Qian X, Jin L, Kulig E, Lloyd R V
Department of Laboratory Medicine and Pathology, Mayo Clinic and Mayo Foundation, Rochester, Minnesota, USA.
Am J Pathol. 1998 Nov;153(5):1475-82. doi: 10.1016/S0002-9440(10)65735-5.
We previously reported loss of expression of p27Kip1 (p27) protein in rat GH3 and mouse GHRH-CL1 pituitary tumor cells compared with normal pituitary (NP). The molecular basis for the loss of expression of p27 protein in GH3 and GHRH-CL1 cells is unknown. To determine the role of p27 gene methylation in the regulation of the expression of this cell cycle protein, the methylation patterns of p27 in normal and neoplastic pituitary cells was analyzed. Inhibition of DNA methyltransferase (DNA-MTase) with 5-aza-2'-deoxycytidine (AZAdC) induced expression of both p27 protein and mRNA when GH3 and GHRH-CL1 cells were treated for 7 days in vitro. DNA methylation correlated inversely with the expression of p27 gene products in NP and pituitary tumor cell lines. Bisulfite genomic sequencing analysis showed that the normally unmethylated cytosines in exon 1 in NP and AtT20 cells were extensively methylated in GH3 and GHRH-CL1 cells. After treatment of GH3 and GHRH-CL1 cells with 10 micromol/L AZAdC, there were decreased numbers of methylated cytosines (by 60% to 90%/o) with variable methylation patterns observed by bisulfite genomic sequencing. Analysis of genomic DNA with methylation-sensitive enzymes showed that all SmaI, HhaI, and AvaI enzyme sites of the p27 gene in exon 1 were methylated in GH3 cells but not in NP, confirming the bisulfite genomic sequencing results. AtT20 cells and a human pituitary null cell adenoma cell line (HP75), which expressed abundant p27, had a methylation pattern similar to the NP. DNA-MTase activity was elevated fourfold in GH3 cells and twofold in GHRH-CL1 cells compared with DNA-MTase activity in NP and AtT20 cells. These results suggest that increased DNA methylation is another mechanism of silencing of the p27 gene in some pituitary tumors and possibly in other types of neoplasms.
我们之前报道过，与正常垂体（NP）相比，大鼠GH3和小鼠GHRH-CL1垂体肿瘤细胞中p27Kip1（p27）蛋白表达缺失。GH3和GHRH-CL1细胞中p27蛋白表达缺失的分子基础尚不清楚。为了确定p27基因甲基化在调控这种细胞周期蛋白表达中的作用，我们分析了正常和肿瘤性垂体细胞中p27的甲基化模式。当GH3和GHRH-CL1细胞在体外处理7天时，用5-氮杂-2'-脱氧胞苷（AZAdC）抑制DNA甲基转移酶（DNA-MTase）可诱导p27蛋白和mRNA的表达。DNA甲基化与NP和垂体肿瘤细胞系中p27基因产物的表达呈负相关。亚硫酸氢盐基因组测序分析表明，NP和AtT20细胞中外显子1中正常未甲基化的胞嘧啶在GH3和GHRH-CL1细胞中被广泛甲基化。用10微摩尔/升AZAdC处理GH3和GHRH-CL1细胞后，亚硫酸氢盐基因组测序观察到甲基化胞嘧啶数量减少（减少60%至90%），甲基化模式各异。用甲基化敏感酶分析基因组DNA表明，GH3细胞中外显子1中p27基因的所有SmaI、HhaI和AvaI酶切位点均被甲基化，而NP中未被甲基化，这证实了亚硫酸氢盐基因组测序结果。AtT20细胞和人垂体无功能细胞腺瘤细胞系（HP75）表达丰富的p27，其甲基化模式与NP相似。与NP和AtT20细胞中的DNA-MTase活性相比，GH3细胞中的DNA-MTase活性升高了四倍，GHRH-CL1细胞中的升高了两倍。这些结果表明，DNA甲基化增加是某些垂体肿瘤以及可能其他类型肿瘤中p27基因沉默的另一种机制。
