Chauhan D, Uchiyama H, Akbarali Y, Urashima M, Yamamoto K, Libermann T A, Anderson K C
Division of Hematologic Malignancies, Dana-Farber Cancer Institute, Boston, MA 02115, USA.
Blood. 1996 Feb 1;87(3):1104-12.
Adhesion of multiple myeloma (MM) cells to bone marrow stromal cells (BMSCs) not only localizes MM cells in the marrow microenvironment, but also triggers interleukin-6 (IL-6) secretion by BMSCs and related MM cell proliferation. In the present study, we characterized the regulation of IL-6 gene expression in BMSCs during MM cell adhesion. Adhesion of ARH-77, HS-Sultan, IM-9, and U266 MM cell lines to BMSCs and BMSC lines (LP 101 and AA 101) triggered 5-through 15-fold and 2-through 4-fold increases in IL-6 secretion, respectively. IL-6 mRNA transcripts were undetectable by Northern blotting in IM-9 MM cells or LP 101 BMSCs cultured alone; however, adherence of IM-9 cells to LP 101 cells induced a transient increase in IL-6 transcripts at 6 hours, followed by peak IL-6 secretion at 24 hours. To confirm increased IL-6 transcription and characterize its regulation, LP101 BMSCs were transiently transfected with full length and deletion fragments of the IL-6 promoter linked to the chloramphenicol acetyltransferase (CAT) reporter gene. Transient transfection of LP101 BMSCs with plasmid containing an intact NF-kappa B site showed a 6.8 +/- 0.4-fold increase in CAT activity triggered by IM-9 MM cell adhesion (n = 3, P < .05). Transfection of LP 101 cells with plasmid containing a single base pair deletion from the NF-kapp B binding motif abolished the MM adhesion-induced increase in CAT activity, whereas transfection with plasmid containing three copies of synthetic NF-kappa B sequence resulted in an 8.1 +/- 0.7-fold increase in CAT activity related to MM adhesion (n = 3, P < .05). These data suggest that the NF-kappa B site is one of the essential regulatory elements for MM cell adhesion-induced IL-6 transcription in BMSCs. Electrophoretic mobility shift assays confirmed the involvement of NF-kappa B activation in regulating MM adhesion-induced IL-6 transcription in BMSCs. Further characterization of the upstream events in the signalling cascade regulating IL-6 may not only delineate mechanisms of IL-6 regulation during paracrine MM cell growth, but also provide new therapeutic strategies based on interruption of IL-6 mediated tumor cell growth.
多发性骨髓瘤（MM）细胞与骨髓基质细胞（BMSC）的黏附不仅使MM细胞定位于骨髓微环境中，还能触发BMSC分泌白细胞介素-6（IL-6）以及相关的MM细胞增殖。在本研究中，我们对MM细胞黏附过程中BMSC内IL-6基因表达的调控进行了表征。ARH-77、HS-Sultan、IM-9和U266 MM细胞系与BMSC及BMSC系（LP 101和AA 101）的黏附分别使IL-6分泌增加了5至15倍和2至4倍。单独培养的IM-9 MM细胞或LP 101 BMSC中，通过Northern印迹法无法检测到IL-6 mRNA转录本；然而，IM-9细胞与LP 101细胞的黏附在6小时时诱导IL-6转录本短暂增加，随后在24小时时出现IL-6分泌峰值。为了证实IL-6转录增加并表征其调控机制，将与氯霉素乙酰转移酶（CAT）报告基因相连的IL-6启动子全长和缺失片段瞬时转染至LP101 BMSC。用含有完整NF-κB位点的质粒瞬时转染LP101 BMSC后，IM-9 MM细胞黏附触发CAT活性增加了6.8±0.4倍（n = 3，P <.05）。用含有NF-κB结合基序单碱基对缺失的质粒转染LP 101细胞，消除了MM黏附诱导的CAT活性增加，而用含有三个合成NF-κB序列拷贝的质粒转染导致与MM黏附相关的CAT活性增加了8.1±0.7倍（n = 3，P <.05）。这些数据表明，NF-κB位点是MM细胞黏附诱导BMSC中IL-6转录的必需调控元件之一。电泳迁移率变动分析证实了NF-κB激活参与调节MM黏附诱导的BMSC中IL-6转录。对调控IL-6的信号级联上游事件的进一步表征不仅可能阐明旁分泌MM细胞生长过程中IL-6调控的机制，还可能提供基于中断IL-6介导的肿瘤细胞生长的新治疗策略。
