Dawson M J, Trapani J A
Cellular Cytotoxicity Laboratory, Austin Research Institute, Austin Hospital, Heidelberg, Australia.
J Cell Biochem. 1995 Jan;57(1):39-51. doi: 10.1002/jcb.240570106.
We have characterized the induction of mRNA and protein products of the human IFI 16 gene in response to IFN-gamma, IFN-alpha, and IFN-beta 2 (IL-6). We demonstrate that the IFI 16 gene product is a novel nucleoprotein expressed in association with the differentiation of myeloid precursor cell lines. In Northern blots, IFI 16 mRNA was increased approximately 25-fold above barely detectable levels in unstimulated promyelocytic HL-60 cells, in response to IFN-gamma. Other myeloid cell lines, U937 and K562, also demonstrated a marked IFN-gamma-inducibility of IFI 16 mRNA. However, all three cell lines were far less responsive to IFN-alpha, and there was no response to IL-6. By comparison, a panel of T and B cell lines demonstrated high constitutive expression of IFI 16 mRNA that was not regulated by these cytokines. Culture of HL-60 cells in medium containing dimethylsulfoxide, retinoic acid, and 1,25 dihydroxyvitamin D3, agents that stimulate the differentiation of HL-60 along myeloid pathways, also caused the induction of IFI 16 mRNA. To characterize the protein product of IFI 16, a monoclonal antibody was raised against a recombinant bacterial protein comprising the amino terminal 159 amino acids of IFI 16 fused to glutathione S-transferase. The antibody, designated 1G7, was used in Western blotting to demonstrate the strong induction of a cluster of proteins of 85-95 kDa in the nuclear extracts of IFN-gamma-treated HL-60. The nuclear localization of IFI 16 antigen was confirmed by immunohistochemical staining of HL-60 cells treated with IFN-gamma, dimethylsulfoxide, and retinoic acid. IFI 16 was also detected in the nuclei of monocytes, neutrophils, and lymphocytes in normal peripheral blood. Database comparisons of the IFI 16 amino acid sequence revealed 51% identity with the recently cloned myeloid cell nuclear differentiation antigen (MNDA), and extensive similarity to protein products of the Gene 200 cluster of IFN-inducible genes, Ifi 202 and Ifi 204. The amino terminal domain of IFI 16 encodes a putative nuclear localization signal, 124PGAQKRKK, which is strongly conserved in MNDA and 204. Nuclear IFI 16 was able to bind double-stranded DNA in vitro and exhibited a similar elution profile from DNA-cellulose as previously observed for MNDA and 204. Therefore, IFI 16 and MNDA are members of a novel family of human DNA-binding proteins whose expression is associated with myeloid cell differentiation induced by cytokines and chemical agents.
我们已对人IFI 16基因的mRNA和蛋白质产物在干扰素-γ、干扰素-α和干扰素-β2（白细胞介素-6）作用下的诱导情况进行了表征。我们证明IFI 16基因产物是一种新型核蛋白，其表达与髓系前体细胞系的分化相关。在Northern印迹分析中，干扰素-γ作用下，未刺激的早幼粒细胞HL-60细胞中IFI 16 mRNA水平从几乎检测不到的水平增加了约25倍。其他髓系细胞系U937和K562也显示出IFI 16 mRNA对干扰素-γ的显著诱导性。然而，这三种细胞系对干扰素-α的反应要弱得多，对白细胞介素-6则无反应。相比之下，一组T和B细胞系显示出IFI 16 mRNA的高组成性表达，且不受这些细胞因子的调节。在含有二甲基亚砜、视黄酸和1,25-二羟基维生素D3的培养基中培养HL-60细胞，这些试剂可刺激HL-60沿髓系途径分化，也会导致IFI 16 mRNA的诱导。为了表征IFI 16的蛋白质产物，制备了一种针对重组细菌蛋白的单克隆抗体，该重组细菌蛋白由IFI 16的氨基末端159个氨基酸与谷胱甘肽S-转移酶融合而成。命名为1G7的抗体用于蛋白质印迹分析，以证明在干扰素-γ处理的HL-60细胞核提取物中85 - 95 kDa的一组蛋白质被强烈诱导。通过对用干扰素-γ、二甲基亚砜和视黄酸处理的HL-60细胞进行免疫组织化学染色，证实了IFI 16抗原的核定位。在正常外周血的单核细胞、中性粒细胞和淋巴细胞的细胞核中也检测到了IFI 16。对IFI 16氨基酸序列进行数据库比较发现，它与最近克隆的髓系细胞核分化抗原（MNDA）有51%的同一性，与干扰素诱导基因200簇的蛋白质产物Ifi 202和Ifi 204有广泛的相似性。IFI 16的氨基末端结构域编码一个假定的核定位信号124PGAQKRKK，在MNDA和204中高度保守。核IFI 16能够在体外结合双链DNA，并且从DNA-纤维素上洗脱的图谱与之前观察到的MNDA和204相似。因此，IFI 16和MNDA是一类新型人类DNA结合蛋白家族的成员，其表达与细胞因子和化学试剂诱导的髓系细胞分化相关。
