Carrington M N, Chedid M, Ting J P, Ward F E
Hum Immunol. 1987 Feb;18(2):151-61. doi: 10.1016/0198-8859(87)90012-7.
Melanoma cell lines treated with or without interferon-gamma were tested for the presence of DR alpha mRNA and protein. The six lines examined fell into three general categories: two that expressed high levels of DR alpha mRNA and protein before and after interferon-gamma treatment, one that expressed very low levels before treatment with interferon-gamma, but was induced to express high levels after interferon-gamma treatment, and three that expressed very low levels before treatment, and were only slightly inducible after treatment with interferon-gamma. The presence of DR-alpha protein on the melanoma cell surface was always positively correlated with the presence of DR alpha mRNA in the cells. Furthermore, in the cell line that was interferon-gamma-inducible, the time at which DR alpha mRNA and protein appeared and the doses of interferon-gamma needed to induce this appearance were directly correlated. Methylation patterns of the DR alpha gene in these cell lines were also studied in order to determine whether the degree of DR alpha gene methylation among the lines correlated with expression of the gene. Digestion of DNA with the restriction enzyme MspI, which recognizes the sequence 5'CCGG3' and 5'CmCGG3', led to the appearance of a 3.1 kb band from all lines tested. Hpa II digestion, which recognizes 5'CCGG3', but not 5'CmCGG3', led to the appearance of 3.1, 4.4, and 6.7 kb bands in all lines tested except for DUMEL 8, which showed only the 3.1 kb band. Interestingly, DUMEL 8 expressed very low levels of DR alpha mRNA and protein before and after interferon-gamma treatment. We conclude that interferon-gamma has a regulatory effect on DR alpha genes of various melanoma cell lines to varying degrees. This may reflect an effect of interferon-gamma on certain subpopulations of melanocytes in vivo. Our data also indicate that partial methylation of the DR alpha gene does not inhibit its expression. Furthermore, interferon-gamma does not appear to induce expression of the DR gene by altering methylation patterns within the region recognized by our probe.
对用或未用γ干扰素处理的黑色素瘤细胞系进行了DRα mRNA和蛋白的检测。所检测的六个细胞系大致分为三类：两个在γ干扰素处理前后均表达高水平的DRα mRNA和蛋白；一个在γ干扰素处理前表达水平极低，但在γ干扰素处理后被诱导表达高水平；三个在处理前表达水平极低，在γ干扰素处理后仅有轻微诱导作用。黑色素瘤细胞表面DR-α蛋白的存在始终与细胞内DRα mRNA的存在呈正相关。此外，在γ干扰素可诱导的细胞系中，DRα mRNA和蛋白出现的时间以及诱导其出现所需的γ干扰素剂量直接相关。还研究了这些细胞系中DRα基因的甲基化模式，以确定各细胞系中DRα基因的甲基化程度是否与该基因的表达相关。用识别序列5'CCGG3'和5'CmCGG3'的限制性内切酶MspI消化DNA，导致所有测试细胞系均出现一条3.1 kb的条带。用识别5'CCGG3'但不识别5'CmCGG3'的Hpa II消化，除DUMEL 8仅显示3.1 kb条带外，所有测试细胞系均出现3.1、4.4和6.7 kb的条带。有趣的是，DUMEL 8在γ干扰素处理前后均表达极低水平的DRα mRNA和蛋白。我们得出结论，γ干扰素对各种黑色素瘤细胞系的DRα基因有不同程度的调节作用。这可能反映了γ干扰素对体内某些黑素细胞亚群的作用。我们的数据还表明，DRα基因的部分甲基化并不抑制其表达。此外，γ干扰素似乎不是通过改变我们探针所识别区域内的甲基化模式来诱导DR基因的表达。
