Patterns of proto-oncogene expression in human glioma cell lines

Previously reported studies have suggested that variations in the pattern of proto-oncogene expression within a specific tumor type may denote an underlying difference in the biology and clinical behavior of those tumors. To more sensitively characterize malignant tumors of the central nervous system, we have used Northern blot hybridization analysis to determine the patterns of expression of seven proto-oncogenes in 20 cell lines established from biopsy specimens of patients with malignant glioma. The following proto-oncogenes are expressed at detectable levels in 30 micrograms of total RNA from most glioma cell lines examined: c-myc (20/20), c-mil/raf-1 (18/18), neu (19/20), c-erbB (19/20), and c-myb (17/20). In contrast, only half of the cell lines expressed detectable c-sis (10/20). In none of the cell lines tested was N-myc (0/20) mRNA detected. Morphologic analysis of these 20 cell lines revealed three different growth patterns: bipolar, epithelial, and pleomorphic-glial. Detectable levels of c-sis mRNA typically occurred with either an epithelial or pleomorphic-glial morphology. The pleomorphic-glial subgroup was also characterized by the expression of glial fibrillary acidic protein.     

胶质瘤细胞的ERCC—2基因表达和氯乙基亚硝脲耐药

目的探讨核苷酸切除修复交叉互补（ＥＲＣＣ）基因表达与肿瘤细胞对氯乙基亚硝脲（ＣＥＮＵ）耐药的关系。方法采用我们已建立的逆转录．聚合酶连锁反应（ＲＴ－ＰＣＲ）定量分析方法,对９株人胶质瘤细胞的ＥＲＣＣ－２基因表达进行测定,并与ＣＥＮＵ细胞毒性试验进行相关分析。结果ＥＲＣＣ－２基因表达与二氯乙基亚硝脲（ＢＣＮＵ）及二氯乙基肉芥子亚硝脲（ＳａｒＣＮＵ）耐药性之间有显著的正相关（ｒ＝０．６７２,Ｐ＝０．０４７和ｒ＝０．７２６,Ｐ＝０．０２８）。结论本研究提示ＥＲＣＣ－２基因表达在胶质瘤对亚硝脲类抗癌药耐药中起重要作用。     

ERCC2诱导表达在人脑胶质瘤细胞对BCNU耐药中的作用

目的：探讨BCNU诱导ERCC2表达在胶质瘤细胞耐药中的作用。方法：选用ERCC2基础表达在蛋白水平相同，而mRNA水平不同的2株人脑胶质瘤细胞T98-G和SK-MG-4（在mRNA水平，T98-G比SK-MG-4高25倍），在体外培养时用BCNU处理，随后采用Western Blot测定肿瘤细胞的ERCC2蛋白水平变化。结果：对BCNU耐药的T98-G在BCNU处理后6h，ERCC2蛋白水平上升6倍，随后逐渐下降，48h回复到基础水平。而对BCNU敏感的SK-MG-4，BCNU处理后ERCC2蛋白水平没有增加。结论：本研究提示，mRNA基础水平与胶质瘤耐药密切相关，在mRNA基础水平较高的肿瘤细胞，BCNU能诱导ERCC2蛋白表达，并直接与肿瘤耐药相关。     

Differential expression of glial fibrillary acidic protein in human glioma cell lines

Summary We have obtained a cDNA fragment to human glial fibrillary acidic protein (GFAP) by immunoscreening a gt11 human brain cDNA library with antibody to bovine GFAP. The highly homologous nucleotide sequence of this clone with that of the mouse GFAP enabled the identification of this cDNA as one encoding GFAP. As this cDNA hybridized with a single major RNA species in Northern blots of RNA from human and mouse brain tissues and gave one or two bands in Southern blots of human genomic DNA, it was considered to be specific for GFAP. Using this cDNA as a probe we investigated the levels of GFAP expression in ten human glioma cell lines. A 3.5-kb GFAP mRNA was detected in five of the ten glioma cell lines, one of which was U-251 MG cell line and the other four were clones derived from the same tumor (CL1, 2, 3, and 4). There was a difference in the amount of GFAP mRNA among U-251 MG and the four clonal cell lines. Quantitative evaluation of this difference by RNA dot blot analysis revealed that the amount of GFAP mRNA expressed in CL3 was about 1/5 and in CL4 about 1/10 the amount expressed in U-251 MG, CL1, and CL2. Semiquantitative Western blot analysis showed that GFAP levels corresponded to the GFAP mRNA levels in these cell lines. By Southern blot analysis of genomic DNA the GFAP gene was similarly detected in all of these cell lines regardless of the level of GFAP expression. Thus, by using a cDNa to human GFAP we have demonstrated the presence of clonal cell lines from human glioma showing different levels of GFAP expression, which may provide a useful basis for further investigations on the regulation of GFAP gene expression in glial cells.     

Characterization of four human malignant glioma cell lines

Summary In this paper, the characterization of four human malignant glioma cell lines is described. The four lines are positive for glial fibrillary acidic protein (GFAP) in variable amounts. One of them, LN 992, is positive for S-100 protein. Myelin basic protein could not be detected in any of the four lines. The four lines had high levels of CNPase activity. The karyotype shows polyploidy for all lines, with modal numbers ranging from 80 to 120 and various numbers of marker chromosomes. Particular attention has been paid to the surface phenotype and a panel of three antiglioma monoclonal antibodies (Mabs), five antimelanoma Mabs, one anti-CALLA Mab, and two anti-HLA-DR Mabs has been used in an antibody-binding radioimmunoassay for the four cell lines. Lines LN 215 and LN 235 are positive with two antiglioma Mabs, LN 992 is negative. The four lines are positive with all five antimelanoma Mabs, except for LN 992 which ist negative with Mab D5. LN 992 and LN 215 are positive with the anti-CALLA Mab N2A12. LN 308 and LN 992 are positive with anti-HLA-DR Mab D4-22. There was no correlation between the in vitro morphology of the lines and the expression of the various biochemical or surface markers. These results stress the heterogeneity of the phenotype of human malignant glioma lines. These lines will be useful tools for further immunologic studies.Supported by grant no. 3.314-082 from the Swiss National Foundation for Scientific Research and in part by grant no. 3.176.82 from the Swiss National Science Foundation and Multiple Sclerosis Society of Switzerland     

C-fos启动子在人胶质瘤细胞株及正常组织的转录活性研究

目的 观察C-fos、TERT、Survivin、E2F1、Cox2及CMV启动子在人胶质瘤细胞株U87、U373、U251中的转录活性以及C-fos、CMV启动子在正常组织中的转录活性. 方法 设计引物并应用PCR法从人胶质瘤基因组中克隆C-fos、TERT、Survivin、E2F1、Cox2启动子.将上述5种启动子以及CMV启动子序列插入到pGL4-Basic报告载体中.将构建的pGL4-Cfos、pGL4-TERT、pGL4-Survivin、pGL4-E2F1、pGL4-Cox2、pGL4-CMV重组质粒和内参质粒pRL-TK瞬时共转染胶质瘤细胞U87、U373、U251,双荧光素酶实验检测各启动子的转录活性;C57BL/6J小鼠10只,分为C-fos组、CMV组,每组5只,分别由尾静脉注射600 μL含40 μg pGL4-Cfos-luc、pGL4-CMV-luc质粒的生理盐水,24 h后检测小鼠心、肝、脾、肺组织中荧光素酶的活性. 结果 各启动子基因序列经测序确认正确;TERT、Survivin、Cox2、E2F1、C-fos启动子的转录活性(相对于CMV启动子)在U87细胞中依次为0.03％、0.33％、0.38％、0.24％、3.73％;在U373细胞中依次为0.95％、65.34％、0.32％、9.07％、70.83％,在U251细胞中依次为0.41％、15.57％、0.15％、0.51％、17.30％;CMV组小鼠心、肝、脾、肺组织荧光素酶活性依次为(93.40 ±75.06)RLU/mg蛋白、(12.33±6.49)RLU/mg蛋白、(22.60±7.50)RLU/mg蛋白、(950.47 ±538.74)RLU/mg蛋白;C-fos组小鼠心、肝、脾、肺组织荧光素酶活性依次为(2.13±1.92)RLU/mg蛋白、(1.11 ±0.95)RLU/mg蛋白、(2.07±1.51)RLU/mg蛋白、(29.24±25.89)RLU/mg蛋白,与CMV组比较差异均有统计学意义(P＜0.05). 结论 C-fos启动子在人胶质瘤细胞株中的转录活性高于TERT、Survivin、E2F1、Cox2,在正常组织中的转录活性低于CMV启动子.     

Hydroxyurea synchronization increases mitotic yield in human glioma cell lines

Summary Karyotypic studies of human gliomas are often limited by a low mitotic index and the appearance of contracted chromosomes that do not exhibit clear banding patterns. The purpose of this study was to investigate the use of hydroxyurea (HU) as a synchronizing agent using established human glioma cell lines as a model system. HU was shown to reversibly inhibit cellular replication in glioma cell lines U-251 MG, D-245MG, D-247MG and D-263MG by flow cytometry on the basis of DNA content. Two-to sixfold increases were demonstrated in the mitotic index of HU-treated cultures exhibiting the greatest percentage of cells in the G2/M phases of the cell cycle. HU, therefore, promises to be an effective agent for use in short term cultures from biopsied human tumors to increase the quality of chromosome preparations in these tumors.     

Robustness of gene expression profiling in glioma specimen samplings and derived cell lines

One of the most promising applications of microarrays is class distinction through gene expression profiling as a diagnostic tool. However, as there is apparent spatial heterogeneity in the morphology of cancer cells within a tumor, it is unclear if tumor sampling can be applied and yield consistent signals. In this report, we examined six brain tumors, four glioblastoma, and two oligodendroglioma biopsies. The six brain tumor tissues from two distinct different classes were dissected in four distinct areas and gene expression was profiled using microarrays. We used hierarchical clustering to compare the variability of gene expression profiles between spatially distinct biopsies of the same tumor as compared to the variability between tumors of the same histologic group. We conclude that, in general, repeat spatially distinct samples are not needed for microarray experiments and the gene expression signatures are robust across the tumor. Predominantly, variation was much greater between samples from different patients than from the multiple samplings of given tumor. Further, we compared biopsy expression profiles to the cell lines derived from those tissues. In general, the tumor cell lines vary greatly from the parental tissues and cluster more strongly with each other than the parental tissue. We select and examine the set of genes altered in expression to allow adaptation to cell culture.     

人脑胶质瘤细胞X线照射后癌基因表达的变化

目的:探讨癌基因表达的变化与辐射性细胞放射损伤的关系.方法:采用免疫组化技术检测人脑星形细胞瘤细胞系经X射线处理前后p53、c-myc、bcl-2基因表达的变化;采用原位杂交方法检测p53基因在mPNA水平表达的变化.结果:①处理组内p53、c-myc基因表达率明显高于对照组(P<0.001),bcl-2表达显著减弱(P<0.05),p53或c-myc与bcl-2基因表达呈显著性负相关(P<0.05).②处理组内p53在mRNA水平表达量明显高于对照组(P<0.05),p53基因在蛋白水平与mRNA水平表达呈显著性正相关(P<0.05),p53基因在蛋白水平与mRNA水平表达呈显著性正相关(P<0.05).结论:p53、c-myc和bcl-2基因在X线诱导的细胞损伤中起着重要的作用.本研究结果为脑胶质瘤的放射治疗和基因治疗提供了一条新线索.     

人脑胶质瘤细胞系miRNA表达谱初步研究

目的 确定部分人脑胶质瘤细胞系miRNA表达谱的初步特征.方法 提取人脑胶质母细胞瘤细胞系U251、TJ861、TJ905、TJ899和A172以及星形细胞瘤细胞系H4细胞的miRNA,miRNA微阵列芯片杂交,扫描后分析差异表达的miRNA.选择差异表达的miR-21设计反义寡核苷酸处理U251细胞后原位杂交和Western blot检查SEPT 7的表达.结果 在胶质瘤细胞系中hsa-miR-21等8种miRNA一致表达上调;hsa-miR-1等18种miRNA一致表达下调.miR-21锁核酸修饰反义寡核苷酸处理U251细胞72h后,原位杂交发现阳性信号集中在细胞核的miR-21表达显著下降,Western blot发现U251细胞中SEPT 7的表达明显上调.结论 miRNA的差异表达可能是胶质瘤的重要分子生物学标签,并在基因表达调控中具有潜在的研究价值.     

Coexpression of stem cell factor and its receptor c-Kit in human malignant glioma cell lines

Stem cell factor (SCF), a hematopoietic growth factor, is the ligand of the tyrosine kinase receptor encoded by the c-kit proto-oncogene. Beside the important role of this receptor-ligand complex in hematopoiesis, gametogenesis and melanogenesis, SCF and its receptor have been shown to be expressed in the brain. We have studied the expression of SCF and c-kit in 20 human malignant glioma cell lines at the mRNA as well as at the protein level. In addition, recombinant human (rh) SCF was tested in [³H]thymidine uptake assays for a mitogenic effect on these cells. SCF and c-Kit proteins were detected in the cytoplasm of glioma cells by alkaline phoshatase-monoclonal anti-alkaline phosphatase immunostaining and Western blot analysis. However, neither SCF nor c-Kit were seen on the cell surface by flow cytometry. Furthermore, none of the proliferation assays showed a mitogenic effect for exogenously added rhSCF. Blocking studies using an anti-SCF antibody failed to demonstrate modulating effects on the growth of selected cell lines. These results suggest that SCF and c-Kit may mediate non-proliferative signals or may employ intracellular mechanisms for autocrine growth regulation of glioma cells.     

Neuritin基因在胶质瘤组织中的表达研究

目的了解neuritin基因在胶质瘤组织中的表达情况,探讨neuritin基因表达与胶质瘤发生发展的相关性。方法应用免疫组织化学测定42例脑组织石蜡标本,包括对照组7例,胶质瘤组25例（以Ⅲ-Ⅳ级为主）和癌旁组织组10例的neuritin表达情况,应用逆转录酶-多聚酶链反应（RT-PCR）方法检测39例脑组织新鲜标本,包括对照组7例,胶质瘤组20例（以Ⅲ-Ⅳ级为主）和癌旁组织组12例的mRNA表达。结果胶质瘤组neuritin表达和mRNA水平表达均明显高于对照组和癌旁组织组,差异有显著性（P〈0.01）,癌旁组织组与对照组间差异均无显著性（P〉0.05）。结论neuritin在胶质瘤组织和正常脑组织、癌旁组织表达存在显著性差异,提示neuritin表达可能与该肿瘤的发生发展相关。     

胶质瘤化疗药物相关标志分子在胶质瘤与正常脑组织中的丰度比较

目的检测并比较常用胶质瘤化疗药物相关标志分子在胶质瘤组织与正常脑组织中的丰度。方法检测并比较MGMT、ERCCl、TopoⅡα和Stathmin在46例不同病理级别胶质瘤标本及6例正常脑组织中的丰度,比较同一病理级别胶质瘤中这些标志分子的变异度。结果正常脑组织中MGMT和ERCCl的丰度显著高于各级别胶质瘤组织（P〈0．05）,而TopoⅡα的丰度则显著低于各级别胶质瘤组织（P〈0．05）,Stathmin的丰度在正常脑组织与各级别胶质瘤中均无显著性差异。这4种标志分子的丰度在不同级别胶质瘤之间差异均无统计学意义,而在同一级别胶质瘤中的变异度均较大。结论在胶质瘤组织中,MGMT、ERCCl、TopoⅡα和Stathmin的丰度在不同个体间波动很大,实行胶质瘤的个体化丰度检测和个体化化疗会更有益。     

Effect of human T lymphotropic retrovirus-I exposure on cultured human glioma cell lines

Summary Four different human tumor cell lines of glial origin have been exposed to a human T lymphotropic retrovirus (HTLV-I). All these cell lines were positive for the glial marker glial fibrillary acidic protein (GFAP). The presence of virus RNA was demonstrated by in situ hybridization using an HTLV-I, SStI-SStI viral insert as probe. Virus expression has been monitored through an indirect immunofluorescence assay using a monoclonal antibody against virus core protein p19. All the four glioma cell lines tested became positive for p19 after 2 weeks of co-cultivation and showed a clear alteration of GFAP expression.Supported in part by the Ministero della Sanità, Projects AIDS and by MURST 40% Grant     

Production of soluble autocrine inhibitory factors by human glioma cell lines

Gliomas in vitro exhibit density-limited growth upon the attainment of confluency, an effect usually attributed to cell-cell contact inhibition. Since gliomas have been demonstrated to secrete an array of soluble factors which can enhance tumor growth, we undertook this study to ascertain whether production of soluble factors by the tumor may also inhibit growth in an autocrine manner, and whether production of such factors is associated with the growth phase of the glioma. We observed that cell-conditioned medium (supernatants) from non-confluent glioma cultures induced growth, while confluent culture supernatants produced pronounced growth suppression. These latter supernatants enhanced proliferation of non-transformed astrocytes. Supernatants derived from all stages of confluency produced inhibition of lymphocyte proliferation. To characterize these factors, dialyzed supernatant was tested and found to continue to produce lymphocyte suppression but no glioma growth limitation. Growth of tumors in indomethacin or in acetylsalicylic acid to abolish prostanoid synthesis abrogated the inhibitory influence on glioma growth but only partially reversed the lymphocyte suppressive capacity. These studies suggest that gliomas do produce a growth phase dependent autocrine inhibitory factor(s), and that the production of these small molecular weight factors is at least partially under control of the cyclooxygenase pathway.     

c-Kit receptor expression in normal human Schwann cells and Schwann cell lines derived from neurofibromatosis type 1 tumors

The growth factor receptor c-Kit has several well-characterized functions during the development of numerous cell types, including red blood cells, mast cells, and melanocytes. Its role in Schwann cells has been described in transformed cells derived from malignant peripheral nerve sheath tumors from patients with neurofibromatosis type 1 (NF1 MPNST; Badache et al. [1998] Oncogene 17:795-800). However, c-Kit functions have not been investigated in normal Schwann cells. We report here that neonatal rat Schwann cells express low c-Kit levels, whereas expression levels for c-Kit are high for Schwann cells derived from MPNST of NF1 patients. In addition, c-Kit expression is not detectable in normal adult human Schwann cells. Although the c-Kit ligand stem cell factor (SCF) induces the phosphorylation of protein kinase B (or Akt) and prevents apoptosis in Schwann cells, SCF has no effect on the proliferation or differentiation of Schwann cells.     

血清剥夺法分离U87细胞系中的胶质瘤干细胞

背景：细胞周期分析已证实,90%以上的干细胞处于G0静止状态,因此采用不含任何生长因子和其他相关营养添加剂的血清剥夺培养基更适合筛选分离肿瘤干细胞。 目的：应用血清剥夺法培养胶质瘤U87细胞,并筛选鉴定该细胞系中的胶质瘤干细胞。 方法：将U87细胞培养在只含有DMEM和L-谷氨酰胺的培养基中培养6 d,筛选出胶质瘤干细胞,接着更换为神经干细胞培养基,观察细胞肿瘤球的形成过程;将肿瘤球接种于血清培养基,观察其在体外的分化特点;免疫荧光鉴定血清剥夺后尚存的细胞、增殖形成的肿瘤球细胞和分化细胞。 结果与结论：应用血清剥夺的方法成功地筛选出了表达CD133的肿瘤干细胞,并能增殖形成肿瘤球;肿瘤球可多向分化,子代细胞表达胶质纤维酸性蛋白和神经元特异性烯醇化酶。说明U87细胞系中存在具有自我更新及多向分化能力的胶质瘤干细胞。     

Telomerase subunits expression variation between biopsy samples and cell lines derived from malignant glioma

Although scientific advances have recognised the prognostic power of telomerase activity in different cancers, as yet there has been no investigation regarding the expression variation of telomerase subunits in glioma tissues and cell lines. In this study, a recurrent anaplastic ependymoma and seven glioblastoma biopsy samples, four cell lines and four controls including two normal brain tissues were analysed for telomerase subunit expression profiles together with telomerase activity. Since telomerase activity is linked to tumourgenesis, the genes were analysed with respect to their expression variation. TEP1 was expressed in all glioma cell lines and 70% of glioblastoma tissues, in addition to the control brain tissues. Tankyrase was expressed in 85% of the glioblastoma tissues and was down-regulated in the recurrent anaplastic ependymoma tissue control cell lines. However, it was expressed in the control tissues. Dyskerin was expressed in all cell lines and tissues apart from U87-MG and NHA cells and the recurrent anaplastic ependymoma tissue. As expected, PARP1 and GAPDH showed constitutive expression throughout all cell lines and tissues since both are known to be housekeeping genes. hTERT was expressed in all glioma cell lines and tissues but was absent in the control cells and tissues. Telomerase activity was absent in IPDDC-A2 cells and 57% of the glioblastoma tissues. These results suggest that hTERT expression and not telomerase activity possibly represents a simple and reliable biological diagnostic tool.