Wahlfors J
Department of Biochemistry and Biotechnology, University of Kuopio, Finland.
Biochem J. 1991 Oct 15;279 ( Pt 2)(Pt 2):435-40. doi: 10.1042/bj2790435.
The ornithine decarboxylase (ODC; EC 4.1.1.17) gene in parental, dexamethasone-resistant and 2-difluoromethylornithine (DFMO)-resistant human IgG-myeloma-cell lines was studied with the aid of methylation-sensitive restriction endonucleases and probes recognizing different parts of the gene. In all cell lines the promoter region of the ODC gene appeared to be heavily methylated, whereas the first long intron was unmethylated. Methylation analyses of several clones from the parental cell line revealed that these cells are heterogeneous with respect to the methylation status of the ODC gene, whereas all clones from DFMO-resistant cell lines displayed the same methylation pattern. Two of the parental clones represented a hypomethylated type very close to that exclusively found among the DFMO-resistant clones with ODC gene amplification. This typical methylation pattern was due to decreased methylation of a few CCGG sequences in the 3'-flanking region of the gene. It is possible that this kind of hypomethylation favours the initiation of the gene-amplification process in certain individual cells. This hypothesis was supported by the finding that no hypomethylation was present in the ODC gene of another human myeloma cell line that had acquired resistance to DFMO without gene amplification. In a dexamethasone-resistant cell line that overproduced ODC mRNA at normal gene dosage there were some minor differences between the methylation pattern of the ODC gene of different clones, but no such hypomethylation could be found in clones from the parental cell line. In dexamethasone-resistant cells the ODC gene was hypomethylated around the two HpaII sites and three CfoI sites in the coding region and also, as well as in cells with amplified ODC sequences, in the 3'-flanking region of the gene. Some hypomethylation in the distant 5'-flanking region was also observed.
借助甲基化敏感的限制性内切酶和识别该基因不同部分的探针，对亲本、地塞米松抗性和2-二氟甲基鸟氨酸（DFMO）抗性的人IgG骨髓瘤细胞系中的鸟氨酸脱羧酶（ODC；EC 4.1.1.17）基因进行了研究。在所有细胞系中，ODC基因的启动子区域似乎高度甲基化，而第一个长内含子未甲基化。对亲本细胞系的几个克隆进行甲基化分析表明，这些细胞在ODC基因的甲基化状态方面是异质的，而来自DFMO抗性细胞系的所有克隆都表现出相同的甲基化模式。亲本克隆中的两个代表了一种低甲基化类型，与在具有ODC基因扩增的DFMO抗性克隆中唯一发现的类型非常接近。这种典型的甲基化模式是由于该基因3'侧翼区域中一些CCGG序列的甲基化减少所致。这种低甲基化可能有利于某些单个细胞中基因扩增过程的启动。这一假设得到了以下发现的支持：另一种对DFMO产生抗性但未进行基因扩增的人骨髓瘤细胞系的ODC基因中不存在低甲基化。在一个以正常基因剂量过量产生ODC mRNA的地塞米松抗性细胞系中，不同克隆的ODC基因甲基化模式之间存在一些细微差异，但在亲本细胞系的克隆中未发现这种低甲基化。在地塞米松抗性细胞中，ODC基因在编码区的两个HpaII位点和三个CfoI位点周围以及在具有扩增的ODC序列的细胞中在基因的3'侧翼区域均发生低甲基化。在较远的5'侧翼区域也观察到了一些低甲基化。
