title: Multiple myeloma cell adhesion-induced interleukin-6 expression in bone marrow stromal cells involves activation of NF-kappa B.
abstract: 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.
authors.affiliation: Division of Hematologic Malignancies, Dana-Farber Cancer Institute, Boston, MA 02115, USA.
authors.forename: D
authors.initials: D
authors.lastname: Chauhan
authors.forename: H
authors.initials: H
authors.lastname: Uchiyama
authors.forename: Y
authors.initials: Y
authors.lastname: Akbarali
authors.forename: M
authors.initials: M
authors.lastname: Urashima
authors.forename: K
authors.initials: K
authors.lastname: Yamamoto
authors.forename: T A
authors.initials: TA
authors.lastname: Libermann
authors.forename: K C
authors.initials: KC
authors.lastname: Anderson
mesh_terms: D001483:Base Sequence; D001853:Bone Marrow / Q000378:metabolism*; D001854:Bone Marrow Cells; D002448:Cell Adhesion / Q000502:physiology; D002478:Cells, Cultured; D018920:Coculture Techniques; D003238:Connective Tissue / Q000378:metabolism*; D003239:Connective Tissue Cells; D005786:Gene Expression Regulation; D006801:Humans; D015850:Interleukin-6 / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D009101:Multiple Myeloma / Q000473:pathology*; D016328:NF-kappa B / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D015850:Interleukin-6; D016328:NF-kappa B; D009363:Neoplasm Proteins; D011993:Recombinant Fusion Proteins