Mufson R A, Myers C, Turpin J A, Meltzer M
Cell Biology Department, Jerome H. Holland Laboratory, American Red Cross, Rockville, MD 20855.
J Leukoc Biol. 1992 Dec;52(6):637-44. doi: 10.1002/jlb.52.6.637.
NF kappa B is a potent mediator of specific gene expression in human monocytes and has been shown to play a role in transcription of the HIV-1 genome in promonocytic leukemias. There is little information available on the response of NF kappa B to cytokines in normal human monocytes. We have used a 32P-labeled oligonucleotide derived from human immunodeficiency virus (HIV-1) long terminal repeat, which contains a tandem repeat of the NF kappa B binding sequence, as a probe in a gel retardation assay to study this transcription factor. Using this assay, we have detected NF kappa B in extracts of nuclei from normal human monocytes. Treatment of normal monocytes with 12-0-tetradecanoyl phorbol-13-acetate (TPA) for 4-24 h caused the complete disappearance of NF kappa B from nuclear extracts of monocytes. A similar result was obtained with the mature monocytic leukemia cell line THP-1. The constitutive transcription factor SP1 was unaffected by addition of TPA. The disappearance of NF kappa B from the nucleus was concentration dependent between 10 and 50 ng/ml of phorbol ester. In THP-1 cells, TPA also induced a new, faster-migrating NF kappa B species not induced in monocytes. Protein kinase C inhibitor staurosporine, but not cyclic nucleotide-dependent protein kinase inhibitor HA-1004, also dramatically reduced constitutive levels of nuclear NF kappa B. Finally, TPA addition to monocytes infected with HIV-1 inhibited HIV-1 replication, as determined by reverse transcriptase assays, in a concentration-dependent manner. These results are in striking contrast to the increase in nuclear NF kappa B and HIV-1 replication induced by phorbol esters in promonocytic leukemia cells U937 and HL-60, and emphasize the importance of studying cytokine regulation of HIV-1 in normal monocytes.
核因子κB是人类单核细胞中特异性基因表达的强效调节因子，且已证实在原单核细胞白血病中它在HIV-1基因组转录过程中发挥作用。关于正常人类单核细胞中核因子κB对细胞因子的反应，目前所知甚少。我们使用了一种来源于人类免疫缺陷病毒（HIV-1）长末端重复序列的32P标记寡核苷酸作为凝胶阻滞试验中的探针来研究这种转录因子，该寡核苷酸包含核因子κB结合序列的串联重复。通过这个试验，我们在正常人单核细胞核提取物中检测到了核因子κB。用12-0-十四酰佛波醇-13-乙酸酯（TPA）处理正常人单核细胞4 - 24小时，导致单核细胞核提取物中的核因子κB完全消失。成熟的单核细胞白血病细胞系THP-1也得到了类似结果。组成型转录因子SP1不受TPA添加的影响。在10至50 ng/ml佛波酯浓度范围内，核因子κB从细胞核中的消失呈浓度依赖性。在THP-1细胞中，TPA还诱导出一种新的、迁移速度更快的核因子κB，而在单核细胞中未诱导出这种核因子κB。蛋白激酶C抑制剂星形孢菌素可显著降低核因子κB的组成型水平，而环核苷酸依赖性蛋白激酶抑制剂HA-1004则无此作用。最后，通过逆转录酶测定发现，向感染HIV-1的单核细胞中添加TPA会以浓度依赖性方式抑制HIV-1复制。这些结果与佛波酯在原单核细胞白血病细胞U937和HL-60中诱导核因子κB增加及HIV-1复制形成鲜明对比，强调了研究正常单核细胞中HIV-1细胞因子调节的重要性。
