Salnikow K, Cosentino S, Klein C, Costa M
Department of Environmental Medicine, New York University Medical Center, New York.
Mol Cell Biol. 1994 Jan;14(1):851-8. doi: 10.1128/mcb.14.1.851-858.1994.
mRNA from normal Chinese hamster embryo (CHE) cells was transcribed to cDNA and subtracted with an excess of mRNA from Chinese hamster embryo cells transformed by nickel compounds. Here we report the recovery of a sequence found to be highly homologous to the mouse thrombospondin 1 gene that was obtained by this subtraction procedure. Since thrombospondin is antiangiogenic, cancer cells expressing high levels of thrombospondin cannot grow in vivo because capillaries will not proliferate to cells secreting thrombospondin. To examine expression of thrombospondin, normal CHE cells were stained with monoclonal antibodies to human thrombospondin. The protein was present abundantly in the cytoplasm of normal cells but at greatly reduced levels in Ni-transformed cells. Analysis of mRNA by Northern (RNA) blot revealed transcripts in normal cells but little thrombospondin mRNA in Ni-transformed cells. Loss of thrombospondin mRNA expression was related to Ni treatment rather than transformation, since Ni-resistant cells also exhibited fewer thrombospondin transcripts than did wild-type cells. Digestion of genomic DNA with various combinations of restriction enzymes revealed thrombospondin gene patterns that were identical in both cell types, suggesting that there were no major deletions or rearrangements of the gene in the nickel-transformed cells. The inactivation of the thrombospondin gene was further investigated by analyzing the promoter activity of this gene linked to a chloramphenicol acetyltransferase (CAT) reporter plasmid that was transfected into normal and Ni-transformed cells. The CAT activity in normal cells was significantly higher than in Ni-transformed cells, suggesting that the promoter region of thrombospondin was less efficiently transcribed in Ni-transformed cells. We studied the consequences of enhanced expression of the retinoblastoma (Rb) gene, a known tumor suppressor gene, on CAT transcription driven by the human thrombospondin promoter. Cotransfection of an expression vector containing the mouse Rb gene greatly enhanced the transcription from the thrombospondin promoter such that the expression was higher in normal cells than in transformed cells.
来自正常中国仓鼠胚胎（CHE）细胞的mRNA被转录成cDNA，并与过量的由镍化合物转化的中国仓鼠胚胎细胞的mRNA进行消减杂交。在此我们报告通过这种消减程序获得了一个与小鼠血小板反应蛋白1基因高度同源的序列。由于血小板反应蛋白具有抗血管生成作用，表达高水平血小板反应蛋白的癌细胞在体内无法生长，因为毛细血管不会向分泌血小板反应蛋白的细胞增殖。为了检测血小板反应蛋白的表达，用抗人血小板反应蛋白的单克隆抗体对正常CHE细胞进行染色。该蛋白在正常细胞的细胞质中大量存在，但在镍转化细胞中的水平大大降低。通过Northern（RNA）印迹分析mRNA发现正常细胞中有转录本，但镍转化细胞中血小板反应蛋白mRNA很少。血小板反应蛋白mRNA表达的丧失与镍处理有关而非转化，因为镍抗性细胞也比野生型细胞表现出更少的血小板反应蛋白转录本。用各种限制性酶组合消化基因组DNA显示两种细胞类型中血小板反应蛋白基因模式相同，这表明在镍转化细胞中该基因没有重大缺失或重排。通过分析与氯霉素乙酰转移酶（CAT）报告质粒连接的该基因的启动子活性，进一步研究了血小板反应蛋白基因的失活情况，该报告质粒被转染到正常细胞和镍转化细胞中。正常细胞中的CAT活性明显高于镍转化细胞，这表明血小板反应蛋白的启动子区域在镍转化细胞中的转录效率较低。我们研究了已知的肿瘤抑制基因视网膜母细胞瘤（Rb）基因表达增强对由人血小板反应蛋白启动子驱动的CAT转录的影响。含有小鼠Rb基因的表达载体的共转染极大地增强了血小板反应蛋白启动子的转录，使得正常细胞中的表达高于转化细胞。
