Identification of genes differentially expressed in V79 cells grown as multicell spheroids
Identification of genes differentially expressed in V79 cells grown as multicell spheroids
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DOI：
10.1080/09553000210122299
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发表时间：
2002-06-01
影响因子：
2.6
通讯作者：
Olive, PL
中科院分区：
文献类型：
--
作者：
Oloumi, A;Lam, W;Olive, PL
目的：中国仓鼠V79细胞呈多细胞球体生长，可增强对电离辐射和依托泊苷的杀伤能力。差异显示技术用于鉴定细胞生长为球体时基因表达的变化。材料和方法：以指数生长的中国仓鼠V79细胞和V79球体外层细胞层为材料，进行差异显示。利用6对不同的引物，共筛选出20条变异条带。使用反向Northerns确认的八个基因在GenBank搜索中显示匹配。抗钙结合蛋白mts1的抗体证实了该蛋白的差异表达。结果：球体外层相对单层表达上调的基因包括：(1)参与细胞增殖、转移、细胞黏附和血管生成的钙结合蛋白mts1(S100A4)；(2)细胞色素C氧化酶II；(3)B-ind1，RAC-1信号的介导者；(4)trm，内质网蛋白。球体中下调的基因有：(5)磷酸甘油酸激酶；(6)与ras相关的GTP结合蛋白ARL-3；(7)MHC III类补体4A；和(8)2，4-二烯酰辅酶A。免疫组织化学证实Mts1和另一种钙结合蛋白钙网状蛋白在V79外球状细胞中相对于单层细胞过表达。表现出接触效应的C6大鼠胶质瘤和SIHA人宫颈癌细胞也表现出mts1或钙网蛋白的上调，而缺乏接触抗性的WiDR结肠癌细胞中这两种钙结合蛋白的表达没有变化。与单层相比，V79球体外层细胞内游离钙水平几乎低两倍。钙离子螯合剂BAPTA-AM对V79单层细胞和外球细胞的DNA损伤程度与依托泊苷相似。结论：当V79细胞生长为球状细胞时，参与钙结合、信号和代谢的基因表达存在差异。细胞内钙离子水平的差异可能是接触效应的基础。
Purpose: Growth of Chinese hamster V79 cells as multicell spheroids leads to an increase in resistance to killing by ionizing radiation and etoposide. Differential display was used to identify changes in gene expression that occur when cells are grown as spheroids.Materials and methods: Differential display was performed using exponentially growing Chinese hamster V79 cells and the outer cell layer of V79 spheroids. Using six different pairs of primers, 20 altered bands were selected. Eight genes, confirmed using reverse Northerns, showed a match in a GenBank search. Antibodies against a calcium-binding protein, mts1, confirmed differential expression of this protein. Intracellular free calcium levels were measured using fluo-3 fluorescence, and the effect of a calcium-binding agent on etoposide resistance was examined using the comet assay.Results: Genes upregulated in the outer cell layer of spheroids relative to monolayers included: (1) mts1 (S100A4), a calcium-binding protein implicated in proliferation, metastasis, cell adhesion, and angiogenesis; (2) cytochrome c oxidase II; (3) B-ind1, a mediator of Rac-1 signaling; (4) TRAM, an endoplasmic reticulum protein. Genes downregulated in spheroids were: (5) phosphoglycerate kinase; (6) ARL-3, a ras-related GTP binding protein; (7) MHC class III complement 4A; and (8) 2,4-dienoyl-CoA. Immunohistochemistry confirmed overexpression of mts1 and another calcium-binding protein, calreticulin, in V79 outer spheroid cells relative to monolayers. C6 rat glioma and SiHa human cervical carcinoma cells that demonstrate a contact effect also showed upregulation of mts1 or calreticulin, while WiDr colon carcinoma cells that lack contact resistance showed no change in expression of either calcium binding protein. Intracellular free calcium levels were found to be almost two times lower in the outer cells of V79 spheroids compared to monolayers. V79 monolayer and outer spheroid cells treated with the calcium chelating agent BAPTA-AM showed a similar level of DNA damage by etoposide.Conclusions : Expression of genes involved in calcium binding, signaling and metabolism are differentially expressed when V79 cells are grown as spheroids. Differences in the levels of intracellular calcium may underlie the contact effect.