively, had no effect. The Δ[Ca²⁺]_i induced by SP was unaffected by 100 μM cadmium or by removal of extracellular calcium ions. Caffeine up to 30 mM had no effect on [Ca²⁺]_i. In contrast, thapsigargin increased resting [Ca²⁺]_i by 92 nM and reduced the Δ[Ca²⁺]_i induced by SP. A pertussis treatment (500 ng/ml-24 h) did not modify the Δ[Ca²⁺]_i induced by SP. We conclude that SP, acting on a NK1 receptor, mobilizes cytosolic calcium from an intracellular calcium pool which can be partially depleted by thapsigargin. © 1994 Wiley-Liss, Inc.     

Localization and biosynthesis of functional thrombomodulin in human megakaryocytes and a human megakaryoblastic cell line (MEG-01)

The localization and biosynthesis of functional thrombomodulin (TM) on the cell surfaces of human platelets, megakaryocytes and a human megakaryoblastic cell line (MEG-01) were investigated. TM was demonstrated on the cell surfaces and in cytoplasms of human platelets, megakaryocytes and MEG-01 by an indirect immunofluorescent technique using monospecific rabbit anti-human TM serum. Immunoelectronmicroscopic analysis revealed that TM was localized in plasma membranes of MEG-01 cells as well as human megakaryocytes. 125I-monoclonal antithrombomodulin IgG binding assay showed that one MEG-01 cell possessed approximately 78,000 TM molecules on its cell surface. Thrombin-dependent protein C activating-cofactor activity was demonstrated on MEG-01 cells. Northern hybridization technique using cDNA probe of TM revealed that poly(A)(+)-RNA from MEG-01 cells showed a single band of 3.8 kb similar to that from human endothelial cells. These data suggest that human megakaryocytes synthesize functional TM, and thereby platelets possess TM on their surfaces. TM on platelets may participate in the activation of protein C at the site of a hemostatic plug.     

全反式维甲酸抑制MDM2基因在胶质瘤细胞SHG-44中的表达

目的 探讨全反式维甲酸对胶质瘤细胞SHG-44中MDM2基因表达的影响,为进一步研究脑胶质瘤的进展机制及基因治疗提供依据.方法 分别利用cDNA微阵列与Western blot技术分析在10μmol/L全反式维甲酸(all-transretinoic acid,ATRA)处理前后的胶质瘤SHG-44细胞MDM2基因和蛋白的差异表达应用免疫组化链霉菌抗生物素蛋白-过氧化酶(Streptavidin-Peroxidase,SP)法检测Ⅱ级与Ⅳ级胶质瘤标本MDM2蛋白的表达.随机选择数个差异基因进行Northern杂交实验,以验证cDNA微阵列的结果.结果 应用cDNA微阵列检测发现,MDM2基因在ATRA处理与未处理的SHG-44细胞之间表达量的比值为0.37,提示ATRA可抑制MDM2基因在SHG-44中的表达.该结果进一步得Northern杂交实验结果的支持.Western blot分析结果显示10μmol/L ATRA处理前后胶质瘤SHG-44细胞之间MDM2蛋白的相对表达量分别为21.40±0.58和14.02±0.35(t=24.728,P=0.000),提示MDM2蛋白在SHG-44中的表达受到ATRA抑制.Ⅱ级和Ⅳ级胶质瘤标本MDM2蛋白的阳性表达率分别为24.00%(6/25)和56.52%(13/23)(X2=5.298,P=0.021),MDM2蛋白的表达随胶质瘤恶性程度的增高而增加.结论 ATRA可抑制SHG-44胶质瘤细胞中MDM2基因的表达,MDM2基因的表达水平与胶质瘤的演进有关.     

Overexpression of human reticulon 3 (hRTN3) in astrocytoma

Neuroendocrine-specific protein (NSP) reticulons (RTN) are endoplasmic reticulum-associated protein complexes, which are localized in the endoplasmic reticulum (ER) and identified as markers for neuroendocrine differentiation. At least 4 different RTN genes have been identified in mammals, but in most cases, the functions of the encoded proteins except mammalian RTN4-A and RTN4-B are still elusive. In the present study, the expression of human reticulon 3 (hRTN3) in a variety of tissues was investigated. Northern blotting hybridization revealed higher expression of hRTN3 in normal brain and some endocrine-related organs such as thyroid relative to other non-endocrine organs. Twelve surgical specimens meeting the histological criteria for astrocytoma grade II, III and IV were investigated by in situ hybridization and immunohistochemical analysis, which demonstrated the overexpression of hRTN3 in astrocytoma tumor cells, while in non-cancerous brain tissues hRTN3 was mainly expressed in neurons and almost no signals in glia cells could be detected. The differential expression of hRTN3 between astrocytoma and non-cancerous tissue may provide insight into the progress of astrocytoma.     

Effect of changes in the CD44 gene on tumour cell invasion in gliomas

H. Bouterfa, M. Janka, E. Meese, S. Kerkau, K. Roosen & J. C. Tonn (1997) Neuropathology and Applied Neurobiology 23, 373–379
Effect of changes in the CD44 gene on tumour cell invasion in gliomas
Cell adhesion is a critical factor in the multistep process of tumour invasion. CD44 is one of the cell surface adhesion molecules responsible for interaction with hyaluronic acid, a component of the CNS extracellular matrix. The aim of the present study was to demonstrate whether alterations in the CD44 gene might account for different invasive behaviour. EcoRI restriction analysis by Southern blot hybridization revealed several additional hybridization signals in tissue specimens of two out of 16 patients with glioblastoma, indicating DNA rearrangements or point mutations, respectively, within the region of the CD44 gene. Expression patterns of CD44 isoforms in these two rearranged gliomas and in 28 other patients with malignant gliomas were analysed by RT-PCR. All cases displayed only the splice variant CD44H, which acts as hyaluronic acid receptor in glioma tumour cells. Tumour cell invasion was studied with Boyden chamber assays using hyaluronic acid as ligand and functional CD44H blocking antibody. Invasion of cells derived from those gliomas carrying the rearranged CD44 gene locus was decreased by about 50% compared with gliomas without rearrangement, indicating that the altered hybridization patterns in the two glioma samples influenced CD44H mediated glioma cell invasion through hyaluronic acid in vitro . Our results on CD44 isoform expression suggest that, in contrast to other solid tumours, gliomas seem to express only the CD44 variant. Genetic alterations within the CD44 gene might alter the binding domain of the receptor and thus account for different invasive behaviour in glioblastomas.     

CD44s、CD44v6在颅内转移瘤与胶质母细胞瘤中的表达研究

目的:探讨粘附分子CD44基因蛋白在颅内转移瘤及胶质母细胞瘤中的表达及其与这些肿瘤侵袭、转移之间的关系。方法:应用标准型CD44(CD44s)和变异型CD44v6基因蛋白单克隆抗体,微波-LSAB免疫组化染色检测20例正常脑组织、35例脑胶质母细胞瘤和30例颅内转移瘤中CD44基因蛋白的表达情况。结果:20例正常脑组织中CD44s和CD44v6表达均为阴性:35例脑胶质母细胞瘤CD44s阳性表达率为100％(35／35),CD44v6表达为阴性:30例颅内转移瘤中CD44s阳性表达率为86.7％(26／30),CD44v6阳性表达率为66.7％(20／30)。CD44v6在颅内转移瘤及脑胶质母细胞瘤的表达差异有显著性(P<0.01)。结论:脑胶质母细胞瘤中CD44S的表达可能与其脑内侵袭过程有关,无CD44v6表达可能与其很少发生颅外转移有关,并有可能成为颅内转移瘤诊治的有用指标之一。     

Localization of a neurectoderm-associated cell surface antigen in the developing and adult rat

The distribution of a neurectoderm-associated carbohydrate antigen (termed D1.1) in tissues of the developing and adult rat was determined using indirect immunofluorescent techniques. The antigen was detected as early as embryonic days 8 and 9 when it was localized to cells within the developing neural plate and neural tube. As the central nervous system (CNS) developed, the anti-D1.1 antibody labeled neuroepithelial cells but not terminally differentiated neurons or glial cells. In addition, the notochord and somatic mesoderm were labeled transiently with the antibody. Outside of the CNS, the antibody labeled dorsal root ganglia neurons, adrenal chromaffin cells and cells of the kidney glomerulus. These tissues were labeled at embryonic day 14 and the labeling persisted in the adult. We used a sensitive immunoautoradiography assay to identify antigenic gangliosides present in extracts of these tissues. The anti-D1.1 antibody recognized a ganglioside of kidney and adrenal glands that has a chromatographic mobility identical to that of the D1.1 antigen previously identified from cell lines and developing cerebellum. However, the antibody bound to a separate and distinct set of gangliosides present in extracts of adult dorsal root ganglia. Thus, the carbohydrate sequence recognized by the antibody can be associated with more than one molecular species of ganglioside. These results demonstrate that within the context of the developing CNS, the D1.1 antigen is a stage-specific embryonic antigen, but, as is the case with other cell surface carbohydrate antigens, is also found on a limited but developmentally unrelated set of tissues in the adult.     

Tumorigenic cell culture lines from a spontaneous VM/Dk murine astrocytoma (SMA)

Summary One of the few spontaneous gliomas in inbred animals, the VM/Dk spontaneous murine astrocytoma (SMA), has seen limited use. Previously restricted to an in vivo system, the SMA was only transplantable intracerebrally (IC) using nonquantifiable suspensions of normal brain and tumor tissue. Prior atempts at establishing permanent tumorigenic SMA cell lines have not succeeded; tumorigenicity was lost during serial in vitro passage. We have established three different cell culture lines from a serially IC-transplanted SMA and two from tumors that arose from intraperitoneal (IP) injection of the IC-transplanted SMA. In contrast to previous cell cultures and transplantable lines of SMA, all five cell lines are not only tumorigenic IC but subcutaneously (SC) as well. Astrocytic features are present in three of five lines to varying degrees, evidenced by in vitro and in vivo morphology, response to dibutyryl cyclic AMP (db-cAMP), and the presence of neuroglial fibers: None of the lines express CNPase, S-100, or GFA proteins in significant amounts. P560, highly tumorigenic and possessing the most astrocytic features of the five lines, extends the use of the spontaneous astrocytoma system of the inbred VM/Dk mouse strain by allowing quantitative in vivo and in vitro experiments.Supported by NCI Grants CA 11898, CA 22790, and USPHS Grant 5T32-AG-00007     

缺氧诱导SHG44细胞拟态血管形成及相关机制

目的探索缺氧对Ⅱ-Ⅲ级人脑星形细胞瘤细胞血管生成拟态(VM)的诱导作用及其相关机制。方法应用化学缺氧法诱导人脑星形细胞瘤SHG44细胞(Ⅱ-Ⅲ级)在三维培养体系中形成血管生成拟态,采用Transwell法检测细胞的迁移和侵袭能力,用Western blot和逆转录聚合酶链反应(RT-PCR)检测血管内皮生长因子(VEGF)、酪氨酸蛋白激酶受体A2(EphA2)、基质金属蛋白酶2(MMP2)、MMP9和血管内皮钙粘蛋白(VE-cadherin)的表达,探讨缺氧诱导血管生成拟态的机制。结果在三维培养体系中,缺氧能诱导SHG44细胞相互连接,相互融合形成管状结构。缺氧组平均每视野管状结构数量为56.80±12.21,对照组为4.20±2.62,两组相比具有统计学差异(P<0.01)。缺氧组细胞侵袭数目为64.56±16.40,细胞迁移数目为178.71±18.81,明显高于对照组(P<0.01),与血管生成拟态的形成呈正相关。VEGF、EphA2、MMP2、MMP9的表达也明显增强,但是VE-cadherin的表达无明显改变。结论缺氧能够诱导SHG44细胞形成血管生成拟态,VEGF-EphA2-MMPs-VM可能是...     

Hyaluronate receptor (CD44) and integrin alpha4 (CD49d) are up-regulated on T cells during MS relapses

A longitudinal study of peripheral blood T cell adhesion molecule expression was performed in 24 relapsing-remitting MS patients. There were 15 relapses in 11 patients during 15 months of observation. In comparison with remission, expression of hyaluronate receptor (CD44) was highly significant, and expression of integrin alpha4 (CD49d, VLA-4) significantly up-regulated during relapses. CD44 and CD49d are putative activity markers and CD44 a potential novel therapeutic target in MS.     

人脑星形细胞肿瘤CD133和巢蛋白阳性细胞的分布特征

目的 探讨CD133 和巢蛋白阳性细胞在人脑星形细胞肿瘤中的分布特征.方法 采集不同级别星形细胞肿瘤标本31 例,免疫组织化学染色检测其胶质瘤干细胞标志物CD133 和巢蛋白表达水平,光学显微镜下观察CD133 和巢蛋白阳性细胞在不同级别星形细胞肿瘤中的分布特征.结果 不同级别肿瘤之间和同一肿瘤不同区域之间CD133 阳性细胞的分布呈现极大变异性,低级别星形细胞肿瘤完全不表达或仅单个细胞表达,高级别星形细胞肿瘤成簇表达,其特异性分布特征为CD133 阳性细胞围绕微血管分布,单个细胞贴附于血管外壁,在肿瘤周围水肿区域亦可呈散在分布而不贴附于血管外壁;簇状或巢状分布的CD133 阳性细胞均位于近血管区域,CD133 阳性细胞形态亦不尽一致,无论是新生发芽的肿瘤性血管内皮细胞还是成熟的血管内皮细胞均不表达CD133.巢蛋白阳性细胞在不同级别肿瘤之间和同一肿瘤不同区域之间具有与CD133 阳性细胞相似的分布特征,但新生发芽的肿瘤性血管内皮细胞巢蛋白表达阳性;变性肿瘤中巢蛋白阳性细胞可呈" 开屏状"分布,而CD133 阳性细胞未见类似分布.结论 CD133 和巢蛋白阳性细胞在人脑星形细胞肿瘤中呈现近微血管分布和富集于肿瘤周围水肿区域的分布特征,新生发芽的肿瘤性血管内皮细胞巢蛋白表达阳性.     

Subependymal giant cell astrocytoma (SEGA): Is it an astrocytoma? Morphological,immunohistochemical and ultrastructural study

Subependymal giant‐cell astrocytoma (SEGA) is a rare intra‐ventricular low‐grade tumor which frequently occurs as a manifestation of tuberous sclerosis complex. The histogenesis of SEGA is controversial and its astrocytic nature has been doubted. First studies suggested the astrocytic nature of SEGA while several recent reports demonstrate its glio‐neuronal nature. In spite of this, in the recently revised WHO classification of the CNS tumors, SEGA has been still included in the group of astrocytomas. We studied nine tuberous sclerosis complex‐associated SEGAs. Patients were 1–18 years old. Eight patients (89%) had a solitary lesion located in the lateral ventricle close to of the head of the caudate nucleus, the remaining patient (11%) had two tumors, one located close to the head of the left caudate nucleus and the other in the central part of the right lateral ventricle. Histologically, tumors were composed of three types of cells: spindle, gemistocytic and ganglion‐like. Four tumors (44%) had a prominent vascularization and three (33%) showed an angiocentric pattern. Calcifications were observed in six cases (66%). By immunohistochemistry, the majority of the tumors were GFAP‐ (9; 100%), neurofilament‐ (8, 89%), neuron‐specific enolase‐ (9, 100%), and synaptophysin‐ (8; 89%) positive. Ultrastructural studies were performed on four cases. In all four there were glial cell processes filled with intermediate filaments. In one case dense core putative neurosecretory granules were appreciable. Our results emphasize the glio‐neuronal nature of SEGA. We suggest moving it into the group of mixed glio‐neuronal tumors under the denomination of subependymal giant cell tumor.     

Perinatal (fetal and neonatal) astrocytoma: a review

Introduction

The purpose of this review is to document the various types of astrocytoma that occur in the fetus and neonate, their locations, initial findings, pathology, and outcome. Data are presented that show which patients are likely to survive or benefit from treatment compared with those who are unlikely to respond.

Materials and methods

One hundred one fetal and neonatal tumors were collected from the literature for study.

Results

Macrocephaly and an intracranial mass were the most common initial findings. Overall, hydrocephalus and intracranial hemorrhage were next. Glioblastoma (GBM) was the most common neoplasm followed in order by subependymal giant cell astrocytoma (SEGA), low-grade astrocytoma, anaplastic astrocytoma, and desmoplastic infantile astrocytoma (DIA). Tumors were detected most often toward the end of the third trimester of pregnancy.

Conclusion

A number of patients were considered inoperable since their tumor occupied much of the intracranial cavity involving large areas of the brain. High-grade astrocytomas were more common than low-grade ones in this review. Fetuses and neonates with astrocytoma have a mixed prognosis ranging from as low as 20 % (GBM) to a high of 90 %. The overall survival was 47/101 or 46 %.

     

Interleukin-6 production by U373 MG, a human astrocytoma cell line: different pathways involved in substance P and lipopolysaccharide activation

Substance P (SP) and lipopolysaccharide (LPS) stimulated interleukin-6 (IL-6) gene expression, as well as IL-6 protein secretion in the human astrocytoma cell line U373 MG. Staurosporine, an inhibitor of protein kinase C (PKC), entirely blocked SP- but not LPS-induced IL-6 release. In addition, the down regulation of PKC inhibited the SP response and only marginally altered LPS activation. Differently from SP, LPS-induced IL-6 release was markedly reduced by W7, a calmodulin antagonist. Moreover, SP interacted in a synergistic manner with LPS. Thus, neural (SP) and bacterial (LPS) mediators stimulate U373 MG IL-6 release via distinct, though not antagonistic, activation pathways.     

基质金属蛋白酶(MMP-2、MMP-9)在人脑星形胶质细胞瘤的表达

目的 探讨基质金属蛋白酶(matrix metalloproteinases，MMP)与人脑星形胶质细胞瘤浸润性生长之间的关系，以及基质金属蛋白酶的阳性表达与星形胶质细胞瘤病理分级的关系。方法 应用免疫组织化学染色法(SP法)检测44例人脑星形胶质细胞瘤组织中MMP-2、MMP-9的表达。结果 Ⅲ、Ⅳ级中MMP-2、MMP-9蛋白的表达显著高于Ⅰ、Ⅱ级，而正常脑组织中无表达；在人脑星形胶质细胞瘤中MMP-2和MMP-9的表达之间无明显相关性。结论 MMP-2、MMP-9的表达与星形胶质细胞瘤的恶性程度有关，其高表达可能与星形胶质细胞瘤的侵袭转移有关。